EIGHTEENTH EDITION

EC I
MA
Mark K. Fung
Brenda J. Grossman
Christopher D. Hillyer
Connie M. Westhof
Technical
Manual
1 8 T H E D I T I O N
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Blood Transfusion Therapy: A Physician’s Handbook, 10th edition
Edited by Karen King, MD
Judd’s Methods in Immunohematology, 3rd edition
By John W. Judd, FIBMS; Susan T. Johnson, MSTM, MT(ASCP)SBB; and Jill Storry, PhD, FIBMS
Antibody Identification: Art or Science? A Case Study Approach
By Janis R. Hamilton, MS, MT(ASCP)SBB; Susan T. Johnson, MSTM, MT(ASCP)SBB;
and Sally V. Rudmann, PhD, MT(ASCP)SBB

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Technical
Manual
1 8 T H E D I T I O N

E di te d b y

Mark K. Fung, MD,

PhD Fletcher Allen
Health Care Burlington,
VT

Brenda J. Grossman, MD, MPH

Washington University School of
Medicine St. Louis, MO

Christopher D. Hillyer,
MD New York Blood
Center New York, NY

Connie M. Westhoff, PhD, SBB

New York Blood Center
New York, NY
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AABB ISBN No. 978-1-56395-888-5

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Cataloging-in-Publication Data

Technical manual / editor, Mark K. Fung—18th ed.

p. ; cm.
Including bibliographic references and index.
ISBN 978-1-56395-888-5
1. Blood Banks—Handbooks, manuals, etc. I. Fung, Mark K. II.
AABB. [DNLM: 1. Blood Banks-laboratory manuals. 2. Blood
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RM172.T43 2014 615’.39—
dc23 DNLM/DLC
 Technical Manual
Authors
Colleen A. Aronson, MT(ASCP)SBB
Aleksandar M. Babic, MD, PhD
Robert A. Bray, PhD
Laura Cooling, MD, MS
Brian R. Curtis, PhD, D(ABMLI),
MT(ASCP)SBB
Robertson D. Davenport, MD
Meghan Delaney, DO, MPH
Gregory A. Denomme, PhD, FCSMLS(D)
Katharine A. Downes, MD
Larry J. Dumont, MBA, PhD
Deborah F. Dumont, MT(ASCP)SBB
Nancy M. Dunbar, MD
Anne F. Eder, MD, PhD
William FitzGerald, LTC USA (Ret)
Susan A. Galel, MD
Howard M. Gebel, PhD
Mary Ghiglione, RN, MSN, MHA
Janis R. Hamilton, MS, MT(ASCP)SBB
Jeanne E. Hendrickson, MD
Eapen K. Jacob, MD
Shweta Jain, MD
Yameena Jawed,
MD
Betsy W. Jett, MT(ASCP), CQA(ASQ)CQM/OE
Brian Johnstone, PhD
Cassandra D. Josephson, MD
Melanie S. Kennedy, MD
Scott A. Koepsell, MD, PhD
Mickey B. C. Koh, MD, PhD
Patricia M. Kopko, MD
Regina M. Leger, MSQA, MT(ASCP)SBB,
CMQ/ OE(ASQ)
Christine Lomas-Francis, MSc, FIBMS
Keith March, MD, PhD
Kim Maynard, BSN, RN, OCN
Catherine A. Mazzei, MD
David H. McKenna Jr, MD
Erin Meyer, DO, MPH
Tania L. Motschman, MS, MT(ASCP)SBB,
CQA(ASQ)
Maria D.L.A. Muniz,
MD Theresa Nester, MD
Mona Papari, MD
Jessica Poisson, MD
Marilyn S. Pollack, PhD
Mark A. Popovsky, MD
Kathleen E. Puca, MD, MT(ASCP)SBB
Glenn Ramsey, MD
Donna M. Regan, MT(ASCP)SBB
Rita A. Reik, MD
Edward R. Samuel, PhD, MSc
Scott Scrape, MD
Ira A. Shulman, MD
James W. Smith, MD,
PhD Steven L. Spitalnik,
MD
Simon Stanworth, FRCP, FRCPath, DPhil
Jill R. Storry, PhD, FIBMS
Garnet Suck, PhD, MSc
Ruth D Sylvester, MS, MT(ASCP)SBB
Sreedhar Thirumala, PhD
Alan Tinmouth, MD, FRCPC,
MSc Christopher A. Tormey, MD
Lance D. Trainor, MD
Wendy Trivisonno
Phyllis S. Walker, MS,
MT(ASCP)SBB Connie M. Westhoff,
PhD, SBB Theresa Wiegmann, JD
Susan L. Wilkinson, EdD, MS, MT(ASCP)SBB
James C. Zimring, MD, PhD
 Acknowledgments
HE 1 8 T H E D I T I O N OF the Technical
T Manual was the work of many dedicated
individuals. In addition to the chapter
authors, I would like to thank my three
associate edi- tors: Brenda Grossman, Chris
Hillyer, and Connie Westhoff. Their efforts
and long hours in revising and rewriting
chapters during the review process made
my job immeasurably
easier.
We would also like to acknowledge the
members of the following committees and
program units for their expert review of chap-
ters, methods, and appendices for the 18th
edition of the Technical Manual.
REVIEWING GROUPS
AABB Representative to ASFA
AATB Representative
Circular of Information Task Force
Clinical Transfusion Medicine Committee
Cord Blood Subsection of the Cellular Thera-
pies Section
Donor Center Accreditation Program Unit
Donor History Task Force
Immunohematology Reference Laboratories
Accreditation Program Unit
Immunohematology Reference Laboratories
Standards Program Unit
Information Systems Committee
Molecular Testing Laboratories Accreditation
Program Unit
Molecular Testing Laboratories Standards Pro-
gram Unit
Novel Therapies and CT Product
Development Subsection of the Cellular
Therapies Section
Patient Blood Management Advisory Group
Perioperative Accreditation Program Unit
Perioperative Standards Program Unit
Product Collection and Clinical Practices
Subsection of the Cellular Therapies
Section Product Manufacturing and Testing
Subsec-
tion of the Cellular Therapies Section
Quality Operations Subsection of the
Cellular
Therapies Section
Quality Systems Accreditation Subcommittee
Regulatory Affairs Subsection of the Cellular
Therapies Section
Relationship Testing Accreditation Program
Unit
Relationship Testing Standards Program Unit
Transfusion-Transmitted Disease Committee
Finally, we would like to thank the
editors, authors, and program unit members
of the 17th and earlier editions of the
Technical Man- ual for selected tables,
figures, methods, and written sections of the
chapters that are valu- able inclusions in the
new edition.
Mark K. Fung, MD, PhD
Editor in Chief
 Preface

T IS W I T H TR E M E N D O U S pleasure that

I we introduce you to the 18th edition of the

AABB Technical Manual. As with all
adjunctive therapies that can reduce the need
for transfusion. As health-care economics
join better patient care in prompting
previous editions, this revision is based on
continued adoption of PBM, readers will
the solid foundation of knowledge gathered
find that the new emphasis is highly
by past contributors to whom we are
relevant to their needs.
indebted. I would like to especially
Other content receiving special
acknowledge the tre- mendous contributions
attention in this edition is that involving
of Drs. Hillyer and Grossman who have
molecular test- ing. An increasing number of
helped guide previous editions. With the
organizations seek the more detailed test
18th edition they both con- clude their
results possible through investments in
tenures as Associate Editors. Along the same
molecular technology. Those in the
lines, I want to welcome Dr. Westhoff who
workforce today need (or soon will need) a
has joined me in this new challenge of
solid understanding of the both the theory
providing an up-to-date comprehensive
and practice of molecular testing systems,
resource of information in the field of
which is found in this volume.
transfu-
Perhaps the most obvious upgrade in
sion medicine and cellular therapies.
the new edition is the relocation of the
This edition of the Technical Manual
methods from the printed pages to electronic
will be most notable for several innovations.
storage medium found inside the back cover.
First, the cellular therapy content has been
By mov- ing the methods into the digital
expanded and reorganized to include many
format, we are able for the first time to give
novel therapies that are moving from the
Technical Manual users methods that are
research setting into the clinical realm. In
already set up as stan- dard operating
addition to updates on the sources of stem
procedures (SOPs)—in a tem- plate that
cells and the transfusion support of stem cell
reflects how procedures would actually be
transplantation, chapters focus on the quality
used in real-life. Users are invited to upload
and regulatory issues associated with cord
the methods to their facility net- works and
banking, novel stem cell therapies using non-
customize them for integration into their
hematopoietic stem cells, and tissue engineer-
existing SOPs.
ing. The new scope will be of great value to
In addition to these particular innova-
the increasing number of professionals who
tions, all chapters have been revised. Some
now include some aspect of cellular therapy in
of the chapter authors have added
their daily responsibilities.
substantial updates in great detail to assist
In a similar manner, the content on
the reader, whether working at the bench or
patient blood management (PBM) reflects
the bedside. They have embraced the task of
the growing scope of what is considered
explaining the issues that face all of us in the
PBM. The traditional topics covered as part
ever-changing world of transfusion
of discus- sions on blood utilization review
medicine and cellular therapies. We
and periop- erative blood recovery are
welcome your comments and feedback on
augmented by detailed content on anemia
the effectiveness of our labors.
management, optimization of coagulation,
and a host of
Mark K. Fung, MD, PhD
Editor in Chief
ix
 Contents

Preface.....................................................................................................ix

QUALITYISSUES

1. Quality Management Systems: Theory and Practice................1

Tania L. Motschman, MS, MT(ASCP)SBB, CQA(ASQ);
Betsy W. Jett, MT(ASCP), CQA(ASQ)CQM/OE; and
Susan L. Wilkinson, EdD, MS, MT(ASCP)SBB
Concepts in Quality......................................................................................2
Practical Application of Quality Management Principles.................................4
Key Points...............................................................................................................29
References.......................................................................................................30
Appendix 1-1. Glossary of Commonly Used Quality Terms...........................33
Appendix 1-2. Code of Federal Regulations Quality-Related References.......35
Appendix 1-3. Suggested Quality Control Performance Intervals for
Equipment and Reagents........................................................................36

2. Facilities, Work Environment, and Safety..................................39

Betsy W. Jett, MT(ASCP), CQA(ASQ)CQM/OE;
Susan L. Wilkinson, EdD, MS, MT(ASCP)SBB; and
Tania L. Motschman, MS, MT(ASCP)SBB, CQA(ASQ)
Facilities. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 40
....
Safety Program . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 42
....
Fire Prevention. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 45
.....
Electrical Safety . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 46
....
Biosafety . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 47
....
Chemical Safety . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 55
......
Radiation Safety . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 60
......
Shipping Hazardous Materials . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 63
....
General Waste Management. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 63
....
Key Points . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 64
......
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 64
......

xi
xii ■ AA BB T E C H N I C A L M A N U A L

Appendix 2-1. Safety Regulations and Recommendations Applicable to

Health-Care Settings...............................................................................68
Appendix 2-2. General Guidelines for Safe Work Practices, Personal
Protective Equipment, and Engineering Controls.......................................70
Appendix 2-3. Biosafety Level 2 Precautions.................................................73
Appendix 2-4. Sample List of Hazardous Chemicals that May Be
Encountered in a Blood Bank.................................................................74
Appendix 2-5. Chemical Categories and How to Work Safely with Them.....76
Appendix 2-6. Incidental Spill Response........................................................78
Appendix 2-7. Managing Hazardous Chemical Spills.....................................81

3. Regulatory Issues in Blood Banking...........................................83

Glenn Ramsey, MD
Biological Products.........................................................................................84
Licensure and Registration..............................................................................84
FDA Inspections......................................................................................................86
Blood-Related Devices...................................................................................87
Hematopoietic Progenitor Cells as Tissues................................................87
Managing Recalls and Withdrawals................................................................89
Medical Laboratory Laws and Regulations.....................................................89
Hospital Regulations and Accreditation..........................................................91
Conclusion......................................................................................................91
Key Points...............................................................................................................91
References.......................................................................................................92

4. Disaster Management.............................................................97
Ruth D. Sylvester, MS, MT(ASCP)SBB; William FitzGerald, LTC USA (Ret);
Wendy Trivisonno; and Theresa Wiegmann, JD
Background.....................................................................................................98
Business Operations Planning..................................................................102
Regulatory Considerations in Emergencies..............................................109
Testing the Disaster Plan..........................................................................111
Summary of Lessons Learned from Recent Disasters...................................112
Conclusion....................................................................................................112
Key Points.............................................................................................................112
References.....................................................................................................113

BLOODDONATIONANDCOLLECTION

5. Allogeneic and Autologous Blood Donor Selection.................117

Anne F. Eder, MD, PhD, and Maria D.L.A. Muniz, MD
Overview of Blood Donor Screening............................................................117
Selection of Allogeneic Blood Donors..........................................................118
Blood-Center-Defined Donor Eligibility Criteria..........................................127
 Table of Contents ■ xiii

Abbreviated DHQ for Frequent Donors........................................................130

Recipient-Specific “Designated” or “Directed” Blood Donation..................130
Key Points.............................................................................................................132
References....................................................................................................132

6. Whole-Blood Collection and Component Processing..........135

Larry J. Dumont, MBA, PhD; Mona Papari, MD;
Colleen A. Aronson, MT(ASCP)SBB; and
Deborah F. Dumont, MT(ASCP)SBB
WB Collection..............................................................................................135
Blood Component Preparation and Processing........................................146
Descriptions of Major Blood Components....................................................148
Blood Component Modification...................................................................154
Quarantine................................................................................................157
Labeling........................................................................................................158
QC of Blood Components.............................................................................159
Key Points.............................................................................................................160
References....................................................................................................161

7. Blood Component Collection by Apheresis..............................167

James W. Smith, MD, PhD
Component Collection..................................................................................167
Instruments and Systems for Donor Apheresis Collections..........................173
Key Points.............................................................................................................176
References....................................................................................................176

8. Infectious Disease Screening.....................................................179

Susan A. Galel, MD
Historical Overview of Blood Donor Screening...........................................179
Donor Screening Tests..................................................................................182
Residual Infectious Risks of Transfusion......................................................192
Screening for Specific Agents.......................................................................194
Pathogen Reduction Technology..................................................................204
Summary.......................................................................................................205
Key Points.............................................................................................................205
References....................................................................................................206

9. Hospital Storage, Monitoring, Pretransfusion

Processing, Distribution, and Inventory Management
of
Blood Components...........................................................213
Nancy M. Dunbar, MD
Blood and Blood Component Storage and Monitoring............................213
Pretransfusion Processing.............................................................................221
Distribution...................................................................................................224
 Inventory Management............................................................................227
xiv ■ A A BB T E C H N ICA L M ANU A L

Key Points.............................................................................................................228
References.....................................................................................................229

BLOODGROUPS

10. Molecular Biology and Immunology in Transfusion

Medicine................................................................................231
James C. Zimring, MD, PhD, and Steven L. Spitalnik, MD
Nucleic Acid Analysis...........................................................................................231
Protein Analysis............................................................................................240
Basic Immunology........................................................................................246
Key Points.............................................................................................................252
References.....................................................................................................253

11. Blood Group Genetics...............................................................255

Christine Lomas-Francis, MSc, FIBMS
Basic Principles of Genetics..........................................................................256
Inheritance of Genetic Traits....................................................................265
Population Genetics..................................................................................274
Relationship Testing.....................................................................................276
Blood Group Gene Mapping.........................................................................277
Chimerism.....................................................................................................278
Blood Group Terminology............................................................................278
Blood Group Genomics.................................................................................279
Key Points.............................................................................................................287
References.....................................................................................................288

12. ABO, H, and Lewis Blood Groups and Structurally

Related Antigens....................................................................291
Laura Cooling, MD, MS
ABO System.................................................................................................291
The H System................................................................................................301
The Lewis System.........................................................................................304
I and i Antigens.............................................................................................306
P Blood Groups/GLOB Collection................................................................309
Key Points.............................................................................................................313
References.....................................................................................................313

13. The Rh System...........................................................................317

Gregory A. Denomme, PhD, FCSMLS(D), and
Connie M. Westhoff, PhD, SBB
Characterization of Rh..................................................................................317
 Terminology..................................................................................................319

Table of Contents ■ xv

Rh Genes and Rh Proteins........................................................................319

Antigens........................................................................................................321
Rh Genotyping..............................................................................................330
Rhnull Syndrome and RhAG Blood Group System.........................................331
Rh Antibodies...............................................................................................331
Technical Considerations for Rh Typing......................................................331
Key Points.............................................................................................................333
References....................................................................................................334

14. Other Blood Group Systems and Antigens...............................337

Jill R. Storry, PhD, FIBMS
The MNS System..........................................................................................337
M (MNS1), N (MNS2), S (MNS3), and s (MNS4)..............................................341
The Lutheran System....................................................................................344
The Kell and Kx Systems.............................................................................345
The Duffy System.........................................................................................349
The Kidd System..........................................................................................351
The Diego System.........................................................................................352
The Yt System.......................................................................................................354
The Xg System.............................................................................................354
The Scianna System......................................................................................354
The Dombrock System.................................................................................354
The Colton System........................................................................................355
The Landsteiner-Wiener System...................................................................355
The Chido/Rodgers System..........................................................................356
The Gerbich System......................................................................................357
The Cromer System......................................................................................357
The Knops System........................................................................................358
The Indian System........................................................................................358
The Ok System.............................................................................................359
The Raph System..........................................................................................359
The John Milton Hagen System....................................................................359
The GILL System.........................................................................................359
The RHAG System.......................................................................................360
The FORS System........................................................................................360
The Jr System...............................................................................................360
The Lan System............................................................................................360
The Vel System.............................................................................................361
Antigens That Do Not Belong to a Blood Group System..............................361
Erythroid Phenotypes Caused by Mutations in Transcription
Factor Genes.............................................................................................362
Key Points.............................................................................................................363
References....................................................................................................363

xvi ■ A A BB T E C H N ICA L M ANU A L

 ANTIGENANDANTIBODYTESTING

15. Pretransfusion Testing..............................................................367

Katharine A. Downes, MD, and Ira A. Shulman, MD
Requests for Transfusion...............................................................................367
Identification of Recipients and Labeling of Blood Specimens.....................368
Specimen Requirements...........................................................................370
Serologic Testing Principles..........................................................................371
Pretransfusion Tests......................................................................................372
Tubeless Methods for Pretransfusion Testing...........................................377
Comparison of Current Testing Results with Previous Records...............378
Donor RBC Unit Testing..............................................................................378
Donor RBC Unit Selection............................................................................378
Compatibility Testing or Crossmatch (Serologic or Computer/
Electronic)................................................................................................379
Interpretation of Antibody Screening and Crossmatch Results....................381
Pretransfusion Orders...............................................................................381
Availability of Compatible Blood........................................................................384
Labeling of Blood and Blood Components with the Recipient’s
Information...............................................................................................384
Special Clinical Situations............................................................................385
Key Points.............................................................................................................387
References.....................................................................................................387

16. Identification of Antibodies to Red Cell Antigens................391

Phyllis S. Walker, MS, MT(ASCP)SBB, and
Janis R. Hamilton, MS, MT(ASCP)SBB
Significance of Alloantibodies......................................................................392
Preanalytical Considerations.........................................................................392
Analytical Phase of Antibody Identification.................................................393
Postanalytical Considerations: Selecting Blood for Transfusion...................419
Key Points.............................................................................................................421
References.....................................................................................................422
Suggested Readings......................................................................................424

17. The Positive Direct Antiglobulin Test and Immune-

Mediated Hemolysis..............................................................425
Regina M. Leger, MSQA, MT(ASCP)SBB, CMQ/OE(ASQ)
The DAT................................................................................................................425
Autoimmune Hemolytic Anemia..................................................................430
Drug-Induced Immune Hemolytic Anemia..............................................440
Key Points.............................................................................................................444
References.....................................................................................................445
Appendix 17-1. Drugs Associated with Immune Hemolytic Anemia............448

Table of Contents ■ xvii

18. Platelet and Granulocyte Antigens and Antibodies..............453

 Brian R. Curtis, PhD, D(ABMLI), MT(ASCP)SBB
Platelet Antigens and Antibodies..................................................................453
Granulocyte Antigens and Antibodies......................................................466
Key Points.............................................................................................................469
References....................................................................................................470

19. The HLA System.......................................................................475

Robert A. Bray, PhD; Marilyn S. Pollack, PhD; and Howard M. Gebel, PhD
Genetics of the MHC....................................................................................476
Biochemistry, Tissue Distribution, and Structure....................................480
Identification of HLA Antigens and Alleles.................................................484
Crossmatching and Detection of HLA Antibodies........................................487
The HLA System and Transfusion................................................................488
HLA Testing and Transplantation.................................................................491
Other Clinically Significant Aspects of HLA................................................493
Summary.......................................................................................................494
Key Points.............................................................................................................494
References....................................................................................................495

C L I N I C A L C O N SI D E R A T I O N S I N
T R A N S F U S I O NP R A C T I C E

20. Hemotherapy Decisions and Their Outcomes......................499

Theresa Nester, MD; Shweta Jain, MD; and Jessica Poisson, MD
Red Cell Transfusion....................................................................................499
Platelet Transfusion......................................................................................507
Plasma Transfusion.......................................................................................517
Cryoprecipitate Transfusion..........................................................................522
Granulocyte Transfusion...............................................................................523
Plasma-Derivative Transfusion.....................................................................526
Key Points.............................................................................................................532
References.....................................................................................................533

21 . Administration of Blood Components......................................545

Kim Maynard, BSN, RN, OCN
Events and Considerations Before Dispensing Components....................545
Blood Component Transportation and Dispensing...................................549
Events and Considerations Before Component Administration...............550
Manual Administration.................................................................................552
Unique Transfusion Settings.........................................................................555
Conclusions...................................................................................................556
Key Points.............................................................................................................556
References.....................................................................................................557
xviii ■ A A B B T EC H N I C AL M ANU AL

22. Perinatal Issues in Transfusion Practice...............................561

 Melanie S. Kennedy, MD; Meghan Delaney, DO, MPH;
and Scott Scrape, MD
HDFN....................................................................................................................561
RhIG......................................................................................................................565
ABO Hemolytic Disease...............................................................................566
Immune Thrombocytopenia......................................................................567
Key Points.............................................................................................................568
References.....................................................................................................569

23. Neonatal and Pediatric Transfusion Practice........................571

Cassandra D. Josephson, MD, and Erin Meyer, DO, MPH
Transfusion in Infants Younger Than 4 Months.......................................571
Transfusion in Infants Older Than 4 Months and Children......................586
Prevention of Adverse Effects of Transfusion in Neonates,
Older Infants, and Children..................................................................589
Key Points.............................................................................................................591
References.....................................................................................................592

24. Patient Blood Management..................................................599

Mary Ghiglione, RN, MSN, MHA, and
Kathleen E. Puca, MD, MT(ASCP)SBB
Definition and Scope of Patient Blood Management....................................599
The Rationale for Patient Blood Management..........................................600
Basic Elements of a PBM Program...............................................................603
Postoperative Strategies................................................................................608
Blood Utilization Review and Changing Physician Behavior.......................610
Program Development..................................................................................612
Key Points.............................................................................................................613
References.....................................................................................................613
Appendix 24-1. Pharmacologic Therapies for Supporting Patient
Blood Management..............................................................................620
Appendix 24-2. Responsibilities for Activity Levels 1, 2, and 3 PBM
Programs...................................................................................................629

25. Transfusion Support for Hematopoietic Stem Cell

Transplant Recipients........................................................631
Christopher A. Tormey, MD, and Jeanne E. Hendrickson, MD
Implications of ABO- and Non-ABO-Antigen-Incompatible
Red Blood Cell Transplantation for Transfusion..................................632
Blood Component Considerations............................................................633
Patients with Neutropenia and Infection that Is Unresponsive to
Antimicrobial Therapy.........................................................................638

Table of Contents ■ xix

Special Processing of Blood Components for HSCT Recipients..................638

Special Considerations for Transfusions in Pediatric HSCT Recipients. .639
Information Portability for HSCT Recipients..........................................640
 Key Points.............................................................................................................640
References....................................................................................................641

26. Therapeutic Apheresis...............................................................645

Robertson D. Davenport, MD
Principles and Modalities..............................................................................645
Indications....................................................................................................647
Anticoagulation.............................................................................................658
Adverse Effects.....................................................................................................658
Vascular Access.....................................................................................................660
Patient Evaluation.........................................................................................660
Key Points.............................................................................................................661
References....................................................................................................662

27. Noninfectious Complications of Blood Transfusion.................665

Catherine A. Mazzei, MD; Mark A. Popovsky, MD;
and Patricia M. Kopko, MD
Hemovigilance..............................................................................................665
Recognition and Evaluation of a Suspected Transfusion Reaction...............666
Delayed Transfusion Reactions.....................................................................686
Fatality Reporting Requirements..................................................................691
Key Points.............................................................................................................691
References....................................................................................................692

28. Approaches to Blood Utilization Auditing................................697

Alan Tinmouth, MD, FRCPC, MSc, and
Simon Stanworth, FRCP, FRCPath, DPhil
Rationale for Monitoring Blood Utilization..................................................698
Types of Transfusion Audits.........................................................................699
Interventions to Change Transfusion Practice..........................................704
Effectiveness of Monitoring and Interventions to Change
Transfusion Practice.................................................................................704
Selecting an Audit Process to Monitor Transfusions...............................705
Conclusions..................................................................................................705
Key Points.............................................................................................................707
References....................................................................................................708
Appendix 28-1. Transfusion Order Form in Use at St. Vincent
Indianapolis Hospital Since 2001.............................................................711

xx ■ AABB T ECHNI C AL M AN U A L

TRANSPLANTATION

29. The Collection and Processing of Hematopoietic

Stem Cells..............................................................................713
Scott A. Koepsell, MD, PhD; Eapen K. Jacob, MD; and
 David H. McKenna Jr, MD
Clinical Utility..............................................................................................713
Determination of Graft Source.................................................................717
Collection/Sources of HSCs..........................................................................718
Processing of HSCs.......................................................................................720
Specialized Cell-Processing Methods...........................................................721
Cryopreservation...........................................................................................722
Shipping and Transport of HSC Cellular Products........................................723
Patient Care...............................................................................................724
Other Regulatory Considerations..............................................................725
Conclusion....................................................................................................725
Key Points.............................................................................................................725
References.....................................................................................................726

30. Umbilical Cord Blood Banking.................................................729

Aleksandar M. Babic, MD, PhD, and Donna M. Regan, MT(ASCP)SBB
Background...................................................................................................729
Donor-Related Issues....................................................................................730
UCB Collection.............................................................................................733
UCB Processing............................................................................................734
Shipment.......................................................................................................737
Receipt of UCB for Transplantation.........................................................738
Thawing and Washing of UCB.....................................................................740
Infusion of UCB............................................................................................741
Economic Issues............................................................................................742
Regulations and Standards........................................................................743
Key Points.............................................................................................................747
References.....................................................................................................747

31. Tissue-Derived Non-Hematopoietic Stem Cell Sources

for Use in Cell-Based Therapies............................................753
Yameena Jawed, MD; Brian Johnstone, PhD;
Sreedhar Thirumala, PhD; and Keith March, MD, PhD
MSC Sources................................................................................................754
Properties of Clinical Relevance...................................................................758
Isolation and Expansion............................................................................758
Standardization of Methods for Isolation and Expansion of
Clinical Product........................................................................................760
Cell Product Banking and Management of Supply Chain and End Use...761
Therapeutic Applications of MSCs...............................................................762
Table of Contents ■ xxi

Current Research and Development: Focus on Cell Culture and

Handling...................................................................................................764
Conclusions and Future Directions...........................................................765
Key Points.............................................................................................................766
References.....................................................................................................766
32. Human Allografts and the Hospital Transfusion Service.....773
Lance D. Trainor, MD, and Rita A. Reik, MD
Tissue Transplantation..............................................................................773
Regulations and Standards........................................................................777
Hospital Tissue Services...............................................................................778
Transfusion Service Support for Organ Transplantation..........................783
Key Points.............................................................................................................783
References.....................................................................................................784

33. Blood and Marrow-Derived Nonhematopoietic Stem Cell

Sources and Immune Cells for Clinical Applications............785
Mickey B. C. Koh, MD, PhD; Edward R. Samuel, PhD, MSc;
and Garnet Suck, PhD, MSc
Immune Cells for Clinical Therapy...............................................................786
Induced Pluripotent Stem Cells................................................................795
Regulatory and Oversight Activities.............................................................796
Conclusion....................................................................................................797
Key Points.............................................................................................................797
References.....................................................................................................798

Index.....................................................................................................803

METHODS

Methods Introduction

1. General Laboratory Methods—Introduction

Method 1-1. Shipping Hazardous Materials
Method 1-2. Monitoring Temperature During Shipment of
Blood Method 1-3. Treating Incompletely Clotted Specimens
Method 1-4. Solution Preparation Procedure
Method 1-5. Serum Dilution Procedure
Method 1-6. Dilution of Percentage Solutions
Procedure Method 1-7. Preparing a 3% Red Cell
Suspension
xxii ■ AA BB T E C H N ICA L M ANU A L

Method 1-8. Preparing and Using Phosphate Buffer

Method 1-9. Reading and Grading Tube Agglutination

2. Red Cell Typing Methods—Introduction

Method 2-1. Determining ABO Group of Red Cells—Slide Test
Method 2-2. Determining ABO Group of Red Cells and Serum—Tube Test
Method 2-3. Determining ABO Group of Red Cells and Serum—Microplate Test
Method 2-4. Initial Investigation of ABO Grouping Discrepancies Procedure
Method 2-5. Detecting Weak A and B Antigens and Antibodies by
Cold Temperature Enhancement
Method 2-6. Confirming Weak A and B Antigens Using Enzyme-Treated
Red Cells
Method 2-7. Confirming Weak A or B Subgroup by Adsorption and Elution
Method 2-8. Testing Saliva for A, B, H, Lea, and Leb Antigens
Method 2-9. Confirming Anti-A1 in an A2 or Weak A Subgroup
Method 2-10. Resolving ABO Discrepancies Caused by Unexpected
Alloantibodies
Method 2-11. Determining Serum Group Without Centrifugation
Method 2-12. Determining Rh (D) Type—Slide Test
Method 2-13. Determining Rh (D) Type—Tube Test
Method 2-14. Determining Rh (D) Type—Microplate Test
Method 2-15. Testing for Weak D
Method 2-16. Preparing and Using Lectins
Method 2-17. Removing Autoantibody by Warm Saline Washes
Method 2-18. Using Sulfhydryl Reagents to Disperse Autoagglutination
Method 2-19. Using Gentle Heat Elution to Test Red Cells with a Positive DAT
Method 2-20. Dissociating IgG by Chloroquine for Antigen Testing of Red Cells
with a Positive DAT
Method 2-21. Using Acid Glycine/EDTA to Remove Antibodies from Red
Cells Method 2-22. Separating Transfused from Autologous Red Cells by
Simple
Centrifugation
Method 2-23. Separating Transfused from Autologous Red Cells in Patients with
Hemoglobin S Disease

3. Antibody Detection, Identification, and Compatibility Testing

— Introduction
Method 3-1. Using Immediate-Spin Compatibility Testing to Demonstrate ABO
Incompatibility
Method 3-2. Saline Indirect Antiglobulin Test Procedure
Method 3-3. Albumin or LISS-Additive Indirect Antiglobulin Test Procedure
Method 3-4. LISS-Suspension Indirect Antiglobulin Test Procedure
Method 3-5. PEG Indirect Antiglobulin Test Procedure
Method 3-6. Prewarming Procedure
Method 3-7. Detecting Antibodies in the Presence of Rouleaux—Saline
Replacement
Method 3-8. Preparing Ficin Enzyme Stock, 1%
w/v Method 3-9. Preparing Papain Enzyme Stock,
1% w/v Method 3-10. Standardizing Enzyme
Procedures
 Table of Contents ■ xxiii

Method 3-11. Evaluating Enzyme-Treated Red Cells

Method 3-12. One-Stage Enzyme Procedure
Method 3-13. Two-Stage Enzyme Procedure
Method 3-14. Performing a Direct Antiglobulin Test
Method 3-15. Antibody Titration Procedure
Method 3-16. Using Sulfhydryl Reagents to Distinguish IgM from IgG Antibodies
Method 3-17. Using Plasma Inhibition to Distinguish Anti-Ch and -Rg from
Other Antibodies with Similar Characteristics
Method 3-18. Treating Red Cells Using DTT or
AET Method 3-19. Neutralizing Anti-Sda with
Urine Method 3-20. Adsorption Procedure
Method 3-21. Using the American Rare Donor Program

4. Investigation of a Positive DAT Result—Introduction

Method 4-1. Cold-Acid Elution Procedure
Method 4-2. Glycine-HCl/EDTA Elution Procedure
Method 4-3. Heat Elution Procedure
Method 4-4. Lui Freeze-Thaw Elution
Procedure Method 4-5. Cold Autoadsorption
Procedure
Method 4-6. Determining the Specificity of Cold-Reactive Autoagglutinins
Method 4-7. Cold Agglutinin Titer Procedure
Method 4-8. Adsorbing Warm-Reactive Autoantibodies Using Autologous Red
Cells
Method 4-9. Adsorbing Warm-Reactive Autoantibodies Using Allogeneic Red
Cells
Method 4-10. Polyethylene Glycol Adsorption
Procedure Method 4-11. Performing the Donath-
Landsteiner Test
Method 4-12. Detecting Drug Antibodies by Testing Drug-Treated Red
Cells Method 4-13. Detecting Drug Antibodies by Testing in the
Presence of Drug

5. Hemolytic Disease of the Fetus and Newborn—Introduction

Method 5-1. Testing for Fetomaternal Hemorrhage—The Rosette Test
Method 5-2. Testing for Fetomaternal Hemorrhage—Modified
Kleihauer-Betke Test
Method 5-3. Using Antibody Titration Studies to Assist in Early
Detection of Hemolytic Disease of the Fetus and Newborn

6. Blood Collection, Component Preparation, and Storage

— Introduction
Method 6-1. Screening Donors for Anemia—Copper Sulfate Method
Method 6-2. Preparing the Donor’s Arm for Blood Collection
Method 6-3. Collecting Blood and Samples for Processing and Compatibility
Tests
Method 6-4. Preparing Red Blood Cells from Whole Blood
Method 6-5. Preparing Prestorage Red Blood Cells Leukocytes Reduced from
Whole Blood
Method 6-6. Using High-Concentration Glycerol to Cryopreserve Red
Cells— Meryman Method
xxiv ■ A A BB T E C H N ICA L M ANU A L

Method 6-7. Using High-Concentration Glycerol to Cryopreserve Red

Cells— Valeri Method
Method 6-8. Checking the Adequacy of Deglycerolization of Red Blood Cells
Method 6-9. Preparing Fresh Frozen Plasma from Whole Blood
Method 6-10. Preparing Cryoprecipitated AHF from Whole
Blood Method 6-11. Thawing and Pooling Cryoprecipitated
AHF Method 6-12. Preparing Platelets from Whole Blood
Method 6-13. Removing Plasma from Platelets (Volume Reduction)

7. Transplantation of Cells and Tissue—Introduction

Method 7-1. Infusing Cryopreserved Hematopoietic
Cells Method 7-2. Processing Umbilical Cord Blood
Method 7-3. Investigating Adverse Events and Infections Following Tissue
Allograft Use

8. Quality Control Methods—Introduction

Method 8-1. Validating Copper Sulfate Solution
Method 8-2. Calibrating Liquid-in-Glass Laboratory Thermometers
Method 8-3. Calibrating Electronic Oral Thermometers
Method 8-4. Testing Refrigerator Alarms
Method 8-5. Testing Freezer Alarms
Method 8-6. Calibrating Centrifuges for Platelet Separation
Method 8-7. Calibrating a Serologic Centrifuge for Immediate Agglutination
Method 8-8. Calibrating a Serologic Centrifuge for Washing and Antiglobulin
Testing
Method 8-9. Testing Automatic Cell Washers
Method 8-10. Monitoring Cell Counts of Apheresis Components
Method 8-11. Counting Residual White Cells in Leukocyte-Reduced Blood
and Components—Manual Method

APPENDICES

Appendix 1. Normal Values in Adults

Appendix 2. Selected Normal Values in Children
Appendix 3. Typical Normal Values in Tests of Hemostasis and Coagulation
(Adults)
Appendix 4. Coagulation Factor Values in Platelet Concentrates
Appendix 5. Approximate Normal Values for Red Cell, Plasma, and
Blood
Volumes
Appendix 6. Blood Group Antigens Assigned to Systems
Appendix 7. Examples of Gene, Antigen, and Phenotype Symbols in
Conventional and International Society of Blood Transfusion
Terminology
Appendix 8. Examples of Correct and Incorrect Terminology
Appendix 9. Distribution of ABO/Rh Phenotypes by Race or
Ethnicity Appendix 10. Example of a Maximum Surgical Blood
Order Schedule Appendix 11. Directory of Organizations
 Abbreviations
AATB American Association of Tissue
cDNA complementary deoxyribonucleic
Banks ACD acid-citrate-dextrose
acid CDRH Center for Devices and
ACE angiotensin-converting enzyme
Radiological
ACOG American College of Obstetricians Health
and Gynecologists
CFR Code of Federal Regulations
ADP adenosine diphosphate
CFU colony-forming unit
AET 2-aminoethylisothiouronium
CGD chronic granulomatous disease
AHF antihemophilic factor
cGMP current good manufacturing practice
AHG antihuman globulin
cGTP current good tissue practice
AHTR acute hemolytic transfusion reaction
cGy centiGray
AIDS acquired immune deficiency
CI confidence interval
syndrome AIHA autoimmune hemolytic
CIDP chronic inflammatory demyelinating
anemia polyneuropathy
ALDH aldehyde dehydrogenase CJD Creutzfeldt-Jakob disease
ALT alanine aminotransferase CLIA Clinical Laboratory
AML acute myelogenous leukemia Improvement Amendments
AMR antibody-mediated rejection CLSI Clinical and Laboratory
ANH acute normovolemic hemodilution Standards Institute
AORN Association of periOperative CML chronic myelogenous leukemia
Registered CMS Centers for Medicare and
Nurses Medicaid
APC antigen-presenting cell Services
aPTT activated partial thromboplastin CMV cytomegalovirus
time ARDP American Rare Donor Program CNS central nervous system
AS additive solution CP2D citrate-phosphate-dextrose-dextrose
ASFA American Society for Apheresis CPD citrate-phosphate-dextrose
ASHI American Society for CPDA-1 citrate-phosphate-dextrose-adenine-1
Histocompatibility and CR complement receptor
Immunogenetics CREG cross-reactive group
ATP adenosine triphosphate CRYO cryoprecipitate (Cryoprecipitated
BCR B-cell receptor AHF) C/T crossmatch/transfusion
BLA biologics license application CV coefficient of variation
BPD biological product deviation DAF decay-accelerating factor
BSA bovine serum albumin or body DAT direct antiglobulin test
surface area
DDAVP deamino-D-arginine vasopressin
BSC biological safety cabinet
DHQ donor history questionnaire
BSL-1 Biosafety Level 1
DHTR delayed hemolytic transfusion reaction
CAP College of American Pathologists
DIC disseminated intravascular
CAS cold agglutinin syndrome
coagulation DMSO dimethylsulfoxide
CBER Center for Biologics Evaluation
DNA deoxyribonucleic acid
and Research
CCI corrected count increment DOT (US) Department of
CD clusters of differentiation Transportation 2,3-DPG 2,3-
CDC Centers for Disease Control diphosphoglycerate
and Prevention DRG diagnosis-related group
DSTR delayed serologic transfusion
HDFN hemolytic disease of the fetus
reaction DTTdithiothreitol and newborn
EACA epsilon aminocaproic acid HES hydroxyethyl starch
EBAA Eye Bank Association of America HHS (US) Department of Health and
ECMO extracorporeal membrane Human Services
oxygenation ECV extracorporeal volume HIT heparin-induced thrombocytopenia
EDTA ethylenediaminetetraacetic acid HIV human immunodeficiency virus
EIA enzyme immunoassay HNA human neutrophil antigen
ELBW extremely low birthweight HPA human platelet antigen
ELISA enzyme-linked immunosorbent HPC hematopoietic progenitor cell
assay EMAs emergency management agencies HPC(A) HPCs from apheresis (HPC,
EPO erythropoietin Apheresis) HPC(C) HPCs from cord blood
FACT Foundation for the Accreditation (HPC, Cord
of Cellular Therapy Blood)
FcR Fc gamma receptor HPC(M) HPCs from marrow (HPC,
FDA Food and Drug Administration Marrow) HSC hematopoietic stem
FFP Fresh Frozen Plasma cell
FMH fetomaternal hemorrhage HSCT hematopoietic stem cell
FNAIT fetal/neonatal transplantation HTLV-I human T-cell
alloimmune lymphotropic virus, type I HTR hemolytic
thrombocytopenia transfusion reaction
FNHTR febrile nonhemolytic transfusion HUS hemolytic uremic syndrome
reaction
IAT indirect antiglobulin test
FTA-ABS fluorescent treponemal antibody
IATA International Air Transport
absorption test
Association ICAM-1 intercellular adhesion
G-CSF granulocyte colony-stimulating factor
molecule-1
GalNAc N-acetylgalactosamine
ID indentification or individual donation
GM-CSF granulocyte-macrophage
colony- stimulating factor Ig immunoglobulin
GMP good manufacturing practice IL-1 interleukin-1 alpha
GPIa glycoprotein Ia IL-1 interleukin-1 beta
GPA glycophorin A IL-2 interleukin-2
GPB glycophorin B IM intramuscular
GPC glycophorin C IND investigational new drug
GPD glycophorin D INR international normalized ratio
GTP good tissue practice iPSCs induced pluripotent stem
GVHD graft-vs-host disease cells IRL immunohematology
Gy Gray reference
laboratory
HAV hepatitis A virus
IS immediate spin
HAZMAT hazardous material
ISBT International Society of
Hb hemoglobin
Blood Transfusion
HBc hepatitis B core
ISO International Organization
antigen HBsAg hepatitis B for Standardization
surface antigen HBV hepatitis B ITP immune thrombocytopenia
virus IU international unit
Hct hematocrit IV intravenous
HCT/Ps human cells, tissues, and cellular IVIG intravenous immune globulin
and tissue-based products
LDH lactate dehydrogenase
HCV hepatitis C virus
LDL low-density lipoprotein
LISS low-ionic-strength saline
LN2 liquid nitrogen
LR leukocyte-reduced
MAC membrane attack complex
QC quality control
2-ME 2-mercaptoethanol
QSE Quality System Essential
MF mixed field
RBCs Red Blood Cells (blood donor unit)
MHC major histocompatibility complex
RFLP restriction fragment length
MNC mononuclear cell polymorphism
MoAb monoclonal antibody rFVIIa recombinant Factor VIIa
MPHA mixed passive hemagglutination Rh Rhesus factor
assay mRNAmessenger ribonucleic acid RHAG Rh-associated
MSC mesenchymal stem cell glycoprotein RhIG Rh Immune
MSDS material safety data Globulin
sheet RIBA recombinant immunoblot assay
MSM male who has sex with another RIPA radioimmunoprecipitation
male NAIT neonatal alloimmune assay RNA ribonucleic acid
thrombocytopenia NAN neonatal RPR rapid plasma reagin (serologic test
alloimmune neutropenia for syphilis)
NAT nucleic acid testing RT room temperature or
NHLBI National Heart, Lung, and reverse transcriptase
Blood Institute SCF stem cell factor
NIH National Institutes of Health SD standard deviation or solvent/detergent
NIPA nonimmunologic protein adsorption SNP single nucleotide polymorphism
NK natural killer SOP standard operating procedure
NMDP National Marrow Donor Program SPRCA solid-phase red cell
NRC Nuclear Regulatory Commission adherence TA transfusion-associated
NRF National Response Framework TACO transfusion-associated
OSHA Occupational Safety and Health circulatory overload
Administration TCR T-cell receptor
p probability TMA transcription-mediated amplification
PAD preoperative autologous TNCs total nucleated cells
(blood) donation TNF- tumor necrosis factor alpha
PBM patient blood management TPE therapeutic plasma exchange
PBS phosphate-buffered saline TPO thrombopoietin
PCH paroxysmal cold TRALI transfusion-related acute lung
hemoglobinuria PCR polymerase chain injury TSE transmissible spongiform
reaction encephalopathy
PEG polyethylene glycol TTP thrombotic thrombocytopenic purpura
PF24 Plasma Frozen Within 24 Hours UCB umbilical cord blood
After Phlebotomy UDP uridine diphosphate
PF24 RT24 Plasma Frozen Within 24 Hours UNOS United Network for Organ
After Phlebotomy Held at Room Sharing USC United States Code
Temperature Up to 24 Hours after
vCJD variant Creutzfeldt-Jakob disease
Phlebotomy
VLBW very low birthweight
PPE personal protective equipment
vWD von Willebrand disease
PRA panel-reactive antibody
vWF von Willebrand factor
PRCA pure red cell aplasia
WAIHA warm autoimmune hemolytic
PRP platelet-rich plasma
anemia WB whole blood or Western blot
PRT pathogen reduction technology
WBC white blood cell
PT prothrombin time or proficiency
testing WHO World Health Organization
PTP posttransfusion purpura WNV West Nile virus
PTT partial thromboplastin time
PVC polyvinyl chloride
QA quality assessment or quality
assurance
 C h a p t e r 1

Quality Management Systems:

Theory and Practice

Tania L. Motschman, MS, MT(ASCP)SBB, CQA(ASQ);

Betsy W. Jett, MT(ASCP), CQA(ASQ)CQM/OE; and
Susan L. Wilkinson, EdD, MS, MT(ASCP)SBB

A PR I MA R Y G O A Lof tr a n s f u s i o
istration (FDA), especially in the current good
n medicine, cellular therapies, and
manufacturing practice (cGMP) and current
clinical
good tissue practice (cGTP) regulations.2-5 The
diagnostics is to promote high standards of
FDA regulations in the Code of Federal
quality in all aspects of patient care and
Regula- tions (CFR) Title 21, Part 211.22
relat- ed products and services. This
require an in- dependent quality control (QC)
commitment to quality is reflected in
or quality as- surance unit that has
standards of practice set forth by the AABB.
responsibility for the overall quality of the
AABB standards use a quali- ty management
facility’s finished product and authority to
system as the framework for quality. A
control the processes that may affect this
quality management system in- cludes the
product.3 (See frequently used CFR quality-
organizational structure, responsi- bilities,
related citations in Appendix 1-2.)
policies, processes, procedures, and
Professional and accrediting organizations
resources established by executive
such as the AABB,6,7 College of American
manage- ment to achieve and maintain
Pa- thologists (CAP), 8 The Joint
quality. (A glos- sary of quality terms used
Commission, 9,10
Clinical and Laboratory
in this chapter is in- cluded in Appendix 1-
Standards Institute (CLSI),11 and Foundation
1.)
for the Accreditation of Cellular Therapy
The establishment of a formal quality
(FACT),12 have also estab- lished requirements
as- surance program is required under the
and guidelines to address quality issues. The
Centers for Medicare and Medicaid
International Organization for
Services (CMS) Clinical Laboratory
Standardization (ISO) quality manage-
Improvement Amend- ments (CLIA)1 and
the Food and Drug Admin-

Tania L. Motschman, MS, MT(ASCP)SBB, CQA(ASQ), Quality Director, Esoteric Business Unit, Laboratory
Corporation of America, Burlington, North Carolina; Betsy W. Jett, MT(ASCP), CQA(ASQ)CQM/OE, Vice
Presi- dent for Quality and Regulatory Affairs, New York Blood Center, New York, New York; and Susan L.
Wilkinson, EdD, MS, MT(ASCP)SBB, Interim Department Head, Analytical and Diagnostic Sciences,
College of Allied Health Sciences, and Associate Professor Emerita, University of Cincinnati, Cincinnati,
Ohio
The authors have disclosed no conflicts of interest.
1
2 ■ AABB T E C HNI CAL M ANUAL
ment standards (ISO 9001) are generic to
any industry and describe the important
mini- mum elements of a quality
management sys- tem.13 The ISO 15189
standards are specific to laboratory
medicine.14 In addition, the Health Care
Criteria for Performance Excellence pub-
lished by the Baldrige Performance
Excellence Program15 provides an excellent
framework for implementing quality on an
organizational level.
The AABB has defined the minimum ele-
ments that must be addressed in its quality
system essentials (QSEs). 16 The AABB
QSEs were developed to be compatible with
ISO 9001 standards, the FDA Guideline for
Quality Assurance in Blood Establishments,5
and other FDA quality system approaches.17,18
CONCEPTS IN QUALITY
Quality Control, Quality Assurance,
and Quality Management
The purpose of QC is to provide feedback to
operational staff about the state of a process
that is in progress. QC tells staff whether to
continue (everything is acceptable) or to
stop until a problem has been resolved
(something is found to be out of control).
Historically, transfusion services and
do- nor centers have used many QC
measures as standard practices in their
operations. Exam- ples include reagent QC;
product QC; clerical checks; visual
inspections; and measure- ments, such as
temperature readings on refrig- erators and
volume or cell counts on finished blood
components.
Quality assurance activities are not tied
to the actual performance of a process.
Rather, they include activities, such as the
develop- ment of documents like standard
operating procedures (SOPs), to ensure
consistent and correct performance of
processes, training of personnel, and
qualification of materials and equipment.
Quality assurance activities also include
retrospective reviews and analyses of
operational performance data to determine
whether the overall process is in a state of
con- trol and to detect shifts or trends that
require attention. Quality assurance
provides infor-
mation to process managers regarding
levels of performance that can be used in
setting pri- orities for process improvement.
Examples of quality assurance activities
in transfusion medicine and cellular
therapies include record reviews, monitoring
of quality indicators, and internal
assessments.
Quality management considers
interrelat- ed processes in the context of the
organization and its relations with customers
and suppliers. It addresses the leadership
role of executive management in creating
a commitment to quality throughout the
organization, the un- derstanding of
suppliers and customers as partners in
quality, the management of human and other
resources, and quality planning.
The quality systems approach
described in this chapter encompasses all of
these activi- ties. It ensures application of
quality principles throughout the
organization and reflects the changing
focus of quality efforts from detec- tion to
prevention.
Juran’s Quality Trilogy
Juran’s Quality Trilogy is one example of a
quality management approach. This model
centers around three fundamental processes
for managing quality in any organization:
planning, control, and improvement.19(p2.5)
The planning process for a new
product or service includes activities to
identify re- quirements, develop product
and process specifications that meet those
requirements, and design the process.
During the planning phase, the facility must
perform the following steps:
1. Establish quality goals for the project.
2. Identify the customers.
3. Determine customer needs and expecta-
tions.
4. Develop product and service
specifications to meet customer,
operational, regulatory, and accreditation
requirements.
5. Develop operational processes for
produc- tion and delivery, including
written proce- dures and resources
requirements.
6. Develop process controls and validate the
process in the operational setting.
 CHAP TER 1 Quality Management Systems: Theory and Practice
The results of the planning process are
re- ferred to as “design output.”13
Once the plan is implemented, the con-
trol process provides a feedback loop for
oper- ations that includes the following:
1. Evaluation of performance.
2. Comparison of performance to goals.
3. Action to correct any discrepancy
between the two.
The control process addresses inputs,
production, and delivery of products and
ser- vices to meet specifications. Process
controls should allow staff to recognize
when things are going wrong and to either
make appropriate adjustments to ensure a
product’s quality or stop the process.
An important goal in quality manage-
ment is to establish a set of controls that
en- sure process and product quality but are
not excessive. Controls that do not add
value should be eliminated to conserve
limited resources and allow staff to focus
attention on those controls that are critical
to the opera- tion.
Statistical tools, such as process
capabili-
ty measurement and control charts, allow a fa-
cility to evaluate process performance during
the planning stage and in operations. These
tools help determine if a process is stable (ie,
in statistical control) and if it is capable of
meeting product and ser vice specifica-
tions.19(p22.19)
Quality improvement is intended to en-
able an organization to attain higher levels
of performance by creating new or better
fea- tures that add value or by removing
deficien- cies in the process, product, or
service. Oppor- tu n i t i es to im prov e m
a y b e re la ted to deficiencies in the
initial planning process; unforeseen factors
discovered on implementa- tion; shifts in
customer needs; or changes in starting
materials, environmental factors, or other
variables that affect the process. Im-
provements must be based on data-driven
analysis; an ongoing measurement and
assess- ment program is fundamental to that
process.
■ 3
Process Approach
In its most generic form, a process includes
all of the resources and activities that
transform an input into an output. An
understanding of how to manage and control
processes in trans- fusion medicine, cellular
therapies, and clini- cal diagnostic activities
is based on this simple equation:
INPUT  PROCESS  OUTPUT
For example, a key process for donor
cen- ters is donor selection. The “input”
includes the individual who presents for
donation and all of the resources required
for that donor’s health screening. Through a
series of activities (a process), including the
verification of the donor’s identity, a
deferral status review, a mini-physical
exam, and a health history questionnaire,
an individual is deemed an “eli- gible
donor.” The “output” is either an eligible
donor who can continue to the next process
(blood collection) or an ineligible donor who
is deferred. When the selection process
results in a deferred donor, the resources
(inputs) asso- ciated with that process do
not continue through the process but
contribute to the cost of quality. One way
that donor centers at- tempt to minimize
this cost is to educate po- tential donors
before screening so that those who are not
eligible do not enter the selection process.
Strategies for managing a process
should address all of its components,
including its in- terrelated activities, inputs,
outputs, and re- sources. Supplier
qualification, formal agree- ments, supply
verification, and inventory control are
strategies for ensuring that the inputs to
a process meet specifications. Personnel
training and competence assess- ment,
equipment maintenance and control,
management of documents and records, and
implementation of appropriate in-process
controls provide assurance that the process
will operate as intended. End-product testing
and inspection, customer feedback, and
outcome measurement provide data to evalu-
ate product quality and improve the process.
These output measurements and quality
4 ■ AABB T E C HNI CAL M ANUAL
indicators are used to evaluate the effective-
ness of the process and process controls.
To manage a system of processes
effec- tively, the facility must understand
how its processes interact and what cause-
and-effect relationships exist between them.
In the donor selection scenario, the
consequences of ac- cepting a donor who is
not eligible reach into almost every other
process in the facility. For example, if a
donor with a history of high-risk behavior is
not identified as such during the selection
process, the donated unit(s) may re- turn
positive test results for one of the viral
marker assays, triggering follow-up
testing, look-back investigations, and
donor deferral and notification
procedures. Components must be
quarantined and their discard docu- mented.
Personnel involved in collecting and
processing the unit(s) are at risk of exposure
to infectious agents. Part of quality planning
is to identify these relationships so that
quick and appropriate corrective action
can be taken when process controls fail.
It is important to remember that opera-
tional processes include not only product
manufacture or service creation, but also
the delivery of a product or service.
Delivery gen- erally involves interaction
with the customer. The quality of that
transaction is critical to customer
satisfaction and should not be over- looked
in the design and ongoing assessment of the
quality management system.
Service vs Production
Quality management principles apply
equally to a broad spectrum of activities,
from those related to processing and
production to those involving interactions
between individuals in delivering a service.
However, different strate- gies may be
appropriate when there are differ- ing
expectations related to customer satisfac-
tion. Although the emphasis in a production
process is on minimizing variation to create
a product that consistently meets
specifications, service processes require a
certain degree of flexibility to address
customer needs and cir- cumstances at the
time of the transaction. In production,
personnel need to know how to maintain
uniformity in day-to-day operations.
In service, personnel need to be able to
adapt a service in a way that meets customer
ex- pectations but does not compromise
quality. To do this, personnel must have
sufficient knowledge and understanding of
interrelated processes to use independent
judgment ap- propriately, or they must have
ready access to higher-level decision
makers.
When one designs quality
management systems for production
processes, it is useful to think of the process
as the driver, with peo- ple providing the
oversight and support need- ed to keep it
running smoothly and effectively. In service,
people are the focus; the underlying process
provides a foundation that enables staff to
deliver safe and effective services that meet
customers’ needs in almost any situa- tion.
Quality Management as an Evolving
Science
The principles and tools used in quality
man- agement today will change as research
pro- vides new knowledge of organizational
behav- ior, technology provides new
solutions, and the transfusion medicine and
cellular thera- pies fields present new
challenges. Periodic as- sessments of the
quality management system will help
identify practices that are no longer effective
or that could be improved through the use of
new technology or new tools.
PRACTICAL APPLICATION OF
QUALITY MANAGEMENT
PRINCIPLES
The remainder of this chapter addresses the
elements of a quality management system
and practical application of quality
management principles to the transfusion
medicine, cellular therapies, and clinical
diagnostics environ- ments. These basic
elements include the fol- lowing:
■ Organization and leadership.
■ Customer focus.
■ Facilities, work environment, and safety.
■ Human resources.
■ Suppliers and materials management.
 CHAP TER 1 Quality Management Systems: Theory and Practice
■ Equipment management.
■ Process management.
■ Documents and records.
■ Information management.
■ Management of nonconforming events.
■ Monitoring and assessment.
■ Process improvement.
Organization and Leadership
The facility should be organized in a manner
that promotes effective implementation and
management of its operational and quality
management system. The structure of the or-
ganization must be documented, and the
roles and responsibilities for the provision of
tests, products, and services must be clearly
defined. These provisions should include a
description of the relationships and avenues
of communi- cation between organizational
units and those responsible for key quality
functions. Each fa- cility may define its
structure in any format that suits its
operations. Organizational trees or charts
that show the structure and relation- ships
are helpful.
The facility should define in writing the
authority and responsibilities of executive
management to establish and maintain the
quality management system. These responsi-
bilities include the following:
■ Establishing a quality policy and associated
quality goals and quality objectives.
■ Providing adequate facilities as well as hu-
man, equipment, and material resources
to carry out the operations of the facility
and the quality management system.
■ Ensuring appropriate design and effective
implementation of new or modified pro-
cesses and procedures.
■ Participating in the review and approval
of
quality and technical policies, processes,
and procedures.
■ Enforcing adherence to operational and
quality policies, processes, and procedures.
■ Overseeing operations and regulatory and
accreditation compliance.
■ Periodically reviewing and assessing
quality
management system effectiveness.
■ 5
■ Identifying designees and defining their
re- sponsibilities when assisting
executive management in carrying out
these duties.
Executive management support for
the quality management system goals,
objectives, and policies is critical to the
program’s success. Executive management
needs to clearly com- municate its
commitment to quality goals and create an
organizational culture that embraces quality
principles.
The individual designated to oversee
the facility’s quality functions should
report di- rectly to executive management.
In addition to having the responsibility to
coordinate, moni- tor, and facilitate quality
system activities, this person should have
the authority to recom- mend and initiate
corrective action when ap- propriate.5 The
designated individual need not perform all
of the quality functions personally. Ideally,
this person should be independent of the
facility’s operational functions. In small fa-
cilities, this independence may not always
be possible and carrying out this function
may re- quire some creativity. Depending on
the orga- nization’s size and scope, the
designated over- sight person may work in
the transfusion service, have laboratory-
wide responsibilities, supervise other
workers (eg, a quality unit), or be part of an
organization-wide quality unit (eg, hospital
quality or risk management). In- dividuals
with dual quality and operational
responsibilities should not provide
quality oversight for operational work that
they have performed.
Quality oversight functions may include
the following5:
■ Review and approval of SOPs and training
plans.
■ Review and approval of validation plans
and results.
■ Review and approval of document
control and record-keeping systems.
■ Audit of operational functions.
■ Development of criteria for evaluating sys-
tems.
■ Review and approval of suppliers.
■ Review and approval of product and
service specifications (eg, the
requirements to be
6 ■ AABB T E C HNI CAL M ANUAL

met in the manufacture, distribution, or Customer Focus

ad- ministration of blood components,
A primary focus for any organization
cellular therapy products, tissues, and
interest- ed in quality is serving the needs of
derivatives).
its custom- ers. Customers have a variety of
■ Review of reports of adverse reactions,
needs and ex- pectations. The most
de- viations in the manufacturing process,
appropriate way to ensure that these needs
nonconforming products and services,
and expectations are met is for the facility
and customer complaints.
and its customers to de- fine them in an
■ Participation in decisions to determine
agreement, a contract, or an- other
whether blood components, cellular
document. Additional information on
thera- py products, tissues, derivatives,
agreements can be found in the “Suppliers
and ser- vices are suitable for use,
and Materials Management” section.
distribution, or recall.
When planning for new or changed
■ Review and approval of corrective action
plans. prod- ucts or services, the facility should
take the customer’s needs and
■ Surveillance of problems (eg, event or inci-
expectations into ac- count. If these
dent reports, Form FDA 483 observations,
changes are determined to be critical to the
or customer complaints) and the effective-
quality or effectiveness of the products and
ness of corrective actions implemented to
services provided by the facility, they should
solve those problems.
be incorporated into the product or service
■ Use of data resources to identify trends
specifications as customer require- ments.
and potential problems before a situation
The facility must have a process to
wors- ens and patients and/or products
manage needs and expectations that are not
are af- fected.
met. For example, for a facility that has
■ Preparation of periodic (as specified by
agreed to deliver leukocyte-reduced
the organization) reports of quality
components dai- ly to one of its customers,
issues, trends, findings, and corrective
processing compo- nents in a manner that
and pre- ventive actions.
ensures adequate leu- kocyte removal is
Quality oversight functions may be critical to this product’s quality. Such an
shared among existing staff, departments, expectation should be incor- porated into the
and facilities or, in some instances, may be product specifications. Daily delivery of
per- formed by an outside firm under a products is a customer need and
contract. The goal is to provide an expectation, but it is not critical to the
independent evalua- tion of the facility’s quality of the manufactured product. The
quality activities to the ex- tent possible. facility should have a process to manage
Policies, processes, and proce- dures this agreed- on expectation and ensure that
should exist to define the roles and the product delivery mechanism meets
responsibilities of all individuals in the this customer need. If this need cannot be
devel- opment and maintenance of these met, the facility should have a process to
quality goals. Quality management system address this failure.
policies and processes should be applicable Once agreements have been made be-
tween the facility and its customers, there
across the entire facility. A blood bank,
should be a means to obtain feedback from
tissue bank, trans- fusion service, or cellular
the customer to ensure that the facility is
therapy product ser- vice need not develop
meeting the customer’s expectations.
its own quality policies if it is part of a
Mecha- nisms for obtaining such feedback
larger entity whose quality management
proactively include satisfaction surveys and
system addresses all of the mini- mum
periodic re- views of agreements. Reactive
requirements. The quality management
feedback is ob- tained through customer
system should address all matters related to
complaints. A review of event data may also
compliance with federal, state, and local
indicate failures to meet customer needs
regu- lations and accreditation standards
and expectations. Data ob- tained through
that are applicable to the organization.
these mechanisms should be evaluated, and
appropriate follow-up actions
 CHAP TER 1 Quality Management Systems: Theory and Practice
must be taken. One such action could be
to change the agreement. Inadequately
address- ing customer concerns or failing to
meet ex- pectations may result in loss of the
customer.
Facilities, Work Environment, and
Safety
The facility should provide a safe workplace
with adequate environmental controls and
emergency procedures to ensure the safety
of patients, donors, staff, and visitors. Space
allo- cation, building utilities, and the
communica- tion infrastructure should
adequately support the facility’s activities.
The facility should be kept clean and well
maintained so that the products and services
provided are not com- promised. Procedures
should be in place to address the following:
■ General safety.
■ Disaster preparedness, response, and re-
covery.
■ Biological safety (eg, protection from
blood-
borne pathogens).
■ Chemical safety.
■ Fire safety.
■ Radiation safety, if applicable.
■ Discard of biologic and other hazardous
substances.
cGMP regulations require quality
plan- ning and control of the physical work
environ- ment, including the following:
■ Adequate space and ventilation.
■ Sanitation and trash disposal.
■ Equipment for controlling air quality and
pressure, humidity, and temperature.
■ Water systems.
■ Toilet and hand-washing facilities.
An evaluation of the infrastructure and its
limitations before implementation of proce-
dures or installation of equipment will help
ensure maximum efficiency and safety. A
more thorough discussion of facilities and
safety can be found in Chapter 2.
■ 7
Human Resources
This element of the quality management
sys- tem is aimed at management of
personnel, in- cluding selection, orientation,
training, com- petence assessment, and
staffing.
Selection
Each facility should have a process to provide
adequate numbers of qualified personnel to
perform, verify, and manage all activities in
the facility. Qualification requirements for
person- nel are determined on the basis of job
respon- sibilities. The selection process should
consid- er the applicant’s qualifications for a
particular position as determined by his or her
educa- tion, training, experience,
certifications, and licensure. For laboratory
testing staff, the stan- dards for personnel
qualifications should be compatible with the
regulatory requirements established under
CLIA.1 Job descriptions are required for all
personnel involved in process- es and
procedures that affect the quality of tests,
products, and services. Effective job de-
scriptions clearly define the qualifications and
responsibilities of the positions as well as their
reporting relationships.
Orientation, Training, and Competence
Assessment
Once hired, employees should be oriented to
their position and the organization’s policies
and procedures and be trained in their new
duties. The orientation program should cover
facility-specific requirements and policies that
address issues such as safety, quality, informa-
tion systems, security, and confidentiality. The
job-related portion of the orientation program
should cover operational issues specific to the
work area. Training should be provided for
each procedure for which employees are re-
sponsible. The ultimate result of the orienta-
tion and training program is to deem new em-
ployees competent to independently perform
the duties and responsibilities defined in their
job descriptions. Time frames should be estab-
lished to accomplish this goal.
Before the introduction of a new test or
service, existing personnel should be trained
8 ■ AABB T E C HNI CAL M ANUAL
to perform their newly assigned duties and
must be deemed competent in these duties.
During orientation and training,
employees should be given the opportunity
to ask ques- tions and seek additional help
or clarification. All aspects of the training
should be docu- mented, and the facility
trainer or designated facility management
representative and em- ployees should
mutually agree on how em- ployees’
competence will be determined.
FDA cGMP training is required for
staff involved in the manufacture of
blood and blood products, including staff
involved in col- lection, testing, processing,
preservation, stor- age, distribution, and
transport. 3 Likewise, cGTP training is
required for personnel in- volved in similar
activities for human cells, tis- sues, and
cellular and tissue-based products
(HCT/Ps).4 Such training should provide
staff with an understanding of the regulatory
basis for the facility’s policies and
procedures as well as the specific
application of the cGMP and cGTP
requirements as described in the facili- ty’s
own written operating procedures. This
training should be provided periodically to
en- sure that personnel remain familiar with
regu- latory requirements.
To ensure that skills are maintained, the
facility should have regularly scheduled com-
petence evaluations of all staff members
whose activities affect the quality of laboratory
testing, manufacture of products, or provision
of products or services.1,5-10 Competence as-
sessment of management personnel should
also be considered.
Depending on the nature of the job du-
ties, competence assessments may include
written evaluations; direct observations of ac-
tivities; reviews of work records or reports, in-
formation system records, and QC records;
testing of unknown samples; and evaluations
of employees’ problem-solving skills.5 For all
testing personnel, CMS requires that each of
the following methods be used, when applica-
ble, for each test system annually1:
■ Direct observations of
– Routine patient test performance (in-
cluding patient preparation, if applica-
ble), specimen handling, processing, and
testing.
– Performance of instrument maintenance
and function checks.
■ Monitoring of the recording and reporting
of test results.
■ Review of intermediate test results or
work- sheets, QC records, proficiency
testing re- sults, and preventive
maintenance records.
■ Assessment of
– Test performance by testing previously
analyzed specimens, internal blind
test- ing samples, or external
proficiency test- ing samples.
– Problem-solving skills.
A formal competency plan that includes a
schedule of assessments, a defined minimum
for acceptable performance, and remedial
measures is one way to ensure appropriate
and consistent competence assessments. As-
sessments need not be targeted to each indi-
vidual test or procedure performed by the em-
ployee; instead, they can be grouped together
to assess similar techniques or methods. How-
ever, any test with unique aspects, problems,
or procedures should be assessed separately to
ensure that staff maintain their competency to
report test results promptly, accurately, and
proficiently.1 Written tests can be used effec-
tively to evaluate problem-solving skills and
cover many topics by asking one or more
ques- tions for each area to be assessed.
CMS re- quires that employees who perform
testing be assessed semiannually during
the first year that patient specimens are tested
and an- nually thereafter.1 Initial training
verification activities may serve as the first of
these com- petence assessments.
The quality oversight personnel
should assist in the development, review,
and approv- al of training programs,
including the criteria for retraining. 5
Quality oversight personnel also monitor
the effectiveness of training pro- grams and
competence evaluations, and they
recommend changes as needed. In addition,
The Joint Commission requires analyses of
ag- gregate competence assessment data to
iden- tify staff learning needs.9
 CHAP TER 1 Quality Management Systems: Theory and Practice
Staffing
Management should have a staffing plan
that describes the number and qualifications
of personnel needed to perform the facility’s
functions safely and effectively. There
should be an adequate number of staff to
perform the operational activities and
support quality management system
activities. Organizations should assess
staffing effectiveness by evaluat- ing human
resource indicators (eg, overtime, staff
injuries, and staff satisfaction) in con-
junction with operational performance indi-
cators (eg, adverse events and patient com-
plaints). The results of this evaluation
should feed into the facility’s human
resource plan- ning process along with
projections based on new or changing
operational needs.
Suppliers and Materials Management
Materials, supplies, and services used as in-
puts to a process are considered critical if
they affect the quality of products and
services be- ing produced. Examples of
critical supplies are blood components,
blood bags, test kits, and reagents. Examples
of critical services are infectious disease
testing, blood component irradiation,
transportation, equipment cali- bration, and
preventive maintenance. The suppliers of
these materials and services may be internal
(eg, other departments within the same
organization) or external (outside ven- dors).
Supplies and services used in the collec-
tion, testing, processing, preservation,
storage, distribution, transport, and
administration of blood components,
cellular therapy products, tissues, and
derivatives that have the potential to affect
quality should be qualified before use and
obtained from suppliers who can meet the
facility’s requirements. Three important ele-
ments of supplier and materials management
are 1) supplier qualification; 2) agreements;
and 3) receipt, inspection, and testing of in-
coming supplies.
Supplier Qualification
Critical supplies and services must be quali-
fied on the basis of defined requirements.
Sim- ilarly, suppliers should be qualified to
ensure
■ 9
that they are reliable sources of materials. The
facility should clearly define requirements or
expectations for its suppliers and share this in-
formation with staff and suppliers. Suppliers’
ability to consistently meet specifications for a
supply or service should be evaluated along
with performance relative to availability,
deliv- ery, and support. The following are
examples of factors that could be considered to
qualify suppliers:
■ Licensure, certification, or accreditation.
■ Supply or product requirements.
■ Supplier-relevant quality documents.
■ Results of audits or inspections.
■ Quality summary reports.
■ Customer complaints.
■ Experience with that supplier.
■ Cost of materials or services.
■ Delivery arrangements.
■ Financial security, market position, and
customer satisfaction.
■ Support after sales.
A list of approved suppliers should
be maintained that includes both primary
suppli- ers and suitable alternatives for
contingency planning. Critical supplies and
services should be purchased only from
suppliers that have been qualified. Once
suppliers are qualified, periodic evaluations
of supplier performance help ensure
suppliers’ continued ability to meet
requirements. Tracking suppliers’ ability to
meet expectations gives the facility valuable
information about the stability of each
suppli- er’s processes and commitment to
quality. Documented failures of supplies or
suppliers to meet defined requirements
should result in immediate action by the
facility, including no- tification of the
supplier, quality oversight per- sonnel, and
management with contracting authority, if
applicable. Supplies may need to be
replaced or quarantined until all quality is-
sues are resolved.
Agreements
Contracts and other agreements define
expec- tations and reflect the concurrence of
the par- ties involved. Periodic reviews of
agreements
10 ■ AABB T E C HNIC AL MANUAL
ensure that the expectations of all parties
con- tinue to be met. Changes should be
mutually agreed upon and incorporated as
needed.
Transfusion and cellular therapy
services should maintain written contracts
or agree- ments with outside suppliers of
critical mate- rials and services, such as
blood components, cellular therapy
products, irradiation, compat- ibility
testing, or infectious disease marker
testing. An outside supplier may be
another department within the same
facility that is managed independently, or it
may be another facility (eg, contract
manufacturer). The con- tracting facility
assumes responsibility for the manufacture
of the product; ensuring the safe- ty, purity,
and potency of the product; and en- suring
that the contract manufacturer com- plies
with all applicable product standards and
regulations. Both the contracting facility
and the contractor are legally responsible
for the work performed by the contractor.
It is important for the transfusion or cel-
lular therapy service to participate in the eval-
uation and selection of suppliers. The service
should review contracts and agreements to en-
sure that all important aspects for their critical
materials and services are addressed. Exam-
ples of issues that could be addressed in an
agreement or contract include the responsibil-
ity for a product or blood sample during ship-
ment; the supplier’s responsibility to promptly
notify the facility when changes have been
made that could affect the safety of blood
components, cellular therapy products, tis-
sues, derivatives, or services for patients; and
the supplier’s responsibility to notify the facili-
ty when information is discovered indicating
that a product may not be considered safe,
such as during look-back procedures.
Receipt, Inspection, and Testing
of Incoming Supplies
Before acceptance and use, critical materials
such as reagents, blood components, cellular
therapy products, tissues, and derivatives
should be inspected and tested (if necessary)
to ensure that they meet specifications for
their intended use. It is essential that supplies
used in the collection, processing, preserva-
tion, testing, storage, distribution, transport,
and administration of blood, components,
tis- sues, and cellular therapy products also
meet FDA requirements.
The facility must define acceptance crite-
ria for critical supplies (see 21 CFR 210.3)3
and must develop procedures to control and
pre- vent the inadvertent use of materials that
do not meet specifications. Corrective action
may include returning the material to the
vendor or destroying it. Receipt and inspection
records enable the facility to trace materials
that have been used in a particular process and
provide information for ongoing supplier
qualifica- tion.
Equipment Management
Equipment that must operate within defined
specifications to ensure the quality of blood
components, cellular therapy products, tis-
sues, derivatives, and services is referred to as
“critical equipment” in the quality manage-
ment system. Critical equipment may include
instruments, measuring devices, and comput-
er systems (hardware and software).
Activities designed to ensure that
equip- ment performs as intended include
qualifica- tion, calibration, maintenance,
and monitor- ing. Calibration, functional
and safety checks, and preventive
maintenance should be sched- uled and
performed according to the manu-
facturer’s recommendations and regulatory
re- quirements of the FDA2-4 and CMS. 1
Written procedures for equipment use and
control should comply with the
manufacturer’s rec- ommendations unless
an alternative method has been validated by
the facility and, in some instances, approved
by the appropriate regula- tory and
accrediting agencies.
When one selects new equipment, it is
important to consider not only the perfor-
mance of the equipment as it will be used
in the facility but also any issues regarding
ongo- ing service and support by the
supplier. There should be a written plan for
installation, oper- ational, and performance
qualifications.6 The plan should provide for
1) installation accord- ing to the
manufacturer’s specifications, 2)
verification of the equipment’s
functionality
 CH A P TE R 1 Quality Management Systems: Theory and Practice
before use by ensuring that the criteria estab-
lished by the manufacturer for its intended use
are met, and 3) assurance that the equipment
performs as expected in the facility’s process-
es. After the equipment is installed, any prob-
lems and follow-up actions taken should be
documented. Recalibration and requalifica-
tion may be necessary if repairs are made that
affect the equipment’s critical operating func-
tions. Recalibration and requalification should
also be considered when existing equipment is
relocated.
The facility must develop a mechanism
to uniquely identify and track all critical
equip- ment, including equipment software
versions, if applicable. The unique
identifier assigned by the manufacturer may
be used, or a unique identification code
may be applied by the transfusion or
cellular therapy service or assigned
through a laboratory-wide or organi- zation-
wide identification system. Maintain- ing a
list of all critical equipment helps in the
control function of scheduling and
performing functional and safety checks,
calibrations, pre- ventive maintenance, and
repair. The equip- ment list can be used to
ensure that all appro- priate actions have
been performed and recorded. Evaluating
and trending equipment calibration,
maintenance, and repair data help the
facility assess equipment functionality,
manage defective equipment, and identify
equipment needing replacement. When
equipment is found to be operating outside
acceptable parameters, the potential
effects on the quality of products or test
results must be evaluated and documented.
Process Management
Written and approved policies, processes,
and procedures must exist for all critical
functions performed in the facility, and these
functions must be carried out under
controlled condi- tions. Each facility should
have a systematic approach for identifying,
planning, and imple- menting new (and
making changes to existing) policies,
processes, and procedures that affect the
quality of the facility’s tests, products, or
services. Such activities should include a re-
view of at least the following:
■ 11
■ Customer needs and expectations.
■ Accreditation and regulatory requirements.
■ Specifications to be met.
■ Risk assessment.
■ Performance measures.
■ Nonconformance analyses.
■ Current knowledge (eg, of other successful
practices).
■ Resource needs (eg, financial, facility, hu-
man, materials, and equipment).
■ Interrelationships of the new or changed
process(es) with other processes.
■ Documents needed for the new or changed
process(es).
The documents developed should be
re- viewed by management personnel with
direct authority over the process and by
quality over- sight personnel before
implementation. Changes in policies,
processes, and proce- dures should be
documented, validated, re- viewed, and
approved. Additional information on
policies, processes, and procedures can be
found in the “Documents and Records”
sec- tion later in this chapter.
Once a process has been implemented,
the facility should have a mechanism to
ensure that procedures are performed as de-
fined and that critical equipment, reagents,
and supplies are used in conformance with
manufacturers’ written instructions and
facili- ty requirements. Table 1-1 lists
elements that constitute sound process
control (among oth- er elements of a quality
management system). A facility using
critical equipment, reagents, or supplies in a
manner that is different from the
manufacturer’s directions should validate
such use. If the activity is covered under
regulations for blood and blood components
or HCT/Ps, the facility may be required to
request FDA ap- proval to operate at
variance to requirements (see 21 CFR
640.1202 or 21 CFR 1271.1554). If a
facility believes that changes to the
manufac- turer’s directions would be
appropriate, it should encourage the
manufacturer to make such changes in the
labeling (ie, the package insert or user
manual).
12 ■ AABB T E C HNIC AL MANUAL

TABLE 1-1. Components of a Quality Management System

Quality System ComponentQuality Functions and Responsibilities

Organization and leadership ■ Organization structure and function

■ Leadership roles and responsibilities, authority, and relationships
■ Establishment of a quality management system
■ Customer needs
■ Planned products and services
■ Documented, followed, and improved policies, processes,
and procedures
■ Quality representative
■ Management reviews
■ Provision of adequate resources
■ Adequate design and effective implementation
■ Conformance with requirements
■ Effective communication
■ Effective process improvement
Customer focus ■ Customer requirements
■ Agreements
■ Customer feedback
Facilities, work environment,
■ Minimal health and safety risks
and safety
■ Design and space allocations
■ Clean work environment
■ Controlled environment
■ Communication and information management systems
■ Storage facilities
■ Health and safety programs
■ Hazard discards
■ Emergency preparedness
Human resources ■ Adequate and qualified staff
■ Job descriptions and qualifications
■ Defined roles and responsibilities for all staff and their
reporting relationships
■ Staff selection
■ New hire orientation
■ Training on the quality system, job-related activities, computer
use, and safety
■ Staff competence
■ Continuing education
■ Staff identifying information
■ End-of-employment activities
Suppliers and materials
■ Supplier qualification
management
■ Qualifying materials
■ Agreement reviews
■ Inventory management
■ Adequate storage conditions
■ Receipt, inspection, and testing of incoming materials and products
■ Acceptance and rejection of materials and products
■ Tracing critical supplies and services
 CH A P TE R 1 Quality Management Systems: Theory and Practice
TABLE 1-1. Components of a Quality Management System (Continued)
Quality System ComponentQuality Functions and Responsibilities
Equipment management
Process management
Documents and records
Information management
Management of nonconforming
events
Monitoring and assessment
Process improvement
■ 13
■ Selection and acquisition
■ Unique identification code
■ Verification of performance
■ Installation, operational, and performance qualification
■ Calibration
■ Preventive maintenance and repairs
■ Retirement
■ Process development
■ Change control
■ Process validation
■ Process implementation
■ Adherence to policies, processes, and procedures
■ Quality control program
■ Inspection of products and services
■ Concurrent creation of records
■ Requirements for critical activities
■ Traceability
■ Standardized formats
■ Document creation
■ Unique identification code
■ Review and approval process
■ Document use and maintenance
■ Change control
■ Record archiving and storage
■ Record retention and destruction
■ Confidentiality
■ Prevention of unauthorized access
■ Data integrity
■ Data backup
■ Alternative system
■ Detection of deviations and nonconformances
■ Complaint file
■ Adverse event reporting
■ Investigations
■ Immediate actions
■ Monitoring and assessment of specified requirements
■ Quality indicators
■ Internal and external assessments
■ Laboratory proficiency testing
■ Data analyses
■ Identifying opportunities for improvement
■ Systems approach to continual improvement
■ Root cause evaluation
■ Corrective action plans
■ Preventive action plans
■ Monitoring for effectiveness
14 ■ AABB T E C HNIC AL MANUAL
Process Validation
Validation is used to demonstrate that a pro-
cess is capable of consistently and reliably
achieving planned results.13 It is critical to
vali- date processes in situations where it is
not fea- sible to measure or inspect each
finished prod- uct or service to fully verify
conformance with specifications. However,
even when effective end-product testing can
be achieved, it is ad- visable to validate
important processes to gen- erate
information that can be used to optimize
performance. Prospective validation is used
for new or revised processes. Retrospective
validation may be used for processes that are
already in operation but were not adequately
validated before implementation.
Concurrent validation is used when required
data cannot be obtained without
performance of a “live” process. If
concurrent validation is used, data are
reviewed at predefined periodic intervals
before full implementation receives final ap-
proval. Modifications to a validated process
may warrant revalidation, depending on the
nature and extent of the change. It is up to
the facility to determine the need for
revalidation on the basis of its understanding
of how the proposed changes may affect the
process.
Test Method Validation
When the laboratory wishes to implement a
nonwaived test using an FDA-approved or
-cleared test system, CLIA requires that the
performance specifications established by the
manufacturer be verified by the laboratory be-
fore it reports patient results.1 At a minimum,
the laboratory must demonstrate that it can
obtain performance specifications compara-
ble to those of the manufacturer for accuracy,
precision, reportable range, and reference in-
tervals (normal values).
If the laboratory develops its own meth-
od, introduces a test system not subject to
FDA approval or clearance, or makes
modifications to an FDA-approved or -cleared
test system, or if the manufacturer does not
provide perfor- mance specifications, then
the laboratory must establish the test
system performance
specifications before reporting patient results. 1
At a minimum, the following must be estab-
lished for the test system:
■ Accuracy.
■ Precision.
■ Reportable range of test results for the
test system.
■ Reference intervals (normal values).
■ Analytical sensitivity.
■ Analytical specificity, including interfering
substances.
■ Any other performance characteristic re-
quired for test performance (eg, specimen
or reagent stability).
Based on performance specifications, the
laboratory must also establish calibration and
control procedures and document all activities
for test method validation. (See 42 CFR
493.1253.1)
Validation Plan
Validation should be planned if it is to be
effec- tive. Development of a validation
plan is best accomplished after one obtains
an adequate understanding of the system or
framework within which the process will
occur. The plan should include conducting
the process as de- signed. Additionally, a
significant amount of effort should be
targeted at attempts to “break” the process
to identify weaknesses and limitations.
Many facilities develop a template for the
written validation plan to ensure that all
aspects are adequately addressed. Although
no single format for a validation plan is re-
quired, most plans include the following
com- mon elements:
■ System description.
■ Purpose or objectives.
■ Risk assessment.
■ Responsibilities.
■ Validation procedures.
■ Acceptance criteria.
■ Approval signatures.
■ Supporting documentation.
 CH A P TE R 1 Quality Management Systems: Theory and Practice
The validation plan should be
reviewed and approved by quality
oversight personnel before the validation
activities are carried out.
Staff responsible for carrying out the
vali-
dation activities should be trained in the pro-
cess before the plan is executed. The
results and conclusions of these activities
may be ap- pended to the approved
validation plan or may be recorded in a
separate document. This documentation
typically contains the follow- ing elements:
■ Expected and observed results.
■ Interpretation of results as acceptable or
unacceptable.
■ Corrective action for and resolution of
un-
expected results.
■ Explanation of and rationale for any devia-
tions from the validation plan.
■ Conclusions and limitations.
■ Approval signatures.
■ Supporting documentation.
■ Implementation timeline.
When a validation process does not
pro- duce the expected outcome, its data
and cor- rective actions must be
documented as well. The responsible
quality oversight personnel should provide
final review and approval of the validation
plan, results, and corrective ac- tions and
determine whether new or modified
processes and equipment may be
implement- ed as planned or implemented
with specified limitations.
Equipment Validation
Validation of new equipment used in a process
should include installation, operational, and
performance qualifications, as follows20:
■ Installation qualification demonstrates
that the instrument is properly installed in
envi- ronmental conditions that meet the
manu- facturer’s specifications.
■ Operational qualification demonstrates
that the installed equipment operates as
in- tended. It focuses on the equipment’s
capa- bility to operate within the
established lim-
■ 15
its and specifications supplied by the
manufacturer.
■ Performance qualification demonstrates
that the equipment performs as expected
for its intended use in the processes
estab- lished by the facility and that the
output meets the facility’s specifications.
It evalu- ates the adequacy of equipment
for use in a specific process that uses the
facility’s own personnel, procedures, and
supplies in a normal working
environment.
Computer System Validation
The FDA considers computerized systems
to include “hardware, software, peripheral
devic- es, networks, personnel, and
documenta- tion.”21 End-user validations of
computer sys- tems and the interfaces
between systems should be conducted in the
environment in which they will be used.
Testing by the com- puter software vendor
or supplier is not a sub- stitute for computer
validation at the facility. End-user
acceptance testing may repeat some of the
validation performed by the developer, such
as load or stress testing and verification of
security, safety, and control features, to eval-
uate performance under actual operating
con- ditions. In addition, the end user should
evalu- ate the ability of personnel to use the
computer system as intended within the con-
text of actual work processes. The hardware
and software interface should be designed so
that staff can navigate successfully and re-
spond appropriately to messages, warnings,
and other functions. If changes to the
comput- er system result in changes to the
way a pro- cess is performed, process
revalidation should also be performed. As
with process validation, quality oversight
personnel should review and approve
validation plans, results, and corrective
actions and should determine wheth- er
implementation may proceed with or with-
out limitations.
For additional information,
facilities should refer to FDA guidance
on computer system validation in the user’s
facility.21 Those who develop their own
software should con- sult Title 21 CFR Part
880 and FDA guidance
16 ■ AABB T E C HNIC AL MANUAL
regarding general software validation princi-
ples.22
Quality Control
QC testing is performed to ensure the proper
functioning of materials, equipment, and
methods during operations. QC performance
expectations and acceptable ranges should
be defined and be made readily available to
staff so they will recognize, and respond
appropri- ately to, unacceptable results and
trends. The frequency for QC testing is
determined by the facility in accordance
with the applicable CMS, FDA, AABB, state,
and manufacturer re- quirements. QC results
should be documented concurrently with
performance.2 Records of QC testing should
include the following:
■ Identification of personnel performing the
test.
■ Identification of reagents (including lot
numbers and expiration dates).
■ Identification of equipment.
■ Testing date and, when applicable, time.
■ Results.
■ Interpretation (eg, meets or fails to meet
es- tablished criteria).
■ Reviews.
Unacceptable QC results must be
investi- gated and corrective action must
be imple- mented, if indicated, before the
QC procedure is repeated or the operational
process is con- tinued. If products or
services have been pro- vided since the last
acceptable QC results were obtained, it may
be necessary to evaluate the conformance
of these products or services. Examples of
QC performance intervals for equipment
and reagents are included in Ap- pendix 1-
3.
Documents and Records
Documentation provides a framework for
understanding and communicating about
processes throughout the organization.
Doc- uments provide a description of or
instruc- tions regarding what is supposed
to happen. Documents describe how
processes are in- tended to work, how
they interact, where
they must be controlled, what their
require- ments are, and how to implement
them. Rec- ords provide evidence of what
did happen (ie, that a process was
performed as intend- ed), and provide
information needed to as- sess product and
service quality. Together, documents and
records are used by quality oversight
personnel to evaluate the effective- ness of
a facility’s policies, processes, and
procedures. ISO 9001 provides an example
of quality system documentation that
includes the following items13:
■ The quality policy and objectives.
■ A description of the interactions between
processes.
■ Documented procedures for the control of
documents, records, and nonconforming
products and for corrective action,
preven- tive action, and internal quality
audits.
■ Records related to the quality
management system, operational
performance, and product or service
conformance.
■ All other “documents needed by the organi-
zation to ensure the effective planning, op-
eration, and control of its processes.”
Written policies, process
descriptions, procedures, work instructions,
job aids, labels, forms, and records are all
part of the facility’s document management
system. They may be paper based or
electronic. A document man- agement
system provides assurance that doc- uments
are comprehensive, current, and
available and that records are accurate and
complete. A well-structured document
man- agement system links policies,
process de- scriptions, procedures, forms,
and records to- gether in an organized and
workable system.
Documents
Documents should be developed in a format
that conveys information clearly and
provides staff with the necessary
instructions and templates for recording
data. The CLSI offers guidance regarding
general levels of documentation11 as well as
detailed instruc- tions on how to write
procedures.23 General types of
documentation are described below.
 CH A P TE R 1 Quality Management Systems: Theory and Practice
PO L I CI E S . Policies communicate the organi-
zation’s highest-level goals, objectives, and in-
tent. The rest of the organization’s documenta-
tion interprets, and provides instructions
regarding implementation of, these policies.
PR OCES SE S. Process documents describe a
sequence of actions and identify
responsibili- ties, decision points,
requirements, and accep- tance criteria.
Process diagrams or flowcharts are often
used for this level of documentation. It is
helpful to show process control points on a
diagram as well as the flow of information
and handoffs between departments or work
groups.
PR O CE D URES , WO R K IN S T RUCT IO NS ,
AND
JO B A I D S . These documents provide step-
by- step directions on performing job tasks
and procedures. Procedures and work
instructions should include enough detail to
perform a task correctly but not so much as
to be difficult to read. The use of
standardized formats helps staff know where
to find specific elements and facilitates
implementation and control. Job aids are
excerpted from an approved docu- ment and
condense information into a short- er, more
readily viewable format. External doc-
uments (eg, from a manufacturer’s manual or
package insert) may also be incorporated
into the facility’s procedures manual by
reference. Relevant procedures should be
available to the staff in each area in which
the corresponding job tasks are
performed.2,5,8
FO R M S . Forms provide templates for captur-
ing data on paper or electronically. These doc-
uments specify the data requirements called
for in SOPs and processes. Forms should be
carefully designed to be easy to use, minimize
the likelihood of errors, facilitate data and in-
formation retrieval, effectively capture out-
comes, and support process traceability. When
it is not immediately evident what data should
be recorded or how to record them, forms
should include instructions for their use.
Forms should indicate units of measure for
recording quantitative data.
LA BEL S . Product labels, such as blood
com- ponent or HCT/P labels, are subject to
many of
■ 17
the requirements in a document management
system. Many facilities maintain a master
set of labels that can be used as a reference
to veri- fy that only currently approved
stock is in use. The accuracy of new stock
labels should be verified before this stock is
put into inventory; comparison against a
master label provides a mechanism for this
verification. Change con- trol procedures
should be established for the use of on-
demand label printers to prevent
nonconforming modifications of label
format or content.
Each facility should have a defined pro-
cess for developing and maintaining docu-
ments. This process should identify basic
ele- ments required for documents;
procedures for review and approval of new
or revised docu- ments; a method for
keeping documents cur- rent; a process for
control of document distri- bution; and a
process for archiving, protecting, and
retrieving obsolete documents. Training
should be provided to the staff responsible
for the content of new or revised documents.
Doc- ument management systems include
these es- tablished processes:
■ Verifying the adequacy of the document
be- fore its approval and issuance.
■ Periodically reviewing, modifying, and
re- approving documents as needed to
keep them current.
■ Identifying changes and revision status.
■ Ensuring that documents are legible,
iden- tifiable, and readily available in the
loca- tions in which they will be used.
■ Retaining and retrieving previous
versions for the required retention period.
■ Preventing unintended use of outdated or
obsolete documents.
■ Protecting documents from alteration,
damage, or unintended destruction.
External documents that are
incorporated by reference become part of
the document management system and
should be identified and controlled. The
facility should have a mechanism to detect
changes to external doc- uments in its
system, such as manufacturers’ package
inserts or user manuals, so that corre-
18 ■ AABB T E C HNIC AL MANUAL
sponding changes to procedures and forms
can be made.
When new or revised policies, process
de- scriptions, procedures, or forms are
added to or replaced in the facility’s manual,
these doc- uments should be marked with
the date on which they were first put into
use (ie, effective date).
One copy of retired documents should
be retained as defined by existing and
applicable standards and regulations.
A master list of all current policies,
pro- cess descriptions, procedures, forms,
and la- bels is useful for maintaining
document con- trol. It should include
document titles, individuals or work
groups responsible for maintaining each
document, revision dates, unique document
identifiers, and the areas in which each
document is used. It should also identify
the number and locations of con- trolled
copies in circulation. Copies of docu-
ments that are used in the workplace should
be identified and controlled to ensure that
none are overlooked when changes are
imple- mented.
Records
Records provide evidence that critical steps
in a procedure have been performed
appropri- ately and that products and
services conform to specified requirements.
Records should be created concurrently with
the performance of each significant step and
should clearly indi- cate the identity of the
individuals who per- formed each step and
when each step was completed. 2,6,7 Data
should be recorded in a format that is clear
and consistent. When forms are used for
capturing or recording data, steps, or test
results, the forms become rec- ords.
The process for managing records
should address the following items:
■ Creation and identification of records.
■ Protection from accidental or
unauthorized modification or destruction.
■ Verification of completeness, accuracy, and
legibility.
■ Storage and retrieval.
■ Creation of copies or backups.
■ Retention periods.
■ Confidentiality.
Records review is an important tool to
help evaluate the effectiveness of the
quality management system. The facility
should de- fine a process and time frames
for records re- view to ensure accuracy,
completeness, and appropriate follow-up.
It should determine how reports and
records are to be archived and how to define
their retention period. Specific requirements
for records to be maintained by AABB-
accredited facilities are included in the
relevant AABB standards.
Record-keeping systems should allow
for ready retrieval of records within time
frames established by the facility and
permit trace- ability of blood components,
cellular therapy products, tissues, and
derivatives as required by federal
regulations.2,4 When copies of rec- ords are
retained, the facility should verify that each
copy contains the complete, legible, and
accessible content of the original record
before the original is destroyed.
If records are maintained
electronically, adequate backups should
exist in case of sys- tem failure. Electronic
records should be read- able for the entire
duration of their retention period. Obsolete
computer software that is necessary to
reconstruct or trace records should also
be archived appropriately. If the equipment
or software used to access archived data
cannot be maintained, the records should be
converted to another format or copied to
another medium to permit continued access.
Converted data should be verified against
the original to ensure completeness and
accuracy. Electronic media such as magnetic
tapes, opti- cal disks, or online remote
servers are widely used for archiving
documents. Records kept in this manner
must meet FDA requirements for electronic
record-keeping.24 Microfilm or mi- crofiche
may also be used to archive records. The
medium selected should be appropriate to
comply with the retention requirements.
Each facility must have a policy for alter-
ing or correcting records. 6 The date of the
changes and the identity of the individual
making each change must be documented. In
 CH A P TE R 1 Quality Management Systems: Theory and Practice ■ 19
data.

some instances, it may also be important to

in- dicate the reason for the change. The
original wording must not be obliterated
in written records; the original may be
crossed out with a single line, but it should
remain legible. Write- overs and scratch-
outs should not be used. Electronic
records must permit tracking of both
original and corrected data and must in-
clude the date and identity of the person
who made the change. There should be a
process for controlling changes. A method
for refer- encing changes to records that is
linked to the original record and a system
for reviewing changes for completeness
and accuracy are es- sential. Audit trails for
changed data in com- puterized systems are
required by the FDA.24
The following issues might be
considered when planning record storage:

■ Storage of records in a manner that

protects them from damage and
accidental or unau- thorized destruction
or modification.
■ Degree of accessibility of records in
propor- tion to frequency of their use.
■ Method and location of record storage re-
lated to the volume of records and the
amount of available storage space.
■ Availability of properly functioning
equip- ment, such as computer hardware,
and software to view archived records.
■ Documentation that all records copied,
transferred to microfiche, or converted to
digital files legitimately replace originals
that are stored elsewhere or destroyed.
■ Retention of original color-coded records
when only black-and-white reproductions
are available.

Considerations for electronic records

in- clude the following:

■ A method of verifying the accuracy of data

entry.
■ Prevention of unintended deletion of data
or access by unauthorized persons.
■ Adequate protection against inadvertent
data loss (eg, when a storage device is
full).
■ Validated safeguards to ensure that a
record can be edited by only one person
at a time.
■ Security of and access to confidential
 that critical data have not been inad- vertently
modified, been lost, or become inaccessible.
The facility should When data are sent manually or electronically
maintain a record of names; from one point to another, a process should be
inclusive dates of in place to ensure that the data accurately and
employment; and reliably reach their final destination in a timely
corresponding signatures, manner.
identifying initials, or Backup versions (eg, disks, tapes, or
identification codes of du- plicate hard copies) of critical data
personnel autho- rized to should be maintained in the event of an
create, sign, initial, or review unexpected loss from the storage medium.
reports and records. It is advisable to store backup or archived
Magnetically coded computer records and databases off-site at
employee badges and other a sufficient distance away to ensure that
computer-related identify- disasters will not affect both the originals
ing methods are generally and the backups. The
accepted in lieu of written
signatures, provided that the
badges or other methods meet
electronic record-keeping
requirements.

Information Management
The quality management
system should en- sure the
confidentiality and
appropriate use of data and
information in both oral and
written communications.
Privacy of patient and donor
records should be addressed
to maintain the security and
confidentiality of such
records.
The system should
prevent unauthorized access,
modification, or destruction
of the data and information.
Individuals who are au-
thorized to make changes to
data should be defined by
name, code, or job
responsibility. Information
systems should be designed
with security features to
prevent unauthorized ac- cess
and use. Systems may include
levels of se- curity defined by
job responsibility and re-
quire the use of security codes
and passwords or, for paper-
based systems, locked
cabinets and keys.
The integrity of data
should be main- tained so that
data are retrievable and usable.
Periodic integrity checks
should be conducted to ensure
20 ■ AABB T E C HNIC AL MANUAL

the
backup storage facility should be secure.
Envi- ronmental conditions in the backup
storage facility should be maintained in a
way that protects and preserves the
equipment and media for the duration of
their storage. Tem- perature and humidity
should be monitored and controlled.
Archival copies of computer operating
systems and applications software required
to view original records should be stored
in the same manner.
The facility should develop and
maintain alternative systems to ensure
access to critical data and information in
the event that com- puterized data or
primary sources of informa- tion are not
available. The backup and recov- er y
procedures for computer downtime
should be defined, and validation
documenta- tion should show that the
backup system works properly. The
associated processes should be tested
periodically to ensure that the backup
system remains effective. Special
consideration should be given to staff
compe- tence and readiness to use the
backup system.

Management of Nonconforming
Events
The quality management system should in-
clude a process for detecting, investigating,
and responding to events that result in
devia- tions from accepted policies,
processes, and procedures or in failures to
meet require- ments, as defined by the
facility, AABB stan- dards, or applicable
regulations.2-4 This pro- cess should be
implemented after, for example, the
discovery of nonconforming products and
services or of adverse reactions to donation,
blood components, cellular ther- apy
products, tissues, or derivatives. The facili-
ty should define how to perform the
following for nonconforming events:

■ Document and classify occurrences.

■ Determine the effect, if any, on the
quality of products or services.
■ Evaluate the effect on interrelated
activities.
■ Analyze the event to understand root
causes.
■ Implement corrective action as appropri-
ate, including notification and recall, on
 corrective action are dis- cussed in the
basis of investigations section on “Process Improve- ment.” A
and root cause anal- summary of each event, investiga- tion,
yses. and any follow-up activities must be
■ Implement prepared. Table 1-2 outlines suggested
preventive actions as com- ponents of an internal event report.
appropri- ate on the Fatalities related to blood collection or
basis of analyses of transfusion or to HCT/Ps must be reported as
aggregate data about soon as possible to the FDA Center for Biolog-
events and their ics Evaluation and Research (CBER). [See
causes. 21 CFR 606.170(b) and 1271.350(a)(i),
■ Report to external agencies when respective- ly.] Instructions for reporting to
required. CBER are available in published guidance 27
■ Evaluate effectiveness and on the FDA website.28 A written follow-up
of the corrective ac- report must be submitted within 7 days of the
tions and preventive fatality and should include a description of
actions taken. any new pro- cedures implemented to avoid
recurrence. AABB Association Bulletin #04-
The CLSI has 06 provides ad- ditional information on these
published a consensus reporting re- quirements, including a form for
stan- dard on reporting do- nor fatalities.29
occurrence Regardless of their licensure and registra-
management that ex- tion status with the FDA, all donor centers,
plores event
management in more
detail.25
Facility personnel
should be trained to
recognize and report
such occurrences. De-
pending on the severity
of an event and risk to
patients, donors, and
products as well as the
likelihood of recurrence,
investigation of con-
tributing factors and
underlying causes may
be warranted. The
cGMP regulations
require investigation
and documentation of
results if a specific
event could adversely
affect patient safety or
the safety, purity, or
potency of blood or
components. 2 ,3 The
cGTP regulations
require similar
activities for deviations
and possible product
contamination or
communi- cable
disease transmission. 4
Tools and ap- proaches
for performing root
cause analysis and
implementing
 CH A P TE R 1 Quality Management Systems: Theory and Practice ■ 21

TABLE 1-2. Components of an Internal Event Report26

ComponentExamples

Who ■ Reporting individual(s)

■ Individual(s) involved (by job title) in committing, compounding, discovering, investi-
gating, and initiating any immediate action
■ Patient or donor identification code
■ Reviewer(s) of report
What ■ Brief description of event
■ Effects on and outcomes for patient, donor, blood component, or tissue
■ Name of component and unit identification number
■ Manufacturer, lot number, and expiration dates of applicable reagents and supplies
■ Immediate actions taken
When ■ Date of report
■ Date and time event occurred
■ Date and time of discovery
■ Date (and time, if applicable) that immediate action was taken

And as applicable, date and time of:

■ Blood component collection, processing steps, and shipping
■ Order for blood component
■ Order for patient testing
■ Patient sample collection, transport, and receipt
■ Test performance and reporting
Where ■ Physical location of event
■ Where in process event was detected
■ Where in process event was initiated
Why and How ■ How event occurred
■ Contributing factors to event
■ Root cause(s)
Follow-up ■ External reports or notifications (eg, regulatory* or accreditation agencies, manufac-
turer, or patient’s physician)
■ Corrective actions
■ Implementation dates
■ Effectiveness of actions taken
■ Linkage to preventive action if appropriate
*All blood establishments (including licensed, registered but unlicensed, and unregistered transfusion services) 2 (21 CFR
606.121) are required to notify the FDA of deviations from cGMP, applicable standards, or established
specifications that may affect the safety, purity, or potency of biological products or otherwise cause the biological
products to be in violation of the Food, Drug, and Cosmetic Act or the Public Health Service Act (21 CFR 600.14).2 The
FDA has identified the following examples as reportable events if components or products are released for
distribution:
■ Arm preparation not performed or performed incorrectly.
■ Units released from donors who are (or should have been) either temporarily or permanently deferred because of
their medical history or a history of repeatedly reactive viral marker tests.
■ Shipment of a unit with repeatedly reactive viral markers.
■ ABO/Rh or infectious disease testing not performed according to the manufacturer’s package insert.
■ Units released from donors for whom test results were improperly interpreted because of testing errors
related to improper use of equipment.
■ Units released before completion of all tests (except as an emergency release).
■ Sample used for compatibility testing that contains incorrect identification.
■ Testing error that results in the release of an incorrect unit.
■ Incorrectly labeled blood components (eg, ABO group or expiration date).
(Continued)
22 ■ AABB T E C HNIC AL MANUAL

TABLE 1-2. Components of an Internal Event Report26 (Continued)

■ Incorrect crossmatch label or tag.

■ Storage of biological products at an incorrect temperature.
■ Microbial contamination of blood components when the contamination is attributed to an error in manufacturing.
Deviations involving distributed HCT/Ps and relating to core cGTP requirements must also be reported to the FDA if
they occurred in the facility or in a facility that performed a manufacturing step for the facility under contract,
agreement, or other arrangement. 4 Each report must contain a description of the HCT/P deviation, information
relevant to the event and the manufacture of the HCT/Ps involved, and information on all follow-up actions that
have been or will be taken in response to the deviation.
CFR = Code of Federal Regulations, FDA = Food and Drug Administration; cGMP = current good manufacturing
practice; HCT/Ps = human cells, tissues, and cellular and tissue-based products; cGTP = current good tissue
practice.

blood banks, and transfusion services must
promptly report biological product devia-
tions—and information relevant to these
events—to the FDA2,30 using Form FDA-3486
when the event 1) is associated with manufac-
turing (ie, collecting, testing, processing, pack-
ing, labeling, storing, holding, or distributing);
2) represents a deviation from cGMP, estab-
lished specifications, or applicable regula-
tions or standards or that is unexpected or un-
foreseen; 3) may affect the product’s safety,
purity, or potency; 4) occurs while the facility
had control of, or was responsible for, the
product; and 5) involves a product that has left
the facility’s control (ie, has been distributed).
Using the same form, facilities must
also promptly report biological product
deviations associated with a distributed
HCT/P if the event represents a deviation
from applicable regulations, standards, or
established specifi- cations that relate to the
prevention of com- municable disease
transmission or HCT/P contamination.
This requirement pertains to events that are
unexpected or unforeseeable but may relate
to the transmission or potential transmission
of a communicable disease or may lead to
HCT/P contamination.4 More in- formation
concerning biological product devi- ation
reporting can be found on the FDA web-
site.31
There must also be a mechanism to re-
port medical device adverse events to the
FDA and the device manufacturer. 8,32 The
Joint Commission encourages reporting of
sentinel events, including hemolytic
transfusion reac- tions involving the
administration of blood or
components having major blood group in-
compatibilities.9,10
Hemovigilance processes also provide the
opportunity to detect, investigate, and re-
spond to adverse transfusion reactions and
events that result in deviations from safe blood
transfusion and collection practices. Adverse
transfusion reactions and events (or incidents)
can be reported voluntarily to the Centers for
Disease Control and Prevention (CDC)
Nation- al Healthcare Safety Network
(NHSN) Hemo- vigilance Module. This
system was developed through a public-
private collaboration that in- cluded the
Department of Health and Human Services
and its agencies, and the private sec- tor,
including AABB, America’s Blood Centers,
and the American Red Cross. The AABB
Center for Patient Safety, a licensed Patient
Safety Or- ganization, works with hospitals to
provide ad- ditional analysis and
benchmarking of hospi- tal transfusion event
reports, while protecting data confidentiality.
AABB also administers the AABB United
States Donor Hemovigilance Program where
blood collectors can report, analyze, and
benchmark adverse donor reac- tions.
Each facility should track reported events
and look for trends. The use of classification
schemes may facilitate trend analysis and typi-
cally involves one or more of the following
cat- egories: the nature of the event, the
process (or procedure) in which the event
occurred, the outcome and severity of the
event, and the contributing factors and
underlying causes. If several events within a
relatively short period involve a particular
process or procedure, that process or
procedure should be further inves-
 CH A P TE R 1 Quality Management Systems: Theory and Practice ■ 23

tigated. The most useful schemes involve

the use of multiple categories for each
event, which allows data to be sorted in a
variety of ways so that patterns that were
previously not obvious can emerge. (See
example in Table 1- 3.) Such sorting or
stratification can result in identification of
situations that require closer monitoring or
problems needing corrective or preventive
action. For smaller facilities that may not
have sufficient data to identify trends,
pooling data with a larger entity (eg, the
labo- ratory or all transfusion services in a
health- care system) or following national
trends from data provided by organizations
such as the AABB, CAP, or The Joint
Commission, may also prove helpful. The
extent of monitoring and the length of time
to monitor processes de- pends on the
frequency and critical aspects of the
occurrences. Reporting and monitoring of
events are essential problem identification
methods for process improvement activities
in a quality management system.
Occasionally, there may be a need for a
fa- cility to deviate from approved
procedures to meet the unique medical needs
of a particular patient. When this situation
arises, a medically indicated exception
should be planned and approved in advance
by the facility’s medical director. The
rationale and nature of the planned
exception should be documented. Careful
consideration should be given to
maintaining a controlled process and
verifying the safety and quality of the
resulting product or service. Any additional
risk to the patient must be disclosed.
Monitoring and Assessment
The quality management system should de-
scribe how the facility monitors and
evaluates its processes. Assessments are
systematic ex- aminations to determine
whether actual activ- ities comply with
planned activities, are imple- mented
effectively, and achieve objectives.
Depending on the focus, assessments can

TABLE 1-3. Example of an Event Classification

Event: A unit of Red Blood Cells from a directed donor was issued to the wrong oncology patient. The unit was not transfused

Event Classification
■ Type of event: patient
■ Procedure involved: issuing products
■ Process involved: blood administration
■ Product involved: Red Blood Cells
■ Location: transfusion service
■ Other factors: directed donor
■ Other factors: oncology patient

Underlying Causes
■ Proximate cause: two patients with similar names had crossmatched blood available
■ Root cause: inadequate procedure for verification of patient identification during issue

Outcome
■ Severity: serious, FDA reportable
■ Patient: no harm, correct product was obtained and transfused
■ Product: no harm, product returned to inventory
■ Donor: not applicable

Successful Barriers
■ Problem detected during the patient identification verification step of blood administration
FDA = Food and Drug Administration.
24 ■ AABB T E C HNIC AL MANUAL
include: 1) evaluation of process outputs (ie,
results); 2) the activities that make up a pro-
cess as well as its outputs; or 3) a group of re-
lated processes and outputs (ie, the system).
Assessments can be internal or
external and can include quality
assessments, peer re- views, self-
assessments, and proficiency test- ing.
Evaluations typically include comparisons of
actual to expected results.
Internal Assessments
Internal assessments may include
evaluations of quality indicator data,
targeted audits of a single process, or system
audits that are broader in scope and may
cover a set of inter- related processes. These
assessments should be planned and
scheduled. The details of who performs the
assessments and how they are performed
should be addressed. Assessments should
cover the quality system and the major
operating systems in the donor center and
transfusion or cellular therapy service.
In addition, there should be a process
for responding to the issues raised as a
result of the assessment, including review
processes and time frames. The results
should be docu- mented and submitted to
management per- sonnel who have
authority over the process as- sessed as well
as to executive management. Management
should develop corrective ac- tion plans
with input from operational staff and
quality oversight personnel for any defi-
ciencies noted in the assessment. Quality
oversight personnel should track progress
to- ward implementation of corrective
actions and monitor them for effectiveness.
To make the best use of these assess-
ments, there should be a process to track,
monitor trends in, and analyze the problems
identified so that opportunities for
improve- ment can be recognized. Early
detection of trends makes it possible to
develop preventive actions before patient
safety, blood, compo- nents, tissues, or
derivatives are adversely af- fected.
Evaluation summaries provide infor-
mation that is useful for addressing
individual or group performance problems
and ensuring the adequacy of test methods
and equipment. Any corrective or preventive
action associated
with assessment results should be reviewed
by executive management.
Quality Indicators
Quality indicators are performance measures
designed to monitor one or more processes
during a defined time and are useful for
evalu- ating service demands, production,
personnel, inventory control, and process
stability. These indicators can be process
based or outcome based. Process-based
indicators measure the degree to which a
process can be consistently performed. An
example of a process-based in- dicator is
turnaround time from blood product
ordering until transfusion. Outcome-based
in- dicators are often used to measure what
does or does not happen after a process is or
is not performed. The number of incorrect
test result reports is an example of such an
indicator. For each indicator, thresholds are
set that repre- sent warning limits, action
limits, or both. These thresholds can be
determined from reg- ulatory or
accreditation requirements, bench- marking,
or internal facility data.
Tools frequently used for displaying
qual- ity indicator data are run charts and
control charts. In a run chart, time is
plotted on the x-axis and values on the y-
axis. In control charts, the mean of the data
and the upper and lower control limits,
which have been calculat- ed from the data,
are added to the chart. Single points outside
the upper and lower control limits result
from special causes. Statistical rules for
interpreting consecutive points out- side 1
standard deviation (SD), 2 SDs, and 3 SDs
should be used to recognize a process that is
out of control. The root cause should be de-
termined, and corrective action should be
ini- tiated, if indicated.
Blood Utilization Assessment
The activities of blood usage review
commit- tees in the transfusion setting are an
example of internal assessment. Guidelines
are avail- able from the AABB for both
adult and pediat- ric utilization review.34-36
Peer review of trans- fusion practices,
required by the AABB, is also required by
The Joint Commission9 for hospi- tal
accreditation, by CMS1 for hospitals to
 CH A P TE R 1 Quality Management Systems: Theory and Practice
qualify for Medicare reimbursement, and by
some states for Medicaid reimbursement.
Transfusion audits provide reviews of
pol- icies and practices to ensure safe and
appro- priate transfusions and are based on
measur- able, predetermined performance
criteria. (See Chapter 28.) Transfusion
services should investigate an adequate
sample of cases (eg, 5% of cases within a
defined time frame or 30 cases, whichever is
larger). Audits assess a fa- cility’s
performance and effectiveness in the
following6:
■ Ordering practices.
■ Patient identification.
■ Sample collection and labeling.
■ Infectious and noninfectious adverse
events.
■ Near-miss events.
■ Usage and discard.
■ Appropriateness of use.
■ Blood administration policies.
■ Ability of services to meet patient needs.
■ Compliance with peer-review recommen-
dations.
■ Critical laboratory results before and after
transfusion.
One method of assessing the blood ad-
ministration process is to observe a predeter-
mined number of transfusions by following
a unit of blood as it is issued for transfusion
and is then transfused.34
Assessments of transfusion safety
policy and practice may include reviews of
transfu- sion reactions and transfusion-
transmitted diseases. The review committee
may monitor policies and practices for
notifying recipients or recipients’ physicians
of recalled products and notifying donors of
abnormal test results. Other assessments
important in transfusion practice include
reviews of policies for in- formed consent,
indications for transfusion, releases of
directed donor units, and outpa- tient or
home transfusions. Additional assess- ments
should include, where appropriate, 1)
therapeutic apheresis; 2) use of blood
recov- ery devices; 3) procurement and
storage of he- matopoietic progenitor cells;
4) perioperative autologous blood
collection; 5) procurement and storage of
tissue; and 6) evaluation of
■ 25
evolving technologies and products, such as
growth factors and cytokines.
External Assessments
External assessments include inspections, sur-
veys, audits, and assessments performed by
those not affiliated with the organization, such
as the AABB, CAP, CMS, COLA, FACT, the
FDA,
The Joint Commission, or state and regional
health departments. Participation in an
exter- nal assessment program provides an
indepen- dent objective view of the facility’s
performance. External assessors often bring
broad-based ex- perience and knowledge of
best practices that can be shared. Such
assessments are increas- ingly being
performed unannounced or with minimal
notification.
In the preparation phase, there is typically
some data gathering and information to sub-
mit to the organization performing the assess-
ment. To prepare, facilities can perform inter-
nal audits and conduct drills to ensure that
staff can answer questions. For most external
assessments, there is an increased emphasis
on observations of the processes and dialogue
with nonmanagement staff, so preparation is
key. During the assessment phase, it is impor-
tant to know who is responsible for the asses-
sors or inspectors while they are in the facility.
Clear descriptions of what information can be
given to these individuals—and in what form
— help the facility through the assessment
or inspection process. After the
assessment, identified issues should be
addressed. Usually, a written response is
submitted.
Proficiency Testing for Laboratories
Proficiency testing (PT) is one means for
deter- mining that test systems (including
methods, supplies, and equipment) are
performing as expected. As a condition for
certification, CMS requires laboratories to
participate successful- ly in an approved PT
program for CLIA- regulated testing. When no
approved PT program exists for a particular
analyte, the lab- oratory must have another
means to verify the accuracy of the test
procedure at least twice annually.1 Some
accrediting agencies may re- quire more
frequent verification of accuracy.
26 ■ AABB T E C HNIC AL MANUAL
PT must be performed using routine
work processes and conditions if it is to
provide meaningful information. PT
samples should generally be handled and
tested in the same way as patient or donor
specimens. However, a CLIA-certified
laboratory is prohibited from discussing the
PT or sending the samples to a laboratory
with a different CLIA number dur- ing the
active survey period, even if the two
laboratories are within the same
organization and that would be the routine
manner for han- dling patient or donor
specimens. Supervisory review of the
summary evaluation report should be
documented along with investiga- tion and
corrective action for unacceptable re- sults.
Quality oversight personnel should
monitor the PT program and verify that
test systems are maintained in a state of
control and appropriate corrective action
is taken when indicated.
Process Improvement
Continual improvement is a fundamental goal
in any quality management system. In transfu-
sion and cellular therapies and clinical diag-
nostics, this goal is tied to patient safety goals
and expectations for the highest quality health
care. The importance of identifying, investi-
gating, correcting, and preventing problems
cannot be overstated. The process of develop-
ing corrective and preventive action plans in-
volves identification of problems and their
causes as well as identification and evaluation
of solutions to prevent future problems. This
process should also include evaluation of near-
miss events and a mechanism for data
collection and analysis as well as follow-up to
evaluate the effectiveness of the actions taken.
Statistical tools and their applications may be
found in publications from the AABB and the
TABLE 1-4. Applicable Joint Commission Performance Improvement Standards9,10
■ The organization collects data to monitor its performance, including the following:
– Blood and blood component use.
– All confirmed transfusion reactions.
■ The organization compiles and analyzes data.
■ The organization improves performance on an ongoing basis.
American Society for Quality.37-39 The Joint
Commission standards for performance im-
provement are outlined in Table 1-4.9,10
Corrective action is defined as action
tak- en to address the root causes of an
existing nonconformance or other
undesirable situa- tion to reduce or
eliminate the risk of recur- rence.
Preventive action is defined as action taken
to reduce or eliminate the potential for a
nonconformance or other undesirable situa-
tion to prevent occurrence. Corrective action
can be thought of as a reactive approach to
ad- dress the root causes of actual
nonconfor- mances, deviations,
complaints, and process failures, whereas
preventive action can be thought of as a
proactive approach to address the
underlying causes of anticipated prob-
lems identified through the analysis of
data and information.40 In contrast, remedial
action is defined as action taken to
alleviate the symptoms of existing
nonconformances or any other undesirable
situation.41 Remedial action, sometimes
called correction, addresses only a
problem’s visible indicator and not the
actual cause. (See comparisons in Table 1-
5.) Effec- tive corrective and preventive
action cannot be implemented until the
underlying cause is determined and the
process is evaluated in re- lationship to
other processes. Pending such evaluation,
it may be desirable to implement interim
remedial action.
Identification of Problems and Their
Causes
Sources of information for process improve-
ment activities include process deviations,
nonconforming products and services, cus-
tomer complaints, QC records, PT, internal
au- dits, quality indicators, and external
assess- ments. Active monitoring programs
may be set
 CH A P TE R 1 Quality Management Systems: Theory and Practice
TABLE 1-5. Comparison of Remedial, Corrective, and Preventive Actions40
Action Problem
Remedial Existent
Corrective Existent
Preventive Nonexistent
up to help identify problem areas. These
pro- grams should be representative of the
facility processes and consistent with
organizational goals, and they should reflect
customer needs. Preparation of a facility
quality report at least annually in which data
from all these sources are aggregated and
analyzed can be valuable to identify issues
for performance improve- ment.
Once identified, problems should be
ana- lyzed to determine their scope,
potential ef- fects on quality management
and operational systems, relative frequency,
and extent of their variation. Such an
analysis is important to avoid tampering
with processes that are mere- ly showing
normal variations or problems with little
effect.
The underlying causes of an undesirable
condition or problem can be identified by an
individual or group. The more complex
the problem and the more involved the
process, the greater the need to enlist a team
and to for- malize the analysis. Three
commonly used tools for identifying
underlying causes in an objective manner
are process flowcharting, use of the
“repetitive why,” and the cause-and- effect
diagram.
PR O CE S S F L OWCH A R T . A process
flowchart gives a detailed picture of the
multiple activi- ties and important decision
points within that process. By examining this
picture, one may identify problem-prone areas.
RE PETITIVE W H Y. The “repetitive why” is
used to work backward through the process.
One repeatedly asks “Why did this happen?”
until 1) no new information can be gleaned; 2)
the causal path cannot be followed because of
missing information; or 3) further investiga-
■ 27
Approach Outcome
Reactive Alleviates symptoms
Reactive Prevents recurrence
Proactive Prevents occurrence
tion is impractical, impossible, or outside
the boundaries of the organization. Use of
the “re- petitive why” prevents the mistake
of inter- preting an effect as a cause.
CA US E-A N D-E F F E CT DIAGRA M. The cause-
and-effect diagram, also known as the
Ishika- wa or fish-bone diagram, uses a
specialized form of brainstorming that
breaks down prob- lems into “bite-sized”
pieces. (An example of a cause-and-effect
diagram is shown in Fig 1-1.) The method
used in the diagram is designed to focus
ideas around the component parts of a
process as well as to give a pictorial
represen- tation of the ideas that are
generated and their interactions. When using
the cause-and-effect diagram, one looks at
equipment, materials, methods,
environment, and human factors.
These three tools identify both active and
latent failures. Active failures are those
that have an immediate adverse effect.
Latent fail- ures are more global actions
and decisions with potential for damage
that may lie dor- mant and become evident
only when triggered by the presence of
localized factors. The key to successfully
determining root causes is to avoid
stopping too soon or getting caught in the
trap of placing blame on an individual.
Most problems, particularly those that are
complex, have several root causes. A
method that can be of use when such
problems occur is the Pareto analysis. A
chart of causes, laid out in order of
decreasing frequency, is pre- pared. Those
that occur most frequently are considered
the “vital few”; the rest are consid- ered the
“trivial many.” This method offers di-
rection on where to dedicate resources for
maximal effect. An example of a Pareto
chart is shown in Fig 1-2.
28 ■ AABB T E C HNIC AL MANUAL
Root Cause Analysis of Failed Test Runs
FIGURE 1-1. Example of a cause-and-effect diagram.
SOP = standard operating procedure.
Identification and Evaluation
of Solutions
Potential solutions to problems are identified
during the creative phase of process
improve- ment. Brainstorming and process
flowcharting can be particularly helpful in
this phase. Benchmarking with other
organizations can also be helpful. Possible
solutions should be evaluated relative to
organizational con- straints and should be
narrowed down to those that are most
reasonable. Individuals who implement the
process are usually the most knowledgeable
about what will work. They should be
consulted when possible solu- tions are
being considered. Individuals with
knowledge of the interrelationships of pro-
cesses who have a more “global” view of the
organization should also be involved in this
step. Solutions may fail if representatives
with those perspectives are not involved.
Potential solutions should be tested be-
fore full implementation and a clear plan
should be created regarding methods,
objec- tives, timelines, decision points,
and algo- rithms for all possible results of a
trial. Large-
scale solutions can be tried on a limited
basis and can be expanded if successful;
small-scale solutions can be implemented
pending an ef- fectiveness evaluation. Data
should be collect- ed to evaluate the
effectiveness of the pro- posed change.
Data can be collected using the methods
employed initially to identify the
problems or methods specially designed
for the trial. Once solutions have been
successful- ly tested, full implementation
can occur. After implementation, data
should be collected at least periodically to
ensure the continuing ef- fectiveness and
control of the changed pro- cess.
Other Process Improvement Methods
Failure modes and effects analysis is a
system- atic stepwise approach for
identifying all pos- sible failures within a
process, product, or service; studying and
prioritizing the conse- quences, or effects, of
those failures; and elim- inating or reducing
the failures, starting with those of highest
priority. Despite their relative complexity,
LEAN and Six Sigma process im-
provement methods from the manufacturing
 CH A P TE R 1 Quality Management Systems: Theory and Practice ■ 29

FIGURE 1-2. Example of a Pareto chart.

industry are finding increasing use in the
health-care setting. LEAN emphasizes speed
and efficiency. Six Sigma emphasizes
precision and accuracy. Six Sigma uses the
data-driven approach to problem solving of
define, mea- sure, analyze, improve, and
control. Applica-
tion of these principles and techniques can
improve performance, reduce costs and
waste, cut time, and eliminate non-value-
added ac- tions. Additional information
about both methods can be found on the
website of the American Society for
Quality.42

KEY POINTS

1. Organization and Leadership. A defined organizational structure in addition to top man- agement’s support and comm
30 ■ AABB T E C HNIC AL MANUAL

2. Customer Focus. Quality organizations should understand and meet or exceed

customer needs and expectations. These needs and expectations should be defined in a
contract, agreement, or other document developed with feedback from the customer.
3. Facilities, Work Environment, and Safety. Procedures related to general safety;
biological, chemical, and radiation safety; fire safety; and disaster preparedness are
required. Space al- location, building utilities, ventilation, sanitation, trash, and hazardous
substance disposal must support the organization’s operations.
4. Human Resources. Quality management of all personnel addresses adequate staffing
levels and staff selection, orientation, training, and competence assessment as well as
specific regulatory requirements.
5. Suppliers and Materials Management. Suppliers of critical materials and services (ie,
those affecting quality) should be qualified, and these requirements should be defined in
con- tracts or agreements. All critical materials should be qualified and then inspected and
test- ed upon receipt to ensure that specifications are met.
6. Equipment Management. Critical equipment may include instruments, measuring devices,
and computer hardware and software. This equipment must be uniquely identified and
op- erate within defined specifications, as ensured by qualification, calibration,
maintenance, and monitoring.
7. Process Management. A systematic approach to develop new, and control changes to, poli-
cies, processes, and procedures includes process validation, test method validation, com-
puter system validation, equipment validation, and QC. Validation must be planned and re-
sults reviewed and accepted.
8. Documents and Records. Documents include policies, process descriptions,
procedures, work instructions, job aids, forms, and labels. Records provide evidence
that the process was performed as intended and allow assessment of product and
service quality.
9. Information Management. Unauthorized access, modification, or destruction of data and
information must be prevented and confidentiality of patient and donor records main-
tained. Data integrity should be assessed periodically and backup devices, alternative sys-
tems, and archived documents maintained.
10. Management of Nonconforming Events. Deviations from facility-defined requirements,
standards, and regulations must be addressed by documenting and classifying occurrences,
assessing effects on quality, implementing remedial actions, and reporting to external agen-
cies as required.
11. Monitoring and Assessment. Evaluation of facility processes includes internal and
external assessments, monitoring of quality indicators, blood utilization assessment,
proficiency testing, and analysis of data.
12. Process Improvement. Opportunities for improvement may be identified from
deviation reports, nonconforming products and services, customer complaints, QC
records, profi- ciency testing results, internal audits, quality indicator monitoring, and
external assess- ments. Process improvement includes determination of root causes,
implementation of corrective and preventive actions, and evaluation of the
effectiveness of these actions.

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 CH A P TE R 1 Quality Management Systems: Theory and Practice
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■ 31
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19. Juran JM, Godfrey AB. Juran’s quality hand-
book. 5th ed. New York: McGraw-Hill, 1999.
20. Food and Drug Administration. Guidance for
industry: Process validations: General princi-
ples and practices ( January, 2011). Silver
Spring, MD: CBER Office of Communication,
Outreach, and Development, 2011.
21. Food and Drug Administration. Guidance for
industry: Blood establishment computer sys-
tem validation in the user’s facility (April,
2013). Silver Spring, MD: CBER Office of
Com- munication, Outreach, and
Development, 2013.
22. Food and Drug Administration. General prin-
ciples of software validation: Final guidance
for industry and FDA staff (January, 2002).
Sil- ver Spring, MD: CBER Office of
Communica- tion, Outreach, and
Development, 2002.
23. Clinical and Laboratory Standards Institute.
Quality Management System: Development
and management of laboratory documents;
approved guideline. 6th ed. (GP02-A6/QMS
02-A6). Wayne, PA: CLSI, 2013.
24. Code of federal regulations. Title 21, CFR Part
11. Washington, DC: US Government Printing
Office, 2014 (revised annually).
25. Clinical and Laboratory Standards Institute.
Management of nonconforming laboratory
events; approved guideline (GP32-A/QMS 11-
A). Wayne, PA: CLSI, 2007.
26. Motschman TL, Santrach PJ, Moore SB. Error/
incident management and its practical appli-
cation. In: Duckett JB, Woods LL, Santrach
PJ, eds. Quality in action. Bethesda, MD:
AABB, 1996:37-67.
27. Food and Drug Administration. Guidance for
industry: Notifying FDA of fatalities related to
blood collection or transfusion (September,
2003). Silver Spring, MD: CBER Office of
Com- munication, Outreach, and
Development, 2003.
28. Food and Drug Administration. Transfusion/
donation fatalities: Notification process for
transfusion related fatalities and donation re-
lated deaths. Silver Spring, MD: Center for
Bio- logics Evaluation and Research, 2007.
[Avail- able at
http://www.fda.gov/BiologicsBloodVac
cines/SafetyAvailability/ReportaProblem/
TransfusionDonationFatalities/default.htm
(accessed August 23, 2013).]
32 ■ AABB T E C HNIC AL MANUAL
29. AABB. Reporting donor fatalities. Association
bulletin #04-06. Bethesda, MD: AABB, 2004.
30. Food and Drug Administration. Guidance
for industry: Biological product deviation
report- ing for blood and plasma
establishments (Oc- tober, 2006). Silver
Spring, MD: CBER Office of
Communication, Outreach, and Develop-
ment, 2006.
31. Food and Drug Administration. Biological
product deviations: Includes human tissue and
cellular and tissue-based product (HCT/P)
reporting (BPDR). Silver Spring, MD: Center
for Biologics Evaluation and Research, 2010.
[Available at http://www.fda.gov/Biologics
BloodVaccines/SafetyAvailability/Reporta
Problem/BiologicalProductDeviations/de
fault.htm (accessed August 23, 2013).]
32. Code of federal regulations. Title 21, CFR Part
803. Washington, DC: US Government Print-
ing Office, 2014 (revised annually).
33. Strong DM, AuBuchon J, Whitaker B,
Kuehnert MJ. Biovigilance initiatives. ISBT
Sci Ser 2008; 3:77-84.
34. Shulman IA, Lohr K, Derdiarian AK,
Picukaric JM. Monitoring transfusionist
practices: A strategy for improving
transfusion safety. Transfusion 1994;34:11-
15.
35. Roback J, Waxman D, for the Clinical Transfu-
sion Medicine Committee and the Transfusion
Medicine Section Coordinating Committee.
Guidelines for patient blood management and
blood utilization. Bethesda, MD: AABB,
2011.
36. Strauss RG, Blanchette VS, Hume H. National
acceptability of American Association of
Blood Banks Hemotherapy Committee
guidelines for auditing pediatric
transfusion practices. Transfusion
1993;33:168-71.
37. Vaichekauskas L. You need the tools to do the
job. In: Walters L, ed. Introducing the big Q:
A practical quality primer. Bethesda, MD:
AABB Press, 2004:181-206.
38. Walters L. So many tools, so little understand-
ing. In: Walters L, Carpenter-Badley J, eds.
S3: Simple Six Sigma for blood banking,
transfu- sion, and cellular therapy.
Bethesda, MD: AABB Press, 2007:9-24.
39. Tague NR. The quality toolbox. 2nd ed. Mil-
waukee, WI: ASQ Quality Press, 2005.
40. Motschman TL. Corrective versus preventive
action. AABB News 1999;21(8):5,33.
41. Russell JP, Regel T. After the quality audit:
Clos- ing the loop on the audit process. 2nd
ed. Mil- waukee, WI: ASQ Quality Press,
2000.
42. American Society for Quality. Learn about
quality. Milwaukee, WI: ASQ, 2013.
[Available at http://asq.org/learn-about-
quality/ (ac- cessed August 23, 2013).]
 CH A P TE R 1 Quality Management Systems: Theory and Practice ■ 33

■ APPENDIX 1-1
Glossary of Commonly Used Quality Terms
Biovigilance Collection and analysis of adverse event data for the purpose of
improving out- comes in the use of blood products, organs, tissues, and
cellular therapies.
Calibration Comparison of measurements performed with an instrument to those
made with a more accurate instrument or standard for the purpose of
detecting, reporting, and eliminating errors in measurement.

Change control Established procedures for planning, documenting, communicating, and exe-
cuting changes to infrastructure, processes, products, or services. Such
proce- dures include the submission, analysis, approval,
implementation, and postimplementation review of the change and
decisions made about the change. Formal change control provides a
measure of stability and safety and avoids arbitrary changes that might
affect quality.
Control chart A graphic tool used to determine whether the distribution of data values
gener- ated by a process is stable over time. A control chart plots a
statistic vs time and helps to determine whether a process is in control or
out of control according to defined criteria (eg, a shift from a central line or
a trend toward upper or lower acceptance limits).

Design output Documents, records, and evidence in any other format used to verify that
design goals have been met. Design output should identify characteristics
of a product or service that are crucial to safety and function and to
meeting regulatory requirements. It should contain or make reference to
acceptance criteria. Exam- ples of design output include standard operating
procedures; specifications for supplies, reagents, and equipment;
identification of quality control require- ments; and results of verification
and validation activities.
End-product test
Verification through observation, examination, or testing (or a combination) that
and inspection
the finished product or service conforms to specified requirements.
Near-miss event An unexpected occurrence that did not adversely affect the outcome but
could have resulted in a serious adverse event.

Process capability Ability of a controlled process to produce a service or product that

fulfills requirements or a statistical measure of the inherent process
variability for a given characteristic relative to design specifications. The
most widely accepted formula for process capability is Six Sigma.
Process control Activities intended to minimize variation within a process to produce a
predict- able output that meets specifications.

Qualification Demonstration that an entity is capable of fulfilling specified requirements

and verification of attributes that must be met or complied with for a
person or thing to be considered fit to perform a particular function. For
example, equipment may be qualified for an intended use by verifying
performance characteristics, such as linearity, sensitivity, or ease of use.
An employee may be qualified on the basis of technical, academic, and
practical knowledge and skills developed through training, education, and
on-the-job performance.

(Continued)
34 ■ AABB T E C HNIC AL MANUAL

■ APPENDIX 1-1
Glossary of Commonly Used Quality Terms (Continued)
Quality assurance Activities involving quality planning, control, assessment, reporting,
and improvement necessary to ensure that a product or service meets
defined qual- ity standards and requirements.
Quality control Operational techniques and activities used to monitor and eliminate causes
of unsatisfactory performance at any stage of a process.

Quality indicators Measurable aspects of processes or outcomes that provide an indication of

the condition or direction of performance over time. Quality indicators are
used to monitor progress toward stated quality goals and objectives.
Quality management The organizational structure, processes, and procedures necessary to ensure
that the overall intentions and direction of an organization’s quality program
are met and that the quality of the product or service is ensured. Quality
manage- ment includes strategic planning, allocation of resources, and other
systematic activities, such as quality planning, implementation, and
evaluation.
Requirement A stated or obligatory need or expectation that can be measured or
observed and that is necessary to ensure quality, safety, effectiveness, or
customer satis- faction. Requirements can include things that the system
or product must do, characteristics that it must have, and levels of
performance that it must attain.
Specification Description of a set of requirements to be satisfied by a product,
material, or process indicating, if appropriate, the procedures to be used
to determine whether the requirements are satisfied. Specifications are
often in the form of written descriptions, drawings, professional standards,
and other descriptive references.

Validation Demonstration through objective evidence that the requirements for a

particular application or intended use have been met. Validation provides
assurance that new or changed processes and procedures are capable of
consistently meeting specified requirements before implementation.
Verification Confirmation, by examination of objective evidence, that specified
requirements have been met.
 CH A P TE R 1 Quality Management Systems: Theory and Practice ■ 35

■ APPENDIX 1-2
Code of Federal Regulations Quality-Related References

Code of Federal Regulations, Title 21

Topic Biologics, Blood Drugs Tissues, HCT/Ps

Personnel 600.10, 606.20 211.25, 211.28 1271.170
Facilities 600.11, 606.40 211.42-58 1271.190
Environmental control 211.42 1271.195
and monitoring
Equipment 606.60 211.63-72, 211.105, 1271.200
211.182
Supplies and reagents 606.65 211.80 1271.210
Standard operating 606.100 211.100-101 1270.31, 1271.180
procedures
Process changes and 211.100-101 1271.225, 1271.230
validation
Quality assurance/quality 211.22
control unit
Label controls 610.60-64, 211.122-130 1271.250, 1271.370
606.120-122
Laboratory controls 606.140 211.160
Records and record 600.12, 606.160 211.192, 211.194, 1270.33, 1271.55
reviews 211.196 1271.270
Receipt, predistribution, 606.165 211.142, 211.150 1271.265
and distribution
Adverse reactions 606.170 211.198 1271.350
Tracking 211.188 1271.290
Complaints 606.170-171 211.198 1271.320
Reporting deviations 600.14, 606.171 1271.350
Storage 640.2, 640.11, 211.142 1271.260
640.25, 640.34,
640.54, 640.69
HCT/Ps = human cells, tissues, and cellular and tissue-based products.
36 ■ AABB T E C HNIC AL MANUAL

■ APPENDIX 1-3
Suggested Quality Control Performance Intervals for Equipment and Reagents*
Equipment and ReagentFrequency of Quality Control

Refrigerators/freezers/platelet storage
Refrigerators

■ Recorder Daily
■ Manual temperature Daily
■ Alarm system board (if applicable) Daily
■ Temperature charts (review daily) Weekly
■ Alarm activation Quarterly
Freezers
■ Recorder Daily
■ Manual temperature Daily
■ Alarm system board (if applicable) Daily
■ Temperature charts (review daily) Weekly
■ Alarm activation Quarterly
Platelet incubators
■ Recorder Daily
■ Manual temperature Daily
■ Temperature charts (review daily) Weekly
■ Alarm activation Quarterly
■ Ambient platelet storage Every 4 hours
Laboratory equipment

Centrifuges/cell washers
■ Speed Quarterly
■ Timer Quarterly
■ Function Yearly
■ Tube fill level (serologic) Day of use
■ Saline fill volume (serologic) Weekly
■ Volume of antihuman globulin dispensed (if applicable) Monthly
■ Temperature check (refrigerated centrifuge) Day of use
■ Temperature verification (refrigerated centrifuge) Monthly
 CH A P TE R 1 Quality Management Systems: Theory and Practice ■ 37

■ APPENDIX 1-3
Suggested Quality Control Performance Intervals for Equipment and Reagents*
(Continued)
Equipment and ReagentFrequency of Quality Control

Heating blocks/waterbaths/view boxes

■ Temperature Day of use
■ Quadrant/area checks Periodically
Component thawing devices Day of use
pH meters Day of use
Blood irradiators
■ Calibration Yearly
■ Turntable (visual check each time of use) Yearly
■ Timer Monthly/quarterly
■ Source decay Dependent on source type
■ Leak test Twice yearly
■ Dose delivery check (with indicator) Each irradiator use
■ Dose delivery verification
– Cesium-137 Yearly
– Cobalt-60 Twice yearly
– Other source As specified by manufacturer
Thermometers (vs NIST-certified or traceable thermometer)
■ Liquid-in-glass Yearly
■ Electronic As specified by manufacturer
Timers/clocks Twice yearly
Pipette recalibration Quarterly
Sterile connecting device
■ Weld check Each use
■ Function Yearly
Blood warmers
■ Effluent temperature Quarterly
■ Heater temperature Quarterly
■ Alarm activation Quarterly

(Continued)
38 ■ AABB T E C HNIC AL MANUAL

■ APPENDIX 1-3
Suggested Quality Control Performance Intervals for Equipment and Reagents*
(Continued)
Equipment and ReagentFrequency of Quality Control

Blood collection equipment

Whole blood equipment

■ Agitators Day of use
■ Balances/scales Day of use
■ Gram weight (vs NIST-certified) Yearly
Microhematocrit centrifuge
■ Timer check Quarterly
■ Calibration Quarterly
■ Packed cell volume Yearly
Cell counters/hemoglobinometers Day of use
Blood pressure cuffs Twice yearly
Apheresis equipment
■ Checklist requirements As specified by manufacturer
Reagents

Red cells Day of use

Antisera Day of use
Antiglobulin serum Day of use
Transfusion-transmissible disease marker testing Each test run

Miscellaneous
Copper sulfate Day of use
Shipping containers for blood and component transport
Twice yearly
(usually at temperature extremes)
*The frequencies listed above are suggested intervals, not requirements. For any new piece of equipment, installation,
oper- ational, and performance qualifications must be performed. After the equipment has been suitably qualified for use,
ongoing QC testing should be performed. Depending on the operational and performance qualification methodology, the
ongoing QC may initially be performed more often than the ultimately desired frequency. Once a record of
appropriate in-range QC results has been established (during either equipment qualification or the ongoing QC), the
frequency of testing can be reduced. At a minimum, the frequency must comply with the manufacturer’s suggested
intervals; if no such guidance is pro- vided by the manufacturer, the intervals given in this table are appropriate
to use.
NIST = National Institute of Standards and Technology, QC = quality control.
 C h a p t e r 2

Facilities, Work Environment,

and Safety

Betsy W. Jett, MT(ASCP), CQA(ASQ)CQM/OE;

Susan L. Wilkinson, EdD, MS, MT(ASCP)SBB; and
Tania L. Motschman, MS, MT(ASCP)SBB, CQA(ASQ)

THE P H YSI C AL WOR K environment
can have a significant impact on the
safety, efficiency, and effectiveness of work
processes and on the quality of work process
outcomes. It should be designed and managed
in a way that meets operational needs and
provides for the safety of staff and visitors.
The layout of the physical space; management
of utilities, such as water and air ventilation;
flow of personnel, materials, and waste; and
ergo- nomic factors should all be considered in
the facility management plan.
In addition to providing adequate
facili- ties, the organization should develop
and im- plement a safety program that
defines policies and procedures for safe
work practices and emergency responses.
Such a program also in- cludes requirements
for training, hazard com- munication, use of
engineering controls, and protective
equipment. All employees are re-
sponsible for protecting their own safety
and the safety of others by adhering to
policies and procedures set forth in the
facility safety pro- gram.
The AABB requires its accredited
facilities to plan, implement, and maintain a
program to minimize risks to the health
and safety of donors, patients, volunteers,
and employees from biological, chemical,
and radiological hazards.1,2 Other
professional and accrediting organizations,
including the College of Ameri- can
Pathologists (CAP), the Clinical and Labo-
ratory Standards Institute, and The Joint
Com- mission, have similar or more
detailed safety program requirements.3-6
US federal regulations and
recommenda- tions intended to protect the
safety of workers and the public in health-
care settings are listed in Appendix 2-1.
Appendix 2-1 also lists rele- vant safety
recommendations of trade and

Betsy W. Jett, MT(ASCP), CQA(ASQ)CQM/OE, Vice President for Quality and Regulatory Affairs, New
York Blood Center, New York, New York; Susan L. Wilkinson, EdD, MS, MT(ASCP)SBB, Interim
Department Head, Analytical and Diagnostic Sciences, College of Allied Health Sciences and Associate
Professor Emerita, Uni- versity of Cincinnati, Cincinnati, Ohio; and Tania L. Motschman, MS,
MT(ASCP)SBB, CQA(ASQ), Quality Director, Esoteric Business Unit, Laboratory Corporation of America,
Burlington, North Carolina
The authors have disclosed no conflicts of interest.

39
40 ■ AABB T E C HNIC AL MANUAL
professional associations. The contents
of these regulations and guidelines are
discussed in more detail in each section of
this chapter. US state and local
government regulations may have
additional safety requirements, in- cluding
architectural and construction safety
considerations.
FACILITIES
Facility Design and Workflow
Effective design and maintenance of facilities
along with the physical organization of work
activities can help reduce or eliminate many
potential hazards. Facility design, workflow,
and maintenance also affect process efficien-
cy, productivity, error rates, employee and cus-
tomer satisfaction, and the quality of products
and services.
During the design phase for a new or
ren- ovated space, the location and flow of
person- nel, materials, and equipment should
be con- sidered in the context of the
processes to be performed. Adequate space
must be allotted for personnel movement,
location of supplies and large equipment,
and private or distrac- tion-free zones for
certain manufacturing tasks (eg, donor
interviewing, record review, and blood
component labeling). The facility must
offer designated “clean” and “dirty” spac- es
and provide for controlled movement of
materials and waste in and out of these
areas to avoid contamination. Chemical
fume hoods and biological safety cabinets
(BSCs) should be located away from drafts
and high-traffic areas. The number and
location of eyewash stations and
emergency showers must also be considered
in planning. Staff handling hazard- ous
materials must have ready access to hand-
washing sinks. In some cases, additional
spe- cial water sources for reagent
preparation must be provided. The location
of very heavy equipment, such as
irradiators, should be tak- en into account to
ensure that the flooring has sufficient load-
bearing capacity.
Laboratories must be designed with
ade-
quate illumination, electrical power, and
con- veniently located electrical outlets.
Emergency backup power sources, such as
uninterrupt-
ible power supplies and backup
generators, should be considered to
ensure that blood components, cellular
therapy products, and critical reagents are
not compromised during power failures. The
National Electrical Code is routinely used as
a national guideline for the design of
essential electrical distribution sys- tems,
with modifications approved by the local
building authority that has jurisdiction.7
Heating, ventilation, and air conditioning
must be adequate for the needs of the facility.
Environmental monitoring systems should be
considered for laboratories that require posi-
tive or negative air pressure differentials or
where air filtration systems are used to control
particle levels. The nationally accepted specifi-
cations for ventilation are published by the
American Society of Heating, Refrigerating,
and Air-Conditioning Engineers.8
Housekeeping
The workplace should be kept clean and free
of clutter. Work surfaces and equipment
should be regularly cleaned and disinfected.
Items that may accumulate dust and debris
should not be stored above clean supplies or
work surfaces. Exits and fire safety equipment
must not be blocked or obstructed in any way.
Receptacles and disposal guidelines for non-
hazardous solid waste and biohazardous,
chemical, and radiation waste should be clear-
ly delineated. Housekeeping responsibilities,
methods, and schedules should be defined for
every work area. Written procedures, initial
training, continuing education of personnel,
and ongoing monitoring of housekeeping ef-
fectiveness are essential to safe operations.
Clean Rooms
Clean-room facilities should be considered
for open processing activities that cannot be
ac- commodated in a BSC. Laboratories that
pro- cess cellular therapy products may
choose to adopt clean-room specifications
and mainte- nance practices to meet the
requirements of the Food and Drug
Administration (FDA) cur- rent good tissue
practice regulations.9
International standards for clean rooms
are published by the International Organiza-
 C H A P T E R 2 Facilities, Work Environment, and Safety ■ 41
presence is permitted.

tion for Standardization and provide

specifica- tions for general manufacturing
applications to limit airborne particulates,
contaminants, and pollutants.10 These
standards also provide guidance for
pharmaceutical and biotechnol- ogy
applications that include methods to as-
sess, monitor, and control
biocontamination.11 Aspects of a
biocontamination-control system include 1)
developing air-sampling plans us- ing
validated equipment; 2) assessing biocon-
tamination of surfaces, textiles, and liquids;
3) evaluating laundering processes; and 4)
main- taining personnel training and work
practices.

Restricted Areas
Hazardous areas should be clearly and uni-
formly identified with warning signs in
accor- dance with federal Occupational
Safety and Health Administration (OSHA)
and Nuclear Regulatory Commission (NRC)
standards so that personnel entering or
working around them are aware of existing
biological, chemi- cal, or radiation
dangers.12-15 Staff members not normally
assigned to these areas should receive
adequate training to avoid endanger- ing
themselves. Risk areas can be stratified. For
example, high-risk areas might include those
that contain chemical fume hoods, BSCs,
and storage areas for volatile chemicals or
radio- isotopes. Technical work areas might
be con- sidered moderate risk and restricted
to labora- tory personnel. Administrative
and clerical areas are generally considered
low risk and not restricted. Guidelines for
restricted access based on biosafety levels
are published by the US Department of
Health and Human Services (DHHS).16
Where possible, functions not re- quiring
special precautions should be separat- ed
from those performed in restricted areas.
Organizations should consider
establish-
ing specific safety guidelines for visitors
with business in restricted areas and
verifying that safety guidelines have been
reviewed before the visitors enter the
area. Casual visitors should not be allowed
in restricted areas. Chil- dren should not be
allowed in areas where they could be
exposed to hazards and should be closely
supervised in those areas where their
 disorder syndromes, or injury, in- cluding
the following17:
Mobile Sites
■ Awkward postures—positions that place
Mobile blood collection stress on the body, such as reaching over-
operations can pre- sent special head, twisting, bending, kneeling, or
challenges. An advance safety squat- ting.
sur- vey of the proposed ■ Repetition—performance of the same
collection site helps en- sure mo- tions continuously or frequently.
that hazards are minimized. ■ Force—the amount of physical effort used
Responsibility for site to perform work.
safety should be as- signed to ■ Pressure points—pressing of the body
an individual with adequate against hard or sharp surfaces.
knowl- edge to recognize ■ Vibration—continuous or high-intensity
safety concerns and the au- hand/arm or whole-body vibration.
thority to address them in a
timely manner. All mobile
personnel should be trained to
recog- nize unsafe conditions
and understand how to
effectively implement
infection-control poli- cies
and procedures in a variety of
settings.
Hand-washing access is
essential at col- lection sites.
Carpeted or difficult-to-
clean surfaces may be
covered using an absorbent
overlay with waterproof
backing to protect them
from possible blood spills.
Portable screens and crowd-
control ropes are helpful in
directing traffic flow to
maintain safe work ar- eas.
Food service areas should be
physically separated from
areas for blood collection and
storage. Blood-contaminated
waste must be either returned
to a central location for
dispos- al or packaged and
decontaminated in accor-
dance with local regulations
for medical waste.

Ergonomics
Consideration in physical
design should be given to
ergonomics and to
accommodations for
individuals covered under the
Americans with Disabilities
Act [42 United States Code
(USC) Sections 2101-12213,
1990]. Several fac- tors may
contribute to employee
fatigue, mus- culoskeletal
42 ■ AABB T E C HNIC AL MANUAL
■ Other environmental factors—extreme high
or low temperatures or lighting that is too
dark or too bright.
Both the total time per work shift and
the length of uninterrupted work periods
can make significant contributions to
physical problems. Actions to correct
problems associ- ated with ergonomics may
include the follow- ing:
■ Engineering improvements to reduce or
eliminate the underlying cause, such as
making changes to equipment, worksta-
tions, or materials.
■ Administrative improvements, such as
pro- viding variety in tasks; adjusting
work schedules and work pace; providing
recov- ery or relaxation time; modifying
work practices; ensuring regular
housekeeping and maintenance of work
spaces, tools, and equipment; and
encouraging exercise.
■ Provision of personal protective
equipment (PPE), such as gloves, knee
and elbow pads, protective footwear, and
other items that employees wear to
protect themselves against injury.
SAFETY PROGRAM
An effective safety program starts with a
well- thought-out safety plan. This plan
identifies the applicable regulatory
requirements and describes how they will be
met. A safety plan includes procedures to:
■ Provide a workplace free of recognized
haz- ards.
■ Evaluate all procedures for potential expo-
sure risks.
■ Evaluate each employment position for
po- tential exposure risks.
■ Identify hazardous areas or materials
with appropriate labels and signs.
■ Educate staff, document training, and
monitor compliance.
■ Apply standard precautions (including
uni- versal and blood and body fluid
precau- tions) to the handling of blood,
body fluids, and tissues.
■ Dispose of hazardous waste appropriately.
■ Report incidents and accidents, and pro-
vide treatment and follow-up.
■ Provide ongoing review of safety
policies, procedures, operations, and
equipment.
■ Develop facility-specific plans for disaster
preparedness and response, and test these
plans at defined intervals (Chapter 4).
Safety programs should consider the
needs of all persons affected by the work
envi- ronment. Most obvious is the safety of
techni- cal staff members, but potential risks
for blood donors, ancillary personnel,
volunteers, visi- tors, housekeeping staff,
and maintenance and repair workers must
also be evaluated. Appropriate provisions
must be applied if these individuals cannot
be excluded from risk areas.
Laboratories should consider appointing
a safety officer who can provide general guid-
ance and expertise.4 Typical duties of a safety
officer are to develop the safety program, over-
see orientation and training, perform safety
audits, survey work sites, recommend chang-
es, and serve on or direct the activities of
safety committees. Facilities using hazardous
chemi- cals and radioactive materials often
assign specially trained individuals to oversee
chemi- cal and radiation protection
programs as needed. 12,15 Five basic elements
must be ad- dressed for each type of hazard
covered in the safety program:
■ Training.
■ Hazard identification and communication.
■ Engineering controls and PPE.
■ Safe work practices, including waste dis-
posal.
■ Emergency response plan.
Management controls should be
estab- lished to ensure that these elements
are imple- mented and maintained and that
they are ef- fective. Management is
responsible for the following:
■ Developing and communicating the writ-
ten plan.
 C H A P T E R 2 Facilities, Work Environment, and Safety ■ 43

■ Ensuring implementation and providing temporary work assignments if significant

adequate resources.
■ Providing access to employee health
servic- es related to prevention strategies
and treatment of exposures.
■ Monitoring compliance and effectiveness.
■ Evaluating and improving the safety plan.
Basic Elements of a Safety Program
Training
Employees must be trained to recognize the
hazards in their workplace and take
appropri- ate precautions. Supervisors are
responsible for assessing and documenting
each employ- ee’s understanding of and
ability to apply safe- ty precautions before
independent work is permitted. Safety
training must precede even
po- tential for exposure exists. Staff
members who do not demonstrate the
requisite understand- ing and skills must
receive additional training. Training should
be provided not only to labo- ratory staff,
but also to housekeeping and oth- er
personnel who may come into contact with
hazardous substances or waste. Table 2-1
lists topics to cover in work safety training
pro- grams.
Hazard Identification
and Communication
Employers are required to provide
information about workplace hazards to
their staff to help reduce the risk of
occupational illnesses and injuries. Staff
need to know what hazardous

TABLE 2-1. Topics to Cover in a Work Safety Training

Program Work safety training programs should ensure that all
personnel:
■ Have access to a copy of pertinent regulatory texts and an explanation of the contents.
■ Understand the employer’s exposure control plan and how to obtain a copy of the written plan.
■ Understand how hepatitis and human immunodeficiency virus (HIV) are transmitted and how often; be
famil- iar with the symptoms and consequences of hepatitis B virus (HBV), hepatitis C virus, and
HIV infection.
■ Know that they are offered vaccination against HBV.
■ Recognize tasks that pose infectious risks, and distinguish them from other duties.
■ Know what protective clothing and equipment are appropriate for the procedures they will perform and
how to use them.
■ Know and understand the limitations of protective clothing and equipment (eg, different types of gloves
are recommended according to the permeability of the hazardous material to be used).
■ Know where protective clothing and equipment are kept.
■ Become familiar with and understand all requirements for work practices specified in standard operating
procedures for the tasks they perform, including the meaning of signs and labels.
■ Know how to remove, handle, decontaminate, and dispose of contaminated material.
■ Know the appropriate actions to take and the personnel to contact if exposed to blood or other
biologic, chemical, or radiologic hazards.
■ Know the corrective actions to take in the event of spills or personal exposure to fluids, tissues, and
contam- inated sharp objects; appropriate reporting procedures; and medical monitoring recommended
when paren- teral exposure may have occurred.
■ Know their right to access to medical treatment and medical records.
■ Know fire safety procedures and evacuation plans.
44 ■ AABB T E C HNIC AL MANUAL
substances they are working with and where
those materials are located in the facility.
This communication is achieved by means
of sign- age, labels on containers, written
information, and training programs.
Engineering Controls and PPE
If the physical workspace cannot be designed
to eliminate the potential for exposure to haz-
ards, appropriate protective gear must be pro-
vided. Engineering controls are physical plant
controls or equipment, such as sprinkler sys-
tems, chemical fume hoods, and needleless
systems, that isolate or remove the hazard
from the workplace.
PPE is specialized clothing or
equipment, such as gloves, masks, and
laboratory coats, worn by employees for
protection against a hazard. Employees
should remove their PPE, such as gloves
and laboratory coats, and should wash
their hands with soap and water when
leaving a laboratory area. General guid-
ance on the use of engineering controls
and PPE is included in Appendix 2-2.
Safe Work Practices
Employees must be trained in how to work
with hazardous materials in ways that
protect themselves, their coworkers, and the
environ- ment. Safe work practices are
defined as tasks performed in a manner that
reduces the likeli- hood of exposure to
workplace hazards. Gen- eral
recommendations for safe work practices are
included in Appendix 2-2.
Emergency Response Plan
When engineering and work practice
controls fail, employees must know how to
respond promptly and appropriately. The
purpose of advance planning is to control a
hazardous sit- uation as quickly and safely
as possible. Regu- lar testing of the
emergency response plan identifies areas for
improvement and builds staff confidence in
their ability to respond ef- fectively in a real
emergency. OSHA requires facilities with
more than 10 employees to have a written
emergency response plan. Verbal
communication of the plan is acceptable for
facilities with 10 or fewer employees.18
Management Controls
Supervisory personnel must monitor safety
practices in their areas of responsibility. Con-
tinuing attention to safety issues should be ad-
dressed in routine staff meetings and training
sessions. Periodic audits performed by a safety
professional increase safety awareness. Man-
agement should seek staff input on the design
and improvement of the facility’s safety plan.
The safety program policies,
procedures, guidelines, and supporting
references should be documented in writing
and made available to all personnel at risk.
These documents should be reviewed on a
regular basis and up- dated as technology
evolves and new informa- tion becomes
available. Work sites and safety equipment
should be inspected regularly to ensure
compliance and response readiness.
Checklists may be helpful for
documenting safety inspections and
assessing safety pre- paredness.3,4,19
Employee Health Services
Hepatitis Prophylaxis
All employees routinely exposed to blood
must be offered hepatitis B virus (HBV)
vaccine if they do not already have HBV-
protective anti- bodies (ie, anti-HBs). OSHA
requires that the vaccine be offered at no cost
to all employees and, if any employee refuses
the vaccine, that the refusal be documented.14
Monitoring Programs
Employers must provide a system for
monitor- ing exposure to certain substances
as defined in the OSHA standard if there is
reason to be- lieve that exposure levels
routinely exceed the recommended action
level.20
Medical First Aid and Follow-Up
When requested by a worker who has sus-
tained known or suspected blood exposure,
monitoring for HBV, hepatitis C virus (HCV),
and human immunodeficiency virus (HIV) in-
 C H A P T E R 2 Facilities, Work Environment, and Safety
fection should be provided with appropriate
counseling. In some states, consent is
required for this voluntary testing; rejection
of offered testing must be documented. The
usual schedule includes immediate tests of
the worker and the source of the potentially
infec- tious material, with follow-up testing
of the worker at intervals after exposure. 13,14
All as- pects of accident follow-up should be
appro- priately documented.
The Centers for Disease Control and
Pre- vention (CDC) has published
recommenda- tions for both pre-exposure
and post-exposure prophylaxis if the
contaminating material is HBV-positive
or if this information is un- known.21 HBV
immune globulin is usually giv- en
concurrently with HBV vaccine in cases of
penetrating injuries. When administered
in accordance with the manufacturer’s
direc- tions, both products are very safe and
carry no documented risk of infection with
HBV, HCV, or HIV.21 Post-exposure
prophylaxis for HIV is continually
evolving; policies are generally based on
Public Health Service recommenda- tions
and current standards of practice.
Reporting Accidents and Injuries
When an injury occurs, relevant information
should be documented, including the date,
time, and place where the injury occurred;
the nature of the injury; descriptions of what
hap- pened from the injured person and any
wit- nesses; and first aid or medical attention
pro- vided. The supervisor should complete
any accident reports and investigation forms
re- quired by the institution’s insurer and
worker’s compensation agencies. Employers
must re- port fatalities and injuries resulting
in the hos- pitalization of three or more
employees to OSHA within 8 hours of the
accident.22
OSHA requires health service
employers with 11 or more workers to
maintain records of occupational injuries
and illnesses requiring a level of care that
exceeds the capabilities of a person trained
in first aid.23 Initial documenta- tion must be
completed within 6 days of the incident.
Records of first aid provided by a
nonphysician for minor injuries, such as cuts
or burns, do not need to be retained. All
logs,
■ 45
summaries, and supplemental records must
be preserved for at least 5 years beyond
the calendar year of occurrence. Medical
records of employees should be preserved
for the du- ration of employment plus 30
years, with few exceptions.24
Latex Allergies
Adverse reactions associated with latex,
pow- dered gloves, or both include contact
dermati- tis, allergic dermatitis, urticaria,
and anaphy- laxis. Medical devices that
contain latex must bear a caution label. The
National Institute for Occupational Safety
and Health (NIOSH) of- fers the following
recommendations to prevent these allergic
reactions25:
■ Make latex-free gloves available as an
alter- native to latex, and encourage the
use of latex-free gloves for activities and
work en- vironments where there is
minimal risk of exposure to infectious
materials.
■ If latex gloves are used, consider
providing reduced-protein and powder-
free gloves.
■ Use good housekeeping practices to
remove latex-containing dust from the
workplace.
■ Use work practices that reduce the chance
of reaction, such as hand washing and
avoiding oil-based hand lotions.
■ Educate workers about latex allergy.
■ Evaluate current prevention strategies.
■ Periodically screen high-risk workers for
la- tex allergy symptoms.
■ If symptoms develop, have workers avoid
direct contact with latex and consult a
phy- sician about allergy precautions.
FIRE PREVENTION
Fire prevention relies on a combination of fa-
cility design that is based on the National Fire
Protection Association (NFPA) Life Safety
Code, which identifies processes to maintain
fire protection systems in good working order,
and fire safe-work practices. 26 The Life Safety
Code includes both active and passive fire-
protection systems (eg, alarms, smoke detec-
tors, sprinklers, exit lights in corridors, and
fire-rated barriers).
46 ■ AABB T E C HNIC AL MANUAL
Training
Fire safety training is recommended at the
start of employment and at least annually
thereafter. Training should emphasize
preven- tion and an employee’s awareness of
the work environment, including how to
recognize and report unsafe conditions, how
to report fires, where the nearest alarm and
fire-containment equipment are located and
how to use it, and what the evacuation
policies and routes are.
All staff members in facilities
accredited by CAP or The Joint
Commission are required to participate in
fire drills at least annually.3,5 In areas where
patients are housed or treated, The Joint
Commission requires quarterly drills on
each shift. Staff participation and under-
standing should be documented.
Hazard Identification and
Communication
Emergency exits must be clearly marked with
an exit sign. Additional signage must be
posted along the exit route to show the
direction of travel if it is not immediately
apparent. All flammable materials should be
labeled with appropriate hazard warnings, and
flammable storage cabinets should be clearly
marked.
Engineering Controls and PPE
Laboratories storing large volumes of flamma-
ble chemicals are usually built with 2-hour fire
separation walls, or with 1-hour separation
walls if there is an automatic fire-
extinguishing system.4 Fire detection and
alarm systems should be provided in
accordance with feder- al, state, and local
regulations. All fire equip- ment should be
inspected on a regular basis to ensure that it is
in good working order. Fire ex- tinguishers
should be readily available, and the staff
should be trained to use them proper- ly.
Housekeeping and inventory management
plans should be designed to control the accu-
mulations of flammable and combustible ma-
terials stored in the facility. In areas where
sprinkler systems are installed, all items
should be stored at least 18 inches below the
sprinkler heads. Facilities should consult local
fire codes, which may require greater clear-
ance.
Safe Work Practices
Emergency exit routes must be clear of any-
thing that would obstruct evacuation efforts.
Exit doors must not be locked in such a way
as to impede egress. Permanent exit routes
must be designed to allow free and
unobstructed exit from all parts of the
facility to an area of safety. Secondary exits
may be required for ar- eas larger than 1000
square feet; facilities should consult local
safety authorities with ju- risdiction, such as
the local fire marshal and NFPA, for
guidance on secondary exits.
Emergency Response Plan
The fire emergency response plan should
en- compass both facility-wide and area-
specific situations. It should describe
reporting and alarm systems; location and
use of emergency equipment; roles and
responsibilities of the staff during the
response; “defend-in-place” strategies; and
conditions for evacuation, evacuation
procedures, and exit routes.5,18
When a fire occurs, the general
sequence for immediate response should be
to 1) rescue anyone in immediate danger;
2) activate the fire alarm system and alert
others in the area;
3) confine the fire by closing doors and shut-
ting off fans or other oxygen sources if possi-
ble; and 4) extinguish the fire with a portable
extinguisher if the fire is small, or evacuate if
it is too large to manage.
ELECTRICAL SAFETY
Electrical hazards, including fire and shock,
may arise from the use of faulty electrical
equipment; damaged receptacles,
connectors, or cords; or unsafe work
practices. Proper use of electrical
equipment, periodic inspection and
maintenance, and hazard recognition
training are essential to help prevent
accidents that may result in electric shock or
electrocu- tion. The severity of shock
depends on the path that the electrical
current takes through the body, the amount
of current flowing
 C H A P T E R 2 Facilities, Work Environment, and Safety
through the body, and the length of time that
current is flowing through the body. Even
low- voltage exposures can lead to serious
injury.27
Training
Safety training should be designed to make
employees aware of electrical hazards
associ- ated with receptacles and connectors.
This training should also help them
recognize po- tential problems, such as
broken receptacles and connectors, improper
electrical connec- tions, damaged cords, and
inadequate grounding.
Hazard Identification and
Communication
The safety plan should address the proper
use of receptacles and connectors.
Equipment that does not meet safety
standards should be marked to prevent
accidental use.
Engineering Controls and PPE
OSHA requires that electrical systems and
equipment be constructed and installed in a
way that minimizes the potential for work-
place hazards. When purchasing equipment,
the facility should verify that it bears the
mark of an OSHA-approved independent
testing laboratory, such as Underwriters
Laborato- ries.28 Adequate working space
should be pro- vided around equipment to
allow easy access for safe operation and
maintenance. Ground- fault circuit
interrupters should be installed in damp or
wet areas.
Safe Work Practices
Electrical safety practices focus on two
factors:
1) proper use of electrical equipment and 2)
proper maintenance and repair of this equip-
ment. Staff should not plug or unplug equip-
ment from an electrical source with wet
hands. Overloading circuits with too many
devices may cause the current to heat the
wires to a very high temperature and
generate a fire. Damaged receptacles and
faulty electrical equipment must be tagged
and removed from service until they have
been repaired and
■ 47
checked for safety. Flexible cords should be
se- cured to prevent tripping and should be
pro- tected from damage from heavy or
sharp ob- jects. Flexible cords should be
kept slackened to prevent tension on
electrical terminals, and cords should be
checked regularly for cut, bro- ken, or
cracked insulation. Extension cords should
not be used in lieu of permanent wiring.
Emergency Response Plan
In case of an emergency in which it is not
pos- sible to decrease the power or
disconnect equipment, the power supply
should be shut off from the circuit breaker.
If it is not possible to interrupt the power
supply, a nonconduc- tive material, such as
dry wood, should be used to pry a victim
from the source of cur- rent.27 Victims must
not be touched directly. Emergency first aid
for victims of electrical shock must be
sought. Water-based fire extin- guishers
should not be used on electrical fires.
BIOSAFETY
The facility must define and enforce
measures to minimize the risk of exposure
to biohazard- ous materials in the
workplace. Requirements published by
OSHA (Blood-borne Pathogens Standard)
and recommendations published by the US
DHHS provide the basis for an effective
biosafety plan.13,14,16
Blood-Borne Pathogens Standard
The OSHA Blood-Borne Pathogens Standard
is intended to protect employees in all
occupa- tions where there is a risk of
exposure to blood and other potentially
infectious materials. It requires the facility
to develop an exposure control plan and
describes appropriate engi- neering controls,
PPE, and work practice con- trols to
minimize the risk of exposure. The standard
also requires employers to provide HBV
vaccinations to any staff members with
occupational exposure, provide medical fol-
low-up care in case of accidental exposure,
and keep records related to accidents and ex-
posures.
48 ■ AABB T E C HNIC AL MANUAL
Standard Precautions
Standard precautions represent the most cur-
rent recommendations by the CDC to reduce
the risk of transmission of blood-borne
patho- gens and other pathogens in
hospitals. Origi- nally published in 1996 in
the Guidelines for Isolation Precautions in
Hospitals, standard precautions build on
earlier recommenda- tions, including body
substance isolation (1987), universal
precautions (1986), and blood and body
fluid precautions (1983).13 Standard
precautions apply to all patient care
activities, regardless of diagnosis, in which
there is a risk of exposure to 1) blood; 2) all
body fluids, secretions, and excretions,
except sweat; 3) nonintact skin; or 4)
mucous mem- branes.
The OSHA Blood-Borne Pathogens
Stan- dard refers to the use of universal
precautions. However, OSHA recognizes
the more recent guidelines from the CDC
and, in Directive CPL 02-02-069, allows
hospitals to use acceptable alternatives,
including standard precautions, as long as
all other requirements in the stan- dard are
met.29
Biosafety Levels
Recommendations for biosafety in laborato-
ries are based on the potential hazards per-
taining to specific infectious agents and the
activities performed.16 Biosafety recommen-
dations include guidance on both
engineering controls and safe work
practices. The four bio- safety levels are
designated in ascending order, with
increasing protection for personnel, the
environment, and the community:
■ Biosafety Level 1 (BSL-1) involves work
with agents of no known or of minimal
potential hazard to laboratory personnel
and the en- vironment. Activities are
usually conducted on open surfaces, and
no containment equipment is needed.
■ BSL-2 work involves agents of moderate
potential hazard to personnel and the
envi- ronment, usually from contact-
associated exposure. Most blood bank
laboratory ac- tivities are considered
BSL-2.
■ BSL-3 includes work with indigenous or
ex- otic agents that may cause serious or
potentially lethal disease as a result of ex-
posure to aerosols (eg, Mycobacterium
tu- berculosis) or by other routes (eg,
HIV) that would result in grave
consequences to the infected host.
Recommendations for BSL-3 work are
designed to contain biohazardous
aerosols and minimize the risk of surface
contamination.
■ BSL-4 applies to work with dangerous or
exotic agents that pose high individual
risk of life-threatening disease from
aerosols (eg, agents of hemorrhagic
fevers or filovi- ruses). BSL-4 is not
applicable to routine blood-bank-related
activities.
The precautions described in this section
focus on BSL-2 requirements. Laboratories
should consult the CDC or National Institutes
of Health guidelines for precautions appropri-
ate for higher levels of containment.
Training
OSHA requires annual training for all
employ- ees whose tasks increase their risk
of infectious exposure.14,29 Training
programs must be tai- lored to the target
group both in level and con- tent. General
background knowledge of bio- hazards,
understanding of control procedures, or
work experience cannot meet the require-
ment for specific training, although an
assess- ment of such knowledge is a first
step in plan- ning program content.
Workplace volunteers require at least as
much safety training as paid staff members
who perform similar functions.
Hazard Identification and
Communication
The facility’s exposure control plan communi-
cates the risks present in the workplace and
describes controls to minimize exposure. BSL-
2 through BSL-4 facilities must have a biohaz-
ard sign posted at the entrance when infec-
tious agents are in use. The sign notifies
personnel and visitors of the presence of infec-
tious agents, provides a point of contact for
 C H A P T E R 2 Facilities, Work Environment, and Safety
the area, and indicates any special protective
equipment or work practices required.
Biohazard warning labels must be
placed on containers of regulated waste;
refrigerators and freezers containing blood
or other poten- tially infectious material; and
other containers used to store, transport, or
ship blood or other potentially infectious
materials. Blood compo- nents that are
labeled to identify their contents and have
been released for transfusion or oth- er
clinical use are exempted.
Engineering Controls and PPE
OSHA requires that hazards be controlled
by engineering or work practices whenever
possi- ble.14 Engineering controls for BSL-2
laborato- ries include limited access to the
laboratory when work is in progress and
BSCs or other containment equipment for
work that may in- volve infectious aerosols
or splashes. Hand- washing sinks and
eyewash stations must be available. The
work space should be designed so that it can
be easily cleaned, and bench tops should be
impervious to water and resistant to
chemicals and solvents.
To help prevent exposure or cross-
con- tamination, work area telephones
can be equipped with speakers to eliminate
the need to pick up the receiver. Computer
keyboards and telephones can be covered
with plastic. Such equipment should be
cleaned on a regu- lar basis and when visibly
soiled.
BSCs are primary containment devices
for handling moderate-risk and high-risk
or- ganisms. There are three types—Classes
I, II, and III—with Class III providing the
highest protection to workers. In addition to
protect- ing personnel during the handling
of biohaz- ardous materials, a BSC may be
used to pre- vent contamination of blood
and cellular therapy products during
open processing steps. A comparison of
the features and appli- cations of the three
classes of cabinets is pro- vided in Table 2-
2.
BSCs are not required by standard
pre- cautions, but centrifugation of open
blood samples or manipulation of units
known to be positive for HBV surface
antigen or HIV are ex-
■ 49
amples of blood bank procedures for which a
BSC could be useful. The effectiveness of the
BSC is a function of directional airflow inward
and downward through a high-efficiency fil-
ter. Efficacy is reduced by anything that dis-
rupts the airflow pattern (eg, arms moving rap-
idly in and out of the BSC, rapid movements
behind an employee using the BSC, down-
drafts from ventilation systems, or open labo-
ratory doors). Care should be taken not to
block the front intake and rear exhaust grills.
Performance of the BSC should be certified
annually.31
Injuries from contaminated needles and
o t her shar p objec t s ( s om etim es c
alle d “sharps”) continued to be a major
concern in health-care settings even after
the Blood- Borne Pathogens Standard went
into effect. In 2001, OSHA revised the
standard to refer to engineered sharps
injury protections and needleless
systems. The revised standard re- quires
14
that employers implement appropriate new
control technologies and safer medical
devices in exposure control plans and that
em- ployers solicit input from their
employees to identify, evaluate, and select
engineering and work practice controls.
Examples of safer de- vices are needleless
systems and self-sheath- ing needles in
which the sheath is an integral part of the
device.
Decontamination
Reusable equipment and work surfaces that
may be contaminated with blood require
daily cleaning and decontamination.
Obvious spills on equipment or work
surfaces should be cleaned up immediately;
routine wipe-downs with disinfectant should
occur at the end of each shift or a different
frequency that provides equivalent safety.
Equipment that is exposed to blood or other
potentially infec- tious material must be
decontaminated before it is serviced or
shipped. When decontamina- tion of all or a
portion of the equipment is not feasible, a
biohazard label stating which por- tions
remain contaminated should be at- tached
before the equipment is serviced or shipped.
 50
■
TABLE 2-2. Comparison of Classes I, II, and III Biological Safety Cabinets*

Category Main Features Intended Use Common Applications

AABB TECHNICAL MAN U AL

Class I Unfiltered room air is drawn into the cabinet. Inward Personal and environmental To enclose equipment (eg,
airflow protects personnel from exposure to materials protection centrifuges) or procedures that may
inside the cabinet. Exhaust is high-efficiency particulate generate aero- sols
air (HEPA) filtered to protect the environment. It
maintains airflow at a minimum velocity of 75 linear feet
per minute (lfpm) across the front opening (face
velocity).
Class II (general— Laminar flow (air moving at a constant velocity in one Personal, environmental, and prod- Work with microorganisms assigned
direc- to
applies to all types of tion along parallel lines) is used. Room air is drawn into uct protection Biosafety Levels 1, 2, or 3
Class II cabinets) the front grille. HEPA-filtered air is forced downward in a
laminar flow to minimize cross-contamination of
materials in the cabinet. Exhaust is HEPA filtered.
Handling of products for which
preven- tion of contamination is
critical, such as cell culture
propagation or manipu- lation of
blood components in an open
system
Class II, A Approximately 75% of air is recirculated after passing See Class II, general See Class II, general
through a HEPA filter. Face velocity = 75 lfpm.
Class II, B1 Approximately 70% of air exits through the rear grille, See Class II, general Allows for safe manipulation of small
is HEPA filtered, and is then discharged from the quantities of hazardous chemicals and
building. The other 30% is drawn into the front grille, is biologics

CH A P T E R 2
HEPA filtered, and is recirculated. Face velocity = 100
lfpm.
Class II, B2 All air is exhausted, and none is recirculated. A supply See Class II, general Provides both chemical and
blower biological
draws air from the room or outside and passes it containment; is more expensive to
through a

Facilities, Work Environment, and Safety

HEPA filter to provide the downward laminar flow. operate because of the volume of
Face con-
velocity = 100 lfpm. ditioned room air being exhausted
Class II, B3 Although similar in design to Type A, the system is See Class II, general Allows for safe manipulation of
ducted small
and includes a negative pressure system to keep any quantities of hazardous chemicals
possi- and
ble contamination within the cabinet. Face velocity = biologics
100 lfpm.
Class III Cabinet is airtight. Materials are handled with rubber Maximum protection to Work with Biosafety Level 4 micro-
gloves personnel
attached to the front of the cabinet. Supply air is HEPA fil- and environment organisms
tered. Exhaust air is double HEPA filtered or may have
one
filter and an air incinerator. Materials are brought in and
out
of the cabinet either through a dunk tank or a double- ■
door
pass-through box that can be decontaminated. Cabinet is
kept under negative pressure.

51
*Data from the US Department of Health and Human Services.30
52 ■ AABB T E C HNIC AL MANUAL
Choice of Disinfectants
The Environmental Protection Agency
(EPA) maintains a list of chemical products
that have been shown to be effective
hospital antimicro- bial disinfectants.32 The
Association for Profes- sionals in Infection
Control and Epidemiology also publishes a
guideline to assist health-care professionals
with decisions involving judi- cious
selection and proper use of specific dis-
infectants.33 For facilities covered under the
Blood-Borne Pathogens Standard, OSHA al-
lows the use of EPA-registered
tuberculocidal disinfectants, EPA-registered
disinfectants that are effective against both
HIV and HBV, a diluted bleach solution to
decontaminate work surfaces, or a
combination of these.29
Before selecting a disinfectant
product, workers should consider
several factors. Among them are the type
of material or sur- face to be treated; the
hazardous properties of the chemical, such
as corrosiveness; and the level of
disinfection required. After a product has
been selected, procedures need to be writ-
ten to ensure effective and consistent
cleaning and treatment of work surfaces.
Some factors to consider for effective
decontamination in- clude contact time,
type of microorganisms, presence of
organic matter, and concentration of the
chemical agent. Workers should review the
basic information on decontamination and
follow the manufacturer’s instructions.
Storage
Hazardous materials must be segregated,
and areas for different types of storage must
be clearly demarcated. Blood must be
protected from unnecessary exposure to
other materials and vice versa. If transfusion
products cannot be stored in a separate
refrigerator from re- agents, specimens, and
unrelated materials, areas within the
refrigerator must be clearly la- beled, and
extra care must be taken to reduce the
likelihood of spills and other accidents.
Storage areas must be kept clean and
orderly; food or drink is never allowed
where biohaz- ardous materials are stored.
PPE
When hazards cannot be eliminated, OSHA re-
quires employers to provide appropriate PPE
and clothing and to clean, launder, or dispose
of PPE at no cost to their employees.14 Stan-
dard PPE and clothing include uniforms, labo-
ratory coats, gloves, face shields, masks, and
safety goggles. Indications and guidelines for
their use are discussed in Appendix 2-2.
Safe Work Practices
Safe work practices appropriate for standard
precautions include the following:
■ Wash hands after touching blood, body
flu- ids, secretions, excretions, and
contaminat- ed items, whether or not
gloves are worn.
■ Wear gloves when touching blood, body
fluids, secretions, excretions, and
contami- nated items, and change gloves
between tasks.
■ Wear a mask and eye protection or a face
shield during activities that are likely to
generate splashes or sprays of blood,
body fluids, secretions, and excretions.
■ Wear a gown during activities that are
likely to generate splashes or sprays of
blood, body fluids, secretions, or
excretions.
■ Handle soiled patient-care equipment in a
manner that prevents exposure; ensure
that reusable equipment is not used for
another patient until it has been cleaned
and repro- cessed appropriately; and
ensure that single-use items are discarded
properly.
■ Ensure that adequate procedures are de-
fined and followed for the routine care,
cleaning, and disinfection of environmen-
tal surfaces and equipment.
■ Handle soiled linen in a manner that pre-
vents exposure.
■ Handle needles, scalpels, and other sharp
instruments or devices in a manner that
minimizes the risk of exposure.
■ Use mouthpieces, resuscitation bags, or
other ventilation devices as an alternative
to mouth-to-mouth resuscitation meth-
ods.
■ Place in a private room patients who are
at risk of contaminating the environment
or
 C H A P T E R 2 Facilities, Work Environment, and Safety
are not able to maintain appropriate hy-
giene (eg, patients with tuberculosis).
Laboratory Biosafety Precautions
Several factors need to be considered when
as- sessing the risk of blood exposures
among lab- oratory personnel. Some of
these factors in- clude the number of
specimens processed, personnel behaviors,
laboratory techniques, and type of
equipment.34 The laboratory direc- tor may
wish to institute BSL-3 practices for
procedures that are considered to be higher
risk than BSL-2. When there is doubt
whether an activity is BSL-2 or BSL-3, the
safety precau- tions for BSL-3 should be
followed. BSL-2 pre- cautions that are
applicable to the laboratory setting are
summarized in Appendix 2-3.
Considerations for the Donor Room
The Blood-Borne Pathogens Standard ac-
knowledges a difference between hospital
pa- tients and healthy donors, in whom the
preva- lence of infectious disease markers is
significantly lower. The employer in a
volun- teer blood donation facility may
determine that routine use of gloves is not
required for phlebotomy as long as the
following condi- tions exist14:
■ The policy is periodically reevaluated.
■ Gloves are made available to those who
want to use them, and their use is not dis-
couraged.
■ Gloves are required when an employee
has cuts, scratches, or breaks in skin;
when there is a likelihood that
contamination will occur; while an
employee is drawing autologous units;
while an employee is per- forming
therapeutic procedures; and dur- ing
training in phlebotomy.
Procedures should be assessed for risks
of biohazardous exposures and risks
inherent in working with a donor or patient
during the screening and donation
processes. Some tech- niques or procedures
are more likely to cause injury than others,
such as using lancets for finger puncture,
handling capillary tubes, crushing vials
for arm cleaning, handling any
■ 53
unsheathed needles, cleaning scissors, and
giving cardiopulmonary resuscitation.
In some instances, it may be necessary
to collect blood from donors known to
pose a high risk of infectivity (eg, collection
of autolo- gous blood or source plasma for
the produc- tion of other products, such as
vaccines). The FDA provides guidance on
collecting blood from such “high-risk”
donors.35,36 The most re- cent regulations and
guidelines should be con- sulted for changes
or additions.
Emergency Response Plan
Table 2-3 lists steps to take when a spill
occurs. Facilities should be prepared to
handle both small and large blood spills.
Good preparation for spill cleanup includes
several elements:
■ Work areas designed so that cleanup is
rela- tively simple.
■ A spill kit or cart containing all necessary
supplies and equipment with instructions
for their use. It should be placed near
areas where spills are anticipated.
■ Responsibility assigned for kit or cart main-
tenance, spill handling, record-keeping,
and review of significant incidents.
■ Personnel trained in cleanup procedures
and procedures for reporting significant
in- cidents.
Biohazardous Waste
Medical waste is defined as any waste
(solid, semisolid, or liquid) generated in the
diagno- sis, treatment, or immunization of
human be- ings or animals in related
research, produc- tion, or testing of
biologics. Infectious waste includes
disposable equipment, articles, or substances
that may harbor or transmit patho- genic
organisms or their toxins. In general, in-
fectious waste should be either incinerated
or decontaminated before disposal in a
sanitary landfill.
If state law allows, blood and compo-
nents, suctioned fluids, excretions, and secre-
tions may be carefully poured down a drain
connected to a sanitary sewer. Sanitary sewers
may also be used to dispose of other potential-
ly infectious wastes that can be ground and
54 ■ AABB T E C HNIC AL MANUAL

TABLE 2-3. Blood Spill Cleanup Steps

■ Evaluate the spill.
■ Wear appropriate protective clothing and gloves. If sharp objects are involved, gloves must be
puncture resistant, and a broom or other instrument should be used during cleanup to avoid injury.
■ Remove clothing if it is contaminated.
■ Post warnings to keep the area clear.
■ Evacuate the area for 30 minutes if an aerosol has been created.
■ Contain the spill if possible.
■ If the spill occurs in the centrifuge, turn the power off immediately and leave the cover on for 30
minutes. The use of overwraps helps prevent aerosolization and contain the spill.
■ Use absorbent material to mop up most of the liquid contents.
■ Clean the spill area with detergent.
■ Flood the area with disinfectant and use it as described in the manufacturer’s instructions. Allow
adequate contact time with the disinfectant.
■ Wipe up residual disinfectant if necessary.
■ Dispose of all materials safely in accordance with biohazard guidelines. All blood-contaminated items
must be autoclaved or incinerated.

flushed into the sewer. State and local health
departments should be consulted about laws
and regulations pertaining to disposal of bio-
logic waste into the sewer.
Laboratories should clearly define
what will be considered hazardous waste.
For exam- ple, in the blood bank, all items
contaminated with liquid or semiliquid
blood are biohazard- ous. Items
contaminated with dried blood are
considered hazardous if there is potential
for the dried material to flake off during
handling. Contaminated sharp objects are
always con- sidered hazardous because of
the risk for per- cutaneous injury. However,
items such as used gloves, swabs, plastic
pipettes with excess liq- uid removed, or
gauze contaminated with small droplets
of blood may be considered nonhazardous
if the material is dried and will not be
released into the environment during
handling.
Guidelines for Biohazardous
Waste Disposal
Employees must be trained before handling
or disposing of biohazardous waste, even if
it is
packaged. The following disposal guidelines
are recommended37:
■ Identify biohazardous waste consistently;
red seamless plastic bags (at least 2 mm
thick) or containers carrying the
biohazard symbol are recommended.
■ Place bags in a protective container with
closure upward to avoid breakage and
leak- age during storage or transport.
■ Prepare and ship waste transported over
public roads according to US Department
of Transportation (DOT) regulations.
■ Discard sharps (eg, needles, broken glass,
glass slides, and wafers from sterile con-
necting devices) in rigid, puncture-proof,
leakproof containers.
■ Put liquids in leakproof, unbreakable con-
tainers only.
■ Do not compact waste materials.
Storage areas for infectious material
must be secured to reduce accident risk.
Infectious waste must never be placed in the
public trash collection system. Most
facilities hire private
 C H A P T E R 2 Facilities, Work Environment, and Safety
carriers to decontaminate and dispose of in-
fectious or hazardous waste. The facility
should disclose all risks associated with
the waste in their contracts with private
compa- nies. The carrier is responsible for
complying with all federal, state, and local
laws for bio- hazardous (medical) waste
transport, treat- ment, and disposal.
Treating Infectious or Medical Waste
Facilities that incinerate hazardous waste
must comply with EPA standards of perfor-
mance for new stationary sources and emis-
sion guidelines for existing sources. 38 In this
regulation, a hospital/medical/infectious waste
incinerator is any device that combusts any
amount of hospital waste or medical/infec-
tious waste.
Decontamination of biohazardous waste
by autoclaving is another common method
for decontamination or inactivation of
blood samples and blood components. The
follow- ing elements are considered in
determining processing time for
autoclaving:
■ Size of load being autoclaved.
■ Type of packaging of item(s) being auto-
claved.
■ Density of items being autoclaved.
■ Number of items in a single autoclave load.
■ Placement of items in the autoclave to al-
low for steam penetration.
It is useful to place a biologic indicator
in the center of loads that vary in size and
con- tents to evaluate optimal steam
penetration times. The EPA provides
detailed information about choosing and
operating such equip- ment.37
For decontamination, material should
be autoclaved for a minimum of 1 hour. For
steril- ization, longer treatment times are
needed. A general rule for decontamination
is to process for 1 hour for every 10 pounds
of waste. Usual- ly, decontaminated
laboratory wastes can be disposed of as
nonhazardous solid wastes. The staff should
check with the local solid waste authority
to ensure that the facility is in com- pliance
with regulations for the area. Waste-
■ 55
containing broken glass or other sharp
items should be disposed of using a method
consis- tent with policies for the disposal
of other sharp or potentially dangerous
materials.
CHEMICAL SAFETY
One of the most effective preventive
measures that a facility can take to reduce
hazardous chemical exposure is to choose
alternative nonhazardous chemicals
whenever possible. When the use of
hazardous chemicals is re- quired,
purchasing these supplies in small quantities
reduces the risks associated with storing
excess chemicals and then dealing with their
disposal.
OSHA requires that facilities using
haz- ardous chemicals develop a written
chemical hygiene plan (CHP) and that the
plan be ac- cessible to all employees. The
CHP should out- line procedures,
equipment, PPE, and work practices that
are capable of protecting em- ployees from
hazardous chemicals used in the facility.15,20
The CHP must also provide assur- ance that
equipment and protective devices are
functioning properly and that criteria to
determine implementation and maintenance
of all aspects of the plan are in place.
Employ- ees must be informed of all
chemical hazards in the workplace and be
trained to recognize chemical hazards,
protect themselves when working with
these chemicals, and know where to find
information about particular hazardous
chemicals. Safety audits and annual reviews
of the CHP are important control steps to
help ensure that safety practices comply
with the policies set forth in the CHP and
that the CHP is up to date.
Establishing a clear definition of what
constitutes hazardous chemicals is some-
times difficult. Generally, hazardous chemicals
pose a significant health risk if an employee is
exposed to them or a significant physical risk,
such as fire or explosion, if they are handled or
stored improperly. Categories of health and
physical hazards are listed in Tables 2-4 and
2-5. The NIOSH Pocket Guide to Chemical
Haz- ards provides a quick reference for many
com- mon chemicals.39
56 ■ AABB T E C HNIC AL MANUAL

TABLE 2-4. Categories of Health Hazards

HazardDefinition

Carcinogens Cancer-producing substances

Irritants Agents causing irritation (eg, edema or burning) to skin or mucous
mem- branes upon contact
Corrosives Agents causing destruction of human tissue at the site of contact
Toxic or highly toxic agents Substances causing serious biologic effects following inhalation, ingestion,
or skin contact with relatively small amounts
Reproductive toxins Chemicals that affect reproductive capabilities, including chromosomal
damages and effects on fetuses
Other toxins Hepatotoxins; nephrotoxins; neurotoxins; agents that act on the
hemato- poietic system; and agents that damage the lungs, skin, eyes,
or mucous membranes

The facility should identify a qualified
chemical hygiene officer to be responsible for
developing guidelines for hazardous materi-
als.20 The chemical hygiene officer is also ac-
countable for monitoring and documenting
accidents and for initiating process change as
needed.
Training
Employees who may be exposed to
hazardous chemicals must be trained before
they begin work in an area in which hazards
exist. If a new employee has received prior
training, it may not be necessary to retrain
the individual, de- pending on the
employer’s evaluation of the new employee’s
level of knowledge. New em- ployee
training is likely to be necessary regard- ing
such specifics as the location of each rele-
vant material safety data sheet (MSDS),
details on chemical labeling, PPE to be
used, and site- specific emergency
procedures.
Training must be provided for each
new physical or health hazard when it is
intro- duced into the workplace but not for
each new chemical that falls within a
particular hazard class.15 For example, if a
new solvent is brought into the workplace
and the solvent has hazards similar to
existing chemicals for which training has
already been conducted, then the employ- er
need only make employees aware of the
new solvent’s hazard category (eg, corrosive
or irritant). However, if the newly
introduced sol- vent is a suspected
carcinogen and carcino- genic hazard
training has not been provided, then new
training must be conducted for em- ployees
with potential exposure. Retraining is
advisable as often as necessary to ensure
that employees understand the hazards
linked to the materials with which they
work, particu- larly any chronic and
specific target-organ health hazards.
Hazard Identification
and Communication
Hazard Communication
Employers must prepare a comprehensive
hazard communication program for all areas
in which hazardous chemicals are used to
complement the CHP and “ensure that the
hazards of all chemicals produced or
imported are classified, and that information
concern- ing the classified hazard is
transmitted to em- ployers and
employees.”15 The program should include
labeling of hazardous chemicals, in-
structions on when and how to post warning
labels for chemicals, directions for
managing MSDS reports for hazardous
chemicals in the facilities, and employee
training. Safety mate- rials made available
to employees should in- clude the following:
 C H A P T E R 2 Facilities, Work Environment, and Safety ■ 57

TABLE 2-5. Categories of Physical Hazards

HazardDefinition

Combustible or flammable
chemicals
Compressed gases
Explosives
Unstable (reactive) chemicals
Water-reactive chemicals
Chemicals that can burn (including combustible and flammable
liquids, solids, aerosols, and gases)
Gases or mixtures of gases in a container under pressure
Unstable or reactive chemicals that undergo violent
chemical changes at normal temperatures and
pressure
Chemicals that could be self-reactive under certain conditions
(shocks, pressure, or temperature)
Chemicals that react with water to release a gas that either is
flammable or presents a health hazard

■ The facility’s written CHP.
■ The facility’s written program for hazard
communication.
■ Identification of work areas where hazard-
ous chemicals are located.
■ Required list of hazardous chemicals and
the relevant MSDS. (It is the responsibility
of the facility to determine which chemi-
cals may present a hazard to employees.
This determination should be based on the
quantity of chemical used; physical prop-
erties, potency, and toxicity of the chemi-
cal; manner in which the chemical is used;
and means available to control the release
of, or exposure to, the chemical.)
Hazardous Chemical Labeling and
Signs
The Hazard Communication Standard re-
quires manufacturers of chemicals and haz-
ardous materials to provide the user with
basic information about the hazards of these
mate- rials through product labeling and the
MSDS.15 Employers are required to provide
employees who are expected to work with
these hazard- ous materials with information
about what the hazards of the materials are,
how to read the labeling, how to interpret
symbols and signs on the labels, and how to
read and use the MSDS.
MSDS forms typically include the follow-
ing:
■ Identification.
■ Hazard(s) identification.
■ Composition/information on ingredients.
■ First-aid measures.
■ Fire-fighting measures.
■ Accidental release measures.
■ Handling and storage considerations.
■ Exposure controls/personal protection in-
formation.
■ Physical and chemical properties.
■ Stability and reactivity.
■ Toxicologic information.
■ Ecologic information.
■ Disposal considerations.
■ Transport information.
■ Regulatory information.
■ Other information.
At a minimum, hazardous chemical
con- tainer labels must include the name
of the chemical, name and address of the
manufac- turer, hazard warnings, symbols,
designs, and other forms of warning to
provide visual re- minders of specific
hazards. The label may refer to any MSDS
for additional information. Labels applied
by the manufacturer must re- main on
containers. The user may add storage
requirements and dates of receipt, opening,
and expiration. If chemicals are aliquotted
into
58 ■ AABB T E C HNIC AL MANUAL
secondary containers, the secondary contain-
er must be labeled with the name of the
chem- ical and appropriate hazard
warnings. Addi- tional information, such
as precautionary measures, concentration
if applicable, and date of preparation, are
helpful but not man- datory.
It is a safe practice to label all containers
with their content, even water. Transfer con-
tainers used for temporary storage need not be
labeled if the person performing the transfer
retains control and intends the containers to
be used immediately. Information regarding
acceptable standards for hazard communica-
tion labeling is provided by the NFPA40 and the
National Paint and Coatings Association.41
Signs meeting OSHA requirements
must be posted in areas where hazardous
chemicals are used. Decisions about where
to post warn- ing signs are based on the
manufacturer’s rec- ommendations
regarding the chemical haz- ards, the
quantity of the chemical in the room or
laboratory, and the potency and toxicity of
the chemical.
MSDS
The MSDS identifies the physical and
chemi- cal properties of a hazardous
chemical (eg, flash point or vapor pressure),
its physical and health hazards (eg, potential
for fire, explo- sion, and signs and
symptoms of exposure), and precautions for
the chemical’s safe han- dling and use.
Specific instructions in an indi- vidual
MSDS take precedence over generic
information in the hazardous materials pro-
gram.
Employers must maintain copies of each
required MSDS in the workplace for each haz-
ardous chemical and ensure that MSDS cop-
ies are readily accessible during each work
shift to employees when they are in their work
areas. When household consumer products
are used in the workplace in the same manner
that a consumer would use them (ie, when the
duration and frequency of use, and therefore
exposure, are not greater than those that the
typical consumer would experience), OSHA
does not require that an MSDS be provided to
purchasers. However, if exposure to such prod-
ucts exceeds that normally found in
consumer applications, employees have a
right to know about the properties of such
hazardous chemi- cals. OSHA does not
require or encourage em- ployers to
maintain an MSDS for nonhazard- ous
chemicals.
Engineering Controls and PPE
Guidelines for laboratory areas in which
haz- ardous chemicals are used or stored
must be established. Physical facilities, and
especially ventilation, must be adequate for
the nature and volume of work conducted.
Chemicals must be stored according to
chemical compat- ibility (eg, corrosives,
flammables, and oxidiz- ers) and in minimal
volumes. Bulk chemicals should be kept
outside work areas. NFPA stan- dards and
others provide guidelines for proper
storage.4,40,42
Chemical fume hoods are
recommended for use with organic solvents,
volatile liquids, and dry chemicals with a
significant inhalation hazard. 4 Although
constructed with safety glass, most fume
hood sashes are not designed to serve as
safety shields. Hoods should be po- sitioned
in an area where there is minimal foot traffic
to avoid disrupting the airflow and com-
promising the containment field.
PPE that may be provided, depending
on the hazardous chemicals used, includes
chem- ical-resistant gloves and aprons,
shatterproof safety goggles, and respirators.
Emergency showers should be
accessible to areas where caustic, corrosive,
toxic, flam- mable, or combustible
chemicals are used.4,43 There should be
unobstructed access, within 10 seconds, to
the showers from the areas where
hazardous chemicals are used. Safety
showers should be periodically flushed
and tested for function, and associated floor
drains should be checked to ensure that
drain traps remain filled with water.
Safe Work Practices
Hazardous material should not be stored or
transported in open containers. Containers
and their lids or seals should be designed to
prevent spills or leakage in all reasonably
an- ticipated conditions. Containers should
be
 C H A P T E R 2 Facilities, Work Environment, and Safety ■ 59

able to safely store the maximum anticipated cumstances of release, and mitigating
volume and be easy to clean. Surfaces fac- tors play a role in determining the
should be kept clean and dry at all times. appro- priate response. The facility’s
When an employee is working with emergency response plan should provide
a chemical fume hood, all materials should guidance on how to determine whether a
be kept at least 6 inches behind the face spill is inci- dental or requires an
opening. The vertical sliding sash should be emergency response.
positioned at the height specified on the ■ Emergency response releases pose a threat
certification sticker. The airfoil, baffles, to health and safety regardless of the
and rear ventilation slot must not be circum- stances surrounding their release.
blocked. Appendix 2-5 lists suggestions These spills may require evacuation of
for working safely with specific chemicals the imme- diate area. The response
typically comes from outside the
Emergency Response Plan immediate release area by personnel
trained as emergency respond- ers. These
The time to prepare for a chemical spill is
spills include those that involve
be- fore it occurs. A comprehensive
immediate danger to life or health,
employee training program should provide
serious threat of fire or explosion, and
each employ- ee with all tools necessary to
high levels of toxic substances.
act responsibly at the time of a chemical
spill. The employee should know response
Appendix 2-7 addresses the
procedures, be able to assess the severity of
management of hazardous chemical spills.
a chemical spill, know or be able to quickly
Spill cleanup kits or carts tailored to the
look up the basic physical characteristics of
specific hazards present should be available
the chemicals, and know where to find
in each area. The kits or carts may contain
emergency response telephone numbers.
rubber gloves and aprons, shoe covers,
The employee should be able to as- sess,
goggles, suitable aspirators, gen- eral
stop, and confine the spill; either clean up
absorbents, neutralizing agents, a broom, a
the spill or call for a spill cleanup team; and
dust pan, appropriate trash bags or cans for
follow procedures for reporting the spill.
waste disposal, and cleanup directions.
The employee must know when to ask for
Chem- ical absorbents, such as clay
assis- tance, when to isolate the area, and
absorbents or spill blankets, can be used
where to find cleanup materials.
for cleaning up a number of chemicals and
Chemical spills in the workplace can be
thus may be easier for employees to use in
categorized as follows44:
spill situations.
With any spill of a hazardous
■ Incidental releases are limited in quantity
chemical, but especially of a carcinogenic
and toxicity and pose no significant
agent, it is es- sential to refer to the MSDS
safety or health hazard to employees.
and contact a des- ignated supervisor or
They may be safely cleaned up by
designee trained to han- dle these spills and
employees familiar with the hazards of
hazardous waste disposal.4 Facility
the chemical involved in the spill. Waste
environmental health and safety
from the cleanup may be classified as
personnel can also offer assistance. The
hazardous and must be dis- posed of in
em- ployer must assess the extent of the
the proper fashion. Appendix 2- 6
employee’s exposure. After an exposure,
describes appropriate responses to inci-
the employee must be given an opportunity
dental spills.
for medical con- sultation to determine the
■ Releases that may be incidental or may
need for a medical examination.
re- quire an emergency response may
Another source of workplace hazards
pose an exposure risk to employees
is the unexpected release of hazardous
depending on the circumstances.
vapors into the environment. OSHA has set
Considerations such as the hazardous
limits for exposure to hazardous vapors
substance properties, cir-
from toxic and hazardous substances.45 The
potential risk as- sociated with a chemical is
determined by the manufacturer and listed
on the MSDS.
60 ■ AABB T E C HNIC AL MANUAL
Chemical Waste Disposal
Most laboratory chemical waste is
considered hazardous and is regulated by the
EPA through the Resource Conservation and
Recovery Act (42 USC §6901 et seq, 1976).
This regulation specifies that hazardous
waste can be legally disposed of only at an
EPA-approved disposal facility. Disposal of
chemical waste into a sani- tary sewer is
regulated by the Clean Water Act (33 USC
§1251 et seq, 1977), and most US states
have strict regulations concerning dis- posal
of chemicals in the water system. Federal
and applicable state regulations should be
consulted when a facility is setting up and
re- viewing its waste disposal policies.
RADIATION SAFETY
Radiation can be defined as energy in the
form of waves or particles emitted and
propagated through space or a material
medium. Gamma rays are electromagnetic
radiation, whereas al- pha and beta emitters
are examples of particu- late radiation. The
presence of radiation in the blood bank,
from either radioisotopes used in laboratory
testing or self-contained blood irra- diators,
requires additional precautions and
training.4,46
Radiation Measurement Units
The measurement unit quantifying the amount
of energy absorbed per unit mass of tissue is
the gray (Gy) or radiation absorbed dose (rad);
1 Gy = 100 rad.
Dose equivalency measurements are
more useful than simple energy measure-
ments because dose equivalency measure-
ments take into account the ability of
different types of radiation to cause biologic
effects. The ability of radiation to cause
damage is as- signed a number called a
quality factor (QF). For example, exposure
to a given amount of al- pha particles (QF =
20) is far more damaging than exposure to
an equivalent amount of gamma rays (QF
= 1). The common unit of measurement
for dose equivalency is the roentgen or
rad equivalent man (rem). Rem is the dose
from any type of radiation that pro-
duces biologic effects in humans equivalent
to 1 rad of x-rays, gamma rays, or beta
rays. To obtain the dose from a particular
type of radia- tion in rem, the number of rad
should be mul- tiplied by the QF (rad × QF
= rem). Because the QF for gamma rays, x-
rays, and most beta par- ticles is 1, the dose
in rad is equal to the dose in rem for these
types of radiation.
Biologic Effects of Radiation
Any harm to tissue begins with the
absorption of radiation energy and
subsequent disruption of chemical bonds.
Molecules and atoms be- come ionized or
excited (or both) by absorbing this energy.
The direct action path leads to ra- diolysis or
formation of free radicals that, in turn, alter
the structure and function of mole- cules in
the cell.
Molecular alterations can cause
cellular or chromosomal changes,
depending on the amount and type of
radiation energy ab- sorbed. Cellular
changes can be manifested as a visible
somatic effect (eg, erythema). Chang- es at
the chromosome level may be manifested as
leukemia or other cancers or possibly as
germ-cell defects that are transmitted to
future generations.
Several factors influence the level of
bio- logic damage from exposure,
including the type of radiation, part of the
body exposed, to- tal absorbed dose, and
dose rate. The total ab- sorbed dose is the
cumulative amount of radi- ation absorbed
in the tissue. The greater the dose, the
greater the potential for biologic damage.
Exposure can be acute or chronic. The low
levels of ionizing radiation likely to oc- cur
in blood banks should not pose any detri-
mental risk.47-50
Regulations
The NRC controls the use of radioactive
mate- rials by establishing licensure
requirements. States and municipalities may
also have re- quirements for inspection,
licensure, or both. The type of license for
using radioisotopes or irradiators depends
on the scope and magni- tude of the use of
radioactivity. US facilities should contact
the NRC and appropriate state agencies for
information on license require-
 C H A P T E R 2 Facilities, Work Environment, and Safety
ments and applications as soon as such
activi- ties are proposed.
Each NRC-licensed establishment
must have a qualified radiation safety
officer who is responsible for establishing
personnel protec- tion requirements and for
proper disposal and handling of radioactive
materials. Specific ra- diation safety policies
and procedures should address dose limits,
employee training, warn- ing signs and
labels, shipping and handling guidelines,
radiation monitoring, and expo- sure
management. Emergency procedures
must be clearly defined and readily
available to the staff.
In 2005, the NRC imposed additional se-
curity requirements for high-risk radioactive
sources, including those used in blood irradia-
tors. The purpose of the increased controls is
to reduce the risk of unauthorized use of ra-
dioactive materials that may pose a threat to
public health and safety. These 2005 measures
include controlled access, approval in writing
of individuals deemed trustworthy and reli-
able to have unescorted access, a system of
monitoring to immediately detect and re-
spond to unauthorized access, and documen-
tation of authorized personnel and monitor-
ing activities. 51 In 2007, a requirement for
fingerprinting was added.52
Exposure Limits
The NRC sets standards for protection against
radiation hazards arising from licensed activi-
ties, including dose limits.12 Such limits, or
maximal permissible dose equivalents, are a
measure of the radiation risk over time and
serve as standards for exposure. The occupa-
tional total effective-dose-equivalent limit is
5 rem/year, the shallow dose equivalent limit
(skin) is 50 rem/year, the extremity dose
equiv- alent limit is 50 rem/year, and the eye
dose equivalent limit is 15 rem/year. 12,47 Dose
limits for an embryo or fetus must not exceed
0.5 rem during pregnancy.12,47,53 Employers are
ex- pected not only to maintain radiation expo-
sure below allowable limits, but also to keep
exposure levels as far below these limits as can
reasonably be achieved.
■ 61
Radiation Monitoring
Monitoring is essential for early detection
and prevention of problems resulting from
radia- tion exposure. Monitoring is used to
evaluate the facility’s environment, work
practices, and procedures and to comply
with regulations and NRC licensing
requirements. Monitoring is accomplished
with the use of dosimeters, bioassays, survey
meters, and wipe tests.4
Dosimeters, such as film or
thermolumi- nescent badges, rings, or both,
measure per- sonnel radiation doses. The
need for dosime- ters depends on the
amount and type of radioactive materials
in use; the facility radia- tion safety officer
determines individual do- simeter needs.
Film badges must be changed at least
quarterly and in some instances
monthly, be protected from high temperature
and humidity, and be stored at work away
from sources of radiation.
Bioassays, such as thyroid and whole
body counting or urinalysis, may be used to
determine whether there is radioactivity inside
the body and if so, how much. If necessary,
bioassays are usually performed quarterly and
after an incident where accidental intake may
have occurred.
Survey meters are sensitive to low
levels of gamma or particulate radiation and
provide a quantitative assessment of
radiation hazard. Survey meters can be used
to monitor storage areas for radioactive
materials or wastes, test- ing areas during or
after completion of a pro- cedure, and
packages or containers of radio- active
materials. Survey meters must be
calibrated annually by an authorized NRC
li- censee. Selection of appropriate meters
should be discussed with the radiation safety
officer.
In areas where radioactive materials
are handled, all work surfaces, equipment,
and floors that may be contaminated
should be checked regularly with a wipe
test. In the wipe test, a moistened
absorbent material (the wipe) is passed
over the surface and then mea- sured for
radiation.
Training
Personnel who handle radioactive materials
or work with blood irradiators must receive
radi-
62 ■ AABB T E C HNIC AL MANUAL
ation safety training before beginning work.
This training should address the presence
and potential hazards of radioactive
materials in the employee’s work area,
general health pro- tection issues,
emergency procedures, and ra- diation
warning signs and labels in use. Instruction
in the following areas is also sug- gested:
■ NRC regulations and license conditions.
■ The importance of observing license
condi- tions and regulations and of
reporting vio- lations or conditions of
unnecessary expo- sure.
■ Precautions to minimize exposure.
■ Interpretation of results of monitoring de-
vices.
■ Requirements for pregnant workers.
■ Employees’ rights.
■ Documentation and record-keeping re-
quirements.
The need for refresher training is deter-
mined by the license agreement between the
NRC and the facility.
Engineering Controls and PPE
Although self-contained blood irradiators
present little risk to laboratory staff and film
badges are not required for routine
operation, blood establishments with
irradiation pro- grams must be licensed by
the NRC.48
The manufacturer of the blood
irradiator usually accepts responsibility for
radiation safety requirements during
transportation, in- stallation, and validation
of the unit as part of the purchase contract.
The radiation safety of- ficer can help
oversee the installation and vali- dation
processes and should confirm that
appropriate training, monitoring systems,
procedures, and maintenance protocols are
in place before use and that they reflect the
man- ufacturer’s recommendations.
Suspected mal- functions must be reported
immediately so that appropriate actions can
be initiated.
Blood irradiators should be located in
se- cure areas so that only trained
individuals have access. Fire protection for
the unit must also be considered. Automatic
fire detection
and control systems should be readily avail-
able in the immediate area. Blood compo-
nents that have been irradiated are not radio-
active and pose no threat to the staff or the
general public.
Safe Work Practices
Each laboratory should establish policies
and procedures for the safe use of
radioactive mate- rials. These policies and
procedures should in- clude requirements for
following general labo- ratory safety
principles, appropriate storage of radioactive
solutions, and proper disposal of radioactive
wastes. Radiation safety can be im- proved
with the following procedures:
■ Minimizing the time of exposure by work-
ing as efficiently as possible.
■ Maximizing the distance from the source
of the radiation by staying as far from the
source as possible.
■ Maximizing shielding (eg, by using a
self- shielded irradiator or wearing a lead
apron) when working with certain
radioactive ma- terials. These
requirements are usually stip- ulated in
the license conditions.
■ Using good housekeeping practices to
min- imize the spread of radioactivity to
uncon- trolled areas.
Emergency Response Plan
Radioactive contamination is the dispersal
of radioactive material into or onto areas in
which it is not intended—for example, the
floor, work areas, equipment, personnel
cloth- ing, or personnel skin. The NRC
regulations state that gamma or beta
radioactive contami- nation cannot exceed
2200 disintegrations per minute (dpm) per
100 cm2 in the posted (re- stricted) area or
220 dpm/100 cm2 in an unre- stricted area,
such as a corridor. For alpha emitters, these
values are 220 dpm/100 cm2 and 22
dpm/100 cm2, respectively.54
If a spill occurs, employees’
contaminated skin surfaces must be washed
several times, and the radiation safety
officer must be noti- fied immediately to
provide further guidance. Others must not
be allowed to enter the area until emergency
response personnel arrive.
 C H A P T E R 2 Facilities, Work Environment, and Safety ■ 63

Radioactive Waste Management substance, Category B” (UN3373). HIV

or HBV in culture are classified as Category
Policies for the disposal of radioactive waste,
A in- fectious substances, but these viruses
whether liquid or solid, should be established
present in a patient blood specimen are
with input from the radiation safety officer.
classified as Category B.
Liquid radioactive waste may be collected
Patient specimens with minimal
into large sturdy bottles labeled with an appro-
likeli- hood of containing pathogens are
priate radiation waste tag. The rules for sepa-
exempt from hazardous materials
ration by chemical compatibility apply. Bottles
regulations if the specimens are properly
must be carefully stored to protect against
packaged and marked. Blood components,
spillage or breakage. Dry or solid waste may
cellular therapy products, and tissue for
be sealed in a plastic bag and tagged as
transfusion or transplantation are not
radiation waste. The isotope, its activity, and
subject to hazardous material regula- tions.
the date on which the activity was measured
Method 1-1 provides additional ship- ping
should be recorded on the bag. Radioactive
instructions for safe transport of these
waste must never be discharged into the
materials. However, the most recent revision
facility’s drain system without prior approval
of the IATA or US DOT regulations should
by the radiation safety officer.
be con- sulted for the most current
classification, packaging, and labeling
SHIPPING HAZARDOUS requirements as well as for limitations on
MATERIALS the volumes of hazardous materials that can
be packaged together in one container.
Hazardous materials commonly shipped by
transfusion medicine, cellular therapy, and
clinical diagnostic services include GENERAL WASTE MANAGEMENT
infectious substances, biologic substances,
Those responsible for safety at a facility
liquid nitro- gen, and dry ice.
must be concerned with protecting the
The US DOT regulations for
environ- ment as well as all staff members.
transporta- tion of hazardous materials are
Every effort should be made to establish
harmonized with the international standards
facility-wide pro- grams to reduce solid
published an- nually by the International Air
wastes, including non- hazardous and,
Transport Asso- ciation (IATA).55,56 These
especially, hazardous wastes (ie,
regulations provide instructions for
biohazardous, chemical, and radioactive
identifying, classifying, pack- aging,
wastes).
marking, labeling, and documenting
A hazardous waste-reduction program
hazardous materials to be offered for ship-
in- stituted at the point of use of the
ment on public roadways or by air.
material achieves several goals. It reduces
Specimens are classified as Category A
the institu- tional risk for occupational
if they are known or likely to contain
exposures to haz- ardous agents, reduces
infectious substances in a form that is
capable of causing permanent disability or “cradle-to-grave” lia- bility for disposal,
life-threatening or fa- tal disease in and enhances compliance with
otherwise healthy humans or an- imals environmental requirements to reduce
when an exposure occurs. The proper pollution generated from daily operations
shipping name for Category A specimens of the laboratory.37,57,58
is “infectious substances, affecting Facilities can minimize pollution of
humans” (UN2814) or “infectious the environment by practicing the “three
substances, affecting animals only” R’s”: re- duce, reuse, and recycle. Seeking
(UN2900). suitable al- ternatives to materials that
Specimens that may contain infectious create hazardous waste and separating
substances but do not have the level of risk hazardous waste from nonhazardous waste
described above are classified as Category can reduce the volume of hazardous waste
B, and the proper shipping name is and decrease costs for its dis- posal.
“biological
64 ■ AABB T E C HNIC AL MANUAL

Changes in techniques or materials to In some states, copper sulfate contaminated

re- duce the volume of infectious waste or
render it less hazardous should be carefully
consid- ered, and employees should be
encouraged to identify safer alternatives
wherever possible.
Facilities should check with state
and local health and environmental
authorities about current requirements for
storage and disposal of a particular
multihazardous waste before creating that
waste. If creating the mul- tihazardous waste
cannot be avoided, the vol- ume of waste
generated should be minimized.
with blood is considered a multihazardous
waste. The disposal of this waste poses
several problems with transportation from
draw sites to a central facility for disposal of
the final con- tainers. State and local
health departments must be involved in
reviewing transportation and disposal
practices where this is an issue, and
procedures must be developed in accor-
dance with state and local regulations as
well as those of the US DOT.

KEY POINTS

Facilities should be designed and maintained in a way that supports the work being done in the physical space. Designing the
The facilities safety program should 1) strive to reduce hazards in the workplace, 2) ensure that staff are trained to handle kno
Safety programs should address fire, electrical, biological, chemical, and radioactive haz- ards that may be found in the facility
For each type of hazard, five basic elements that must be covered are 1) training; 2) hazard identification and communication;
Management controls ensure that the safety program is implemented, maintained, and ef- fective. Management is responsible
2) ensuring implementation of the plan and providing adequate resources for this imple- mentation, 3) providing access to em

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PA:
68 ■ AABB T E C HNIC AL MANUAL
■ APPENDIX 2-1
Safety Regulations and Recommendations Applicable to Health-Care Settings
Agency/Organization
Federal Regulations and
Recommendations
Nuclear Regulatory Commission
Occupational Safety and Health
Administration
Department of Transportation
Environmental Protection Agency
(EPA)
Centers for Disease Control and
Prevention
Food and Drug Administration
Reference Title
10 CFR 20 Standards for Protection Against Radia-
tion
10 CFR 36 Licenses and Radiation Safety Require-
ments for Irradiators
Guide 8.29 Instructions Concerning Risks from
Occupational Radiation Exposure
29 CFR 1910.1030 Occupational Exposure to Bloodborne
Pathogens
29 CFR 1910.1020 Access to Employee Exposure and Medi-
cal Records
29 CFR 1910.1096 Ionizing Radiation
29 CFR 1910.1200 Hazard Communication Standard
29 CFR 1910.1450 Occupational Exposure to Hazardous
Chemicals in Laboratories
49 CFR 171-180 Hazardous Materials Regulations
EPA Guide for Infectious Waste Manage-
ment
Guideline for Isolation Precautions in
Hospitals
21 CFR 606.3-606.171 Current Good Manufacturing Practice for
Blood and Blood Components
21 CFR 630.6 General Requirements for Blood, Blood
Components, and Blood Derivatives
21 CFR 640.1-640.120 Additional Standards for Human Blood
and Blood Products
21 CFR 211.1-211.208 Current Good Manufacturing Practice for
Finished Pharmaceuticals
21 CFR 1270 Human Tissue Intended for Transplanta-
tion
21 CFR 1271 Human Cells, Tissues, and Cellular
and Tissue-Based Products
 C H A P T E R 2 Facilities, Work Environment, and Safety ■ 69

■ APPENDIX 2-1
Safety Regulations and Recommendations Applicable to Health-Care
Settings (Continued)
Agency/Organization
Trade and Professional
Organizations
National Fire Protection
Association (NFPA)
National Paint and Coatings
Association
International Air Transport
Association
CFR = Code of federal regulations.
Reference Title
NFPA 70 National Electrical Code
NFPA 70E Electrical Safety Requirements for
Employee Workplaces
NFPA 101 Life Safety Code
NFPA 99 Standards for Health Care Facilities
NFPA 704 Standard for Identification of the Hazards
of Materials for Emergency Response
Hazardous Materials Identification
System Implementation Manual
Dangerous Goods Regulations
70 ■ AABB T E C HNIC AL MANUAL

■ APPENDIX 2-2
General Guidelines for Safe Work Practices, Personal Protective Equipment, and
Engineering Controls
UNIFORMS AND LABORATORY COATS

Personnel should wear closed laboratory coats or full aprons over long-sleeved uniforms or gowns when
they are exposed to blood, corrosive chemicals, or carcinogens. The material of required coverings
should be appropriate for the type and amount of hazard exposure. Plastic disposable aprons may be
worn over cotton coats when there is a high probability of large spills or splashing of blood and
body fluids; nitrile rubber aprons may be preferred when caustic chemicals are poured.

Protective coverings should be removed before the employee leaves the work area and should be discarded or
stored away from heat sources and clean clothing. Contaminated clothing should be removed promptly,
placed in a suitable container, and laundered or discarded as potentially infectious. Home laundering of
gar- ments worn in Biosafety Level 2 areas is not permitted because unpredictable methods of transportation
and handling can spread contamination and home laundering techniques may not be effective.1

GLOVES
Gloves or equivalent barriers should be used whenever tasks are likely to involve exposure to hazardous materials.

TYPES OF GLOVES
Glove type varies with the task:
■ Sterile gloves: for procedures involving contact with normally sterile areas of the body.
■ Examination gloves: for procedures involving contact with mucous membranes, unless otherwise
indicated, and for other patient care or diagnostic procedures that do not require the use of
sterile gloves.
■ Rubber utility gloves: for housekeeping chores involving potential blood contact, instrument cleaning and
decontamination procedures, and handling concentrated acids and organic solvents. Utility gloves may
be decontaminated and reused but should be discarded if they show signs of deterioration (eg, peeling,
cracks, or discoloration) or if they develop punctures or tears.
■ Insulated gloves: for handling hot or frozen material.

GUIDELINES FOR USE

The following guidelines should be used to determine when gloves are necessary1:
■ For donor phlebotomy when the health-care worker has cuts, scratches, or other breaks in his or her skin.
■ For phlebotomy of autologous donors or patients (eg, therapeutic apheresis procedures or intraoperative
red cell collection).
■ For persons who are receiving training in phlebotomy.
■ When handling open blood containers or specimens.
■ When collecting or handling blood or specimens from patients or donors known to be infected with a
blood- borne pathogen.
■ When examining mucous membranes or open skin lesions.
■ When handling corrosive chemicals and radioactive materials.
■ When cleaning up spills or handling waste materials.
■ When the likelihood of exposure cannot be assessed because of lack of experience with a procedure or situation.
 C H A P T E R 2 Facilities, Work Environment, and Safety ■ 71

■ APPENDIX 2-2
General Guidelines for Safe Work Practices, Personal Protective Equipment,
and Engineering Controls (Continued)
The Occupational Safety and Health Administration (OSHA) does not require the routine use of gloves by
phle- botomists working with healthy prescreened donors or the changing of unsoiled gloves between
donors if gloves are worn.1,2 Experience has shown that the phlebotomy process is low risk because
donors typically have low rates of infectious disease markers. Also, exposure to blood is rare during
routine phlebotomy, and other alternatives can be used to provide barrier protection, such as using a
folded gauze pad to control any blood flow when the needle is removed from the donor’s arm.
Employers whose policies and procedures do not require routine gloving should periodically reevaluate the
potential need for gloves. Employees should never be discouraged from using gloves, and gloves
should always be available.

GUIDELINES FOR USE

Guidelines for the safe use of gloves by employees include the following3,4:
■ Securely bandage or cover open skin lesions on hands and arms before putting on gloves.
■ Change gloves immediately if they are torn, punctured, or contaminated; after handling high-risk
samples; or after performing a physical examination (eg, on an apheresis donor).
■ Remove gloves by keeping their outside surfaces in contact only with outside and by turning the glove
inside out while taking it off.
■ Use gloves only when needed, and avoid touching clean surfaces such as telephones, doorknobs, or
com- puter terminals with gloves.
■ Change gloves between patient contacts. Unsoiled gloves need not be changed between donors.
■ Wash hands with soap or other suitable disinfectant after removing gloves.
■ Do not wash or disinfect surgical or examination gloves for reuse. Washing with surfactants may
cause “wicking” (ie, enhanced penetration of liquids through undetected holes in the glove). Disinfecting
agents may cause deterioration of gloves.
■ Use only water-based hand lotions with gloves, if needed; oil-based products cause minute cracks in latex.
FACE SHIELDS, MASKS, AND SAFETY GOGGLES

Where there is a risk of blood or chemical splashes, the eyes and mucous membranes of the mouth
and nose should be protected.5 Permanent shields fixed as a part of equipment or bench design are
preferred (eg, splash barriers attached to tubing sealers or centrifuge cabinets). All barriers should be
cleaned and disin- fected on a regular basis.

Safety glasses alone provide impact protection from projectiles but do not adequately protect eyes
from bio- hazardous or chemical splashes. Full-face shields or masks and safety goggles are
recommended when per- manent shields cannot be used. Many designs are commercially available;
eliciting staff input on comfort and selection can increase use.
Masks should be worn whenever there is danger from inhalation. Simple, disposable dust masks are
adequate for handling dry chemicals, but respirators with organic vapor filters are preferred for areas
where noxious fumes are produced (eg, for cleaning up spills of noxious materials). Respirators
should be fitted to their wearers and checked annually.
(Continued)
72 ■ AABB T E C HNIC AL MANUAL

■ APPENDIX 2-2
General Guidelines for Safe Work Practices, Personal Protective Equipment, and
Engineering Controls (Continued)
HAND WASHING

Frequent, thorough hand washing is the first line of defense in infection control. Blood-borne
pathogens gen- erally do not penetrate intact skin, so immediate removal reduces the likelihood of
transfer to a mucous mem- brane or broken skin area or of transmission to others. Thorough washing
of hands (and arms) also reduces the risks from exposure to hazardous chemicals and radioactive
materials.
Employees should always wash their hands before leaving a restricted work area or using a biosafety
cabinet, between medical examinations, immediately after becoming soiled with blood or hazardous
materials, after removing gloves, or after using the toilet. Washing hands thoroughly before touching
contact lenses or apply- ing cosmetics is essential.
OSHA allows the use of waterless antiseptic solutions for hand washing as an interim method.2 These
solu- tions are useful for mobile donor collections or in areas where water is not readily available for
cleanup pur- poses. If such methods are used, however, hands must be washed with soap and
running water as soon as possible thereafter. Because there is no listing or registration of acceptable hand-
wipe products similar to the one that the Environmental Protection Agency maintains for surface
disinfectants, consumers should request data from the manufacturer to support advertising claims.

EYEWASHES

Laboratory areas that contain hazardous chemicals must be equipped with eyewash stations.3,6
Unobstructed access within a 1- second walk from the location of chemical use must be provided for
these stations. Eye- washes must operate so that both of the user’s hands are free to hold open the
eyes. Procedures and indica- tions for use must be posted, and routine function checks must be
performed. Testing eyewash fountains weekly helps ensure proper function and flushes out stagnant
water. Portable eyewash systems are allowed only if they can deliver flushing fluid to the eyes at a rate
of at least 1.5 liters per minute for 15 minutes. They should be monitored routinely to ensure the
purity of their contents.
Employees should be trained in the proper use of eyewash devices, although prevention—through
consistent and appropriate use of safety glasses or shields—is preferred. If a splash occurs, the
employee should be directed to keep his or her eyelids open and to use the eyewash according to
procedures, or the employee should go to the nearest sink and direct a steady, tepid stream of water
into his or her eyes. Solutions other than water should be used only in accordance with a
physician’s direction.
After eyes are adequately flushed (many facilities recommend 15 minutes), follow-up medical care should
be sought, especially if pain or redness develops. Whether washing the eyes is effective in preventing
infection has not been demonstrated, but it is considered desirable when accidents occur.
1. Code of federal regulations. Title 29, CFR Part 1910.1030. Fed Regist 1991;56:64175-82.
2. Occupational Safety and Health Administration. Enforcement procedures for the occupational exposure to bloodborne pathogens.
OSHA Instruction CPL 02-02-069. Washington, DC: US Government Printing Office, 2001.
3. Clinical laboratory safety: Approved guideline. 3rd ed (GP17-A3). Wayne, PA: Clinical and Laboratory Standards Institute, 2012.
4. Medical glove powder report. (September 1997) Rockville, MD: Food and Drug Administration, 2009. [Available at
http://www.fda. gov/MedicalDevices/DeviceRegulationand Guidance/GuidanceDocuments/ucm113316.htm (accessed
January 6, 2013).]
5. Inspection checklist: General laboratory. Chicago: College of American Pathologists, 2012.
6. American national standards for emergency eyewash and shower equipment. ANSI Z358.1-2009. New York: American National
Stan- dards Institute, 2009.
 C H A P T E R 2 Facilities, Work Environment, and Safety ■ 73

■ APPENDIX 2-3
Biosafety Level 2 Precautions
Biosafety Level 2 precautions as applied in the blood establishment setting include at least the following1,2:
■ High-risk activities are appropriately segregated from lower-risk activities, and the boundaries are clearly
defined.
■ Bench tops are easily cleaned and are decontaminated daily with a hospital disinfectant approved by the
Envi- ronmental Protection Agency.
■ Laboratory rooms have closable doors and sinks. An air system with no recirculation is preferred
but not required.
■ Workers are required to perform procedures that create aerosols (eg, opening evacuated tubes,
centrifuging, mixing, or sonicating) in a biological safety cabinet or equivalent or to wear masks and
goggles in addition to gloves and gowns during such procedures. (Note: Open tubes of blood should not
be centrifuged. If whole units of blood or plasma are centrifuged, overwrapping is recommended
to contain leaks.)
■ Gowns and gloves are used routinely and in accordance with general safety guidelines. Face shields or
their equivalents are used where there is a risk from splashing.
■ Mouth pipetting is prohibited.
■ No eating, drinking, smoking, applying cosmetics, or manipulating contact lenses occurs in the work
area. All food and drink are stored outside the restricted area, and laboratory glassware is never
used for food or drink. Personnel are instructed to avoid touching their face, ears, mouth, eyes, or
nose with their hands or other objects, such as pencils and telephones.
■ Needles and syringes are used and disposed of in a safe manner. Needles must never be bent,
broken, sheared, replaced in a sheath, or detached from a syringe before being placed in puncture-
proof, leakproof containers for controlled disposal. Procedures are designed to minimize exposure
to sharp objects.
■ All blood specimens are placed in well-constructed containers with secure lids to prevent leaking
during transport. Blood is packaged for shipment in accordance with regulatory agency requirements for
etiologic agents or clinical specimens, as appropriate.
■ Infectious waste is not compacted and is decontaminated before its disposal in leakproof containers.
Proper packaging includes double, seamless, tear-resistant, orange or red bags that are enclosed in
protective car- tons. Both the cartons and the bags inside display the biohazard symbol. Throughout
delivery to an incinera- tor or autoclave, waste is handled only by suitably trained persons. If a waste
management contractor is used, the agreement should clearly define the respective responsibilities
of the staff and the contractor.
■ Equipment to be repaired or submitted for preventive maintenance, if potentially contaminated with
blood, must be decontaminated before its release to a repair technician.
■ Accidental exposure to suspected or actual hazardous material is reported to the laboratory
director or responsible person immediately.
1. Clinical laboratory safety: Approved guideline. 3rd ed (GP17-A3). Wayne, PA: Clinical and Laboratory Standards Institute, 2012.
2. Fleming DO. Laboratory biosafety practices. In: Fleming DO, Richardson JH, Tulis JJ, Vesley DD, eds. Laboratory safety, principles,
and practices. 2nd ed. Washington, DC: American Society for Microbiology Press, 1995:203-18.
74 ■ AABB T E C HNIC AL MANUAL

■ APPENDIX 2-4
Sample List of Hazardous Chemicals that May Be Encountered in a Blood Bank
ChemicalHazard

Ammonium chloride Irritant

Bromelin Irritant, sensitizer
Calcium chloride Irritant
Carbon dioxide, frozen (dry ice) Corrosive
Carbonyl iron powder Oxidizer
Chloroform Toxic, suspected carcinogen
Chloroquine Irritant, corrosive
Chromium-111 chloride hexahydrate Toxic, irritant, sensitizer
Citric acid Irritant
Copper sulfate (cupric sulfate) Toxic, irritant
Dichloromethane Toxic, irritant
Digitonin Toxic
Dimethyl sulfoxide Irritant
Dry ice (carbon dioxide, frozen) Corrosive
Ethidium bromide Carcinogen, irritant
Ethylenediaminetetraacetic acid Irritant
Ethyl ether Highly flammable and explosive, toxic, irritant
Ficin (powder) Irritant, sensitizer
Formaldehyde solution (34.9%) Suspected carcinogen, combustible, toxic
Glycerol Irritant
Hydrochloric acid Highly toxic, corrosive
Imidazole Irritant
Isopropyl (rubbing) alcohol Flammable, irritant
Liquid nitrogen Corrosive
Lyphogel Corrosive
2- Mercaptoethanol Toxic, stench
Mercury Toxic
Mineral oil Irritant, carcinogen, combustible
Papain Irritant, sensitizer
Polybrene Toxic

Sodium azide Toxic, irritant, explosive when

heated Sodium ethylmercurithiosalicylate (thimerosal) Highly toxic, irritant
 C H A P T E R 2 Facilities, Work Environment, and Safety ■ 75

■ APPENDIX 2-4
Sample List of Hazardous Chemicals that May Be Encountered in a Blood Bank
(Continued)
ChemicalHazard

Sodium hydrosulfite Toxic, irritant

Sodium hydroxide Corrosive, toxic
Sodium hypochlorite (bleach) Corrosive
Sodium phosphate Irritant, hygroscopic
Sulfosalicylic acid Toxic, corrosive
Trichloroacetic acid Corrosive, toxic
Trypsin Irritant, sensitizer
Xylene Highly flammable, toxic, irritant
76 ■ AABB T E C HNIC AL MANUAL

■ APPENDIX 2-5
Chemical Categories and How to Work Safely with Them
Chemical CategoryHazardPrecautionsSpecial Treatment

Acids, alkalis, and Irritation, severe burns, During transport, protect Store concentrated
corro- sive tissue damage large containers acids in acid safety
compounds with plastic or rubber cabinets. Limit
bucket carriers. volumes of con-
During pouring, wear centrated acids to 1
eye protection and liter per container.
chemi- cal-resistant- Post cautions for
rated gloves and materi- als in the
gowns as area.
recommended. Report changes in
Always add acid to appearance
water, never add (perchloric acid may
water to acid. be explosive if it
When working with becomes yellowish or
large jugs, have one brown) to chemical
hand on the neck and safety officer.
the other hand at the
base, and position
them away from the
face.
Acrylamide Neurotoxic,
Wear chemically rated Store in a chemical
carcino- genic,
gloves. cabi- net.
absorbed
Wash hands
through the skin
immediately after
exposure.
Compressed gases Explosive Label contents.
Transport using hand
Leave valve safety
trucks or dollies.
covers on until use.
Place cylinders in a
Open valves slowly for
stand or secure them
use.
to pre- vent tipping
Label empty tanks.
over.
Store in well-
ventilated separate
rooms.
Do not store oxygen
close to combustible
gas or solvents.
Check connections for
leaks using soapy
water.
 C H A P T E R 2 Facilities, Work Environment, and Safety ■ 77

■ APPENDIX 2-5
Chemical Categories and How to Work Safely with Them (Continued)
Chemical CategoryHazardPrecautionsSpecial Treatment

Flammable solvents Classified according to Use extreme caution Make every attempt to
flash point—see mate- when handling. replace hazardous
rial safety data Post “No Smoking” materials with less haz-
sheet, classified signs in working ardous materials
according to volatility area. Store containers larger
Keep a fire extinguisher than 1 gallon in a
and solvent cleanup kit flam- mable solvent
in the room. storage room or a
Pour volatile solvents fire safety cabinet.
under a suitable hood. Ground metal containers
Use eye protection and by connecting the can
chemical-resistant neo- to a water pipe or
prene gloves when ground connection. If
pouring. the recipient container
No flame or other is also metal, it should
source of possible be electrically con-
ignition should be in nected to the delivery
or near areas where container during pour-
flammable solvents ing.
are being poured.
Label as “flammable.”
Liquid nitrogen Freeze injury, The tanks should be
severe burns to Use heavy insulated securely supported to
skin or eyes gloves and goggles avoid being tipped
when working with over.
liq- uid nitrogen. The final container of
liq- uid nitrogen
(freezing unit) must
be securely
supported to avoid
being tipped over.
78 ■ AABB T E C HNIC AL MANUAL

■ APPENDIX 2-6
Incidental Spill Response*
ChemicalsHazardsPPEControl Materials

Acids If inhaled, causes Acid-resistant gloves Acid neutralizers or

Acetic severe irritation. Apron and coveralls absorbent material
Hydrochloric Contact causes burns to Goggles and face Absorbent boom
Nitric skin and eyes. shield Acid-resistant foot Leakproof
Perchloric Spills are corrosive. covers containers
Sulfuric Fire or contact with Absorbent pillow
Photographic chemi- metal may produce Mat (cover drain)
cals (acidic) irritating or Shovel or paddle
poisonous gas.
Nitric, perchloric, and
sulfuric acids are
water-reactive oxidiz-
ers.
Bases and caustics
Potassium hydroxide Spills are corrosive. Gloves, impervious Base control/neutralizer
Sodium hydroxide Fire may produce irritat- apron or coveralls Absorbent pillow
Photographic ing or poisonous gas. Goggles or face shield, Absorbent boom
chemicals (basic) impervious foot covers Drain mat
Leakproof
container Shovel
Chlorine
or paddle
Bleach Inhalation can cause Gloves (double set of 4H
Sodium hypochlorite respiratory irritation. undergloves and butyl Chlorine control powder
Liquid contact can or nitrile overgloves), Absorbent pillow
pro- duce irritation impervious apron or Absorbent material
of the eyes or coveralls Absorbent boom
skin. Goggles or face shield Drain mat
Toxicity is caused by Impervious foot covers Vapor barrier
alkalinity, possible (neoprene boots for Leakproof container
chlorine gas genera- emergency response Shovel or paddle
tion, and oxidant releases)
prop- erties. Self-contained
breathing apparatus
(emergency response
Cryogenic gases
releases)
Carbon dioxide
Nitrous oxide Contact with liquid Full face shield or Hand truck (to
Liquid nitrogen nitro- gen can gog- gles, neoprene transport cylinder
produce frost- bite. boots, gloves outdoors if
Release can create an (insulated to provide necessary)
oxygen-deficient protection from the Soap solution (to check
atmo- sphere. cold) for leaks)
Nitrous oxide has anes- Putty (to stop minor pipe
thetic effects. and line leaks)
 C H A P T E R 2 Facilities, Work Environment, and Safety ■ 79

■ APPENDIX 2-6
Incidental Spill Response* (Continued)
ChemicalsHazardsPPEControl Materials

Flammable gases Simple asphyxiant Face shield and Hand truck (to
Acetylene (displaces air). goggles, neoprene transport cylinder
Oxygen gases Inhaled vapors have an boots, double set of outdoors if needed)
Butane anesthetic potential. gloves, coveralls with Soap solution (to check
Propane Flammable gases pose hood and feet for leaks)
an extreme fire and
explo- sion hazard.
Release can create
an oxygen-
deficient
atmosphere.
Flammable Absorbent material
liquids Vapors are harmful if Gloves (double set of Absorbent boom
Acetone inhaled (central ner- 4H undergloves and Absorbent pillow
Xylene vous system depres- butyl or nitrile Shovel or paddle (non-
Methyl alcohol toluene sants). overgloves), metal, nonsparking)
Ethyl alcohol Liquid is harmful if impervious apron or Drain mat
Other alcohols absorbed through the coveralls, goggles or Leakproof container
skin. face shield,
Substances are impervious foot
extremely flammable. covers
Liquid evaporates to
form flammable
Aldehyde neutralizer or
vapors.
Formaldehyde and absorbent
glutaraldehyde Vapors are harmful Absorbent boom
4% formaldehyde if inhaled; liquids Gloves (double set of Absorbent pillow
37% formaldehyde are harmful if 4H undergloves and Shovel or paddle
10% formalin absorbed through butyl or nitrile over- (nonsparking)
2% glutaraldehyde skin. gloves), impervious Drain mat
Substances are irritants apron or coveralls, Leakproof container
to skin, eyes, and goggles, impervious
respiratory tract. foot covers
Formaldehyde is a sus-
pected human carcino-
gen.
37% formaldehyde
should be kept away
from heat, sparks, and
flames.

(Continued)
80 ■ AABB T E C HNIC AL MANUAL

■ APPENDIX 2-6
Incidental Spill Response* (Continued)
ChemicalsHazardsPPEControl Materials

Mercury Mercury and mercury Gloves (double set of Mercury vacuum or

Cantor tubes vapors are rapidly 4H undergloves and spill kit
Thermometers absorbed in respira- butyl or nitrile Scoop
Barometers tory tract, gastrointesti- overgloves), Aspirator
Sphygmomanometers nal (GI) tract, or skin. impervious apron or Hazardous waste contain-
Mercuric chloride Short-term exposure coveralls, goggles, ers
may cause erosion of impervious foot covers Mercury indicator powder
respi- ratory or GI Absorbent material
tracts, nau- sea, Spatula
vomiting, bloody Disposable towels
diarrhea, shock, Sponge with amalgam
head- ache, or Vapor suppressor
metallic taste.
Inhalation of high con-
centrations can cause
pneumonitis, chest
pain, dyspnea, cough-
ing, stomatitis,
gingivi- tis, and
salivation.
Avoid evaporation of
mercury from tiny
globules by quick and
thorough cleaning.
*This list of physical and health hazards in not intended as a substitute for the material safety data sheet (MSDS)
informa- tion. In case of a spill or if any questions arise, always refer to the chemical-specific MSDS for more
complete information.
 C H A P T E R 2 Facilities, Work Environment, and Safety
■ APPENDIX 2-7
Managing Hazardous Chemical Spills
ActionsInstructions for Hazardous Liquids, Gases, and Mercury
Deenergize.
Isolate, evacuate, and
secure the area.
Have the appropriate per-
sonal protective equipment
(PPE).
Contain the spill.
Confine the spill.
Neutralize the spill
Clean up the spill.
■ 81
Liquids: For 37% formaldehyde, deenergize and remove all sources of igni-
tion within 10 feet of spilled hazardous material. For flammable
liquids, remove all sources of ignition.
Gases: Remove all sources of heat and ignition within 50 feet for
flammable gases.
Remove all sources of heat and ignition for nitrous oxide release.
Isolate the spill area and evacuate everyone from the area surrounding
the spill except those responsible for cleaning up the spill. (For mercury,
evacuate within 10 feet for small spills or 20 feet for large spills.)
Secure the area.
See Appendix 2-2 for recommended PPE.
Liquids or mercury: Stop the source of spill if possible.
Gases: Assess the scene; consider the circumstances of the release
(quantity, location, and ventilation). If circumstances indicate that it is an
emergency response release, make appropriate notifications; if the release
is determined to be incidental, contact the supplier for assistance.
Liquids: Confine the spill to the initial spill area using appropriate control
equipment and material. For flammable liquids, dike off all
drains. Gases: Follow the supplier’s suggestions or request outside
assistance.
Mercury: Use appropriate materials to confine the spill (see Appendix 2-
6). Expel mercury from the aspirator bulb into a leakproof container, if
applicable.
Liquids: Apply appropriate control materials to neutralize the chemical (see
Appendix 2-6).
Mercury: Use a mercury spill kit if needed.
Liquids: Scoop up solidified materials, booms, pillows, and any other
materi- als. Put used materials into a leakproof container. Label the
container with the name of the hazardous materials. Wipe up residual
material. Wipe the spill area surface three times with a detergent solution.
Rinse the areas with clean water. Collect the supplies used (eg, goggles
or shovels) and remove gross contamination; place equipment to be
washed and decontaminated into a separate container.
Gases: Follow the supplier’s suggestions or request outside assistance.
Mercury: Vacuum up the spill using a mercury vacuum, or scoop up
mercury paste after neutralization and collect the paste in a designated
container. Use a sponge and detergent to wipe and clean the spill
surface three times to remove absorbent. Collect all contaminated
disposal equipment and put it into a hazardous waste container. Collect
supplies and remove gross contam- ination; place equipment that will be
thoroughly washed and decontaminated into a separate container.
(Continued)
82 ■ AABB T E C HNIC AL MANUAL

■ APPENDIX 2-7
Managing Hazardous Chemical Spills (Continued)
ActionsInstructions for Hazardous Liquids, Gases, and Mercury

Dispose. Liquids: Dispose of material that was neutralized as solid waste. Follow
the facility’s procedures for disposal. For flammable liquids, check with
the facil- ity safety officer for appropriate waste determination.
Gases: The manufacturer or supplier will instruct the facility about disposal
if applicable.
Mercury: Label with appropriate hazardous waste label and Department of
Transportation diamond label.
Report. Follow appropriate spill documentation and reporting procedures.
Investigate the spill; perform a root cause analysis if needed. Act on
opportunities for improving safety.
 C h a p t e r
Regulatory Issues in Blood Banking
Glenn Ramsey, MD
IN THE UN I T ED S T ATE S , when fed-
I eral laws are enacted, they are published
chronologically as statutes and placed into the
appropriate 1 of 50 subject areas (titles) of the
United States Code (USC).1 The government
agency responsible for the law—for blood-
related laws, the Food and Drug Administra-
tion (FDA)—then writes regulations (rules) to
enforce the law. Proposed rules for public
comment and final rules, along with back-
ground and interpretive information, are pub-
lished chronologically in the daily Federal
Reg- ister.2 The current edition of all rules is
collated annually in the appropriate title of the
Code of Federal Regulations (CFR); Title 21
addresses food and drugs, and Title 42 focuses
on health care.3
Some rules have associated guidance
documents to provide current thinking about
regulatory topics. Guidance documents
con- tain recommendations, not
requirements, but they are usually followed
by industry. Memo- randa to blood
establishments were issued be- fore 1998,
but some are still active references. These
publications can be accessed from the FDA
blood and blood products webpage.4
Glenn Ramsey, MD, Medical Director, Blood Banks, Northwestern Memorial Hospital and Ann & Robert H.
Lurie Children’s Hospital of Chicago, and Department of Pathology, Feinberg School of Medicine, Northwest-
ern University, Chicago, Illinois
The author has disclosed no conflicts of interest.
3
The FDA regulates drugs and medical de-
vices under the Food, Drug, and Cosmetic Act
(USC Title 21, Sections 301-399d). 5 Blood
products are defined as drugs because they are
intended to cure, mitigate, treat, or prevent
disease (21 USC 321).6,7 The law requires
blood product manufacturers to register with
the FDA, obtain biologics licenses, and follow
cur- rent good manufacturing practice (cGMP)
reg- ulations. It also prohibits adulteration
and misbranding, authorizes inspections, and
de- fines civil and criminal penalties for
violations. The act controls the use of
unapproved drugs and devices during their
investigational phas- es and public health
emergencies.
Medical devices include instruments,
in- vitro reagents, and their parts and
accessories for the diagnosis and treatment
of disease. The FDA classifies devices as
Class I, II, or III, in as- cending order of
associated risk [21 USC 360(c)].8,9 Class I
devices have no potential un- reasonable
risk. Class II devices must meet
performance standards beyond basic FDA
reg- ulations. Some Class I devices and most
Class II devices must be cleared by the FDA
based on substantial equivalence with
another device
83
84 ■ AABB T E C HNIC AL MANUAL
already on the market. This process is
called 510(k) clearance, referring to the
applicable section of the act describing the
submission of applications to the FDA for
such devices [21 USC 360(k)].8 Class III
devices pose potential unreasonable risk
and require specific pre- market approval
from the FDA, as do unprece- dented new
devices before their class is deter- mined.
BIOLOGICAL PRODUCTS
Biological products come from living
sources and include blood, blood
components, deriva- tives, therapeutic
serum, and vaccines appli- cable to the
prevention or treatment of dis- ease. They
are regulated by Section 351 (42 USC 262)
of the Public Health Service Act, which
addresses licensure, labeling, inspec- tions,
recalls, and penalties for violations in
producing biological drugs. This set of
regula- tions provides the core of federal law
that is most specific to blood products.10
Section 361, Regulations to Control
Communicable Diseas- es (42 USC 264),
grants the Surgeon General inspection and
quarantine powers to prevent the spread of
infectious diseases and is in- voked in the
regulation of tissues (see below).11 The
FDA’s key regulations enforcing the Food
and Drug Act and the Public Health Ser-
vice Act are in Title 21 of the CFR, Food
and Drugs, and in particular, Parts 200
through 299 for drugs, 600 through 680 for
biologicals, 800 through 898 for devices,
and 1270 through 1271 for tissues (Table 3-
1).12 The FDA website has links to pertinent
laws in the USC and reg-
ulations in the CFR.13
Within the FDA, the Center for Biologics
Evaluation and Research (CBER) regulates
blood products and most other biological
therapies.14 The Center for Devices and Radio-
logical Health (CDRH) regulates most medical
devices, but CBER retains primary jurisdiction
over medical devices used with blood and cel-
lular products.15 The FDA’s Office of
Regulatory Affairs (ORA) has responsibility
for all field op- erations, which include
inspections and inves- tigations.
As part of the development process for
FDA regulations and guidance documents,
several forums are offered for input from
the public and regulated groups.16 Proposed
rules and draft guidance documents are
published with an invitation for written
comments, which are filed in public
dockets.17 When final rules are published in
the Federal Register, the accompanying
commentaries offer response to key
questions. The FDA also receives peti-
tions to write or change regulations.
Expert opinions on current issues are
sought from several advisory committees,
including the FDA’s committees on blood
products and on cellular, tissue, and gene
therapies, and the Department of Health
and Human Services (DHHS) Committee on
Blood Safety and Avail- ability. Public
meetings hosted by the FDA on selected
topics provide an additional opportu- nity
for input.
Facilities may apply to CBER for
approval of exceptions to the regulations
for blood products or alternative
procedures. These ap- provals are
periodically published, although the
circumstances for these approvals may not
apply to other facilities.18
LICENSURE AND
REGISTRATION
Blood establishments are classified into three
categories by the FDA: 1) licensure and regis-
tration for interstate commerce, 2) registration
for manufacturers not involved in interstate
commerce, and 3) exemption from registration
for transfusion services as described in the
regulations.19 Licensed and/or registered facil-
ities are inspected by the FDA. For transfusion
services that perform minimal manufacturing
and are certified for reimbursement by the
Centers for Medicare and Medicaid Services
(CMS), the FDA accepts CMS-sanctioned
labo- ratory inspections and approval. 20
However, the FDA still has jurisdiction and
may conduct its own inspections if warranted.
All military blood facilities, including
transfusion services, register with and are
inspected by the FDA.21
Facilities use the Biologics License
Appli- cation (BLA, Form 356h) to apply
for their establishment and product
licenses for interstate commerce.22
Subsequent license amendments are based
on the potential for
 CH A P T E R 3 Re g u l a t o r y I s s u es I n B l o o d B a n k i n g ■ 85

TABLE 3-1. Regulations of Interest in Title 21 of the CFR (Food and Drugs)

Topic Section Topic Section

FDA general Donor notification 630.6
Enforcement 1-19 Blood product 640
standards
Research and 50-58 Donor eligibility 640.3
development
Labeling 201 Blood collection 640.4
cGMP for drugs 210-211 Blood testing 640.5
Biological products 600-680 Red Blood Cells 640.10-.17
General 600 Platelets 640.20-.27
Licensing 601 Plasma 640.30-.34
cGMP for blood 606 Cryoprecipitated AHF 640.50-.56
components
Personnel, 606.20-.65 Exceptions, alternatives 640.120
resources
Standard operating Medical devices 800-898
procedures 606.100

Labeling 606.120-.122 Adverse events 803

Compatibility testing 606.151 Hematology and 864
pathology
Records 606.160-.165 Tissues
Adverse reactions 606.170 Tissues for transplan- 1270
tation
Product deviations 606.171 Cellular and tissue-based 1271*
products
Establishment 607 General provisions 1271.1-.20
registration
General standards 610 Registration and 1271.21-.37
products
Donor testing 610.40 Donor eligibility 1271.45-.90
Donor deferral 610.41 cGTP 1271.145-.320
Look-back 610.46-.48 Section 361 provi- 1271.330-.440
sions†
Dating periods 610.53
*The following citations represent Subparts A, B, C, D, and E-F, respectively.
†
Autologous and related donors (see text).
CFR = Code of Federal Regulations, FDA = Food and Drug Administration, cGMP = current good manufacturing
practice; cGTP = current good tissue practice.
86 ■ AABB T E C HNIC AL MANUAL
adverse effects of the changes.23
Unlicensed blood products, whether
manufactured by a li- censed or an
unlicensed facility, may be shipped
across state lines for a medical emer- gency
if necessary, but this shipping should be
unscheduled and infrequent. 24 The shipping
facility must retain documentation of the
emergency for FDA review.
Facilities must register annually with the
FDA if they collect, manufacture, prepare, or
process blood products (Form FDA 2830).25,26
Transfusion services are exempt from this re-
quirement if they are approved for reimburse-
ment by CMS and their preparation activities
are basic, such as preparing Red Blood Cells
from whole blood, converting unused plasma
to recovered plasma, pooling components, re-
ducing leukocytes with bedside filters, or col-
lecting blood only in emergencies. However,
facilities that routinely collect blood (includ-
ing autologous units) or perform such proce-
dures as irradiation, washing, laboratory leu-
kocyte reduction, freezing, deglycerolization,
and rejuvenation must register as community
or hospital blood banks. Transfusion services
acting as depots for forwarding products to
other hospitals must register as distribution
centers. Clinical laboratories performing in-
fectious disease testing required for blood do-
nors must register unless they are CMS ap-
proved. If blood irradiation is performed
outside the blood bank or transfusion service,
such as in a nuclear medicine department,
that facility must register as well.
FDA INSPECTIONS
New facilities are generally inspected within
1 year of establishment by a team from
CBER and ORA. Subsequent routine
inspections are generally performed by ORA
every 2 years or earlier depending on the
facility’s compliance history.
ORA publishes online manuals and
in- structions for FDA investigators for
inspec- tions in general and for inspections
of licensed and unlicensed blood banks in
particular.25,26 The blueprints for blood bank
inspections are the general regulations for
cGMP and drugs (21 CFR 210-211) and
the specific require-
ments for blood components (21 CFR
606).27 Routine inspections address the
FDA’s five re- quired layers of blood safety
—donor screen- ing, donor testing, product
testing, quarantin- ing, and monitor i ng
and inv e stigating problems.
Investigators review the following
operational systems that create these safety
layers: quality assurance, donor
eligibility, product testing,
quarantine/inventory man- agement, and
production and processing.
Full inspections of all systems are desig-
nated Level I. After a favorable inspection pro-
file, facilities with four or five systems some-
times have streamlined Level II inspections of
three systems. (Focused inspections for prob-
lems or fatalities need not follow these for-
mats.) Within each system, the investigators
review standard operating procedures, per-
sonnel and training, facilities, equipment cali-
bration and maintenance, and records. Specif-
ic requirements for individual systems and
processes are discussed in detail in their re-
spective chapters of this book.
If the investigator judges that
significant objectionable practices,
violations, or condi- tions are present under
which a drug or device is or could be
adulterated or injurious to health, these
observations are written and pre- sented to
the facility (Form 483). Investigators are
instructed to seek and record the manage-
ment’s intentions to make corrections.25
How- ever, most facilities also respond in
writing im- mediately with questions or
corrective actions. Final determination that
a violation has oc- curred is made by ORA.
The FDA can take a number of steps in
re- sponse to a violation, including no action
at all. For significant violations, the FDA may
is- sue a warning letter, which is an advisory
ac- tion to provide the facility with the
opportuni- ty for voluntary compliance.
Enforcement actions are categorized as
administrative, judi- cial, or recall.
Administrative actions include formal
citations of violation and—for licensed
facilities—suspension or revocation of a li-
cense. Judicial actions range from seizures of
products to court injunctions, civil monetary
penalties, and criminal prosecution. The FDA
may also conduct recalls if necessary. ORA’s
 CH A P T E R 3 Re g u l a t o r y I s s u es I n B l o o d B a n k i n g
Regulatory Procedures Manual provides details
on available sanctions.28
BLOOD-RELATED DEVICES
CBER has lead responsibility in
collaboration with CDRH for equipment
marketed for trans- fusion and the collection
and processing of blood products,
hematopoietic stem cells, and
autotransfusions.29,30 These devices include
di- agnostic tests for infectious diseases in
blood donors and pretransfusion testing in
patients. Blood bank computer software
programs are CBER-approved devices.
Most blood-related devices are in Class II
and cleared by 510(k) equivalence. Examples
of Class I devices are copper sulfate solutions
for hemoglobin screening, blood grouping
view boxes, and heat sealers. Human immuno-
deficiency virus (HIV ) and hepatitis B and
C virus donor tests are regulated as Class II or
III devices. Screening tests for blood
donations are regulated as BLAs. BLAs and
Class III de- vices require specific
premarket approval. Each category of device
is assigned a code, and all cleared or approved
vendors and products for that code are
searchable in the Establish- ment
Registration and Device Listing data- base
on the CDRH website.31
Serious adverse events related to
medical devices must be reported (21 CFR
803).32 User facilities must report deaths or
serious inju- ries in which a device was or
may have been a factor. Serious injury is
defined as being life threatening, causing
permanent impairment or damage, or
needing medical or surgical in- tervention.
Reports of serious injuries are sent to the
device maker using FDA Medwatch
Form 3500A within 10 working days of
the event, and deaths must also be reported
to the FDA. In years when a Form 3500A
report is submitted, an annual summary
must be sent to the FDA by January 1 of the
following year (Form 3419). 22 Users may
voluntarily report other device-related
adverse events to the FDA (Form 3500). 22
All possible adverse events, whether
reported or not, must be investigated and
their records must be kept on file for 2
years.
■ 87
HEMATOPOIETIC PROGENITOR
CELLS AS TISSUES
Unmanipulated marrow cell transplants are
regulated by the Health Resources and
Servic- es Administration of DHHS, which
also over- sees marrow and cord blood
donations and transplant procedures
coordinated by the Na- tional Marrow Donor
Program. Other hemato- poietic progenitor
cells (HPCs) collected be- fore May 25,
2005 are regulated by FDA rules for tissue
transplants in 21 CFR 1270.12. The FDA’s
regulations in 21 CFR 1271 for human cells,
tissues, and cellular and tissue-based
products (HCT/Ps) apply to HPCs collected
on or after May 25, 2005.12,33,34
HCT/P manufacturers must comply with
rules for 1) facility registration and
product listing; 2) donor eligibility, including
testing for communicable diseases; and 3)
production ac- cording to current good
tissue practice (cGTP) regulations (Table 3-
1). Registrants using FDA Form 3356 report
each type of HPC they man- ufacture in
three categories: 1) HCT/Ps as de- scribed in
21 CFR 1271.10(a), 2) biologics, and
3) drugs. Table 3-2 outlines how the FDA de-
fines, regulates, and (when applicable) ap-
proves each type of HPC. Basic peripheral
blood stem cells (PBSCs) from autologous or
first- or second-degree related donors are in
the first category; these products are regulated
mainly with regard to preventing transmission
of communicable diseases or contamination
(Public Health Service Act, Section 361, 42
USC 264).11 Typical PBSCs from unrelated
donors are biologics, and the FDA is
considering li- censure requirements for them.
Since October 20, 2011, unrelated umbilical
cord cells must be licensed or used under an
investigational new drug application (see
Chapter 30). Any HPCs that are more than
minimally manipu- lated (eg, by expansion,
property alteration, or gene therapy) or used
for a different function (eg, tissue
engineering) are categorized as a drug and
must have premarket approval.
The FDA specifies required infectious dis-
ease tests for tissue donors and posts lists
of tests that have been licensed or cleared
for this indication.45 The elements of cGTP
are analo- gous to those of cGMP for blood
products. The
TABLE 3-2. US Regulations for Manufacturers of Hematopoietic Progenitor Cells35,36

88
Key Regulations (21 CFR FDA Premarket Licensure, FDA Compliance Program
Type of HPC Product Jurisdiction Category except as noted) Approval, or Clearance? Manual37,38
Unmanipulated marrow Health Resources and Ser- 42 US Code 274(k) No Not applicable
■
vices Administration
HPCs* collected before May 25, Tissues, 21 CFR 1270 1270, 1271 Subparts A-B No 7341.002A39
2005 (before

AABB TECHNICAL MAN U AL

May 25, 2005) (see Table 3-1)
Autologous or allogeneic related- PHS Act Section 361: 1271.10(a)§ (must meet No 7341.00237
HCT/Ps‡ all
donor† HPCs criteria); 1271 A-F 7342.007 Addendum40
(imported products)
Unrelated-donor peripheral blood PHS Act Section 351: 1271 A-D Planned 7341.00237
HPCs Biologics
Unrelated-donor umbilical cord cells Section 351: Biologics 1271 A-D Yes (after October 20, 7341.00237
2011):
Licensure Guidance41 licensure or IND application
for
IND Guidance42 nonqualifying products
HPCs with altered characteristics or Cellular and Gene Therapy, 1271 A-D Yes: IND and BLA 7345.84843
different function (including somatic CBER
cell therapy), or HPCs combined
with
drug
HPCs combined with medical device CBER and CDRH, as deter- 1271 A-D Yes: investigational device 7382.84544
mined exemption and BLA
*From manipulated marrow, peripheral blood, and umbilical cord blood (includes donor lymphocyte infusions).
†
First- or second-degree relative.
‡
As defined by 2005 tissue regulations [21 CFR 1271.3(d)]
§
21 CFR 1271.10(a) as applied to Section 361 (see full rule for details) requires that HPCs be 1) minimally manipulated (biological characteristics unaltered), 2) for homologous
use only (similar function as original tissue), 3) not combined with another article (except water; crystalloids; or sterilizing, preserving, or storage agents with no new safety
concerns), and 4) used in autologous transfusion or in allogeneic transfusion of donations from a first- or second-degree relative.
HPC = hematopoietic progenitor cell, CFR = Code of Federal Regulations, FDA = Food and Drug Administration, PHS = Public Health Service, HCT/Ps = human cells, tissues, and
cellular and tissue-based products, IND = investigational new drug, CBER = Center for Biologics Evaluation and Research, CDRH = Center for Devices and Radiological Health, BLA =
biologics license application.
 CH A P T E R 3 Re g u l a t o r y I s s u es I n B l o o d B a n k i n g
Circular of Information for the Use of
Cellular Therapy Products is jointly
written by the AABB and other
organizations for users of these products.46
The AABB and the Founda- tion for
Accreditation of Cellular Therapy set
voluntary standards and accredit facilities for
collection and processing of HPCs (see Chap-
ters 29 and 30 for information on these stan-
dards and accreditation procedures).
The FDA tissue regulations do not
apply to facilities that receive, store, and
administer tissues but do not perform any
manufacturing steps. However, The Joint
Commission (TJC) has hospital standards
for handling and trac- ing tissues and
investigating adverse events (TS.03.01.01
to 03.03.01) (see Chapter 32 for information
on these standards).47
MANAGING RECALLS AND
WITHDRAWALS
The FDA’s requirements for monitoring and
investigating problems with drugs extend to
the time after a product’s release. When blood
banks discover after distribution that a blood
product was in violation of rules, standards,
specifications, or cGMP regulations, they must
report the problem to the FDA and their con-
signee. These problems comprise a subset of
biological product deviations (BPDs)—events
in which the safety, purity, or potency of a dis-
tributed blood product may be affected—all of
which must be reported to the FDA.48
A recall is defined as the removal or
cor- rection of a marketed product that is in
viola- tion of the law (21 CFR 7.3).49 The
FDA classi- fies recalls by severity. Most
blood component recalls are in Class III,
not likely to cause ad- verse health
consequences. Class II recalls are for
products that may cause temporary ad-
verse effects or remotely possible
serious problems. Class I recalls involve a
reasonable probability of serious or fatal
adverse effects. All recalls are published
by the FDA and in- volve about 1 in 5800
blood components is- sued in the United
States.50
Market withdrawals are required for
products found to be in minor violation of
the law. Problems beyond the control of the
man- ufacturer, such as postdonation donor
infor-
■ 89
mation, are often in this category.
Withdrawals are much more common than
recalls but are not published.
Recommendations for managing
com- mon notices have been published in
the Unit- ed States and Canada.50,51
Transfusion services need to have
procedures and training for rapid responses
as advised when recalls and with- drawals
are received along with records of ac- tions,
reviews, and follow-up actions, as indi-
cated. Accreditation requirements of the
AABB (Standard 7.1) and the College of
American Pa- thologists (CAP) (Checklists
TRM.42120 and TRM.42170) address
aspects of this topic.52,53
Most of these blood components are
transfused before a notice is received of
their nonconformance. In some recent blood
guid- ance documents on infectious
diseases, the FDA has included
recommendations on whether or not to
notify the recipient’s physi- cian about
transfused units. In cases of possi- ble
recent infectious disease exposure in
donors or transfusion recipients, the
serocon- version window periods for tests
should be consulted for scheduling
prospective testing or reviewing
retrospective results, such as for a donor
who has been retested since an expo-
sure.50
The most common reasons for BPDs
and blood component recalls are given in
Table 3-
3. Look-backs investigations on units from do-
nors found after donation to have HIV or hep-
atitis C virus are discussed in Chapter 5.
MEDICAL LABORATORY LAWS
AND REGULATIONS
CMS regulates all US medical laboratories un-
der the Clinical Laboratory Improvement
Amendments [CLIA, 42 USC 263(a) and 42
CFR 493] to Section 353 of the Public Health
Service Act.54,55
The law and regulations establish the
re- quirements and procedures for
laboratories to be certified under CLIA as
both a general re- quirement and a
prerequisite for receiving Medicare and
Medicaid payments.
To be certified, laboratories must
have adequate facilities and equipment,
superviso- ry and technical personnel with
training and
90 ■ AABB T E C HNIC AL MANUAL
TABLE 3-3. Most Common Reasons for Biological Product Deviations and Blood Component
Recalls
Biological Product Deviations (Ranked by number
Blood
of Component
events)
Malaria travel/residence
preparation Variant Creutzfeldt-Jakob disease travel/residence Storage temperature
Postdonation illness
Tattoo
Donor deferral missed via incorrect donor
identification
Biological product deviations shown are from licensed establishments. 48 Blood component recalls are compiled from Food
and Drug Administration Enforcement Reports.50 Market withdrawals are not included in public recalls data. 50
experience appropriate to the complexity of
testing, a quality management system (see
Chapter 1), and successful ongoing perfor-
mance in CMS-approved proficiency testing
(PT).56 All laboratories must register with
CMS, submit to inspection by CMS or its
designees, and obtain recertification every 2
years.
All laboratory tests are rated for
complexi- ty by the FDA for CMS as
waived, moderate complexity, and high
complexity. Waived tests are simple and
easily performed with limited technical
training. Examples include over-the- counter
tests, urinalysis dipsticks, copper sul- fate
specific-gravity hemoglobin screens, spun
microhematocrits, and some simple
devices for measuring hemoglobin.
Laboratories that perform only waived tests
register with CMS for a certificate of
waiver. The Centers for Dis- ease Control
and Prevention provides techni- cal and
advisory support to CMS for laboratory
regulation and has published practice recom-
mendations for waived testing sites.57
Nonwaived tests are classified as being
of moderate or high complexity based on a
scor- ing system of needs for training,
preparation, interpretive judgment, and
other factors (42 CFR 493.17).54 The CDRH
website provides the searchable CLIA
database on the complexity levels of
specific tests.31
Blood banks and transfusion services
have three pathways to obtain a CLIA
certifi-
Recalls (Ranked by number of units)
Collection sterility and arm
Production according to current good manufacturing
practice
Donor suitability
Product quality control
cate to perform testing: 1) certificate of
com- pliance: approval via state health
department inspections using CMS
requirements; 2) certif- icate of
accreditation: approval via a CMS-
approved accrediting organization; and
3) CMS-exempt status: licensure programs
for nonwaived laboratories in New York
and Washington States that are accepted by
CMS.58 The CLIA regulations delineate
general requirements for facilities; quality
systems, in- cluding quality assurance and
quality control systems; and management
and technical per- sonnel qualifications.
High-complexity tests require more
stringent personnel qualifica- tions.
Immunohematology laboratories have
standards for blood supply agreements, com-
patibility testing, blood storage and
alarms, sample retention, positive
identification of blood product
recipients, investigation of transfusion
reactions, and documentation (42 CFR
493.1103 and 493.1271).54 Viral and syphi- lis
serologic tests are part of the immunology
requirements. CMS has published
guidelines
for conducting surveys (inspections).59
CMS has approved six laboratory accredi-
tation organizations with requirements that
meet CMS regulations: the AABB,
American Osteopathic Association, American
Society for Histocompatibility and
Immunogenetics (ASHI), CAP, COLA
(formerly the Commission on Office
Laboratory Accreditation), and The
 CH A P T E R 3 Re g u l a t o r y I s s u es I n B l o o d B a n k i n g ■ 91

Joint Commission. 60 The Joint Commission
has cooperative agreements with ASHI, CAP,
and COLA to accept their laboratory
accredita- tions in facility surveys.61
AABB and CAP can coordinate joint
sur- veys by facilities seeking both types of
accredi- tation. CMS may perform its own
follow-up surveys to validate those of the
accreditation organizations.
CMS requires successful PT for ongoing
laboratory certification of nonwaived testing.
Within each laboratory section, CMS regula-
tions specify tests and procedures (regulated
analytes) that must pass approved PT if the
laboratory performs them. The CMS website
has a list of approved PT providers. 62 PT is dis-
cussed in Chapter 1.
laboratory specimens and transfusion recipi-
ents (NPSG.01.01.01, .01.03.01), checking
blood products in the Universal Protocol pre-
procedure verification process (“time-out”)
(UP.01.01.01), and assessing transfusion
appro- priateness (MS.05.01.01).47 The Joint
Commis- sion includes hemolytic transfusion
reactions in its Sentinel Events reporting
program.64
Facilities also should be familiar with
all relevant state and local laws and
regulations, including professional licensure
requirements for medical and laboratory
personnel. In some situations, facilities
providing products or ser- vices in other
states must comply with local regulations
in the customer’s location.

HOSPITAL REGULATIONS AND

ACCREDITATION
CMS approves hospitals for Medicare reim-
bursement through state surveys or accredita-
tion programs from The Joint Commission, the
American Osteopathic Association, and DNV
Healthcare. These inspections cover CMS
regulations for blood administration and the
evaluation of transfusion reactions [42 CFR
482.23(c)].63 The Joint Commission has stan-
dards for preventing misidentification of
CONCLUSION
Blood components and other blood products
are regulated as pharmaceutical agents, and
blood banks and transfusion services are
also regulated as medical laboratories. These
many requirements make compliance
complex. However, close regulation
underscores the rec- ognized importance to
public health of safe, effective blood
products provided via good laboratory
practices using the quality manage- ment
systems described in Chapter 1 and the
procedures discussed throughout this
manual.

KEY POINTS

The FDA regulates the manufacture and use of drugs and medical devices, including blood components, blood-derived bio
The FDA requires licensure and registration for blood manufacturers engaged in interstate commerce and registration alone
Licensed and registered blood facilities are inspected by the FDA every 2 years, with a focus on current cGMP (21 CFR 21
HPCs for transplantation are regulated as tissues by the FDA, with an emphasis on prevent- ing transmissions of infections
92 ■ AABB T E C HNIC AL MANUAL

5. The FDA requires drug (and blood) manufacturers to conduct recalls or market
withdrawals when noncompliance is found after products are issued, such as for donor
malaria risk or potentially compromised component sterility. Transfusion services
should have procedures for evaluating and managing the potential impact of these
recalls and withdrawals on pa- tients transfused with such products.
6. All US medical laboratories must be approved by CMS every 2 years. Laboratory
regulations emphasize the need for adequate facilities and qualified personnel
commensurate with the complexity of testing, and they require ongoing successful
performance in proficiency test- ing by CMS-approved vendors. Laboratory approval by
CMS is granted via inspections per- formed by CMS-approved accrediting agencies or state
health departments.
7. Health-care facilities also have CMS regulations for their activities, and The Joint
Commis- sion and other organizations accredit many hospitals for CMS compliance.
CMS and The Joint Commission have requirements for monitoring transfusion
practices, evaluating ad- verse transfusion reactions, and preventing mistransfusions.
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32. Code of federal regulations. Title 21, CFR Part
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ing Office, 2014 (revised annually).
94 ■ AABB T E C HNIC AL MANUAL
33. Tissues and tissue products. Silver Spring,
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34. Halme DG, Kessler DA. Regulation of stem-
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35. Guidance for industry: Regulation of human
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36. Guidance for industry: Current good tissue
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37. 7341.002—Inspection of human cells, tissues,
and cellular and tissue-based products (HCT/
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38. Compliance programs (CBER). Silver Spring,
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39. 7341.002A—Inspection of tissue establish-
ments. (December 1, 2007) Silver Spring, MD:
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41. Guidance for industry: BLA for minimally
ma- nipulated, unrelated allogeneic
placental/um- bilical cord blood intended for
hematopoietic and immunologic
reconstitution in patients with disorders
affecting the hematopoietic system. (March
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Communication, Outreach, and De-
velopment, 2009. [Available at http://www.
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42. Guidance for industry and FDA staff: IND ap-
plications for minimally manipulated, unrelat-
ed allogeneic placental/umbilical cord blood
intended for hematopoietic and immunologic
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fecting the hematopoietic system. (March
2014) Silver Spring, MD: CBER Office of
Com- munication, Outreach, and
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ucts. (October 1, 2010) Silver Spring, MD:
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vember 16, 2013).]
44. Inspection of medical device manufacturers.
Program 7382.845. (February 2, 2011) Silver
Spring, MD: Food and Drug Administration,
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calDevices/DeviceRegulationandGuidance/
GuidanceDocuments/ucm072753.htm (ac-
cessed November 16, 2013).]
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45. Testing HCT/P donors for relevant communi-
cable disease agents and diseases. Silver
Spring, MD: CBER Office of Communication,
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cines/SafetyAvailability/Tissue Safety/ucm
095440.htm (accessed November 8, 2013).]
46. AABB, America’s Blood Centers, American
As- sociation of Tissue Banks, American
Red Cross, American Society for Apheresis,
Ameri- can Society for Blood and Marrow
Transplan- tation, College of American
Pathologists, Foundation for the
Accreditation of Cellular Therapy,
ICCBBA, International Society for Cellular
Therapy, Joint Accreditation Commit- tee,
National Marrow Donor Program, and
Netcord. Circular of information for the use
of cellular therapy products. Bethesda,
MD: AABB, 2013. [Available at
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8, 2013).]
47. 2012 Comprehensive accreditation manual for
hospitals: The official handbook (CAMH).
Oakbrook Terrace, IL: The Joint Commission,
2012.
48. Biological product deviations. Silver Spring,
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SafetyAvailability/ReportaProblem/Biologi
calProductDeviations/default.htm (accessed
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49. Code of federal regulations. Title 21, CFR Part
7.3. Washington, DC: US Government
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50. Ramsey G. Blood component recalls and mar-
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51. Recommendations for the notification of re-
cipients of a blood component recall.
(March 21, 2012) St. John’s, NL and
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sources/guidelines/recall-recipient-notifica
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52. Levitt J, ed. Standards for blood banks and
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AABB, 2014.
53. College of American Pathologists Laboratory
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(re- vised annually).
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54. Code of federal regulations. Title 42, CFR Part
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ing Office, 2013 (revised annually).
55. United States code. Title 42, USC Part 263a.
56. Rauch CA, Nichols JH. Laboratory accredita-
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845-58.
57. Howerton D, Anderson N, Bosse D, et al.
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guidelines for laboratories and laboratory ser-
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under CLIA. Baltimore, MD: Centers for
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cessed November 18, 2013).]
 C h a p t e r 4

Disaster Management

Ruth D. Sylvester, MS, MT(ASCP)SBB;

William FitzGerald, LTC USA (Ret);
Wendy Trivisonno; and Theresa Wiegmann, JD
MU CH H AS B E E N learned over the
past decade about preparing for and re-
sponding to disasters. A disaster is defined
by the United Nations International Strategy
for Disaster Reduction as a serious event
that dis- rupts the normal functioning of a
community or society and that exceeds the
affected area’s ability to cope.1 Disasters can
be small, only affecting a single business, or
large, encom- passing an entire region.
Whether a disaster involves natural forces
(eg, hurricanes, earth- quakes, floods, or
pandemic influenza) or is the result of
human activity (eg, terrorism or industrial
accidents), organizations around the world
have put forth tremendous resourc- es to
counteract and mitigate the threats relat-
ed to these events.
The goal of this chapter is to provide
an overview of disaster management,
which en- compasses much more than just
preparing for a disaster. This chapter
addresses the entire cycle of disaster
management, including elements
often overlooked in traditional disaster
plans. This chapter is more strategic than
procedural. Many of the specific and
tactical information and procedures
mentioned in this chapter are described in
detail in the AABB Disaster Opera- tions
Handbook, which should be considered a
complementary text to this chapter.2
Even though this chapter focuses
primar- ily on emergency management
strategies used in the United States, many of
the methods can also be adapted for use in
other countries. Hospital-based blood
banks and transfusion services should note
that many of the activities discussed in this
chapter are intended for stand-alone
organizations (eg, blood collec- tion
centers) and may already be addressed in a
hospital’s overall disaster plan. However,
hospital staff members should review both
their hospital and departmental disaster
plans to ensure that blood-related issues
(including those pertaining to cellular and
related biolog- ic therapies) are sufficiently
covered.

Ruth D Sylvester, MS, MT(ASCP)SBB, Director, Regulatory Services, America’s Blood Centers,
Washington, District of Columbia; William FitzGerald, LTC USA (Ret), Senior Advisor, Biomedical
Preparedness, American Red Cross, Washington, District of Columbia; Wendy Trivisonno, Director,
Strategic Biologic Sourcing and Logistics, Blood Centers of America, West Warwick, Rhode Island; and
Theresa Wiegmann, JD, Director of Public Policy, AABB, Bethesda, Maryland
The authors have disclosed no conflicts of interest.

97
98 ■ AABB T E C HNIC AL MANUAL
BACKGROUND
Disaster Management Cycle
Disaster management includes plans for, re-
sponses to, and recovery from disasters.
Plan- ning by its nature is cyclic and, as with
many activities, future responses to disasters
can be improved by learning from past
events. The di- saster management cycle
includes four func- tional areas: mitigation,
preparedness, re- sponse, and recovery.3
Organizations should consider each of these
areas when creating an effective disaster
management program.
■ Mitigation efforts focus on making perma-
nent changes to plants and property to re-
duce the overall exposure to various known
hazards. Mitigation strategies typically in-
volve the “where” and “how” of building
physical structures and protecting employ-
ees and assets (eg, building code require-
ments, flood plain analysis, and storm shel-
ters). Mitigation techniques also involve
taking relatively minor steps to reduce loss
or injury (eg, fastening bookshelves or file
cabinets to a wall).
■ Preparedness focuses on areas of risk that
cannot be addressed through mitigation ef-
forts alone. Emergencies are dynamic and
complex events, and careful preparation ef-
forts are needed to reduce the loss of life
and property while sustaining business
opera- tions. Preparation efforts begin with
a thor- ough risk analysis of all known
hazards (both natural and human) that
could affect an or- ganization (eg, fires,
severe storms, earth- quakes, pandemics,
and terrorism). Based on the risk
assessment, planning efforts should focus
on the people and organiza- tional systems
that are most likely to be af- fected.
Effective preparation involves a con- tinual
improvement process whereby the disaster
plan is routinely tested by a series of real or
simulated events (drills) through which
gaps are identified and remedied.
■ Response takes place during an
emergency and typically involves time-
sensitive action steps taken by the staff to
protect life and property and to stabilize
the organization (eg, communication with
staff, evacuation
procedures, and responses to customers
and the media). Effective response strate-
gies center on clearly outlined processes
or procedures that can be practiced
during drills and be implemented at any
time of the day or night (eg, during night
and week- end shifts). These strategies
are also based on well-defined lines of
authority.
■ Recovery efforts begin once the initial re-
sponse actions have taken place. These ef-
forts focus on restoring critical systems (eg,
communications, water, power, and
sewage) to resume and maintain business
opera- tions. Recovery efforts complete the
cycle of disaster management by providing
insights into additional mitigation strategies
that can be deployed for future
emergencies.
Risk Assessment
The cornerstone of an effective disaster man-
agement program is a thorough assessment of
the known hazards that can affect the organi-
zation. Risk can be defined as the potential
losses (physical, economic, and social) associ-
ated with a hazard and can be described in
terms of expected probability and frequency,
causative factors, and locations or areas affect-
ed.4 The goal of risk assessment is to identify
and reduce the number of potential risks asso-
ciated with a hazard given its probability of
oc- currence and scope. Potential hazards can
be grouped in different ways, such as natural
vs man-made or internal vs external to an
organi- zation. Using the latter categorization,
hazards that are generated externally include
natural events, such as earthquakes, floods,
wildfires, pandemics, and hurricanes, as well
as human events, such as terrorism or
industrial acci- dents, including chemical,
hazardous materi- al, and nuclear power plant
emergencies.
Another class of potential hazards to con-
sider involves events that may occur
internal- ly at the organization. Examples
of internal hazards include internal
flooding caused by burst water pipes or
fire sprinkler malfunc- tions, fires, natural
gas leaks, workplace vio- lence, and
hazardous spills. Each of these haz- ards
may involve service disruptions and
evacuations of staff, donors, and patients.
An
 CH APT E R 4 Disaster Management ■ 99

example of a question to answer in a risk operations at different levels of government

as- sessment of internal disasters is, what
is the potential risk to stored blood
components and patients if the hospital’s
blood bank becomes flooded or must be
relocated because of smoke damage from
a fire?
There are many approaches to conduct-
ing an effective risk assessment. This text
does not provide guidance on a specific risk
assess- ment process. However, a sample
risk assess- ment chart is provided in
Table 4-1, which highlights the major
functional areas and haz- ards to consider.
For instance, a facility located in an area of
seismic activity would list earth- quakes as
a potential external hazard and would
need to determine the immediate ef- fect of
an earthquake on its operational status (to
humans, property, and business) and the
resources needed to recover and restore
oper- ations. Conversely, an organization
located far from hurricane and flood zones
would reflect that situation in its risk
assessment chart. The AABB Disaster
Operations Handbook contains a list of
common hazards, including impact factors
as well as preparedness and response
strategies.2
Emergency Operations in the
United States
It is important for staff members to under-
stand the relationships between emergency
and an organization’s disaster management
program. An understanding of the entire spec-
trum of emergency operations allows an orga-
nization to communicate effectively when
working with emergency management profes-
sionals during a disaster event or a drill.
Disasters are inherently local events
re- quiring citizens, local organizations, and
first responders to work together. Key to
effective emergency response is the
integrated plan- ning for disasters that
starts at the national level and extends to
the local emergency man- agement agency
(EMA). State and national support systems
are available to assist if a local municipality
is overwhelmed by a disaster and with the
development of local response plans and
training. These national and local systems
include the following:
■ National Incident Management System
(NIMS). The NIMS is a nationally
accepted system for helping responders
from all lev- els of government, various
jurisdictions, and the private sector to
communicate and coordinate their
response efforts.5 The NIMS uses a
unified approach for incident
management that offers standard com-
mand and management structures and
em- phasizes preparedness, mutual aid,
and re- source management.
Organizations should

TABLE 4-1. Risk Assessment Chart with Sample Data4

Recovery
Probability of Human Property Business Resources
Occurrence Effect Effect Effect Needed Total
High Low High Low High Low High Low High Low
51 51 51 51 51
External Hazards
Earthquake 5 3 4 5 4 21
Hurricane 1 1 1 2 2 7
Internal Hazards
Flooding 4 1 4 3 2 14
Workplace violence 2 5 2 4 1 14
100 ■ AABB T EC HNIC AL MANUAL
be familiar with NIMS protocols so that
they are prepared to communicate effec-
tively with responding organizations [eg,
police and fire departments and the
Federal Emergency Management Agency
(FEMA)]. Online training courses are
available on the FEMA website.6
■ National Response Framework (NRF). The
NRF is a federally operated all-hazards re-
sponse plan that is activated in response to
a presidentially declared disaster under the
Robert T. Stafford Disaster Relief and
Emer- gency Assistance Act (amended June
2006).7,8 Under this act, the federal govern-
ment, coordinated by the Department of
Homeland Security (DHS), provides per-
sonnel, assets, and financial support to
state and local governments that are over-
whelmed during a major disaster. The need
for blood is covered under the medical and
public health section of the NRF (Emergen-
cy Support Function No. 8).8(p33),9 Blood col-
lection and hospital facilities should be fa-
miliar with the NRF to request and receive
assistance during major emergencies.
■ National Terrorism Advisory System
(NTAS). The DHS assesses threats posed
by terror- ists and communicates those
threats to the US public, government
agencies, and the private sector through
the NTAS alerts.10 This system replaces
the color-coded Homeland Security
Advisory System (HSAS) used in the
past. The NTAS uses only two tiers of
alerts:
– Imminent Threat Alerts warn of
“credi- ble, specific, and impending
terrorist threat[s] against the United
States.”
– Elevated Threat Alerts warn of
credible, but not specific, threats
against the Unit- ed States.
NTAS alerts are brief, clearly articulating
what can be done to prevent and even, if
possible, mitigate a threat’s impact and
re- spond if necessary. Unlike the
previously used HSAS that issued
ongoing warnings, NTAS alerts are
issued for a specific period and expire
once that period has ended.
■ National Disaster Medical System (NDMS).
The NDMS is a cooperative asset-sharing
program directed by the Department of
Health and Human Services (DHHS).
The NDMS augments local medical care
when an emergency exceeds the scope of
a com- munity’s health-care system. The
NDMS consists of more than 9000
medical and support personnel from
federal, state, and local governments and
the private sector as well as civilian
volunteers.11
■ AABB Interorganizational Task Force on
Domestic Disasters and Acts of Terrorism
(AABB Disaster Task Force). The AABB
Disaster Task Force provides support to
blood collection and transfusion facilities
during major emergencies, including dur-
ing NRF activation under the Stafford Act.
Facilities should review the AABB Disaster
Task Force response plan and integrate its
response processes into their organization-
al disaster plans.2
■ EMAs. EMAs are local, tribal, state, and fed-
eral agencies tasked with helping
individu- als and organizations deal with
all cycles of disaster management. These
agencies are staffed by both full-time and
volunteer per- sonnel (emergency
managers) and use standard methods
when responding to a disaster.11
Major Disaster Management Factors
for Blood and Transfusion
In conjunction with comprehensive disaster
planning strategies, blood collection and
hos- pital facilities should consider the
disaster management factors, described in
this section, that are key for blood and blood
components during all phases from
collection to transfu- sion.12-16
Most of the blood and blood
components used for transfusion in the
United States are collected, processed,
tested, and stored at re- gional blood centers
and must be delivered to hospitals before
transfusion. During an emer- gency, this
“just-in-time” delivery system re- sults in
the need for close coordination be- tween
blood centers and the hospitals they serve.
This involves robust communication and
information systems, transportation and
logistics support, and critical utilities, such
as fuel and electrical power, to ensure that
blood
 C H A P TE R 4 Disaster Management ■ 101
Disas- ter Task Force. However, delivering blood from

can be transported and stored at required

tem- peratures.
Emergency management personnel
are often unaware of issues related to the
collec- tion, processing, storage, and
distribution of blood and blood
components and may as- sume that blood
is collected and stored direct- ly in hospitals.
As a result, blood-related issues (eg, the
need for transportation and logistics
support) are often overlooked during real
and simulated disasters. A continuing
process of education for emergency
managers at both the local and national
levels is imperative to in- crease their
awareness of issues related to blood.
Blood centers should pursue active par-
ticipation in EMA planning activities
and participate in activities of the
emergency oper- ations center to ensure
continuous represen- tation of blood-related
issues.
Historically, an average of about 200
units of blood are needed by victims of most
disas- ters. However, the public response to
disasters has traditionally resulted in
increases in blood donations regardless of
the true medical need for transfusions. A
sudden increase in unneed- ed blood
donations can disrupt both local and
national blood supplies. Efforts should be
made to establish the medical need for blood
during a disaster and communicate this need
to national blood organizations and the
AABB Disaster Task Force, blood donors,
and the public through a clear and
consistent messag- ing strategy.12-16
In disasters resulting in injuries that
do require blood transfusions to treat
victims, the available local blood inventory
is the primary source for the initial
treatments. Because blood collected in
response to a disaster takes 24 to 48 hours to
process, it is critical for the lo- cal blood
supply to remain at ample levels to treat
victims of potential hazards as defined in the
organizational risk assessment. The AABB
Disaster Task Force recommends
maintaining a 5- to 7-day supply [combined
inventory at the blood collector and
hospital(s) it serves] to address potential
disasters.2 Efforts are made to resupply the
affected facilities if needed from
neighboring blood collection centers or
through national support by the AABB
 System Pre- paredness Program has
developed an exten- sive set of materials to
outside the immediate help hospitals prepare to respond to
vicinity to the affected disasters including “When Healthcare
hospital might take 12 to 24 Resources are Scarce,” a set of guidelines
hours. to assist health-care facilities in planning
Disasters resulting from for events that would overwhelm the
the use of biolog- ic, chemical, system’s resources, including blood and
radiologic, or nuclear agents blood components.19, 20
may result in widespread donor Finally, certain disasters may affect a
deferrals and the potential blood collector’s ability to collect and
quarantine of blood process blood during the entire event, thus
components al- ready in the straining the local blood supply for days or
manufacturing process or weeks. For instance, as a hurricane
storage until the nature and approaches, a blood collector may suspend
effect of the agent can be collections for a few days before and after
determined. Radiologic-nuclear the storm has passed,
attacks also would dramatically
increase the demand for
platelets, other blood
components, and stem cells for
several weeks following an
event. In addition, in the event
of an influenza pandem- ic,
blood supplies may be severely
strained for weeks due to high
staff absenteeism and donor
shortages as these individuals
become ill, need to stay home
to take care of loved ones, or
fear exposure to influenza in
public places. An in- fluenza
pandemic could also severely
affect the availability of needed
supplies as manu- facturers
face similar absenteeism and
trans- portation difficulties.17,18
Hospitals and transfusion services should
develop blood shortage
management plans to
maximize the probability of
optimal blood dis- tribution
and use during a severe
shortage. It is critical that
detailed blood triage plans
devel- oped by hospital
transfusion services, man-
agement staff, and
physicians who make
transfusion decisions be
available and be reg- ularly
reviewed in advance of a
severe disaster. Blood centers
should work with their
hospital customers to ensure
that they have triage plans
in place. The Minnesota
Department of Health’s
Minnesota Healthcare
102 ■ AABB T EC HNIC AL MANUAL
resulting in a significant loss of expected
col- lections. Elective surgeries typically
are post- poned during this period, helping
alleviate the strain on supply. However,
lessons learned from previous disasters
indicate that blood collection and
transfusion facilities should prepare for
the potential acute shortage of blood
components in the days following a hur-
ricane or disaster of similar magnitude,
paying special attention to the supply of
platelets. Fa- cilities should augment
supplies before a pre- dictable hazard (eg, a
hurricane or severe win- ter storm) and
contact the AABB Disaster Task Force for
support if routine supply channels are
inadequate to bolster supplies before or af-
ter the event.
BUSINESS OPERATIONS
PLANNING
Business operations planning is generally a
three-step process: assembling a planning
team; analyzing the current situation, includ-
ing hazards and capabilities; and developing
a plan to continue operations in an
emergency. Once planning has been
completed, imple- mentation of the plan
(which includes train- ing, testing,
evaluating, and revising the plan) can
proceed.21,22
Disaster Planning Team
The first step in disaster planning is to identify
the person or team of people in an organiza-
tion who are responsible for emergency opera-
tions planning. The number of people in-
volved will depend on the size and complexity
of the operation. Assistance will be needed
from key people, such as the organization’s ac-
countant, attorney, human resources staff, and
insurance agent. In addition, establishing a re-
lationship with local EMAs can be vital to the
success of a disaster plan in an actual emer-
gency.
Once the responsible person has been
identified or the team has been formed, a
proj- ect plan should be developed to
identify the key steps and a timeline for
completing the di- saster plan. Business
continuity planning has proliferated over the
past 5 years as organiza-
tions have seen human and natural disasters
wreak havoc in the United States and
abroad. References, training materials, and
toolkits with step-by-step instructions and
templates are widely available on the
Internet and should be acquired and used
when developing a di- saster plan.21-25
Analysis of Current Situation
The success of disaster planning depends on
a risk analysis of the current situation (see
sec- tion on “Risk Assessment”). This
assessment includes determining the hazards
to which an organization is most vulnerable,
the probabili- ty of each hazard, and the
anticipated effect of each event on human
and physical resources and on business
operations.4,21 Effective pre- vention
strategies can be used for risks such as fires,
power interruptions, facility security
breaches, and workplace violence. What
can- not be prevented should be mitigated.
For ex- ample, conducting essential
operations away from floodplains, having
multiple sources (but not necessarily
multiple suppliers) of critical supplies, and
relocating vital records (includ- ing critical
information technology resources) to upper
floors in flood-prone areas are all mit-
igation strategies. Plans should be developed
for events that have a high likelihood of
occur- ring as well as low-probability events
with po- tentially catastrophic consequences
(eg, Cate- gory 5 hurricanes or pandemic
influenza).
Continuity of Operations Plan
A Continuity of Operations Plan (COOP) is
designed to ensure continued operation of
essential functions in the event of an
emergen- cy or disaster.21,25 A COOP should
cover the emergencies that are most likely to
occur or that could have a significant effect.
A COOP should address at least the areas
identified in Table 4-2. These elements are
discussed in this section.
Hospital-based blood banks and transfu-
sion services may be covered by the
overall hospital or laboratory COOP;
however, the var- ious COOPs should be
reviewed to ensure that issues specific to
blood components and transfusion are
sufficiently covered. Such
 C H A P TE R 4 Disaster Management ■ 103

TABLE 4-2. Essential Elements of a Continuity of Operations Plan19,20,23

1. Identify essential processes and functions required for continued operations.
2. Develop decision trees for implementing the Continuity of Operations Plan.
3. Consider alternate facility options.
4. Establish safety and security plans for staff facilities and fleet vehicles.
5. Identify and ensure protection and survivability of vital records and databases.
6. Review insurance coverage and ensure that it is adequate for potential risks.
7. Establish minimum cash reserves necessary to continue operations for 90 days.
8. Develop an emergency communications plan.
9. Establish a chain of command and an order of succession for decision-making authority.
10. Develop a plan for dealing with the news media.
11. Identify designated spokespersons, and train them to handle risk and emergency communications.
12. Plan to maintain supplies and logistical support for operations.
13. Evaluate utility needs and develop contracts or memoranda of agreement to ensure replenishment
and restoration of essential utilities.
14. Review information technology systems and develop redundancies to ensure that vital systems and
their supporting subsystems remain operational during an emergency.

15. Identify staffing issues, including essential personnel and key contacts necessary to carry out
essential functions as well as employee compensation and benefits during and after the
disaster.
16. Develop procedures for transitioning back to normal operations after a disaster.

plans are required by the AABB in its COOP Decision Trees

Standards for Blood Banks and Transfusion
Services as well as by The Joint
Commission.26,27
Essential Functions
Essential functions are those that must be
car- ried out to ensure business continuity,
such as the storage of blood and blood
components and their transportation and
delivery to hospi- tals. Even though blood
collections, compo- nent preparation, and
testing may be inter- rupted, the continued
viability of a blood center depends on its
ability to provide hospi- tal customers with
blood. Blood can be im- ported from outside
the affected area to en- sure an adequate
inventory until collections can resume.
Likewise, hospitals must be cer- tain that
they can receive, store, and transfuse blood
to patients in need.
COOP operations and decisions are best
thought out in advance during normal opera-
tions. Flowcharts and decision trees that list
options can be developed to guide leaders
during emergency operations. Such flow
charts and decision trees can be developed
by groups and refined using lessons learned
from exercises and real-world events. An
example of a decision tree developed to
support blood bank operations by the
California Blood Bank Society is available
on the Internet.28
Alternate Facilities
Alternate facilities that can be used to
contin- ue operations should be identified
during the planning phase of a COOP.
Thought should be given to the need for
equipment, supplies, in- formation
technology support, and other
104 ■ AABB T EC HNIC AL MANUAL
items at the alternate location. Consideration
should also be given to practical locations
for alternate facilities. For example, an
alternate facility across the street will most
likely be sim- ilarly affected by an external
disaster. If fund- ing is not available to pay
for an alternate facility, the organization
should consider es- tablishing formal
agreements with other orga- nizations in the
area to provide alternate space if one facility
is incapacitated.
Security and Safety
In a disaster, security of critical resources—
ranging from the facility and fleet to staff and
information systems—can become a major
concern.22 For example, crowd control may be-
come an issue if large numbers of donors gath-
er at collection locations or in the event of
special screening requirements during a pan-
demic influenza outbreak. In natural disasters
in which fuel shortages occur, the security of
fuel supplies can become a major challenge. A
COOP should include the measures that will
be necessary to ensure the security of all criti-
cal resources.
Planners should review their normal op-
erations security plan and determine whether
the plan adequately addresses potential
threats and hazards to the local area. Coordi-
nation with EMA officials can provide
infor- mation on threat or risk assessments.
Planners should consider the
implementation of mea- sures to increase
the security of their facilities, staff,
volunteers, and donors during changes in
the NTAS.10
Lastly, planners should ensure that the
fa-
cility complies with all local building codes
and applicable Occupational Safety and
Health Administration (OSHA) regulations.
The actions implemented should be dis-
cussed with the local EMA and the insurance
company, and any suggestions that they pro-
vide should be addressed.22
Vital Records
The protection of vital records is especially
critical in an industry that depends on
records
to document the safety and availability of
blood components. In addition to donor rec-
ords, the organizational human resource
files, legal records, payroll records, and
financial records (such as accounts
receivable and ac- counts payable) are
critical to a business’s sur- vivability during
a disaster.22,29 Records of in- surance policies,
bank account numbers, and supplies of
blank checks must be available during
emergency operations to ensure conti- nuity
of operations. Corporation records, stra-
tegic plans, and research-related records
must also be maintained. Redundancy of
computer- ized records should be
established by periodi- cally by backing
records up on a duplicate server. Geographic
considerations must be taken into account in
identifying off-site stor- age locations. Not
all records are maintained electronically, so
provisions to safely keep pa- per copies of
records must be considered, es- pecially if
alternate facility operations are im-
plemented.
Insurance
Adequate insurance is essential to the
surviv- ability of any business. The two
major types of insurance coverage for blood
collection and transfusion facilities are
property and liability. Key provisions of
these policies include valua- tions of
property; perils covered, such as flood- ing,
wind, and power interruptions; deduct-
ibles; and how to file claims. Business
interruption insurance can cover loss of in-
come. Additional background information
on relevant insurance policies is available
from a variety of resources, including the
Small Busi- ness Administration and
insurance compa- nies.24,30
Cash Reserves
The single most crucial element for the
surviv- ability of a business during a disaster
is access to cash that can provide support
until normal operations can be resumed.31,32
Cash reserves equal to several months’ worth
of operational expenses should be accrued
during normal operations.
 C H A P TE R 4 Disaster Management ■ 105
through

Emergency Communications Plan

Clear, quick, and effective communication is
vital to protecting life and property during a
disaster, and communication systems are of-
ten the first assets to be strained during an
emergency. Telecommunication systems can
quickly become overloaded or jammed;
com- puter systems may be disrupted
because of a loss of power; and the staff
may become sepa- rated, resulting in general
confusion and the inability to make rapid
decisions during and after a disaster.
Organizations should invest heavily in
ensuring that the staff can commu- nicate
both internally and externally and should
develop an Emergency Communica- tions
Plan (ECP). No single definition or all-en-
compassing set of procedures exists for an
ECP, but organizations should consider the
fol- lowing areas when designing one.

IDENT I FYING INT E RNAL AND EXTERNAL

AUDIE N CES FOR THE EC P. Blood
collection and hospital facilities should
identify and map the essential internal
departments and per- sonnel that need to
communicate both during a disaster
(response) and after a disaster (re- covery).
All employees need to be included in the
ECP because all employees will need to re-
ceive information regarding the response
pro- cedures and the operational status of
the facil- ity (eg, when to report back to
work).
External audiences need to be
identified and assessed for their ability to
communicate with an organization (eg,
vendors must have an ECP for
communications with an organiza- tion).
For instance, a hospital blood bank or
transfusion service should identify all
essential departments and personnel at its
blood sup- plier(s). Conversely, blood
centers should identify the key
departments and personnel at hospitals or
the testing providers that need to be
contacted during and after an emergency.

KEY CO NTACTS . Key lists should be main-

tained and periodically updated of all of the
critical internal and external personnel, de-
partments, and organizations that need to
dis- seminate or receive information during
an emergency. The contact lists should be
readily available both electronically (eg,
 primary authority, clearly defined lines of
decision- making authority, delegation, and
smart phone applications) communi- cation should be established
and in print (eg, on laminated among the lead- ership team and employees.
cards) for emergency
ST RATE GIE S FO R CO M M UNICAT I O N RE -
response per- sonnel to use
DUNDANCY AND CO M PAT IBILIT Y. A cr
during a disaster. iti-
Internal contact lists cal challenge for organizations during real
should include es- sential or simulated disasters is the ability (or
personnel and key leaders. inability) to communicate using routine
External contact lists should methods. These methods quickly become
include customers, suppli- overloaded, requiring organizations to use
ers, and vendors of critical alternate com- munication methods. It is
supplies; the AABB Disaster vital to identify and test these redundant
Task Force; personnel at state communication chan- nels before a
and lo- cal EMAs; and other disaster because significant
key business resources, such
as insurance agents, utility
service repre- sentatives,
legal representatives, and
banking personnel. Facilities
should consider collect- ing
redundant contact
information for each person
in their contact lists (ie,
business, mo- bile, and home
telephone numbers; e-mail
ad- dresses; and text
messaging number). For or-
ganizational contacts (eg,
blood bag suppliers, delivery
services, and electrical utility
provid- ers), facilities should
obtain alternate direct
(“back door”) contact
information to bypass
standard automated
answering systems dur- ing
an emergency.
Human resources staff and legal counsel
should be consulted to ensure
that they sup- port the
collection of staff contact
informa- tion. In some cases,
the staff may be unwilling to
provide personal contact
information due to privacy
concerns.
CHAIN OF CO MM AND.
Leadership is critical for
maintaining control and
direction during a disaster.
Organizations should clearly
define and communicate to
all personnel and exter- nal
partners who will be (or who
are) in charge of a given
event.4(p6) In addition to a
106 ■ AABB T EC HNIC AL MANUAL
delays can occur in switching to alternate
methods during an event, resulting in the po-
tential loss of life and property.
No single method is used by organiza-
tions to design communications redundancy.
Local and state jurisdictions use different
types of emergency communications
equip- ment (eg, fire and police personnel
use radios) and have different local
telecommunications infrastructures (eg, cell
towers) and capacities. Organizations should
acquire multiple redun- dant
communications technologies and iden- tify
the order in which they should be used
during an emergency. Alternative
communica- tion technologies are
described in the AABB Disaster
Handbook.2
COMMUNICATION WITH LOCAL, STATE, AND
NAT I ONAL E M ERG E NCY RES P ONS E AG
EN -
CIES. In addition to communicating with
routine suppliers and vendors, facilities
should establish and maintain relationships
with local, state, tribal, and national
emergen- cy response organizations as
shown in Fig 4-1 (see also section on
Participating in Disaster Drills with EMAs).
In particular, blood collection and trans-
fusion facilities should identify agencies that
FIGURE 4-1. Communication with external agencies.
EMAs = emergency management agencies.
can assist with obtaining 1) transportation
support for components, critical supplies,
and staff; 2) power restoration; 3) fuel for
backup generators; 4) fuel for fleet and
essential staff vehicles; 5) communication
support; 6) securi- ty if staff or property is
threatened; and 7) oth- er utilities support
(eg, telecommunications and Internet).
Facilities should educate these agencies on
how the blood supply operates both
locally and nationally (eg, the use of
“just-in-time” delivery methods and the
need to deliver blood from regional blood
centers to hospitals) and should request
that they be deemed a critical health entity
or critical infra- structure entity in the
emergency response structure.8
Blood centers and hospitals should work
with the local EMA to ensure their inclusion
in local emergency communications
systems. These systems may include
ongoing videocon- ferences or conference
calls, blast e-mail lists, and 800-MHz radio
networks. Furthermore, blood centers
should seek admission to any pri- ority
networks established by local hospitals to
improve communications during an incident.
Periodically, management should
pro- vide informational briefings to
members of the EMA and the public health
department about
 C H A P TE R 4
the mission of the organization, the scope
of its operations, and the effect on local
health- care facilities if the blood center or
hospital is not operational. Management
should also be- come familiar with the
NIMS and NRF docu- ments and should
consider enrolling in FEMA online
educational courses30 or disaster train- ing
courses provided by the state or local com-
munity. Management should request that the
EMA provide informational briefings to
key staff of the blood center or hospital.
During ex- changes with the EMA,
management should discuss resource issues
(eg, transportation as- sistance, fuel support,
storage, security, inclu- sion in EMA
notification systems, and assign- ment of a
blood center or hospital liaison to the
EMA). Management should invite the EMA to
participate in its disaster exercises and ac-
tively encourage the EMA to include the
blood center or hospital in its disaster
exercise pro- grams.
National assistance during disasters (eg,
coordination of national media messages
and resupply of blood components to
affected fa- cilities) is provided through the
AABB Disaster Task Force in coordination
with other national and federal
organizations. Along with local planning
efforts, blood collection and transfu- sion
facilities are encouraged to integrate the
task force response system into their
disaster plans. The response system is
described in the AABB Disaster Operations
Handbook on the AABB website.2
WO RKING WITH PU BLIC M E DIA. Creating
and disseminating a clear and consistent
mes- sage about the status of the local and
national blood supply is a critical
component of an ECP.12-16 Unnecessary
donor surges can be pre- vented, or donations
of specific blood compo- nents (eg, group O
red cells or platelets) can be requested by
working with public media out- lets to
effectively convey messages about the
status of the blood supply following a
disaster. The AABB Disaster Task Force
has developed media-related resources for
facilities, includ- ing boilerplate press
releases that are available in the AABB
Disaster Operations Handbook.2
Only individuals who have received
train- ing in risk and emergency
communication
Disaster Management ■ 107
should speak with members of the media.
Spe- cific activities should take place
before, dur- ing, and after each interview.33
For instance, written background
information should be provided to a
reporter before an interview, and a
spokesperson should focus the most salient
points of the message into “sound bites”
that can be quoted in a story. Another useful
tech- nique when communicating with the
media is message mapping.34 Message maps
help com- municators develop and
synthesize complex information by
identifying three key messages to be
delivered in three short sentences with a
total of 27 words. This process
accommodates both broadcast and print
media. Most impor- tant, a spokesperson
should never speak off the record with a
reporter. Many other tips and techniques can
be used to ensure that the cor- rect messages
are conveyed. One resource for such tips is
the Northwest Center for Public Health
Practice in collaboration with Public
Health—Seattle and King County.35
Logistics
During the development of a COOP, facility
planners must place heavy emphasis on the
management of daily operations, including
making available the supplies and
equipment required to perform essential
functions related to 1) recruiting of donors,
2) collecting donated blood, 3)
manufacturing and testing blood
components, and 4) delivering the
components to the hospital for transfusion to
patients. Dur- ing a disaster, the normal
logistical systems may not be available or
capable of providing the necessary support.
Key logistical issues to address in a COOP
are described below.
TRANSPORTATI ON. Past disaster and
terror- ist events have clearly demonstrated
the vul- nerability of transportation
systems. Blood centers should coordinate
with their local EMAs to ensure that
vehicles engaged in col- lecting or
distributing blood are duly autho- rized as
emergency support vehicles and are granted
the appropriate clearances to operate on the
roads. In addition, memoranda of un-
derstanding, agreements, or statements of
un- derstanding should be established with
the
108 ■ AABB T EC HNIC AL MANUAL
appropriate EMAs regarding access to
the roads and use of law enforcement,
National Guard, and state militia personnel
or vehicles to deliver blood. Under the
provisions of the NRF, DHHS can ask the
Department of Trans- portation to assist with
the movement of blood by air, rail, water,
and motor vehicle.8 Such re- quests should
be directed to the AABB Disaster Task
Force.2
“ J U S T- IN-TIM E” S Y S T EM S. M a ny blood
centers and hospitals have adopted just-in-
time supply systems to minimize expenses
as- sociated with the storage of large
quantities of supplies, maintenance of
storage facilities, staffing, and outdated
products. Blood centers and hospitals
should determine the critical items
required for their business operations, the
vendor’s resupply capability (including
manufacturing schedule and transport time-
lines), the shelf life of each supply item,
and any storage requirements (eg, special
environ- mental factors).36
STORAG E . Planners should consider the
stor- age capacity of their customers and
identify lo- cal storage companies that
could provide an emergency storage site.
Contingency contracts or contingency
clauses in existing contracts with
customers and ice and dry-ice suppliers
should be considered. Temporary storage
space may be provided by commercial
con- tainers, including prevalidated
refrigerated containers that could be placed
at the blood center, depending on space
and electrical power. The local EMA may
permit storage of consumables at municipal
disaster supply fa- cilities at no cost.
CO NT RACTS . All vendor contracts
should identify the process for emergency
deliveries of supplies, equipment, and
services. The ven- dor or supplier should
have a disaster plan that demonstrates the
ability to meet a facili- ty’s supply
requirements in a disaster. It is ad- visable
to require vendors to have disaster plans
for their operations. Facilities might also
develop contracts containing noncompete
agreements with local competitor
organiza- tions for implementation during
disasters.
BIOHAZ AR DS. In the aftermath of a
disaster, the disposal of biohazardous
material may be- come a serious problem
for a blood center or hospital. Planners
should incorporate appro- priate language
in their biohazard material disposal
contracts that will ensure that ven- dors
establish contingency plans to meet the
facility’s requirements. The blood center
or hospital should coordinate with the EMA
and fire or safety agencies and should
provide in- formation on the types of
biohazardous wastes that it generates.
Organizations that have irra- diators should
provide local fire or safety agen- cies with a
detailed building plan that includes
photographs of the ingress routes to the
device and of the device as well as the
manufacturer’s specifications and contact
information. Affect- ed facilities should
contact the AABB Disaster Task Force2 if
local and state EMA authorities cannot
provide this assistance.
Utilities
Blood centers and hospitals rely on utility
ser- vices (eg, electricity, water, fuel, sewage
dis- posal, telecommunications, and
Internet) from a variety of sources, such as
municipali- ties or publicly or privately
owned companies. Planners should establish
contingency sup- port plans with their
service providers. These plans should
identify emergency points of contact at the
utility service provider and in- clude their
telephone numbers and e-mail ad- dresses.
Most utility services have priority lists for
service restoration during emergencies.
During the planning process (and not during
or after an emergency), planners should
solicit the support of the local EMA and
hospitals to ensure that their organization
will be designat- ed to receive priority
restoration of services.
Staffing
Blood centers and hospitals depend on their
highly trained, competent, and motivated
staff and volunteers to be successful in their
mis- sion of recruiting blood donors and
collecting, manufacturing, testing, and
distributing blood components. Management
should promote disaster preparedness to
staff and volunteers
 C H A P TE R 4
and should encourage them to complete
fami- ly preparedness plans.8.22
ES SE NT IAL PE R S ON NEL . During the
emer- gency planning process, the
management team should identify staff
members and vol- unteers (if applicable)
who are responsible for the vital services
required to maintain busi- ness operations.
Each department should de- velop a “smart
book” that describes its essen- tial
functions, essential personnel who
accomplish them, vital information and
rec- ords, contract information, and
customer in- formation.
Employees who are deemed essential
be- cause of their responsibilities must
receive ex- tensive training in the facility’s
emergency plans and participate in disaster
training exer- cises to refine operational
procedures under emergency conditions.
Consideration should be given to
establishing alternate work sites and/or
implementing telework programs to
mitigate the effects of the loss of the
blood center or hospital on operations. The
human resources department should work
with man- agement to ensure that
employee-training plans provide for the
succession of personnel into positions held
by designated essential personnel should
the incumbent become un- available.
Management should also plan for housing
and feeding essential personnel and
providing them with fuel to help them travel
to and from work when the local
infrastructure is not operational.23
CROSS-FUN CTION AL TRA I NIN G.
Planners,
in close coordination with the human
resourc- es department and, if applicable,
any collec- tive bargaining units, should
develop a cross- functional training
program that identifies personnel whose
routine duties do not sup- port the facility’s
essential functions during a disaster
response. These reassigned personnel may
serve as drivers, public affairs representa-
tives, and recruiters or perform other
nonreg- ulated duties. The human
resources depart- ment and management
should make the necessary modifications
to the job descrip- tions of the affected
employees or volunteers, develop training
plans, and conduct initial and
Disaster Management ■ 109
refresher training as required.
Management may need to negotiate with
collective bargain- ing units to modify
existing contracts.
HU M A N R E SO UR C E S IS SU E S . Of i m por -
tance to employees—beyond the safety of
loved ones and availability of food and shel-
ter—are issues such as salary continuation,
flexible work schedules, implementation of
telework procedures, and benefits.8,23 Because
these issues are influenced by many factors,
having a predeveloped decision matrix can be
particularly helpful during times of crisis.
REGULATORY CONSIDERATIONS IN
EMERGENCIES
The blood collection and transfusion
commu- nity is highly regulated, and even
slight chang- es to processes and procedures
can have far- reaching effects on facilities
and patients, especially during an
emergency. Blood collec- tion and
transfusion facilities should carefully
consider regulatory issues in their disaster
management plans.
Federal Agencies Involved
The blood community is regulated by
numer- ous agencies. The Center for
Biologics Evalua- tion and Research
(CBER) of the Food and Drug
Administration (FDA) has primary re-
sponsibility for ensuring the safety, purity,
and potency of all biologic products,
including blood and blood components. The
Center for Devices and Radiological Health
(CDRH), also part of the FDA, regulates
medical devices, including radiation-
emitting devices. The Department of
Transportation regulates the movement of
cargo, including blood, blood components,
and progenitor cells, through the
transportation network. OSHA ensures the
safety of the US workforce. Likewise, the
mis- sion of the Environmental Protection
Agency is to protect human health and the
environ- ment. The Centers for Medicare
and Medicaid Services under the Clinical
Laboratory Im- provement Amendments of
1988 oversees pa- tient (as distinct from
donor) testing at blood
110 ■ AABB T EC HNIC AL MANUAL

and physicians should

centers, hospital blood banks, and
transfusion services.
Consideration must be given to the
regula- tions and requirements of each of
these agen- cies when developing
emergency response plans. Such regulatory
considerations generally fall into three
categories: effect on available blood
components, effect on donors, and po-
tential consequences for operations.

Effects on Components in
Available Inventory
All blood centers and hospitals must have
written procedures to follow in an
emergency. These procedures should
address the provi- sion of emergency
electrical power and con- tinuous
temperature monitoring during stor- age.
These procedures should address the
potential effects of unsuitable storage condi-
tions, such as extreme (high or low)
tempera- tures or exposure to smoke or
water.37 Consid- eration should also be given
to nontraditional emergencies that may
affect blood, such as a radiologic event or
exposure to a biologic or chemical agent.38
Blood components that have
potentially been exposed in an event
should be quaran- tined, and a
determination of component suit- ability
should be made before the components are
returned to inventory. When the quality,
safety, purity, or potency of a component is
in question, CBER should be consulted.
An ex- ception or variance may be
needed to use components in such
situations.39 In addition, the AABB Disaster
Task Force is available to assist facilities
with questions about a compo- nent’s
safety, purity, or potency during a
disaster.2
All blood released for use during a
disas- ter should be fully tested, including
for infec- tious diseases. An exception to
full testing should be made in the event
that blood sup- plies are exhausted,
resupply is not possible, and blood is
needed immediately to save lives. Blood-
testing samples should be retained for
retrospective testing after a disaster.
Thor- ough documentation of the
circumstances of the disaster is essential,
 effect must be estimated, and donors must be
be notified regarding deferred appropriately. As with com- ponent
what tests have and suitability, CBER and the AABB Disas- ter
have not been Task Force should be consulted about po-
completed on the tential donor deferral issues.
blood.39
In very extreme circumstances, the FDA Potential Consequences on
may Operations
issue emergency
guidance to help ensure Emergencies involving power outages, floods,
an adequate blood or structural damage to facilities disrupt nor-
supply. For example, on mal operations. Disasters that do not directly
Sep- tember 11, 2001, affect the blood center’s or hospital’s
the FDA issued the physical infrastructure can still disrupt
Policy Statement on operations. For example, a pandemic
Urgent Collection, influenza outbreak may cause severe staffing
Shipment, and Use of shortages with up to a 40% absenteeism
Whole Blood and Blood rate.17 Collections should be performed only
Compo- nents Intended by facilities that routinely collect blood.
for Transfusion to Facilities that collect only autol- ogous
Address Blood Supply blood should not attempt to collect allo-
Needs in the Current geneic blood during a disaster.
Disaster Situation.40
Blood collection
facilities should closely
follow all developments
regarding do- nor
deferrals and all
emergency FDA
guidance during
disasters.

Effects on Donors
Disasters, particularly
emergencies involving
potential infectious
diseases or hazardous
chemicals, can affect
donor eligibility. Infec-
tious and chemical agents
should be evaluated for
1) transmissibility by
blood transfusion, 2)
potential to harm
recipients, and 3)
potential for an
asymptomatic interval of
infectivity or toxicity
after exposure but before
or after any donor illness.
If an agent is determined
to be transmissible, a
deferral period that
ensures an adequate
period of safety from
infectivity or adverse
 C H A P TE R 4
Adequate staffing can be challenging
im- mediately before, during, and after an
emer- gency while staff members tend to
their fami- lies and personal needs. Only
staff trained in regulated functions should
perform these functions. The FDA
recommends that blood establishments
have adequate backup person- nel in the
event of a disaster and that more than one
backup person be trained for each critical
function. This training should be docu-
mented.41 Volunteers can be used to
perform nonregulated functions, such as
predonation education and maintenance of
the canteen. Likewise, appropriate licensure
is required to safely operate fleet vehicles.
Emergency plans should include lists of
staff trained in multiple functions. For
exam- ple, staff members who have
transferred from one area to another (and
who have maintained competency in the
prior area) could perform the initial function
with appropriate certifica- tion and any
necessary commercial vehicle li- censes.
Equipment and supplies should also
be assessed for exposure to water, humidity,
and temperature extremes. CDRH has
published an information guide about the
effects of di- sasters on medical devices; it
can be useful in planning as well as
responding to disasters.42
Records Management
Vital records must be secured and
maintained. These documents include
records of donors, donations, manufacturing,
testing, quality as- surance, and product
disposition. If these rec- ords are damaged
or lost during a disaster, blood in inventory
at that time may require quarantine and
recall. Efforts should be made to retrieve
and preserve any damaged records. CBER
should be consulted to determine the
disposition of inventory when records are
lost.
TESTING THE DISASTER PLAN
Continual improvement of a disaster plan is
critical for maintaining a high state of readi-
ness for future emergencies, and participation
in disaster drills is one way to achieve this
goal. By simulating the conditions of a real
disaster,
Disaster Management ■ 111
a facility can identify and correct deficiencies
and gaps in its disaster plan.
Internal and External Disaster Drills
Drills can be conducted internally (eg, a fire
drill or simulated hazardous spill cleanup) or
in conjunction with other organizations.
Mul- tiple-organization disaster drills are
typically conducted by local or state EMAs.
Unfortu- nately, blood-related issues are
often over- looked in the exercise scenarios
developed by these agencies because many
of the planners are unaware of how blood is
collected, stored, and distributed to
hospitals. Blood collection facilities should
develop relationships with EMAs, as
described in the “Major Disaster
Management Factors for Blood and Transfu-
sion” section.
Participating in Disaster Drills with EMAs
The National Strategy for Homeland Security
directed the establishment of a national exer-
cise strategy. Homeland Security Directive 8
directed the DHS to establish a National Exer-
cise Program (NEP).43 In addition to full-scale,
integrated, national-level exercises, the NEP
provides tailored exercise activities that serve
as the primary DHS vehicle for training na-
tional leaders and staff. The NEP enhances
collaborations among partners at all levels of
government for assigned homeland security
missions. National-level exercises provide the
means to conduct “full-scale, full-system
tests” of collective preparedness, interopera-
bility, and collaboration across all levels of
government and the private sector.44 The
program also incorporates elements to allow
identification of any implications of changes
to homeland security strategies, plans, tech-
nologies, policies, and procedures.
Blood centers and hospitals should
seek inclusion in disaster drills and
exercises by con- tacting their local EMAs.
Participation in local, state, and federal
exercises increases a facility’s preparedness.
This participation also increases awareness
by the emergency preparedness offi- cials at
all levels of the critical role that blood
centers play in providing the blood
compo-
112 ■ AABB T EC HNIC AL MANUAL
nents required by victims of a disaster and
the resources that are required to do so.
Including Blood Issues in Drills
Disaster drills range from informal discussions
about improving response methods to full-
scale, real-time exercises that involve
hundreds of organizations. Each method can
be used by a single organization or in
conjunction with mul- tiple organizations.
When an organization par- ticipates in drills
with other organizations (ie, full-scale
exercises), it is important for blood collection
and transfusion facilities to “inject” blood-
related scenarios into the drills during the
planning stages. For example, drills should
address 1) the need to transport blood from a
blood center to a hospital (or hospitals) to treat
victims when local roads have been damaged
or destroyed and 2) the effect of an unknown
bio- logic agent release on the local blood
supply, donors (eg, need to defer donors), or
both. Ad- ditional information on the basic
types of drills can be found on the FEMA
Emergency Manage- ment Institute website.45
After-Action Review
Perhaps the most important aspect of a well-
executed drill or actual disaster is the after-
ac- tion review process. During this process,
par- ticipants evaluate what worked well and
what did not work well, with the aim of
identifying lessons learned and
implementing corrective actions to improve
future responses.
SUMMARY OF LESSONS
LEARNED FROM RECENT
DISASTERS
Blood collection facilities and hospitals
around the world have dealt with significant
KEY POINTS
Planning is the key to success during a disaster. Disaster planning is not a one-time event; it requires continual review, exercis
Continuity of operations planning provides a framework for facilities to maintain or restore the critical functions necessary to
disasters in recent years. Hurricanes, tsuna-
mis, earthquakes, tornados, fires, industrial
accidents, and terrorism have tested
emergen- cy response systems.
Organizations have used the learning points
from these events to fur- ther sharpen their
disaster plans. Many of these lessons can be
found in the AABB Disas- ter Operations
Handbook.2 Blood collection and
transfusion facilities are encouraged to share
the major learning points from real or
simulated disasters that they have
experienced with AABB through the AABB
National Blood Exchange at 1.800.458.9388
or by e-mail to nbe@aabb.org. AABB is
collecting such lessons and sharing them with
facilities around the world.
CONCLUSION
In the wake of disasters—such as the Septem-
ber 11, 2001, attacks on the World Trade Cen-
ter and Pentagon; the rail transit bombings in
Great Britain, Spain, and Boston; and the dev-
astating natural disasters that occur every year
—organizations around the world have fo-
cused significant attention and resources on
strengthening their disaster plans. Given the
heightened awareness of the need for organi-
zations to be prepared, both the public and
members of the media are holding organiza-
tions to the highest standards of excellence for
ensuring that all necessary planning has been
done to protect both life and property from
known threats. Blood collection and transfu-
sion facilities are not immune to this reality
and should therefore strive to maintain the
highest standards of disaster preparedness.
Above all else, the most important benefit of
careful and continual planning is assurance
that blood and blood components are avail-
able to patients when and where needed.
 C H A P TE R 4 Disaster Management ■ 113

3. The cornerstone of an effective disaster plan is a thorough risk assessment of the

probable hazards and their impact on critical operations. Risks can include internal
events, such as a broken pipe that floods a laboratory, or external events, such as
tornadoes and earthquakes.
4. Facilities should use the resources of the AABB Interorganizational Task Force on
Domestic Disasters and Acts of Terrorism in disaster management planning (eg,
Disaster Operations Handbook) and incorporate the task force’s national response
procedures into local plans.
5. The ability to communicate during a disaster determines much of the success of a
response; facilities need well-developed and routinely tested communication plans.
6. Hospitals should have emergency plans for triage of blood components in disasters in
which demand exceeds supply (eg, a severe pandemic or nuclear or biologic attack).
Blood centers should work with their hospital customers to ensure that such plans are in
place.
7. Adequate cash reserves and insurance are essential for a blood center to survive a major
di- saster.
8. Coordination with local emergency management officials before a disaster is critical to
suc- cess during that disaster.
9. Regulatory considerations during a disaster include the effects on the blood
components on the shelf, the donors in the affected area, and operations.
10. Facilities should routinely reinforce their disaster plan by conducting information
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 C h a p t e r 5

Allogeneic and Autologous Blood

Donor Selection

Anne F. Eder, MD, PhD,

and Maria D.L.A. Muniz,
MD

TH E F O R E MOST R E SP ON SIBILITY
of blood centers is to maintain a safe
and adequate blood supply. The selection
of ap- propriate blood donors is essential to
protect their health during and following
donation, and to ensure the safety, quality,
identity, puri- ty, and potency of the donated
blood compo- nents to protect the
transfusion recipient. Key elements of the
selection process, as part of the overall
approach to blood safety, are edu- cation,
the donor health history questionnaire, a
focused physical examination, infectious
disease testing (see Chapter 8), and
manage- ment of postdonation information.
This chapter describes the current feder-
al regulations, accreditation requirements,
and medical considerations related to screen-
i ng bl oo d do nors be fore their blo o d is
collected and tested for various blood-borne
diseases.1-3
OVER VIEW OF BLOOD DONOR
SCREENING
Blood centers provide prospective blood
do- nors with information on the donation
pro- cess and potential donation-related
adverse effects and instruct them not to
donate if they may be infected with blood-
borne pathogens. The screening process
includes a focused physical examination
and direct questioning about specific risk
behaviors, medications, travel, and other
factors that potentially affect recipient or
donor safety. The questions ad- dress risks
related to infectious diseases for which
sensitive tests are currently performed [eg,
hepatitis B and C viruses and human im-
munodeficiency virus (HIV)], for which
tests are not universally used (eg, Chagas
disease), and for which licensed screening
tests are not yet available (eg, babesiosis
and malaria).

Anne F. Eder, MD, PhD, Executive Medical Officer, American Red Cross, National Headquarters, Biomedical
Services Medical Office, Holland Laboratory, Rockville, Maryland; and Maria D.L.A. Muniz, MD, Clinical
Pathologist, Allegheny Health Network, Pittsburgh, Pennsylvania
The authors have disclosed no conflicts of interest.

117
118 ■ AABB T EC HNIC AL MANUAL
If individuals are instructed not to donate
blood for others because of their health histo-
ry, reactive test results, behavioral risks, or
medical reasons, they may be added to a confi-
dential deferral list to prevent future dona-
tions. Deferrals may be for a defined interval
of time or an indefinite period, or they may
be permanent with no potential for
reinstate- ment as a blood donor in the future.1
In addi- tion, blood centers are required to
manage in- formation received after the
donation that could affect the safety, quality
or potency of the donated blood components
and the future eligibility of the donor, and keep
records of de- ferred individuals.2
The criteria to evaluate individuals
who are donating blood for their own use
(ie, autol- ogous donation) may be less
stringent than those for allogeneic donation.
However, the fo- cus remains on providing
the safest possible blood for transfusion to
the donor-patient and on evaluating the risks
that the collection pro- cedure poses to his
or her health.
The blood center must determine donor
eligibility prior to collection and on the day
of collection, in accordance with federal
and state regulations and voluntary
accreditation standards. Specific criteria
used to select do- nors are established by the
Food and Drug Ad- ministration (FDA)
through the Code of Feder- al Regulations
(CFR), guidance documents, and
memoranda to industry.2 The AABB also
develops professional standards for donor
se- lection with which accredited blood
establish- ments must comply.1
An industry-wide effort led by AABB
to streamline and standardize donor
screening culminated with the release by the
FDA, in Oc- tober 2006, of a final guidance
document rec- ognizing the donor history
questionnaire (DHQ) as an effective
donor screening tool that provides licensed
and unlicensed facilities with a way to meet
all FDA requirements.4 The DHQ is
currently used by most blood centers in the
United States.5,6 The references in this
chapter are to DHQ Version 1.3 (May
2008), which the FDA officially recognized
as accept- able DHQ documentation in May
2010.4,7
Medical directors of blood collection fa-
cilities are responsible for determining donor
eligibility policies on issues that are not
cov- ered by regulations or standards. 3,8-11
Conse- quently, medical decisions regarding
the same issue may differ among blood
centers or even among physicians at the
same blood center. Considerable variability
exists in national and international practices
for determining donor eligibility, which
reveals the inherent uncer- tainty in risk
assessment.8
A precautionary approach attempts to
eliminate the risk of known or potential
trans- fusion-transmitted diseases from the
blood supply but also results in the
disqualification of large segments of the
healthy population. Prospective donors
who are deferred from blood donation
may be disappointed, angry, or confused
about the relevance that certain questions
have to their health or ability to do- nate
blood for transfusion to others. Blood
center staff should be able to explain the in-
tended purpose of AABB and FDA
require- ments as well as their center-
specific eligibility screening practices.
The most frequently asked questions
about federal regulations defining blood do-
nor eligibility are available to the public in the
“Questions about Blood” section of the FDA
website.12 Questions about the interpretation
or underlying rationale of AABB Standards
for Blood Banks and Transfusion Services
should be directed to the AABB Standards
Depart- ment (standards@aabb.org);
responses and discussion of selected issues
are posted on the AABB website.
SELECTION OF ALLOGENEIC
BLOOD DONORS
Registration and Donor Identification
In the United States, blood components for
al- logeneic transfusion are typically
collected from volunteer, nonremunerated
donors; oth- erwise, they must be labeled as
being from paid donors.2
Prospective blood donors should
provide an acceptable form of identification,
and each donor must be properly identified
by the col- lection staff before each
donation. Acceptable forms of
identification include government-
 CHAP TER 5 Allogeneic and Autologous Blood Donor Selection
issued documents, such as a driver’s license
or passport, or a blood-center-issued donor
card with a unique alphanumeric code.
Many blood collectors no longer use social
security numbers for donor identification
because of regulations restricting their use
and privacy concerns.
Accurate records are essential to
identify all prior donations from any given
donor, in- cluding whether the donor has
ever given blood under a different name, so
that the link with all prior donations within
the blood sys- tem is maintained. Accurate
records are also essential to ensure that the
donor can be con- tacted in the weeks
following the donation and informed of test
results or other relevant infor- mation from
the current donation, if neces- sary. Blood
centers must make reasonable at- tempts to
notify the donor within 8 weeks of the
donation if any test results disqualify the
individual from continued donation.2
Blood centers often require donors to
provide an ad- dress and telephone number
where they can be reached during this
interval for counseling or other follow-up, if
necessary. Accurate do- nation records must
be kept by the donor cen- ter for the
requisite amount of time, according to
current regulations and standards.1,2
Notably, there is currently no national
registry of deferred blood donors in the
United States, and individuals who are
deferred by one blood center may be
eligible in another blood system. The
available evidence suggests that such donor
deferral registries are not nec- essary
because they do not contribute to blood
safety and do not prevent the release of
unsuit- able components.13
Educational Materials and
Donor Consent
US blood centers provide all prospective
blood donors with information about blood
dona- tion during the informed consent
process. The DHQ educational materials
incorporate all necessary elements required
by federal regula- tions and AABB
Standards, and blood centers have added
elements to protect both blood donors and
transfusion recipients.1(pp15-16,60-63),4 At each
encounter, the blood center staff
■ 119
should explain the collection procedure to
the donor in terms that the donor
understands and document the donor’s
consent, which in- dicates that the donor has
considered all the educational materials and
has had an oppor- tunity to ask
questions.1(p16) The donor should be informed
about possible adverse reactions to the
collection procedure and the infectious
disease tests that will be performed on his or
her donated blood components. The donor
should also be informed of the notification
process for positive test results, any
reporting requirements to federal or state
health author- ities, and the possibility of
inclusion in the do- nor center’s deferral
registry and subsequent deferral from future
donation. The individual should agree not to
donate if his or her blood could pose a risk
to the blood supply. Donors should also be
informed if investigational tests or other
research may be performed on sam- ples or
information collected during the blood
donation. Finally, the limitations of the tests
to detect early infections and the possibility
that a test may not be performed if samples
are not adequate should be explained to the
donor.
No one should donate blood for the pur-
pose of receiving free infectious disease test-
ing. Information about alternative HIV test
sites or public health options to obtain HIV
tests should be provided to all prospective
donors.
All prospective donors must be able
to give informed consent and provide an
accu- rate health history. They should be
given an opportunity to ask questions and
they have the right to withdraw from the
process at any time. Blood donation
centers should docu- ment donors’ consent
to have their blood col- lected and tested.
Blood centers must comply with
applica- ble state laws to obtain permission
from par- ents or guardians for minors (ie,
16- or 17-year olds) or legally incompetent
adults.1(p16) More- over, AABB Standard 5.2.2
requires that collec- tion facilities have a
process to provide infor- mation about the
donation process to parents or legally
authorized representatives of donors when
parental permission is required.1(p16)
Donor centers should also establish
poli- cies on accommodating individuals
who are
120 ■ AABB T EC HNIC AL MANUAL

not fluent in English or are illiterate, are
vision or hearing impaired, or have other
physical disabilities. Many blood centers
try to make reasonable accommodations for
donors’ spe- cial needs. However, centers
must also ensure that the collection
procedure does not pose undue risk to
donors or staff members and that the
informed consent process is not com-
promised. The final authority for such
deci- sions rests with the donor center’s
physician who supervises donor
qualification and phle- botomy. 1(p1)
Donor Qualification by Focused
Physical Examination and
Hemoglobin or Hematocrit
Measurement
Qualification procedures for blood donation
include a focused physical examination (eg,
taking the donor’s temperature and
inspecting his or her arm) and a hemoglobin
or hemato-
crit measurement. These tests have potential
implications for the potency or safety of the
collected component (Table 5-1). Additional
requirements apply to apheresis donors, who
must meet the weight and hemoglobin or he-
matocrit requirements approved by the FDA
for the automated collection device.
Prior to phlebotomy, the collection
staff inspect the donor’s antecubital skin,
which must be free of lesions and evidence
of injec- tion drug use, such as multiple
needle punc- tures (eg, small scars lined up
in “tracks”) or sclerotic veins. Scars or
pitting on the forearm associated with
frequent blood donation should not be
mistaken for evidence of injec- tion drug
use. Common and mild skin disor- ders (eg,
poison ivy rash) are not a cause for deferral
unless there are signs of localized bac- terial
superinfection or the condition inter- feres
with proper skin disinfection in the ante-
cubital site before phlebotomy.

TABLE 5-1. Physical Examination and Requirements for Allogeneic and Autologous Whole-Blood Donation

Allogeneic Autologous
AABB Reference Standard 5.4.1A; Title AABB Standard 5.4.4; Title 21,
21, CFR Part 640.3 CFR
Part 640.3
Age Conform to applicable state law or
16 years Alternate requirements defined by
Blood pressure No requirement in AABB standards, systolic and blood center’s medical director (AABB
diastolic blood pressure “within normal limits” Standard 5.4.4).
[Title 21, CFR Part 3(2)].
Pulse No requirement in AABB standards or CFR.
Whole blood Maximum of 10.5 mL/kg of donor weight,
vol-
ume collected including samples.
Donation interval 8 weeks after whole blood donation; 16
weeks
after 2-unit red cell collection; 4 weeks after
infrequent plasmapheresis; and 2 days after
plasma-, platelet-, or leukapheresis.
Temperature 37.5 C (99.5 F) if measured orally or Deferral for conditions presenting
equivalent risk
if measured by another method. of bacteremia (AABB Standard
5.4.4.4).
Hemoglobin 12.5 g/dL (38%). >11 g/dL (33%).
(hematocrit)
CFR = Code of Federal Regulations.
 CHAP TER 5 Allogeneic and Autologous Blood Donor Selection ■ 121
hemoglobin.1(p27)

The FDA defines the minimum

accept- able hemoglobin concentration for
both men and women as 12.5 g/dL, and the
essentially equivalent hematocrit
requirement of 38%.2 This requirement is
controversial and is peri- odically debated
because normal hemoglobin values are
influenced by gender, race, nutri- tional
status, and other factors.14,15 In particu- lar, a
single acceptance criterion for men and
women is contentious because normal
hemo- globin values for women are lower
than those for men. However, this
requirement has re- mained unchanged for
more than 30 years. Hemoglobin or
hematocrit screening may pre- vent
collection of blood from a donor with sig-
nificant anemia, which could have
implica- tions for the health of the donor as
well as the potency of the collected
component.
A hemoglobin level lower than the 12.5
g/dL cutoff is the most common reason for
disqual- ification of potential blood donors.
This single deferral criterion can be
problematic for both male and female
donors. Some female donors with
hemoglobin levels below the 12.5 g/dL
cutoff are actually in the normal range. In
con- trast, some male donors with a
hemoglobin level above 12.5 g/dL may, in
fact, be anemic. Furthermore, hemoglobin
screening does not ensure that the donor has
an adequate store of iron.16,17 Debate
continues on the minimum hemoglobin
requirement for blood donation, frequent
donors’ iron status, and allowable do- nation
intervals.18
Donor hemoglobin screening may ensure
a minimum content of hemoglobin in a unit of
Red Blood Cells (RBCs), but currently neither
the FDA nor the AABB define potency stan-
dards for RBC units prepared from whole-
blood collection. If a donor’s hemoglobin level
is 12.5 g/dL, a 500-mL whole-blood collection
is expected to yield about 62.5 g of
hemoglobin per unit of RBCs, but
determining the final content of hemoglobin
in an RBC unit pre- pared from whole blood
is not required. AABB Standards require
apheresis RBC units to be prepared using a
method known to ensure a fi- nal component
containing a mean hemoglo- bin level of 60
g of hemoglobin, with 95% of the units
sampled containing more than 50 g of
 approximately 0.2 to 0.5 g/dL, and the vast
majority of deferred donors have hemo-
In general, neither the globin or hematocrit values near the cutoff
FDA nor the AABB specifies value. For capillary-sample-based
the test method, specimen methods, the most likely source of
type (cap- illary, venous preanalytical error is the sampling
blood), or acceptable perfor- technique, and testing must be performed in
mance characteristics for tests compliance with the manufac- turer’s
used for hemo- instructions.
globin/hematocrit screening.
One exception is that a Health History Assessment—DHQ
capillary sample collected The AABB DHQ is now used by most blood
from an ear- lobe puncture is centers in the United States, although its use is
not an acceptable specimen not currently mandated by the FDA ( Table 5-
for hemoglobin/hematocrit 2).19 Alternative procedures for collecting
screening for allo- geneic or required information from blood donors are
autologous donors.1(pp19,60) subject to FDA review and acceptance. In ad-
Most US blood centers use dition, the FDA acknowledges that the DHQ
fingerstick capillary samples
for hemoglobin
determination. These sam-
ples tend to give slightly
higher hemoglobin values
than venous samples.
The methods to measure
hemoglobin or hematocrit are
generally selected for their
ease of use in the mobile
blood collection setting. The
copper sulfate density method
(Method 6-
1) is still an acceptable
screening tool in blood
centers in the United States
but has been largely
replaced by methods such as
spectro- photometric
measurement of
hemoglobin with portable
devices (eg, HemoCue Donor
Hb Checker, HemoCue AB,
Angelholm, Sweden; DiaSpect
Hemoglobin, DiaSpect
Medical AB, Uppsala,
Sweden) or hematology
analyzers. The point-of-care
methods that use portable
devices yield quantitative
hemoglobin results, with a
coefficient of variation (CV) of
1.5%.15 A typical automated
analyzer measures hemo-
globin levels in a venous
sample with a CV
1.2%.15 Most quantitative
methods currently in use
reliably measure
hemoglobin levels within
122 ■ AABB T EC HNIC AL MANUAL
TABLE 5-2. Donor History Questionnaire and Eligibility Requirements for Allogeneic Blood Donations19
Donor History QuestionnaireBrief Description of Eligibility Criteria
1. Are you feeling healthy and well today?
2. Are you currently taking an antibiotic?
3. Are you currently taking any other
medica- tion for an infection?
4. Are you now taking or have you ever
taken any medications on the Medication
Deferral List?
5. Have you read the educational materials?
6. In the past 48 hours have you taken
aspirin or anything that has aspirin in it?
7. Female donors: Have you been pregnant
or are you pregnant now? (Males: Check
“I am male.”)
8. In the past 8 weeks have you donated
blood platelets or plasma?
The prospective donor shall appear to be in good health and
shall be free of major organ disease (eg, heart, liver,
lungs), cancer, or abnormal bleeding tendency, unless
determined eligible by the medical director.
Variable, temporary deferral criteria as defined by the medi-
cal director.
Variable, temporary deferral if treated for active infections,
as defined by the medical director.
No deferral for prophylactic (preventive) use of antibiotics
(eg, tetracycline for acne).
Variable, temporary deferral if treated for active infections.
Examples:
Potent teratogens (potential for harm to unborn children):
■ Finasteride (Proscar, Propecia), isotretinoin (Absorica,
Accutane, Amnesteem, Claravis, Myorisan, Sotret,
Zena- tane): Defer for 1 month after last dose.
■ Dutasteride (Avodart, Jalyn): Defer for 6 months after
last dose.
■ Acitretin (Soriatane): Defer for 3 years after last dose.
■ Etretinate (Tegison): Defer indefinitely.
Potential vCJD risk (but no documented cases of transmis-
sion):
■ Bovine insulin manufactured in the United Kingdom: Defer
indefinitely.
N/A
Medications that irreversibly inhibit platelet function pre-
clude use of the donor as sole source of platelets:
■ Defer for at least 36 hours after ingestion of aspirin.
■ Defer for use of other medications as defined by the
facil- ity’s medical director.
Defer if donor has been pregnant within the last 6 weeks.
Donation interval requirements:
■ Defer for 8 weeks after whole blood donation.
■ Defer for 16 weeks after 2-unit red cell collection.
■ Defer for 4 weeks after infrequent plasmapheresis.
■ Defer 2 days after plasmapheresis, plateletpheresis,
or leukapheresis.
 CHAP TER 5 Allogeneic and Autologous Blood Donor Selection
TABLE 5-2. Donor History Questionnaire and Eligibility Requirements for Allogeneic Blood Donations19
(Continued)
Donor History QuestionnaireBrief Description of Eligibility Criteria
9. In the past 8 weeks have you had
any vaccinations or other shots?
10. In the past 8 weeks have you had
contact with someone who had a
smallpox vaccina- tion?
11. In the past 16 weeks have you
donated a double unit of red cells using
an apheresis machine?
12. In the past 12 months have you had
a
blood transfusion?
13. In the past 12 months have you
had a transplant such as organ,
tissue, or bone marrow?
14. In the past 12 months have you
had a graft such as bone or skin?
■ 123
No deferral for receipt of toxoids or synthetic or killed
viral, bacterial, or rickettsial vaccines listed below if the
donor is symptom free and afebrile.
■ Anthrax, cholera, diphtheria, hepatitis A, hepatitis B,
influ- enza, Lyme disease, paratyphoid, pertussis, plague,
pneu- mococcal polysaccharide, polio (Salk/injection),
Rocky Mountain spotted fever, tetanus, or typhoid (by
injection).
No deferral for receipt of recombinant vaccines (eg. human
papillomavirus vaccine).
No deferral for receipt of intranasal live attenuated flu vac-
cine.
Defer for 2 weeks for receipt of the following live
attenuated viral and bacterial vaccines:
■ Measles (rubeola), mumps, polio (Sabin/oral),
typhoid (oral), or yellow fever.
Defer for 4 weeks for receipt of the following live
attenuated viral and bacterial vaccines:
■ German measles (rubella) or chicken pox (varicella zos-
ter).
Defer for 12 months unless otherwise indicated by
medical director for receipt of other vaccines, including
unlicensed vaccines.
Refer to current FDA guidance.
Defer for 16 weeks after 2-unit red cell collection.
Defer for 12 months for receipt of blood, components,
human tissue, or plasma-derived clotting factor concen-
trates.
Defer indefinitely for receipt of human (cadaveric) dura
mater.
(Continued)
124 ■ AABB T EC HNIC AL MANUAL

TABLE 5-2. Donor History Questionnaire and Eligibility Requirements for Allogeneic Blood Donations19
(Continued)

Donor History QuestionnaireBrief Description of Eligibility Criteria

15. In the past 12 months have you come
into
contact with someone else’s blood?
16. In the past 12 months have you had
an accidental needle-stick?
Defer for 12 months
■ From the time of mucous membrane exposure to blood.
■ For nonsterile skin penetration with instruments or
equip- ment contaminated with blood or body fluids
other than the donor’s own.

17. In the past 12 months have you had

sexual contact with anyone who has
HIV/AIDS or has had a positive test for the
HIV/AIDS virus?
18. In the past 12 months have you had
sexual contact with a prostitute or anyone
else who takes money or drugs or other Defer for 12 months from the time of sexual contact
payment for sex? with an individual with HIV infection or at high risk of
19. In the past 12 months have you had HIV infection.
sexual contact with anyone who has ever
used nee- dles to take drugs or steroids, or
anything not prescribed by their doctor?
20. In the past 12 months have you had
sexual contact with anyone who has
hemophilia or has used clotting factor
concentrates?
21.Female donors: In the past 12 months
have you had sexual contact with a male
who has ever had sexual contact with
another male? (Males: check “I am Defer for 12 months for sexual contact or for having
male.”) lived with an individual who meets any of the
following criteria:
22. In the past 12 months have you had
■ Has acute or chronic hepatitis B (positive hepatitis B
sexual contact with a person who has
sur- face antigen test).
hepatitis?
■ Has symptomatic hepatitis C.
23. In the past 12 months have you lived ■ Is symptomatic for any other viral hepatitis.
with a person who has hepatitis? Defer for 12 months from the time of nonsterile skin
penetra- tion with instruments or equipment contaminated
with blood or bodily fluids other than the donor’s own,
24. In the past 12 months have you including for tat- toos or permanent makeup unless,
had a tattoo? applied by a stage-regu- lated entity with sterile needles
25. In the past 12 months have you had and ink that is not reused.
ear or body piercing? Defer for 12 months or for longest applicable period for
the following situations:
26. In the past 12 months have you had
■ Following the completion of treatment for syphilis or
or been treated for syphilis or
gon- orrhea.
gonorrhea?
■ For a reactive screening test for syphilis where no
confir- matory testing was performed.
■ For a confirmed positive test for syphilis (FDA reentry
pro- tocol after 1 year applies).
 CHAP TER 5 Allogeneic and Autologous Blood Donor Selection ■ 125

TABLE 5-2. Donor History Questionnaire and Eligibility Requirements for Allogeneic Blood Donations19
(Continued)

Donor History QuestionnaireBrief Description of Eligibility Criteria

27. In the past 12 months have you Defer for 12 months.

been in juvenile detention, lockup, jail,
or prison for more than 72 hours?
28. In the past three years have you been Malaria:
out- side the United States or Canada? ■ Defer for 3 years after departure from malaria-endemic
area(s) for individuals(s) who have lived for longer
than 5 consecutive years in a country(ies) with areas
consid- ered endemic by the Malarial Branch, Centers
for Disease Control and Prevention, US Department
of Health and Human Services.
■ Defer for 12 months after departure from malaria-
endemic area(s) for other individuals who have traveled
to an area where malaria is endemic.
29. From 1980 through 1996, did you
spend time that adds up to three (3)
months or more in the United Kingdom?
(Review list of coun- tries in the UK.)
30. From 1980 through 1996, were you
a member of the US military, a civilian
military employee, or a dependent of a
member of the Defer indefinitely for risk of vCJD, as defined in most recent
U.S. military? FDA guidance.
31. From 1980 to the present, did you
spend time that adds up to five (5) years
or more in Europe? (Review list of
countries in Europe.)
32. From 1980 to the present, did you
receive a blood transfusion in the United
Kingdom? (Review list of countries in the
UK.)
33.From 1977 to the present, have you
received
money, drugs, or other payment for sex? Defer indefinitely donors:
34. Male donor: From 1977 to the
■ Excluded by current FDA regulations and recommenda-
present, have you had sexual contact tions for the prevention of HIV transmission by blood
with another male, even once? (Female and components.
donors: Check “I am female.”) ■ With present or past clinical or laboratory evidence
35. Have you EVER had a positive test for of infection with HCV, HTLV, or HIV or as excluded
by cur- rent FDA regulations.
the HIV/AIDS virus?
■ For use of a needle to administer nonprescription drugs.
36. Have you EVER used needles to
take drugs, steroids, or anything not
prescribed by your doctor?

(Continued)
126 ■ AABB T EC HNIC AL MANUAL
TABLE 5-2. Donor History Questionnaire and Eligibility Requirements for Allogeneic Blood Donations19
(Continued)
Donor History QuestionnaireBrief Description of Eligibility Criteria
37. Have you EVER used clotting
factor concentrates?
38. Have you EVER had hepatitis?
39. Have you EVER had malaria?
40. Have you EVER had Chagas disease?
41. Have you EVER had babesiosis?
42. Have you EVER received a dura
mater (or brain covering) graft?
43. Have you EVER had any type of
cancer, including leukemia?
44. Have you EVER had any problems
with your heart or lungs?
45.Have you EVER had a bleeding condition
or a blood disease?
46. Have you EVER had sexual contact
with anyone who was born in or lived in
Africa?
47. Have you EVER been in Africa?
48. Have any of your relatives had
Creutzfeldt- Jakob disease?
vCJD = variant Creutzfeldt-Jakob disease; N/A = not applicable; HIV = human immunodeficiency virus; AIDS =
acquired immunodeficiency syndrome; FDA = Food and Drug Administration; UK = United Kingdom; HCV = hepatitis C
virus; HTLV = human T-cell lymphotropic virus.
■ Defer as directed by the medical director donors
with abnormal bleeding tendency.
■ Defer for 12 months for receipt of blood,
components, human tissues, or plasma-derived
clotting factor concen- trates.
Defer indefinitely for history of viral hepatitis after
11th birthday.
Defer for 3 years after becoming asymptomatic prospective
donors who have had a diagnosis of malaria.
Defer indefinitely for a history of babesiosis or Chagas
disease.
Defer indefinitely for receipt of human (cadaveric) dura
mater.
Variable deferral criteria, as defined by medical director.
Examples:
■ Nonhematologic cancer: Defer for 5 years after
comple- tion of treatment.
■ Local skin cancer, in situ cancer: No deferral if
treated completely with excision and healed.
■ Hematologic cancer (eg, leukemia): Defer indefinitely.
Variable deferral criteria, as defined by medical
director.
Variable deferral criteria, as defined by medical director.
Defer indefinitely, as excluded by current FDA
regulations and recommendations for the prevention of
HIV transmis- sion by blood and blood components.
The questions for risk of HIV group O can be deleted
from the DHQ if the blood center is using an HIV
antibody test that has been approved by FDA to
include a claim for detec- tion of HIV group O.
Defer indefinitely for family history of Creutzfeldt-Jakob
disease.
 CHAP TER 5 Allogeneic and Autologous Blood Donor Selection
documents contain questions related to the
following issues not addressed by any FDA
re- quirement or recommendation: cancer;
organ, tissue, or marrow transplants; bone or
skin grafts; and pregnancy. However, AABB
recom- mends that blood centers implement
the DHQ materials, including the following
documents, in their entirety:
■ Blood donor educational materials.
■ Full-length DHQ.
■ User brochure, including glossary and
ref- erences.
■ Medication Deferral List.
The use of the DHQ flow charts is option-
al. All DHQ documents that the FDA has rec-
ognized as acceptable are available on the
FDA website. 20 The most current version is
also available on the AABB website.19
The wording, order, and text of the
DHQ questions should not be changed. A
collection facility may make minor changes
to the time frame of a question on the DHQ
to make the question more restrictive so that
its use results in the deferral of more donors.
Blood centers may choose to include
additional questions as long as they place
these questions at the end of the DHQ. The
DHQ documents are intended to be self-
administered by the donor, but blood
centers may choose to use direct oral
questioning, self-administration, or a combi-
nation of both methods to administer the
DHQ.
If AABB standards or FDA regulations
do not address specific medical conditions that
a blood center has chosen to include in the
DHQ, the blood center must develop standard
operating procedures (SOPs) for determining
the criteria for acceptance or deferral of a do-
nor. A rational approach to donor health histo-
ry assessment attempts to balance the need to
take appropriate precautions to protect the
blood supply with avoiding unnecessarily re-
strictive policies that disqualify large segments
of the population without contributing to ei-
ther recipient or donor safety. 3 Decisions
about donor eligibility should be based on
available evidence regarding the risk that the
■ 127
medical condition or history poses to the
blood donor and transfusion recipients.
If a potential exists for risk to the
recipient or donor, the effectiveness and
incremental benefit of screening donors by
questioning should be evaluated, especially
in light of oth- er safeguards that protect the
donor or other transfusion practices that
mitigate potential risks to the recipient. If
the blood center re- ceives information
after the donation that would have been
cause for deferral had it been reported at
the time of donation, then any subsequent
actions, such as product retrieval, market
withdrawal, or consignee notification,
should be commensurate with the potential
hazard and likelihood of possible harm to
the recipient. The center’s approach to
develop- ing donor deferral criteria should
take into ac- count evidence as it becomes
available to modify those decisions. Some
of these issues that allow for medical
judgment and for which questions exist in
the DHQ can be explored further with the
donor, but each donor center must develop
and follow its own SOPs.3
BLOOD-CENTER-DEFINED
DONOR ELIGIBILITY CRITERIA
Unlike questions about potential risks to
transfusion recipients, selection criteria di-
rected primarily at protecting donor safety
are left to the discretion of the blood center’s
med- ical director. Consequently, practice
varies at different blood centers.3,8 AABB
Standards re- quires that prospective donors
appear to be in good health and be free of
major organ disease (eg, diseases of the
heart, liver, or lungs), can- cer, or abnormal
bleeding tendency, unless de- termined
eligible by the medical director.1(p61) The
rationale for each deferral for medical
conditions should be carefully considered
be- cause even temporary deferrals
adversely af- fect the likelihood that
individuals will return to donate blood.21
Donor centers have successfully eliminated
some health-related questions and removed
screening require- ments, such as assessment
of pulse, without a deleterious effect on
donor health or dona- tion-related
reactions.4,22 These findings sup- port the
conclusion that some arbitrary and
128 ■ AABB T EC HNIC AL MANUAL

defined
rigid selection criteria, ostensibly intended
to protect donors, are unnecessary and can
be safely eliminated.

Cancer
Each year in the United States, blood centers
receive hundreds of reports of cancer in
indi- viduals who had donated blood.
Direct transmission of cancer through
blood transfusion—although
biologically plausible—has not yet been
documented to occur even though people
with cancer fre- quently donate blood
before discovering their diagnosis. 23 A
retrospective study examined the
incidence of cancer among patients in
Denmark and Sweden who received blood
from donors with subclinical cancer at the
time of donation.24 Of the 354,094
transfusion recipients, 12,012 (3%) were
exposed to blood components from
precancerous donors, yet there was no
excess risk of cancer among these recipients
compared with recipients of blood from
donors without cancer.24 These data indi-
cate that cancer transmission by blood
collect- ed from blood donors with incident
cancer, if it occurs at all, is so rare that it
could not be de- tected in a large cohort of
transfusion recipi- ents that included the
total blood experience of two countries over
several years.
In considering the future eligibility of
do-
nors with cancer, some degree of caution
is warranted to allow sufficient time for
donors to recover after chemotherapy or
other treat- ment. There are currently no US
federal regu- lations or professional
standards regarding the criteria that should
be used to evaluate donors with a history of
cancer. For this reason, a blood center’s
medical director has consider- able
flexibility in determining donor eligibility
policies.
Almost all licensed blood centers
current- ly accept donors who report
localized cancers after treatment, with no
deferral period. These cancers include skin
cancer (eg, basal cell or squamous cell
carcinoma) and carcinoma in situ (eg,
cervical) that have been fully excised and
are considered cured. Most blood centers
defer individuals with a history of a solid
organ or nonhematologic malignancy for a
 2) may affect the hemostatic efficacy of
period after completion their blood and its suitability for transfusion
of treatment provided to others.3
that the donor remains Plasma components and Cryoprecipitated
symptom free without Antihemophilic Factor should contain
relapse. The deferral ade- quate amounts of functional
period following coagulation fac- tors and should not contain
comple- tion of significant inhibi- tory or prothrombotic
treatment for factors. Similarly, platelet components
nonhematologic cancer intended as the sole source for patients
ranges from 1 to 5 should contain platelets that have adequate
years.24 Hematologic function and are not irre- versibly impaired
malig- nancy typically by the presence of inhibitors. Individuals
results in permanent with a history of significant bleeding
deferral from complications are usually counseled to avoid
allogeneic blood blood donation. However, screening donors
donation, although for such a history does not prevent the rare
some US blood but serious thrombotic or hemorrhagic
centers reportedly complications in otherwise healthy blood
accept adults if they donors.
have been successfully Individuals with hemophilia, clotting fac-
treated for childhood tor deficiencies, or clinically significant inhibi-
leukemia or tors—all of which are manifested by variable
lymphoma and have
had a long (eg, 10- to
20-year) cancer-free
period following
completion of
treatment. These
various deferral
policies are currently
defensible but should be
reevaluated if new in-
formation becomes
available about the
poten- tial for cancer
transmission through
blood transfusion.

Bleeding Conditions or Blood Diseases

Bleeding conditions and
blood diseases have the
potential to affect donor
safety as well as product
potency, and blood
centers must de- fine
their procedures for
handling donors with
hematologic disorders.
In general, prospective
donors should be
evaluated for bleeding
con- ditions or blood
diseases that 1) place
the do- nors at risk of
bleeding or thrombosis
as a re- sult of the
collection procedure or
 CHAP TER 5 Allogeneic and Autologous Blood Donor Selection
bleeding tendencies—require deferral for
both donor safety and product potency
consider- ations. The exception is Factor
XII deficiency, which is not associated with
either bleeding or thrombosis.
Carriers of autosomal-recessive or
sex- linked recessive mutations in clotting
factors usually are not at risk of bleeding.
They typi- cally have decreased factor
levels but are ac- cepted by most blood
centers because of the normal, wide
variability in clotting factor ac- tivity
levels (50%-150%) compared to the
much lower relative activity that is
necessary to maintain hemostasis (5%-30%).3
Individuals with von Willebrand disease are
typically de- ferred by most blood centers,
although some centers may allow
individuals with mild dis- ease and no
history of bleeding to donate red cells.
Individuals taking anticoagulants to treat or
prevent venous thromboembolism are de-
ferred for both donor safety and product
po- tency considerations.
Heart and Lung Conditions
Cardiovascular disease is common in the Unit-
ed States, affecting an estimated 80 million (1
in 3) adults.25 Prospective blood donors are
asked if they have ever had problems with
their heart or lungs as a donor safety measure,
but the criteria for accepting donors with a
history of heart or lung disease are defined by
each blood center.
The collective, published experience
with autologous donation by patients
scheduled for cardiac procedures has
demonstrated that ad- verse effects are not
more frequent than in do- nors without a
history of cardiac disease.26-30 Despite the
relative frequency of cardiovascu- lar
disease in the adult population, vasovagal
reactions occur in only about 2% to 5% of
whole-blood donations by healthy donors
and are actually more likely to occur among
young, healthy adolescents than older
adults.31
A rational approach to screening
donors with a history of cardiac disease
allows the ac- ceptance of donors who are
asymptomatic on the day of donation, have
been medically eval- uated, and report no
functional impairment or limitations on
daily activity after being diag-
■ 129
nosed or treated for cardiac disease. Some do-
nor centers advise individuals to wait at least 6
months after a cardiac event, procedure, or di-
agnosis. These centers then allow these indi-
viduals to donate if they have been asymptom-
atic and able to perform their usual daily
activities during this interval.
Causes for deferral may include
recent symptoms, limitations on activity or
function- al impairment resulting from
unstable angina, recent myocardial
infarction, left main coro- nary disease,
ongoing congestive heart failure, or severe
aortic stenosis.3
Medications
The DHQ and Medication Deferral List con-
tain the requirements for deferrals for specific
medications as stipulated by the AABB and
the FDA. These requisite medication deferrals
fall into five broad categories:
1. Potent teratogens that pose potential harm
to unborn children (although there have
been no documented cases of adverse
fetal outcomes related to transfusions
from donors taking these medications).
2. Anticoagulants and antiplatelet agents
that affect component potency (plasma or
platelet components only).
3. Potential variant Creutzfeldt-Jakob disease
risk from bovine insulin manufactured in
the United Kingdom (although there have
been no documented cases of transfusion
trans- mission from donors taking bovine
insulin).
4. Human-pituitary-derived growth
hormone, which theoretically increases
the risk of Creutzfeldt-Jakob disease
(although there have been no documented
cases of transfu- sion transmission from
donors taking these growth hormones).
5. Antibiotics or antimicrobials to treat an
infection that could be transmitted
through blood transfusion (excluding
preventive antibiotics for acne, rosacea,
and other chronic conditions).
Although blood centers may add
medica- tions whose use requires donor
deferral to the
130 ■ AABB T EC HNIC AL MANUAL
Medication Deferral List, many have chosen
to use the list as developed by the FDA and
the AABB or have added only a few
drugs. The DHQ task force has encouraged
blood centers to fully consider the reasons
behind each local deferral and avoid
unnecessary deferral prac- tices.3
FDA’s pregnancy risk categories, which
are designed to assess the benefit vs risk ratio
if drugs are taken during pregnancy, are often
in- appropriately used for blood donor
selection. For example, categories D and X
include some commonly used drugs (eg, oral
contraceptives and anticholesterol agents) that
may be con- traindicated in pregnancy but
pose negligible, if any risk, to any transfusion
recipient.
Local medication deferrals are often
based on concerns about the reason for the
potential donor’s use of the medication and
his or her underlying medical condition
rather than on any inherent threat posed by
residual medication in the collected blood
compo- nent. Most drugs used by donors
pose no harm to recipients, and many
factors should be considered when
evaluating the potential risk of a drug’s use
by a donor (eg, the medica- tion’s half-life,
mean and peak plasma concen- tration,
residual concentration in blood com-
ponent, and dilution when transfused to a
recipient).
ABBREVIATED DHQ FOR
FREQUENT DONORS
Blood centers have recognized for years that
frequent and repeat donors must answer the
same questions at every donation about re-
mote risk factors that are not likely to
change—a situation that leaves many
dedicat- ed donors dissatisfied with the
donation expe- rience. A small number of
US blood centers have received approval
from the FDA and im- plemented an
abbreviated DHQ (aDHQ) in 2003 for
donors who have successfully com- pleted
the full-length DHQ on at least two sep-
arate occasions and given one or more dona-
tions within the past 6 months.32
The AABB aDHQ, which was developed
and validated by the same task force as the
full-length DHQ, has been officially recog-
nized by the FDA as “acceptable” and can
be implemented by blood establishments
using the corresponding full-length
DHQ. 33 Two “capture questions” about
new diagnoses or treatments since the last
donation replace 17 previous questions
about remote risks (eg, blood transfusion,
Chagas disease, and babe- siosis). Although
the aDHQ is only somewhat shorter than the
DHQ and its use is restricted to frequent
donors, it may improve donors’ ex-
perience.
RECIPIEN T-SPE CIFIC
“DESIGNATED” OR “DIRECTED”
BLOOD DONATION
Exceptional Medical Need
In certain clinical circumstances, a patient
may benefit from blood components
collected from a specific donor, such as 1) a
patient with multiple antibodies or with
antibodies to high- incidence antigens who
requires units from donors whose red cells
are negative for the corresponding antigens
or 2) an infant with neonatal alloimmune
thrombocytopenia who requires antigen-
negative platelets from his or her mother.
Frequent donation by a specific donor for
a specific patient with a medical need
requires that the donor center have a
procedure that typically calls for both a
request from the pa- tient’s physician and
approval by the donor center’s physician.
The donor must meet all allogeneic donor
selection requirements, with the exception of
donation frequency, provided that they are
examined and certified by a phy- sician [21
CFR 640.3(f )]. For frequent blood donors,
the CFR allows centers to qualify a do- nor
by testing the first donation in each 30-day
period. 2 In emergency medical situations,
blood components can be released before in-
fectious disease test results are available
pro- vided that the units are labeled and
managed in accordance with the CFR.
Testing on the units must be completed as
soon as possible after release or shipment,
and results must be promptly reported to the
hospital or transfu- sion service.2
 CHAP TER 5 Allogeneic and Autologous Blood Donor Selection
Directed Blood Donations
The use of directed donations has decreased
over the last 10 years, but the ongoing
demand from patients for transfusions from
specific donors during scheduled surgeries
likely still reflects the skewed perception
among the gen- eral public of the risk for
HIV associated with blood transfusion. Most
blood centers and hospitals surmount the
associated collection, storage, and tracking
logistical difficulties to provide this service.
Directed donations have higher viral
marker rates than volunteer donations, most-
ly but not entirely reflecting the higher
preva- lence of first-time donors among the
former group.34 There is no evidence that
directed do- nations are safer to use than
donations from volunteer community
donors. On the contrary, some concerns
persist that directed donors may feel
unduly pressured to give blood, which
could compromise blood safety.
Directed donors must meet the same cri-
teria as voluntary donors, and their blood can
be used for other patients if not needed by the
individual for whom the donations were ini-
tially intended. If collection of whole blood is
required from a directed donor more than once
in an 8-week period, the CFR requires that the
donor be examined and certified to be in good
health by a physician on the day of do- nation
[21 CFR 640.3(f )].
The donor center should clearly
commu- nicate its directed-donation
procedures so that the expectations
regarding availability of directed-donor
units are known to the order- ing physician
and patient. The communica- tion required
includes defining the mandated interval
between collection of the blood and its
availability to the patient, mentioning the
possibility that the patient will identify
donors who are not ABO compatible or not
otherwise acceptable blood donors, and
defining the policy for release of donor-
directed units for transfusion to other
patients.
Autologous Blood Donations
Autologous donations have declined dramati-
cally in the United States since the 1990s.
Wan-
■ 131
ing interest in autologous donations may re-
flect the decline in viral risk associated with
allogeneic blood transfusion and,
consequent- ly, the minimal medical benefit
and increased cost of autologous blood.35,36
In general, the use of preoperative
autolo- gous blood donation alone provides
only a rel- atively small benefit in reducing
the probabili- ty of allogeneic transfusion
and may actually increase the risk of lower
postoperative he- matocrits, with
enhanced risk of ischemic complications
after surgery. Preoperative au- tologous
donations may still be used in
conjunction with other blood conservation
methods, such as acute normovolemic
hemo- dilution, perioperative blood
recovery, and pharmacologic strategies.
(See further discus- sion in Chapter 24.)
Patients identified as candidates for au-
tologous donation are evaluated by the
donor center. The following criteria for
autologous donations are specified by the
FDA, AABB, or both:
■ A prescription or order from the patient’s
physician.
■ Minimum hemoglobin concentration of
11 g/dL or hematocrit of 33%.
■ Collection at least 72 hours before the an-
ticipated surgery or transfusion.
■ Deferral for conditions presenting a risk
of bacteremia.
■ Use only for the donor-patient if labeled
“autologous use only.”
Contraindications to autologous blood
donation should be defined by the blood
cen- ter and may include medical conditions
asso- ciated with the greatest risk from
blood dona- tion, such as 1) unstable
angina, 2) recent myocardial infarction
or cerebrovascular accident, 3) significant
cardiac or pulmonary disease with ongoing
symptoms but without an evaluation by the
treating physician, or 4) untreated aortic
stenosis.37 Both the ordering physician and
the donor center physician need to
carefully balance the risks of the col- lection
procedure against any perceived bene- fit to
the patient-donor.
132 ■ AABB T EC HNIC AL MANUAL

KEY POINTS

The DHQ and associated documents were developed by an AABB task force, and their use is recognized by the FDA as an adeq
The most current versions of the DHQ and associated documents are available on the AABB website, and all DHQ documents t
Prospective blood donors are informed of the risks of blood donation, clinical signs and symptoms associated with HIV infectio
To be accepted for allogeneic blood donation, individuals must feel healthy and well on the day of donation and must meet all A
In certain clinical circumstances (eg, rare phenotype or antigen-negative Red Blood Cell units needed), a patient may benefit fro
The ongoing demand from patients to choose specific donors to provide blood for their transfusions during scheduled surgeries

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9. Eder A, Goldman M, Rossmann S, et al. Selec-
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10. Strauss RG. Rationale for medical director ac-
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11. Reik RA, Burch JW, Vassallo RR, Trainor L.
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13. Cable R, Musavi F, Notari E, Zou S, ARCNET
Research Group. Limited effectiveness of
donor deferral registries for transfusion-
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the blood hemoglobin concentration? Blood
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15. Cable RG. Hemoglobin determination in
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16. Simon TL, Garry PJ, Hooper EM. Iron stores
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17. Cable RG, Glynn SA, Kiss JE, et al. Iron defi-
ciency in blood donors: The REDS-II Donor
Iron Status Evaluation (RISE) study. Transfu-
sion 2012;52:702-11.
18. Triulzi DJ, Shoos KL. AABB Association
Bulle- tin #12-03: Strategies to monitor, limit
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21. Custer B, Schlumpf KS, Wright D, et al.
NHLBI Retrovirus Epidemiology Donor
Study-II. Do- nor return after temporary
deferral. Transfu- sion 2011;51:1188-96.
22. Germain M, Delage G, Grégoire Y, Robillard
P. Donation by donors with an atypical pulse
rate does not increase the risk of cardiac
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23. Eder AF. Blood donors with a history of
cancer. In: Eder AF, Bianco C, eds. Screening
blood do- nors: Science, reason, and the
donor history questionnaire. Bethesda, MD:
AABB Press, 2007:77-92.
24. Edgren G, Hjalgrim H, Reilly M, et al. Risk of
cancer after blood transfusion from donors
with subclinical cancer: A retrospective cohort
study. Lancet 2007;369:1724-30.
25. AHA Statistics Committee and Stroke
Statistics Subcommittee. Heart disease and
stroke statistics—2007 Update. Circulation
2007;115: e69-171.
26. Kasper SM, Ellering J, Stachwitz P, et al. All
ad- verse events in autologous blood donors
with cardiac disease are not necessarily caused
by blood donation. Transfusion 1998;38:669-
73.
27. Mann M, Sacks HJ, Goldfinger D. Safety of
au- tologous blood donation prior to elective
sur- gery for a variety of potentially high risk
pa- tients. Transfusion 1983;23:229-32.
28. Klapper E, Pepkowitz SH, Czer L, et al.
Confir- mation of the safety of autologous
blood donation by patients awaiting heart or
lung transplantation. A controlled study using
he- modynamic monitoring. J Thorac
Cardiovasc Surg 1995;110:1594-9.
29. Dzik WH, Fleisher AG, Ciavarella D, et al.
Safe- ty and efficacy of autologous blood
donation before elective aortic valve
operation. Ann Thorac Surg 1992;54:1177-
80.
30. Popovsky MA, Whitaker B, Arnold NL.
Severe outcomes of allogeneic and autologous
blood donation: Frequency and
characterization. Transfusion 1995;35:734-7.
31. Eder AF, Dy BA, Kennedy J, Notari E, et al.
The American Red Cross donor
hemovigilance program: Complications of
blood donation re- ported in 2006. Transfusion
2008;48:1809-19.
32. Kamel HT, Bassett MB, Custer B, et al.
Safety and donor acceptance of an
abbreviated do-
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hepatitis C virus, hepatitis B virus and human
46:1745-55.
T-lymphotropic virus marker rates for directed
33. Food and Drug Administration. Guidance for
versus volunteer blood donations to the Amer-
industry: Implementation of acceptable ab- ican Red Cross during 2005 to 2010. Transfu-
breviated donor history questionnaire and ac-
sion 2013;53:1250-6.
companying materials for use in screening fre-
35. Brecher ME, Goodnough LT. The rise and fall
quent donors of blood and blood components.
of preoperative autologous blood donation.
(May, 2013) Silver Spring, MD: CBER Office
Transfusion 2002;42:1618-22.
of Communication, Outreach, and
36. Schved JF. Preoperative autologous blood do-
Develop- ment, 2013. [Available at
nation: A therapy that needs to be scientifical-
http://www.fda.gov/
ly evaluated. Transfus Clin Biol 2005;12:365-
BiologicsBloodVaccines/GuidanceComplian
9.
ceRegulatoryInformation/Guidances/Blood/
37. Goodnough LT. Autologous blood donation.
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virus,
 C h a p t e r 6

Whole-Blood Collection and

Component Processing

Larry J. Dumont, MBA, PhD; Mona Papari,

MD; Colleen A. Aronson, MT(ASCP)SBB; and
Deborah F. Dumont, MT(ASCP)SBB

TH E C O L L E C T I O N OF whole blood WB COLLECTION

(WB) from the donor and subsequent
processing of the donation into components
requires careful attention to proper tech-
niques to ensure the optimal care of both the
donor and the recipient. The classical,
manu- ally intense component-preparation
methods are undergoing significant changes
as more automation is incorporated by
developers, from hands-off expression of
components to completely hands-off
methods for the entire process. Differences
are also recognized in methods for platelet
preparation by the “buffy coat” intermediary
process and the “platelet-
rich plasma” (PRP) intermediary process.
This chapter describes WB collection
and component preparation and processing
that may take place at the blood center or
the hos- pital-based collection facility.
The phlebotomy staff involved in WB
collec- tion must be trained in proper
collection tech- niques to minimize
contamination of donated units and
phlebotomy-related local complica- tions,
such as hematoma or nerve injury, using
locally approved standard operating proce-
dures. Phlebotomy must be performed only
af- ter the donor has been found to be
eligible for blood donation and the
following items have been properly labeled
using a unique dona- tion identification
number (DIN) label: the blood donor record,
primary and satellite con- tainers, and
sample tubes. A final check of ap- propriate
labeling before phlebotomy helps ensure
that the donor history data, laboratory data,
and other manufacturing data are associ-
ated with the correct blood components. The

Larry J. Dumont, MBA, PhD, Associate Professor, Geisel School of Medicine at Dartmouth, Lebanon, New
Hampshire; Colleen A. Aronson, MT(ASCP)SBB, Regional Director, Transfusion Services, ACL
Laboratories, Rosemont, Illinois; Mona Papari, MD, Medical Director, LifeSource, Rosemont, Illinois; and
Deborah F. Dumont, MT(ASCP)SBB, Research Technologist, Center for Transfusion Medicine Research,
Dartmouth Hitchcock, Lebanon, New Hampshire
L. Dumont has disclosed financial relationships with Advanced Preservation Technologies, New Health
Sci- ences, Fenwal, Terumo BCT, Haemonetics/Hemerus, Advanced Cell Technology, BioMed/CitraLab,
and EryDel. M. Papani, C. Aronson, and D. Dumont have disclosed no conflicts of interest.

135
136 ■ AABB T EC HNIC AL MANUAL
final check may be documented by having
the staff initial the donor record.
Blood Containers
Desirable Properties
WB collection containers must be approved
by the appropriate regulatory authority [eg,
the Food and Drug Administration (FDA);
Japa- nese Ministry of Health, Labor and
Welfare; or Health Canada] and must be CE
marked for use in Europe. The containers
should be ster- ile, pyrogen free, and
identified by a lot num- ber. They should
also list an expiry date and include other
label data required by regulatory authorities.
The desired properties of storage
contain- ers include being easily formed and
processed; flexible; kink resistant;
transparent; and resis- tant to damage
throughout the anticipated use, including
component processing, storage, and
transfusion. In addition, the container’s
material must be compatible with
sterilization by gamma irradiation, ethylene
oxide, electron beam, and/or steam.
The gas (oxygen, carbon dioxide, and
wa- ter) permeability properties should be
appro- priate for the intended use. Gas
exchange and water loss before use is often
limited by an overwrap or pouch. After
removal from a pouch, the collection set
should be used with- in the time prescribed
by the manufacturer. Pouches should be
labeled with the date and time that they are
opened, and unused containers should be
stored within closed pouches.
Blood containers are usually made of
thermoplastics, such as polyvinylchloride
(PVC), ethylvinylacetate, polyolefins (polyeth-
ylene or polypropylene), or fluoropolymers.
The material formulation determines the
physical properties that are to be matched to
the use requirements, such as high gas perme-
ability for platelet storage or low glass transi-
tion temperature for freezing.
The glass transition temperature of PVC
bags is about –25 C to –30 C. At and below
these temperatures, the container is brittle
and should be treated as if it were made of
glass and fragile enough to break during trans-
port and handling. PVC is rigid at room tem-
perature and requires the addition of a plasti-
cizer such as di-(2-ethylhexyl) phthalate
(DEHP). DEHP not only imparts flexibility to
the PVC but also has been shown to protect
red cells against hemolysis during storage. Be-
cause of concerns over possible toxicity of
DEHP, alternative plasticizers, such as butyryl-
trihexyl-citrate, have been made available in
some collection sets. Because of the protective
effect of DEHP on red cells, finding an equally
effective substitute for DEHP has been chal-
lenging. This has recently been reviewed by
Simmchen.1
Blood collection and processing sets are
generally latex free. The manufacturer’s label
should be consulted to verify the absence of
latex before use in patients with a latex allergy.
Configurations, Anticoagulants, and
Additive Solutions
Collection and storage systems come in
differ- ent configurations based on the
intended pro- cessing methods (manual or
automated) and the number and types of
final components to be prepared from a unit
of WB. Blood collec- tion systems may be
intended for final processing with a classical
combination of manual and mechanical
methods (eg, centrif- ugation, manual bag
transfers, and manual ex- pression of
contents) or may be designed for use in a
fully automated system of blood com-
ponent preparation. There will be
differences in the configurations based on
the method of choice for preparing platelets
(buffy coat or PRP processes). Blood
collection systems may also include in-line
filters for removal of leuko- cytes from WB,
Red Blood Cell (RBC) units, and/or
platelets. Approved anticoagulants in- clude
anticoagulant citrate dextrose solution
Formula A (ACD-A) or Formula B (ACD-B),
an- ticoagulant citrate phosphate dextrose
solu- tion (CPD), anticoagulant citrate
phosphate double-dextrose solution (CP2D),
and citrate phosphate dextrose adenine
solution (CPDA-
1) (see Tables 6-1 and 6-2). RBCs anticoagulat-
ed with CPDA-1 have a shelf life of 35 days in
the United States. The use of additive
solutions (ASs) enables extension of RBC
shelf life to
 C H A P TE R 6 Whole-Blood Collection and Component Processing ■ 137

TABLE 6-1. Anticoagulant-Preservative Solutions for Collection of 450-mL Whole Blood*

Variable CPD CP2D CPDA-1

pH 5.0-6.0 5.3-5.9 5.0-6.0
Ratio (mL solution to blood) 1.4:10 1.4:10 1.4:10
FDA-approved shelf life (days) 21 21 35
Content (mg in 63 mL)
Sodium citrate, dehydrate 1660 1660 1660
Citric acid, anhydrous 188 206 188
Dextrose, monohydrate 1610 3220 2010
Monobasic sodium phosphate, 140 140 140
monohydrate
Adenine 0 0 17.3
*Data were supplied and verified by manufacturers.
CPD = citrate-phosphate-dextrose; CP2D = citrate-phosphate-dextrose-dextrose; CPDA-1 = citrate-phosphate-dextrose-
adenine; FDA = Food and Drug Administration.

42 days in the United States and to 56 days in
some other jurisdictions. Four ASs are current-
ly approved in the United States, and others
are approved in other regions (Table 6-3).2
Containers have integrally attached tub-
ing that permits aseptic/closed transfers be-
tween bags. This tubing is marked with repeat-
ing serial numbers and may be sealed at
several locations with either a dielectric (heat)
sealer or a metal clip (grommet) to prepare ap-
proximately 13 to 15 segments. These seg-
ments may be used later for ABO/Rh typing,

TABLE 6-2. Anticoagulant-Preservative Solutions for Collection of 500-mL Whole Blood*

Variable CPD CP2D CPDA-1

pH 5.0-6.0 5.3-5.9 5.0-6.0
Ratio (mL solution to blood) 1.4:10 1.4:10 1.4:10
FDA-approved shelf life (days) 21 21 35
Content (mg in 70 mL)
Sodium citrate, dehydrate 1840 1840 1840
Citric acid, anhydrous 209 229 300
Dextrose, monohydrate 1780 3570 2230
Monobasic sodium phosphate, phosphate, 155 155 155
monohydrate
Adenine 0 0 19.3
*Data were supplied and verified by manufacturers.
CPD = citrate-phosphate-dextrose; CP2D = citrate-phosphate-dextrose-dextrose; CPDA-1 = citrate-phosphate-dextrose-
adenine; FDA = Food and Drug Administration.
138 ■ AABB T EC HNIC AL MANUAL

TABLE 6-3. RBC Additive Solutions Currently in Routine Use around the World* †

AS-1
Adsol AS-3 AS-5 AS-7
Fresensius- Nutricel Optisol SOLX
Constituents Kabi Haemon- Terumo Haemon- PAGGSM
(mM) SAGM (Fenwal) etics BCT etics MAP MacoPharma

NaCl 150 154 70 150 - 85 72

NaHCO3 - - - - 26 - -
Na2HPO4 - - - - 12 - 16
NaH2PO4 - - 23 - - 6 8
Citric acid 2 - - 1
Na3-citrate - - 23 - - 5 -
Adenine 1.25 2 2 2.2 2 1.5 1.4
Guanosine - - - - - - 1.4
Dextrose 45 111 55 45 80 40 47
(glucose)

Mannitol 30 41 - 45.5 55 80 55
pH 5.7 4.6 - 7.2 5.8 5.5 8.5 5.7 5.7
Density (g/mL) 1.02 1.012 1.005 ‡
1.01 ‡
1.02
Osmolality 347 - 383 462 418 393 237 ‡
270 - 310
(mOsm)

Anticoagulant CPD CPD CP2D CPD CPD ACD CPD

FDA-licensed No Yes Yes Yes Yes No No
Countries used Europe United United United United Japan Germany
United States States States States
Kingdom Canada Europe
Australia
Canada
New
Zealand
Adapted from Sparrow.2
*Data were supplied and verified by manufacturers.
†
Brand names and primary sources are listed for each solution. Formulations may be available through other sources.
‡
Not reported.
SAGM = saline, adenine, glucose, and mannitol; AS = additive solution; MAP = mitogen-activated protein; PAGGSM =
phos- phate-adenine-glucose-guanosine-saline-mannitol; NaCl = sodium chloride; NaHCO 3 = sodium bicarbonate;
Na2HPO4 = sodium hydrogen phosphate; NaH2PO4 = monosodium phosphate; Na3-citrate = trisodium citrate; FDA =
Food and Drug Administration.
 C H A P TE R 6 Whole-Blood Collection and Component Processing
crossmatching, antigen typing, investigation
of adverse events of transfusion, or other ap-
propriate laboratory tests. A unique number
is imprinted on each segment. A printed
DIN is wrapped around the segment and is
archived for investigative purposes in case
any transfu- sion reactions occur.
Containers also contain integral access
ports for open connection of infusion sets or
other spike entry. Such open access limits
the outdate from the time of entry to reduce
the risk of septic transfusion reactions
caused by bacteria. This period may range
from 24 hours for RBCs held at 1 to 6 C to 4
hours for RBCs held at room temperature,
cryoprecipitate, plasma, and platelets.
Base labels and any additional labels
that are placed directly on the container
must use approved adhesives. In accordance
with the FDA guideline issued in 1985 for
the uniform labeling of blood and blood
components, only those substances that are
FDA approved as “indirect food additives”
may be used in adhe- sives and coating
components for labels placed over the base
label.3 The FDA has addi- tional standards
for labels that are applied di- rectly on
plastic blood containers. Because of a lack
of sufficient space for attaching labels di-
rectly to the container, tie tags may be used
as an appropriate alternative, particularly for
items that do not have to be directly affixed
to the container. National regulatory require-
ments should be verified when selecting
labels and establishing labeling policies.
Diversion Pouch
Collection systems intended for the prepara-
tion of platelets must include a diversion
pouch immediately after the access nee-
dle.4(p22) Diversion of the first few milliliters
of blood into a pouch has been shown to
reduce the risk of bacterial contamination of
the blood unit by capturing bacteria from the
skin (also known as a skin plug). 5 The
diversion pouch should be isolated by a heat
seal or met- al clip prior to any collection
into the primary collection container. After
the pouch is isolat- ed, blood in the pouch
may be used for donor testing.
■ 139
Container Modification by
Sterile Connecting Device
Blood containers may be modified by using
a sterile connecting device cleared or
approved by the appropriate regulatory body
for that purpose.6 FDA-approved indications
for the use of a sterile connecting device are
present- ed in Table 6-4. Use of such a
device is increas- ing, primarily to obtain a
sample from aphere- sis platelets for
bacterial contamination testing, attaching a
leukocyte reduction filter to prepare
prestorage leukocyte-reduced (LR) RBCs
and platelets, and preparing prestorage
pooled platelets and pooled cryoprecipitate.
Recently available computer software
permits tracking of lot numbers (eg, for
wafers, bags, and filters) and product codes,
as well as other steps that involve the
scanning of bar codes.
Donor and Blood Component
Identification
Identification of blood components and test
results to maintain association and linkage to
the blood donor is critical to ensure the trans-
fusion recipient’s safety by permitting look-
back investigations and product withdrawals if
indicated. Before phlebotomy, the donor is
asked to present appropriate identification.
Donor-identifying information commonly in-
cludes the donor’s first name, middle name or
initial, last name, and birth date. A donor’s So-
cial Security number is less commonly used as
an identifier because of concerns about identi-
ty theft. Contact information is necessary for
future recruitment and notification of the do-
nor about abnormal test results.
The blood component identity (ID) pro-
cess uniformly uses both a bar-coded and an
eye-readable, unique DIN that is assigned to
each sample tube and each component pre-
pared from the donation. The donor history
form and blood sample tubes are similarly
la- beled with the unique number for each
dona- tion. Electronic records of the
donation are also assigned the same number.
The DIN should be verified on the donation
record, col- lection set primary and
secondary containers, and sample tubes
before blood collection can
140 ■ AABB T EC HNIC AL MANUAL
TABLE 6-4. Use of Sterile Connecting Devices for Modification of Collection Containers 6
UseComment
To add a new or smaller needle to a blood
collection set
To prepare components
To pool blood products
To prepare an aliquot for pediatric use and
divided units
To connect additional saline or anticoagulant
lines during an automated plasmapheresis
procedure
To attach processing solutions
To add an FDA-cleared leukocyte reduction filter
To remove samples from blood product
containers for testing
RBC = Red Blood Cell; FDA = Food and Drug Administration; SOPs = standard operating procedures.
proceed as well as during and after the
collec- tion.
Phlebotomy
Vein Selection
The phlebotomist selects one arm for
phlebot- omy after inspecting both of the
donor’s arms. The phlebotomist checks for
the presence of a prominent, large, firm vein
in the antecubital fossa to permit a single,
readily accessible phlebotomy site that is
devoid of scarring or skin lesions. A blood
pressure cuff inflated at a pressure of
between 40 and 60 mm Hg or a tourniquet is
applied to enlarge the vein. Pal- pating the
vein is also recommended before the
phlebotomy site is prepared.
The first choice for the phlebotomy site is
the well-anchored median vein, which is cen-
trally located in the antecubital fossa. The sec-
ond choice is the cephalic vein that lies
laterally
If a needle is added during the procedure, an
approved device to weld liquid-filled tubing should be
used.
Examples include adding a fourth bag to make
cryo- precipitate, adding solution to the RBC unit, or
adding an in-line filter.
Appropriate use of a sterile connecting device to
pool platelets prepared from whole blood
collection may obviate potential contamination
from the spike and port entries commonly used.
FDA provides specific guidance if this activity is
con- sidered to involve manufacturing of new
products.
SOPs are required, although prior approval from the
FDA is not required.
Examples include washing and freezing RBCs.
The purpose is to prepare prestorage leukocyte-
reduced RBCs.
The label may need revision if the product’s cell
count is affected.
(shoulder side) and is often superficial. The
third choice is the basilic vein, which lies on
the underside of the antecubital fossa; the
basilic vein is not well anchored and may roll
during phlebotomy. Excessive probing with the
needle should be avoided to prevent nerve
injury.
Although the Occupational Safety and
Health Administration provides an
exemption from the requirements to wear
gloves during phlebotomy of volunteer
blood donors, some blood collection centers
require their employ- ees to wear gloves for
phlebotomy of all alloge- neic and
autologous donors.
Disinfection Methods for Venipuncture
Site
Specific instructions in the product insert for
the use of approved agents should be
followed for arm and phlebotomy site
preparation (Method 6-2). These methods
provide surgical
 C H A P TE R 6 Whole-Blood Collection and Component Processing
cleanliness, but none of the methods can
achieve an absolutely aseptic site.
Approximately 50% of donors have no
bacterial colonies on culture of the
venipunc- ture site after disinfection with
povidone io- dine or isopropyl alcohol plus
iodine tincture. Bacterial growth with low
colony numbers (1 to 100) occurs in about
half of the donors. More than 100 colonies
is rare (1%) in such cultures. 7 Bacteria
residing deep within skin layers are not
accessible to disinfectants. Therefore, if skin
tissue enters the collection bag, it can result
in contamination. In one study, pigskin
epidermal cells were detect- able in the
lavage fluid in 1 out of 150 punc- tures. 8
Whether human skin fragments or epi-
dermal cells are carried into the bag during
routine blood donation has not been studied
in detail. Nonetheless, AABB Standards for
Blood Banks and Transfusion Services
(Stan- dards)4(p22) requires collection
containers that divert the first few milliliters
of blood into a special pouch that retains
skin plugs to pre- vent contamination when
platelet compo- nents will be prepared from
the collection. Diversion of the first aliquot
of blood passing through the needle has
been shown to reduce the proportion of
blood products containing viable bacteria.9
Blood Collection Process
Method 6-3 describes steps for blood collec-
tion and sample collection for processing and
compatibility tests. The average time to collect
500 mL of blood is less than 10 minutes. A
draw time longer than 15 to 20 minutes may
not be suitable for collecting platelets or plas-
ma for transfusions.
The collection bag should be
periodically mixed during the collection to
ensure uniform distribution of anticoagulant.
Devices are available to automatically mix
the unit during collection.
Samples for donor testing may be
collect- ed from the diversion pouch after
the pouch is isolated either by clamping or
sealing the entry line. Samples for testing
may also be aseptical- ly obtained from the
collection bag with either an integrally
attached sample container or a
■ 141
sampler that is sterile and connected to the
bag. If insufficient volume is available for test
samples, a second phlebotomy may be per-
formed immediately after the blood collection.
Care should be taken to avoid diluting sample
tubes.
Blood collection containers now include
safety devices, such as a sliding sheath
needle guard, to prevent accidental needle-
stick inju- ries. These devices allow
retraction of the nee- dle into a safety guard
at the completion of blood collection.
Capping of needles should be avoided to
prevent injuries.
Volume of Blood Collected
AABB Standards permits collection of 10.5
mL of blood per kilogram of the donor’s
weight for each donation, including the
blood unit and all samples for testing. 4(p60) In
North America and Europe, the volume of
blood collected during routine phlebotomy
is typically either 450 ± 10% (405-495 mL)
or 500 mL ± 10% (450-
550 mL). The volume may be different in
other regions and may be as low as 200 to
250 mL. For general blood banking
applications, the volume of blood collected
can be determined from the net weight in
grams collected divided by the density of
WB (1.053 g/mL).
Empirical estimates may be used for the
density of RBC components as reported by
Burstain et al.10 The theoretical density may
also be calculated from the densities of the
red
cells (DRBC), density of the suspending
medium (DS), and measured hematocrit (H,
L/L): den- sity of RBC component = (DRBC –
DS ) × H + DS × (1 – H).
Collection volumes must be within the
manufacturer’s specified range to ensure the
correct anticoagulant-to-WB ratio. High-vol-
ume allogeneic collections should be
discard- ed. Low-volume allogeneic
collections should be relabeled as “RBCs
Low Volume” (Table 6-5). Plasma and
platelets from low-volume units should be
discarded.
Low-volume autologous collections
may be retained if they are approved by a
physi- cian. Plasma from low-volume units
has a higher citrate concentration than
plasma from high-volume units and should
be discarded.
142 ■ AABB T EC HNIC AL MANUAL
TABLE 6-5. Weight and Volumes of Routine-Volume, Low-Volume, and Overweight Whole Blood
Collections*
450 mL Collection Bag
Low-Volume Collection Volume
Weight
Routine-Volume Collection Volume
Weight
Overweight Collection Volume
Weight
*A density of 1.053 g/mL was used for conversion calculations. The volume and weight of anticoagulant and bag(s)
are not accounted for in this table. Low-volume Red Blood Cell products must be labeled as low-volume
products.
The evidence indicates that the volume
collected does not affect in-vivo red cell sur-
vival. Red cell recovery (average) after 21
days of storage in CPD was reported to be
accept- able (approximately 80%) when only
300 g (285 mL) of blood was collected. 11
Recovery was more than 90% when 400 g (380
mL) of blood was collected and stored for 21
days. Similarly, as much as 600 g (570 mL) of
blood collected in a standard 450-mL CPD
container and stored for 21 days had normal
in-vivo red cell recovery. Undercollected units
(275 mL or 290 g) in CPDA-1 stored for 35
days had a mean 24-hour survival rate of
88%.12 These data show that for undercollected
and overcol- lected units, variability in the
amount collect- ed does not adversely affect
red cell storage for 21 to 35 days.
Volumes of other components may also
be determined gravimetrically by dividing the
net weight in grams by the appropriate density
in g/mL. Table 6-6 provides generally accepted
densities and, for some, the applicable regula-
tory documents provide a specific value.13,16
Often, the specific gravity (ratio of density
component to density water) is used for this
purpose, assuming that the density of water is
1.00 g/mL.
Donor Care After Phlebotomy
Immediately after collection, the needle is
withdrawn into a protective sleeve to prevent
accidental injuries. Local pressure is applied
500 mL Collection Bag
300-404 mL Volume 333-449 mL
316-425 g Weight 351-473 g
405-495 mL Volume 450-550 mL
427-521 g Weight 474-579 g
>495 mL Volume >550 mL
>521 g Weight >579 g
by hand to the gauze placed directly over the
venipuncture site while the donor’s arm is
kept elevated. Pressure is applied until
hemostasis is achieved, which may require
more time for donors who are taking
anticoagulants or anti- platelet drugs. Once
hemostasis is evident, a bandage or tape
may be applied.
Postphlebotomy care includes
observing the donor for signs or symptoms
of reactions. If the donor tolerates a sitting
position without problems, he or she may
proceed to the can- teen area and is
encouraged to drink fluids and wait until
being released. Local or state laws may
specify the amount of time that the donor
must wait after the donation and before leav-
ing the collection area. The donor is encour-
aged to drink more fluids and refrain from
heavy exercise for the next 4 hours, avoid
alco- hol ingestion for several hours, and
refrain from smoking for 30 minutes. The
donor is also instructed to apply local
pressure to the phlebotomy site if any
bleeding recurs and to call the blood center
if the bleeding does not stop with pressure.
A telephone number is provided so that the
donor can report if he or she feels that the
donated unit should not be used, has any
reactions, or experiences any signs or
symptoms of infection.
Adverse Donor Reactions
In donor follow-up surveys, minor reactions,
such as dime-sized hematomas at the
phlebot- omy site, are reported by as many
as one-third
 C H A P TE R 6 Whole-Blood Collection and Component Processing ■ 143

TABLE 6-6. Density of Principal Blood Cells and Components

Blood Cell or Component Specific Gravity Source*

Cell Council of Europe13
Platelet 1.058
Monocyte 1.062
Lymphocyte 1.070
Neutrophil 1.082
Red cell 1.100
Components
Red cells collected by automated 1.06† FDA guidance14
methods with additive solution
Red cells collected by automated 1.08† FDA guidance14
methods without additive solution
Apheresis Platelets 1.03 FDA guidance15
Plasma Dependent on manufacturer of
plasma or plasma derivatives
Octapharma AG‡ 1.026
CSL Plasma §
1.027
Fenwal (Amicus Cell Separator) II
1.027
Whole blood 1.053† FDA guide for inspection of blood
banks16
*Specific gravity (SG) is the density relative to water. Assuming a density of water of 1000 g/mL, the values of SG and
density are equal.
†
See text for alternate methods to determine density of whole blood and Red Blood Cell components.
‡
Octapharma AG, Lachen, Switzerland.
§
CSL (previously ZLB) Plasma, Boca Raton, FL.
II
Fenwal, Lake Zurich, IL.
FDA = Food and Drug Administration.

of all donors.17 Adverse reactions occur at the
time of donation or are reported later in about
3.5% of donations. The American Red Cross
observed a similar rate of 4.35% (435 per
10,000 donations) in 4.3 million donations in
2007 (see Table 6-7).18 Reactions that need
medical care after the donor has left the dona-
tion site occur in 1 in 3400 donors. A popula-
tion-based European study found the rate of
complications leading to long-term morbidity
or disablement to be 5/100,000 donations and
2.3/100,000, respectively.19 Donor hemovigi-
lance programs instituted by the American
Red Cross also recorded a lower rate of
compli- cation rates for WB collections than
with apheresis methods for platelet and 2-
unit RBC collection, with minor
presyncopal reactions and small hematomas
representing most of the reactions. Major
reactions were slightly more common for
WB collection (7.4/10,000) compared with
plateletpheresis (5.2/10,000) and 2-unit red
cell apheresis (3.3/10,000).20
Systemic Reactions
Vasovagal reaction complex includes dizzi-
ness, sweating, nausea, vomiting, weakness,
144 ■ AABB T EC HNIC AL MANUAL

TABLE 6-7. Postcollection Adverse Event Rates in Calendar Year 2007 Based on 4,348,686
Whole Blood Collections in the American Red Cross System*

Adverse Event Number Rate per 10,000 Collections

Minor reactions
Presyncope 120,561 277.2
Hematoma (small) 61,029 140.3
Citrate (minor) †
31 0.1
Other (minor) 165 0.4
Allergic (minor) 30 0.1
Subtotal 181,816 418.1
LOC and major reactions
LOC (<1 minute) 4,269 9.8
LOC (>1 minute) 591 1.4
Prolonged recovery 842 1.9
LOC with injury 438 1.0
Other (major) 82 0.2
Citrate (major)† 3 0.0
Allergic (major) 2 0.0
Subtotal 6,227 14.3
Major phlebotomy-related reactions
Hematoma (large) 387 0.9
Nerve irritation 314 0.7
Arterial puncture 449 1.0
Subtotal 1,150 2.6
All adverse events 189,193 435.1
Outside medical care 1,814 4.2
Adapted with permission from Benjamin et al.18
*Citrate reactions after WB donations represent errors in classification.
†
95% confidence interval includes 1.0.
LOC = loss of consciousness; ND = not determined.

apprehension, pallor, hypotension, and tion, intravenous fluid administration in the

brady- cardia. In severe cases, syncope and
convul- sions may be observed. The pulse
rate is often low during vasovagal reactions,
but the rate is often high during volume
depletion. Some do- nors with severe
reactions or with prolonged recovery times
may need short-term observa-
emergency room, or both. A protocol for
tele- phone follow-up of donors who have
experi- enced severe reactions is helpful to
assess the donors for any residual
symptoms. Donor re- actions after WB
donation do not predict accu- rately the
possibility of recurrent syncope in
 C H A P TE R 6 Whole-Blood Collection and Component Processing
returning donors, although they reduce the
likelihood of future donations.21
Approximately 60% of systemic reactions
occur in the canteen area.17 The main predic-
tors of immediate and delayed vasovagal
and presyncopal reactions are young age,
low esti- mated blood volume, and first-time
donation status.22,23 Ingestion of
antihypertensive medi- cations does not
appear to be a risk factor.24 Approximately
15% of reactions occur away from the
donation site, usually within 1 hour of
donation.17 In donors experiencing a reac-
tion, an injury to head, face, or extremity
may occur. The staff should be vigilant to
detect re- actions early and prevent injuries
as much as possible. Deferral strategies for
young donors with estimated low blood
volume (<3.5 liters) and physiologic
strategies to minimize donor reactions in
young donors are aimed at im- proving
donor safety.21 Donor education, envi-
ronmental controls, instructions to donors to
drink water right before donation, dietary in-
terventions, and muscle tension are all effec-
tive in reducing the rate of adverse
reactions, especially in young donors, in
whom a 20% re- duction has been
observed.25,26
In case of a vasovagal reaction,
phleboto- my should be stopped, and the
donor should be placed in a recumbent
position as soon as the reaction is suspected.
Applying cold wet towels to the donor’s
neck and shoulder area and loosening the
donor’s clothes can assist in symptom
management.
Oral fluid intake before and soon after
nonautomated WB donation appears to re-
duce the frequency of systemic reactions.17
Coffee ingestion can reduce the frequency
of reactions, but it may also reduce blood
flow during collection because of its
vasoconstric- tive effect.
Bruise or Hematoma
Both symptoms are common after phleboto-
my but generally do not prevent donors
from donating again.
Fatigue
First-time donors and females are more
likely to report fatigue after donation. Donor
return
■ 145
is reduced by one-third among donors
experi- encing this symptom.17
Local Nerve Injury
Nerve injuries may be unavoidable because
nerves cannot be palpated. In 40% of cases
of nerve injuries, phlebotomy is performed
with- out difficulty.17 Donors may complain
of sen- sory changes away from the
phlebotomy site, such as in the forearm,
wrist, hand, upper arm, or shoulder. These
injuries are usually tran- sient, and recovery
almost always occurs; how- ever, in 7% of
injured donors, recovery may take 3 to 9
months.17 In severe cases, referral to a
neurologist may be indicated.
Arterial Puncture
Presence of bright red blood, rapid
collection (within 4 minutes), and a
pulsating needle suggest arterial puncture.
The hematoma rate is higher with arterial
puncture. When punc- ture is recognized
early, the needle should be pulled out
immediately, and local pressure should be
applied for an extended period. Most donors
recover quickly and completely, but some
might present with waxing and wan- ing
hematomas and should be evaluated for
pseudoaneurysm by ultrasound studies.
Upper-Extremity Deep Vein Thrombosis
Only one case of this thrombosis has been
re- ported in the literature.17 Symptoms
include pain; antecubital fossa tenderness;
swelling of the arm; and a prominent,
palpable, cord-like thickening of the
thrombosed vein. Medical referral of donors
who are experiencing deep vein thrombosis
should not be delayed so that treatment can
begin promptly.
Postdonation Mortality
The FDA requires blood establishments to
re- port deaths caused by blood donation.
For fis- cal years 2008-2012, the FDA
received 50 re- ports of postdonation
fatalities for automated and manual
collections, of which only 11 fol- lowed WB
donation. After medical review, one case
was ruled out, and in the remaining
146 ■ AABB T EC HNIC AL MANUAL

separa- tion and hard spin of the PRP. Plasma for fur-
10 cases, there was no evidence of a causal
re- lationship between blood donation and
the donors’ demise.27 Most deaths that take
place after blood donation are coincidentally
related to the donation rather than caused by
it.

BLOOD COMPONENT
PREPARATION AND
PROCESSING
Improvements and innovations continue to
be made to WB collection and its processing
into components. Apheresis is growing in
impor- tance for collection of all major
components, as discussed in Chapter 7.
Single WB units are collected in plastic
containers containing anti- coagulant;
typically CDP, CP2D, or CPDA-1 (Methods
6-3, 6-4, and 6-5). Innovations in this area
include scales for monitoring the collec- tion
volume plus automatic mixing and devic- es
to add anticoagulant at a fixed ratio as the
blood is withdrawn from the vein.
Methods and devices are available to
pro-
cess WB in a variety of manners for WB
leuko- cyte reduction, separation of plasma,
and sep- aration of red cells and platelets.
Important secondary processing includes
leukocyte re- duction, irradiation to prevent
graft-vs-host disease (GVHD), and pathogen
reduction.
For preparation of platelets from WB,
two primary methods are available (Method
6-12): preparation from buffy coats and
preparation from PRP. The buffy coat
method is employed in many regions but is
not currently cleared in the United States. In
short, non-leukocyte- reduced WB units are
first centrifuged under a high g-force (ie,
hard or heavy spin) and plas- ma, and red
cells and buffy coats are removed for further
processing. Buffy coats from 4 or 5 units are
pooled with 1 unit of plasma and then
centrifuged under a low g-force (ie, soft or
light spin) to separate the platelet
concentrate for additional processing, such
as leukocyte reduction. These processes are
often associat- ed with extended holding of
the WB and buffy coats for 8 to 24 hours at
20 to 24 C.28
Preparation of platelets from PRP
begins
with a soft spin of the WB, followed by
 closed system when various connections are
ther processing is performed, such as pooling or sampling,
removed from the thereby maintaining the product’s original ex-
platelet pellet, which is piry date.
held undisturbed for 30
to 60 minutes before WB Handling Before Component
being resuspended.29 Processing—Timing and Temperatures
Leuko- cyte reduction The temperature conditioning and handling
may occur at the PRP or of WB immediately after collection is
final platelet determined by downstream processing
concentrate stage, requirements for component preparation. In
depending on the device some cases, this may mean that collections
system used. Platelet at mobile blood drives or fixed collection
concentrates are labeled sites should be trans- ported as soon as
and stored as individual possible to the central com- ponent
units or, if cleared by preparation laboratory. With other
regulatory authorities, processing methods, transportation may not
pooled and stored as be as urgent.
transfusable doses.30 Requirements for cooling and
Compared to the transporta- tion methods are quite variable,
PRP preparation meth- and the speci- fications of the device
od, the buffy coat manufacturer should be
method yields more
plasma, greater red cell
loss, better initial white
blood cell (WBC)
reduction before
filtration, and moderate
reduction in viable
bacteria in the platelets.
Plastic collection
containers, satellite bags,
and integrally attached
tubing that are
hermetically sealed allow
component manu-
facturing to take place in
a closed system. The
blood container should
not be entered before
issue except for the
purposes of blood collec-
tion or transfer of
components to a different
container. The container
material should not have
any adverse effect on the
safety, purity, or potency
of the blood.
Components prepared
with an open system
require a reduction in
their expiration time
from the time that the
system was opened. The
use of approved ster- ile
connection devices
maintains a functional- ly
 C H A P TE R 6 Whole-Blood Collection and Component Processing
carefully followed. By the time phlebotomy
is completed, the blood has air cooled to
about 30 C.31 If such units are left at the
ambient tem- perature, the cooling rate is
quite slow and about 6 hours more are
needed for units to reach 25 C.31 To cool
blood more rapidly, units are typically placed
in specific storage envi- ronments. Some
centers use cooling plates that provide rate-
controlled cooling toward 20 C. These
cooling plates contain 1, 4-butane- diol that
has a melting temperature of 20 C and
serves as a heat absorber. With the cooling
plates, about 2 hours are needed for the col-
lected blood to reach 20 C.31
Many WB leukocyte reduction systems
al- low filtration at ambient temperature begin-
ning soon after collection and lasting up to 24
hours. WB filtration may also be started and/
or completed at refrigerator temperatures. WB
destined for platelet preparation should not be
cooled to less than 20 C. Platelets may need to
be separated from WB within 8 hours in some
regions or with some collection systems. Other
methods are approved or are under consider-
ation for up to a 24-hour hold of WB at 20 to
24 C before separation of plasma and platelets.
All processes for preparing platelets by the
buffy coat methods have an extended hold of
WB at 20 to 24 C for approximately 2 to 24
hours following collection and before the sep-
aration of the buffy coat and plasma. 13 The
pooled buffy coat is often held undisturbed for
approximately 2 to 18 hours at 20 to 24 C be-
fore separation of the platelet concentrate.
The details of these hold times are
gener-
ally adjusted to ease the operational logistics
for the blood center. For example, morning
collections are held until the afternoon when
plasma is separated and buffy coats
prepared. The buffy coats are held overnight
as either in- dividual units or pools, and the
platelets are separated the following morning.
Likewise, af- ternoon collections may be
held overnight, the buffy coats are prepared
the next morning, and platelets are prepared
in the afternoon.
WB that will not be used to prepare plate-
lets should be cooled to refrigerator tempera-
ture as soon as possible; this is often accom-
plished by placing the unit on wet ice or other
appropriate cooling media. The allowable time
■ 147
window for separation of plasma from WB is
quite variable from region to region. FFP
should be separated from the WB and frozen
within the time frame specified in the direc-
tions for use of the blood collection, process-
ing, and storage system. In the United States,
plasma that is separated from WB collection
and placed in the freezer within 8 hours can be
labeled “FFP”; if it is within 8 to 24 hours
after collection of WB (manual or apheresis
meth- od), it can be labeled as “Plasma Frozen
Within 24 Hours After Phlebotomy.” In
Europe, if the WB is refrigerated, plasma may
be separated within 18 hours; if the WB is held
at 20 to 24 C for platelet production, plasma
can be separat- ed within 24 hours, frozen, and
labeled as FFP.
Shipping containers and cooling methods
used to transport and hold WB and compo-
nents must be sufficiently validated according
to local and national regulations and guide-
lines. Validation is generally performed with
various quantities of units in the transport
container and a fixed amount of ice or gel-
pack to determine the number of units that
can be stored and transported while maintain-
ing the desired temperature. Portable temper-
ature monitors are available to record temper-
ature continuously.
Separation by Centrifugation
All current methods for separation and
prepa- ration of the three major blood
components— RBCs, platelets, and plasma
—rely on one or more centrifugation steps.
As mentioned above, the first step may be a
high g-force cen- trifugation followed by a
low g-force step for the buffy coat platelet
preparation method (the opposite of the PRP
platelet separation method) or an automated
sequence of steps in recently developed
systems. Centrifuges should be properly
validated, maintained, and calibrated in a
way that is consistent with local and
regional guidelines. Centrifuge methods for
blood component preparation should be
calibrated or checked in a systematic
manner to verify the processing conditions.
The major variables that affect the recov-
ery of cells from WB by differential
centrifuga- tion are rotor size, centrifuge
speed, duration
148 ■ AABB T EC HNIC AL MANUAL
of centrifugation, and acceleration/decelera-
tion protocol. Published papers often refer to
relative centrifugal force (g-force) that is de-
rived from the radius of the centrifuge rotor
and its revolutions. For a given centrifuge,
the rotor size is generally not variable.
Therefore, the other two variables
(centrifuge speed and duration) can be
altered in a stepwise fashion in a simplex
strategy to determine the optimal conditions
for preparing PRP.32 The simplex strategy
can also be used to identify the opti- mal
conditions of centrifugation for platelet
concentrates when quality control (QC) data
show that the platelet counts in the platelet
concentrates are not satisfactory. Method 8-
4 describes a process of functional
calibration of the centrifuge to maximize
platelet yield. The device manufacturer
should be consulted for recommendations
on validating the perfor- mance of
automated systems.
On the day of preparation, some platelet
units may contain clumps composed of plate-
let aggregates.33 In routine practice, visual in-
spection is adequate to determine the degree
of clumping subjectively and ensure that units
with excessive clumping are not released for
labeling. Most of the clumps seen on day 0
dis- appear on day 1 of storage with
continuous ag- itation, particularly those
showing light to moderate clumping.33 The
temperature at which platelets are prepared
may influence clumping; platelets prepared at
24 C appear to show the least amount of
clumping compared to those prepared at less
than 24 C.33 Visual in- spections after the
platelet concentrates are prepared have shown
an absence of visible red cells in the vast
majority of units, which im- plies that the units
contain fewer than 0.4 × 109 red cells.
Generally, the number of red cells in a unit of
platelets does not exceed 1.0 × 109.34
Blood Component Division
Following separation by centrifugation,
com- ponents must be carefully divided into
sepa- rate containers for further processing.
Many laboratories use manual expressers for
this purpose. Automated and semiautomated
de- vices, available in some regions, control
the rate of expression, monitor component
weights, add storage solutions, clamp and
seal tubing, and perform other useful
functions that assist in the consistent
preparation of blood components. Some
systems also com- bine nearly all of the
functions, including cen- trifugation,
component expression, filtration, sealing,
and additive addition, without relying on
operator interventions. The systems and
methods used for these steps, whether
predominantly manual or fully automated,
should be validated by the user.
Blood component extractors are avail-
able for making components in a semiauto-
mated manner. After primary centrifugation,
WB is placed in the extractor, and a pressure
plate creates an outflow of components from
the container. Outflow can occur from the
top or bottom of the container, depending on
the device.
When a cellular interface is detected by
an optical sensing device, outflow tubing is
au- tomatically clamped at an appropriate
level. Such devices may improve the
standardization of components, but they are
not widely used in the United States. An
automated system is available in Europe that
performs multiple functions to prepare
pooled platelet concen- trates derived from
WB by the buffy-coat method. Steps that are
automated include pooling, rinsing,
centrifugation, transfer, filtra- tion, and
sealing.
DESCRIPTIONS OF MAJOR
BLOOD COMPONENTS
WB
The minimum hematocrit of WB units in anti-
coagulant-preservative, such as CPD, is usual-
ly approximately 33%. WB is most often sepa-
rated into components and is rarely used for
transfusion directly. Severe hemorrhage cases,
such as those resulting from trauma, may ben-
efit from fresh WB transfusions when platelets
are not available.35 Labile coagulation factors
diminish and platelets may activate and devel-
op a storage lesion as the storage interval in-
creases.
WB in ACD, CPD, or CP2D has an
expira- tion date of 21 days when stored at 1 to
6 C; the
 C H A P TE R 6 Whole-Blood Collection and Component Processing
maximum storage time for units in CPDA-1
is 35 days. WB may be reconstituted by
combin- ing RBCs with thawed plasma to
achieve a de- sired hematocrit level—for
example, when used for neonatal exchange
transfusions. Ad- ditional information about
the preparation of reconstituted WB is
provided in Chapter 9.
RBCs
RBCs in anticoagulant-preservative CPD or
CPD2 have a shelf life of 21 days at 1 to 6 C
with a hematocrit of 65% to 85%, or 35 days
in CPDA-1 with a hematocrit of <80%. The
addi- tion of additive solution (Table 6-3)
reduces the hematocrit to approximately
55% to 65%. Additive solutions maintain
the red cells dur- ing storage (for example,
reduce hemolysis) and extend the expiry
date to 42 days or 56 days, depending on
regulatory approvals. He- molysis at the end
of storage should be less than 1% in the
United States or less than 0.8% in the
European Union (EU).
Hemoglobin content per unit varies be-
cause of differences between donors and pro-
cessing specifics. For example, more hemo-
globin is generally lost when buffy coat
preparation methods are used than with PRP-
type methods.
Hemoglobin content per unit may be
more precisely controlled with automated
col- lections. Total hemoglobin content is
not di- rectly regulated in the United States
but has a lower limit of 45 g per unit or 40 g
per unit for leukoreduced RBCs in the EU.36
Some experts have advocated for
standardizing the amount of hemoglobin per
RBC unit at 50 g.37 Depend- ing on local
practice, RBCs stored for less than 7 to 10
days are issued for neonatal or pediatric
transfusions, and some neonatologists may
prefer RBCs without additive solutions (see
Chapter 23).
RBCs that are labeled as low-volume
units are made available for transfusion
when 300 to 404 mL of WB is collected into
an anticoagu- lant volume calculated for 450
± 45 mL, or when 333 to 449 mL of WB is
collected into an anticoagulant volume
calculated for 500 ± 50 mL. Although the
resulting RBCs may be trans-
■ 149
fused, other components, such as platelets,
plasma, and cryoprecipitate, should not be
prepared from low-volume units.
RBCs may be subject to several secondary
processing steps, for example, leukocyte re-
duction, gamma or x-ray irradiation to
prevent GVHD, and pathogen reduction.
The FDA re- quires leukoreduced units to
contain <5 × 106 WBCs per unit and retain
>85% of the preleu- koreduced RBC
content. The EU regulations call for <1 ×
106 WBCs per unit.
Retention segments are held at the blood
center for 7 days after the expiration date of
the unit containing red cells and at the
hospi- tal transfusion service for 7 days after
the transfusion. Hemolysis identified in a
seg- ment by visual inspection often does
not cor- relate with the presence of
hemolysis in the unit. In one study,
approximately three- fourths of the visual
assessments did not agree with the
hemoglobin levels measured by chemical
methods, indicating a high false- positive
rate with visual assessment.38
Visual inspection of RBC units can
detect white particulate matter, abnormal
color caused by bacterial contamination,
hemoly- sis, and clots. Abnormal color
caused by bacte- rial contamination may be
observed in the bag, where some segments
appear to be darker than others because of
oxygen consumption in the bag. The bag
content may look purple with or without
hemolysis, and large clots may be evident.
The unit can be centrifuged to facili- tate
inspection of the supernatant in the case of
suspected bacterial contamination, and vi-
sual inspection of the supernatant may
reveal murky, brown, or red fluid.39
However, visual inspection will not detect all
contaminated units.
Blood clots in RBC units are often too
small to be detected by visual inspection.
Clots are sometimes revealed during
transfusion when they clog the filter or in
the component laboratory when the units are
filtered through a leukocyte reduction filter.
Units known to have clots should not be
released for transfu- sion.
150 ■ AABB T EC HNIC AL MANUAL
Platelets
The two major methods used in preparing
platelets from WB, buffy coat, and PRP, are
de- scribed above and in Method 6-12. Single
units of platelets are normally suspended in 40
mL to 70 mL of plasma, although studies have
shown good recovery and survival rates when
platelets are stored in plasma volumes of 35 to
40 mL.40,41 Buffy-coat-derived platelets are
nearly always pooled with the addition of
1 unit of plasma from one of the donor units or
the addition of a platelet additive solution. In
the United States, no platelet additive solu-
tions are approved for use with platelets pre-
pared from WB. Platelets prepared by the PRP
or the buffy-coat methods can be further pro-
cessed to reduce leukocytes by using a leuko-
cyte reduction filter as described in Method 6-
12.
Platelets have demonstrated superior
in-vivo recovery when stored between 20
and 24 C.42 However, at this storage
temperature, contaminating bacteria can
proliferate during storage and result in septic
transfusion reac- tions, some of which are
fatal. Therefore, platelet shelf life is limited
to 5 days in the United States, 3 days in
Japan, 4 days in Ger- many, and up to 7 days
in most EU and other countries. These limits
are primarily deter- mined by regulatory risk
assessments of the likelihood of septic
transfusion reactions.
Practices to prevent and detect bacterial
contamination have been effectively imple-
mented for apheresis platelets and prestorage
pooled platelets from WB. However,
develop- ing effective and affordable
detection methods for single-unit platelets
from WB has proved challenging, although
several manufacturers are working to
develop an effective testing so- lution.
Pathogen-reduction technologies are
anticipated by many to be the best general
protection but have been slow to gain
regula- tory approval and be implemented in
the field. Plateletsare
metabolically active
throughout the storage period. They use
both glycolysis and oxidative
phosphorylation to produce adenosine
triphosphate (ATP), a re- quirement to
maintain cell integrity and func- tion. The
product of glycolysis in platelets, lac-
tic acid, is buffered by bicarbonate and
exogenous buffers in additive solutions (eg,
phosphate). These buffer systems are
limited, and lactic acid can cause the pH of
the stored platelets to decline to less than 6.2
at 22 C, a level that results in unacceptably
low in-vivo recoveries.43 Use of oxidative
phosphorylation does not result in acid
production, and it is a much more efficient
pathway for ATP produc- tion that, in turn,
reduces the need for glycoly- sis to run at a
high rate—again limiting acid production.
Modern platelet storage contain- ers permit
oxygen to enter the storage bag to support
oxidative phosphorylation and car- bon
dioxide escape; the latter maintains the
bicarbonate buffer function. This advance in
storage containers has allowed the extended
storage of platelets beyond 3 days. The use
of acetate, a component of many platelet
addi- tive solutions, as a metabolic fuel also
contrib- utes positively to pH control
because it con- sumes protons and is
metabolized through oxidative
phosphorylation.44
Because of their metabolic requirements,
platelets must be continuously agitated
during storage. This action keeps platelets
suspended in the storage media, ensuring
effective ex- change of oxygen, carbon
dioxide, and lactic acid between the platelets
and the suspending media. Long periods of
static storage of plate- lets interrupts this
dynamic and can result in lactic acid
production with a decrease in pH.45 During
transport to hospitals from the blood center
or long-distance air transport, when
components are exchanged between blood
centers, platelets are not agitated. Instead,
they are double wrapped and secured in a
shipping system. In-vitro studies have
shown that platelets are not damaged when
they are stored without agitation for 24
hours.
Platelets must be stored and shipped at
20 to 24 C. Shipping, temporary equipment
failures, and power outages present
challenges to meeting this requirement.
Platelets main- tained in-vitro function when
they are stored at 37 C for 6 hours followed
by room-temperature storage without
agitation for an additional 18 hours.46
Several studies have demonstrated negative
effects on in-vivo platelet recovery and
survival when platelets are stored at tem-
 C H A P TE R 6 Whole-Blood Collection and Component Processing ■ 151

peratures <20 C.47,48 Thus, proper steps stored at –18 C or colder. FFP that is stored at
should be taken to maintain the required –65 C may be stored for longer than 12
range of temperatures during storage at the months, but such storage requires FDA ap-
blood cen- ter and during transport. proval.4(pp56-57) Rapid freezing of plasma can
be accomplished using a blast freezer, dry
Plasma ice, or a mixture of dry ice with either
ethanol or anti- freeze. Plasma should be
Plasma preparations are defined and
thawed at 30 to 37 C in a waterbath or by
regulated through a dizzyingly broad
using an FDA-cleared de- vice. When a
combination of collection methods, storage
waterbath is used, the compo- nent should
temperatures, freezing methods, secondary
be placed in a protective plastic overwrap.
processing, tim- ing, and storage after
Thawing of larger units of FFP col- lected
thawing. These specifi- cations are covered
by apheresis may require more time. FFP,
in an array of standards, rules, and
once thawed, has a shelf life of 24 hours at 1
guidelines overlaid with various re-
to 6 C. Thawed plasma held longer than 24
quirements of the country where the plasma
hours must be relabeled as Thawed Plasma,
is prepared and/or used. Major, although not
and it can be stored for an additional 4 days
ex- haustive, sources of this information
at 1 to 6 C.
include the US Code of Federal Regulations,
In the past several years, many blood
FDA guid- ance documents, the US Circular
cen- ters in the United States have increased
of Informa- tion,49 the AABB Standards, and
the amount of WB collected from 450 mL to
EU directives. The definitions and
500 mL, resulting in a larger amount of
requirements of the coun- try where the
plasma per unit. A unit can contain 500 mL
plasma is prepared should al- ways be
to 800 mL of plasma when collection is
consulted.
performed by auto- mated (single-donor)
Plasma is prepared from WB
plasmapheresis.
collections and by apheresis. Plasma is
The Council of Europe defines “plasma,
generally frozen to maintain factor activity
fresh frozen” as prepared from either WB or
and provide an ex- tended shelf life. Frozen
collected by apheresis. Plasma freezing must
plasma is thawed for clinical use and may be
be initiated within 6 hours of collection, within
maintained at 1 to 6 C for some time prior to
18 hours if the WB is held at 1 to 6 C, or
use. Frozen plasma is also the source of
within 24 hours if WB or apheresis plasma is
cryoprecipitate and cryopre- cipitate-
rapidly conditioned to 20 to 22 C following
reduced plasma. There are several methods
collection. Freezing must be completed within
available for pathogen reduction of plasma
1 hour to less than –30 C. Plasma, fresh frozen
that may be applied depending on na- tional
has an ex- piry time of 36 months if held at
regulatory approvals. Plasma may be used
less than –25 C or 3 months if held at –18 C to
for preparation of specific plasma protein
–25 C.13 The Council of Europe does not
products through fractionation processes.
address specific thawing methods or treatment
The descriptions below are based on the
of the plasma following thawing, including
prevailing guidance documents and
expiry dating.
regulations of the FDA and Council of
AABB requires4(p17) interventions to
Europe.
mini- mize the preparation of high-plasma
volume components (apheresis platelets,
FFP
plasma prod- ucts, and WB) for transfusion
In the United States, FFP is plasma collected from donors who are at risk of developing
either from a single unit WB collection or by HLA antibodies.50 These products should be
apheresis. FFP must be placed in the freezer collected from males, never-pregnant
within 8 hours of collection; within 6 hours females, or parous fe- male donors who test
if anticoagulated with ACD; or as directed negative for HLA anti- bodies.
by the manufacturer’s instructions for use of FFP contains normal amounts of all
the blood collection, processing, and storage coag- ulation factors, antithrombin, and
sys- tem. FFP has a shelf life of 12 months ADAMTS13.
when
152 ■ AABB T EC HNIC AL MANUAL
Quarantine FFP
Quarantine plasma was introduced to
increase the viral safety of plasma. The
Council of Eu- rope notes that quarantine
FFP can be re- leased from quarantine after
the donor returns to the blood center and has
repeatedly nega- tive test results for, at a
minimum, hepatitis B and C viruses, HIV-1,
and HIV-2 beyond a min- imum quarantine
period that is greater than the diagnostic
window period for viral infec- tion, typically
6 months. With the use of nucle- ic acid
tests for viral screening, this window period
for quarantine FFP may be reduced.51
Plasma Frozen within 24 Hours After
Phlebotomy
The FDA defines plasma that is frozen within
24 hours of collection as Plasma Frozen within
24 Hours After Phlebotomy (PF24). Once
thawed, PF24 has a shelf life of 24 hours at 1
to 6 C. Thawed plasma held longer than 24
hours must be relabeled as Thawed Plasma,
which can be stored for an additional 4 days at
1 to 6 C.
Plasma Frozen within 24 Hours After
Phlebotomy Held at Room Temperature
up to 24 Hours After Phlebotomy
The FDA defines Plasma Frozen within 24
Hours After Phlebotomy Held at Room Tem-
perature up to 24 Hours After Phlebotomy
(PF24RT24) as apheresis plasma that is held
for up to 24 hours after collection at room
tem- perature and then stored at less than –18
C. Once thawed, PF24 and PF24RT24 have a
shelf life of 24 hours at 1 to 6 C. Thawed
plasma held for longer than 24 hours must be
relabeled as Thawed Plasma and can be stored
for an addi- tional 4 days at 1 to 6 C. A similar
product pre- pared from WB held at room
temperature for more than 8 hours (ie,
overnight hold) may be defined in the future if
FDA approves an over- night-hold WB
process.
Thawed Plasma
The FDA defines Thawed Plasma (which is
not licensed by the FDA) as FFP, PF24, or
PF24RT24 that has been thawed and held at
1 to 6 C for >24 hours. Thawed Plasma may
be held at 1 to 6 C for up to 5 days after
thawing. Stable Factor II, fibrinogen, and
reduced amounts of other factors have been
observed in Thawed Plasma. Thawed Plasma
prepared from FFP and stored for 5 days
contains re- duced levels of Factor V (>60%)
and Factor VIII (>40%). ADAMTS13 levels
are well maintained for 5 days at 1 to 6 C in
Thawed Plasma.
Plasma Cryoprecipitate Reduced
Plasma cryoprecipitate reduced (United States)
or plasma, fresh frozen cryoprecipitate
deplet- ed (Europe) is a byproduct of
cryoprecipitate preparation. In the United
States, plasma cryo- precipitate reduced
must be refrozen within 24 hours at less than
–18 C. The storage tem- peratures and
expiration that apply to FFP ap- ply to this
component in both the United States and
Europe. The product contains a normal level
of Factor V (85%). Even after the removal
of cryoprecipitate, the product has a
fibrinogen level of about 200 mg/dL.52 The
lev- els of the following coagulation factors
have been demonstrated to be normal in
plasma cryoprecipitate reduced: Factor I,
Factor VII, Factor X, antiplasmin,
antithrombin, protein C, and protein S.
Levels of Factor VIII, the von Willebrand
factor (vWF) antigen, vWF activity,
fibrinogen, and Factor XIII are decreased.53
Liquid Plasma
In the United States, liquid plasma for
transfu- sion can be separated from WB at
any time during storage and stored at 1 to 6
C for up to 5 days after the WB’s expiration
date.
Recovered Plasma
Blood centers often convert plasma and
liquid plasma to an unlicensed component,
“recov- ered plasma (plasma for
manufacture),” which is usually shipped to a
fractionator and pro- cessed into derivatives,
such as albumin and/ or immune globulins.
To ship recovered plas- ma, the collecting
facility must have a “short supply
agreement” with the manufacturer. Be-
cause recovered plasma has no expiration
 C H A P TE R 6 Whole-Blood Collection and Component Processing
date, records for this component should be
re- tained indefinitely. Storage conditions
for re- covered plasma are established by the
frac- tionator. FFP used as human plasma
for fractionation in Europe must comply
with the applicable European
Pharmacopoeia guide- lines.
Pathogen-Reduced Plasma
Plasma can be treated to inactivate microbial
agents for pathogen reduction. Four such
methods are available and in use in Europe:
the methylene blue, psoralen (amotosalen),
ri- boflavin, and solvent/detergent
treatments.
Methylene blue (approximately 0.085
mg/ unit of plasma) can be added to thawed
FFP, followed by activation using white
light. After removal of methylene blue with
a filter (resid- ual concentration: 0.3 µmol),
plasma can be frozen. Methylene-blue-
treated plasma con- tains approximately
15% to 20% less Factor VIII and fibrinogen
than untreated plasma.
Plasma prepared from WB or by
automat- ed methods can be treated with 15
mL of amo- tosalen per 250 mL of plasma,
followed by illu- mination with ultraviolet A
light (320-400 nm) with 3.0 J/cm2. After
amotosalen is removed by exposing treated
plasma to an adsorption de- vice, the unit is
frozen for storage at –18 C. Av- erage
activity values for coagulation and anti-
thrombotic factors are reported to be within
reference ranges for untreated plasma.
Plasma prepared by apheresis or from
single WB units (volume range 170-360
mL) can be treated with the Mirasol system
by add- ing 35 mL of riboflavin (vitamin
B2) followed by illumination for 6 to 10
minutes. Immedi- ately after illumination,
the plasma can be re- leased or frozen below
–30 C for 2 years. Resid- ual RBC levels up
to 15 × 109 per liter have been qualified to
result in successful pathogen re- duction and
leukocyte inactivation. Coagula- tion and
anticoagulation proteins are well pre- served
in plasma treated with the Mirasol
system.54,55
Solvent/detergent-treated plasma (SD
plasma) is prepared from a pool of plasma
from many donors (no more than 2500) that
undergoes treatment with 1% tri-n-butyl
■ 153
phosphate and 1% Triton X-100 for pathogen
reduction. This treatment has been shown to
significantly inactivate lipid-enveloped virus-
es. SD plasma is manufactured in facilities that
can manage large-scale production rather
than in blood centers. Each unit contains 200
mL of plasma that is stored frozen at –18 C
with an expiration date of 12 months.56 All co-
agulation factors are reduced by 10% in SD
plasma, except for Factor VIII, which is re-
duced by 20%.57 Also, SD plasma contains
50% less functional protein S in comparison to
the nontreated FFP.58 The product is labeled
with the ABO blood group and, once thawed,
should be used within 24 hours. SD plasma is
available in Europe and has been recently ap-
proved in the United States.59
Cryoprecipitated Antihemophilic
Factor
Cryoprecipitated antihemophilic factor (AHF),
or simply “cryoprecipitate” in Europe, is pre-
pared from FFP. Cold-insoluble protein that
precipitates when FFP is thawed to 1 to 6 C is
collected by centrifugation; supernatant plas-
ma is transferred into a satellite container; the
precipitate is resuspended in a small amount
of residual plasma, generally 15 mL; and the
precipitate is refrozen as described in Method
6-11. FFP can be thawed to prepare cryopre-
cipitated AHF by placing the FFP in a
refrigera- tor (at 1 to 6 C) overnight or in a
circulating waterbath at 1 to 6 C. An alternate
method that uses microwaves for thawing has
been de- scribed. The cryoprecipitated AHF is
placed in a freezer within an hour of removal
from the refrigerated centrifuge and can be
stored at
–18 C for 12 months from the original collec-
tion date. In Europe, thawing is to be per-
formed at 2 to 6 C, and the product can be
stored for up to 36 months below –25 C and
for 3 months at –18 to –25 C.
AABB Standards4(pp28-29) requires that cryo-
precipitated AHF contain at least 80 interna-
tional units (IU) of Factor VIII and 150 mg
of fibrinogen per unit, although the average
fi- brinogen content is generally 250 mg.60
Euro- pean standards are at least 70 IU/unit
of Factor VIII, 140 mg/unit of fibrinogen, and
100 IU/unit
154 ■ AABB T EC HNIC AL MANUAL
of vWF. More current preparations are
report- ed to have much higher amounts of
fibrinogen (median, 388 mg/unit). 61
Cryoprecipitated AHF also contains the
vWF ristocetin cofactor activity
(approximately 170 units/bag), Factor XIII
(approximately 60 units/bag), and fibro-
nectin. Rapid freezing of FFP is found to in-
crease the Factor VIII yield in
cryoprecipitated AHF.62 ADAMTS13 levels
are normal in cryo- precipitated AHF.63 Anti-
A and anti-B are known to be present in
cryoprecipitated AHF, but the combined
amount of these antibodies from the unit of
plasma is only 1.15% of the total.64
Thawed cryoprecipitated AHF should
be used as soon as possible but may be
held at room temperature (20-24 C) for 6
hours as sin- gle units or a pool prepared as
a closed system using an approved sterile
connecting device or for 4 hours if pooling
was with an open system. Pooling may be
accomplished with the aid of a diluent, such
as 0.9% sodium chloride (USP), to facilitate
removal of material from individual bags.
At room temperature, the mean declines
of Factor VIII levels at 2, 4, and 6 hours are
ap- proximately 10%, 20%, and 30%,
respectively.65 Cryoprecipitated AHF from
blood groups A and B has higher levels of
Factor VIII compared to that derived from
blood group O donors (about 120 vs 80 IU per
bag, respectively).66 Thawed cryoprecipitate
should not be refrozen.
Granulocytes
Although it is possible to prepare granulocytes
from fresh WB, current practice is to collect
granulocytes by apheresis. Refer to Chapter 7
for information on automated collections.
BLOOD COMPONENT
MODIFICATION
Prestorage Leukocyte Reduction by
Filtration
For RBCs that are leukocyte reduced, the FDA
requires a residual number of <5.0 × 106 leuko-
cytes per unit. The Council of Europe requires
that the residual number be <1 × 10 6 per unit.
In the United States, leukocyte reduction by
filtration of RBCs should result in a compo-
nent that contains at least 85% of the
original red cell content. The Council of
Europe’s stan- dards require a minimum of
40 g of hemoglo- bin to be present in each
unit after leukocyte reduction.13
For WB-derived platelets, AABB Stan-
dards requires that the leukocyte reduction
process ensures that 95% of the platelet units
sampled contain <8.3 × 105 leukocytes per
unit, at least 75% of the units sampled contain
5.5 × 1010 platelets, and at least 90% of the
units sampled have a pH of 6.2 at the end of
the al- lowable storage period.4(p29) The
number in the Council of Europe’s standards
is <0.2 × 106 re- sidual leukocytes per unit
of platelets from WB.13
In practice, approximately 1% of RBC
components do not achieve the levels of <1 ×
106 residual leukocytes in the component.
Sickle cell trait of red cells is the most
common cause of filter failure. Approximately
50% of the RBC units with sickle cell trait fail
to filter. Although the other 50% pass through
the filter, the residual leukocyte content may
be higher than the allowable limits.67
Prestorage leukocyte reduction is
general- ly performed soon after WB
collection and is always performed within 5
days of collection. In-line WB filters are
available in collection sets that permit
preparation of LR RBCs and FFP. WB filters
that spare platelets are now available as well.
If WB is collected without the in-line
leukocyte reduction filter, a filter can be
attached to the tubing by an FDA-cleared
ster- ile connecting device. The number of
platelets in LR platelet concentrates is
generally lower than in non-LR platelet
concentrates.
Methods that measure residual leuko-
cytes include Nageotte hemocytometry and
flow cytometry (see Method 8-11). In a
multi- center study, flow cytometry gave
better re- sults than Nageotte
hemocytometry when freshly prepared
samples (within 24 hours) were tested. For
instance, at a concentration of 5 white cells
per µL for RBC units, the intersite
coefficient of variation was 4.9% for flow
cy- tometry and 54% for Nageotte
hemocytome- try. In general, Nageotte
hemocytometry tends to underestimate the
number of white cells compared to flow
cytometry.68 A new semiau-
 C H A P TE R 6 Whole-Blood Collection and Component Processing
tomated methodology offers to reduce the
technical burden associated with both Na-
geotte and flow cytometry methods.69
Cryopreservation
RBCs
Glycerol is the most commonly used cryo-
preservative agent and is added in either high
or low concentration to RBCs within 6 days of
collection. Two commonly used protocols for
the high-glycerol method are described in
Methods 6-6 and 6-7.
Frozen RBCs must be stored at
tempera- tures colder than –65 C and expire
after 10 years. Rare frozen units may be
used beyond the expiration date, but only
after medical re- view and approval based
on the patient’s needs and availability of
other rare compatible units. The units should
be handled with care because the containers
may crack if they are bumped or handled
roughly. The containers can also crack
during transportation.
The units should be thawed at 37 C and
generally take about 10 minutes to thaw com-
pletely. Glycerol must be removed after thaw-
ing and before transfusion. This removal is
generally accomplished by instruments that
allow the addition and removal of sodium
chloride solutions. In most cases, addition of
the glycerol and its removal
(deglycerolization) require the system to be
opened; for this rea- son, thawed and
deglycerolized units can be stored only for 24
hours at 1 to 6 C. The final solution in which
cells are suspended is 0.9% sodium chloride
and 0.2% dextrose. Dextrose provides
nutrients and has been shown to sup- port
satisfactory posttransfusion viability for 4 days
of storage after deglycerolization.70 For QC,
determining the volume of red cells in the unit
after deglycerolization and examining the last
wash for hemolysis are recommended (see
Method 6-8).
Recently, automated addition of glycerol
to RBCs and removal from RBCs in a closed
system has become possible. With this sys-
tem, glycerol is added within 6 days of WB
col- lection. Postthaw red cells prepared in this
manner are suspended in additive solution 3
(AS-3) and can be stored for 14 days at 4 C.70
■ 155
Postwash units have a hematocrit of 51% to
53% and contain a mean of about 9.0 × 106
leu- kocytes per unit.71
Platelets
Cryopreservation of platelets is not widely
available because the procedures for cryo-
preservation are complex and not routinely
practiced at most blood centers. Several
cryo- protectants have been described for
platelet cryopreservation72,73 However, 5% or
6% di- methyl sulfoxide (DMSO) is most
commonly used, mainly for autologous
platelet transfu- sions in patients who are
refractory to alloge- neic platelets.
Cryopreserved platelets can be stored for at
least 2 years. After thawing, the platelet
recovery rate in vitro is about 75%, which
may be reduced further if thawed plate- lets
are centrifuged to remove the DMSO be-
fore transfusion. The in-vivo recovery rate
af- ter transfusion of thawed, DMSO-
reduced platelets is about 35% to 42%. 74 In
vivo, the platelets that survive after
transfusion are he- mostatically effective.75
Irradiation
Cellular blood components can be irradiated
for prevention of GVHD. Frozen plasma
com- ponents, such as FFP and
cryoprecipitated AHF, are generally not
irradiated because they are considered
noncellular components. In addition, the
small number of T lymphocytes present in
the components may not survive the freeze-
thaw cycle.
The irradiation sources in use include
gamma rays—from either cesium-137 or co-
balt-60 sources—and x-rays produced by
radi- ation therapy linear accelerators or
stand- alone units. Both sources achieve
satisfactory results in rendering T
lymphocytes inactive. Freestanding
instruments that allow the use of each of
these sources are commercially avail- able
for blood bank use. The US Nuclear Regu-
latory Commission requires an increased
num- ber of controls for the radioactive
material license that is needed for a gamma
irradiator and its source onsite.76 The
increased controls are designed to reduce the
risk of unauthor- ized use of radioactive
materials. The licensee
156 ■ AABB T EC HNIC AL MANUAL

be irradiated only
is required to secure any area where radioac-
tive source is present, and only approved
indi- viduals may access the site to perform
their duties.
In the United States, the radiation dose
to the center of the irradiation field must be
at least 25 gray (Gy) [2500 centigray (cGy)]
and no more than 50 Gy (5000 cGy).77
During dosime- try, the delivered dose of 25
Gy is targeted to the internal midplane of the
container. More- over, the minimum
delivered dose to any por- tion of the blood
components must be at least 15 Gy in a fully
loaded canister.4(p25) The Euro- pean standard
requires a higher dose, with no part of the
component receiving less than 25 Gy and a
maximum dose to any part of the component
of 50 Gy.13
Each instrument must be routinely
moni- tored to ensure that an adequate dose
is deliv- ered to the container that houses the
blood components during irradiation. Dose
map- ping is used to monitor the
instrument’s func- tion. For this purpose,
irradiation-sensitive films or badges that
monitor the delivered dose are used for QC
of the irradiators. Several systems consisting
of irradiation films or badges are
commercially available.77 Verifica- tion of
the delivered dose must be performed
annually for the cesium-137 source and
semi- annually for the cobalt-60 source. For
x-ray ir- radiators, the dosimetry should be
performed in accordance with the
manufacturer’s recom- mendations. Dose
verification is also required after major
repairs or relocation of the irradia- tor. For
gamma irradiators, the turntable oper- ation,
timing device, and lengthening of irradi-
ation time caused by source decay should
also be monitored periodically.
Another important quality assurance
step
is the demonstration that the product that
was irradiated received the desired amount
of irra- diation. For that reason, irradiation-
sensitive labels are used to demonstrate that
irradiation of each batch of units was
accomplished; this is a requirement in
Europe.13
In the United States, RBCs may be
irradi- ated up to the end of their storage
shelf life. The postirradiation expiration date
is 28 days or the original expiration date,
whichever is earlier. In Europe, RBCs may
 shortest ex- piration date of the pooled units
up to day 28 following determines the expiration date of the pool.
collection. Irradiated In the United States, each pool prepared
cells may not be stored from LR platelets must have <5.0 × 106
longer than the earlier residual leukocytes. The approved pooling
of 14 days set also al- lows sampling of the pool for
postirradiation or 28 detection of bac- terial contamination. A
days postcol- lection. record of the unique DIN for each individual
Platelets may be member of the pool must be available. The
irradiated until their pool must be labeled with the approximate
expiration date, and total volume, ABO/Rh type of the units in
their postirradiation the pool, and number of units in the pool.
expi- ration date is the Many countries in Europe prepare
same as the original prestorage pools of buffy-coat platelets that
expira- tion date. are preserved in platelet additive solution or
Irradiation of RBCs in the plasma from one of the units from
followed by storage which platelets are prepared.79 Instruments
does result in a decrease that au- tomate the pooling process are
in the percentage of increasingly used in Europe. In a few
recovery after countries in Europe, systems for prestorage
transfusion. In addition, pooling of buffy-coat-
an in- creased efflux of
potassium from red
cells causes the
potassium levels to rise
approxi- mately twofold
compared to
nonirradiated units.
Platelets are not
damaged by an irradia-
tion dose as high as 50
Gy.78

Pooling
Platelets that are pooled
have an expiration time
of 4 hours from when
the system was opened
for pooling. A closed
system for prestorage
pooling of platelets has
been li- censed by the
FDA. This system
permits the storage of
pooled platelets for up
to 5 days from the time
of WB collection. Four
to six LR or non-LR
platelet units that are
ABO identical can be
pooled using a set
consisting of a multi-
lead tubing manifold for
sterile connection. If
non-LR units are
pooled, they are then
filtered as part of the
pooling process. The
 C H A P TE R 6 Whole-Blood Collection and Component Processing
derived platelets followed by pathogen-
reduc- tion treatment have become
available. The lat- ter systems are not yet
licensed by the FDA.
Cryoprecipitated AHF units may be
pooled immediately before transfusion in an
“open” system; the pool has an expiration
time of 4 hours at 20 to 24 C storage.
Prestorage pools can also be prepared in an
“open” sys- tem and stored for 12 months at
–18 C as de- scribed in Method 6-11. After
thawing, the product expires in 4 hours.
Prestorage pools prepared with the use of an
FDA-cleared ster- ile connecting device are
stored for 12 months at –18 C; postthaw
expiration time is 6 hours. The number of
units pooled may vary and can consist of 4,
5, 6, 8, or 10 units. Prestorage pools must
be placed in a freezer within 1 hour. The
potency of the pool is calculated by
assuming that each unit in the pool con-
tains 80 IU of coagulation Factor VIII and
150 mg of fibrinogen multiplied by the
number of units in the pool. If normal saline
is used to rinse the bags during preparation
of the pool, the amount of saline in the pool
must be stat- ed on the label.
Volume Reduction (Platelets)
Volume-reduced platelets may be needed
for patients in whom a reduced amount of
plasma is desired to prevent cardiac
overload, to mini- mize ABO antibody
infusion, or for intrauter- ine transfusion.
Method 6-13, describes vol- ume reduction
by centrifugation. Platelet concentrate
volume can be reduced to 10 to 15 mL/unit.
In-vitro properties such as platelet
morphology, mean platelet volume,
hypotonic shock response, synergistic
aggregation, and platelet factor 3 activity,
appear to be main- tained in the volume-
reduced platelets stored for 5 days.80 The in-
vitro recovery rate of plate- lets is about
85% after the volume-reduction step.
Platelets from WB that are volume re- duced
by centrifugation (580 × g for 20 min- utes)
from an approximate volume of 60 mL to
between 35 and 40 mL yield a high platelet
count (>2.3 × 109/L).81 Lowering the pH of
platelets prepared from WB avoids platelet
ag- gregates visible to the unaided eye
(macroag- gregates).82 The addition of 10%
ACD-A to
■ 157
platelets to lower the pH before
centrifugation can help with resuspension of
high-concentra- tion platelets and avoid
aggregation.
Apheresis platelets can also be volume
re- duced during the collection process. In
early trials, collection of apheresis platelets
in ap- proximately 60 mL showed good in-
vitro plate- let characteristics and function.
In-vivo autol- ogous recovery was at least
equivalent to that of control apheresis
platelets at standard con- centrations when
the storage time of 1, 2, or 5 days was
adjusted based on the platelet con-
centration.83,84 For apheresis platelets, the
vol- ume reduction from 250 mL to 90 mL
by cen- trifugation has been shown to cause
a mild increase in platelet activation and an
im- paired aggregation response to
adenosine di- phosphate but not to collagen.
In these experi- ments, the platelet count
before the volume reduction was 1.0 ×
109/mL; after the reduc- tion, the count was
1.9 × 109/mL.85
Recent developments include refine-
ments in collection protocols for apheresis
in- struments that result in the collection of
plate- lets in a high concentration that may,
therefore, obviate the need for volume
reduc- tion. Platelet collections were at
concentra- tions as high as 3.0 to 4.0 ×
109/L.83,86 Further- more, the highly
concentrated platelets may be suspended in a
platelet additive solution with the
autologous plasma at a ratio of 5:1 to 3:1.
Platelet units prepared in this manner
contain much lower amounts of plasma
com- pared to standard apheresis platelets. 80
Re- cently, the FDA has approved additive
solu- tions for apheresis platelets.
Posttransfusion platelet increments have
been satisfactory after transfusion of
volume- reduced platelets.80 However, the
overall in- vivo survival data for volume-
reduced plate- lets are limited. If an open
system is used, the maximum allowable
storage time is 4 hours. The maximum
allowable storage time has not been
established for closed systems.
QUARANTINE
All units of blood collected should be
immedi- ately placed in quarantine in a
designated area until donor information and
donation records
158 ■ AABB T EC HNIC AL MANUAL
have been reviewed, the current donor infor-
mation has been compared to the previous
in- formation, the donor’s previous deferrals
have been examined, and all laboratory
testing has been completed.87 Because of the
limited amount of time after collection that is
avail- able for component separation, WB
units may be separated into components
before all of the earlier processes have been
completed. Sepa- rated components are
quarantined at the ap- propriate temperature
until all of the suitabili- ty steps have been
completed and reviewed. Often, physical and
electronic quarantine are used
simultaneously.
Certain blood components from
previous donations by donors whose more
recent dona- tions test positive for infectious
disease also require quarantine and
appropriate disposi- tion, as do units
identified as unsuitable for transfusion
because of postdonation informa- tion.
Other components may need to be quar-
antined so that the QC samples can be taken
and analyzed. For instance, if a sample is
ob- tained for bacterial contamination
testing, the component is held in quarantine
for some pre- set amount of time and then
released if the test results are negative.
A thorough understanding of the
quaran- tine process is needed to prevent
erroneous release of unsuitable blood
components. Components may be removed
from the quar- antine area, labeled, and
released for distribu- tion if all of the donor
information, previous donor records, and
current test results are sat- isfactory. Some
nonconforming autologous blood
components may be released for autolo-
gous use only.
Some blood components require emer-
gency release because they have a very short
storage time. Such is the case for granulocytes.
Emergency release requires physician approv-
al and a label or tie tag to indicate that testing
was incomplete at the time of release.
Despite the widespread use of software
to control manufacturing processes,
instances of failure of quarantine and release
of unsuitable products continue to be
reported to the FDA. For instance, during
fiscal year 2011, the FDA received 54,947
reports of blood and plasma
product deviations. Of these reports, 4258
(7.7%) involved QC and distribution errors.88
LABELING
Blood component labeling is a highly
regulat- ed activity, and the documents that
describe the requirements are listed below.
Readers are advised to consult these
documents and spe- cific national
regulations for details.
The FDA requirements for labeling of
blood and components are detailed in the
Guidelines for Uniform Labeling of Blood and
Blood Components published in 1985.3 The
FDA approved the International Society of
Blood Transfusion (ISBT) 128 symbology,
Ver- sion 1.2.0, in 2000 and Version 2.0.0 in
2006.89 Detailed requirements are described in
the Code of Federal Regulations (Title 21,
CFR Parts 606.120, 606.121, and 606.122).
AABB
Standards requires that accredited facilities
use ISBT 128 labels.4(p12)
The FDA rule that requires all blood
com- ponents to be labeled with a bar-coded
label became effective on April 26, 2006.
The rule re- quires that, at a minimum, the
label contain the following bar-coded
information: 1) the unique facility identifier
(eg, registration num- ber), 2) lot number
relating to the donor, 3) product code, and 4)
ABO group and Rh type of the donor. These
pieces of information must be present in
eye-readable and machine-read- able format.
The rule applies to blood centers that collect
and prepare blood components. The rule
also applies to hospital transfusion services
that prepare pooled cryoprecipitate and/or
prepare divided units or aliquots of RBCs,
platelets, and plasma for pediatric use.
Another major part of labeling in the
United States is the information circular. The
circular must be made available to everyone
involved in the transfusion of blood compo-
nents. The Circular of Information for the Use
of Human Blood and Blood Components is
pro- duced by AABB, America’s Blood
Centers, the American Red Cross, and the
Armed Services Blood Program and is
recognized as accept- able by the FDA.49 The
circular provides im- portant information about
each blood compo-
 C H A P TE R 6 Whole-Blood Collection and Component Processing
nent and should be consulted for
information not included in this chapter.
Special message labels may also be af-
fixed to blood component containers. The la-
bels may include one or more of the following
indications: 1) hold for further manufactur-
ing, 2) for emergency use only, 3) for autolo-
gous use only, 4) not for transfusion, 5) irradi-
ated, 6) biohazard, 7) from a therapeutic
phlebotomy, and 8) screened for special fac-
tors [eg, HLA type or cytomegalovirus (CMV)
antibody status]. ISBT 128 allows incorpora-
tion of special attributes of the component,
such as CMV antibody status.
Additional information on the container
can be conveyed using a tie tag. Tie tags are
es- pecially useful for autologous and
directed do- nations. Tie tags include the
patient’s identify- ing information, name of
the hospital where the patient will be
admitted for surgery, date of surgery, and
other information that may be helpful to the
hospital transfusion service.
Each component must also bear a
unique
DIN that can be traced back to the blood do-
nor. If components are pooled, a pool
number must allow tracing to the individual
units with- in a pool.
For groups planning to implement ISBT
128, an important source of information is
ICCBBA (formerly known as the International
Council for Commonality in Blood Banking
Automation). This organization’s website fea-
tures updates and a revised list of product
codes.90
Immediate benefits of ISBT 128 include
the following:
■ Uniform labels applied on blood compo-
nents manufactured by different
collection centers.
■ Better traceability of components.
■ Improved self-checking features per char-
acter.
■ Encoding of entire ASCII character set
that includes alphanumeric and special
charac- ters.
■ Improved accuracy through reduction in
the number of misreads during scanning
of the bar-coded information.
■ 159
■ Double-density coding of numeric
charac- ters, which permits encoding of
more infor- mation in a given space.
■ Larger number of product codes, which
al- lows more detailed descriptions of
blood components.
■ Enhanced scanning, which permits more
facile auditing of movements of blood
com- ponents from one location to
another.
■ Ability to add information for autologous
donations.
■ Ability to read more than one bar code
with a single sweep (concatenation).
■ Less laborious importation of “foreign”
in- ventory into “own” inventory via the
avail- ability of uniform systems for
DINs and product codes.
In the future, ISBT 128 is expected to
per- mit information transfer by
radiofrequency ID tags or other means of
electronic data trans- mission.
QC OF BLOOD COMPONENTS
A quality management system is critical for
es- tablishing a current good manufacturing
prac- tice-compliant operation (see Chapter
1). Test- ing of blood components is
necessary to ensure the safety, purity, and
potency of the product and verify
compliance with national regulatory
requirements, such as those of FDA. These
requirements are minimum standards, and
an individual manufacturer may establish
more stringent ones. QC failures can serve
as an indicator of unexpected suboptimal re-
agents or materials. Furthermore, QC data
can reveal previously unrecognized
variations from validated procedures and
processes. A timely detection system
provides a proactive approach to early
identification and resolution of a
manufacturing problem.
Equipment QC is necessary to ensure that
blood components achieve desired
properties in a consistent manner. Suggested
QC steps for critical equipment in
component laboratories are described in
Chapter 1 and are listed in Ap- pendix 1-3.
Limitations of QC are demonstrated, for
example, in QC failures that are due to poor
160 ■ AABB T EC HNIC AL MANUAL

sampling techniques and failures that may
be ascribed to donor-related variables that
can- not be controlled. Examples of donor-
associ- ated variables include occult donor
bactere- mia or viremia and leukocyte
reduction filter failure resulting from the
presence of sickled red cells.
National regulatory authorities provide
specific minimum requirements for QC
testing of blood products. These may differ
from country to country, and the most recent
guid- ance documents and regulations
should be re- viewed. For certain blood
components, it may
be impractical to perform the QC steps. For
ex- ample, the FDA and Council of Europe
do not require QC of bedside leukocyte
reduction fil- ters.
A statistical process control approach
has been suggested for QC of blood compo-
nents.15,91,92 Such an approach is expected to
provide a definition of product conformance
to a standard with a given probability. This
ap- proach also allows a limit to be
established for nonconformance, facilitates
implementation of corrective actions, and
permits QC to be in- dividualized for
different blood components.

KEY POINTS

1. Modern blood containers are composed of soft plastic and identified by a lot number.
They should be pyrogen-free and flexible yet tough and both kink- and scratch-
resistant. During frozen storage, each plastic container has a glass transition
temperature below which it be- comes brittle and susceptible to breakage during
transportation.
2. The initial 35 to 45 mL of blood drawn is allowed to collect into a diversion pouch at
the col- lection tubing. The pouch reduces bacterial contamination of collected blood
by diverting bacteria from the skin that may have otherwise entered the collection.
Blood in this pouch may be used for laboratory tests.
3. The average rate of adverse donor reactions after donation is 3.5% to 4.5%. Most reactions
are mild and require no further medical care. These reactions can be systemic (eg, fainting)
or local (eg, hematoma). About 1 in 3400 donors experience a reaction after leaving the do-
nation site and may need medical care. Deferral of low-blood-volume (less than 3.5 L) do-
nors may be helpful in reducing the risk of reactions, especially in young donors.
4. During centrifugation for component preparation, primary variables that affect cell
separa- tion and cell recovery are rotor size, centrifuge speed, and duration. The
platelet-rich plas- ma method is used in the United States for platelet concentrate
preparation, and the buffy- coat method is more common in Canada and Europe.
5. LR RBCs and platelets are not to exceed 5.0 × 106 residual WBCs per transfusion dose in
the United States or 1.0 × 106 residual WBCs per transfusion dose in Europe.
6. In plasma prepared from WB (by manual or apheresis methods) and labeled as “Plasma
Fro- zen Within 24 Hours After Phlebotomy,” the levels of all the coagulation factors are
similar to those of FFP except for some decrease in coagulation Factor VIII.
7. The radiation dose must be 25 to 50 Gy with a minimum delivered dose to any portion
of the blood components of 15 Gy in the United States. RBCs may be irradiated up
until the end of their storage shelf life; the postirradiation expiration date is 28 days
after collection or the original expiration date, whichever is sooner. The European
standard requires that no part of the component receive less than 25 Gy, a maximum
dose to any part of the component of 50 Gy, and irradiation of RBCs only until day 28,
with storage for no longer than 14 days after irradiation or 28 days after collection,
whichever is earliest. Platelet shelf life is not reduced after irradiation.
8. Bar-coded and eye-readable container labels are increasingly using the ISBT
symbology (ISBT 128). ISBT 128 offers a number of advantages over the Codabar
symbology, including identification of the manufacturer throughout the world, more
product codes, better accu-
 C H A P TE R 6 Whole-Blood Collection and Component Processing
racy as a result of reduced misreads during scanning, and enhanced conveyance of other
la- beling information.
9. Various approaches for QC of blood components have been promulgated by the AABB,
Council of Europe, and FDA.
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2. Sparrow RL. Time to revisit red blood cell
addi- tive solutions and storage conditions: A
role for “omics” analyses. Blood Transfus
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3. Food and Drug Administration. Guideline for
the uniform labeling of blood and blood
components. (August 1985) Silver Spring,
MD: CBER Office of Communication,
Outreach, and Development, 1985. [Available
at http://www.
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6. Food and Drug Administration. Guidance for
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 C h a p t e r 7
Blood Component Collection
by Apheresis
James W. Smith, MD, PhD
“A P H E R E S I S ,” “ P H E R E S I S ,”
“hema-
pheresis,” and all of the various terms
used to refer to automated blood component
collection procedures are derived from the
Greek word “aphairos,” meaning “to take
from.” Specifically, whole blood is
separated into components during
collection, the desired component is
removed/modified, and the re- maining
components are returned to the do- nor or
patient. Centrifugal and membrane- based
apheresis techniques were under
development in the late 19th and early 20th
centuries. By the 1970s, multiple improved
technologies had emerged and apheresis
pro- cesses advanced rapidly.
Today, centrifugal technique is primarily
used in the United States, whereas membrane
filtration is used in other parts of the world
(pri- marily Europe and Japan) for donor
apheresis.
Early versions of automated, computer-
ized, and centrifugal techniques facilitated
large-scale donations of platelets, plasma,
and granulocytes. As the technology
continued to evolve, equipment, disposables,
and software became increasingly
sophisticated, and now
James W. Smith, MD, PhD, Medical Director, Oklahoma Blood Institute, Oklahoma City,
Oklahoma The author has disclosed no conflicts of interest.
the collection of various combinations of
com- ponents is possible (see Table 7-1).
This chap- ter provides a discussion of the
technology and instrumentation used during
donor apheresis, with special consideration
given to the associated regulatory
requirements.
COMPONENT COLLECTION
The collection of components by apheresis
fol- lows many of the same rules and
guidelines that apply to whole-blood
donation. Like whole-blood donors,
apheresis donors must be given sufficient
information to enable them to give informed
consent to donate blood. Al- though the
apheresis collection and prepara- tion
processes differ from those used for whole-
blood-derived components, the storage and
transportation requirements and several
quality-control steps are the same for both
processes. Another similarity is that the
facility must maintain written procedures
and proto- cols for all types of collections
used and must keep records of each
procedure as required by AABB Standards
for Blood Banks and Transfu-
167
168 ■ AABB T EC HNIC AL MANUAL

TABLE 7-1. Components that Can Be Collected with Various Instruments

Instrument GRAN PLT cRBC 2-RBC PLASMA cPLASMA

Fenwal ALYX X X X
Fenwal Amicus X X X
Fenwal Autopheresis C X
Fresenius AS104 X
TerumoBCT (COBE) X X X
Spectra
TerumoBCT Spectra Optia X
TerumoBCT Trima V-4 X X X X
TerumoBCT Trima Accel X X X X
Haemonetics Cymbal X
Haemonetics MCS+ X X X
LN9000
Haemonetics MCS+ X X X
LN8150
Haemonetics PCS-2 X
*Concurrent collection refers to the ability to collect more than one type of product.
GRAN = granulocytes; PLT = plateletpheresis (single, double, triple); cRBC = concurrent* 1 unit of Red Blood Cells
(RBCs); 2-RBC = double unit of RBCs; PLASMA = 1 unit of plasma; cPLASMA = concurrent plasma; V-4 =
software version 4.

sion Services.1(pp62-67) The circumstances that

are unique to apheresis collection are ad-
dressed in the sections that follow.
Platelets
Apheresis is used to obtain platelets from
vol- unteer donors, patients’ family
members, or donors with HLA or platelet-
antigen-compati- ble phenotypes. By design,
apheresis proce- dures are intended to collect
large numbers of platelets from an
individual, thereby providing a more potent
product with fewer donor expo- sures for the
patient. AABB Standards requires that an
apheresis platelet component contain at least
3 × 1011 platelets in 90% of sampled
units.1(pp28-29)
With newer technology and more effi-
cient processes, higher yields of platelets
may be obtained from one donor, and the
original apheresis unit may be split into
multiple units,
each of which must meet minimum
standards. Some instruments are
programmed to calcu- late the platelet yield
based on the donor’s he- matocrit, platelet
count, height, and weight.
For alloimmunized patients who do not
respond to random allogeneic platelets,
trans- fusions of platelets from an apheresis
donor selected on the basis of a compatible
platelet crossmatch or that are matched for
HLA anti- gens may be the only way to
achieve a satisfac- tory posttransfusion
platelet increment. In the United States, the
use of apheresis platelets has been steadily
increasing over the past 25 years. It is
estimated that 90% of platelets transfused in
the United States are apheresis platelets.2
Donor Selection and Monitoring
Plateletpheresis donors may donate more
fre- quently than whole-blood donors but
must
 CH A P T E R 7 Blood Component Collection by Apheresis ■ 169
separator device (which may be more or less than the 500-
mL or 600-
meet all of the other criteria for whole-blood
donation. The interval between donations
should be at least 2 days, and donors should
not undergo plateletpheresis more than
twice in a week or 24 times in a rolling 12-
month pe- riod.1(p20),3 If the donor donates a
unit of whole blood or if it becomes
impossible to return the donor’s red cells
during plateletpheresis, at least 8 weeks
should elapse before a subse- quent
plateletpheresis procedure unless the
extracorporeal red cell volume (ECV) is less
than 100 mL. Platelets may be collected
from donors who do not meet these
requirements only if the component is
expected to be of par- ticular value to a
specific intended recipient and a physician
certifies in writing that the do- nor’s health
will not be compromised by the donation.
Donors who have taken antiplatelet
medications that irreversibly inhibit platelet
function are deferred for specific intervals
be- fore donation (48 hours for
aspirin/aspirin- containing medications and
piroxicam, 14 days for clopidogrel and
ticlopidine) because apheresis platelets are
often the sole source of platelets given to a
patient.1(p61),3
A platelet count is not required before
the
first apheresis collection; however, triple
col- lections of platelets may not be drawn
from first-time donors unless a qualifying
platelet count is obtained on a sample
collected before the procedure.3 If the
donation interval is less than 4 weeks, many
facilities prefer that the donor’s platelet
count be above 150,000/µL be- fore
plateletpheresis occurs to prevent a post-
donation count of less than 100,000/µL.
AABB Standards permits qualification of a
donor with a platelet count from a sample
collected immediately before the procedure
or one ob- tained either before or after the
previous pro- cedure.1(p21) Exceptions to
these laboratory cri- teria should be approved
in writing by the apheresis program
physician based on docu- mented medical
need.
The Food and Drug Administration
(FDA) specifies that the total volume of
plasma col- lected should be no more than
500 mL (or 600 mL for donors weighing
more than 175 lb) or the volume described
in the labeling of the au- tomated blood cell
 compatible with the recipient’s plasma and
be crossmatched.1(pp37-38) In such cases, a
mL volume previously sample of donor blood is attached to the
mentioned). The plate- let con- tainer for compatibility testing. In some
count of each unit should be in- stances, it may be desirable for the donor
kept on record but need not plas- ma to be ABO compatible with the
be written on the component recipient’s red cells (eg, if the recipient is a
la- bel. Units containing less child or an ABO-mismatched allogeneic
than 3.0 × 1011 plate- lets progenitor cell transplant recipient). In the
should be labeled with the United States, to be considered leukocyte
actual platelet count.3 reduced, apheresis platelets must contain
It is possible to collect less than 5 × 106 leuko- cytes per unit and
plasma concur- rently with platelets must meet the specifications of the
platelets. Such collection is apheresis device manufacturer.3 In Europe,
dis- cussed in more detail in the guideline for
the “Plasma” section below.
Vasovagal and
hypovolemic reactions are
rare in apheresis donors but
may occur. Pares- thesias
(tingling sensations) and
other reac- tions to citrate
anticoagulant are not uncom-
mon. (Similar citrate toxicity
reactions in recipients are
discussed along with whole
blood transfusions in Chapter
27.) Serious re- actions occur
less often among apheresis
do- nors than whole blood
donors.4

Laboratory Testing
Tests for ABO group, Rh
type, unexpected allo-
antibodies, and transfusion-
transmitted dis- eases must
be performed by the
collecting fa- cility in the
same manner as for other
blood components. Each unit
must be tested unless the
donor is undergoing repeated
procedures to support a
specific patient, in which
case testing for infectious
disease markers needs to be
repeated only at 30-day
intervals.5
If red cells are visible in
a product, the he- matocrit
should be determined. AABB
Stan- dards state that if the
component contains more
than 2 mL of red cells, the
red cells must be ABO
170 ■ AABB T EC HNIC AL MANUAL
leukocyte-reduced components is fewer than
1 × 106 leukocytes per unit.
Record-Keeping
Complete records must be kept on each proce-
dure. All adverse reactions occurring during
collection procedures (or transfusion) must be
documented along with the results of thor-
ough investigations. Records of all laboratory
findings and collection data must be periodi-
cally reviewed by a knowledgeable physician
and must be found to be within acceptable
limits. FDA guidelines require a periodic re-
view of donor records to monitor platelet
counts.3 Facilities must have policies and pro-
cedures in place to ensure that donor red cell
loss during each procedure does not exceed
acceptable limits.1(p18)
Plasma
Apheresis devices can be used to collect
plas- ma for transfusion or as Source Plasma
for subsequent manufacturing. Recently, the
FDA approved apheresis devices for the
collection of plasma held at 1 to 6 C within
8 hours and frozen within 24 hours after
phlebotomy and plasma held at room
temperature for up to 24 hours and frozen
within 24 hours after phle- botomy.
The FDA has provided guidance with
re- gard to the volume of plasma that may be
col- lected using automated devices. A
distinction is made between infrequent
plasmapheresis, in which the donor
undergoes plasmapheresis no more
frequently than once every 4 weeks, and
serial plasmapheresis (or Source Plasma
collection, the process to collect plasma for
fractionation into plasma components), in
which the donation is more frequent than
once every 4 weeks. For donors in
infrequent plasmapheresis programs, donor
selection and monitoring requirements are
the same as those for whole-blood donation.
Plasma ob- tained by these processes is
intended for direct transfusion.
For serial plasma (Source Plasma)
collec- tion using either automated
instruments or manual techniques, the
following principles apply6:
1. Donors must give consent for the proce-
dure, and they must be observed closely
during the procedure. Emergency medical
care must always be available.
2. Red-cell losses related to the procedure,
including samples collected for testing,
should be monitored so that no more than
200 mL of red cells are removed in 8
weeks. If the donor’s red cells cannot be
returned during an apheresis procedure,
hemapher- esis or whole-blood donation
should be deferred for 8 weeks.
3. For manual collection systems, a mecha-
nism must exist to ensure safe reinfusion
of the autologous red cells.
4. In manual procedures for donors weighing
110 to 175 lb, no more than 500 mL of
whole blood should be removed at one time
or no more than 1000 mL during a session
or within a 48-hour period. The limits for
donors who weigh  175 lb are 600 mL and
1200 mL, respectively. For automated pro-
cedures, the allowable volume has been
determined for each instrument by the
FDA.
5. At least 48 hours should elapse between
successive procedures. Donors should not
undergo more than two procedures within
a 7-day period.
6. At the time of initial plasmapheresis and at
4-month intervals for donors undergoing
serial (large-volume) plasmapheresis
(donors undergoing plasmapheresis more
often than once every 4 weeks), serum or
plasma must be tested for total protein
and for serum protein electrophoresis or
for quan- titative immunoglobulins.
Results must be within normal limits.
7. A qualified licensed physician, knowledge-
able about all aspects of hemapheresis,
must be responsible for the program.
For manual collection systems, a
process that is rarely used in the United
States at pres- ent, requirements are outlined
in the Code of Federal Regulations6 and
have been summa- rized in previous editions
of this Technical Manual.
 CH A P T E R 7 Blood Component Collection by Apheresis
Red Cells and Multicomponent
Donations
Both AABB Standards and FDA guidance
doc- uments address the removal of red cells
by au- tomated apheresis methods. A
guidance docu- ment issued in 2001 by the
FDA finalized recommendations for the use
of automated apheresis equipment to collect
the following7:
■ Single units of RBCs and plasma.
■ Single units of RBCs and platelets.
■ Single units of RBCs, platelets, and plasma.
■ Double units of RBCs only.
The guidance document includes FDA
regulations requiring that equipment
perform and be used in the manner for
which it was de- signed to collect or process
blood and compo- nents. Standard operating
procedures, includ- ing device
manufacturers’ instructions for use and
maintenance of current records, are de-
scribed. The sections below summarize the
in- formation in the guidance document.
Donor Selection and Monitoring
The FDA requires that an adequate
hemoglo- bin level be determined by a
quantitative method for predonation
hemoglobin or the hematocrit of donors
undergoing double-RBC collection. The
procedure is limited to persons who are
larger and have a higher hematocrit than the
minimum standards for whole-blood
donations. For males, the minimum weight is
130 lb, and the minimum height is 5 1. For
fe- males, the minimum weight is 150 lb,
and the minimum height is 5 5. The
minimum hema- tocrit is 40% for both
genders. Donors who are shorter than the
minimum height or who weigh less than the
minimum weight, as estab- lished by the
FDA in device operator manuals, should be
further evaluated. These donors must also
meet all relevant FDA criteria for al-
logeneic or autologous whole-blood
donation. Donors who have given a single
unit of RBCs with platelets, plasma, or both
should be deferred for at least 8 weeks. The
exception is when a donor serves as a
plateletpheresis do- nor or a donor of
platelets with plasma by-
■ 171
products within 8 weeks and the ECV of the
procedure is <100 mL. Donors should be de-
ferred for at least 16 weeks after a double-
RBC donation. If an apheresis procedure is
discon- tinued before completion and absolute
red cell loss is <200 mL, the donor may donate
again within 8 weeks if he or she meets all
donor eli- gibility criteria.
If a donor has a second red cell loss of
<100 mL during a subsequent donation within
8 weeks of a previous donation, the donor
should be deferred for 8 weeks. If the total
ab- solute red cell loss within 8 weeks is
>300 mL, the donor should be deferred for
16 weeks from the date of the last red cell
loss. If an apheresis procedure is
discontinued and the absolute red cell loss is
>200 mL but <300 mL, the donor should be
deferred for 8 weeks. If an apheresis
procedure is discontinued and total absolute
red cell loss is >300 mL, the donor should
be deferred for 16 weeks.
Saline infusion is used to minimize vol-
ume depletion.
Quality-Control Issues
The FDA has promulgated quality-control
(QC) programs for RBC unit collection by
apheresis. There are two phases:
■ In Phase I QC, 100 consecutive RBC units
are tested to determine the expected or tar-
get red cell volume in accordance with the
specifications in the device operator’s man-
ual. Target values are compared with the
actual values to determine product accept-
ability. If the QC results are satisfactory,
which includes that at least 95% of the
units meet product specifications, the
establish- ment may proceed to Phase II.
■ Phase II QC consists of monthly testing of
a representative sample of 50 units of the
manufactured product from each collec-
tion center. At least 1 unit from a single
RBC protocol or both units from a double-
RBC protocol device used at the center
should be included in the testing. At least
95% of the products tested should meet
product specifications as described in the
device operator’s manual.
172 ■ AABB T EC HNIC AL MANUAL
Record Requirements
US blood establishments must update their
blood establishment registrations and
product listing forms with the FDA to
collect RBCs us- ing automated methods.
Automated RBC col- lection has substantial
potential to have an ad- verse effect on the
identity, strength, quality, purity, or potency
of a product. Blood estab- lishments that are
approved to manufacture RBCs with one
manufacturer’s device and that want to use
another manufacturer’s device in- stead
must submit a prior approval supple- ment
and must receive FDA approval before
distribution of the product manufactured on
the new device. The FDA requires these
estab- lishments to make available a number
of rec- ords and forms regarding RBC or
multicompo- nent unit collection for FDA
inspection. These records and forms include
documents ad- dressing donor consent,
donor eligibility, product collection, and
product QC.7
Granulocytes
The use of granulocyte transfusions has been
controversial for a number of years. Analysis
of randomized controlled trials of granulocyte
transfusions in adults has indicated that an ac-
ceptable minimum dose (>1 × 1010 granulo-
cytes/day) and crossmatch compatibility (no
recipient antibodies to granulocyte antigens
have a major impact on the effectiveness of
such transfusions.8 Recently, there has been
renewed interest in granulocyte transfusion
therapy because much larger cell doses may be
obtained from donors who have received re-
combinant colony-stimulating factors.
Agents Administered to Increase Yields
AABB Standards requires that 75% of
granulo- cyte components contain at least 1
× 1010 gran- ulocytes,1(p30) although the
optimal therapeutic dose in adult patients is
unknown. For infants and children, a dose of
10 to 15 mL/kg may provide an adequate
number of granulocytes per dose. To collect
this number of cells in a unit of
granulocytes, one must administer drugs or
sedimenting materials to the donor.
Sedimenting agents enhance granulocyte
har-
vest by increasing sedimentation of the
RBCs, thereby enhancing the interface in the
collec- tion device and resulting in minimal
RBC con- tent in the final product. The
donor’s consent to the procedure must
provide permission for any of these drugs or
sedimenting agents to be used.
H Y D ROX YE TH Y L STA RCH . A common
sedi- menting agent, hydroxyethyl starch
(HES) causes red cells to aggregate, thereby
sedi- menting them more completely.
Because HES can be detected in donors as
long as a year af- ter infusion, AABB
Standards requires facilities performing
granulocyte collections to have a process to
control the maximum cumulative dose of
any sedimenting agent administered to a
donor within a given interval.1(p23) HES is a
colloid that acts as a volume expander.
Donors who receive HES may experience
headaches or peripheral edema because of
expanded cir- culatory volume. A boxed
warning from the FDA exists for its use due
to increased mortali- ty and severe renal
injury in certain patient populations.9
C O R T IC OS TER O IDS . Corticosteroids
can double the number of circulating
granulo- cytes by mobilizing granulocytes
from the marginal pool. The common
protocol is to use 60 mg of oral prednisone
in a single or divided dose before donation
to collect large numbers of granulocytes
with minimal systemic steroid activity.
Another protocol uses 8 mg of oral
dexamethasone. Donors should be
questioned about their relevant medical
history before they use systemic
corticosteroids. Hyperten- sion, diabetes,
cataracts, or peptic ulcer can be relative or
absolute contraindications to corti- costeroid
use.
GROW TH FACTORS. Although not a licensed
indication, granulocyte colony-stimulating
factor (G-CSF) can effectively increase granu-
locyte yields. Hematopoietic growth factors
given alone can result in the collection of up to
4 to 8 × 1010 granulocytes per apheresis proce-
dure. Typical doses of G-CSF are 5 to 10
µg/kg given 8 to 12 hours before granulocyte
collec- tion. Preliminary evidence suggests
that in- vivo recovery and survival of these
granulo-
 CH A P T E R 7 Blood Component Collection by Apheresis
cytes are excellent and that growth factors
are well tolerated by donors.
Laboratory Testing
Testing for ABO and Rh group, red cell anti-
bodies, and infectious disease markers is re-
quired on a sample drawn at the time of phle-
botomy. Red cell content in granulocyte
products is inevitable; the red cells should be
ABO compatible with the recipient’s plasma. If
>2 mL of red cells are present, the component
should be crossmatched for Rh compatibility
and HLA compatibility. 1(pp37-38)
Storage and Infusion
Granulocyte function deteriorates rapidly
dur- ing storage, and concentrates should be
trans- fused as soon as possible after
preparation. AABB Standards mandates a
storage tem- perature of 20 to 24 C for no
longer than 24 hours.1(p55) Agitation during
storage is undesir- able. Irradiation is
required for products to be administered to
immunodeficient recipients and is indicated
for nearly all recipients be- cause their
primary diseases are likely to in- volve
deficiencies in their immune systems. Use of
a microaggregate or leukocyte reduc- tion
filter is contraindicated because it re- moves
the collected granulocytes.
INSTRUMENTS AND SYSTEMS FOR
DONOR APHERESIS
COLLECTIONS
This section provides descriptions of equip-
ment that is available for use in the United
States for the collection of blood components
by automated techniques. A brief description
is given of each instrument; more detailed
information can be found in other resourc-
es.10,11 This section does not include the
CS3000 and CS3000+ (Fenwal, Lake Zurich,
IL) because the company has discontinued the
sale of these devices in the United States (al-
though disposables for the devices are still
available at the time of this writing). These
two devices are capable of collecting RBCs,
plate- lets, concurrent plasma, and
granulocytes.
■ 173
Plasma
Fenwal Autopheresis C
The Autopheresis C (Fenwal) is an instrument
designed to collect plasma only.11 It uses a
rotating cylindrical filter to separate the plas-
ma from the cellular elements of blood. Be-
cause of the high efficiency of the rotating fil-
ter, the filter is small and the system’s ECV is
approximately 200 mL. The Autopheresis C is
a single-access system, and saline replacement
can be administered. It is considered an open
system and can collect several units of plasma.
According to the FDA definition, an open sys-
tem requires that plasma outdates 4 hours af-
ter thawing. Variances can be granted that al-
low 24-hour outdates, but these units cannot
be relabeled as Thawed Plasma.
Haemonetics PCS-2
The PCS-2 (Haemonetics, Braintree, MA), a
simplified version of the MCS Plus, is
designed for plasma collection.12 The PCS-2
uses a blow- molded (grenade-shaped)
centrifuge bowl to separate plasma from
cellular elements. De- pending on the degree
of cell reduction re- quired, one of three
versions of the PCS-2 bowl can be used:
standard, filter core, or high sepa- ration
core. The standard bowl uses centrifu- gal
force to remove the plasma from the top of
the bowl. To increase cell reduction, the
filter core and high separation core bowls
allow plasma to pass through the core,
which is cov- ered with a filter membrane.13-
15
Use of this de- vice results in the greatest
cell reduction, but it has not been released in
the United States at the time of this writing.
The ECV of the PCS-2 is variable
depending on the hematocrit of the donor
and ranges from 491 mL (38% hemato- crit)
to 385 mL (50% hematocrit). The PCS-2 is
a single-access system, and saline replace-
ment can be administered. It is considered
an open system. As noted for the Auto-C, a
vari- ance can be granted allowing 24-hour
out- dates of plasma processed with this
device, but these units may not be relabeled
as Thawed Plasma. The PCS-2 can collect
several units of plasma at a time.
174 ■ AABB T EC HNIC AL MANUAL
Equipment for Concurrent Plasma
Collection
Plasma can be collected as a concurrent
prod- uct during collection of apheresis
platelets or automated RBCs. The amount
that can be col- lected is determined by the
volume of plate- lets, red cells, or both, that
are being collected and by the maximum
volume that can be re- moved from the
donor. Equipment capable of concurrent
plasma collection includes Hae- monetics
MCS+ LN9000, Haemonetics MCS+ LN8150,
Fenwal Amicus, Fenwal ALYX, Teru- moBCT
(COBE) Spectra (TerumoBCT, Lake- wood,
CO), TerumoBCT Trima, and Teru- moBCT
Trima Accel.
Platelets
TerumoBCT (COBE) Spectra
The TerumoBCT (COBE) Spectra is capable
of collecting single, double, or triple
apheresis platelet units as well as concurrent
plasma, de- pending on the size and platelet
count of the donor.10,16-19 This device uses a
dual-stage (dif- ferent radius) channel to
collect leukocyte- reduced platelets.
Leukocyte reduction to <5 × 106 White
Blood Cells (WBCs) can be obtained in
approximately 85% of the collections for
software versions that are lower than 5.0.20
Use of software versions 5.0 and 7.0 can
more con- sistently result in the collection of
products containing <1.0 × 106 WBCs with
the leukocyte- reduction system (LRS),
which uses a cone in the centrifuge and
saturated, fluidized, particle- bed filter
technology to remove residual WBCs
leaving the second stage of the channel.10,16-19
The Spectra is capable of single- or double-
access procedures. The ECV of the single-
access kit is 361 mL and of the double-
access kit is 272 mL. The Spectra is being
replaced by the more efficient Trima or
Trima Accel.11
TerumoBCT Trima and Trima Accel
The TerumoBCT Trimas were designed as
automated donor collection machines for
platelets, plasma, and RBCs only. The Teru-
moBCT Trima (Version 4) uses a smaller,
mod-
ified, dual-stage channel and LRS cone to
con- sistently collect leukocyte-reduced
platelets (<1.0 × 106 WBCs). To increase
platelet yields, the Trima Accel (Versions
5.0, 5.01, and 5.1) uses a single-stage,
doughnut-shaped channel and larger LRS
cone to consistently collect leu- kocyte-
reduced platelets.21 The Trimas use sin- gle-
access kits only. The ECV of the Trimas is
182 to 196 mL. They are capable of
collecting single, double, or triple units of
apheresis platelets as well as concurrent
plasma and RBCs, depending on donor size,
platelet count, and hematocrit.21-23
Fenwal Amicus
The Amicus is capable of collecting single,
double, or triple apheresis platelets as well
as concurrent plasma and RBCs (single-
access kit only), depending on the donor’s
size, platelet count, and hematocrit.10,16,22,24
The Amicus uses centrifugal force and a
double compart- ment belt wrapped around
a spool to separate the platelets. The
platelets accumulate in the collection
chamber and are transferred to the final
collection bags at the end of the proce- dure.
The Amicus is capable of single- or double-
access procedures. The ECV of the double-
access kit is 210 mL, and that of the single-
access kit is 209 mL. The ECV of the
whole-blood bag is adjustable. Consistent
leu- kocyte reduction is accomplished
without ex- ternal filtration, and this process
is approved by the FDA for submission of a
prior approval supplement.
Haemonetics MCS+ LN9000
The Haemonetics MCS+ LN9000 system is
ca- pable of conducting several different
apheresis procedures, including apheresis
platelet col- lection. It uses the Latham
conical bowl, plas- ma-controlled
hematocrit, and plasma surge technique to
build up the platelets and float them off the
bowl with rapid plasma infusion. Although
this technique results in leukocyte- reduced
platelets, the use of an inline leuko- cyte
reduction filter ensures consistency in
leukocyte reduction.10,25-28 The LN9000 uses
a single-access kit, and the ECV ranges
from 480 mL (38% hematocrit) to 359 mL
(52% he-
 CH A P T E R 7 Blood Component Collection by Apheresis
matocrit). The LN9000 is capable of
collecting single, double, or triple units of
apheresis platelets as well as concurrent
plasma, de- pending on donor size and
platelet count.10,25-28
RBCs
TerumoBCT Trima and Trima Accel
As previously mentioned, the Trima can col-
lect a single unit of RBCs concurrently with
platelets.11,23,29 A kit is available to collect a
double unit of RBCs with or without
concur- rent plasma, depending on the
donor’s size and hematocrit. The Trima uses
the single- stage channel for the double-RBC
collection, and saline is returned to the
donor during the collection. The addition of
the preservative solution and leukocyte
reduction by filtration are performed
manually and off-line after the collection is
complete for both the single and double
units of RBCs.
Fenwal ALYX
The Fenwal ALYX was developed as an auto-
mated, double-RBC collection system only.
Recently, it has been approved to collect con-
current plasma as well. The ALYX uses a
rigid, cylinder-shaped chamber in the
centrifuge to separate the plasma from the
cells. The plasma is collected in one bag, and
the red cells are collected in a separate bag.
During reinfusion, the plasma and saline are
returned to the do- nor. When the collection is
complete, the ALYX automatically adds the
preservative solution and pumps the red cells
through an inline leu- kocyte reduction filter
into the final storage bags. The ALYX uses a
single-access kit only, and its ECV is
approximately 110 mL. The ALYX is capable
of collecting 2 units of RBCs or 1 unit of
RBCs and concurrent plasma at a time,
depending on donor size and hemato- crit.11,28-
30
Fenwal Amicus
As mentioned previously, the Fenwal
Amicus can collect a single unit of RBCs
concurrently with plateletpheresis, but only
with the single-
■ 175
access kit.11,31 The addition of the preservative
solution and leukocyte reduction by filtration
are performed manually and off-line after the
collection is complete.
Haemonetics Cymbal
The Cymbal is a collection system that uses an
expanding, variable-volume bowl. The system
has a relatively small ECV when compared to
the Haemonetics MCS+ LN8150 and collects
a double-RBC unit.32
Haemonetics MCS+ LN8150
The Haemonetics MCS+ LN8150 was
designed as a donor instrument only for the
collection of RBCs and concurrent plasma.
The LN8150 uses the blow-molded bowl that
is also used for plasma collection. It uses a
single-access kit and its ECV varies,
depending on the do- nor’s hematocrit, from
542 mL (38% hemat- ocrit) to 391 mL (54%
hematocrit). The LN8150 is capable of
collecting a single or double unit of RBCs
with or without concurrent plasma, depending
on the donor’s size and hemat- ocrit.11,29,33 The
addition of preservative and leukocyte
reduction by filtration are per- formed
manually and off-line after the collec- tion is
complete.
Granulocytes
TerumoBCT (COBE) Spectra
The Spectra is capable of collecting granulo-
cytes.11,34,35 It uses a doughnut-shaped, single-
stage channel in the centrifuge to isolate the
granulocytes that are continuously collected
in the final storage bag. It uses a double-
access kit only, and its ECV is 285 mL.
TerumoBCT Spectra Optia
The Spectra Optia system (not to be confused
with the Spectra system above) used mainly
for therapeutic apheresis procedures has re-
cently been approved for granulocyte collec-
tions.36 Its ECV is 191 mL, as it is modified
from the MNC protocol.
176 ■ AABB T EC HNIC AL MANUAL

Fresenius AS 104 Haemonetics MCS+ LN9000

The Fresenius AS 104 is capable of collecting
several components, including granulo-
cytes.11,37 It uses a doughnut-shaped, single-
stage channel to separate the granulocytes.
The granulocytes build up in the centrifuge
and are harvested into the final collection bag
intermittently. The AS 104 uses a double-
access kit, and its ECV is 175 mL.
The LN9000 can also be used to collect
granu- locytes. It uses the conical Latham
bowl for separation, and the buffy coat is
then trans- ferred to one of two bags. The
red cells settle to the bottom of the bag
before being returned to the donor. The
LN9000 switches from one bag to another to
return the red cells. A single- or double-
access kit can be used for granulocyte
collection, and the ECV ranges, based on
the hematocrit of the donor, from 480 mL
(38% hematocrit) to 359 mL (52%
hematocrit).

KEY POINTS

Apheresis components must meet many of the same basic regulatory requirements (eg, do- nor consent, storage conditions, an
The majority of platelets collected and transfused in the United States are apheresis-derived platelets.
Plasma can be collected by apheresis for transfusion or as Source Plasma for subsequent manufacturing. The FDA provides g
In multicomponent donations, a variety of components or combinations of components may be collected with apheresis techn
Red cells can be removed concurrently with other components, or a double RBC unit may be collected.
Several instruments and systems have been developed and/or adapted for apheresis collec- tion of blood components that use
Granulocyte collection differs from that of other components. Specific techniques should be used and certain factors must be

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Prospec- tive comparison of
plateletapheresis using four apheresis
systems on the same donors. J Clin Apher
1999;14:163-70.
17. Perseghin P, Mascaretti L, Riva M, et al. Com-
parison of plateletapheresis concentrates pro-
duced with Spectra LRS version 5.1 and LRS
Turbo version 7.0 cell separators. Transfusion
2000;40:789-93.
18. Zingsem J, Glaser A, Weisbach V. Evaluation
of a platelet apheresis technique for the
prepara- tion of leukocyte-reduced platelet
concen- trates. Vox Sang 1998;74:189-92.
19. Zingsem J, Zimmermann R, Weisbach V, et al.
Comparison of COBE white cell-reduction and
standard plateletapheresis protocols in the
same donors. Transfusion 1997;37:1045-9.
20. Maresh S, Randels M, Strauss R, et al.
Compar- ison of plateletapheresis with a
standard and an improved collection
device. Transfusion 1993;33:835-7.
21. McAteer M, Kagen L, Graminske S, et al.
Trima Accel improved platelet collection
efficiency with the merging of single stage
separation technology with leukoreduction
performance of the LRS chamber (abstract).
Transfusion 2002;42(Suppl):37S.
22. Burgstaler EA, Winters JL, Pineda AA. Paired
comparison of Gambro Trima Accel vs Baxter
Amicus single-needle plateletapheresis. Trans-
fusion 2004;44:1612-20.
23. Elfath MD, Whitley P, Jacobson MS, et al.
Eval- uation of an automated system for the
collec- tion of packed RBCs, platelets, and
plasma. Transfusion 2000;40:1214-22.
24. Yockey C, Murphy S, Eggers L, et al.
Evaluation of the Amicus separator in the
collection of apheresis platelets. Transfusion
1999;38:848
25. Valbonesi M, Florio G, Ruzzenenti MR, et al.
Multicomponent collection (MCC) with the
latest hemapheresis apparatuses. Int J Artif Or-
gans 1999;22:511-15.
26. Paciorek L, Holme S, Andres M, et al. Evalua-
tion of the continuous filtration method with
double platelet products collected on the
MCS+ (abstract). J Clin Apher 1998;13:87.
27. Ford K, Thompson C, McWhorter R, et al.
Eval- uation of the Haemonetics MCS+
LN9000 to produce leukoreduced platelet
products (ab- stract). J Clin Apher
1996;11:104.
178 ■ AABB T EC HNIC AL MANUAL
28. Rose C, Ragusa M, Andres M, et al.
Evaluation of the MCS + LN9000 in-line
leukoreduction filter (abstract). Transfusion
1996;36(Suppl):85.
29. Picker SM, Radojska SM, Gathof BS.
Prospec- tive evaluation of double RBC
collection using three different apheresis
systems. Transfus Apher Sci 2006;35:197-
205.
30. Snyder EL, Elfath MD, Taylor H, et al.
Collec- tion of two units of leukoreduced
RBCs from a single donation with a portable
multiple-com- ponent collection system.
Transfusion 2003; 43:1695-705.
31. Moog R, Frank V, Müller N. Evaluation of a
concurrent multicomponent collection sys-
tem for the collection and storage of WBC-
reduced RBC apheresis concentrates. Transfu-
sion 2001;41:1159-64.
32. Nussbaumer W, Grabmer C, Maurer M, et al.
Evaluation of a new mobile two unit red cell
apheresis system (abstract). J Clin Apher
2006; 21:20.
33. Smith JW. Automated donations: Plasma, red
cells, and multicomponent donor procedures.
In: McLeod BC, Szczepiorkowski ZM,
Wein- stein R, Winters JL, eds. Apheresis:
Principles and practice. 3rd ed. Bethesda,
MD: AABB Press, 2010:125-40.
34. Worel N, Kurz M, Peters C, Höcker P. Serial
granulocyte apheresis under daily
administra-
tion of rHuG-CSF: Effects on peripheral blood
counts, collection efficiency, and yield. Trans-
fusion 2001;41:390-5.
35. Dale DC, Lises WC, Llewellyn C, et al. Neutro-
phil transfusions: Kinetics and functions
of neutrophils mobilized with granulocyte
colo- ny-stimulating factor and
dexamethasone. Transfusion 1998;38:713-
21.
36. Food and Drug Administration. Substantially
equiva lent 510 ( k) devi ce infor m
ation, BK130065 summary. TerumoBCT
Spectra Op- tia. Silver Spring, MD: FDA,
2013. [Available at://
http://www.fda.gov/BiologicsBloodVac
cines/BloodBloodProducts/ApprovedProd
ucts/SubstantiallyEquivalent510kDeviceIn
formation/ucm390957.htm (accessed March
31, 2014).]
37. Kretschmer V, Biehl M, Coffe C, et al. New fea-
tures of the Fresenius blood cell separator
AS104. In: Agishi T, Kawamura A,
Mineshima M, eds. Therapeutic
plasmapheresis (XII): Pro- ceedings of the
4th International Congress of Apheresis of
the World Apheresis Association and the
12th Annual Symposium of the Japa- nese
Society for Apheresis, 3-5 June 1992, Sap-
poro, Japan. Utrecht, the Netherlands: VSP
BV, 1993:851-5.
 C h a p t e r 8

Infectious Disease Screening

Susan A. Galel, MD

B L OOD C O M P O N E N T S , LIKE all

United States. Initially, donors were
other medications in the United States,
screened only for syphilis. In the 1960s,
are regulated by the Food and Drug
studies showed that more than 30% of
Adminis- tration (FDA). The FDA requires
patients who received multiple transfusions
medication manufacturers to verify the
developed posttransfu- sion hepatitis
suitability of every raw material in their
(PTH).2 Studies in the early 1970s found
products.1 For biologic pharmaceuticals, the
that hepatitis B virus (HBV) ac- counted for
donor is the key ingredi- ent whose
only 25% of PTH cases.2 Both HBV and non-
suitability must be scrutinized.
A, non-B (NANB) hepatitis occurred more
Blood banks test a sample of blood
frequently in recipients of blood from
from each donation to identify donors and
commercial (paid) blood donors than in
donated components that might harbor
recip- ients of blood from volunteer donors.
infectious agents. This screening process is
By the mid-1970s, implementation of
critically im- portant because many blood
sensitive tests for hepatitis B surface antigen
components (eg, red cells, platelets, plasma,
(HBsAg) and a nearly universal changeover
and cryoprecipi- tate) are administered
to a volunteer do- nor supply resulted in a
intravenously to recipi- ents without
dramatic reduction in the incidence of both
pasteurization, sterilization, or other
HBV and NANB PTH. Still, NANB PTH
treatments to inactivate infectious agents.
continued to occur in approx- imately 6% to
Thus, infectious agents in a donor’s blood at
10% of multitransfused pa- tients.2,3
the time of donation that are not de- tected
In the absence of a specific test for the
by the screening process can be trans- mitted
causative agent of NANB PTH,
directly to recipients.
investigators searched for surrogate markers
that could be used to identify donations
HISTORICAL OVERVIEW OF associated with NANB hepatitis. The
BLOOD DONOR SCREENING presence of antibody to hepatitis B core
antigen (anti-HBc) and/or the presence of
Table 8-1 shows the progression over time of elevated alanine aminotransfer- ase in blood
donor testing for infectious diseases in the donors were shown to be associ-

Susan A. Galel, MD, Associate Professor of Pathology, Stanford University School of Medicine, and Medical
Director of Clinical Services, Stanford Blood Center, Palo Alto, California
The author has disclosed a financial relationship with Roche Molecular Systems.

179
 180
TABLE 8-1. Changes in US Donor Testing for Infectious Diseases ■

Year First
Implemented Screening Test Comments

AABB T ECHNICAL M A NUAL

1940s-1950s Syphilis The syphilis test was mandated by FDA in 1950s.
1970s HBsAg The first-generation test was available in 1970, and a higher-sensitivity test was required in
1973.
1985 Antibody to HIV (anti-HTLV-III) The initial name for HIV, the virus that causes AIDS, was HTLV-III. The first test for
antibody to HIV was called “anti-HTLV-III.”
1986-1987 ALT and anti-HBc ALT and anti-HBc were recommended by AABB as surrogate tests for NANB hepatitis. These
tests were initially not licensed by FDA for donor screening. AABB’s recommendation for
donor ALT testing was dropped in 1995 after antibody testing for HCV was in place. Anti-
HBc was licensed and required by the FDA in 1991.
1988 Anti-HTLV-I Although HTLV-I infection is usually asymptomatic, a small percentage of infected individuals
develop leukemia, lymphoma, or a neurologic disease.
1990 Antibody to HCV, Version 1 (anti-HCV HCV was identified as the cause of most cases of NANB hepatitis.
1.0)
1991 Anti-HBc Anti-HBc was previously recommended by AABB as a surrogate screen for NANB hepatitis. It
was required by the FDA in 1991 as an additional screen for HBV.
1992 Anti-HCV 2.0 This version had improved ability to detect antibody to HCV.
1992 Anti-HIV-1/2 The new HIV antibody tests had improved ability to detect early infection and an expanded
range of detection that included HIV-2 in addition to HIV-1.
1996 HIV-1 p24 antigen test This test was found to detect HIV-1 infection 6 days earlier than the antibody test. FDA
permitted discontinuation of HIV-1 p24 antigen testing with the implementation of a
licensed HIV-1 nucleic acid test.
1997-1998 Anti-HTLV-I/II The new HTLV antibody tests detected HTLV-II in addition to HTLV-I.
1999 HIV-1 and HCV nucleic acid tests to These tests were implemented initially as investigational assays and were licensed by FDA
detect in

CH A P T E R 8
HIV and HCV RNA 2002. They detect infection earlier than antibody or antigen assays.
2003 West Nile virus nucleic acid test to detect This test was implemented initially as an investigational assay and was licensed by FDA
during
WNV RNA 2005-2007. Testing of individual donations, rather than minipools, at times of increased
WNV
activity in the region was recommended by the AABB in 2004 and the FDA in 2009.

Screening
Infectious Disease
2004 Sampling of platelet components to Testing was recommended by the AABB in 2004. Some tests are approved by FDA as
detect quality-
bacterial contamination control tests. Since 2011, AABB has accepted only FDA-approved tests or those validated to
have
equivalent sensitivity.
2006-2007 Antibody to Trypanosoma cruzi This test was approved by FDA as a donor screen late in 2006, and widespread testing
was imple-
mented in 2007. The rarity of seroconversion in US residents led to endorsement in FDA
2010
guidance of one-time donor screening.
2007-2008 HBV nucleic acid test (detects HBV DNA) This test was initially implemented as part of automated multiplex assays that detect HIV
RNA, ■
HCV RNA, and HBV DNA simultaneously. HBV DNA screening was explicitly
recommended by
FDA guidance issued in October 2012.

181
HBsAg = hepatitis B surface antigen; AIDS = acquired immune deficiency syndrome; FDA = Food and Drug Administration; HIV = human immunodeficiency virus; HTLV = human T-cell
lym- photropic virus; ALT = alanine aminotransferase; HBc = hepatitis B core antigen; HCV = hepatitis C virus; NANB = non-A, non-B; HBV = hepatitis B virus; RNA = ribonucleic acid;
WNV = West Nile virus; DNA = deoxyribonucleic acid.
182 ■ AABB T EC HNIC AL MANUAL
ated with an increased risk of NANB PTH. 4-7
However, concerns about the nonspecific na-
ture of these tests led to a delay in their imple-
mentation for donor screening.
The concept of surrogate testing was re-
visited in the early 1980s when concerns
arose about the transmission of AIDS by
transfu- sions before the identification of its
causative agent. In an effort to reduce the
potential transmission of AIDS by
transfusion, some blood banks implemented
donor testing for anti-HBc (because this
antibody was highly prevalent in
populations at increased risk of AIDS) or
donor screening for inverted CD4/ CD8 T-
cell ratio (an immune abnormality found in
both AIDS patients and those with the pre-
AIDS lymphadenopathy syndrome).8 The
leaders of most blood banks, however, be-
lieved that the risk of transmitting AIDS by
transfusion was too low to warrant surrogate
interventions.9 After the human
immunodefi- ciency virus (HIV) was
isolated and identified as the causative agent
of AIDS, a donor screen- ing test for
antibody to this agent was rapidly developed
and implemented in 1985.
Once the HIV antibody test became
avail-
able and cases of HIV were recognized in
both prior donors and transfusion recipients,
it be- came clear that the risk of transmitting
HIV via blood transfusion had been greatly
underesti- mated.10 The HIV experience
highlighted the fact that an infectious agent
associated with a lengthy asymptomatic
carrier state could be present in the blood
supply for years without being recognized.
In the wake of this realization, the ap-
proach to donor screening was extended be-
yond known agents. Current donor history
evaluations include screening for, and exclu-
sion of, donors with, an increased risk of
expo- sure to blood-borne or sexually
transmitted diseases. The intention is to
reduce the likeli- hood that the blood supply
will contain other, as-yet-unidentified agents
that are potentially transmissible by blood.
Rare transmissions of HIV persisted
even after implementation of donor testing
due to a delay of weeks or months between
the time a person is infected with HIV and
the time the screening test for HIV antibody
shows positive
results.11 Blood donated during this “window
period” can contain infectious HIV but is not
detected by the donor screening tests.
The most straightforward means of pro-
tecting the blood supply from window-
period donations is to exclude potential
donors with an increased likelihood of
exposure to HIV. The FDA initially
recommended that blood banks provide
informational materials to do- nors listing
HIV risk activities and requesting that
individuals not donate if they had en- gaged
in these activities. Later, in 1990, the FDA
recommended asking each donor directly
about each risk activity. In 1992, the FDA
is- sued comprehensive guidance describing
this questioning process.
In the years since the discovery of HIV, the
risk of transfusion-transmitted disease has
been progressively reduced through a
variety of measures:
1. Use of donor education and questioning
to minimize window-period donations
and to screen for infections for which no
tests are available.
2. Shortening of the window period for spe-
cific agents by improving and/or adding
tests to detect earlier stages of infection.
3. Use of questions and tests that exclude
donors at increased risk of blood-borne or
sexually transmitted infections.
4. Surveillance for transfusion-transmissible
diseases and implementation of new
donor screening tests, when available.
The approach used to screen potential
donors for a specific agent depends on
wheth- er specific risk factors are
identifiable and whether donor screening
tests are available. Table 8-2 lists the types
of screening approach- es used for different
infectious agents.
DONOR SCREENING TESTS
The donor infectious disease tests required by
the FDA are specified in Title 21, Section
610.40, of the Code of Federal Regulations
(CFR).1 The process of amending the CFR is
slow; therefore, the FDA initially communi-
 C H A P T E R 8 Infectious Disease Screening ■ 183

TABLE 8-2. Approaches to Donor Screening

Approach Context for Use Example(s)

Questioning only Infectious agents with defined Malaria, prions
risk
factors and no sensitive and/or
specific test
Testing only Donor test is available, but no West Nile virus
ques-
tion to distinguish individuals at
risk
of infection
Questioning and testing Agents for which there are both Human immunodeficiency
identified risk factors and effective virus, hepatitis B and C viruses
tests
Use of blood components that Agents with a high prevalence in Cytomegalovirus
test
negative for specific recipients donors but for which an
identifiable
subset of recipients can benefit
from blood components that test
negative

cates changes in its recommendations by
issu- ing guidance publications. Although
FDA guidance documents do not constitute
legal requirements, they define the expected
stan- dard of practice in the United States.
The AABB also issues recommendations to
the blood banking community, and these are
communi- cated either by Association
Bulletins or by in- clusion in AABB
Standards for Blood Banks and Transfusion
Services. AABB recommenda- tions and
standards do not have the force of law,
except that one US state (California) has
incorporated some AABB standards into
state law.
AABB standards are often considered
throughout the United States as defining a
standard of practice in the blood banking
community and therefore are widely imple-
mented. Since 1985, the FDA and AABB
have issued a series of recommendations
and/or standards for additional screening
tests in ad- dition to the long-standing donor
screens for syphilis and HBsAg. Table 8-1
summarizes the chronology of changes in
donor infectious dis- ease testing, and Table
8-3 lists the donor
screening tests that are currently performed
by US blood banks.
Logistics of Testing
All infectious disease testing for donor
qualifi- cation purposes is performed on
samples col- lected at the time of donation
and sent to the donor testing laboratory. In
addition, platelet components are tested for
bacterial contami- nation typically by the
component manufac- turing facility.
Laboratories that perform FDA-mandat-
ed donor testing must be registered with the
FDA as biologics manufacturers because this
“qualification of raw materials” is considered
part of the blood component manufacturing
process. The infectious disease tests and test-
ing equipment used to screen donors must be
approved (licensed or cleared) for this purpose
by the FDA Center for Biologics Evaluation
and Research. The tests must be performed
exact- ly as specified in the manufacturers’
package inserts. Tests and test platforms that
are ap- proved only for diagnostic use may not
be used for screening whole blood donors.
184 ■ AABB T EC HNIC AL MANUAL

TABLE 8-3. Blood Donor Screening Tests Performed in the United

States
Agent Marker Detected Screening Test Method Supplemental Assays*

HBV ■ Hepatitis B surface ChLIA or ■ Positive HBV DNA

EIA antigen (FDA)†
■ Neutralization (FDA)
■ IgM and IgG antibody to ChLIA or
EIA hepatitis B core antigen

■ HBV DNA‡ TMA or PCR

HCV ■ IgG antibody to HCV ChLIA or ■ Positive HCV RNA

EIA peptides (FDA)†
■ RIBA (FDA)§
■ HCV RNA‡ TMA or PCR

HIV-1/2 ■ IgM and IgG antibody to ChLIA or ■ Positive HIV RNA

EIA HIV-1/2 (FDA)†
■ HIV-1: IFA or Western
blot (FDA)
■ HIV-2: EIA (FDA)

■ HIV-1 RNA‡ TMA or PCR

HTLV-I/II ■ IgG antibody to HTLV-I/II ChLIA or EIA IFA, Western blot, RIPA,
and
line immunoblot

Syphilis ■ IgG or IgG + IgM antibody Microhemagglutination or T. pallidum antigen-specific

to Treponema pallidum EIA immunofluorescence or
antigens agglutination assays

or

■ Nontreponemal serologic Solid-phase red cell adher- T. pallidum antigen-specific

test for syphilis (eg, ence or particle immunofluorescence or
rapid plasma reagin) agglutination agglutination assays

WNV ■ WNV RNA‡ TMA or PCR

Trypanosoma ■ IgG antibody to T. cruzi ChLIA or EIA ESA (FDA)
cruzi (one RIPA
time)||

*Supplemental assays with “(FDA)” are FDA-approved supplemental assays. Other supplemental assays listed are
not required but may be useful for donor counseling.
†
Positive results on some nucleic acid tests are approved by FDA as providing confirmation for reactive HBsAg,
HIV anti- body, and HCV antibody serology tests. If a nucleic acid test result is negative, serologic supplemental
test(s) must be performed.
‡
Screening for DNA or RNA in the United States is usually performed on minipools of 6 to 16 donor samples.
§
As of 2013, RIBA is not available. FDA variance may be obtained to use a second licensed screening test as an
alternative.
||
T. cruzi antibody testing may be limited to one-time testing of each donor.
HBV = hepatitis B virus; ChLIA = chemiluminescent immunoassay; EIA = enzyme immunoassay; DNA = deoxyribonucleic
acid, FDA = Food and Drug Administration; Ig = immune globulin; TMA = transcription-mediated amplification; PCR =
poly- merase chain reaction; HCV = hepatitis C virus; RNA = ribonucleic acid; RIBA = recombinant immunoblot assay;
HIV-1/2 = human immunodeficiency virus types 1 and 2; IFA = immunofluorescence assay; HTLV-I/II = human T-
cell lymphotropic virus, types I and II; RIPA = radioimmunoprecipitation assay, WNV = West Nile virus; ESA =
enzyme strip assay.
 C H A P T E R 8 Infectious Disease Screening ■ 185
Trypanoso-

Serologic Testing Process

Most of the serologic screening tests (assays
for the detection of antibody or antigen) are
enzyme immunosorbent assays or
chemilumi- nescent immunoassays.
Typically, the process involves performing
each required screening test once on each
donor sample. If the screen- ing test is
nonreactive, the test result is consid- ered
negative (ie, there is no evidence of infec-
tion). If a test is reactive during the first
round of testing (“initially reactive”), the
package in- sert for the test typically
requires that the test be repeated in
duplicate. If both of the repeat results are
nonreactive, the final interpretation is
nonreactive or negative, and the unit may be
used. If one or both of the repeat results are
re- active, the donor sample is characterized
as “repeatedly reactive,” and the blood unit
is not permitted to be used for allogeneic
transfu- sion. (In the case of cellular therapy
products, there are some circumstances in
which repeat- edly reactive donations may
be used. See “Considerations in Testing
Donors of Human Cells, Tissues, and
Cellular and Tissue-Based Products” later in
this chapter.)
The infectious disease tests that are ap-
proved for donor screening have
performance characteristics chosen to make
them highly sensitive. They are designed to
detect almost all infected individuals and
minimize false- negative results. However,
to achieve this sen- sitivity, the assays also
react with samples from some individuals
who are not infected (false- positive results).
Because the blood donor population is
preselected by questioning to be at low risk
of infection, the vast majority of re-
peatedly reactive results in donors do not
rep- resent true infections. To determine
whether a repeatedly reactive screening
result represents a true infection rather than
a false-positive re- sult, additional, more
specific testing may be performed on the
donor sample.
FDA requires that repeatedly reactive
do-
nor specimens be further evaluated by FDA-
approved supplemental/confirmatory assays
when such assays are available.1 FDA has
ap- proved confirmatory assays for HBsAg,
HIV type 1 (HIV-1) antibody, hepatitis C
virus (HCV) antibody, and antibody to
ma cruzi. The HCV antibody confirmatory
test (the recombinant immunoblot assay)
recently became unavailable, but blood
centers can ob- tain FDA approval to use an
alternative HCV supplemental testing
pathway.12 If no licensed confirmatory assay
is available, unlicensed supplemental tests or
retests of the donor sample using a second
licensed screening test may be helpful for
donor counseling. Table 8-3 displays the
available confirmatory and sup- plemental
assays.
A donation that is repeatedly reactive
on a screening test is not permitted to be
used for allogeneic transfusion, regardless
of the results of confirmatory or
supplemental testing. Syphilis is the only
agent for which negative results on
supplemental tests can, in some cir-
cumstances, enable use of a screening test-
reactive unit. (This applies only to
donations screened using a nontreponemal
assay.) This situation is discussed more
fully in the “Syphi- lis” section.

Nucleic Acid Testing

Nucleic acid testing (NAT) was implemented
to reduce the window periods described
above. The process of screening donor sam-
ples for viral nucleic acid [ribonucleic acid
(RNA) or deoxyribonucleic acid (DNA)] is
somewhat different from the serologic
screen- ing process. NAT requires the
extraction of nu- cleic acid from donor
plasma followed by use of a nucleic acid
amplification test to amplify and detect viral
genetic sequences.
The test systems that were initially
devel- oped in 1999 to screen donors for
HIV and HCV RNA were semiautomated
and had insuf- ficient throughput to allow
individual testing of each donor sample.
Testing of seroconver- sion panels showed
little loss of sensitivity if donor plasma
samples were tested in small pools
[minipools (MPs)] because levels of HIV and
HCV RNA are typically high in the blood
of infected individuals and NAT assays are
exqui- sitely sensitive.
Thus, in the initially approved NAT
donor screening systems, MPs of 16 to 24
donor sam- ples were prepared and tested
together. If a pool tested negative, all
donations contribut-
186 ■ AABB T EC HNIC AL MANUAL

ing to that pool were considered negative for rus (WNV) activity is high in a specific geo-
HIV and HCV RNA. If a pool showed graphic area. Circulating levels of viral RNA
reactivity on the nucleic acid test, further are often low during WNV infection. When
testing of smaller pools and, ultimately, donor samples are combined into MPs, the
individual sam- ples was performed to RNA from a WNV-infected sample may
determine which dona- tion was responsible become diluted to below the detectable level.
for the reactive test result. Donations that It has been esti- mated that MP-NAT screening
were nonreactive on this addi- tional testing for WNV RNA may fail to detect 50% or more
could be released for transfu- sion. of infected do- nations.14-16 Therefore, both the
Donations that were reactive at the indi- FDA and AABB have recommended ID-NAT
vidual sample level were considered screening for WNV RNA at times of high
positive for viral nucleic acid and could not WNV activity in a region.16,17
be released for transfusion.
In recent years, fully automated NAT sys- Implications of Reactive Test Results
tems have been developed. The two automat-
A repeatedly reactive result on a screening
ed test platforms that are approved by the FDA
test (or individually reactive NAT result)
for donor screening use multiplex assays that
typically results in mandatory discarding of
detect HIV RNA, HCV RNA, and HBV DNA
the reactive donation. Most blood bank
in one reaction chamber. Reactive donations
computer systems prevent labeling and/or
are subjected to discriminatory testing to
release of products from donations with
identify which virus is present. These systems
reactive test results. A re- active test result
are ap- proved for testing of individual
may also indicate that the do- nor should be
donations and pools of 6 to 16 donor samples,
prohibited from making future donations,
depending on the platform. The availability of
because many infections are per- sistent.
fully automat- ed NAT platforms raises the
Furthermore, past donations may also be
possibility of mov- ing to routine screening of
considered suspect because the exact date of
individual donor samples [individual donation
onset of a donor’s infection cannot be deter-
(ID) screening], rather than testing of pools
mined.
(MP-NAT).
Both the FDA and AABB have issued
However, it has been estimated that ID-
rec- ommendations regarding whether
NAT screening would minimally increase
reactive test results affect a donor’s
de- tection of infected donors, whereas the
eligibility for future donations, components
associ- ated testing cost would be
from prior donations should be retrieved
significantly higher than with MP-NAT.13
(and if so, how far back in time), and
Furthermore, it is not clear that ID-NAT
patients who previously received
screening of the entire US blood supply
components from that donor should be noti-
would be logistically feasible us- ing the
fied. These recommendations are often
available platforms. An additional con- cern
guided by the results of supplemental or
is that donors might be deferred for false-
confirmatory testing performed on the donor
positive results more frequently with ID-
sample. Many of these recommendations
NAT screening than pooled screening.
have evolved over time.
In contrast to serologic testing policies,
Because these recommendations are
repeat testing is not permitted by the FDA
complex, blood bank laboratories typically
for an individually reactive NAT sample to
create checklists that list each of the actions
deter- mine whether the initially reactive
to be performed after a specific reactive test
result rep- resents a true-positive result. If an
re- sult is obtained. Staff use these checklists
individual (unpooled) specimen is reactive
to document completion of each action as
on a NAT screen for HIV, HCV, or HBV, the
they perform it.
FDA requires that the corresponding blood
Table 8-416-37 lists federal regulations,
component be discarded and the donor be
FDA guidance documents, AABB standards,
deferred indefi- nitely.
and AABB Association Bulletins with
ID-NAT screening rather than MP-NAT
recommen-
screening is recommended when West Nile
vi-
TABLE 8-4. FDA, CMS, and AABB Recommendations for Blood Donor Testing and Actions Following Reactive Results*

Topics

Donor Donor Product Recipient Donor

Document Agent/Test Testing Management Retrieval Notification Reentry

Title 21, CFR Part 610.40 HIV-1/2, HBV, HCV, HTLV-I/II, and X
Syphilis
Title 21, CFR Part 610.41 HIV-1/2, HBV, HCV, HTLV-I/II, and X
Syphilis

CH A P T E R 8
Title 21, CFR Parts 610.46, 610.47, HIV and HCV X X
and
610.48
Title 42, CFR Part 482.2724 HIV and HCV X X
FDA guidance, October 201226 HBV X X X

Screening
Infectious Disease
FDA guidance, November 2011 25
HBV (vaccine) X
FDA guidance, December 2010 27
HCV X X
FDA guidance, December 2010 29
Trypanosoma cruzi X X X X
FDA guidance, May 2010 28
HIV and HCV X X X X X
FDA guidance, May 2010 30
Anti-HBc X
FDA guidance, November 2009 17
WNV X X X
FDA guidance, August 200931 HIV-1 group O X X X
FDA guidance, July 200932 Parvovirus B19†
FDA guidance, June 200533 WNV X X X
■
FDA guidance, October 2004 34
NAT for HIV-1 and HCV X
(Continued)

187
 188
TABLE 8-4. FDA, CMS, and AABB Recommendations for Blood Donor Testing and Actions Following Reactive Results* (Continued)

Topics

Donor Donor Product Recipient Donor ■

Document Agent/Test Testing Management Retrieval Notification Reentry

FDA guidance, August 199735 HTLV X X X

AABB T ECHNICAL M A NUAL

FDA memorandum, July 1996 36
HBsAg and anti-HBc ‡
X
FDA memorandum, December 1991 37
Syphilis X X X§ X§ X
FDA memorandum, December 1987 23
HBsAg X X X
AABB Standard 5.8.5 18(p31)
Required donor tests for HIV, HCV, X
HBV, HTLV, WNV, and syphilis
AABB Standards 5.1.5.1, 5.1.5.1.1, 5.1.5.2, Bacteria X X||
18(pp11,34,86)
and 7.1.3
Association Bulletin #10-0520 Bacteria X X||
Association Bulletin #13-02 16
WNV X
Association Bulletin #05-02 19
Bacteria X X
Association Bulletin #04-07 21
Bacteria X X X
Association Bulletin #99-9 22
HTLV X X
*Recommendations in effect as of March 2014. Blood centers may be bound by additional requirements, such as specifications in recovered plasma contracts.
†
Plasma for further manufacture only.
‡
Memorandum also includes recommendations regarding HCV and HTLV, but these recommendations have been superseded by subsequent documents.
§
Not recommended.
||
Co-components of current donation.
FDA = Food and Drug Administration; CMS = Centers for Medicare and Medicaid Services; CFR = Code of Federal Regulations; HIV-1/2 = human immunodeficiency virus, types 1
and 2; HBV = hepatitis B virus; HCV = hepatitis C virus; HTLV-I/II = human T-cell lymphotropic virus, types I and II; anti-HBc = antibody to hepatitis B core antigen; WNV =
West Nile virus; NAT = nucleic acid testing; HBsAg = hepatitis B surface antigen.
 C H A P T E R 8 Infectious Disease Screening
dations regarding management of blood do-
nors with reactive test results, retrieval of
other components, and notification of prior
recipi- ents. These regulations and
recommenda- tions are described briefly
below.
Donor Eligibility
FDA regulation Title 21, CFR Part 610.41,
ad- dresses donors with reactive screening
test re- sults. FDA guidance documents and
AABB As- sociation Bulletins contain more
detailed recommendations regarding
additional test- ing, donor eligibility, and
donor counseling for these and other tests.
Donors should be noti- fied of any test
results that affect their eligibili- ty or that
could have important implications for their
health. Blood banks should have sys- tems
that prevent future collections from ineli-
gible donors and the release of any compo-
nents inadvertently collected from such
individuals.
For donors deferred for reactive screening
tests, Title 21, CFR Part 610.41, provides for
re- instatement by means of FDA-defined
requali- fication algorithms. The FDA has
issued guid- ance documents (listed in Table 8-
4) that define reentry pathways for donors
deferred for reactivity on HIV, HCV, HBsAg,
anti-HBc, serologic syphilis, and
HIV/HCV/HBV NAT tests. Most of the
pathways require that the do- nor have
negative results on specified tests af- ter a
defined waiting period. Blood banks that
desire to reenter donors must follow the FDA-
defined algorithms explicitly.
Retrieval of Prior Donations and
Notification of Prior Recipients
(“Look-Back”)
The FDA and AABB offer guidance with
regard to the appropriate management of
previously collected blood components from
donors whose current donation is repeatedly
reactive (or, in the case of NAT, individually
reactive) on an infectious disease screening
test. These rec- ommendations address the
concern that at the time of the previous
donation, the donor could have been in the
window period of an early in-
■ 189
fection even though the screening test
results were negative.
For HIV and HCV tests, the algorithms
for managing prior donations and recipients
of prior donations are spelled out in Title 21,
CFR Parts 610.46 and 610.47. These
requirements are replicated in the Centers
for Medicare and Medicaid Services
regulations (Title 42, CFR Part 482.27) to
ensure hospital transfusion ser- vice
compliance with recipient notification
requirements. For other agents,
recommenda- tions for the management of
previously donat- ed components may be
found in the FDA guidance documents or
AABB Association Bul- letins (or both) listed
in Table 8-4.
In most cases, the FDA and AABB
recom- mend retrieval and quarantine of any
remain- ing components from prior
donations of that donor. It is essential that
the retrieval of in- date components be
initiated immediately af- ter the repeatedly
reactive result is obtained. This approach
prevents transfusion of these components
while confirmatory testing is per- formed.
The FDA requires initiation of retriev- al
within 3 calendar days of a reactive HIV or
HCV test and within 1 week of reactive
HBsAg, anti-HBc, or anti-HTLV screening
tests. If con- firmatory test results on the
current donation are negative, the FDA, in
some circumstances, permits rerelease of the
prior donations. In many cases, some or all
of the components from prior donations will
have been trans- fused. For some infectious
agents, the FDA and AABB recommend that
the recipients of prior donations from
confirmed positive do- nors be notified of
their possible exposure to the infectious
agent.
Recommendations for notification of re-
cipients of prior donations (“look-back”) are
usually issued by the AABB, FDA, or both at
the time a new test is implemented, but these
rec- ommendations may evolve as
confirmatory tests become available or
medical treatments are developed for the
infection in question. Look-back is required by
law only for HIV and HCV tests (Title 21,
CFR Parts 610.46 and 610.47). For an HIV
look-back investigation in- volving a deceased
prior recipient, the next of kin must be
notified. The CFR spells out spe- cific
timelines for component retrieval and
190 ■ AABB T EC HNIC AL MANUAL
recipient notification. It also specifies how far
back in time (ie, to which donations) the re-
trieval and notification should extend. For oth-
er agents, such as WNV and T. cruzi, recom-
mendations regarding retrieval and recipient
notification are included in FDA guidance
documents and AABB Association Bulletins.
Table 8-4 indicates which of these documents
address product retrieval or recipient notifica-
tion.
In the absence of published guidance, it
is
not always obvious whether or when it is ap-
propriate to notify prior recipients of their
possible exposure to infection. If there is no
confirmatory assay available, it may be diffi-
cult to determine whether a repeatedly reac-
tive screening test result for a donor represents
a true infection. Furthermore, if there is no ef-
fective treatment for that infection, there may
be no obvious benefit to the recipient of being
told that he or she might have been exposed.
There could, however, be a public health bene-
fit from such a notification. Specifically, a re-
cipient who is alerted of a potential exposure
can be tested and, if the results are positive,
take precautions to avoid further spread of the
infection.
Cytomegalovirus Testing of Products
for Immunocompromised Recipients
Some common infections cause relatively
in- nocuous illnesses in immunocompetent
indi- viduals but can cause severe disease in
immu- nocompromised patients. Such is the
case with cytomegalovirus (CMV).
CMV is a lipid-enveloped DNA virus in
the Herpesviridae family. Like other
herpesvi- ruses, CMV causes lifelong
infection, typically in a latent state, with the
potential for reactiva- tion. Primary CMV
infection in immunologi- cally competent
individuals is mild, with symptoms ranging
from none to an infectious mononucleosis-
type syndrome. In immuno- compromised
patients, however, both primary infection
and reactivation disease can be over-
whelming and even fatal. CMV can be
trans- mitted by blood transfusion, primarily
through intact white cells contained in
cellular blood components. Frozen/thawed
plasma
components do not appear to transmit CMV
infection. Immunocompromised patients
who are at increased risk of transfusion-
transmit- ted disease include fetuses, low-
birthweight premature infants who are born
to CMV-sero- negative mothers, and CMV-
seronegative re- cipients of solid-organ or
allogeneic hemato- poietic cell transplants
from seronegative donors.38
The majority of blood donors have had
prior exposure to CMV, indicated by the fact
that they have CMV antibodies. Therefore, it
would not be possible to produce an adequate
supply of blood if all CMV-antibody-positive
donations were discarded.
It is possible, however, to minimize
CMV transmission to patients at risk of
severe CMV disease, such as those
described above. These patients should be
supported with cellular blood components
that have a reduced risk of transmitting
CMV. These reduced-risk options include
using blood components from donors who
are CMV antibody negative or compo- nents
that have been effectively leukocyte re-
duced. The literature suggests that these two
methods have similar but not identical
effica- cy, with an estimated transmission
risk by seronegative components of 1% to
2% vs a risk of 2% to 3% with leukocyte-
reduced compo- nents.38-40 A recent study,
however, found no CMV transmissions among
100 carefully mon- itored allogeneic
hematopoietic cell transplant recipients who
received CMV-untested, leuko- cyte-reduced
components.41 Because many at- risk
patients receive leukocyte-reduced com-
ponents and are monitored closely for CMV
infection, treated early with anti-CMV
drugs, or both, it is difficult to measure a
benefit from also providing CMV-
seronegative components to these patients.
Autologous Donations
The FDA requires infectious disease testing
of autologous donations that are shipped
from one facility to another. If the receiving
facility does not permit autologous
donations to be crossed over to the general
inventory, the FDA requires testing of only
the first donation in each 30-day period
[Title 21, CFR Part
 C H A P T E R 8 Infectious Disease Screening ■ 191

610.40(d)]. The labeling of the unit must be
consistent with its testing status. Units from
donors with repeatedly reactive tests must
be labeled with biohazard labels. Some
hospitals have policies that prohibit
acceptance of au- tologous units with
positive results on some tests because there
is a potential for an infec- tious unit to be
transfused to the wrong pa- tient. The
AABB has warned that refusal of test-
positive units could be interpreted as vio-
lating the Americans with Disabilities Act.42
Considerations in Testing Donors
of Human Cells, Tissues, and
Cellular and Tissue-Based
Products
Both the questions and tests required by
FDA to screen donors of human cells,
tissues, and cellular and tissue-based
product (HCT/Ps) differ from those for
blood donors, and screen-
ing requirements vary by type of tissue.
These requirements are spelled out in Title
21, CFR Part 1271 and an August 2007
FDA guidance document and are
summarized in Table 8- 5.43,44 The time
frames for testing HCT/P do- nors are also
specified in these documents.
In most cases, the samples for
infectious disease testing must be obtained 7
days before or after the tissue donation.
However, samples of peripheral blood
hematopoietic progenitor cells or marrow
may be tested up to 30 days before donation.
Autologous tissues and re- productive
tissues from sexually intimate part- ners
may be exempt from some testing re-
quirements.
Blood bank laboratories that test
samples from HCT/P donors must take care
to check package inserts for HCT/P testing
methods; a package insert may require a
different testing method for HCT/P donors
than for blood

TABLE 8-5. FDA Testing Requirements for HCT/Ps (as of March 2014)

Tissue TypeAgentTests

HIV Antibody to HIV-1 and HIV-

2* HIV-1 RNA*

HBV Hepatitis B surface antigen*

All tissues Antibody to hepatitis B core
antigen*

HCV Antibody to hepatitis

C* HCV RNA*

For donors of viable leukocyte-rich Treponema pallidum FDA-cleared screening or diagnostic

HCT/Ps (eg, hematopoietic progenitor
cells or semen) also test for the
follow- ing in addition to the above:
For donors of reproductive tissues,
test for the following in
addition to the above:
test HTLV-I/II Antibody to HTLV-I/II*
CMV FDA-cleared screening test for anti-
CMV (total IgG and IgM)
Chlamydia trachomatis FDA-licensed, approved, or cleared diag-
nostic test

Neisseria gonorrhea FDA-licensed, approved, or cleared diag-

nostic test

*These tests must be FDA licensed for donor screening.

HCT/Ps = human cells, tissues, and cellular and tissue-based products; HIV = human immunodeficiency virus; RNA =
ribo- nucleic acid; HBV = hepatitis B virus; HCV = hepatitis C virus; FDA = Food and Drug Administration; HTLV =
human T-cell lymphotropic virus; CMV = cytomegalovirus; Ig = immune globulin.
192 ■ AABB T EC HNIC AL MANUAL
donors. For example, NAT for most types of
HCT/P donors must be performed on individ-
ual donor samples, and MP-NAT is not permit-
ted for most HCT/P donor categories.
In some cases, FDA regulations permit
the use of HCT/P donations that are reactive
on infectious disease screening tests. These
exceptions are listed in Title 21, CFR Part
1271.65. FDA has issued specific labeling,
stor- age, and notification requirements for
these tissues.
Testing of HCT/P donors for WNV
RNA and antibody to T. cruzi is not required
by FDA as of March 2014. However, FDA
has indicated that it considers WNV to be a
“relevant com- municable disease,” and
draft guidance docu- ments indicate that
FDA is considering a re- quirement to test at
least some HCT/P donors for these agents.44
International Variations in Donor
Testing
Although this chapter focuses on infectious
disease screening in the United States, the
general approach to donor screening is
similar in other countries. However, the
specific donor questions and tests vary from
country to coun- try based on the regional
epidemiology of in- fections and tests
available. For example, most countries
where WNV is not endemic do not test for
this agent, although they may question
donors about travel to WNV-affected coun-
tries. Countries where HBV is
hyperendemic cannot exclude donations
from individuals who test positive for anti-
HBc without ad- versely affecting the
adequacy of their blood supply. The AABB
Standards Program Unit in conjunction with
the AABB Subcommittee for the Evaluation
of International Variances con- siders
variance applications from facilities in
countries where national practices and avail-
able tests differ from those in the United
States.
RESIDUAL INFECTIOUS RISKS OF
TRANSFUSION
Despite donor screening, blood components
may still transmit infections. The residual
risk of transmission varies according to the
inci-
dence of the infection in the donor
population and the nature of the donor
screening process- es in place.
Agents for Which Blood Is Tested
Transfusion transmission of HIV, HCV, and
HBV is now so rare that the rate of
transmis- sion cannot be measured by
prospective clini- cal studies. The risk can
only be estimated by theoretical modeling.
One theoretical source of risk is a virus
strain that the current test kits do not detect.
The Centers for Disease Control and
Preven- tion and the test manufacturers
conduct sur- veillance for such emerging
strains. Over time, the FDA has required
that test manufacturers expand their
detection capabilities to include new strains.
A second potential cause of trans- mission is
a quarantine failure (ie, a blood bank’s
failure to quarantine a unit that tests
positive). Quarantine errors are thought to
be rare in blood banks that use electronic
systems to control blood component
labeling and re- lease because these systems
are designed to prevent the release of any
unit with incom- plete testing or a reactive
test result. Erroneous releases appear to
occur more frequently in blood banks that
rely on manual records and quarantine
processes.45
The primary cause of residual transmis-
sions is thought to be donations from
individ- uals in the window period of early
infection, before test results are positive.
Figure 8-1 dis- plays the sequence in which
different types of donor screening tests
demonstrate reactivity. Over time, the
window periods have been shortened by
implementation of donor screening tests that
detect earlier infections. However, because
no test gives a positive re- sult immediately
after an individual acquires an infection, a
window period remains. With NAT, the
average duration of the window peri- od is
estimated to be 9.0-9.1 days for HIV and
7.4 days for HCV.13,46 The window period for
HBV is longer, as discussed in the “HBV”
sec- tion below.
The likelihood that a blood donation has
been obtained from a donor in the window pe-
 C H A P T E R 8 Infectious Disease Screening
FIGURE 8-1. Time sequence of the appearance of various markers of infection.
riod can be estimated mathematically using
the incidence-window period model46:
Probability a donation was made during
the window period = length of window
period × incidence of infections in the
donor population
The incidence of infections in donors
can be calculated from the observed number
of donors with a negative test result for one
do- nation but a positive result for a
subsequent donation (ie, seroconverting
donors). This method measures incidence
rates only in re- peat donors and does not
permit assessment of the likelihood that
first-time donors might be in the window
period.
Other methods permit measurement of
new infection rates in both first-time and re-
peat donors using tests that differentiate new
from established infections. Such tests
include nucleic acid tests (donor blood that
contains HIV or HCV RNA but not
antibody most likely represents a very early
infection) and “sensitive/less sensitive”
antibody testing.13,46-49 When these alternative
methods have been used, new HIV and HCV
infections were two to four times more
common among first-time donors than
among repeat donors.46-48 Howev-
■ 193
er, both of these donor populations have sig-
nificantly lower infection rates than the
gener- al population. The continued
importance of using donor questioning to
select donors with a low incidence of
infection is explored in more detail in the
“HIV” section below.
The current estimated risks of HIV,
HCV, and HBV transmission in donors,
based on window-period and incidence
calculations, are shown in Table 8-6.
Agents for Which No Donor Screening
Tests Are Available
Essentially any infectious agent that can
circu- late in the blood of an apparently
healthy per- son could be transmitted by
transfusion. It is impossible to estimate the
risk of transmission for each of the
infectious agents for which do- nors are not
tested. However, transfusion- transmitted
infections are rarely identified. The
infections that are most likely to be recog-
nized as transmitted by transfusion are those
that have a distinctive clinical presentation
and are otherwise rare in the United States.
The likelihood that an infection will be
recog- nized as transmitted by transfusion is
en- hanced if the infection is usually
associated with a behavioral risk that the
transfusion
194 ■ AABB T EC HNIC AL MANUAL

TABLE 8-6. Estimated Risks of Transfusion-Transmitted Infection in the United States Based on
Window-Period and Incidence Estimates*

Incidence per 105 Infectious Window Residual Risk per

Study Period Agent Person/Years Period (days) Donated Unit
2007-200846* HIV 3.1 9.1 1:1,467,000
2007-2008 *46
HCV 5.1 7.4 1:1,149,000
2009-2011 50†
HBV 1.6 26.5-18.5 1:843,000 to
1:1,208,000
*HIV and HCV risk estimates are based on minipool nucleic acid testing in pools of 16.
†
HBV risk estimates are based on minipool nucleic acid testing in pools of 16 using the Novartis Ultrio
Plus assay. The range indicated for the HBV window period reflects uncertainty regarding the minimum
infectious dose of HBV (1 copy in 20 mL plasma vs 10 copies in 20 mL).
HIV = human immunodeficiency virus; HCV = hepatitis C virus; HBV = hepatitis B virus.

recipient lacks (eg, when malaria develops
in a transfusion recipient who has not
traveled outside the United States).
If a life-threatening agent is recognized as
a potential threat to the blood supply, both the
AABB and FDA typically consider whether a
donor screening question could be used to ex-
clude potentially exposed donors in the ab-
sence of a donor screening test. Donor ques-
tioning regarding travel to and residence in
endemic areas is currently the only means of
protecting the US blood supply from malaria
and variant Creutzfeldt-Jakob disease (vCJD).
Most infectious agents, however, do not have
such clear geographic risk areas. In general, it
is difficult to design donor questions that are
both sensitive (ie, detect most infected indi-
viduals) and specific (ie, exclude only infected
individuals).
An alternative method of protecting the
blood supply from infectious agents is
pathogen inactivation or reduction. Heat
inactivation, solvent/detergent treatment,
nanofiltration, chromatography, cold ethanol
fractionation, and other approaches have
been used with remarkable success to inacti-
vate or remove residual pathogens in plasma
derivatives. Pathogen reduction systems for
cellular blood components are not available
in the United States as of March 2014,
although systems for platelet components
are in use in
some other countries. Pathogen reduction
sys- tems are discussed in the “Pathogen
Reduc- tion Technology” section later in this
chapter.
The AABB Transfusion-Transmitted Dis-
eases Committee published an extensive re-
view of infectious agents that are possible
threats to the blood supply.51 Potential
mitiga- tion strategies were discussed for
each agent, including the documented or
theoretical effi- cacy of pathogen reduction
processes. AABB keeps these materials up
to date and adds ma- terials for new
potential threats as they are identified.52 The
agents deemed to pose the highest threat
from either a scientific or public perspective
are briefly discussed in this chap- ter. (See
the 2009 supplement to TRANSFU- SION
and updates on the AABB website for a
more thorough review of these potential
infec- tious risks.51,52)
SCREENING FOR SP ECIFIC AG E
NTS
HIV
HIV-1, a lipid-enveloped, single-stranded
RNA virus, was identified in 1984 as the
causative agent of AIDS, and blood donor
screening for antibodies to this virus was
implemented in the United States in 1985. In
1992, donor screening tests were modified to
include de-
 C H A P T E R 8 Infectious Disease Screening
tection of antibodies to HIV-2, a closely
related virus identified initially in West Africa.
HIV can be transmitted sexually, paren-
terally, and from infected mothers to their
in- fants. Although heterosexual and vertical
spread of HIV predominate in some parts of
the world, new HIV cases in the United
States continue to be concentrated in men
who have sex with men (MSM) and
individuals with high-risk heterosexual
contact (defined as contact with an
individual who is HIV positive or in an
identified risk group for HIV, such as MSM
or injection drug users).53
Current donor screening for HIV includes
NAT testing for HIV-1 RNA and serologic
test- ing for antibodies to HIV. The antibody
tests approved for donor screening detect both
im- mune globulin M (IgM) and IgG antibody
to both HIV-1 and HIV-2. Some of the
approved tests also detect antibody to the HIV-
1 group O, a strain of HIV-1 found primarily
in Central and West Africa. If the antibody test
used by a donor center does not have an HIV-1
group O detection claim, the donor center
must use donor questioning to exclude
individuals who have resided in, received
medical treatment in, or had sex partners from
HIV-1 group O en- demic areas.31
The average lag time from HIV-1
exposure to test detection (window period)
is currently estimated to be 9-9.1 days for
MP-NAT.13,46 Based on window-period and
incidence-rate calculations, the current risk
of acquiring HIV from transfusion is
estimated to be approxi- mately 1 in 1.5
million units (Table 8-6).
In the United States, blood donor screen-
ing questions exclude very broadly defined
populations at increased risk of HIV. Given the
short delay of only days between exposure and
detection of infection by NAT, experts have
questioned whether donor interviews and ex-
clusion of donors with increased risk remain
medically necessary.54 The continued impor-
tance of a low-risk donor population becomes
evident if different HIV incidence figures are
entered into the blood safety calculation. For
example, HIV incidence rates as high as 1% to
8% have been observed in some high-risk pop-
ulations, such as young urban MSM.55,56 If an
individual from a population with a 1% inci-
■ 195
dence of HIV donates blood, the likelihood
that this individual is in the window period
and that the component will transmit HIV
can be calculated as follows:
Risk that the donation is in window period =
length of window period × incidence of
infec- tion in donor population = (9.0
days/365 days/ year) × (1/100 person-years)
= 1/4100.
This is the likelihood that a unit from
this high-risk donor would harbor HIV but
be missed by the current donor screening.
This risk is clearly much higher than the
estimated HIV transmission risk of 1 in 1.5
million for a unit of blood obtained from the
current donor population. Thus, despite the
short window period with current testing,
inclusion of do- nors with a high risk of HIV
would have a pro- foundly adverse impact
on blood safety. Ac- cordingly, questioning
of donors for risk and temporarily excluding
those at increased risk to minimize window-
period donations contin- ue to be critical for
preserving blood safety.
Despite the efficacy of current donor
questioning approaches, there has been great
interest in developing a more specific donor-
screening algorithm for MSM that would
exclude only individuals who are truly at in-
creased risk of HIV. Although more specific
do- nor screening criteria for MSM are
desirable, the FDA states that no algorithm has
yet been developed that reliably identifies
MSM whose HIV risk is as low as that of
current blood donors.57
FDA’s rationale for permanently
excluding MSM from donation has been less
clear. Most other high-risk populations are
excluded only temporarily (for 1 year after
the risk activity). The FDA’s rationale for
excluding MSM be- yond 1 year appears to
be based on two con- cerns. One concern is
that there are other po- tentially transfusion-
transmissible infections that are more
prevalent in MSM populations (such as
human herpesvirus 8, the causative agent of
Kaposi sarcoma), and donors are not tested
for all of these infections.54 The other
concern is “quarantine release errors,” or the
risk that a blood center might inadvertently
release a unit with a positive test result.
196 ■ AABB T EC HNIC AL MANUAL
However, based on current electronic
controls used by most US blood centers,
release errors appear to be extremely rare.
Mathematical modeling suggests that
the deferral period for MSM could be
shortened without a substantial increase in
risk to recipi- ents.45 A US Department of
Health and Human Services (DHHS) advisory
committee conclud- ed in 2010 that available
scientific data were inadequate to support
any specific alternative policy.58 DHHS
subsequently issued requests for proposals to
evaluate alternative ap- proaches to donor
screening that could main- tain the current
level of blood safety.
HBV
HBV is a lipid-enveloped, double-stranded
DNA virus. Like HIV, HBV is transmitted par-
enterally, sexually, and perinatally. Jaundice is
noted in only 25% to 40% of adult cases and
in a smaller proportion of childhood cases. A
large percentage of perinatally acquired cases
result in chronic infection, but most HBV in-
fections acquired in adulthood are cleared.
HBV is highly prevalent in certain parts of the
world, such as Eastern Asia and Africa, where
perinatal transmission and resultant chronic
infection have amplified infection rates in the
population. In the United States, the incidence
of acute HBV infection has decreased dramati-
cally with the implementation of routine vac-
cination programs. Perinatal screening and
newborn prophylaxis have also been effective
in reducing perinatal transmission.
During HBV infection, DNA and viral
en- velope material (HBsAg) are typically
detect- able in circulating blood. Antibody to
the core antigen is produced soon after the
appear- ance of HBsAg, initially in the form
of IgM an- tibody, followed by IgG. As
infected individuals produce antibody to the
surface antigen (anti- HBsAg), the HBsAg is
cleared.
The FDA requires donor screening for
HBsAg, HBV DNA, and total anti-HBc (IgM
and IgG antibody). Measurements of HBV
inci- dence in donors have been complicated
by the transience of HBsAg and false-positive
results on the HBsAg test.48,50 Published
estimates of the infectious window have varied
because of
differences in the sensitivity of various HBV
as- says and lack of certainty regarding the
level of virus in a blood component that is
required for infectivity.50,59 Recent
publications provide window-period
estimates for different poten- tial infectious
doses of virus (eg, 10 copies/20 mL of
plasma vs 1 copy/20 mL of plasma). The
infectious window prior to a positive result
on the Abbott PRISM (Abbott Laboratories,
Ab- bott Park, IL) HBsAg test has been
estimated to be 30 to 38 days.59 With the
addition of HBV DNA testing in MPs of 16,
the window period is estimated to have been
reduced to 18.5 to 26.5 days.50 Using these
MP testing estimates, US HBV transfusion-
transmission risk has been estimated to be
between 1 in 843,000 dona- tions to 1 in 1.2
million donations (Table 8-6).50 Donor
screening for HBV DNA can be of value at
a variety of points in infection. HBV DNA
may be detected during the infectious
window period before HBsAg detection;
how- ever, DNA levels may be low and
could be be- low the limits of detection of
MP-NAT as- says.50,59 Later in infection,
following the clearance of HBsAg, HBV NAT
may detect per- sistent (ie, “occult” HBV)
infection.60 Such in- fections are interdicted
in the United States by the donor screening
test for anti-HBc. HBV NAT testing can
also detect acute HBV infec- tions in
individuals who have previously been
vaccinated.61,62 Such individuals may never
de- velop detectable HBsAg, but they may
have detectable DNA. The infectivity of
such dona- tions is not known because these
units contain vaccine-induced antibodies to
HBsAg in addi- tion to the virus. Routine
HBV DNA screening of US blood donations
detects at least some of
these infections.
HCV
HCV is a lipid-enveloped, single-stranded
RNA virus. HCV was shown to be the cause
of up to 90% of cases previously called NANB
transfu- sion-related hepatitis.63 The majority
of HCV infections are asymptomatic. However,
HCV infection is associated with a high risk of
chro- nicity, which can result in liver cirrhosis
and hepatocellular carcinoma.
 C H A P T E R 8 Infectious Disease Screening
HCV is thought to be transmitted
primari- ly through blood exposure. In the
United States, about 55% of HCV infections
are associ- ated with injection drug use or
receipt of transfusion before 1992, but the
risk factors for the remainder of the
infections are not clear.64 Sexual and vertical
transmissions are uncom- mon, although
coinfection with HIV may in- crease
transmission rates by these routes.
Current donor screening for HCV in-
cludes NAT testing for HCV RNA and
serologic testing for antibodies to HCV. The
average window period between exposure
and detec- tion of infection by MP-NAT is
estimated to be
7.4 days.13 The serologic test detects only IgG
antibody, a relatively late marker of infection.
Therefore, there may be a significant lag (1.5
to 2 months) between detection of RNA and
de- tection of antibody.65 Donor questioning
has limited potential to exclude individuals
who may be harboring HCV infection because
a large proportion of infected individuals are
asymptomatic and have no identifiable risk
factors. Despite this limitation, the current es-
timated US risk of HCV transmission by trans-
fusion is extremely low—approximately 1 in
1.1 million (Table 8-6).46
Human T-Cell Lymphotropic Virus,
Types I and II
Human T-cell lymphotropic virus type I
(HTLV-I) is a lipid-enveloped RNA virus. It
was the first human retrovirus identified,
isolated in 1978 from a patient with
cutaneous T-cell lymphoma. A closely
related virus, HTLV-II, was later isolated
from a patient with hairy cell leukemia.
Both viruses are highly cell associat- ed,
infect lymphocytes, and cause lifelong in-
fections, but most of these infections are
asymptomatic. Approximately 2% to 5% of
HTLV-I-infected individuals develop adult
T- cell leukemia/lymphoma after a lag of 20
to 30 years. A smaller percentage develop a
neuro- logic disease called HTLV-associated
myelopa- thy or tropical spastic paraparesis.
HTLV-II disease associations remain
unclear. Both in-
■ 197
fections are thought to be spread through
blood, sexual contact, and breast feeding.
HTLV-I infection is endemic in certain
parts of the world, including regions of Japan,
South America, the Caribbean, and Africa. In
the United States, infections are found in im-
migrants from endemic areas, injection drug
users, and the sexual partners of these individ-
uals. Approximately one-half of the HTLV
infections in US blood donors are from HTLV-
II.66,67
The only FDA-approved donor tests for
HTLV infection are screening assays for IgG
antibody to HTLV-I and HTLV-II. Units that
are reactive on the screening assay may not
be re- leased for transfusion. Because there
is no FDA-approved confirmatory assay and
the majority of reactive donor samples are
thought to represent false-positive results,
the FDA does not require permanent
deferral of donors after one reactive
donation. Donors are permanently excluded
only if their sample re- acts again on a
subsequent donation (Title 21, CFR Part
610.41). Unlicensed supplemental tests,
such as immunoblots or immunofluo-
rescence assays, may be very helpful for
coun- seling donors about the likelihood that
the screening test reactivity represents a true
in- fection. Some of these supplemental
assays can also differentiate between HTLV-
I and HTLV-II infection.66,67 Rather than use
unli- censed supplemental tests, some blood
cen- ters retest reactive samples on a
different FDA- licensed donor screening
assay; samples with nonreactive results on
the alternative screen- ing assay are most
likely false-positive. Do- nors whose
samples are reactive on the second screen,
however, must be counseled and de- ferred.35
Risk estimates for transfusion-transmit-
ted HTLV are somewhat uncertain, given
the absence of well-defined window periods
for the current HTLV antibody tests and the
ab- sence of confirmatory assays to
definitively measure true case rates in
donors.48 Like CMV, HTLV is thought to be
transmitted only by white-cell-containing
blood components and not by frozen/thawed
plasma components.66,67
198 ■ AABB T EC HNIC AL MANUAL
Syphilis
Syphilis is caused by the spirochete
bacterium Treponema pallidum. Donor
screening for syphilis has been performed
for more than 60 years. Donors were
initially screened by non- treponemal
serologic tests that detect anti- body to
cardiolipin (eg, rapid plasma reagin). In
recent years, however, most blood banks
have begun using tests that detect specific
an- tibodies to T. pallidum because these
tests can be performed with automated
testing instru- ments.
The vast majority of reactive donor test
results do not represent active cases of
syphi- lis. Most reflect either biologic false-
positive results or persistent antibody in
previously treated individuals (the latter are
detected by treponemal-specific antibody
screening tests). The FDA has permitted use
of additional, treponemal-specific,
confirmatory assays (eg, fluorescent
treponemal antibody absorption test) to
guide the management of both donors and
components. Specifically, the FDA has
permitted release of units from donors who
have reactive nontreponemal screening test
results and negative treponemal
confirmatory results if the units are labeled
with both test results.1,37 If, however, the
result of the trepo- nemal-specific
confirmatory test is positive or if no
additional testing is performed, the com-
ponent may not be used and the donor must
be deferred for at least 12 months.
The current value of donor screening for
syphilis is controversial.68-70 Although numer-
ous cases of transfusion-transmitted syphilis
were reported before World War II, no cases
have been reported in the United States for
more than 40 years. The low transmission
risk is probably related to a declining
incidence of syphilis in donors as well as the
limited surviv- al of the T. pallidum
spirochete during blood storage.
One issue that has been considered is
whether the syphilis screen improves blood
safety by serving as a surrogate marker of
high- risk sexual activity. However, studies
have demonstrated that donor screening for
syphi- lis does not provide incremental value
in de- tecting other blood-borne and
sexually trans-
mitted infections, such as HIV, HBV, HCV, or
HTLV.70
Other Bacteria
Bacterial contamination of blood
components (mainly platelets) continues to
cause some transfusion-related fatalities.71
Bacteria are re- portedly present in
approximately 1 in 3000 cellular blood
components.72 The source of the bacteria can
be either the donor’s skin or asymptomatic
bacteremia in the donor.
The level of bacteria in components just
after collection is generally too low to detect
or to cause symptoms in the recipient.
However, bacteria can multiply during
component stor- age, particularly in platelet
components, which are kept at room
temperature. Bacteria proliferate to a lesser
extent in refrigerated Red Blood Cells, and
septic reactions occur much less commonly
with these components. To re- duce the risk
of septic transfusion reactions associated
with platelets, the AABB imple- mented a
requirement for processes to limit and detect
bacterial contamination in all platelet
components in 2004. (Since 2009 the
requirement has been to “limit and detect or
inactivate” bacterial contamination in
platelet components.18(p11))
To limit blood component contamina-
tion by bacteria from donor skin, two
elements of the blood collection process are
critical. Be- fore venipuncture, the donor
skin must be carefully disinfected using a
method with demonstrated efficacy. Most of
these methods involve iodophors,
chlorhexidine, or alcohol.73 Second,
diversion of the first 10 mL to 40 mL of
donor blood, which can contain skin and ap-
pendages, away from the collection
container (eg, into a sample pouch) further
reduces the likelihood that skin
contaminants will enter the component.73,74
Since 2008, the AABB has required that
collection sets with diversion pouches be
used for all platelet collections, in- cluding
whole-blood collections from which
platelets are made.18(p22)
A variety of technologies are available for
detection of bacteria in platelet components.
AABB Standards requires blood centers to use
a bacteria detection method that is approved
 C H A P T E R 8 Infectious Disease Screening
by the FDA or validated to provide sensitivity
equivalent to FDA-approved methods. None
of these methods is sensitive enough to detect
bacteria immediately after collection. All
methods require a waiting time for bacteria
contaminants to multiply before the compo-
nent is sampled.
The process most commonly used in the
United States to screen apheresis platelets is
a culture-based system that requires the
plate- let component to be stored for 24
hours before sampling. After that time, a
sample is with- drawn and inoculated into
one or more cul- ture bottles. The bottles are
then incubated in the culture system. Some
blood centers con- tinue to hold the
component during the first 12 to 24 hours of
culture and release it for use only if the
culture is negative at the end of that time. In
all cases, the culture is continued for the
shelf life of the unit. If the culture becomes
positive after the component is released, the
blood center attempts to retrieve it. If the
com- ponent has not been transfused,
resampling of the product for culture is very
informative be- cause approximately two-
thirds of the initially positive signals are
determined to be caused by either
contamination of the bottle (and not the
component) or false signals from the cul-
ture system.73,74All positive cultures should
be tested to determine the identity of the
organ- ism. If a true-positive result is related
to an or- ganism that is not a skin
contaminant, the do- nor should be notified
and advised to seek medical consultation.19
Other methods approved in the United
States for platelet quality control testing
early in the storage period include a culture-
based system with a one-time-point readout
and an optical scanning system. All of the
methods are approved for testing leukocyte-
reduced apheresis platelets, and some are
approved for testing pools of leukocyte-
reduced, whole- blood-derived platelets.
These methods are not generally used for
routine screening of individual (unpooled)
whole-blood-derived platelet concentrates.
Low-technology meth- ods for screening
platelets just before issue— such as visually
inspecting the platelets for swirling or
testing them for low glucose or pH—lack
both sensitivity and specificity and
■ 199
do not fulfill the AABB standard for
bacteria detection.18(p11),20,73 However, two
FDA-cleared point-of-issue assays can be
used for bacteria detection testing of platelet
concentrates that are pooled immediately
before issue.
Since the implementation of routine bac-
terial screening of apheresis platelets, the fre-
quency of FDA-reported fatalities from con-
taminated apheresis platelets has declined.71
However, some contaminated apheresis plate-
lets escape detection by this early testing, pre-
sumably because bacterial concentrations are
below limits of detection at the time of sam-
pling; thus, septic, and even fatal, reactions do
still occur. AABB has recommended consider-
ation of policies to further reduce the risk
of bacterially contaminated platelets.75 The
point-of-issue assays mentioned above are
cleared by FDA as adjunct (time-of-issue) tests
for apheresis platelets that have been screened
by another method. In a large clinical trial, one
of these assays detected nine bacterially con-
taminated components among 27,620 aphere-
sis platelets (1 in 3069 components)
previously screened by an early-storage
culture-based as- say.76 There were also 142
false-positive results. As of March 2014, point-
of-issue retesting of apheresis platelets had not
been widely imple- mented in the United
States.
All of the above methods provide incom-
plete assurance of bacteria detection, and
none is practical for screening the red cell
in- ventory. Pathogen-reduction methods,
which impair proliferation of bacteria in the
blood component, could theoretically be
used to re- duce the risk of septic reactions
from bacteria in blood components. Indeed,
in some regions outside the United States,
pathogen reduction has replaced bacteria
detection testing for platelets, but these
technologies are not approved for use in the
United States as of March 2014.
Infections Transmitted by Insect
Vectors
Until recently, malaria was the only vector-
transmitted disease that was widely recog-
nized as having the potential for secondary
transmission by transfusion in the United
200 ■ AABB T EC HNIC AL MANUAL
States. Malaria is rare in the United States,
and the blood supply has been effectively
protect- ed by questions that exclude donors
who have recently traveled to or resided in
malaria- endemic regions. In the past
decade, however, other vector-transmitted
infections have been recognized as threats to
the US blood supply, and these infections
are targeted by the newest blood donor
screening assays.
WNV
WNV is a lipid-enveloped RNA virus in the
Fla- viviridae family. First detected in the
United States in 1999, it subsequently
spread through- out North America,
appearing in annual epi- demics every
summer and autumn. Birds are thought to be
the primary reservoir for WNV, with
infection spreading to humans through
mosquito bites. Approximately 80% of
human cases are asymptomatic; 20% are
associated with a self-limited febrile illness;
and less than 1% are associated with severe
neuroinvasive disease, such as
meningoencephalitis or acute flaccid
paralysis.
Transfusion transmission of WNV was
first recognized in 2002 and traced to blood
donations containing viral RNA in the ab-
sence of antibody. Thus NAT, rather than sero-
logic testing, was required to protect the blood
supply. Donor screening was widely imple-
mented in 2003 using investigational NAT as-
says. Donor tests for WNV RNA are now ap-
proved by FDA and required by both the FDA
and AABB.17,18(pp31,32) The predominant method
of testing is in MPs.
However, as discussed in the “NAT” sec-
tion above, circulating levels of viral RNA are
frequently low during WNV infection, and a
donor sample containing a low concentration
of RNA may escape detection if it is diluted in
a minipool. Therefore, both the AABB and
FDA recommend testing of individual donor
sam- ples, not minipools, when WNV activity
is high in a particular collection region.16,17
Regional WNV activity is monitored through
active communications between neighboring
blood collection agencies about viremic
donors de- tected as well as by public health
reports of
clinical WNV cases and animal and mosquito
surveillance in the area.
Two documented cases of WNV
transmis- sion by transfusion were traced to
donations screened by MP-NAT during
periods when neighboring blood collection
facilities were screening donors by ID-
NAT.77 The most re- cent reported
transmission was traced to a do- nation
containing WNV antibody and a low level
of virus that was not reproducibly detect-
able even by ID-NAT.78 It is possible that
some transmissions have escaped
recognition be- cause most WNV infections
lack distinctive symptoms.
T. cruzi
T. cruzi, the protozoan parasite that causes
Chagas disease, is endemic in parts of
Mexico, Central America, and South
America. It is transmitted to humans by an
insect vector, the reduviid bug. Acute
infection is usually self- limited but may be
severe in immunocompro- mised patients.
Most infections become chronic but
asymptomatic. Decades after the initial
infection, 10% to 40% of infected indi-
viduals develop late-stage manifestations,
including intestinal dysfunction or cardiac
disease, which can be fatal. Transfusion
trans- mission of T. cruzi from the blood of
chronical- ly infected, asymptomatic donors
has been re- ported in endemic areas.
A blood donor screening enzyme
immu- noassay for antibodies to T. cruzi was
ap- proved by the FDA for US use in
December 2006. Although not initially
required by the FDA, the test was widely
implemented by US blood centers during
2007. Supplemental test- ing of reactive
specimens using an FDA- licensed enzyme
strip assay or unlicensed
radioimmunoprecipitation assay (RIPA) is
very helpful for guiding donor counseling.
Based on the results of the latter assay,
about 25% of reactive US donors appear to
be truly infect- ed.79,80 All donors whose
samples are reactive on the screening assay,
however, must be per- manently deferred,
regardless of the results of the supplemental
testing, pending FDA ap- proval of a reentry
algorithm.29
 C H A P T E R 8 Infectious Disease Screening
The vast majority of US donations with
reactive T. cruzi screening test results and
pos- itive supplemental results are from
donors born in T. cruzi-endemic areas. Other
con- firmed-positive donors appear to have
con- genitally acquired infections (ie, the
donor’s mother is from a T. cruzi-endemic
area), and only a small number of donor
infections ap- pear to have been acquired
from vector expo- sure within the United
States (autochthonous cases). In the first 2
years of US donor screen- ing, no donor
seroconversions were identi- fied. 79,80 In
December 2010, the FDA issued guidance
recommending one-time screening of every
US donor for T. cruzi.29
Before implementation of donor screen-
ing, seven cases of transfusion-transmitted
T. cruzi had been identified in the United
States and Canada; all of the cases with
avail- able data were linked to platelet
transfusions. Since implementation of donor
screening, RIPA-positive donors have been
identified, and recipients of their prior
donations have been notified and tested.
Thus far, only two prior recipients (of
platelets from one donor) appear to have
positive test results. Thus, de- spite reported
transmission rates of 10% to 20% from
components from infected donors in
endemic areas, no T. cruzi transmissions by
red cells in the United States have been
docu- mented to date. The lower infectivity
of US red cell components compared to
those in endem- ic areas may be at least
partly attributable to more frequent use of
fresh whole blood in en- demic areas.
Babesia
Babesia are intraerythrocytic parasites.
Cases of transfusion-transmitted babesiosis
are be- ing identified with increasing
frequency, and some have been fatal.71,81,82
There is no FDA- approved donor screening
test for this infec- tion.
Human Babesia infections are zoonotic,
usually acquired through the bite of an
infect- ed tick. In the northeastern and
Midwestern United States, the most
common Babesia spe- cies is B. microti. The
vector is Ixodes scapular- is, the same tick
that transmits Lyme disease.
■ 201
Reported human infections with B. microti
are becoming more frequent. In the western
Unit- ed States, Babesia infections appear to
be less common, and a different species, B.
duncani, predominates. The vector for B.
duncani has not been clearly defined.
Babesia infection is usually
asymptomat- ic, even though parasites can
circulate for months to years. In some
individuals, however, Babesia infection
presents as a severe malaria- like illness that
can be fatal. Immunocompro- mised,
elderly, and asplenic patients are at in-
creased risk of severe disease.
Babesia infection is diagnosed when the
intraerythrocytic parasites are seen on a
blood smear. If a patient is suspected of
having ac- quired the infection by
transfusion, donors of the patient’s
components can be recalled and tested for
antibodies to Babesia using an im-
munofluorescence assay; the presence of
high-titer antibody in the donor is suggestive
of recent infection. Most of the donors
impli- cated in transfusion-transmitted cases
have been residents of endemic areas,
although some were residents of
nonendemic areas who were apparently
exposed to Babesia during travel to endemic
areas.82
Studies in Connecticut have found B.
mi- croti antibodies in approximately 1% of
blood donors, suggesting that B. microti
infections are highly prevalent in the state’s
population and grossly underrecognized.83
In the absence of FDA-approved tests
for blood donor screening, there have been
public discussions of potential interventions
to re- duce transfusion risk in the most
highly en- demic regions.81,84 Clinical trials
of some inves- tigational serologic and
nucleic acid tests are currently under way.85
Malaria
Malaria is caused by an intraerythrocytic
para- site of the genus Plasmodium.
Infection is transmitted to humans through a
mosquito bite. Five species account for most
human in- fections: P. falciparum, P. vivax,
P. malariae,
P. ovale, and P. knowlesi.
No FDA-approved test is available to
screen US blood donations for malaria
infection.
202 ■ AABB T EC HNIC AL MANUAL
Screening is accomplished solely by donor
questioning. Donors are excluded
temporarily from donating blood after
traveling to malaria- endemic areas, residing
in malaria-endemic countries, or after
recovery from clinical ma- laria. Donor
questioning has been remarkably effective at
preventing transfusion-transmit- ted malaria
in the United States, with only six cases
reported between 1999 and 2012. All six
cases were linked to donors with a history of
residence in Africa; on re-interview, five of
the six donors were verified to have met
accep- tance criteria, but one had emigrated
less than 3 years before donation.86,90
This level of transfusion safety has been
achieved at substantial cost in terms of donor
loss; malaria-related questions have excluded
hundreds of thousands of otherwise accept-
able US donors annually. Although travel to
Mexico has accounted for the largest propor-
tion of deferred donors, the risk of acquiring
malaria during tourist travel to Mexico is ex-
ceedingly low.91 The FDA recently issued
guid- ance redefining malaria-endemic areas as
only those for which chemoprophylaxis is
recom- mended.92 With this new definition,
travel to many popular tourist locations will no
longer be considered a malarial risk. However,
the new guidance adds a complex algorithm
for evaluating travel by donors who have lived
for more than 5 years in malaria-endemic
coun- tries because of a concern of partial
immunity in such donors.
Some other countries that exclude
donors
after travel to malaria-endemic areas permit
reinstatement of these donors if they test
neg- ative for malaria antibodies 4 to 6
months after completion of travel. In the
absence of an ap- proved assay, such a “test-
in” reinstatement strategy has not been
accepted by the FDA.
Other Vector-Transmitted Infections
There are many other vector-transmitted in-
fections that could be secondarily
transmitted by transfusion. These agents and
potential intervention strategies are
reviewed in the publicly available AABB
emerging infectious disease resources.51,52
Two of these agents, dengue and chikungunya
viruses, have received
recent attention because donations contain-
ing viral nucleic acid have been documented
during epidemics outside the continental
United States. Dengue activity has also been
documented within the southern United
States and Hawaii.93 The degree to which
these agents threaten the US blood supply is
unclear.94
Prions
Prions are proteinaceous infectious particles
that induce disease by triggering conforma-
tional changes in naturally occurring protein
counterparts. These agents cause fatal infec-
tions of the nervous system called
“transmissi- ble spongiform
encephalopathies” (TSEs).
Classical CJD is a TSE with both a
sporad- ic form and familial forms and an
incidence of about 1 per million. CJD has been
transmitted by infusion or implantation of
products from infected central nervous system
tissues. Blood components do not appear to
transmit classi- cal CJD. Nevertheless, blood
donations are not accepted from donors who
are at increased risk of this disease.95
Another TSE, vCJD, does appear to be
transmissible by blood transfusion. This
TSE is caused by the same prion that causes
bovine spongiform encephalopathy (BSE),
also known as “mad cow disease.” As of
March 2014, four cases of vCJD
transmission by transfusion had been
reported in the United Kingdom, the area in
which BSE was most endemic. In addition,
one latent vCJD infection was identified in a
patient with hemophilia in the United King-
dom who died of other causes. This patient
had received UK-plasma-derived Factor
VIII, including material from a donor who
later developed vCJD, suggesting that vCJD
might have been transmitted by clotting
factor con- centrates.51,52 vCJD infection is
extremely rare in the United States. The few
reported cases have been in individuals who
most likely acquired their infections
elsewhere, and no US transfusion-
transmitted cases have been reported.
There are no FDA-approved donor
screening tests for prion infections. Blood
do- nors in the United States are screened
solely by questioning and are excluded if
they have an
 C H A P T E R 8 Infectious Disease Screening
increased risk of either CJD or vCJD. CJD
ex- clusions are based on family history of
the dis- ease, receipt of human growth
hormone de- rived from pituitary glands, or
receipt of a dura mater tissue graft. vCJD
exclusions are for resi- dence in the United
Kingdom or Europe dur- ing specified times
when BSE was endemic, re- ceipt of a
transfusion in the United Kingdom or
France, or receipt of UK bovine insulin.95 It
is thought that plasma derivative
manufacturing processes remove substantial
amounts of TSE infectivity.95
In-Process Screening for Plasma
Derivatives
Commercial plasma derivatives are prepared
from large pools of plasma derived from
thou- sands of donors. Before the
incorporation of specific pathogen-reduction
processes, con- tamination of these large
pools with viral agents was common. Today,
plasma derivative manufacturing processes
incorporate meth- ods—such as prolonged
heat or solvent/deter- gent (SD) treatment—
that remove or inacti- vate most known
pathogens. SD treatment inactivates lipid-
enveloped agents, such as HIV, HCV, and
HBV. Pathogen infectivity may also be
reduced by nanofiltration, chromatog- raphy,
or cold ethanol fractionation, which are used
in the production of certain products. Not all
infectious agents, however, are re- moved or
inactivated by these processes.
One agent that can persist in plasma-
derivative products is human parvovirus
B19. This small, nonenveloped DNA virus
is ex- tremely resistant to physical
inactivation. Acute infection is typically
mild and self- limited; clinical
manifestations include “fifth disease”
(erythema infectiosum) and polyar-
thropathy. Acute infection is associated with
transient red cell aplasia that may be
clinically significant in immunodeficient
individuals and those with underlying
hemolytic process- es. The aplasia in
immunodeficient individu- als can be
prolonged. Intrauterine infection is
associated with severe fetal anemia and hy-
drops fetalis.
Parvovirus B19 infection is very common;
most adults have antibodies to this agent,
indi-
■ 203
cating previous exposure. Levels of viral
DNA during acute infection may exceed 1012
IU/mL, decreasing over weeks to months in
associa- tion with antibody production.
Viral DNA, mostly at low
concentrations, has been detected in
approximately 1% of blood donations and in
essentially all lots of pooled plasma
derivatives. Transmission of parvovirus B19
by transfusion has been linked only to blood
components or plasma products that contain
high concentrations of viral DNA; only one
transmission has been documented with a
product containing less than 104 IU/mL.52
Currently, there is no FDA-approved test
to screen fresh blood donations for parvovirus
B19 infection. However, plasma-derivative
manufacturers require screening of incoming
plasma units for the presence of high-titer par-
vovirus B19. This is accomplished by
perform- ing NAT on pools of samples from
plasma units, with sensitivity adjusted to
detect only units with a high concentration of
virus. By ex- cluding high-titer units from the
plasma pools, the final titer in the plasma pool
is kept below 104 IU/mL.
Other Agents
The AABB maintains a publicly accessible
electronic resource containing expert analyses
of emerging infectious disease (EID) agents
that have received attention as potential
threats to the US or global blood supply.52 This
digital resource contains up-to-date fact
sheets on a variety of agents. Each fact sheet
includes information about clinical manifesta-
tions and epidemiology of infection, evidence
of transfusion transmissibility, and analyses
of the potential effectiveness of various
mitigation strategies (eg, donor questioning,
serologic testing or NAT, or pathogen reduc-
tion). Readers are encouraged to use this rich
resource.
One agent that has received recent
atten- tion is hepatitis E virus (HEV), a
small, nonen- veloped, single-stranded RNA
virus. HEV is thought to be primarily
transmitted through food and water sources.
However, recent stud- ies have demonstrated
asymptomatic viremia
204 ■ AABB T EC HNIC AL MANUAL
in blood and plasma donors, and transfusion
transmission has been documented. 52,96 As a
nonenveloped agent, HEV is not susceptible
to SD treatment. NAT screening of donors
and/or heat inactivation of plasma pools
may be miti- gation strategies if this agent is
deemed to pose a clinically important threat.
PATHOGEN REDUCTION
TECHNOLOGY
Donor screening reduces, but cannot
eliminate, the infectious risks of blood
transfusion. The ef- ficacy of blood donor
testing is limited by a number of factors,
including the following:
1. It is not logistically feasible to test donors
for every infection that is conceivably
transmissible by transfusion.
2. For every test, there is a lag time
(window period) between when a person
becomes infected and when the test
detects infec- tion.
3. Every test has limited sensitivity (concen-
tration of the target marker that can be
detected by the test).
4. Developing a donor test is a long, multi-
phase process that includes identification
of the infectious agent, selection of the
type of test that would be effective in
interdict- ing infectious donations (eg,
serology vs NAT), development of a test
suitable for donor screening, performance
of clinical trials of the test, and regulatory
approval. During this development
process, infec- tions can be transmitted.
Pathogen reduction technology (PRT)
provides an attractive alternative to relying
on donor testing to interdict all infectious
dona- tions. PRT processes reduce the
infectivity of residual pathogens in blood
components. This approach could reduce the
transmission of in- fectious agents for which
there are no donor screening tests and
further reduce the residual transmission
risks of known agents. PRT could
theoretically enable discontinuation of some
testing that is currently performed (eg, CMV
testing and bacterial testing of platelets), and
some PRT methods could obviate the need
for irradiation, potentially offsetting some of
the cost of PRT.
As discussed above, PRT is now an essen-
tial component of the plasma-derivative man-
ufacturing process. SD treatment can also be
applied to pools of plasma for transfusion. An
SD-treated pooled plasma product has been
approved for use in the United States.
No PRT processes are approved in the
United States for treatment of individual
units of plasma or for cellular blood
components, although some technologies
are in use outside the United States.
Processes that are available or in
development have been recently re- viewed
in detail and are summarized in Table 8-
7.51,52,97,98
The benefit to be gained from PRT in
the United States is primarily the mitigation
of emerging pathogens and platelet-
associated bacterial sepsis. Currently, the
quantifiable in- fectious risks of transfusion
in the United States are low. Therefore, it is
critically impor- tant to demonstrate that
PRT treatments do not introduce new
hazards to patients. Rigor- ous preclinical
and clinical studies are re- quired for US
regulatory approval of PRT. Toxi- cology
studies are critical because most of these
agents interact with nucleic acid, raising the
theoretical potential of carcinogenicity and
mutagenicity. Treated products should be
assessed for neoantigen formation and the
im- pact of the PRT on the final product’s
clinical efficacy. The evaluation process for
PRT in North America was the subject of a
recent con- sensus conference.97,99
Documented transmission of new infec-
tious agents for which there are no donor
screening tests could influence the
risk/bene- fit assessment of PRTs. It is
important, though, to keep in mind that there
are limits to the viral loads that are
inactivated by these processes, and not all
agents are inactivated by PRTs. Pooled SD
plasma, for example, would have a reduced
risk of transmitting most infectious agents
but a potentially increased risk of trans-
mitting an agent that lacks a lipid envelope.
 C H A P T E R 8 Infectious Disease Screening ■ 205

TABLE 8-7. Pathogen Reduction Technologies for Transfusable Blood Components

Component Technology Manufacturer

Plasma: commercially prepared pools Solvent/detergent treatment Octapharma
Plasma: individual units ■ Amotosalen (psoralen) + UV light Cerus
■ Riboflavin (vitamin B2) + UV Terumo BCT
light
■ Methylene blue + light Macopharma
Platelets ■ Amotosalen (psoralen) + UV light Cerus
■ Riboflavin (vitamin B2) + UV Terumo BCT
light
■ UV light Macopharma
Red Blood Cells ■ Frangible nucleic acid crosslinker Cerus
■ Riboflavin (vitamin B2) + UV Terumo BCT
light

SUMMARY Current quantifiable risks of infectious

The current level of safety of blood compo-
nents is based on two critical elements of
do- nor screening: donor questioning, which
is the sole method of screening for certain
agents, such as malaria and prions, and
donor testing. Testing must be performed
carefully and in ac- cordance with
manufacturers’ instructions, and facilities
must have robust systems for quarantining
components of donations that test positive
and for retrieving prior donations from
donors whose samples have tested posi-
tive.
disease transmission are very low; the estimat-
ed risk of HIV transmission by transfusion in
the United States is approximately 1 in 1.5
mil- lion units, the risk of HCV transmission is
ap- proximately 1 in 1.1 million units, and the
risk of HBV transmission is approximately 1
in 800,000 to 1 in 1.2 million units.46,50
However, it is critical to remain vigilant for
evidence of new infectious agents and to
implement miti- gation measures as quickly as
feasible. PRT may, in the future, provide some
protection against emerging infectious agents
for which no screening is in place.

KEY POINTS

Infectious disease screening of donors is accomplished by 1) questioning potential donors and excluding those with an incr
There is a delay between the time when an individual is exposed to an infection and the time when the donor screening test
The estimated window period with MP-NAT of donor samples is less than 10 days for HIV and HCV and less than 28 days
The residual risk of transfusion-transmitted infection is a function of the length of the win- dow period and the incidence of
206 ■ AABB T EC HNIC AL MANUAL

5. Based on window-period and incidence calculations, the current risk of HIV

transmission by transfusion in the United States is approximately 1 in 1.5 million units,
the risk of HCV transmission is approximately 1 in 1.1 million units, and the risk of
HBV transmission is ap- proximately 1 in 800,000 to 1 in 1.2 million units.
6. There are no donor screening tests approved by the FDA for malaria or vCJD. Donor
ques- tioning about potential exposure is the sole means of protecting the US blood
supply from these diseases.
7. AABB requires blood banks to have processes that limit and detect or inactivate
bacteria in platelet components. Pathogen reduction methods have replaced bacteria
testing in some regions outside the United States.
8. Infections transmitted to humans by vectors are increasingly recognized as a potential
source of transfusion-transmitted infection. These include WNV, T. cruzi, Babesia
species, and dengue virus.
9. PRTs may reduce the transmission of infectious agents for which there are no donor
screen- ing tests, and PRTs may further reduce the residual transmission risks of known
agents. PRT- treated plasma derivatives and pooled plasma are available in the United
States. Some PRT systems are available outside the United States for treatment of
individual platelet and plas- ma components, but as of early 2014, these were not
approved for US use.
10. Blood banks must have processes in place to ensure that donations with positive test
results are not released for transfusion. In some circumstances, 1) prior donations from
those do- nors must also be retrieved and quarantined, and 2) recipients of prior
donations must be notified of their possible exposure to infection.
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clini- cal relevance of HTLV type I and II in
transfu- sion medicine. Transfus Med Rev
1997;11:173- 9.
68. Orton S. Syphilis and blood donors: What we
know, what we do not know, and what we need
to know. Transfus Med Rev 2001;15:282-92.
69. Katz LM. A test that won’t die: The serologic
test for syphilis. Transfusion 2009;49:617-19.
70. Zou S, Notari EP, Fang CT, et al. Current value
of serologic test for syphilis as a surrogate
marker for blood-borne viral infections among
blood donors in the United States. Transfusion
2009;49:655-61.
71. Food and Drug Administration. Fatalities re-
ported to FDA following blood collection and
transfusion: Annual summary for fiscal year
2012. Silver Spring, MD: CBER Office of
Com- munication, Outreach, and
Development, 2013. [Available at
http://www.fda.gov/down
loads/BiologicsBloodVaccines/SafetyAvail
ability/ReportaProblem/TransfusionDona
tionFatalities/UCM346856.pdf (accessed Jan-
uary 14, 2014).]
72. Hillyer CD, Josephson CD, Blajchman MA, et
al. Bacterial contamination of blood compo-
nents: Risks, strategies, and regulation: Joint
ASH and AABB educational session in
transfu- sion medicine. Hematology Am Soc
Hematol Educ Program 2003:575-89.
73. Ramirez-Arcos SM, Goldman M,
Blajchman MA. Bacterial infection:
Bacterial contamina- tion, testing and post-
transfusion complica- tions. In: Hillyer CD,
Silberstein LE, Ness PM, et al, eds. Blood
banking and transfusion med- icine: Basic
principles and practice. 2nd ed.
Philadelphia: Churchill Livingstone, 2007:
639- 51.
74. Eder AF, Kennedy JM, Dy BA, et al. Limiting
and detecting bacterial contamination of
apheresis platelets: Inlet-line diversion and
increased culture volume improve component
safety. Transfusion 2009;49:1554-63.
75. AABB. Recommendations to address residual
risk of bacterial contamination of platelets. As-
sociation Bulletin #12-04. Bethesda, MD:
AABB, 2012.
76. Jacobs MR, Smith D, Heaton WA, et al.
Detec- tion of bacterial contamination in
prestorage
 C H A P T E R 8 Infectious Disease Screening
culture-negative apheresis platelets on day
of issue with the Pan Genera Detection
test. Transfusion 2011;51:2573-82.
77. Centers for Disease Control and Prevention.
West Nile virus transmission via organ trans-
plantation and blood transfusion—Louisiana,
2008. MMWR Morb Mortal Wkly Rep
2009;58: 1263-7.
78. Centers for Disease Control and Prevention.
Fatal West Nile virus infection after
probable transfusion-associated transmission
—Colora- do, 2012. MMWR Morb Mort
Wkly Rep 2013; 62:622-4.
79. Otani MM, Vinelli E, Kirchhoff LV, et al.
WHO
comparative evaluation of serologic assays for
Chagas disease. Transfusion 2009;49:1076-82.
80. Food and Drug Administration. Blood Prod-
ucts Advisory Committee meeting: 94th meet-
ing, April 1, 2009, Rockville, MD. Silver
Spring, MD: CBER Office of
Communication, Out- reach, and
Development, 2009. [Available at
http://www.fda.gov/downloads/Advisory
Committees/CommitteesMeetingMaterials/
BloodVaccinesandOtherBiologics/BloodProd
uctsAdvisoryCommittee/UCM155597.pdf (ac-
cessed December 8, 2010).]
81. Gubernot DM, Nakhasi HL, Mied PA, et al.
Transfusion-transmitted babesiosis in the
United States: Summary of a workshop.
Trans- fusion 2009;49:2759-71.
82. Herwaldt BL, Linden JV, Bosserman E, et al.
Transfusion-associated babesiosis in the Unit-
ed States: A description of cases. Ann Intern
Med 2011;155(8):509-19.
83. Johnson ST, Cable RG, Tonnetti L, et al.
Sero-
prevalence of Babesia microti in blood donors
from Babesia-endemic areas of the northeast-
ern United States: 2000 through 2007.
Transfu- sion 2009;49:2574-82.
84. Food and Drug Administration. Blood Prod-
ucts Advisory Committee, July 26, 2010,
Gaith- ersburg, MD. Silver Spring, MD:
CBER Office of Communication, Outreach,
and Develop- ment, 2010 [Available at
http://www.fda.gov/
downloads/AdvisoryCommittees/Commit
teesMeetingMaterials/BloodVaccinesand
OtherBiologics/BloodProductsAdvisoryCom
mittee/UCM225388.pdf (accessed December
8, 2010).]
85. Moritz ED, Johnson ST, Winton C, et al. Pro-
spective investigational blood donation
screening for Babesia microti. Transfusion
2013; 53(Suppl):13A.
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86. Mali S, Steele S, Slutsker L, Arguin PM.
Malaria surveillance—United States, 2007.
MMWR Surveill Summ 2009;58:1-16.
87. Eliades MJ, Shah S, Nguyen-Dinh P, et al. Ma-
laria surveillance—United States, 2003.
MMWR Surveill Summ 2005;54:25-39.
88. Purdy E, Perry, E, Gorlin, J. et al.
Transfusion- transmitted malaria:
Unpreventable by cur- rent donor
guidelines? Transfusion 2003;43
(Suppl):79A.
89. Gonzalez C, Layon A, Arguin P, et al. Transfu-
sion-transmitted falciparum malaria from an
asymptomatic donor. Transfusion 2011;51
(Suppl):197A.
90. Mali S, Tan KR, Arguin PM. Malaria surveil-
lance—United States, 2009. MMWR Morb
Mortal Wkly Rep 2011;60:1-15.
91. Spencer B, Steele W, Custer B, et al. Risk
for malaria in United States donors
deferred for travel to malaria-endemic
areas. Transfusion 2009;49:2335-45.
92. Food and Drug Administration. Guidance for
industry: Recommendations for donor ques-
tioning, deferral, reentry and product man-
agement to reduce the risk of transfusion-
transmitted malaria. (August 2013) Silver
Spring, MD: CBER Office of Communication,
Outreach and Development, 2013. [Available
at http://www.fda.gov/downloads/Biologics
BloodVaccines/GuidanceComplianceRegula
toryInformation/Guidances/Blood/UCM
080784.pdf (accessed November 14, 2013).]
93. Anez G, Rios M. Dengue in the United States of
America: A worsening scenario? Biomed Res
Int 2013;2013:678645.
94. Petersen LR, Busch MP. Transfusion-transmit-
ted arboviruses. Vox Sang 2010;98:495-503.
95. Food and Drug Administration. Guidance for
industry: Revised preventive measures to re-
duce the possible risk of transmission of
Creutzfeldt-Jakob disease (CJD) and variant
Creutzfeldt-Jakob disease (vCJD) by blood and
blood products. (May 2010) Silver Spring,
MD: CBER Office of Communication,
Outreach, and Development, 2010. [Available
at http://
www.fda.gov/downloads/biologicsbloodvac
cines/guidancecomplianceregulatoryinfor
mation/guidances/UCM213415.pdf (ac-
cessed December 4, 2013).]
96. Slot E, Hogema BM, Riezebos-Brilman A, et al.
Silent hepatitis E virus infection in Dutch
blood donors, 2011 to 2012. Euro Surveill 2013;
18.
212 ■ AABB T EC HNIC AL MANUAL

97. Webert KE, Cserti CM, Hannon J, et al. Pro-

99. Klein HG, Anderson D, Bernardi MJ, et al.
ceedings of a consensus conference: Pathogen Pathogen inactivation: Making decisions
inactivation-making decisions about new about new technologies. Report of a consen-
technologies. Transfus Med Rev 2008;22:1-34. sus conference. Transfusion 2007;47:2338-47.
98. AuBuchon J, Prowse C, eds. Pathogen
inactiva- tion: The penultimate paradigm shift.
Bethes- da, MD: AABB Press, 2010.
 C h a p t e r 9
Hospital Storage, Monitoring,
Pretransfusion Processing,
Distribution, and Inventory
Management of Blood Components
Nancy M. Dunbar, MD
B L OOD COMPONENT MANU FAC-
T UR I NG is highly regulated and must
comply with Food and Drug Administration
(FDA) current good manufacturing practice
(cGMP) regulations from the time of
collection to product issue.1,2 The intent of
cGMP is to maintain the safety, purity,
potency, quality, and identity of blood
components. Collection and transfusion
facilities must develop poli- cies, processes,
and procedures and retain rec- ords to
demonstrate compliance with regula- tions
for storage, monitoring, pretransfusion
processing, and distribution of blood
compo- nents. These policies, processes, and
proce- dures must also comply with
additional regu- lations (Clinical Laboratory
Improvement Amendments and state
laboratory statutes) and policies and
standards applicable to blood banking from
voluntary laboratory accrediting
organizations (eg, College of American
Pathol- ogists, AABB,3 The Joint
Commission, and American Osteopathic
Association).
Nancy M. Dunbar, MD, Assistant Professor, Dartmouth-Hitchcock Medical Center, Lebanon, New
Hampshire The author has disclosed no conflicts of interest.
BLOOD AND BLOOD COMPONENT
STORAGE AND MONITORING
General Considerations
Transport and storage requirements must be
followed when blood components are trans-
ferred from the collection site to the process-
ing facility, from the supplier to the blood
bank, or from the blood bank to the patient.
Storage requirements and expiration dates
vary by product type and are based on
factors such as in-vitro red cell metabolism
for Red Blood Cell (RBC) components in
various stor- age solutions or coagulation
protein stabiliza- tion for plasma products
(Table 9-1). Failure to adhere to these
storage and expiration require- ments can
result in decreased product potency and/or
safety.
Temperature requirements during trans-
port of blood components differ from those
during storage.4 Shipping from the supplier
to
213
214 ■ AABB T EC HNIC AL MANUAL
the hospital blood bank is considered trans-
port, and applicable temperature require-
ments must be met. When blood
components are issued from the blood bank
to the patient care area, maintenance of
appropriate tem- perature requirements
allows for the possibili- ty of returning the
component to inventory if it is not
transfused.
Refrigerators, freezers, and platelet
incu- bators for blood and blood component
storage can be equipped with continuous-
tempera- ture-monitoring devices to allow
detection of temperature deviations before
products are af- fected. Automated
electronic monitoring de- vices that are
available include: 1) weekly pen and chart
recorders, 2) sets of hard-wired or radio-
frequency temperature-recording devic- es,
and 3) centralized temperature-monitor- ing
systems. Thermometers or thermocouples
should be strategically placed in the equip-
ment for optimal temperature monitoring. If
an automated temperature-recording device
is not used, temperatures of the blood
storage environment must be recorded
manually ev- ery 4 hours. This requirement
includes ambi- ent room temperature
monitoring of platelets that are not stored in
a platelet chamber or in- cubator.
Recorded temperatures should be
checked
daily to ensure proper operation of the equip-
ment and recorder. Deviations from acceptable
temperature ranges should be documented and
explained (including any actions taken), dated,
and initialed by the person noting the devia-
tion.
Most product-storage devices are
equipped with audible alarms to alert
personnel that tem- perature ranges are
approaching unacceptable levels. Central
alarm monitoring allows facili- ties that do
not have personnel in the vicinity of the
equipment to alert designated staff at
another location when an alarm is activated.
Because platelets must be gently agitated
dur- ing storage, typically using horizontal
flatbed or elliptical rotators, alarm systems
should also emit alerts when the platelet
agitator has malfunctioned.
Transfusion services may locate blood
storage refrigerators in other areas of the hos-
pital to allow immediate access to blood in
emergency situations. Such a practice re-
quires that the same blood component
storage monitoring standards be met in these
other ar- eas.
If an equipment failure occurs and pre-
vents acceptable temperature ranges from
be- ing maintained, the facility should have
poli- cies, processes, and procedures in
place to relocate blood and blood
components. The secondary storage location
may be another on- or off-site refrigerator or
freezer, qualified storage boxes, or coolers
used with a validated process that has been
shown to maintain re- quired storage
temperatures during storage. Because the
safety, purity, potency, and quality of the
blood components could be affected by
delays in relocation to a secondary storage
lo- cation, it is recommended that the
relocation occur before upper or lower
acceptable storage temperatures are
exceeded. This can be ac- complished by
setting the alarm points of the storage
devices so that an alarm sounds before the
unacceptable tempature limit is reached.
Some facilities may use temperature-
monitoring indicators for each blood
compo- nent container. Such indicators
monitor the liquid temperature of the
immediate inner bag, not the liquid core
temperature in the unit, which may be
cooler. Policies, processes, and procedures
should specify how the facility will
determine the disposition of blood com-
ponents when using temperature-monitoring
indicators.
Specific Considerations
Holding blood components that have been
dispensed from the blood bank to other
hospi- tal areas before transfusion is
considered to be “storage.” If the blood
components are not kept in a monitored
device, they must be stored in containers
(eg, boxes or coolers) vali- dated to maintain
the correct temperature during storage.
Red Blood Cells
RBC components are stored in plastic bags
of different types with a variety of added
antico- agulants and additive solutions that
modify
TABLE 9-1. Requirements for Storage, Transportation, and Expiration 3(pp51-59)

Item No.
Component Storage Transport Expiration1 Additional Criteria

Whole Blood Components
1 Whole Blood
1-6 C
If intended for room
tempera- ture components,
then store at 1-6 C within 8
hours after collection
CHAP TER 9
Cooling toward 1-10 C. ACD/CPD/CP2D: 21 days
If intended for room CPDA-1: 35 days
tempera- ture components,
cooling toward 20-24 C

2 Whole Blood Irradiated
Red Blood Cell Components
3 Red Blood Cells (RBCs)
4 Deglycerolized RBCs
5 Frozen RBCs
40% Glycerol
6 RBCs Irradiated
Inventory
Storage, Processing, Distribution, and
1-6 C 1-10 C Original expiration or 28 days
from date of irradiation,
which- ever is sooner
1-6 C 1-10 C ACD/CPD/CP2D: 21 days
CPDA-1: 35 days
Additive solution: 42 days
Open system: 24 hours
1-6 C 1-10 C Open system: 24 hours
Closed system: 14 days or
as
FDA approved
–65 C if 40% glycerol or Maintain frozen state 10 years Frozen within 6 days of
as col-
FDA approved (A policy shall be developed lection unless rejuvenated
if
rare frozen units are to be Frozen before Red Blood
Cell ■
retained beyond this time) expiration if rare unit
1-6 C 1-10 C Original expiration or 28 days
from date of irradiation,

215
which-
ever is sooner
(Continued)
 216
TABLE 9-1. Requirements for Storage, Transportation, and Expiration3(pp51-59) (Continued)

Item
No. Component Storage Transport Expiration1 Additional Criteria
7 RBCs Leukocytes Reduced 1-6 C 1-10 C ACD/CPD/CP2D: 21 days ■
CPDA-1: 35 days
Additive solution: 42 days
Open system: 24 hours

AABB T ECHNICAL M A NUAL

8 Rejuvenated RBCs 1-6 C 1-10 C CPD, CPDA-1: 24 hours AS-1: freeze after rejuvena-
tion
9 Deglycerolized Rejuvenated 1-6 C 1-10 C 24 hours or as approved by
RBCs FDA
10 Frozen Rejuvenated RBCs –65 C Maintain frozen state CPD, CPDA-1: 10 years
AS-1: 3 years
(A policy shall be developed
if
rare frozen units are to be
retained beyond this time)
11 Washed RBCs 1-6 C 1-10 C 24 hours
12 Apheresis RBCs 1-6 C 1-10 C CPDA-1: 35 days
Additive solution: 42 days
Open system: 24 hours
13 Apheresis RBCs Leukocytes 1-6 C 1-10 C CPDA-1: 35 days
Reduced Additive solution: 42 days
Open system: 24 hours
14 Platelets 20-24 C with continuous As close as possible to 24 hours to 5 days, Maximum time without
depending
gentle agitation 20-24 C2 on collection system agitation: 24 hours
Platelet Components
15 Platelets—Irradiated 20-24 C with continuous As close as possible to No change from original expi- Maximum time without
gentle agitation 20-24 C2 ration date agitation: 24 hours
16 Platelets—Leukocytes 20-24 C with As close as possible Open system: 4 hours Maximum time without
Reduced continuous gentle to 20-24 C2 Closed system: No change in agitation: 24 hours
agitation expiration
17 Pooled Platelets Leukocytes 20-24 C with continuous As close as possible to 4 hours after pooling or 5 Maximum time without

CHAP TER 9
days
Reduced gentle agitation 20-24 C2 following collection of the old- agitation: 24 hours
est unit in the pool3
18 Pooled Platelets 20-24 C with continuous As close as possible to Open system: 4 hours
(in open system) gentle agitation 20-24 C2
19 Apheresis Platelets 20-24 C with continuous As close as possible to 24 hours or 5 days, Maximum time without

Inventory
Storage, Processing, Distribution, and
depending
gentle agitation 20-24 C2 on collection system agitation: 24 hours
20 Apheresis Platelets 20-24 C with continuous As close as possible to No change from original expi- Maximum time without
Irradiated gentle agitation 20-24 C2 ration date agitation: 24 hours
21 Apheresis Platelets Leuko- 20-24 C with continuous As close as possible to Open system: within 4 Maximum time without
hours
cytes Reduced gentle agitation 20-24 C2 of opening the system agitation: 24 hours
Closed system: 5 days
22 Apheresis Platelets Platelet 20-24 C with continuous As close as possible to 5 days Maximum time without
Additive Solution Added gentle agitation 20-24 C2 agitation: 24 hours
Leukocytes Reduced
Granulocyte Components
23 Apheresis Granulocytes 20-24 C As close as possible to 24 hours Transfuse as soon as
possi-
20-24 C ble; Standard 5.28.10
applies3(45)
24 Apheresis Granulocytes 20-24 C As close as possible to No change from original Transfuse as soon as
possi- ■
Irradiated 20-24 C expiration date ble; Standard 5.28.10
applies3(45)

217
(Continued)
 218
TABLE 9-1. Requirements for Storage, Transportation, and Expiration3(pp51-59) (Continued)

Item
No. Component Storage Transport Expiration1 Additional Criteria
25 Cryoprecipitated AHF –18 C Maintain frozen state 12 months from original Thaw the FFP at 1-6 C ■
collection Place cryoprecipitate in the
freezer within 1 hour after
removal from refrigerated

AABB T ECHNICAL M A NUAL

centrifuge
Plasma Components
26 Cryoprecipitated AHF (after 20-24 C As close as possible to Single unit: 6 hours Thaw at 30-37 C
thawing) 20-24 C
27 Pooled Cryoprecipitated AHF –18 C Maintain frozen state 12 months from earliest Thaw the FFP at 1-6 C
(pooled before freezing) date of collection of Place cryoprecipitate in the
product
in pool freezer within 1 hour after
removal from refrigerated
centrifuge
28 Pooled Cryoprecipitated AHF 20-24 C As close as possible to Pooled in an open system: Thaw at 30-37 C
(after thawing) 20-24 C 4 hours
If pooled using a sterile
connection device: 6 hours
29 Fresh Frozen Plasma (FFP)5 –18 C or –65 C Maintain frozen state –18 C: 12 months from Place in freezer within
collection 8 hours of collection or as
–65 C: 7 years from stated in FDA-cleared opera-
collection tor’s manuals/package
inserts
Storage at –65 C
requires
FDA approval if product is
stored for longer than
12 months
30 FFP (after thawing)5 1-6 C 1-10 C If issued as FFP: 24 hours Thaw at 30-37 C or using
an FDA-cleared device
31 Plasma Frozen Within 24 –18 C Maintain frozen state 12 months from collection
Hours After Phlebotomy

CHAP TER 9
(PF24)5
32 Plasma Frozen Within 24 1-6 C 1-10 C If issued as PF24: 24 hours Thaw at 30-37 C or using
an
Hours After Phlebotomy FDA-cleared device
(after thawing)5
33 Plasma Frozen Within 24 –18 C or colder Maintain frozen state 12 months from collection

Inventory
Storage, Processing, Distribution, and
Hours After Phlebotomy Held
At Room Temperature Up To
24 Hours After Phlebotomy
(PF24RT24)
34 Plasma Frozen Within 24 1-6 C 1-10 C If issued as PF24RT24: Thaw at 30-37 C or using
an
Hours After Phlebotomy Held 24 hours FDA-cleared device
At Room Temperature Up To
24 Hours After Phlebotomy
(after thawing)
35 Thawed Plasma5 1-6 C 1-10 C 5 days from date product Shall have been collected
was
thawed or original expiration, and processed in a closed
whichever is sooner system
36 Plasma Cryoprecipitate –18 C Maintain frozen state 12 months from collection
Reduced
37 Plasma Cryoprecipitate 1-6 C 1-10 C If issued as Plasma Cryopre- Thaw at 30-37 C
Reduced (after thawing) cipitate Reduced: 24 hours ■

219
(Continued)
 220
TABLE 9-1. Requirements for Storage, Transportation, and Expiration3(pp51-59) (Continued) ■
Item
No. Component Storage Transport Expiration1 Additional Criteria
38 Thawed Plasma Cryoprecipi- 1-6 C 1-10 C If issued as Thawed Shall have been collected

AABB T ECHNICAL M A NUAL

Plasma
tate Reduced Cryoprecipitate Reduced: 5 and processed in a closed
days from date product system
was
thawed or original expiration,
whichever is sooner
39 Liquid Plasma 1-6 C 1-10 C 5 days after expiration of 21 CFR 610.53(c) applies
Whole Blood
40 Recovered Plasma, liquid Refer to short supply Refer to short supply Refer to short supply Requires a short supply
or agree- agree- agree-
frozen ment ment ment agreement4
Tissue and Derivatives
41 Tissue Conform to source facility’s Conform to source facility’s Conform to source facility’s 21 CFR 1271.3(b),
written instructions written instructions written instructions 1271.3(bb), and
21 CFR 1271.15(d) apply
42 Derivatives Conform to manufacturer’s Conform to manufacturer’s Conform to manufacturer’s
written instructions written instructions written instructions
1
If the seal is broken during processing, components stored at 1 to 6 C shall have an expiration time of 24 hours, and components stored at 20 to 24 C shall have an expiration
time of 4 hours, unless otherwise indicated. This expiration shall not exceed the original expiration date or time.
2
21 CFR 600.15(a).
3
Storage beyond 4 hours requires an FDA-cleared system.
4
21 CFR 601.22.
5
These lines could apply to apheresis plasma or whole-blood-derived plasma.
 C H A P T E R 9 Storage, Processing, Distribution, and Inventory
the cellular and protein environment. The
storage temperature must be maintained at
1 to 6 C throughout the duration of stor-
age.3(pp51-53) Changes that may occur during
storage directly affect how long RBC units
may be stored. Product approval by the FDA
re- quires in-vivo labeling studies
demonstrating that at least 75% of the
transfused red cells are present in circulation
24 hours after transfu- sion with less than
1% hemolysis.
During ex-vivo storage of RBC units, bio-
chemical and morphologic changes to the
red cells occur that have been termed the
“storage lesion.” These changes include cell
membrane shape change and
microvesiculation; de- creased pH,
adenosine triphosphate, and 2,3-
diphosphoglycerate; and increased
lysophos- pholipids, potassium, and free
hemoglobin.5 Although these ex-vivo
observations are well described, there is
currently insufficient evi- dence to conclude
that storage duration corre- lates with
clinical outcomes.6,7 One situation where
evidence supports the use of fresher RBCs is
large-volume transfusions (>25 mL/ kg) in
neonates.8
Platelets
Platelet storage conditions and shelf life are af-
fected by morphologic and functional changes
during storage. Metabolic changes include the
glycolytic production of lactic acid and the
oxi- dative metabolism of free fatty acids,
which re- sults in the production of carbon
dioxide. Platelet pH is maintained above 6.2
via buffer- ing of lactic acid by bicarbonate
and promo- tion of oxidative metabolism
facilitated by the diffusion of oxygen and
carbon dioxide across a gas-permeable storage
bag during gentle agi- tation.9 The maximum
time for platelets to be without agitation is 24
hours.3(pp53-55)
Platelet shelf life also is limited because
of
an increased risk of bacterial growth due to
room-temperature storage. Blood banks and
transfusion services are required to have
methods to detect or inactivate bacteria in all
platelet components.3(p11)
■ 221
Granulocytes
Granulocytes are fragile, deteriorate rapidly in
vitro, and should be transfused as soon as pos-
sible after receipt from the supplier. Granulo-
cytes are stored at 20 to 24 C, should not be
ag- itated, and must never be leukocyte
reduced.
PRETRANSFUSION
PROCESSING
Thawing Plasma and Cryoprecipitate
Fresh Frozen Plasma (FFP), Plasma Frozen
within 24 Hours After Phlebotomy (PF24),
and Plasma Frozen within 24 Hours After
Phlebot- omy Held at Room Temperature up to
24 Hours After Phlebotomy (PF24RT24) must
be thawed at 30 to 37 C using a waterbath or
other FDA-approved device. Thawing in a
waterbath requires the frozen component to be
in a plas- tic overwrap before insertion into the
water to prevent contamination of the
container entry ports. Thawed plasma products
(FFP, PF24, and PF24RT24) are stored at 1 to
6 C and expire 24 hours after thawing.
These products must be relabeled as
“Thawed Plasma” if they are stored for longer
than 24 hours. Although not licensed by the
FDA, Thawed Plasma is included in the
AABB Standards for Blood Banks and
Transfusion Services3(p28) and the Circular of
Information for the Use of Human Blood and
Blood Compo- nents.10 Thawed Plasma is
stored at 1 to 6 C and expires 5 days after it
was originally thawed. Facilities may label
such products as “Thawed Plasma” at the
initial time of thaw- ing. By maintaining a
Thawed Plasma invento- ry, transfusion
services may decrease wastage of thawed
plasma products.11
Levels of labile coagulation factors (Fac-
tor V and Factor VIII) and stable factors are
well above 50% of immediate post-thaw
levels in Thawed Plasma that has been
stored for up to 5 days.12 Thawed Plasma
does, however, contain reduced
concentrations of Factor V, Factor VII, and
Factor VIII. For this reason, Thawed Plasma
is not suitable for single-factor replacement
when antihemophilic factor de- rivatives are
unavailable.
222 ■ AABB T EC HNIC AL MANUAL
Cryoprecipitate is thawed at 30 to 37 C,
is gently resuspended, and can be pooled for
ease of transfusion using small quantities of
0.9% sodium chloride, injection (USP) to
rinse the contents of the bag into the final
container (Method 6-11). Thawed
cryoprecipitate is stored at 20 to 24 C and
expires within 4 hours of pooling if it is
pooled in an open system or within 6 hours
for single units or units pooled using an
FDA-cleared sterile connecting de- vice.10
Thawing and Deglycerolizing RBCs
RBC units may be frozen and stored for up
to 10 years following the addition of
glycerol as a cryopreservation agent
(Methods 6-6 and 6-7).13,14 Frozen units can
be thawed using a 37 C dry heater or 37 C
waterbath. After being thawed, the glycerol
must be removed before the component is
transfused. Commercial in- struments for
batch or continuous-flow wash- ing are
available for deglycerolization. The
manufacturer’s instructions should be fol-
lowed to ensure maximal red cell recovery
and minimal hemolysis. Measurement of
free he- moglobin in the final wash can be
used to con- firm adequate free hemoglobin
removal and as a surrogate marker for
adequate deglyceroliza- tion (Method 6-8).
Integrally attached tubing must be filled
with the deglycerolized red cells and sealed
appropriately so that a segment may be de-
tached and available for crossmatch testing.
The shelf life of Deglycerolized RBCs de-
pends on the type of system used. Closed-
system devices allow storage for up to 14
days, but components prepared using open
systems expire within 24 hours of
deglycerolization.
Platelet Gel Production
Platelet gel is produced when thrombin and
calcium are added to platelet-rich plasma to
produce a glue-like substance for surgical
ap- plication.15 This product is typically
prepared at the bedside immediately before
use. Facili- ties involved in the production of
this compo- nent should refer to the current
edition of the AABB Standards for
Perioperative Autologous Blood Collection
and Administration for guid-
ance and quality oversight of this
manufactur- ing process.
Irradiation
Irradiation of cellular components is
intended to prevent transfusion-associated
graft-vs- host disease (TA-GVHD), which is
caused by proliferation of donor T
lymphocytes. People at increased risk of
TA-GVHD include pro- foundly
immunocompromised patients, re- cipients
of intrauterine transfusion, patients
undergoing marrow or peripheral blood stem
cell transplantation, and recipients of
cellular components from blood relatives or
donors selected for HLA compatibility.
Sources of radiation include gamma
rays (cesium-137 or cobalt-60
radioisotopes) and x-rays. The required
gamma radiation dose needed to prevent
proliferation of donor T lymphocytes in the
recipient is a minimum of 25 Gy (2500
cGy/rad) to the central point of the blood
container and 15 Gy (1500 cGy/rad) to any
other part of the container. Confirmation
that the blood container has received an ade-
quate radiation dose can be achieved with
the use of commercially available
radiographic film labels.
Irradiation is associated with damage to
the red cell membrane, which may result in in-
creases in extracellular free hemoglobin and
potassium during product storage. For this
reason, the expiration date of irradiated RBCs
is 28 days after irradiation or the original expi-
ration date, whichever is earlier.
Hospital transfusion services may pur-
chase irradiated blood components from
their supplier or perform irradiation within
the blood bank using approved and
monitored ra- diation devices. Hospitals that
perform their own irradiation may irradiate
their inventory on demand or in batches.
Maintenance of a dual inventory (irradiated
and nonirradiated) requires policies and
procedures to ensure that transfusion
recipients receive the appro- priate
component for their clinical situation.
Poststorage Leukocyte Reduction
Poststorage leukocyte reduction can be per-
formed by the blood bank before issuing a
 C H A P T E R 9 Storage, Processing, Distribution, and Inventory
component using a leukocyte reduction filter
attached by a sterile connection. It can also
be performed at the bedside during
transfusion using a blood-administration
filter designed for this purpose. Leukocyte
reduction filters are designed to remove
>99.9% of white cells (3-log reduction) and
meet the AABB standard of less than 5 × 106
leukocytes in 95% of sam- pled units for
RBCs and Apheresis Plate- lets. 3(p24) The
manufacturer’s instructions must be followed
for the filtration device used to achieve
acceptable leukocyte reduction.
Prestorage leukocyte reduction is
general- ly the preferred method for
leukocyte reduc- tion because it prevents
accumulation of cyto- kines during product
storage. Quality control of bedside filtration
is challenging, and the process has been
associated with hypotensive transfusion
reactions.16
Volume Reduction
Volume reduction results when plasma and
additive solutions are partially removed
from RBC or platelet components following
centrif- ugation. This process may be used
to aggres- sively manage volume in patients
at risk of transfusion-associated circulatory
overload, reduce exposure to plasma
proteins or addi- tives, or achieve a target
hematocrit level.
Volume reduction of platelets is
described in Method 6-13. The speed of
centrifugation may affect the degree of
platelet loss. Higher g forces are associated
with better platelet reten- tion but raise the
theoretical concern of plate- let damage and
activation as platelets are forced against the
container wall. When plate- let volumes are
reduced, platelets should rest at room
temperature for 20 to 60 minutes fol- lowing
centrifugation and before resuspension in
remaining plasma or added saline. The
manufacturer’s instructions must be
followed regarding the minimum volume
necessary to maintain proper air exchange
across the gas- permeable platelet-storage
bag. The shelf life of volume-reduced
platelets is 24 hours for components stored
at 1 to 6 C and 4 hours for those stored at 20
to 24 C.
■ 223
Washing
Cellular components are typically washed to
remove plasma proteins. Washing can also be
performed to remove glycerol from frozen
RBC units after thawing. Indications for
washing RBC or platelet components include a
patient history of severe allergic reactions to
compo- nents containing plasma, the presence
of anti- bodies against immune globulin A
(IgA) in an IgA-deficient recipient when IgA-
deficient cel- lular components are not
available, the pres- ence of maternal anti-
HPA-1a (eg, when using maternal blood for a
neonatal transfusion), and the need for
complement removal.
Washing is accomplished with the use
of 1 to 2 L of sterile normal saline
(preferably using automated equipment). As
with volume re- duction, washed platelets
should rest at room temperature without
agitation between cen- trifugation. Up to
20% of the red cell yield or 33% of the
platelet yield may be lost during washing.
Because washing creates an “open system”
and removes anticoagulant-preserva- tive
solutions, washed RBC units expire 24
hours after washing and washed platelet
units expire 4 hours after washing. It is
recommend- ed that hospitals performing
washing comply with the manufacturer’s
recommendations re- garding minimum
volumes needed for com- ponent storage
bags to maintain optimal stor- age
conditions.
Pooling
Certain blood components (whole-blood-
derived platelets, cryoprecipitate, or RBCs
and plasma to produce reconstituted whole
blood) may need to be pooled to provide
clinically ef- fective transfusion therapy
without the need to transfuse multiple single
components.
Pooled whole-blood-derived platelets
may contain a significant number of red cells,
and, therefore, ABO compatibility and risk for
RhD alloimmunization are recipient factors
that must be considered. If whole-blood-
derived platelets are pooled using an open sys-
tem, the expiration time is 4 hours from the
start of pooling. A commercially available,
FDA- approved, prestorage, whole-blood-
derived,
224 ■ AABB T EC HNIC AL MANUAL
platelet pooling system allows storage for up
to 5 days and the ability to perform culture-
based bacterial testing.17 When this system is
used, the pool maintains the expiration date
of the earliest collected component in the
pool.
Single cryoprecipitate units are pooled
af-
ter thawing in a manner similar to that used
for platelets. The expiration time of cryopre-
cipitate pools depends on the method used
for pooling. Cryoprecipitate pooled in an
open system expires within 4 hours of
pooling. Thawed single concentrates and
pooled con- centrates using sterile
connecting devices ex- pire 6 hours after
thawing. Thawed cryopre- cipitate is stored
at 20 to 24 C. As an alternative, the blood
center may pool single concentrates before
freezing.
Reconstituted whole blood consists of
RBCs combined with ABO-compatible FFP.
This product can be used for neonatal ex-
change transfusion. The conventional ap-
proach is to combine group O RBCs (Rh com-
patible with the neonate) and group AB FFP to
achieve a 50% ± 5% hematocrit of the final
product. The volumes of the two components
before pooling can be adjusted to achieve a
desired hematocrit level. Following recombi-
nation, the product can be stored at 1 to 6 C
for up to 24 hours.
Current FDA uniform guidelines should
be followed when pooled products are la-
beled.18 A unique pool number should be af-
fixed to the final container, and all units in
the pool must be documented in electronic
or manual records.
Aliquoting
Patients requiring low-volume transfusions
may receive aliquots of smaller volumes de-
rived from the original unit via an FDA-
cleared sterile connecting device or integrated
transfer bags. Available products designed for
use with sterile connecting devices include
transfer packs, smaller-volume bags, and
tubing with integrally attached syringes.
The expiration date of the aliquot and
minimum residual volumes that must be
maintained depend on the storage container
used. Hospital transfusion services must de-
velop policies and procedures for aliquot
preparation and storage that comply with
manufacturer specifications. The use of ali-
quots for neonatal transfusion has been shown
to result in a decreased number of donor expo-
sures.19 The process of preparing aliquots for
small-volume transfusion in neonates and
children is discussed in greater detail in Chap-
ter 23. Lower-volume components (split units)
may also be prepared for adult patients who
require slow rates of transfusion due to con-
cerns about fluid overload. Split units are rec-
ommended when the component volume can-
not be transfused at a rate that ensures
completion of the transfusion within 4 hours.
DISTRIBUTION
Inspection
Inspection is a critical control point in blood
component manufacturing and must occur
before shipping, upon receipt, and before is-
sue for transfusion. Proper documentation of
this process includes 1) date of inspection,
2) donor identification number, 3)
description of any visual abnormalities, 4)
action(s) taken, and 5) identity of the staff
member performing the inspection. Visible
abnormalities may in- clude discoloration of
the segments, compo- nent, or supernatant
fluid or the presence of visible clots,
particulate matter, or other for- eign bodies.
Detection of any such abnormali- ties should
result in product quarantine for further
investigation that may include return- ing
the component to the supplier.
If a component is determined to be bacte-
rially contaminated, the component
manufac- turer must be notified so that an
immediate investigation can take place.
Other compo- nents prepared from that
collection should be quarantined until the
investigation is com- plete. If the component
(or co-component) has been transfused, the
recipient’s attending physician should be
notified, and consultation with the medical
director is recommended.
 C H A P T E R 9 Storage, Processing, Distribution, and Inventory
Shipping
Blood components may be transported be-
tween blood centers, hospitals, and blood
cen- ters. All containers used to transport
blood components must be qualified before
use to ensure that the proper component
transport temperature is maintained. The
shipping tran- sit time, mode of transport,
and climatic con- ditions must also be
validated. All components should be
inspected upon receipt to confirm
appropriate transport conditions, component
appearance, and expiration date. Any devia-
tion from routine shipping or component
con- ditions should be reported to the
shipping fa- cility and documented
according to each location’s policies,
processes, and procedures.
Whole Blood, RBCs, and
Thawed Plasma Products
Whole blood, RBCs, and thawed plasma
prod- ucts must be transported at a
temperature of 1 to 10 C. A variety of
options exist for maintain- ing transport
temperature, including bagged wet ice,
commercial cooling packs, and spe- cially
designed containers. All transport cool- ers
must be qualified to maintain the transport
temperature when packed using a validated
process.
Blood components transported at 1 to
10 C and stored at 1 to 6 C may need to be
tempo- rarily removed from those
temperatures for entry into inventory,
irradiation, or other pro- cessing. The
maximum number of units that can be
manipulated before the component reaches
an unacceptable temperature should be
determined and not exceeded. Validation of
this process may be accomplished using
man- ual temperature monitoring indicators
affixed to the blood components or
electronic devices that can measure the
temperature of the blood components
without opening the bag.
Platelets, Thawed Cryoprecipitate,
and Granulocytes
Platelets, thawed cryoprecipitate, and granu-
locytes must be transported at a temperature
of 20 to 24 C. All transport coolers must be
qualified to maintain this transport tempera-
■ 225
ture when packed using a validated process.
For platelets, the maximum time without
agi- tation is 24 hours.
Frozen Components
Frozen components should be packaged to
minimize breakage and maintain a frozen
state. Dry ice in a suitable container is
routine- ly used for shipping these
components. All transport coolers must be
qualified to main- tain the transport
temperature when packed using a validated
process.
Receiving
The receiving facility should notify the ship-
ping facility and document any deviation
from usual shipping container packing or
appear- ance of the shipped blood
components. Any blood component not
complying with the fa- cility’s policies,
processes, and procedures should be
quarantined. Only after investiga- tion of the
deviation and determination that the
component meets acceptance criteria may
the component be removed from quarantine
and released into the general inventory.
Blood components should be fully trace-
able from collection to final disposition.
Elec- tronic or manual records indicating
compli- ance with policies, processes, and
procedures should be generated and
maintained for the applicable record-
retention time. Any devia- tion must be
recorded, and blood components not
meeting requirements should be quaran-
tined. Deviations must be investigated to de-
termine appropriate product disposition and
possible corrective action. The results of any
corrective action should be reported to the
blood supplier as needed. Inventory
manage- ment should consist of routine
determination that all blood components are
accounted for and transfused or
appropriately discarded.
Product Testing
Before transfusion, the ABO group of all
units and Rh type of any units labeled “Rh
negative” must be confirmed by serologic
testing for all red-cell-containing components
(RBCs, whole blood, and granulocytes). Any
typing discrep-
226 ■ AABB T EC HNIC AL MANUAL

must be resolved before issue.

ancies identified must be reported
immediate- ly to the supplier and resolved
before the prod- uct is dispensed for
transfusion.

Issuance of Components
Ensuring that the correct blood component
is transfused to the correct patient is
paramount for transfusion safety. All
requests for blood components must contain
two independent identifiers so that the
intended recipient can be uniquely identified.
Recipient compatibility- testing records must
also be reviewed. Current testing results
must be compared with histori- cal records,
if available, and any discrepancies must be
resolved before product selection.
Personnel must visually inspect and
doc- ument that the selected product is
acceptable for use. This inspection must
include confir- mation that the product does
not have an ab- normal color or appearance
and that the con- tainer is intact. Once
selected for transfusion, the blood
component must have an attached label or
tie tag that contains the intended re- cipient’s
two independent identifiers, dona- tion
identification number, and compatibility test
result interpretation, if performed.
At the time of issue, there must be a
final check of each unit that includes the
following:

1. The intended recipient’s two independent

identifiers, ABO group, and Rh type.
2. The donation identification number,
donor
ABO group, and, if required, Rh type.
3. The interpretation of the crossmatch test
results, if performed.
4. Special transfusion requirements (eg,
cyto-
megalovirus-reduced-risk, irradiated, or
antigen-negative components), if applica-
ble.
5. The expiration date and, if applicable, time.
6. The date and time of issue.

The transfusion service must confirm

that the recipient identifying information,
transfu- sion request, testing records, and
blood com- ponent labeling and
compatibility are accu- rate and in
agreement. Any discrepancies identified
 RBCs.
Final identification 4. Documentation indicates that the compo-
of the transfusion re- nent has been inspected and is acceptable
cipient and blood for reissue.
component rests with
the transfusionist. This Individual unit temperature indicators
individual identifies the or temperature-reading devices can be used
patient and donor unit to determine the acceptability of products
and certifies that the for re- turn to inventory. Blood and blood
identifying information compo- nents may also be transported or
on forms, tags, and la- stored in qualified containers using a
bels is in agreement. validated process that has been shown to
maintain acceptable temperatures for a
Documentation defined interval. If time frames are used to
determine the acceptability of a product’s
The patient’s medical return to inventory, the time frame must be
record must include validated by the individual fa- cility. The
proper documentation validation should demonstrate that for the
of all transfusions. For defined period, the appropriate tem- perature
each transfusion, this of the product has been maintained.
documentation must
contain the transfusion
order, consent for
transfusion, component
name, donation iden-
tification number, date
and time of transfu-
sion, pre- and
posttransfusion vital
signs, volume
transfused,
identification of the
trans- fusionist, and, if
applicable, any
transfusion- related
adverse events.

Return of Blood
Components
and Reissue
The transfusion service
may receive back into
inventory units that
meet acceptance
specifica- tions. These
conditions include the
following:

1. The primary container has not been

opened.
2. The component has
been maintained at
the appropriate
temperature.
3. At least one sealed
segment remains
inte- grally attached
to the container of
 C H A P T E R 9 Storage, Processing, Distribution, and Inventory
Components meeting the acceptance
cri- teria may be returned to the general
blood inventory and reissued. Components
not meeting the acceptance criteria must be
quar- antined for further investigation or
discarded in a biohazard container to
prevent inadver- tent return to inventory.
INVENTORY MANAGEMENT
General Considerations
A sufficient number of ABO- and Rh-
compati- ble units should be available to
meet routine hospital needs, allow for
unanticipated in- creases in utilization due to
emergency situa- tions, and minimize
component outdating. Factors that influence
determination of blood bank component
inventory levels include his- toric usage
patterns, outdate rates, and dis- tance from
suppliers. Inventory levels should be
periodically evaluated in response to insti-
tutional changes that may affect component
usage, including expansion of inpatient beds
or operating rooms; implementation of new
surgical procedures; or changes in hospital
guidelines or medical practice that may
influ- ence transfusion behavior.
The blood bank should also maintain a
reserve of universally compatible RBCs for
emergency use and have a reliable emergency
delivery system to ensure adequate availability
of blood components in unexpected situations
when demand exceeds supply. A disaster plan
should be developed and tested periodically
(see Chapter 4).
Inventory levels should be monitored dai-
ly to facilitate timely ordering from blood sup-
pliers and maintain adequate inventory levels.
This can be particularly challenging for plate-
lets due to their 5-day shelf life. Inventory-
management plans should also take into con-
sideration desirable inventory levels of special
products, such as cytomegalovirus-seronega-
tive, leukocyte-reduced, and irradiated com-
ponents. Antigen-negative RBC units and
HLA-matched or HLA-selected platelets are
typically ordered on an as-needed basis from
suppliers.
■ 227
Surgical Blood Ordering Practices
Component outdate rates are influenced by
surgical ordering practices. For example,
when RBC units are crossmatched for
surgical pa- tients, the shelf life of the unit is
shortened if the component is unused. When
crossmatch- to-transfusion (C:T) ratios are
monitored, a C:T ratio of >2.0 may indicate
excessive order- ing of crossmatched blood.
One approach to reducing excessive
C:T ratios is to identify procedures that do
not typ- ically require blood, and use this
information to develop guidelines for the
use of type and screen units instead of
crossmatched units. Maximum surgical
blood order schedules for common elective
procedures can also be de- veloped based on
local transfusion utilization patterns.20 This
practice is particularly useful in hospital
transfusion services that lack the ability to
perform electronic crossmatching. These
schedules can also be applied to pa- tients
undergoing elective surgery who are known
to have clinically significant alloanti- bodies
and require crossmatch-compatible, antigen-
negative blood. Once a surgical blood-
ordering schedule has been estab- lished, the
transfusion service routinely cross- matches
the predicted number of units for each
patient undergoing the designated pro-
cedures. Routine orders may need to be
modi- fied for patients with anemia,
bleeding disor- ders, or other conditions in
which increased blood use is anticipated. As
with other circum- stances that require rapid
availability of blood components, the
transfusion service staff should be prepared
to provide additional blood components if
the need arises.
Emergent Transfusion
If transfusion is deemed medically necessary
and blood is released before pretransfusion
testing is complete, the records must contain
a signed statement from the requesting
physi- cian indicating that the clinical
situation was sufficiently urgent to require
the release of blood components before
completion of compatibility testing. If there
has been time to confirm the recipient’s
ABO group, the transfusion service should
issue ABO- and
228 ■ AABB T EC HNIC AL MANUAL

Rh-compatible blood. If the patient’s ABO
group is unknown, group O RBCs should be
is- sued.
A more detailed discussion of emergent
transfusion that covers clinical concerns
rath- er than inventory management can be
found in Chapter 15.
Massive Transfusion
Massive transfusion can be defined as the ad-
ministration of 8 to 10 RBC units to an adult
patient in less than 24 hours, acute adminis-
tration of 4 to 5 RBC units in 1 hour, or ex-
change transfusion of an infant.
To ensure the ability to accurately inter-
pret ABO group testing results, the patient
specimen should be obtained for testing as
early as possible during massive transfusion.
If the patient ABO type cannot be
determined,
continued support with group O RBCs is
rec- ommended. Unexpected and significant
usage of group O RBCs in the setting of
massive transfusion should be considered
when deter- mining component inventory
levels. In mas- sive transfusion situations
where large amounts of blood may be
required, policies may be developed to
provide D-positive RBCs to select patients,
such as all adult males and postmenopausal
females.
Many hospitals have developed
massive- transfusion protocols to
standardize the re- sponse to hemorrhage.21,
22
These protocols are designed to rapidly
provide blood compo- nents in a balanced
ratio of plasma and plate- lets to RBCs,
particularly when laboratory test- ing is not
rapid enough to guide transfusion support.
Additional studies are needed to clar- ify
whether the use of these protocols is associ-
ated with improved patient outcomes.

KEY POINTS

Ensuring that the correct blood component is transfused to the correct patient is para- mount for transfusion safety. It must be
Visual inspection of the blood component is a critical control point in the manufacturing process and must occur before shipp
Refrigerators, freezers, and platelet incubators for blood component storage must be moni- tored to ensure that proper storage
Temperature requirements during transport of blood components differ from those during storage. Blood components held out
Acceptable time frames for returning blood components to inventory after issue should be validated by individual facilities. In
Thawed FFP, PF24, and PF24RT24 expire within 24 hours of thawing. These products may be labeled as “Thawed Plasma” to
 C H A P T E R 9 Storage, Processing, Distribution, and Inventory
REFERENCES
1. Food and Drug Administration. Drugs; current
good manufacturing practice in manufacture,
processing, packing, or holding. (June 19,
1963) Fed Regist 1963;133:6385-7.
2. Code of federal regulations. Title 21, CFR
Parts 210 and 211. Washington, DC: US
Government Printing Office, 2014 (revised
annually).
3. Levitt J, ed. Standards for blood banks and
transfusion services. 29th ed. Bethesda, MD:
AABB, 2014.
4. Nunes E. Transport versus storage: What is
the difference? AABB News 2013;15(2):4-5.
5. Klein HG, Spahn DR, Carson JL. Red blood
cell transfusion in clinical practice. Lancet
2007; 370:415-26.
6. van de Watering L. Red cell storage and prog-
nosis. Vox Sang 2011;100:36-45.
7. Zimrin AB, Hess JR. Current issues to the
transfusion of RBCs. Vox Sang 2009;96:93-
103.
8. Strauss RG. Data-driven blood banking prac-
tices for neonatal RBC transfusions. Transfu-
sion 2000;40:1528-40.
9. Shrivastava M. The platelet storage lesion.
Transfus Apher Sci 2009;41:105-13.
10. AABB, American Red Cross, America’s Blood
Centers, Armed Services Blood Program. Cir-
cular of information for the use of human
blood and blood components. Bethesda, MD:
AABB, 2013.
11. Werhli G, Taylor NE, Haines, AL, et al.
Institut- ing a thawed plasma procedure: It just
makes sense and saves cents. Transfusion
2009;49: 2625-30.
12. Tholpady A, Monson J, Radovancevic R, et al.
Analysis of prolonged storage on coagulation
Factor (F)V, FVII, and FVIII in thawed
plasma: Is it time to extend the expiration date
beyond 5 days? Transfusion 2013;53:645-50.
13. Meryman HT, Hornblower M. A method for
freezing and washing RBCs using a high glyc-
erol concentration. Transfusion 1972;12:
14556.
14. Valeri CR, Ragno G, Pivacek LE, et al. A
multi- center study of in vitro and in vivo
values in
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human RBCs frozen with 40-percent (wt/vol)
glycerol and stored after deglycerolization for
15 days at 4 degrees C in AS-3: Assessment of
RBC processing in the ACP 215. Transfusion
2001;41:933-9.
15. Borzini P, Mazzucco L. Platelet gels and releas-
ates. Curr Opin Hematol 2005;12:473-9.
16. Cyr, M, Hume H, Sweeney JD, et al. Anomaly
of the des-Arg9-bradykinin metabolism
associat- ed with severe hypotensive reaticon
during blood transfusions: A preliminary
report. Transfusion 1999;39:1084-8.
17. Benjamin RJ, Kline L, Dy BA, et al. Bacterial
contamination of whole-blood-derived plate-
lets: The introduction of sample diversion and
prestorage pooling with culture testing in the
American Red Cross. Transfusion 2008;48:
2348-55.
18. Food and Drug Administration. Guidance: In-
dustry consensus standard for the uniform la-
beling of blood and blood components using
ISBT 128 version 2.0.0, November 2005.
(Sep- tember 22, 2006) Silver Spring, MD:
CBER Of- fice of Communication, Outreach,
and Devel- opment, 2006.
19. Liu EA, Mannino FL, Lane TA. Prospective,
randomized trial of the safety and efficacy of a
limited donor exposure transfusion program
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1994;125:92- 6.
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guideline for anticipated blood usage during
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Massive transfusion protocols for patients
with sub- stantial hemorrhage. Transfus Med
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 C h a p t e r 1 0

Molecular Biology and

Immunology in Transfusion
Medicine

James C. Zimring, MD, PhD, and Steven L. Spitalnik, MD

I N THIS CHAPTER, the fundamental

principles for analyzing deoxyribonucle-
ic acid (DNA), ribonucleic acid (RNA), and
protein are described. In transfusion medi-
cine, these techniques are used to 1) detect
in- fectious pathogens; 2) predict the
phenotype of red cell, platelet, and
neutrophil antigens; 3) detect and identify
red cell and platelet anti- bodies; 4)
determine HLA type; and 5) perform
relationship testing. In addition to describing
the principles behind these analyses, this
chapter describes potential problems with
the assay systems. This chapter also
addresses the basic mechanisms by which
the immune sys- tem initiates a response to
foreign antigens.
Entire textbooks have been written to
de- scribe these processes, and it is outside
the scope of this chapter to provide detailed
and comprehensive explanations. Instead,
this chapter focuses on topics immediately
rele- vant to the practice of transfusion
testing.
Moreover, this chapter predominantly focuses
on humoral immunity because the role of cel-
lular immunity in transfusion medicine re-
mains unclear and is rarely, if ever, of conse-
quence in the pathophysiology of hemolytic
transfusion reactions. This chapter does not
provide specific assay protocols; rather, it
seeks to provide an understanding of the sci-
entific principles underlying the molecular bi-
ological and immunological assays used in
transfusion medicine.
NUCLEIC ACID ANALYSIS
The clinical utility of nucleic acid analysis
in transfusion medicine lies in detecting
infec- tious pathogens and genotyping blood
donors and recipients. The human genome is
encod- ed in polymers of DNA, and,
likewise, many pathogens utilize DNA to
encode their ge- nome. Alternatively, many
viral pathogens

James C. Zimring, MD, PhD, Director, Transfusion Medicine Research, Puget Sound Blood Center Research
Institute, Seattle, Washington, and Steven L. Spitalnik, MD, Professor of Pathology and Cell Biology, and
Director of Clinical Laboratories, Columbia University, New York, New York
J. Zimring has disclosed financial relationships with Immucor Inc and Haemonetics. S. Spitalnik has
disclosed no conflicts of interest.

231
232 ■ AABB T EC HNIC AL MANUAL
encode their genome as RNA. In addition,
be- cause normal flora may have a
substantial ef- fect on human biology, DNA
and RNA analysis may become important to
monitor normal nonpathological and
commensal microorgan- isms. With the
possible exception of prion- associated
disorders, either DNA or RNA en- codes all
known genetic material relevant to
transfusion medicine.
Basic Chemistry and Structure of
Nucleic Acids
DNA is a nucleic acid polymer consisting of
long chains of nucleotides linked together.1
Nucleotides consist of a pentose (a carbohy-
drate with five carbon atoms), a phosphate
group attached to the fifth carbon atom (C5) of
the pentose, and a base group attached to C1
[Fig 10-1(A)]. Variations in the chemistry of
the base group give rise to the four different
nucle-
FIGURE 10-1. Chemical structure of nucleic acids and DNA.
otides comprising DNA: the purines
(adenine and guanine) and pyrimidines
(cytosine and thymine). In addition, C3 of
the pentose is modified by a hydroxyl group
[Fig 10-1(A)]. The individual nucleotides
form DNA poly- mers when the phosphate
group on C5 of one nucleotide forms a
covalent bond with the free hydroxyl group
on C3 of another nucleotide [Fig 10-1(B)].
DNA molecules vary from each other based
on the sequence of nucleotides that are
incorporated into the polymer. Each DNA
strand has a terminal 5' end that contains a
free phosphate (attached to C5) and a termi-
nal 3' end that has a free hydroxyl group (at-
tached to C3).
The human genome consists of double-
stranded DNA. The bases contained within a
single strand of DNA form hydrogen bonds
to complementary bases on another strand of
DNA. In particular, thymidine (T) binds to
ad-
CH A P T E R 10 Molecular Biology and Immunology in Transfusion Medicine
enine (A), and guanine (G) binds to cytosine
(C). When two strands have complementary
sequences, they can hybridize to form a
double-stranded molecule [Fig 10-1(C)].
The two complementary strands hybridize
such that the 5' and 3' ends have opposite
orienta- tions and form a double helix in
which the phosphodiester backbone is on the
outside of the helix and the hydrogen-bond-
paired bases are on the inside.
When genes are expressed, the DNA
en- coding a given gene is transcribed into
RNA [Fig 10-1(C)]. The structure of RNA is
similar to that of DNA with the following
exceptions: 1) ribonucleotides have an
additional hydroxyl group on C2 of the
pentose sugar (thus form- ing ribose), 2)
uracil (U) replaces thymine (T), and 3) RNA
coding for gene products is typically single-
stranded (although double- stranded RNA
has important regulatory roles). Several
classes of RNA exist in human cells; the
type described in this paragraph, which is
subsequently translated into protein, is
messenger RNA (mRNA). When a gene is ex-
pressed, the transcription machinery
unwinds the DNA double helix, synthesizes
a mRNA strand of complementary
sequence, and rehy- bridizes the DNA in its
wake [Fig 10-1(C)]. In this way, the RNA is
essentially a copy of the sequence found in
the DNA. RNA is synthe- sized in the 5' to
3' direction, and, thus, only a single strand
of the DNA is transcribed into RNA by any
given run of a polymerase. After synthesis
in the nucleus, RNA is processed and
exported to the cytoplasm, where ribosomes
translate it into protein.
Isolation of Nucleic Acids
The first step in most DNA and RNA analyses
is the isolation of nucleic acids. All nucleated
cells of an individual contain identical genom-
ic DNA, with some notable exceptions (eg, re-
arranged genes in mature T and B cells). Ge-
nomic DNA can be isolated from readily
obtainable cellular sources, such as peripheral
blood leukocytes and buccal swabs. In con-
trast, mRNAs are distinct in different cell pop-
ulations because their expression patterns are
■ 233
important in defining the phenotype of these
cells.
For mRNA analysis, then, the choice of
starting cell types is critical. Multiple manu-
facturers offer reagents that simplify and
expe- dite isolation of cellular DNA and/or
mRNA as well as for purifying viral nucleic
acids from plasma. Depending on the type
of testing to be performed, the quantity and
quality of the nu- cleic acids isolated may be
important, as de- termined by the relative
purity of DNA or RNA and the absence of
contaminating protein.
Hybridization-Based Methods of
Nucleic Acid Detection
Before the advent of techniques that allowed
the amplification of a specific nucleic acid
se- quence (see Polymerase Chain Reaction
be- low), detection of nucleic acids
depended on hybridization-based methods.
Probes with a particular sequence of
nucleotides were syn- thesized and labeled
with one of a variety of detectable markers.
The probe could then hy- bridize to
complementary sequences of DNA or RNA,
allowing the detection and quantifica- tion
of the corresponding complementary tar-
get. This could be done in the solid phase
(ie, Southern and Northern blots), the fluid
phase (ie, RNAse protection assay or S1
protection assay) or as a result of enzymatic
extension (ie, primer extension assay).2-5
However, compared to more recent
amplification-based techniques,
hybridization-based methods have limited
sensitivity and are less amenable to automa-
tion. Although hybridization-based methods
are useful in basic research and still play a
mi- nor role in some diagnostic laboratories,
most analyses of nucleic acids are
performed using amplification-based
methods.
The Polymerase Chain Reaction
Nucleic acid detection and analysis were revo-
lutionized by the invention of the polymerase
chain reaction (PCR). PCR was the first ampli-
fication-based technique for generating nucle-
ic acid fragments for direct analysis.6 The
concept of amplification-based systems has
234 ■ AABB T EC HNIC AL MANUAL
grown to include many other techniques and
applications.
A PCR reaction requires 1) a DNA
sample to be analyzed; 2) gene-specific
primers; 3) a thermostable DNA polymerase;
and 4) compo- nents that allow the DNA
polymerase to enzy- matically synthesize
DNA, including nucleo- tides (A, T, C, and G)
and the proper salts and buffer. The PCR
reaction involves repeat cycles of
heating/cooling (thermocycling), allowing
exponential DNA amplification of the frag-
ment of interest. This reaction is carried out in
a thermocycler that rapidly changes tempera-
ture with accuracy and precision. The thermo-
cycling reaction involved is 1) heat denatur-
ation of double-stranded DNA, 2) cooling to
FIGURE 10-2. Graphic overview of the polymerase chain reaction.
allow primer annealing, and 3) extension
and synthesis of DNA on the primer strand.
This procedure continues for multiple
cycles, typi- cally 20 to 40, depending on
the abundance of the template and the
required sensitivity of the assay.
An overview of the PCR process is pre-
sented in Fig 10-2. This example begins
with a single copy of a double-stranded
DNA tem- plate. The DNA is denatured by
heating to near boiling (typically at 94-95
C), which disrupts the hydrogen bonds
between complementary bases, thereby
separating the two strands. The temperature
is then lowered (annealing reac- tion) to
allow gene-specific primers to anneal to
their complementary target. Typically, an
CH A P T E R 10 Molecular Biology and Immunology in Transfusion Medicine
annealing temperature of 5 C below the
melt- ing temperature of the target DNA-
primer complex increases specificity to the
correct target sequence. The exact melting
tempera- ture of a given target DNA primer
complex de- pends on the primer length,
abundance of guanines and cytosines in the
sequence (ie, GC content), and any
mismatches with the target. At this point,
the temperature is raised to that at which the
DNA polymerase functions opti- mally
(typically 72 C), and the primers are ex-
tended along the length of the DNA by the
in- corporation of the correct
complementary nucleotides by DNA
polymerase. Thus, at the end of extension,
there are two copies of the DNA. This
process repeats itself with denatur- ing,
annealing, and extension. With each sub-
sequent cycle, an exponential increase in
DNA copy number occurs. PCR results in a
geomet- ric expansion of a selected DNA
“amplicon,” defined as the sequence that is
flanked by the two chosen primers.
PCR Considerations
Although PCR is a robust and reliable
method of detecting nucleic acids, as with
all methods, technical problems can affect
PCR and other amplification-based
techniques.
Specimen Processing and
Template Degradation
DNA is stable and can usually withstand some
variations in storage temperature and han-
dling before being processed for genomic
analysis. Exceptions include samples in which
the target DNA is present in low quantity, such
as fetal typing from maternal plasma and viral
testing. RNA is far less stable than DNA and
is susceptible to autocatalytic degradation by
RNAse enzymes found in many biological
specimens.
Inhibitors
PCR amplification depends on the enzymatic
activity of DNA polymerase and can be
inhibit- ed by any substance that negatively
affects synthesis by DNA polymerase. Heparin
can in- hibit PCR in some situations, and
hemoglobin
■ 235
or lactoferrin released from erythrocytes or
leukocytes also inhibit PCR.7,8 Most analytic
systems are optimized such that the risk of
in- terference by an inhibitor is minimized,
but care should be taken not to deviate from
es- tablished protocols because such
deviations may introduce unintended
inhibitory sub- stances. Amplifying
ubiquitous target se- quences present in all
samples (ie, conserved genomic DNA or
housekeeping genes, such as hemoglobin)
and/or spiking the specimen with an internal
positive control can be used to assess
whether inhibitors are present.
Primer Design
Inferior performance of primers should not be
a concern in the context of commercially
avail- able tests. Nevertheless, a thorough
under- standing of primer design is important
in trou- bleshooting assays, and primer design
is a key component in developing PCR-based
assays for new targets. Although ideal primers
hy- bridize to a target that is found in only one
lo- cation in the entire genome, given the com-
plexity of genomic DNA, this is not always
possible. Primer annealing to unintended tar-
gets can occur, resulting in ongoing consump-
tion of primers with each cycle and the poten-
tial to amplify unintended targets.
In addition to cross annealing to unin-
tended genomic sites, primers can anneal to
each other and form a short amplicon. For
ex- ample, if the 3' ends of two primers bind
to each other, the polymerase fills in the 5'
over- hangs [Fig 10-3(A)]. A primer
modified in this way may still be able to
anneal to its genomic target sequence.
However, the additional bas- es added to the
3' end no longer anneal to the genomic
target and may prohibit proper ex- tension.
This “primer-dimer formation” can take
place between two different primers in a
reaction or two molecules of the same
primer [Fig 10-3(B)]. Primers can also form
hairpin loops and anneal to themselves [Fig
10-3(C)]. A self-complementary sequence in
a primer may allow this to occur,
particularly in primers over 20 bp in length,
and this reaction is fa- vored because of its
intramolecular nature. If self-annealing
results in a 5' overhang, the
236 ■ AABB T EC HNIC AL MANUAL
PCR Primers
5’ 3’
5’
5’ 3’
A. B.
5’ 3’
3’ 5’ 5’
3’
5’ 3’ 5’
3’ 5’
3’
FIGURE 10-3. Potential problems in primer design that may inhibit polymerase chain reaction (PCR).
molecule can self-prime with the polymerase
and extend the 3' end to be complementary
to the 5' end, increasing the tendency of the
primer to self-anneal. The presence of the
ad- ditional sequence prevents amplification
of the authentic target amplicon.
Contamination between Specimens
One of the greatest strengths of PCR is its
abili- ty to amplify very small amounts of
genetic material. In theory, single-copy
sensitivity can be achieved. In practice,
detection of 10 copies of DNA or fewer is not
uncommon, depending on the sensitivity of the
assay readout. This level of sensitivity also
makes the assay sus- ceptible to false-positive
results due to con- tamination from specimens
being analyzed or amplicons generated in
previous amplifica- tion reactions. Beginning
with just 10 mole- cules of DNA, 30 rounds of
amplification in a PCR reaction yields more
than 1 × 1010 ampli- cons. Thus, if only
0.0000001% of a previous reaction is
inadvertently introduced onto a pi- pette used
to set up a subsequent reaction, a false-positive
result may occur.
To minimize the possibility of contamina-
tion from previously generated amplicons and
a false-positive signal, PCR laboratories rou-
tinely process samples in only one direction.
Normal Amplification
3’ 5’
3’
C.
5’
3’
3’
5’
3’ 5’
3’
5’
Using this approach, DNA extraction is
sepa- rate from testing, PCR reactions are
assembled in one room and amplified in a
second room, and downstream analysis (if
required) is per- formed in a third room.
There should be no retrograde flow, and no
materials or instru- ments used in the
amplification or analysis (post-PCR) rooms
should make their way into the PCR setup
(pre-PCR) room. Pipette filter tips that
minimize carry-over contamination and
sample aerosols are routinely used.
Other ways to minimize potential con-
tamination include the addition of deoxyuri-
dine triphosphate (dUTP) to PCR reactions.
Polymerases incorporate dUTP in place of de-
oxythymidine triphosphate (dTTP), and the
added enzyme uracil-DNA glycosylase (UNG)
specifically cleaves DNA containing uracil but
not normal DNA or RNA.9 Thus, adding UNG
to PCR reactions destroys contaminating am-
plicons from previous amplifications but not
native DNA in the specimen. The UNG is heat
inactivated during the initial denaturation
PCR step, allowing amplification of the new
sample.
Reverse-Transcriptase PCR
When amplification and analysis of mRNA
is required, a reverse-transcriptase (RT)
enzyme
CH A P T E R 10 Molecular Biology and Immunology in Transfusion Medicine
that synthesizes DNA from an RNA
template is used.10,11 Like DNA polymerase,
RT synthesizes in the 5' to 3' direction and
requires the an- nealing of a primer to
initiate transcription. When RNA is
transcribed, the resulting DNA is a single-
stranded complementary copy of the RNA
and is referred to as “complementary DNA”
(cDNA). The cDNA is then a suitable
substrate for PCR, as described above.
Transcription-Mediated Amplification
and Nucleic Acid Sequence-Based
Amplification
Since the advent of PCR, several additional
nu- cleic acid amplification strategies have
been devised. Among the most useful are
two relat- ed techniques, transcription-
mediated ampli- fication (TMA) and nucleic
acid sequence- based amplification
(NASBA).12,13 Although TMA and NASBA
have some differences (de- scribed in this
section), they are conceptually similar and,
thus, are described together (see Fig 10-4).
FIGURE 10-4. Schematic overview of transcription-mediated amplification (TMA) and nucleic acid
sequence- based amplification.
RNA = ribonucleic acid; cDNA = complementary deoxyribonucleic acid; mRNA = messenger RNA;
RNAse = ribonuclease.
■ 237
TMA plays a large role in nucleic acid
test- ing for human immunodeficiency virus
(HIV), hepatitis C virus, and West Nile virus
(ie, using the Chiron/Gen-Probe system). Both
tech- niques use RNA as the amplification
target. The reaction contains two primers, RT,
DNA polymerase, RNAse H, and T7
polymerase. The reaction begins when a
specific downstream primer (Primer 1)
hybridizes to the 3' end of the target RNA and
RT synthesizes a cDNA copy (Fig 10-4, Step
1). Primer 1 not only con- tains a
complementary sequence at its 3' end that
hybridizes to the target RNA, but the 5' end
also encodes a bacteriophage T7 promot- er.
The RNA template is degraded by RT in the
TMA assay or by RNAse H in an additional
step with NASBA (Fig 10-4, Step 2). A
second 5' up- stream primer (Primer 2) then
binds to the newly synthesized cDNA (Fig 10-
4, Step 3) and utilizes DNA polymerase to
synthesize a double-stranded DNA molecule
(Fig 10-4, Step 4). This molecule has a T7
promoter at one end, and T7 polymerase then
drives transcrip- tion of RNA (Fig 10-4, Step
5). Numerous RNA
238 ■ AABB T EC HNIC AL MANUAL
transcripts are synthesized from a single DNA
template. These new RNA molecules can reen-
ter the amplification cycle with Primer 2 initi-
ating reverse transcription, followed by RNA
degradation and subsequent synthesis of DNA
using Primer 1 and DNA polymerase. This
leads to additional amplification with ongoing
cycles of transcription and template synthesis.
One distinct advantage of NASBA compared
to PCR is that repeat nucleic acid denaturation
is not required. Therefore, NASBA amplifies
RNA sequences in an isothermal reaction that
does not require a thermocycler.
In addition to the amplification methods
described above, several other techniques
use nucleic acid polymerizing or ligating
enzymes to amplify DNA and/or RNA.
These include strand displacement
amplification (SDA) and the ligase chain
reaction (LCR).14-16 There are also
probe/signal-amplification methods, such as
the Cleavase Invader, branched DNA, and
hybrid capture assays. Although these
techniques have been adapted to detect
pathogens, they are not widely used in
transfu- sion medicine and are not described
further here.17
Detection of Nucleic Acid
Amplification Products
PCR, RT-PCR, TMA, and NASBA amplify
spe- cific nucleic acid sequences. However,
the am- plified sequences must still be
detected. Tradi- tionally, this has been
accomplished by separating the
amplification reactions by gel
electrophoresis in the presence of a fluores-
cent dye (eg, ethidium bromide), which
makes the nucleic acids visible in the gel
under ultra- violet light. This allows
visualization of the bands and, if appropriate
standards are in- cluded in the gels, provides
a molecular weight by which one can
distinguish amplicons of the correct size
from cross-reactive products of different
sizes. To confirm that the amplified nucleic
acids have the correct sequence, the
amplification products can be digested with
restriction endonucleases and the sizes of
the resulting molecules determined. Because
the amplicon sequence is known, one can
predict the correct restriction sites [this is
known as
“restriction fragment length polymorphism”
(RFLP) analysis]. Even greater specificity can
be achieved by hybridization analysis. After
electrophoresis, the amplified products are
transferred to nitrocellulose paper, which is
then hybridized with DNA probes specific for
the amplified sequences.
Each of these approaches requires gel
electrophoresis, which is time consuming,
in- compatible with the throughput needs of
donor-testing laboratories, and not easily
automated. Moreover, it requires opening
tubes containing amplification reactions and
manipulating the products, which increases
the risk of contamination of reagents and
false-positive results in subsequent samples.
More advanced techniques for detecting
amplification products rely on the chemistry
of fluorescent molecules. Probes that
fluoresce only when the correct amplicon is
present are included in the amplification
reactions. By in- cluding a fluorescence
spectrophotometer in the thermocycler, the
generation of amplifica- tion products can
be measured at each cycle. This approach,
called “real-time PCR,” is high- ly sensitive;
much more quantitative than gel
electrophoresis-based methods; and, by
using multiple reporter dyes with distinct
fluores- cence spectra, makes detection of
multiple targets in a single reaction feasible
(ie, multi- plex nucleic acid amplification).
In addition, including fluorescent probes in
the reaction allows analysis without ever
opening the tube containing the
amplification products; this de- creases the
risk of laboratory contamination with
amplicons and subsequent false-positive
results.
Two of these fluorescent methods rely on
the juxtaposition of a fluorescent molecule
with a quencher molecule that prevents fluo-
rescence. In the TaqMan system, a
sequence- specific probe has the fluorescent
molecule at one end and the quencher at the
other. When the probe hybridizes to its
target, it essentially blocks the DNA
polymerase that is synthesiz- ing the next
round of DNA by extending from the
primer. When the DNA polymerase en-
counters the hybridized probe, it degrades
the probe, thus separating the fluorescent
and quencher molecules [Fig 10-5(A)].
Because
CH A P T E R 10 Molecular Biology and Immunology in Transfusion Medicine ■ 239

FIGURE 10-5. Methods of detection by sequence-specific probes during real-time polymerase chain reaction.
Pol = polymerase enzyme.
240 ■ AABB T EC HNIC AL MANUAL
this occurs only when the probe hybridizes
to its target, fluorescence is generated as a
func- tion of amplicon generation.
A second approach uses molecular bea-
cons that also have a fluorescent molecule on
one end of a DNA probe and a quencher mole-
cule on the other end. In this case, the se-
quence-specific probe is flanked by two se-
quences that are complimentary to each other
and form a hairpin loop, thus juxtaposing the
fluorescent molecule with its quencher. How-
ever, when the probe hybridizes to its target,
the hairpin loop unfolds, separating the
quencher from the fluorescent molecule and
allowing fluorescence to occur [Fig 10-5(B)].
A third approach uses two molecules
that do not fluoresce unless they are in close
prox- imity to each other. These molecules
are linked to two separate DNA probes that
hy- bridize to adjacent sequences on the
ampli- con. If the amplicon is present, the
probes an- neal in such a way that the two
molecules are in close proximity, providing
a fluorescent sig- nal [Fig 10-5(C)].
A fourth method uses SYBR® green dye,
which fluoresces when bound to double-
stranded DNA. SYBR® green is not sequence
specific and, thus, detects all amplicons. How-
ever, authentic amplicons can be distin-
guished from cross-reactive products by melt-
ing curve analysis. Because the melting curve
is a function of amplicon size and GC content
and the size and sequence of the correct am-
plicon is known, the melting curve can be used
to confirm the identity of the amplicon.
As with PCR, these fluorescent-probe
techniques can be applied to other
amplifica- tion technologies, such as TMA.
Analysis of Single-Nucleotide
Polymorphisms
The vast majority of blood group antigens
con- sist of single-nucleotide
polymorphisms (SNPs). The gene product is
present in most people, but the difference
that determines the identity of the blood
group antigen is a small change in sequence,
often a single nucleotide.
Some SNPs destroy or create
recognition sites for restriction
endonucleases. In these
cases, PCR-amplified material can be
digested by restriction enzymes and then
analyzed by agarose electrophoresis to
observe the result- ing fragment sizes. RFLP
analysis has low throughput and depends on
the presence of a restriction enzyme
recognition site associated with the SNP in
the gene of interest.
Several different methods for detecting
SNPs consist mostly of modifications of PCR
or array technologies. With these methods,
prim- ers or probes are engineered such that
hybrid- ization depends on the presence of
the correct SNP. Both multiplex PCR and
DNA array sys- tems can determine the
genotype of blood group antigens in
individual specimens.17-19 These systems
offer the advantage of higher throughput and
automated readout while avoiding the need
for complex interpretation.
Genotyping of red cell antigens may be
more efficient than traditional serologic typ-
ing. In addition, when a patient has received
multiple RBC units and it is not possible to
dis- tinguish the patient’s own red cells from
trans- fused red cells, genotyping the
patient’s DNA may be the only reliable way
to predict the pa- tient’s red cell phenotype.
However, occasion- ally the genotype may
not correlate with the phenotype. It is worth
noting that genotyping typically focuses on
known polymorphisms but does not provide
the entire sequence of the gene or its
regulatory regions. Therefore, genotyping
might not predict phenotype when
1) new, hitherto unknown, polymorphisms in
the coding region alter protein structure; 2)
new polymorphisms in the coding,
promoter, or other regulatory regions
prevent gene ex- pression despite the
presence of the correct coding sequence; or
3) changes alter the epit- ope (ie,
modification by bacterial enzymes) when
epitopes depend on posttranslational
modification.
PROTEIN ANALYSIS
Nucleic acid analysis, as described in the
“Nu- cleic Acid Analysis” section above,
detects the presence of DNA or RNA that
encodes genom- ic material and/or measures
gene expression at the RNA level. However,
due to regulation of protein translation, the
presence of mRNA
CH A P T E R 10 Molecular Biology and Immunology in Transfusion Medicine
does not always correlate with the presence
of the encoded protein. In addition, the
detec- tion of antibodies, which are proteins,
cannot be determined by detecting or
measuring nu- cleic acids. Accordingly,
measuring protein- protein and/or protein-
carbohydrate interac- tions provides relevant
biological information in transfusion
medicine that may not be ob- tainable by
nucleic acid analysis.
Fluid-Phase Assays (Agglutination-
Based Methods)
Depending on antibody isotype,
immunoglob- ulins have 2 to 10 antigen-
binding sites per molecule. Each antibody
can bind more than one target molecule,
allowing antibodies to cross-link antigens
present in multiple copies on particles, such
as red cells or beads. This cross-linking can
aggregate particles that have the relevant
antigen on their surface, a process known as
“agglutination.” Agglutination is an old, but
generally reliable, serologic method for
detecting antibody-antigen interactions and
is used extensively in transfusion medi- cine.
Agglutination can be detected by
several
methods. The antigen copy number and den-
sity varies depending on the blood group.
Ag- glutination is used for serological
crossmatch- ing (donor red cells incubated
with recipient plasma or serum), screening
for unexpected antibodies (reagent red cells
of known blood group antigen composition
incubated with patient plasma or serum),
and blood group antigen phenotyping of the
donor or recipient (test red cells incubated
with monoclonal anti- bodies or reagent-
quality antisera of known specificity).
Because red cells are easily visible due to
their red color, several systems were devised
to detect agglutination. These include 1)
tube testing in which agglutination is
visually de- tected by the adhesion of red
cells to one an- other in the postcentrifuge
pellet, 2) passive agglutination in microtiter
plates where agglu- tination is visualized by
the spread pattern of red cells in individual
wells, and 3) gel testing in which
unagglutinated red cells pass through a
matrix but agglutinated complexes are re-
■ 241
tained at the top or within the matrix due to
their greater size.20 In addition to blood bank
serology, red cells can be used to detect anti-
bodies against other antigens by linking
these particular antigens to the red cell
surface. Ag- glutination-based assays can
also be engi- neered using particles other
than red cells, such as latex beads. Each of
these techniques uses the same basic
principles and has broad applicability in
clinical diagnostic testing.
Although agglutination reactions are
very sensitive and easy to perform, the
formation of agglutinates depends on the
proper stoichio- metric ratio of antibody to
antigen. When the ratio of antigen to
antibody is in a range such that
agglutination readily occurs, it is referred to
as the “zone of equivalence.” In this situa-
tion, each arm of the antibody binds to a dif-
ferent particle, and a network (or lattice) of
linked particles results in agglutination [Fig
10-6(B)]. A false-negative result is generated
if the stoichiometric ratio is outside the zone
of equivalence at either extreme.
A prozone effect can occur when such a
high concentration of antibody is present
that the likelihood of an antibody being able
to bind to two separate particles or red cells
is di- minished [Fig 10-6(A)]. This is seen
most com- monly with non-red-cell-based
agglutination assays, such as the rapid
plasma reagin screen- ing test for syphilis
serology. Although prozone effects are
unusual in classical red cell serolo- gy, they
have been observed when titers of red cell
antibodies are very high. In particular, dis-
crepant reverse ABO typing due to prozone
ef- fects has been reported. 21,22 Diluting the
se- rum being tested and using EDTA
containing diluents decreases the likelihood
of prozone effects. One reason why prozone
effects are uncommon in red cell serology is
that the use of antihuman globulin (AHG)
largely over- comes these problems.
However, a secondary “prozone-like” effect
occurs if there is inade- quate washing
before adding AHG.23 In this case, residual
immune globulin G (IgG) in so- lution binds
the AHG and competes for AHG binding to
IgG on the red cells. This may occur with
very high titers of blood group antibodies or
even in the presence of highly increased lev-
els of polyclonal antibodies, as in
hypergam-
242 ■ AABB T EC HNIC AL MANUAL
FIGURE 10-6. Effects of relative concentrations of antigen and antibody on the outcome of
agglutination reactions.
maglobulinemia. In this setting, additional
washing steps remove the problem.23
In theory, false-negative agglutination re-
actions can occur due to a postzone effect in
which the antigen is in excess [Fig 10-6(C)].
In this situation, every antibody binds to multi-
ple epitopes on the same particle, thereby pre-
venting the cross-linking that leads to aggluti-
nation. This could occur if a large excess of
red cells were used. This problem is easily
con- trolled by careful attention to methods
and is not common in transfusion medicine.
Solid-Phase Assays
Various solid-phase assays exist that utilize
the same general principle. As opposed to
fluid- phase assays, where the reaction
occurs in a solution or suspension, the
antigen or anti- body being studied in solid-
phase assays is im-
mobilized on a solid matrix. The analyte is
then incubated with the coated solid phase,
and adherence of the analyte to the solid
phase is measured. Several combinations of
adherence and detection approaches have
been described.
Solid-Phase Red Cell
Adherence Techniques
SOL I D-PH A S E AS SAYS FOR PH E N OT YPIN G
RE D CEL L S . Antibodies specific for known
blood group antigens are coated onto round-
bottom microtiter plates [Fig 10-7(A)]. Cells
being analyzed are added to the microtiter
plate wells and allowed to adhere. If no bind-
ing occurs (negative reaction), the red cells all
cluster together as a “button” at the bottom of
the well. In contrast, specific binding results in
diffusion of the red cells over the surface of
the
CH A P T E R 10 Molecular Biology and Immunology in Transfusion Medicine ■ 243

FIGURE 10-7. Schematic representation of (A) phenotyping red cells and (B) detecting antibodies by solid-
phase assay.

well (positive reaction). A positive reaction in- antibodies against platelet antigens using the
dicates the presence of the antigen on the red approaches described above.24
cells being tested.
SOLI D-PH ASE ASSAYS FOR DE TECT ING AN TI Enzyme-Linked Immunosorbent Assay
BO DI E S TO RE D CE LL AN TI GE NS . An-
Enzyme-linked immunosorbent assay
tigen-coated particles, consisting of red
(ELISA), also referred to as “enzyme
cells or red cell fragments, are coated onto
immunoassay,” can detect antibodies and
microti- ter plate wells [Fig 10-7(B)].
antigens. A signal is generated via an
Patient serum is then added to each well,
enzyme linked to a secondary antibody or
followed by incuba- tion and then washing.
antigen that converts a substrate to a
If the patient serum has antibodies against
measurable product (eg, a color change or a
the red cell antigens coated on the well,
chemiluminescent reaction). For this reason,
then the antibodies bind to the red cells or
ELISAs have considerable signal
their fragments. Indicator red cells coated
amplification and are significantly more
with antihuman IgG are then add- ed. A
sensitive than fluid- phase agglutination or
positive reaction is demonstrated by dif-
SPRCAs. In most cases, ELISAs use
fuse adherence of the indicator red cell to
purified or recombinant antigens or
the well, whereas a negative reaction is
antibodies, depending on the analyte de-
demon- strated by clustering of indicator
tected. However, intact red cells can be used
cells in a but- ton.
to screen for red cell antibodies, referred to
as the “enzyme-linked antiglobulin test.”25
Solid-Phase Assays for Platelet Testing
DET E CT ION OF AN TI BODIES BY EL IS A ( I N-
Solid-phase red cell adherence (SPRCA) tech-
D I RE CT EL ISA ) . To detect antibodies
nology has also been adapted to detect anti-
against a given antigen, the antigen is coated
gens on platelets, such as HPA-1a, as well as
onto mi- crotiter plate wells [Fig 10-8(A)].
The test sam- ple is then added, incubated,
and washed. If
244 ■ AABB T EC HNIC AL MANUAL
FIGURE 10-8. Schematic representation of (A) indirect enzyme-linked immunosorbent assay (ELISA), (B)
sandwich ELISA, and (C) competitive ELISA.
antibodies against the antigen are present,
they bind to the antigen-coated well and the
bound antibodies are detected by incubating
with anti-Ig (eg, anti-IgG) linked to an
enzyme (eg, alkaline phosphatase or
horseradish per- oxidase). After further
washing, enzyme sub- strate is added and is
converted to a detectable color if enzyme is
present. Quantification is performed using a
standard curve and a spec- trophotometer to
measure the absorbance at the wavelength
appropriate for that enzyme/ substrate
product. In some cases, samples may need
to be diluted to ensure that they yield
absorbance values in the linear range of the
assay.
D E TE CTIO N O F A N T I GE NS B Y S A NDW
I CH
EL IS A. In sandwich ELISA, two separate
anti- bodies are used that bind different
epitopes on the same target antigen; the
antibodies bind
the target without interfering with each
other. Typically, this is accomplished by
monoclonal antibodies. Microtiter plate
wells are coated with one antibody, the
“capture antibody” [Fig 10-8(B)]. The
specimen is then incubated in the well; if the
antigen is present, it binds to the solid-phase
antibody coated in the well. The plate is then
washed and incubated with the second
antibody, which is linked to a re- porter
enzyme (the “conjugate”). Because the
second antibody is specific for the target
anti- gen, it binds to the well only if antigen
was bound to the capture antibody. After
addition- al washing, enzyme substrate is
added and is converted to a detectable color
if enzyme is present.
DETECTION OF ANT I GENS BY CO M PETI-
TIV E EL ISA. Competitive ELISA is similar to
indirect ELISA in that the target antigen is
CH A P T E R 10 Molecular Biology and Immunology in Transfusion Medicine
bound to the microtiter plate well. The test
sample is incubated with antibody specific
for the target antigen and this mixture is
added to the well [Fig 10-8(C)]. If no
antigen is in the specimen, then the reagent
antibodies bind to the solid-phase antigen.
However, if antigen is present in the
specimen, it combines with the reagent
antibodies and prevents them from binding
to the solid-phase antigen. Therefore, as the
amount of soluble antigen in the speci- men
increases, the amount of reagent anti- body
that is free to bind to the solid-phase an-
tigen on the well decreases. Similarly, as the
signal weakens, the amount of soluble
antigen in the specimen rises.
Competitive ELISA is also used for
anti- body detection. In this case, the test
sample along with a labeled reagent
antibody is added to an antigen-coated well.
Both patient anti- body and labeled reagent
antibody compete for antigen-binding sites
in the well. Again, a higher signal is
generated if the antibody level in the sample
is low or absent. Although more difficult to
optimize, competitive ELISAs have an
advantage over sandwich ELISAs in not re-
quiring two separate antibodies against
differ- ent epitopes on the target antigen.
TE CHNICAL PROBLEMS WITH ELISA S . Typ-
ically, ELISAs are straightforward and
robust. Although low signals are possible
due to enzymatic inhibitors in the sample
and false- positive signals can occur due to
enzymatic activity, proper controls and
washing prevent these problems. Problems
also occur if the amount of the antigen being
measured ex- ceeds the amount of antibody
present. This phenomenon, called the “hook
effect,” leads to underestimates of the
concentration of the antigen. Like the
prozone effect (see “Fluid- Phase Assays”
section above), excess amounts of the
analyte can cause the signal to decrease in
some sandwich ELISAs in which the analyte
and detection antibody are added simultane-
ously. Hook effects can be overcome by
dilut- ing the analyte. Finally, some patients
have hu- man antimouse antibodies that
cross-link the capture antibody and the
detection antibody in sandwich ELISAs,
resulting in very high sig- nals for
essentially all analytes.
■ 245
Protein Microarrays
Microarray technology has dramatically in-
creased the number of substrates that can be
simultaneously assayed by solid-phase
meth- ods. By spotting numerous different
protein substances on a small chip, a single
specimen can be assayed for binding activity
to multiple (in some cases thousands)
substances simul- taneously. For example,
patient serum can be tested for antibodies to
blood-group antigens by making a
microarray chip with different blood-group
antigens and then incubating it with patient
serum.
Although microarray approaches are
very promising, like ELISA, they require
that the an- tigens being tested be spotted on
a chip in a pure form that maintains the
structural con- formation required for
recognition. In the cas- es of carbohydrate
antigens or linear protein epitopes, this is
readily achieved. However, with multipass
transmembrane proteins that require
membrane insertion for proper con-
formation (eg, the Rh antigens), it is
consider- ably more difficult. The
application of protein microarrays to blood-
bank serology is still in early stages of
development, and it remains unclear to what
extent it will mature into a useful diagnostic
platform.
Western Blotting
The ELISA techniques described above can
be highly sensitive. However, because the
anti- gens used may not be pure (eg, lysates
of tissue culture grown viruses), false-
positive results are possible due to cross-
reactivity with other components in the
antigen preparation. West- ern blotting is
conceptually similar to ELISA, except that
instead of coating a well with the antigen,
the antigen mixture (eg, viral lysate) is first
separated by high-resolution protein elec-
trophoresis (typically using polyacrylamide
gels). The separated proteins are then trans-
ferred to nitrocellulose paper (or another
suit- able medium), which serves as the
solid phase to be probed with an antibody-
containing pa- tient sample. In this case, one
can determine the molecular weight of the
antigens recog- nized by the antibodies.
246 ■ AABB T EC HNIC AL MANUAL
Although not often used clinically, other
methods can be used to separate antigens
based on physical properties other than size.
For example, isoelectric focusing separates
proteins of different charges over a pH gradi-
ent. Because the likelihood of having a cross-
reactive antigen with the same physical char-
acteristics as the authentic antigen is small,
Western blotting provides more specificity
than ELISA. Thus, Western blots have been
used as confirmatory tests for serologic
screening assays to detect infectious agents,
such as HIV. Similar techniques were devel-
oped using recombinant, or otherwise puri-
fied, proteins for detecting other infectious
disease agents, such as hepatitis C virus.
Flow Cytometry
Flow cytometry revolutionized the analysis of
cell populations. The basic principle is that an-
tibodies against cell-surface molecules are la-
beled with fluorescent tags. Cells are incubat-
ed with the antibodies, and these “stained”
cells are then passed through a flow cytometer.
The individual cells are exposed to lasers that
excite the fluorochromes, causing fluorescent
emissions detected by sensors in the flow cy-
tometer. The amount of fluorescence is deter-
mined on a cell-by-cell basis, allowing both the
quantification of the number of cell-surface
molecules and the visualization of small popu-
lations of cells in a complex mixture.
Clinical flow cytometry has been applied
primarily to the diagnosis of neoplasia,
partic- ularly for hematologic malignancies.
Howev- er, flow cytometry can also be
applied to red cell serology. For example, red
cells can be phenotyped using labeled
antibodies of known specificity.
Alternatively, antibody screens or
crossmatching can be performed by mixing
red cells of known phenotype with pa- tient
antisera and then staining the cells with a
secondary antihuman Ig (eg, anti-IgG) that
is coupled to a fluorescent molecule.
However, flow cytometry has yet to be
widely applied to human transfusion
medicine and is currently utilized mostly in
experimental systems.
BASIC IMMUNOLOGY
The process by which the immune system
generates antibodies against foreign antigens
and yet maintains tolerance to self-antigens is
complicated and elegant, with multiple cellu-
lar players and intricate regulation. The mech-
anistic details of this process fall outside the
scope of this chapter but can be found else-
where. This section covers antibody structure,
function, and role in transfusion complica-
tions.
Antibody Structure
At its simplest, an antibody is a tetramer of
two identical heavy chains and two identical
light chains (Fig 10-9). Each heavy and light
chain contains a variable region, the part of
the mol- ecule that varies from antibody to
antibody and binds antigen, and a constant
region. Two light-chain families (kappa and
lambda) are found in humans, and any given
antibody has either two identical kappa or
two identical lambda light chains. Likewise,
the two heavy chains in an antibody
molecule are identical and differ depending
on isotype.
Immunoglobulins treated with the en-
zyme papain can be digested into two func-
tional fragments. The Fab fragment consists
of the heavy- and light-chain variable
regions, the light-chain constant region, and
one heavy-chain constant region domain.
The Fab fragment binds antigen but does not
activate effector mechanisms. In contrast,
the Fc frag- ment, consisting only of heavy-
chain constant regions, activates effector
mechanisms, allow- ing destruction of the
antibody target. Fc con- stant regions differ
based on antibody isotype and subclass.
There are five different antibody isotypes
— IgM, IgG, IgE, IgA, and IgD—determined
by the constant region of the heavy chain.
Anti- body isotypes can differ in the number
of anti- gen-binding sites per molecule and the
poten- cy of their effector functions [Fig 10-
10(A)]. The number of antigen-binding sites
per anti- body affects binding avidity for
antigen. For example, IgM is expressed early
in an immune response before the onset of
affinity matura- tion. However, IgM has high
avidity because it
CH A P T E R 10 Molecular Biology and Immunology in Transfusion Medicine ■ 247

FIGURE 10-9. General structure of a monomeric immunoglobulin.

FIGURE 10-10. Immune globulin (Ig) isotypes (A), IgG subclasses, and their relative activation of
complement and binding to Fc-gamma receptors (FcRs) (B).
248 ■ AABB T EC HNIC AL MANUAL

consists of five Ig molecules held together IgE antibodies bind to Fc receptors on

by an additional protein (the J chain) and mast cells and induce histamine release when
exten- sive disulfide binding, resulting in 10 they encounter antigen; thus, IgE antibodies
antigen- binding sites. The high avidity of are the predominant cause of allergic and ana-
IgM compen- sates for its typically low phylactic responses (ie, Type I hypersensitivi-
affinity for antigen. Treatment with ty). IgD primarily remains membrane bound
dithiothreitol (DTT) can de- stroy IgM on the B-cell surface, with only minimal levels
binding because it reduces disulfide bonds, in serum, and its functions remain unclear.
and DTT treatment is used to distin- guish
IgM from IgG antibodies in the laborato- ry. The Role of Fc Receptors in Target
IgM potently activates complement by Destruction
changing its three-dimensional structure after
The Fc regions of antigen-bound IgGs are rec-
antigen binding. Although an IgM-specific
ognized by the gamma family of Fc receptors
Fc receptor has long been suspected and was
(FcRs). At least four FcRs have been de-
re- cently cloned, its function is not yet fully
scribed to date, each with subtly different
un- derstood. In general, IgM (and some
properties that can have opposite functions.
IgG) is known to cause hemolysis during
For example, FcR2a and FcR3 promote
transfusion reactions and autoimmune
phagocytosis of targets. Due to the relatively
hemolytic anemia. IgG antibodies are
low affinity of these receptors, monomeric IgG
important in mature humoral immune
does not engage FcR2a and FcR3, making
effector function and are di- vided into four
them specific for targets bound by multiple
subclasses: IgG1, IgG2, IgG3, and IgG4.
antibodies. In contrast, FcR2b is an inhibitory
Each subclass has a different con- stant
receptor that prevents phagocytosis. FcR1 has
region and a different capacity to activate
an unusually high affinity and binds mono-
complement and/or interact with Fc recep-
meric IgG. The result is that Fc R1 binds IgG
tors on phagocytes [Fig 10-10(B)]. IgG1 and
whether or not it is bound to a target; the func-
IgG3 are generally the most potent in these
tion of this activity is currently unclear.
re- gards, IgG2 only weakly activates
Thus, FcR biology is complicated by the
complement, and IgG4 largely lacks effector
fact that any given IgG-bound cell or
activity. Consis- tent with these observations,
particle may simultaneously activate
patients with iso- lated IgG4 subclass red
multiple, poten- tially antagonistic,
cell autoantibodies typically do not exhibit
receptors. This is further complicated by the
hemolysis. In contrast,
fact that each of the four different IgG
IgG1, IgG2, and IgG3 red cell antibodies can
subclasses (IgG1, IgG2, IgG3, and IgG4) has
induce hemolysis.
a different affinity for the different FcRs
IgA is the primary antibody isotype se-
(see Fig 10-11). A mixture of IgG1-IgG4 may
creted at mucosal surfaces; therefore, it is
bind a particle or cell bearing a foreign
largely responsible for neutralizing
antigen, and the net effect on enhancing or
pathogens encountered in the
in- hibiting phagocytosis will depend on the
gastrointestinal, genitouri- nary, and
rela- tive binding of different IgG subclasses
respiratory tracts. Although IgA ex- ists in
and in- teractions with different FcRs.
either monomeric or dimeric forms (a dimer
Thus, direct binding of Fc domains to Fc Rs
is shown in Fig 10-10), it is often mono-
can promote red cell clearance in many
meric in serum. IgA is further divided into
cases but does not always do so.
IgA1 and IgA2 subclasses (not shown). In
the IgA di- meric form, IgA monomers are
The Role of Complement in Target
connected by the J chain, similar to IgM. In
Cell Destruction and Opsonization
rare instances, IgA red cell antibodies cause
hemolysis. Most antiglobulin (ie, Coombs) In addition to serving as ligands for FcRs,
reagents do not typi- cally detect IgA; thus, the Fc regions of IgG antibodies can activate
the potential presence of IgA red cell com-
antibodies must be considered when
analyzing a patient with hemolysis and
negative direct antiglobulin test-results.
CH A P T E R 10 Molecular Biology and Immunology in Transfusion Medicine ■ 249

FIGURE 10-11. Mechanisms of red cell destruction by antibody binding. Upon binding (A), an immune
globulin G (IgG) represents a ligand for Fc-gamma receptors (FcRs) on phagocytes (B). If the red cell
avoids FcR- mediated phagocytosis, opsonization may be increased by activation of complement with
deposition of C3b (C). if the combined opsonization of FcR binding and C3b is not sufficient to mediate
clearance, completion of the complement cascade may lead to insertion of the membrane attack
complex (MAC) into the red cell surface, resulting in lysis (D). In reality, these processes likely occur
simultaneously, with the ultimate outcome being the aggregate effect of competing pathways.
CR = complement receptor.

plement. The complement system consists
of a cascade of proteases that, once
activated, amplifies the initial signal,
leading to the pro- duction of a large number
of effector mole- cules. Although there are
several complement- activation pathways,
this discussion focuses on the “classical
pathway” initiated by Fc re- gions.
IgM is highly efficient in activating
com- plement. However, to avoid
indiscriminant ac- tivation, IgM undergoes a
conformational shift after binding antigen,
thereby exposing com- plement-binding
sites in the heavy-chain con- stant region.
This interaction is so potent that, in theory, a
single antigen-bound IgM is suffi- cient to
lyse a target. In contrast, IgG does not
require a conformational change to bind
com- plement, but complement activation
requires clustered binding of multiple IgG
molecules to the same target. This prevents
indiscriminate activation of complement by
unbound circu- lating IgG.
Once activated, the complement system
provides at least two distinct mechanisms of
target destruction. The first involves target op-
sonization by complement components. Dur-
ing early events in complement activation, C3
covalently attaches to the antigen surface by
thioester bonds, providing multiple copies of
C3b, which are recognized by the complement
receptor 1 (CR1) and CRIg on phagocytes.
When a phagocyte encounters a C3b-coated
molecule, it ingests and destroys it. C3b also
rapidly degrades sequentially into iC3b, C3c,
and C3dg. Because C3dg is recognized by
CR2, which is not on phagocytes, complement
can degrade past the point of promoting
phagocy- tosis. In the second mechanism,
downstream of C3 activation, the cascade
assembles the membrane attack complex
(MAC). The MAC consists of complement
proteins C5b-C9 ar- ranged into a structure
resembling a hollow tube inserted into the
membrane of the target cell. This nonselective
channel between the
250 ■ AABB T EC HNIC AL MANUAL
inside of a target cell and its external environ-
ment results in osmotic lysis of the target (if it
is sensitive to osmotic shock).
Specific Outcomes of MAC Assembly,
C3 Opsonization, and Fc
Opsonization of Antibody-Coated
Red Cells
The effector mechanisms induced by
antibody binding have different effects on
bacteria, vi- ruses, particles, and various
human tissues. In general, once an IgG
antibody binds to a red cell, the target cell
may undergo FcR-mediated phagocytosis by
phagocytes (Fig 10-11). If the antibody
initiates the complement cascade, C3b
deposition on the red cell surface contrib-
utes to opsonization, leading to phagocytosis
mediated by CR1 and CRIg. Finally, if
comple- ment activation is complete, MAC
insertion causes red cell lysis. The relative
contributions of each pathway vary based on
the relative amounts of antibody isotype and
subclass and on the nature of the antigen
(eg, antigen densi- ty or linkage to
cytoskeleton). The sections be- low describe
what is known about these pro- cesses with
regard to red cell destruction and the clinical
manifestations of hemolysis.
Extravascular Hemolysis
Consumption of antibody- and/or C3b-
bound red cells by phagocytes in the
reticuloendothe- lial system (RES;
predominantly in the spleen and liver) is
referred to as “extravascular” he- molysis
because the red cells are destroyed outside
of their normal compartment, the in-
travascular space. This process is also com-
monly referred to as a “delayed hemolytic
transfusion reaction” (DHTR) because it
often occurs days after transfusion in
contrast to “intravascular” hemolysis (see
below), which is recognized acutely during
the transfusion [eg, an acute hemolytic
transfusion reaction (AHTR)]. The delayed
kinetics of DHTRs are due to the milder
clinical manifestations of ex- travascular
hemolysis and/or the need for the implicated
antibodies to develop or increase their titer
before the onset of significant red cell
destruction.
The term “hemolysis” in this context
can cause confusion for health-care
providers not accustomed to blood bank
terminology be- cause they typically think
of hemolysis as the rupture of red cells
within the circulation (ie, intravascular
hemolysis). In contrast, in extra- vascular
hemolysis, the red cells hemolyze in the
digestive compartment (ie, lysosomes) of
phagocytes. This is a very important distinc-
tion because phagocytes in the RES
consume a substantial number of senescent,
autologous red cells each day in the normal
process of red cell turnover. Thus, red cell
consumption in this fashion uses a pathway
that evolved spe- cifically to break down
and recycle red cell contents (eg,
hemoglobin and iron) in a man- ner that
avoids tissue damage.
This does not mean, however, that extra-
vascular removal of antibody-coated red
cells is biologically equivalent to clearance
of nor- mal, senescent red cells. On the
contrary, DHTRs can cause substantial
morbidity and occasional mortality. Indeed,
in murine mod- els of incompatible
transfusion, rapid clear- ance of antibody-
coated red cells induces systemic
inflammation and cytokine storm.
Nonetheless, extravascular hemolysis is dis-
tinctly different from intravascular
hemolysis in which red cell contents are
directly released into the circulating blood.
It is not clear why some red cell antibod-
ies preferentially promote opsonization and
phagocytosis instead of osmotic lysis by the
MAC. Substantial evidence indicates that the
antibody type and/or the topographical ar-
rangement of the target antigen on the red
cells are important. In addition, although
complement may be activated, the aggregate
opsonization of red cells by C3b and antibody
may result in phagocytosis before MAC-
induced lysis occurs. Consistent with these ex-
planations is the observation that extravascu-
lar hemolysis is typically induced by IgG red
cell antibodies, whereas intravascular hemoly-
sis is typically induced by IgM red cell
antibod- ies. The latter is much more efficient
at fixing complement and promoting MAC
formation.
CH A P T E R 10 Molecular Biology and Immunology in Transfusion Medicine
Intravascular Hemolysis
In some cases of incompatible transfusion,
the MAC rapidly assembles and lyses the
red cells before C3b and/or IgG
opsonization can induce phagocytosis.
Because these red cells lyse while still
circulating, this is termed “in- travascular
hemolysis.” In addition, because antibody-
mediated intravascular hemolysis occurs at a
brisker pace than extravascular he- molysis
(or, at least, is more quickly noticed due to
dramatic signs and symptoms), this he-
molytic transfusion reaction is called
“acute.”
As discussed above, AHTRs are typically
caused by IgM antibodies, which efficiently
ac- tivate complement, leading to rapid forma-
tion of the MAC. Although an IgM-specific Fc
receptor has been described (ie, FcR), it is
predominantly expressed on nonphagocytic
lymphocytes; thus, it is unlikely to promote
phagocytosis of IgM-coated red cells. None-
theless, complement activation by IgM red cell
antibodies can produce opsonization by C3b,
leading to CR1- and CRIg-mediated phagocy-
tosis. Taken together, then, it is not surprising
that IgMs predominantly induce intravascular
hemolysis.
Intravascular hemolysis, unlike
extravas- cular hemolysis, does not normally
occur at any appreciable level. The release
of red cell contents directly into the
circulation can be highly toxic, with free
hemoglobin inducing perhaps the greatest
insult. Although much free hemoglobin is
scavenged by haptoglobin, this system is
easily overwhelmed. AHTRs of- ten result
in tea-colored urine (ie, hemoglo- binuria)
and can induce renal dysfunction. Moreover,
the signs and symptoms of AHTRs can be
very dramatic, including disseminated
intravascular coagulation, shock, and death.
This type of reaction most often occurs as a
result of a clerical error that leads to an
ABO- incompatible transfusion, and many
current practices have evolved to prevent
ABO-incom- patible AHTRs.
Nonhemolytic Red Cell Antibodies
Given the redundant pathways leading to the
destruction of antibody-coated red cells, it is
not surprising that transfusions of cross-
■ 251
match-incompatible red cells produce hemo-
lysis. However, somewhat surprisingly, the
vast majority of red cell antibodies are not
hemo- lytic. For some blood-group antigens,
hemoly- sis is never, or only very rarely,
observed fol- lowing incompatible
transfusion (eg, JMH, Chido, and Rodgers
antigens). Indeed, approx- imately 1% of
healthy blood donors have posi- tive direct
antiglobulin test results, indicating that IgG
autoantibodies are bound to their own red
cells, yet there is no evidence of he- molysis
in these donors. Even for antigens known to
be involved in antibody-mediated hemolysis
(eg, in the Rh, Kell, Kidd, Duffy, and Ss
systems), hemolysis is variable. Indeed, in
patients mistakenly transfused with ABO-
incompatible Red Blood Cell (RBC) units,
no clinically significant hemolysis occurs in
50% of cases, even for this robustly
hemolytic anti- gen/antibody combination.
Several explanations may account for the
lack of hemolysis during incompatible trans-
fusions. For antigens that are essentially
never involved in hemolysis, the antigen
density or surface topography may prevent
hemolysis. For antigens that are variably
involved in he- molysis, idiosyncrasies in a
given recipient’s antibodies (ie, titer, affinity,
isotype, or IgG subclass) may play a role.
Based on these properties, different
antibodies (of the same antigenic specificity)
may have different ca- pacities for activating
complement. This is the rationale for
including the anti-C3 compo- nent in the
antiglobulin (Coombs) reagent: it provides
information on whether an antibody can fix
complement. A number of different ge-
netic polymorphisms and/or deficiencies
may also regulate hemolysis vs red cell
survival on a patient-by-patient basis,
including: perturba- tions in complement,
complement-regulatory proteins, and allelic
polymorphisms in FcRs. Thus, in some
cases, regulation of hemolysis may be
independent of the nature of the anti- body.
From a practical standpoint, crossmatch-
incompatible RBCs may be issued for
transfu- sion if the offending entity is a
“clinically insig- nificant antibody,”
especially if the antigen is of very high
frequency and antigen-negative blood is
difficult or impossible to obtain. The
252 ■ AABB T EC HNIC AL MANUAL

blood bank must be prepared to address ap- Summary of Efferent Immunity

propriate concerns from health-care provid-
ers managing these patients. Also from a
prac- tical standpoint, although clinically
significant antibodies may have variable
hemolysis in dif- ferent patients, there are
only very limited means of predicting
whether hemolysis will occur in a given
recipient. Thus, transfusion of crossmatch-
incompatible RBC units should not be
issued for clinically significant antibod- ies
unless this lifesaving procedure is specifi-
cally requested by the managing physician.
If compatible RBC units are unavailable, it
might be determined that hemolysis
occurring over several days (eg, in the case
of a DHTR) is less dangerous than the
consequences of the pa- tient’s severe
anemia. Transfusion with a unit that is
antigen matched to the patient for com-
mon, clinically significant blood-group anti-
gens should be considered. Close and
frequent communication between managing
physi- cians and the blood bank is required
in these cases.
In aggregate, when an antibody binds a red
cell, multiple pathways are activated that
can lead to cell destruction. Complement
activa- tion promotes phagocytosis through
the opso- nizing properties of C3b and direct
red cell lysis through assembly of the MAC.
The pres- ence of IgG Fc domains promotes
red cell phagocytosis by ligating FcRs on
the phago- cyte surface. The relative
contributions of these different pathways
vary depending on the nature of the target
antigen and the prop- erties of the cognate
antibodies. Moreover, the immune-activation
events that occur and the toxicity of the
released RBC units can produce a scenario
in which the negative effects of im- mune
destruction of transfused red cells go far
beyond simply losing the efficacy of the
trans- fused red cells. Indeed, substantial
toxicity can occur, leading to morbidity and,
in some cases, mortality. Should the reader
desire more fine mechanistic details on
immunobiology and the immune response,
additional sources are available.26,27

KEY POINTS

Hybridization-based methods can be used to detect genes, gene products, and polymor- phisms. However, compared to amplif
Amplification-based methods (PCR, TMA, NASBA, SDA, and LCR) are highly sensitive but are also susceptible to problems
The presence of nucleic acids predicts, but does not always equate with, expression of the corresponding protein or antigen str
Analysis of protein expression detects the actual gene product(s). Therefore, it does not suf- fer from the problems of nucleic
Protein analysis is less sensitive than nucleic acid testing because no amplification is in- volved, but protein analysis is also le
Methods of detecting protein suffer from nonamplification-based artifacts (ie, heterophilic antibodies, prozone effects, and ho
There can be substantial variability in detecting antigens and antibodies based on different methods.
Immunoglobulins can cause destruction of red cells by several different mechanisms, based largely on the antigen recognized
CH A P T E R 10 Molecular Biology and Immunology in Transfusion Medicine ■ 253

9. Most IgG antibodies that cause red cell destruction induce extravascular hemolysis by
pro- moting consumption of red cells by phagocytes (through Fc receptors and/or
complement- based opsonization). This typically presents as a delayed hemolytic
transfusion reaction.
10. IgM antibodies (and in some rare cases IgG) that cause red cell destruction typically
induce intravascular hemolysis through complement activation to the membrane attack
complex. This typically presents as an acute hemolytic transfusion reaction.
11. Despite the serious nature of hemolysis due to incompatible transfusion, many antibodies
to red cell antigens are clinically insignificant and do not result in destruction of red cells.
Incompatibility is best avoided whenever possible, but when compatible blood is not avail-
able, incompatible RBC units can be used when the antigens are known to be clinically in-
significant. Such maneuvers must be carefully considered on a-case-by-case basis and with
extensive communication with the clinicians who are requesting the blood products.
REFERENCES

1. Alberts B, Bray D, Lewis J, et al. Molecular

biol- ogy of the cell. 3rd ed. New York:
Garland Sci- ence, 1994:98-105.
2. Southern EM. Detection of specific sequences
among DNA fragments separated by gel elec-
trophoresis. J Mol Biol 1975;98:503-17.
3. Alwine JC, Kemp DJ, Stark GR. Method for
de- tection of specific RNAs in agarose gels
by transfer to diazobenzyloxymethyl-paper
and hybridization with DNA probes. Proc Natl
Acad Sci U S A 1977;74:5350-4.
4. Melton DA, Krieg PA, Rebagliati MR, et al.
Effi- cient in vitro synthesis of biologically
active RNA and RNA hybridization probes
from plas- mids containing a bacteriophage
SP6 promot- er. Nucleic Acids Res
1984;12:7035-56.
5. Berk AJ, Sharp PA. Sizing and mapping of
early adenovirus mRNAs by gel
electrophoresis of S1 endonuclease-digested
hybrids. Cell 1977; 12:721-32.
6. Mullis KB, Faloona FA. Specific synthesis of
DNA in vitro via a polymerase-catalyzed
chain reaction. Methods Enzymol
1987;155:335-50.
7. Masukawa A, Miyachi H, Ohshima T, et al.
[Monitoring of inhibitors of the polymerase
chain reaction for the detection of hepatitis C
virus using the positive internal control].
Rinsho Byori Jap 1997;45:673-8.
8. Al-Soud WA, Radstrom P. Purification and
characterization of PCR-inhibitory compo-
nents in blood cells. J Clin Microbiol 2001;39:
485-93.
9. Pang J, Modlin J, Yolken R. Use of modified
nu- cleotides and uracil-DNA glycosylase
(UNG) for the control of contamination in the
PCR- based amplification of RNA. Mol Cell
Probes 1992;6:251-6.
10. Temin HM, Mizutani S. RNA-dependent DNA
polymerase in virions of Rous sarcoma virus.
Nature 1970;226:1211-13.
11. Baltimore D. RNA-dependent DNA poly-
merase in virions of RNA tumour viruses. Na-
ture 1970;226:1209-11.
12. Compton J. Nucleic acid sequence-based am-
plification. Nature 1991;350:91-2.
13. Kwoh DY, Davis GR, Whitfield KM, et al.
Tran- scription-based amplification system and
de- tection of amplified human
immunodeficien- cy virus type 1 with a bead-
based sandwich hybridization format. Proc
Natl Acad Sci U S A 1989;86:1173-7.
14. Walker GT, Fraiser MS, Schram JL, et al.
Strand displacement amplification—an
isothermal, in vitro DNA amplification
technique. Nucleic Acids Res 1992;20:1691-6.
15. Walker GT, Little MC, Nadeau JG, Shank DD.
Isothermal in vitro amplification of DNA by a
restriction enzyme/DNA polymerase system.
Proc Natl Acad Sci U S A 1992;89:392-6.
16. Wu DY, Wallace RB. The ligation
amplification reaction (LAR)—amplification
of specific DNA sequences using sequential
rounds of tem- plate-dependent ligation.
Genomics 1989;4: 560-9.
17. Denomme GA, Van Oene M. High-
throughput multiplex single-nucleotide
polymorphism analysis for red cell and
platelet antigen geno- types. Transfusion
2005;45:660-6.
18. Bugert P, McBride S, Smith G, et al.
Microarray- based genotyping for blood
groups: Compari- son of gene array and 5'-
nuclease assay tech- niques with human
platelet antigen as a mod- el. Transfusion
2005;45:654-9.
254 ■ AABB T EC HNIC AL MANUAL
19. Hashmi G, Shariff T, Seul M, et al. A flexible
ar- ray format for large-scale, rapid blood
group DNA typing. Transfusion 2005;45:680-
8.
20. Langston MM, Procter JL, Cipolone KM,
Stron- cek DF. Evaluation of the gel system
for ABO grouping and D typing. Transfusion
1999;39: 300-5.
21. Voak D. Observations on the rare phenome-
non of anti-A prozone and the non-specific
blocking of haemagglutination due to C1
com- plement fixation by IgG anti-A
antibodies. Vox Sang 1972;22:408-19.
22. Judd WJ, Steiner EA, O’Donnell DB,
Oberman HA. Discrepancies in reverse ABO
typing due to prozone. How safe is the
immediate-spin crossmatch? Transfusion
1988;28:334-8.
23. Salama A, Mueller-Eckhardt C. Elimination of
the prozone effect in the antiglobulin reaction
by a simple modification. Vox Sang 1982;42:
157-9.
24. Procter JL, Vigue F, Alegre E, et al. Rapid
screening of platelet donors for PIA1 (HPA-
1a) alloantigen using a solid-phase microplate
im- munoassay. Immunohematology
1998;14:141- 5.
25. Leikola J, Perkins HA. Enzyme-linked
antiglob- ulin test: An accurate and simple
method to quantify red cell antibodies.
Transfusion 1980; 20:138-44.
26. Kindt TJ, Osborne BA, Goldsby RA. Kuby
im- munology. 6th ed. New York: WH
Freeman and Company, 2007.
27. Murphy K, Travers P, Walport M. Janeway’s
im- munobiology. 7th ed. New York: Garland
Sci- ence, 2008.
 C h a p t e r 1 1
Blood Group Genetics
Christine Lomas-Francis, MSc, FIBMS
T H E S C I E N C E O F genetics is the
study of heredity—that is, the mecha-
nisms by which particular characteristics are
passed from parents to offspring. This
chapter describes the genetics of blood
groups. The term “blood group” can be
applied to any de- tectable, variable
characteristic of a compo- nent of the blood
including platelet and white cell groups,
serum groups, red cell enzymes, and
hemoglobin variants. In this chapter, the
term “blood group” applies primarily to anti-
gens on the surface of the red cell membrane
that are defined serologically by an antibody.
Platelet and white cell blood groups are dis-
cussed in Chapter 18.
That blood groups are inherited charac-
teristics was first shown by von Dungern
and Hirszfeld in 1910, 10 years after
Landsteiner’s discovery of the ABO blood
group. Blood groups became an ideal tool
for geneticists be- cause they could be
identified by specific anti- bodies in simple
hemagglutination tests and, once identified,
their inheritance could easily be followed in
family studies. Red cell antigens were (and
still are) valuable as markers (de- tectable
characteristics to recognize a gene’s
presence) in genetic and anthropologic stud-
ies as well as in relationship testing.
Christine Lomas-Francis, MSc, FIBMS, Technical Director, Laboratory of Immunohematology and Genomics,
New York Blood Center, New York, New York
The author has disclosed no conflicts of interest.
The detection of inherited differences
on the red cells from different people is the
basis of safe blood transfusion. Therefore,
an under- standing of the principles of
human genetics (including the patterns of
inheritance and the language or terminology
in use) is an impor- tant aspect of
immunohematology and trans- fusion
medicine. This chapter outlines the
fundamental principles of genetics as they
ap- ply to blood group antigens and relates
them to examples relevant to transfusion
medicine. This requires the use of numerous
genetic terms; each term, when first used or
when fully described, will be in bold text
and usually is closely followed by a
definition.
Technical advances in genetics and mo-
lecular biology have ushered in the age of
mo- lecular genetics, when genes are
routinely se- quenced and inheritance and
disease are studied at the nucleic acid
level.1,2 These ad- vances have provided an
understanding of the regulatory elements
and genes that control the expression of
blood groups so that the pres- ence or
absence of blood groups can be pre- dicted
through DNA-based analysis3 with the
potential to revolutionize transfusion medi-
cine and the care of patients. Knowledge of
the fundamental principles of classical
genetics
255
256 ■ AABB T EC HNIC AL MANUAL
aids understanding of the molecular aspects
of individual blood groups.
BASIC PRINCIPLES OF
GENETICS
Gregor Mendel established the basic tech-
niques of genetic analysis when, in 1865, he
published his classic breeding experiments
with pea plants. Mendel’s observations led
him to conclude that there is a “factor” or
unit of inheritance, now known as a gene,
that is passed from one generation to
another ac- cording to two simple rules: the
principles of independent segregation and
independent as- sortment (see “Inheritance
of Genetic Traits” section below).
Cytology studies, toward the end of the
19th century showed that each living cell
has a characteristic set of chromosomes in
the nu- cleus. In the early part of the 20th
century, it was realized that chromosomes
carry genes. Biochemical studies showed
that the chromo- somal material is primarily
made up of nucleic acids and associated
proteins.1,2
Many excellent texts offer a greater in-
sight into classical genetics.4 The
fundamental principles of genetics outlined
in this chapter are intended to serve as a
review of inheritance and expression of
blood group antigens.
Genes (Alleles) and Chromosomes
A gene is a segment of deoxyribonucleic acid
(DNA) that encodes a particular protein. A
gene is the basic unit of inheritance of any
trait (defined as a genetically determined
characteristic or condition), including blood
group antigens, that is passed from parents to
offspring. Genes are arranged on chromo-
somes with each gene occupying a specific lo-
cation known as the gene locus. A locus may
be occupied by one of several alternative
forms of the gene called alleles. For example,
the gene that encodes the protein carrying the
Jka antigen is an alternative form (allele) of the
one that encodes the Jk b antigen. The terms
“gene” and “allele” can be used interchange-
ably. Based on internationally accepted gene
and allele terminology, the written gene or al-
lele name is italicized—for example, RHD
for the gene encoding the RhD protein. The
name usually is not italicized when the word
“gene” or “allele” follows the name: for
example, “RHD gene,” or “RHD allele.” The
Internation- al Society of Blood Transfusion
(ISBT) Working Party on Red Cell
Immunogenetics and Blood Group
Terminology5,6 has developed allele ter-
minology for use in transfusion medicine.
For alleles encoding polymorphic common
anti- gens, the name is based on the ISBT
antigen name: eg, FY*01 or JK*02 refer to the
alleles en- coding the Fya and Jkb antigens,
respectively. Alternatively, where letters are
commonly used, symbols such as FY*A or
JK*B are accept- able. A genotype may also
be written as the italicized antigen, eg, Fya
or Jkb when the geno- type has been inferred
from testing by hemag- glutination.
Chromosomes are the gene-carrying
structures that are visible during nuclear divi-
sion in the nucleus of the cell; they contain the
genetic material (DNA) necessary to maintain
the life of the cell and the organism. A human
somatic cell contains 46 chromosomes that
make up 23 pairs; each pair has one paternally
and one maternally derived chromosome. In
males and females, 22 of the pairs are
homolo- gous chromosomes (a pair of
chromosomes in which males and females
carry equivalent genes) and are referred to as
the autosomes (any chromosome that is not a
sex chromo- some). The remaining pair is
nonhomologous and consists of the sex
chromosomes that de- termine a person’s sex
(gender). The sex-deter- mining chromosomes
of the male are X and Y, whereas females have
two X chromosomes. The karyotype
represents the chromosome complement of a
person; this is written as “46,XY” and
“46,XX” for a normal male and fe- male,
respectively. The hereditary information
carried by the chromosomes is passed from a
parent cell to a daughter cell during somatic
cell division and from parents to offspring
(children) by the gametes during reproduc-
tion.
Chromosomes are best studied during
cell division (mitosis; see “Mitosis” section
be- low) when they become discrete
structures in the nucleus and can be
visualized by various
 CH A P T E R 1 1 Blood Group Genetics ■ 257

microscopic techniques. All chromosomes

have some common morphologic features
but differ in other characteristics, including
size, location of the centromere, and staining
prop- erties. Each chromosome has two
sections or arms that are joined at a central
constriction called the centromere (Figs
11-1 and 11-2). Chromosomes are
distinguished by their length and the
position of the centromere. These
characteristics serve as the basis for
numbering the autosomes 1 through 22 such
that chromosome 1 is the largest and
chromo- some 22 is the smallest. An
internationally rec- ognized terminology is
used to describe chro- mosomes. The
chromosomal arms are of different length,
although the difference be- tween the arms
of chromosome 1 is not obvi- ous (Fig 11-
2). The “p” (or petite) arm is the shorter and,
in diagrams, is at the top of the
chromosome. The longer arm is termed the
“q” arm. Thus, the short arm of chromosome
1 is referred to as “1p,” and the long arm of
chro- mosome 4 is “4q.” The terminal
portion of a chromosome is referred to as
“ter”; “pter” and “qter” indicate the terminus
of the short (p) and long (q) arms,
respectively.
FIGURE 11-2. Morphology and banding pattern of
Staining techniques provide a more de-
a Giemsa-stained human chromosome 1. The
tailed means to distinguish individual chro-
locations of the genes controlling the expression
mosomes. Selected dyes do not stain
of antigens for the Rh (RH), Scianna (SC), Duffy
chromo- somes uniformly and, depending
(FY), Knops (KN), and Cromer (CROM) blood
on the dye used, different banding patterns
group systems are shown.
are obtained so that each human
chromosome has a unique banding pattern.
As shown in Fig 11-2,
Giemsa staining reveals a specific pattern of
dark (G) and light (reverse or R) bands. Quina-
crine, used to stain chromosomal preparations
for fluorescence microscopy, results in fluores-
cent bands equivalent to the dark G bands
seen in light microscopy. The G bands are het-
erochromatin (condensed DNA) and the light-
er bands are euchromatin, which is involved in
FIGURE 11-1. Diagram of a metaphase chromosome.
At the metaphase stage of the cell cycle, the the transcription of DNA to mRNA. The bands
chromosomes have condensed and become visible are numbered from the centromere outward.
by light microscopy. As shown in the diagram, Using chromosome 1 as an example, the re-
metaphase chromosomes have replicated in gion closest to the centromere on the short or
preparation for cell division so that each
chromosome consists of two sister chromatids
connected by the centromere. The telomere is the
end or terminal part of a chromosome.
258 ■ AABB T EC HNIC AL MANUAL
long arm is numbered 1p1 or 1q1,
respectively. With greater resolution, there
is further distinction into subbands (eg, 1p11
and 1p12) that, in turn, can be subdivided
(eg, 1p11.1 and 1p11.2). Genes can be
individually mapped to a specific band
location (Fig 11-2), and the chromosomal
location of the genes encoding the 34 red
cell systems5-9 are shown in Table 11-1.
Cell Division
As a cell divides, the chromosomes replicate
and each daughter cell receives a full
comple- ment of genetic material. In
somatic cells, this occurs through mitosis; in
reproductive cells a similar process called
“meiosis” takes place. A feature common to
both types of cell division is that, before the
start of the process, each chromosome
replicates to form two identical daughter
chromatids attached to each other through
the centromere (Fig 11-1).
Mitosis
Somatic cells divide for growth and repair by
mitosis (Fig 11-3). Through this process, a
sin- gle cell gives rise to two daughter cells
with identical sets of chromosomes. The
daughter cells, like the parent cell, are diploid
(2N); that is, they contain 46 chromosomes in
23 pairs and have all the genetic information
of the parent cell.
Meiosis
Meiosis occurs only in germ cells that are in-
tended to become gametes (sperm and egg
cells). Somatic cells are diploid (2N), whereas
gametes are haploid [having half the chromo-
somal complement of somatic cells (1N)].
Mei- osis is a process of cell division and
replication that leads to the formation of
haploid gametes. During meiosis, diploid cells
undergo DNA replication, followed by two
cycles of cell divi- sion to produce four
haploid gametes (see Fig 11-4). Because
sperm and egg cells fuse at fer- tilization, the
gametes must be haploid. If each gamete
carried a diploid (2N) set of 46 chro-
mosomes, the resulting zygote would have 92
chromosomes, which would be incompatible
with life.
Meiosis ensures genetic diversity
through two mechanisms: independent
assortment and crossing-over. Through
independent as- sortment, each daughter cell
randomly re- ceives either maternally or
paternally derived homologous
chromosomes. Chromosomal crossing-over
involves exchange of genetic material
between homologous chromosome pairs.
Such shuffling of genetic material en- sures
diversity and produces genetically unique
gametes that fuse to produce a unique
zygote.
X Chromosome Inactivation
(Lyonization)
Females have two copies of X-borne genes in
their somatic cells, while males have only one
copy of X-borne genes. Because most X-borne
genes do not have a homolog on the Y chro-
mosome, there is a potential imbalance in the
dosage of X-borne genes between males and
females. This difference is compensated for by
X chromosome inactivation, (also called
ly- onization), a process through which most
of the genes on one of the two X chromosomes
in each female somatic cell are inactivated at a
very early stage of embryonic development.17
It is a matter of chance whether the maternal
or paternal X chromosome is inactivated in
any one cell, but once inactivation has oc-
curred, all descendants of that cell will have
the same inactive X chromosome. Some X-
borne genes escape inactivation; the first gene
found to escape inactivation was XG, the gene
encoding the antigens of the Xg blood group
system. Like XG, most of the genes that escape
inactivation are located on the extreme tip of
the short arm of the X chromosome, but sever-
al are clustered in regions on the short and
long arms of the chromosome.18(pp359-370),19
The XK gene, which encodes the Kx
blood group system, is the only other X-borne
gene known to encode red cell antigens.
Changes or deletions in XK result in McLeod
phenotype red cells that lack Kx antigen and
have reduced expression of Kell antigens. 20,21
The XK gene, unlike XG, is subject to X
chromosome inacti-
 CH A P T E R 1 1 Blood Group Genetics ■ 259

TABLE 11-1. Blood Group Systems

Gene Product and Associated Blood

ISBT System Gene Name ISBT Chromosome Component Name Group Antigens
Name (Number) (HGNC)* Location [CD Number] [Null Phenotype]

ABO ABO 9q34.2 Glycosyltransferase, A; B; A,B; A1

(001) (ABO) carbohydrate [Group O]
MNS MNS 4q31.21 Glycophorin A (GPA) M, N, S, s, U, He, Mia,
(002) (GYPA [CD235a] Vw, and 38 more
[En(a–); U–; MkMk]
GYPB Glycophorin B (GPB)
GYPE) [CD235b]
P1PK P1 22q13.2 Galactosyltransferase, P1, Pk, NOR
(003) (A4GALT) carbohydrate
Rh RH 1p36.11 RhD [CD240D] D, G, Tar
(004) (RHD RhCE [CD240CE] C, E, c, e, V, Rh17,
RHCE) and 45 more
[Rhnull]
Lutheran (005) LU 19q13.32 Lutheran glycoprotein; Lua, Lub, Lu3, Lu4, Aua,
(LU) B-cell adhesion mole- Aub, and 14 more
cule [CD239] [Recessive Lu(a–b–)]
Kell KEL 7q34 Kell glycoprotein K, k, Kpa, Kpb, Ku,
Jsa,
(006) (KEL) [CD238] Jsb, and 28 more
[K0 or Knull]
Lewis LE 19p13.3 Fucosyltransferase, Lea, Leb, Leab, Lebh,
(007) (FUT3) carbohydrate ALeb, BLeb
(Adsorbed from [Le(a–b–)]
plasma)
Duffy FY 1q23.2 Duffy glycoprotein Fya, Fyb, Fy3, Fy5, Fy6
(008) (DARC) [CD234] [Fy(a–b–)]
Kidd JK (SLC14A1) 18q12.h3 Human urea trans- Jka, Jkb, Jk3
(009) porter (HUT) [Jk(a–b–)]
Kidd glycoprotein
Diego DI 17q21.31 Band 3, Anion Dia, Dib, Wra, Wrb,
Wda,
(010) (SLC4A1) Exchanger 1 Rba, and 16 more
[CD233]
Yt YT 7q22.1 Acetylcholinesterase Yta, Ytb
(011) (ACHE)
Xg XG (XG) Xp22.33 Xga glycoprotein Xga
(012) (MIC2) Yp11.2 CD99 (MIC2 product) CD99
Scianna (013) SC 1p34.2 Erythroid membrane- Sc1, Sc2, Sc3, Rd,
and
(ERMAP) associated protein 3 more
(ERMAP) [Sc:–1,–2,–3]

(Continued)
260 ■ AABB T EC HNIC AL MANUAL

TABLE 11-1. Blood Group (Continued)

Systems
Gene Product and Associated Blood
ISBT System Gene Name ISBT Chromosome Component Name Group Antigens
Name (Number) (HGNC)* Location [CD Number] [Null Phenotype]

Dombrock (014) DO 12p12.3 Do glycoprotein; ART Doa, Dob, Gya, Hy, Joa
4 +
(ART4) [CD297] 3 more
[Gy(a–)]
Colton CO 7p14.3 Aquaporin 1 (AQP1) Coa, Cob, Co3, Co4
(015) (AQP1) [Co(a–b–)]
Landsteiner- LW 19p13.2 LW glycoprotein LWa, LWab, LWb
Intra-
Wiener (ICAM4) cellular adhesion [LW(a–b–)]
mole-
(016) cule 4 (ICAM4)
[CD242]
Chido/ Rodgers CH/RG (C4A, 6p21.32 Complement compo- Ch1, Ch2, Rg1 + 6
(017) C4B) nent: more
C4A; C4B [Ch–Rg–]
H H 19q13.33 Fucosyltransferase, H
(018) (FUT1) carbohydrate [CD173] [Bombay (Oh)]
Kx XK Xp21.1 XK glycoprotein Kx
(019) (XK) [McLeod phenotype]
Gerbich (020) GE 2q14.3 Glycophorin C (GPC) Ge2, Ge3, Ge4, and 8
(GYPC) [CD236] more
Glycophorin D (GPD) [Leach phenotype]
Cromer (021) CROM 1q32.2 DAF Cra, Tca, Tcb, Tcc, Dra,
(CD55) [CD55] Esa, IFC, and 11 more
[Inab phenotype]
Knops KN 1q32.2 CR1 Kna, Knb, McCa, Sla,
Yka,
(022) (CR1) [CD35] and 4 more
Indian IN 11p13 Hermes antigen [CD44] Ina, Inb, and 2 more
(023) (CD44)
Ok OK 19p13.3 Neurothelin, basigin Oka, OKGV, OKGM
(024) (BSG) [CD147]
Raph RAPH (CD151) 11p15.5 Tetraspanin MER2
(025) [CD151] [Raph–]
JMH JMH 15q24.1 Semaphorin 7A JMH and 5 more
(026) (SEMA7A) [CD108] [JMH–]
I GCNT2 6p24.2 Glucosaminyltransfer- I
(027) (IGNT) ase, carbohydrate [I– or i adult]
Globoside (028) GLOB 3q26.1 Transferase, carbohy- P
(B3GALNT1) drate [P–]
(Gb4, globoside)
 CH A P T E R 1 1 Blood Group Genetics ■ 261

TABLE 11-1. Blood Group (Continued)

Systems
Gene Product and Associated Blood
ISBT System Gene Name ISBT Chromosome Component Name Group Antigens
Name (Number) (HGNC)* Location [CD Number] [Null Phenotype]

Gill GIL 9p13.3 Aquaporin 3 (AQP3) GIL

(029) (AQP3) [GIL–]
Rh-associated RHAG 6p21.3 Rh-associated glyco- Duclos, Ola, DSLK†,
glycoprotein protein RHAG4
(030) [CD241] [Rhnull (regulator
type)]
Forssman10 FORS 9q34.2 Globoside 3--N- FORS1
(031) (GBGT1) acetylgalactosaminyl-
transferase 1
Forssman glycolipid

JR11,12 JR 4q22.1 Jr glycoprotein Jra

(032) (ABCG2) ATP-binding cassette, [Jr(a−)]
sub-family G, member
2 (ABCG2) [CD338]
Lan13 LAN 2q36 Lan glycoprotein Lan
(033) (ABCB6) ATP-binding cassette, [Lan−]
sub-family B, member
6 (ABCB6)
Vel14-16 VEL 1p36 Small integral Vel
(SMIM1) mem- brane [Vel−]
protein 1
(SMIM1)
*If the genetic information is obtained by blood group typing, the gene name is the italicized form of the of the blood
group system ISBT name. For example, SLC14A1 would be written as JK*A and JK*B or Jka and Jkb.
†
Provisionally numbered by the ISBT because of limited genetic evidence.
ISBT = International Society of Blood Transfusion; HGNC = Human Gene Nomenclature Committee; ATP = adenosine tri-
phosphate.

vation, with the result that a female who is a
carrier (a person who carries one gene for a
re- cessive trait and one normal gene) of a
gene that is responsible for the McLeod
phenotype can have a dual population of Kx–
(McLeod phenotype) and Kx+ (non-McLeod)
red cells. Flow cytometry, using selected Kell
antibodies, shows the weakening of Kell
antigens on red cells of the McLeod phenotype
and demon- strates two red cell populations in
carrier fe- males. This mixed-cell population
reflects the randomness of whether the
maternal or pater- nal X chromosome is
inactivated in any single somatic cell lineage.
Genotype and Phenotype
The genotype of a person is the
complement of genes inherited from his or
her parents; the term is frequently also used
to refer to the set of alleles at a single gene
locus. The phenotype is the observable
expression of the genes in- herited by a
person and reflects the biologic activity of
the gene(s). Thus, the presence or absence of
antigens on the red cells, as deter- mined by
serologic testing, represents the phe- notype.
The presence or absence of antigens on the
red cells predicted by DNA-based test- ing
represents the genotype. Sometimes the
262 ■ AABB T EC HNIC AL MANUAL
FIGURE 11-3. Diagram showing mitosis.
genotype can be predicted from the pheno-
type; for example, when a person’s red cells
are reactive with anti-Jka and anti-Jkb, which is
a Jk(a+b+) phenotype, a JK*A/JK*B genotype
can be inferred. Frequently, the phenotype pro-
vides only a partial indication of the genotype;
for example, red cells that are group B reflect
the presence of a B gene, but the genotype
may be B/B or B/O. For decades, family
studies were often the only way to determine a
person’s genotype, but now that most antigens
and phenotypes can be defined at the DNA
level, family studies to determine genotype
can be mostly replaced by DNA analysis (see
“Blood Group Genomics” section below).
Alleles
A gene at a given locus on a chromosome
may exist in more than one form, that is, be
allelic. Each person has two alleles for a
trait, one that is maternally derived and
another that is pa- ternally derived. At the
simplest level, the ABO gene locus can be
considered to have three al- leles: A, B, and
O. With three alleles, there are six possible
genotypes (A/A, A/O, A/B, B/B, B/O, and
O/O). Depending on the parental contri-
bution, a person could inherit any combina-
tion of two of the alleles and express the
corre- sponding antigens on their red cells.
For example, inheritance of A/A and A/O
would
 CH A P T E R 1 1
FIGURE 11-4. Diagram showing meiosis.
result in group A red cells, A/B would result in
group AB red cells, B/B and B/O would result
in group B red cells, and O/O would result in
group O red cells.
When identical alleles for a given locus
are present on both chromosomes, the
person is said to be “homozygous” for the
particular allele. A person who is
hemizygous for an al- lele has only a
single copy of an allele instead of the
customary two copies; an example is the
deletion of one RHD in a D-positive pheno-
type. When different (ie, not identical)
alleles are present at a particular locus, the
person is
Blood Group Genetics ■ 263
said to be “heterozygous.” For example, a
per- son with K–k+ red cells is homozygous at
the KEL locus for the allele (KEL*02)
encoding the k antigen. A person who is
heterozygous for KEL*01 and KEL*02
(KEL*01/02 genotype), would have red cells
that are K+k+.
Antigens that are encoded by alleles at the
same locus are said to be “antithetical”
(mean- ing “opposite”); thus, K and k are a
pair of anti- thetical antigens. It is incorrect to
refer to red cells that are K–k+ or Kp(a–b+) as
being homo- zygous for the k or Kp b antigen;
rather, it should be said that the cells have a
double
264 ■ AABB T EC HNIC AL MANUAL
dose of the antigen and that they are from a
person who is homozygous for the allele.
Genes are allelic whereas antigens are anti-
thetical.
The quantity of antigen expressed (anti-
gen density) is influenced by whether a per-
son is heterozygous or homozygous for an al-
lele; the antigen density is generally greater
when a person is homozygous. In some blood
group systems, this difference in antigen den-
sity is manifested by antibodies giving stron-
ger reactions with cells that have a double
dose of the antigen. Red cells with the
Jk(a+b–) phenotype, encoded by a JK*A/A
genotype, have a double dose of the Jka
antigen and often are more strongly reactive
with anti-Jka than those that are Jk(a+b+) and
have a single dose of the antigen. Similarly,
M+N– red cells tend to be more strongly
reactive with anti-M than are M+N+ red cells.
Antibodies that are weakly reactive may not
be detected if they are tested with red cells
expressing a single dose of the antigen. This
observable difference in strength of reaction,
based on homozygosity or hetero- zygosity for
an allele, is termed the “dosage ef- fect.”
Polymorphism
For blood group genetics, polymorphism re-
fers to the occurrence in the population of
ge- nomes with allelic variation (two or
more alleles at one locus) producing
different phe- notypes, each with
appreciable (greater than 1%) frequency.
Some blood group systems (Rh and MNS
for example) are highly polymorphic and
have many more alleles at a given locus than
other systems such as Duffy, and Colton.5
An allele that is polymorphic in one popula-
tion is not necessarily polymorphic in all
pop- ulations; for example, the FY allele
associated with silencing of Fyb in red cells
(FY*02N.01) is polymorphic in populations
of African ethnici- ty with a prevalence of
greater than 70%, but this allele is not found
in other populations. A gene polymorphism
may represent an evolu- tionary advantage
for a population, and a polymorphic
population is likely to adapt to evolutionary
change more rapidly than if the
population had genetic uniformity. It is not yet
understood what, if any, evolutionary advan-
tages were derived from the extensive poly-
morphism displayed by red cell antigens, but
many publications associate resistance to, or
susceptibility for, a particular disease with a
particular blood type.22
The difference between two alleles is the
result of a permanent change in the DNA.
An event that leads to the production of an
altered gene and a new allele or
polymorphism that did not exist in the
biologic parents is referred to as a
“mutation.” The mutation rate of ex-
pressed genes resulting in a new phenotype
has been estimated to be less than 10-5 (<1 in
100,000) in humans, and a mutation has to
oc- cur in the germ cells (gametes) to be
inherited.
A mutation can occur spontaneously or
be brought about by agents such as radiation
(eg, ultraviolet rays or x-rays) or chemicals. A
mutation may occur within a gene or in the
intergenic regions. It may be silent—that is,
have no effect on the encoded protein—or it
may alter the gene product and potentially
cause an observable effect in the phenotype.
In the context of an allele that encodes a pro-
tein that carries red cell antigens, any geneti-
cally induced change must be recognized by a
specific antibody before an allele can be said
to encode a new antigen.
Numerous genetic events that generate
red cell antigens and phenotypes have been
identified. The event can occur at the level
of the chromosome (eg, deletion or
transloca- tion of part of a chromosome), the
gene (eg, deletion, conversion, or
rearrangement), the exon (eg, deletion or
duplication), or the nu- cleotide (eg,
deletion, substitution, or inser- tion). The
mechanism that has given rise to most
diversity in the human genome is single
nucleotide polymorphism (SNP)—that is, a
single nucleotide change in the DNA.23-25 Ac-
cordingly, the majority of polymorphic
blood group antigens are the result of
SNPs.3,26,27 DNA analysis to predict the red
cell phenotype is discussed in more detail in
the section on “Blood Group Genomics.”
 CH A P T E R 1 1
INHERITANCE OF GENETIC
TRAITS
A genetic trait is the observed expression of
one or more genes. The inheritance of a trait
(and red cell antigens) is determined by
whether the gene responsible is located on an
autosome or on the X chromosome (sex-
linked) and whether the trait is dominant or
recessive.
Pedigrees
A family study follows the inheritance of a ge-
netic characteristic—for example, an allele en-
coding the expression of a red cell antigen—as
it is transmitted through a kinship. A diagram
that depicts the relationship of family mem-
bers and shows which family members express
(are affected), or do not express, the trait un-
der study is termed a pedigree. A review of a
pedigree should reveal the pattern or type of
inheritance for the trait, or antigen, of interest.
The person who first caused the family to be
investigated is considered the index case and
is often referred to as the proband or
proposi- tus/proposita (male singular form
or gender unknown/female singular form);
propositi is the plural form regardless of
gender. Details of the conventions and the
symbols used for the construction of pedigrees
are provided in Figs 11-5 and 11-6.
Autosomal Dominant Inheritance
An antigen (or any trait) that is inherited in an
autosomal dominant manner is always ex-
pressed when the relevant allele is present, re-
gardless of whether a person is homozygous or
heterozygous for the allele. The antigen ap-
pears in every generation and occurs with
equal frequency in both males and females. A
person who carries an autosomal dominant
trait, on average, transmits it to half of his or
her children. The pedigree in Fig 11-7 demon-
strates autosomal dominant inheritance and
shows that the B allele is dominant over O.
Blood Group Genetics ■ 265
FIGURE 11-5. An example of a pedigree. Males
are denoted by squares and females by circles, and
each different generation in a pedigree is
identified by Roman numerals. Persons in each
generation are identified by Arabic numerals;
the numbering is sequential from left to right,
with the eldest child for each family unit being
placed on the left of any series of siblings.
Closed symbols represent family members
affected by the trait, whereas open symbols
are unaffected members.
Autosomal Codominant Inheritance
Blood group antigens that appear to be auto-
somal dominant may be encoded by alleles
that are inherited in a codominant manner
— that is, when two different alleles are
present (the heterozygous condition), the
products of both alleles are expressed. Thus,
when red cells have the S+s+ phenotype, the
presence of one allele encoding S and
another allele en- coding s [or an S/s
(GYPB*S/s) genotype] can be inferred.
Autosomal Recessive Inheritance
A trait with autosomal recessive inheritance
is expressed only in a person who is
homozygous for the allele and has inherited
the recessive al- lele from both parents.
When a person inherits a single copy of a
recessive allele in combina- tion with a
silent or deleted (null) allele—that is, a
nonfunctioning allele or one that encodes a
product that cannot be detected—the reces-
sive trait is expressed and the person
appears to be homozygous. It is difficult or
impossible
266 ■ AABB T EC HNIC AL MANUAL

to distinguish such a combination from

FIGURE 11-6. Symbols, and their significance,
used in the construction of pedigrees.
homo- zygosity for the recessive allele
through sero- logic testing, but DNA-based
testing can usu- ally make this distinction.
A mating between two heterozygous
car- riers results in one in four of the
children being homozygous for the trait. The
parents of a child who is homozygous for a
recessive trait must be obligate carriers of
the trait. If the fre- quency of the recessive
allele is low, the condi- tion is rare and
usually found only among sib- lings
(brothers and sisters) of the person and not
in other relatives. The condition is not found
in preceding or successive generations
unless consanguineous mating (ie,
between blood relatives) occurs. When a
recessive allele is rare, the parents of an
affected person are most likely
consanguineous because a rare al- lele is
more likely to occur in blood relatives than
in unrelated persons in a random popu-
lation. When a recessive trait is one that is
common, consanguinity is not a prerequisite
for homozygosity; for example, the O allele
of the ABO system, although recessive, is
not rare, and persons who are homozygous
for O are easily found in the random
population.

FIGURE 11-7. Autosomal dominant inheritance of the ABO alleles. Based on the ABO groups of his
children, I-1 would be expected to have a B/O rather than a B/B genotype (showing that the B allele is
dominant over O) because two of his children (II-6 and II-7) are group O and must have inherited an O
allele from their father (I-1) in addition to the O allele inherited from their mother (I-2). Similarly, II-2 and
II-3 are B/O, based on the ABO type of their children, showing the dominance of B over O.
 CH A P T E R 1 1
In blood group genetics, a recessive
trait or condition almost always means that
red cells express a null phenotype [eg, the
Lu(a–b–)
or Rhnull or O phenotypes] because of
homozy- gosity for a “silent” or “amorphic
gene” that either results in no product or
encodes a de-
fective product. The family in Fig 11-8
demon- strates the inheritance of a recessive
silent LU gene, which in the homozygous
state results in the Lu(a–b–) phenotype. The
proband, II-3, who was multitransfused, was
identified be- cause of the presence of anti-
Lu3 (an antibody to a high-prevalence
Lutheran antigen) in his plasma. Because his
Lu(a–b–) phenotype is the result of
recessive inheritance, any potential donors
in the family can be found by testing his
siblings. About 25%, or one in four of the
offspring of the mating between I-1 and I-2,
is expected to have the Lu(a–b–) phenotype;
in this case, only the proband has Lu(a–b–)
red cells.
FIGURE 11-8. Autosomal recessive inheritance. The offspring of II-3, the Lu(a–b–) proband, and II-
4, his Lu(a+b+) wife, demonstrate that Lu is recessive to Lua and Lub and that the presence of the silent
Lu allele is masked by the product of Lua or Lub at the phenotype level.
Blood Group Genetics ■ 267
Sex-Linked Inheritance
A sex-linked trait is one that is encoded by a
gene located on the X or Y chromosome. The
Y chromosome carries few functional genes
and discussion of sex-linked inheritance
generally is synonymous with inheritance of
X-borne genes. In females with two X
chromosomes the inheritance of X-borne
genes, like the inheri- tance of genes carried
on the autosomes, can be dominant or
recessive. Males, in contrast, have one X
chromosome (always maternally derived) and
one Y chromosome (always pa- ternally
derived) and are hemizygous for genes on
the X or Y chromosome because only one
chromosome (and thus one copy of a gene) is
present. Most X-borne genes do not have a
homolog (a similar sequence of DNA) on the
Y chromosome. As a consequence, in-
heritance of an X-borne dominant trait is the
same in males and females. However, an X-
borne trait that is recessive in females is
268 ■ AABB T EC HNIC AL MANUAL

expressed by all males who carry the gene
for the trait. The most striking feature of
both dominant and recessive X-linked
inheritance is that there is no male-to-male
transmission; that is, the trait is never
transmitted from fa- ther to son.
Sex-Linked Dominant Inheritance
A trait encoded by an X-borne allele that has
sex-linked dominant inheritance is
expressed by hemizygous males and by both
heterozy- gous and homozygous females. A
male passes his single X chromosome to all
of his daugh- ters and all daughters will
express the condi- tion or trait. When a
female is heterozygous for an allele that
encodes a dominant trait, each of her
children, whether male or female, has a 50%
chance of inheriting the trait. When a fe-
male is homozygous for an X-borne allele
with dominant inheritance, the encoded trait
is ex- pressed by all her children.
The Xga antigen (Xg blood group system)
is encoded by an allele on the X chromosome
and is inherited in a sex-linked dominant
manner. The first indication that the Xga
anti- gen is X-borne came from the
observation that the prevalence of the
Xg(a–) and Xg(a+) phe- notypes differed
noticeably between males and females; the
Xga antigen has a prevalence of 89% in
females and only 66% in males.5
Figure 11-9 shows the inheritance of
the Xga antigen in a three-generation family.
In generation I, the father (I-1) is Xg(a+)
and has transmitted Xga to all his daughters
but to none of his sons. His eldest daughter
(II-2), for example, must be heterozygous
for Xga/Xg; she received the allele encoding
the Xga antigen from her Xg(a+) father and
a silent allele, Xg, from her Xg(a–) mother.
II-2 has transmitted Xga to half her children,
regardless of whether they are sons or
daughters.
Sex-Linked Recessive Inheritance
A trait encoded by an X-borne recessive
allele is carried, but not expressed, by a
heterozy- gous female. A male inherits the
trait from his

FIGURE 11-9. Sex-linked dominant inheritance. The Xga antigen is encoded by an allele on the tip of the
short arm of the X chromosome. This family demonstrates the sex-linked dominant inheritance of the Xga
antigen.
 CH A P T E R 1 1
mother, who is usually a carrier (or could be
homozygous for the trait if she is the offspring
of a male who expresses the trait and a carrier
female). An affected male transmits the trait to
all of his daughters, who in turn transmit the
trait to approximately half of their sons. There-
fore, the prevalence of the expression of an X-
borne recessive trait is much higher in males
than in females. A carrier female who mates
with a male lacking the trait transmits the trait
to one-half of her daughters (who will also be
carriers) and to one-half of her sons (who will
be affected). If mating is between an affected
male and a female who lacks the trait, all of
the sons will lack the trait and all of the
daughters will be carriers. If an X-borne
recessive trait is rare in the population, the
trait is expressed al- most exclusively in
males.
The XK gene encodes the Kx protein and
demonstrates X-borne recessive inheritance.
FIGURE 11-10. Sex-linked recessive inheritance. This family demonstrates that a sex-linked trait
that is recessive in females will be expressed by any male who inherits the trait. Homozygosity for
such a trait is required for it to be expressed in females. The trait skips one generation and is
Blood Group Genetics ■ 269
Mutations in XK result in red cells with the
McLeod phenotype; such red cells lack Kx
and have reduced expression of Kell
antigens (McLeod syndrome). McLeod
syndrome is as- sociated with late-onset
clinical or subclinical myopathy,
neurodegeneration, and central nervous
system manifestations as well as with
acanthocytosis and, frequently, compensated
hemolytic anemia. More than 30 different
XK gene mutations associated with a
McLeod phenotype have been found.
Different XK mutations appear to have
different clinical ef- fects and may account
for the variability in the prognosis.28
Sequencing of XK to determine the specific
type of mutation in individuals with
McLeod phenotypes has clinical prognos-
tic value. McLeod syndrome is an X-linked
re- cessive condition and, as demonstrated
by the family in Fig 11-10, is found only in
males.
carried through females.
270 ■ AABB T EC HNIC AL MANUAL

The Principles of Independent
Segregation and Independent
Assortment
The passing of a trait from one generation to
the next follows certain patterns or
principles. The principle of independent
segregation re- fers to the separation of
homologous chromo- somes and their
random distribution to the gametes during
meiosis. Only one member of an allelic pair
is passed on to the next genera- tion, and
each gamete has an equal probability of
receiving either member of a parental ho-
mologous allelic pair; these chromosomes
are randomly united at fertilization and thus
seg- regate independently from one
generation to the next. The family in Fig 11-
11 demonstrates the independent segregation
of the ABO alleles on chromosome 9.
The principle of independent assort-
ment states that alleles determining various
traits are inherited independently from each
other. In other words, the inheritance of one
allele (eg, a B allele, on chromosome 9, encod-
ing B antigens) does not influence the inheri-
tance of another allele (eg, an M allele, on
chromosome 4, encoding M antigens). This is
demonstrated by the family in Fig 11-11.
Linkage and Crossing-Over
Linkage is the physical association between
two genes that are located on the same chro-
mosome and are inherited together.
Examples include RHD and RHCE encoding
the antigens of the Rh system, which are
both on chromo- some 1 and are linked loci
that do not assort independently.
Crossing-over is the exchange of genetic
material between homologous chromosome
pairs (Fig 11-4). In this process, a segment
from one chromatid (and any associated
genes) changes places with the
corresponding part of the other chromatid
(and its associated genes); the segments are
rejoined, and some genes will have switched
chromosomes. Thus, crossing-over is a
means to shuffle genetic ma- terial. Because
crossing-over can result in new

FIGURE 11-11. Independent segregation and independent assortment are illustrated by the inheritance of blood
group alleles in one family. Parental ABO alleles were randomly transmitted (independent segregation), and
each child has inherited a different combination. The family also illustrates that the alleles encoding
antigens of the ABO and MNS blood group systems are inherited independently from each other.
 CH A P T E R 1 1 Blood Group Genetics ■ 271

gene combinations on the chromosomes in- first recognized example of autosomal

volved, it is also referred to as
recombination, and the rearranged
chromosomes can be re- ferred to as
recombinants. Crossing-over and
recombination, using chromosome 1 as an
ex- ample, are explained in Fig 11-12.
Two gene loci carried by the same chro-
mosome that are not closely linked are re-
ferred to as being syntenic. For example, the
loci for RH and FY, both located on chromo-
some 1, are syntenic because the distance be-
tween them (RH on the short arm and FY on
the long arm) is great enough for them to un-
dergo crossing-over and to assort indepen-
dently.
The frequency of crossing-over
involving two genes on the same
chromosome is a mea- sure of the distance
[measured in centimor- gans (cM)] between
them; the greater the dis- tance between two
loci, the greater the probability that
crossing-over (and recombi- nation) will
occur. In contrast, genes located very close
together (linked) tend to be trans- mitted
together with no recombination. The degree
of crossing-over between two genes can be
calculated by analyzing pedigrees of
families informative for the genes of interest
and observing the extent of recombina-
tion.The traditional method of linkage
analysis requires the use of lod (logarithm of
the odds) scores.29 Linkage analysis was the
basis through which chromosomes were
mapped and the relative position and
distance between genes established. Linkage
between Lutheran (LU) and ABH secretion
(SE or FUT2) was the
linkage in humans and is explained in Fig
11-13.
Although crossing-over occurs readily be-
tween distant genes, rare examples of recom-
bination have been documented for genes that
are very closely linked or adjacent on a chro-
mosome. Such genes include those encoding
the MN (GYPA) and Ss (GYPB) antigens on
chromosome 4 and are reviewed by Dan-
iels.18(pp96-142)
Linkage Disequilibrium
Genes at closely linked loci tend to be
inherit- ed together and constitute a
haplotype (a combination of alleles at two
or more closely linked loci on the same
chromosome). The al- leles encoding the
MNS antigens are inherited as four
haplotypes: MS, Ms, NS, or Ns. Because
linked genes do not assort independently,
the antigens encoded by each of these
haplotypes have a different prevalence in the
population than would be expected by
random assort- ment. If M and S were not
linked, the expected prevalence for M+ and
S+ in the population would be 17% (from
frequency calculations), whereas the actual
or observed prevalence (obtained from
testing and analyzing families) of the MS
haplotype is 24%.18(pp96-142) This con- stitutes
linkage disequilibrium, which is the
tendency of specific combinations of alleles
at two or more linked loci to be inherited
togeth- er more frequently than would be
expected by chance.

FIGURE 11-12. Crossing-over and recombination. In the diagram, chromosome 1 is used as an example.
The very closely linked RH genes, RHD and RHCE, are located near the tip of the short arm of
chromosome 1. The loci for FY and KN are on the long arm of the chromosome and are not linked.
During meiosis, crossing-over occurs between this homologous chromosome pair, and portions of the
chromosome break and become rejoined to the partner chromosome. Crossing-over of the long arm of
chromosome 1 results in recombination
between the loci for FY and KN such that the gene encoding the Fyb antigen now travels with a gene that
encodes the Sl(a–) phenotype of the Knops system.
272 ■ AABB T EC HNIC AL MANUAL
FIGURE 11-13. Linkage between LU and SE. I-2 is homozygous for Lub and se and must transmit these
alleles to all her offspring. I-1 is doubly heterozygous (Lua/Lub and Se/se). He has transmitted Lub with
Se and Lua with se, showing linkage between Lu and Se. Several such informative families would need to be
analyzed to statistically
confirm linkage.
Gene Interaction and Position Effect
Alleles that are carried on the same chromo-
some are referred to as being in cis position
whereas those on opposite chromosomes of
a homologous pair are in trans position.
Alleles that are in cis and linked are always
inherited together on the same chromosome,
whereas genes in trans segregate
independently.
Historically, the Rh blood group system
was used to explain the meaning of cis and
trans. For example, the DCe/DcE genotype
was described as having C and e alleles in
cis in the DCe haplotype, with c and E
alleles in cis in the partner DcE haplotype,
whereas C and E and also c and e are in
opposing haplotypes and are in trans. In this
alignment, C and e, for exam- ple, are
always inherited together, but C and E are
not. The preceding explanation, which im-
plies that one gene encodes C and c antigens
while another linked gene encodes E and e
an- tigens, was based on the Fisher-Race
theory of three genes at the RH locus. In
contrast, mo- lecular analysis indicates that
only one gene (RHCE), with four alleles
(RHCE*Ce, RHCE*cE, RHCE*ce, and
RHCE*CE), encodes one protein that carries
the CcEe antigens. Thus, for Rh,
DCe is the haplotype, and the RHD allele is in
cis to the RHCE*Ce allele.
The expression of red cell antigens may
be modified or affected by gene or protein
interactions that manifest primarily as re-
duced antigen expression. One example in
which the haplotype on one chromosome af-
fects the expression of the haplotype on the
paired chromosome is commonly referred to
as the “position effect” and can be
observed with Rh antigen expression. When
a Ce haplo- type (note the absence of RHD)
is in trans to a D antigen-encoding
haplotype, the expression of D is
dramatically reduced and a weak D
phenotype can result. When the same D-en-
coding haplotype is inherited with either ce
or cE, D antigen is normally expressed. The
cause of this reduced antigen expression is
not known, but may involve differences in
gene expression levels or altered assembly
of pro- teins in the membrane. In the
presence of the Kell system antigen Kpa,
expression of other Kell system antigens
encoded by the same al- lele is suppressed to
varying degrees (cis-mod- ifier effect). This
is best observed in persons who have a
silenced K0 (a Kellnull gene) in trans.
The amino acid change that results in the ex-
pression of Kpa adversely affects trafficking of
 CH A P T E R 1 1
the Kell glycoprotein to the red cell surface, so
that the quantity of Kpa-carrying Kell
glycopro- tein that reaches the red cell surface
is greatly reduced.
Suppressor or modifier genes affect the
expression of another gene, or genes. For ex-
ample, KLF1, located on chromosome
19p13.3-p13.12, encodes erythroid Krüppel-
like factor, which is a transcription factor es-
sential for terminal differentiation of red cells.
Singleton et al30 first discovered that heterozy-
gosity for nucleotide changes in KLF1 is re-
sponsible for the dominant Lu(a–b–) pheno-
type,5 which is also known as the In(Lu)
phenotype. This heterozygosity is character-
ized by reduced expression of antigens in the
Lutheran system (Lumod) and for P1, Inb and
AnWj antigens.
In kind, the transcription factor GATA-1,
encoded by the X-borne GATA-1 gene, is es-
sential for erythroid and megakaryocyte differ-
entiation. Changes in this gene were associat-
ed with the X-linked type of Lumod that initially
presented as an Lu(a–b–) phenotype.31 Sero-
logic differentiation of these Lumod
phenotypes from the true Lu(a–b–) (Lunull)
phenotype can be challenging, yet has
clinical value. Now that the molecular basis
of these phenotypes is un- derstood,
sequencing of the relevant genes can be
used to make the distinction.
In the past, independent, unidentified
modifier or regulator genes were postulated
to be the basis of several null or variant
pheno- types when a silent or inactive
(nonfunction- ing) gene was not evident. For
example, the regulator type (as opposed to
the amorph
type) of Rhnull was shown through family stud-
ies to result from a gene not at the RH locus.
This phenotype is now known to result from
various silencing changes in RHAG, a gene lo-
cated on chromosome 6 (and thus indepen-
dent of RH). RHAG encodes the Rh-
associated glycoprotein RhAG, which is
required in the red cell membrane for Rh
antigen expression. Similarly, molecular
analysis indicates that the modifying gene
that causes the Rhmod pheno-
type is a mutated RHAG.5
Several red cell antigens require the inter-
action of the products of two or more indepen-
dent genes for their expression. Assignment of
Blood Group Genetics ■ 273
the high-prevalence antigen Wrb to the
Diego blood group system took many years
because the only red cells that lacked Wrb
were rare MNS null phenotypes [En(a–) and
MkMk] and variants, yet Wra, the antigen
that is antitheti- cal to Wrb was clearly
independent of MNS. This anomaly was
resolved when it was under- stood that GPA
(or more precisely, amino acids 75 to 99),
which carries MN antigens, must be present
in the red cell membrane for Wrb ex-
pression. The Wra/Wrb polymorphism is en-
coded by DI and carried on band 3, whereas
GPA is the product of GYPA, a gene that is
in- dependent of DI. An absence of RhD and
RhCE
protein (Rhnull) results in red cells that lack
LW antigens and lack or have reduced
expression of U and S or s antigens, again
demonstrating the interaction in the
membrane of the prod-
ucts of two or more independent blood
group genes.
The sequential interaction of genes at
several loci is required for the expression of
ABO, H, Lewis, and I antigens on red cells
and in secretions. These antigens are
carbohydrate determinants carried on
glycoproteins or gly- colipids, and the genetics
of these antigens is more complex than that of
protein-based anti- gens. The carbohydrate
antigens are carried on oligosaccharide chains
that are assembled by the stepwise addition of
monosaccharides. ABO genes and the genes
encoding the other carbohydrate-based
antigens do not encode membrane proteins but
do encode an enzyme, a glycosyltransferase,
that catalyzes the se- quential transfer of the
appropriate immuno- dominant
monosaccharide. Each monosac- charide
structure is transferred by a separate
glycosyltransferase, such that two genes are
re- quired for a disaccharide, three for a
trisaccha- ride, and so on. An inactivating
change at one locus can prevent or modify the
expression of the other gene products. The
product encoded by the H gene is the
biosynthetic precursor for A and B antigen
production; if the H gene is si- lenced, A or B
antigens cannot be produced. A mutated A or
B allele may result in a glycosyl- transferase
that is inactive or that causes more or less
antigen to be expressed. Details on the
biosynthesis of the ABO, H, Lewis, and I anti-
gens may be found in Chapter 12.
274 ■ AABB T EC HNIC AL MANUAL
POPULATION GENETICS
Population genetics is the study of the
distri- bution patterns of genes and of the
factors that maintain or change gene (or
allele) frequen- cies. A basic understanding
of population ge- netics, probability, and the
application of sim- ple algebraic
calculations is important for relationship
(identity) testing. In transfusion medicine,
the knowledge can be applied to clinical
situations such as predicting the likeli- hood
of finding compatible blood for a patient
who has made antibody(ies) to red cell anti-
gens. It may be helpful to define three com-
monly used words so that their appropriate
use is understood. “Frequency” is used to
de- scribe prevalence at the genetic level—
that is, the occurrence of an allele (gene) in
a popula- tion. “Prevalence” is used to
describe the oc- currence of a permanent
inherited character- istic at the phenotypic
level—for example, a blood group—in any
given population. “Inci- dence” is used
when describing the rate of oc- currence in a
population of a condition that changes over
time, such as a disease, and is thus not
suitable for use with blood groups.
Phenotype Prevalence
The prevalence of a blood group antigen or
phenotype is determined by testing red cells
from a large random sample of people of the
same race or ethnicity with a specific antibody
and calculating the percentage of positive and
negative reactions. The larger the cohort being
tested, the more statistically significant is the
result. The sum of the percentages for the
prevalence of the phenotypes should equal
100%. For example, in the Duffy blood group
system, the prevalence in a random popula-
tion of African ethnicity for the Fy(a+b–),
Fy(a–b+), Fy(a+b+), and Fy(a–b–) phenotypes
is 9%, 22%, 1%, and 68%, respectively;
togeth- er, these percentages total 100%. If the
red cells from 1000 donors of European
ethnicity are tested with anti-c, and 800 of the
samples are positive and 200 are negative for
the Rh anti- gen c, the prevalence of the c+
phenotype is 80% and that of the c– phenotype
is 20%. Thus, in this donor population,
approximately 20% of ABO-compatible units
of blood, or 1 in 5,
should be compatible with serum from a pa-
tient who has made anti-c.
Calculations for Antigen-Negative
Phenotypes
When blood is provided for a patient with
anti- bodies directed at one or more red cell
anti- gens, a simple calculation can be used
to esti- mate the number of units that need to
be tested to find the desired antigen
combina- tion. To calculate the prevalence
of the com- bined antigen-negative
phenotype, the preva- lences of each of the
individual antigens are multiplied together
because the antigens are inherited
independently of each other. An ex- ception
occurs when the antigens are encoded by
alleles that are closely linked and are inher-
ited as haplotypes (M, N, S, s) or reside on
the same carrier protein (C, c, E, e). If a
patient with antibodies to K, S, and Jka
antigens re- quires 3 units of blood, for
example, the preva- lence of the antigen-
negative phenotype and the number of units
that need to be tested to find them can be
calculated as follows:
■ The prevalence of: K– donors = 91%; S–
do- nors = 48%; Jk(a–) donors = 23%
■ The percentage of donors negative for each
antigen is expressed as a decimal and mul-
tiplied: 0.91(K–) × 0.48(S–) × 0.23[Jk(a–)]
= 0.10
■ 0.10 expressed as a % = 0.10 × 100% = 10%
■ 10% expressed as occurrence = 10%/100%
= 1/10
■ Thus, approximately 1 in 10 ABO-compati-
ble RBC units are expected to be K– S–
Jk(a–)
The patient in question requires 3 units,
so on average, 30 units would need to be
tested to fulfill the request. Based on these
calcula- tions, a hospital transfusion service
would be able to determine the likelihood of
having the requested units in house.
The prevalence of a particular antigen
(or phenotype) can vary with race,5 and the
preva- lence for a combined antigen-
negative pheno- type calculation should be
selected on the ba- sis of the predominant
race found in the donor population.
 CH A P T E R 1 1 Blood Group Genetics ■ 275

Allele (Gene) Frequency blood group genetics, and its use is demon-
strated below. In a population of European
The allele frequency is the proportion of one
ethnicity, the frequencies of the two alleles
allele relative to all alleles at a particular gene
en- coding K or k can be determined as
locus in a given population at a given time.
follows:
This frequency can be calculated from the
prevalence of each phenotype observed in a Frequency of the K allele = p
population. The sum of allele frequencies at Frequency of the k allele = q
any given locus must equal 100% (or 1 in an Frequency of the KK genotype = p2
al- gebraic calculation) in the population Frequency of the Kk genotype = 2pq
sample tested. The genotype frequency in a
popula- tion is the number of individuals with Frequency of the kk genotype = q2
a given genotype divided by the total number The K antigen is expressed on the red cells of
of indi- viduals in population. 9% of people of European ethnicity; there-
fore:
p2 + 2pq = the frequency of people who carry K
The Hardy-Weinberg Equilibrium and are K+
Gene frequencies tend to remain constant Thus, p2 + 2pq = 0.09
from generation to generation in any
relatively large population unless they are q2 = 1 – (p2 + 2pq) = the frequency of people
influenced by factors such as selection, who carry kk and are K–
mutation, migration, or nonrandom mating, q2 = 1 – 0.09
any of which would have to be rampant to
q2 = 0.91
have a discernible ef- fect. According to the
principles proposed by the British q = 0.91
mathematician Hardy and the Ger- man
physician Weinberg, gene frequencies reach q = 0.95 = the frequency of k
equilibrium. This equilibrium can be ex-
pressed in algebraic terms by the Hardy- Because the sum of the frequencies of both
Wein- berg formula or equation: al- leles must equal 1.00:
p+q=1
p2 + 2pq + q2 = 1
p=1–q
If two alleles, classically referred to as p = 1 – 0.95
A and a, have gene frequencies of p and q, p = 0.05 = the frequency of K
the homozygotes and heterozygotes are
present in the population in the following
Once the allele frequencies for K and k have
proportions:
been calculated, it is possible to calculate the
percentage of k+ (both K+k+ and K–k+) and
AA = p2 Aa = 2pq aa = q2
K+ (both K+k– and K+k+) people:
In such a two-allele system, if the gene Prevalence of k+ = 2pq + q2
frequency for one allele, say p, is known, q can
= 2(0.05 × 0.95) + (0.95)2
be calculated by p + q = 1.
The Hardy-Weinberg equation permits = 0.9975 × 100
the estimation of genotype frequencies from = a calculated preva-
the phenotype prevalence in a sampled lence of 99.75% (the
popu- lation and, reciprocally, allows the observed prevalence
determina- tion of genotype frequency and of the k+ phenotype is
phenotype prevalence from the gene 99.8%)
frequency. The equation has a number of
applications in
276 ■ AABB T EC HNIC AL MANUAL

Prevalence of K+ = 2pq + p2 when applied to a two-allele situation, are

= 2(0.05 × 0.95) + (0.05)2
= 0.0975
= 0.0975 × 100 = a
calcu- lated
prevalence of K+ of
9.75% (the obser- ved
prevalence of the K+
phenotype is 9%)
The Hardy-Weinberg equation also can be
used to calculate the frequencies of the three
possible genotypes KK, Kk, and kk from the
gene frequencies K (p) = 0.05 and k (q) = 0.95:
p2 + 2pq + q2 = 1
Frequency of KK = p2 =
0.0025 Frequency of Kk =
2pq = 0.095 Frequency of kk
= q2 = 0.9025
If antibodies are available to test for the
products of the alleles of interest (in this
exam- ple, anti-K and anti-k), the allele
frequencies also can be obtained by direct
counting as demonstrated in Table 11-2. The
allele fre- quencies obtained by direct
testing are the ob- served frequencies for
the population being sampled, whereas those
obtained by gene fre- quency calculations
(above) are the expected frequencies. The
various calculations above,
rel- atively simple; the calculations for three
or more alleles are much more complex and
be- yond the scope of this chapter.
For a given population, if the prevalence
of one genetic trait, such as a red cell antigen,
is known, the Hardy-Weinberg equation can
be applied to calculate allele and genotype fre-
quencies. The Hardy-Weinberg equilibrium
principle is valid when the population is suffi-
ciently large that chance alone cannot alter an
allele frequency and when the mating is ran-
dom. A lack of selective advantage or disad-
vantage of a particular trait and other influ-
encing factors, such as mutation or migration
in or out of the population, are assumed to be
absent when the Hardy-Weinberg equilibrium
principle is applied. When all of these condi-
tions are met, the gene pool is in equilibrium
and allele frequencies do not change from one
generation to the next. If the conditions are
not met, changes in allele frequencies may oc-
cur over a few generations and may explain
many of the differences in allele frequencies
between populations.
RELATIONSHIP TESTING
Polymorphisms are inherited characteristics
or genetic markers that can distinguish be-
tween people. The blood groups with the

TABLE 11-2. Allele Frequencies of K and k Calculated Using Direct Counting (assuming the
absence of null alleles)

Phenotype No. of Persons No. of Kk Alleles K k

K+k– 2 4 4
K+k+ 88 176 88 88
K–k+ 910 1820 0 1820
Totals 1000 2000 92 1908
Allele frequency 0.046 0.954
A random sample of 1000 people tested for K and k antigens, have a total of 2000 alleles at the
Kk locus because each person inherits two alleles, one from each parent. Therefore, the two persons with
a K+k– phe- notype (each with two alleles) contribute a total of four alleles. To this are added 88 K
alleles from the K+k+ group, for a total of 92 K alleles or an allele frequency of 0.046 (92 ÷ 2000). The
frequency of the k allele is 0.954 (1908 ÷ 2000).
 CH A P T E R 1 1
greatest number of alleles (greatest polymor-
phism) have the highest power of
discrimina- tion and are the most useful for
determining relationships. Blood is a rich
source of inherit- ed characteristics that can
be detected, includ- ing red cell, HLA, and
platelet antigens. Red cell and HLA antigens
are easily identifiable, are polymorphic, and
follow Mendelian laws of inheritance. The
greater the polymorphism of a system, the
less chance there is of finding two people
who are identical. The extensive
polymorphism of the HLA system alone
allows the exclusion of over 90% of falsely
accused men in cases of disputed paternity.
Serologic methods of identity testing have
been surpassed and replaced by DNA-based
assays32 (referred to as DNA fingerprinting,
DNA profiling, or DNA typing) that were pio-
neered by Jeffreys and colleagues.33,34 Tandem-
ly repeated sequences of DNA of varying
lengths occur predominantly in the noncoding
genomic DNA, and they are classified into
groups depending on the size of the repeat re-
gion. The extensive variation of these tandem-
ly repeated sequences between individuals
makes it unlikely for the same number of re-
peats to be shared by two individuals, even if
these individuals are related. Minisatellite
[also referred to as variable number of tandem
repeats (VNTR)] loci have tandem repeat units
of nine to 80 base pairs, whereas microsatellite
[also referred to as short tandem repeat [STR)]
loci consist of two to five base pair tandem re-
peats.35 Microsatellites and minisatellites are
reviewed by Bennett.36
Assays for VNTR and STR sequences in-
volve the electrophoretic separation of DNA
fragments according to size. DNA profiling
in- volves amplification of selected,
informative VNTR and STR loci using locus-
specific oligo- nucleotide primers with the
subsequent mea- surement of the size of the
PCR products. Hundreds of STR loci have
been mapped throughout the human
genome, and many have been applied to
identity testing. Analysis of different STR
loci (usually at least 12) is used to generate
a person’s DNA profile that is virtu- ally
guaranteed to be unique to that person (or to
two identical twins). DNA fingerprinting is
a powerful tool not only for identity testing
and
Blood Group Genetics ■ 277
population genetics but also for monitoring
chimerism after marrow transplantation.37,38
STR analysis also has been used to monitor
pa- tients for graft-vs-host disease after
organ transplantation, particularly after a
liver trans- plant.39
In a case of disputed paternity, if an al-
leged father cannot be excluded from
paterni- ty, the probability of his paternity
can be calculated. The calculation compares
the probability that the alleged father
transmitted the paternal obligatory genes
with the proba- bility that any other
randomly selected man from the same racial
or ethnic group transmit- ted the genes. The
result is expressed as a like- lihood ratio
(paternity index) or as a percent- age. The
AABB has developed standards and
guidance documents for laboratories that
per- form relationship testing.40
BLOOD GROUP GENE MAPPING
Gene mapping is the process through
which a gene locus is assigned to a location
on a chro- mosome. The initial mapping of
blood group genes was accomplished by
testing many fam- ilies for selected red cell
antigens. Pedigrees were analyzed for
evidence of recombination between the
genes of interest to rule out or es- tablish
linkage of a blood group with another
marker, with a known chromosomal
location.
The gene encoding the antigens of the
Duffy blood group system was the first to be
assigned to a chromosome, by showing that
the gene is linked to an inherited deformity
of chromosome 1. More recently,
recombinant DNA methods were used to
establish the phys- ical locations of genes,
and today, with se- quencing of the human
genome, determining the location of a gene
involves a computer da- tabase sequence
search. The Human Genome Project
(http://www.ornl.gov/sci/tech resourc
es/Human_Genome/home.shtml) has result-
ed in construction of a physical gene map
in- dicating the position of gene loci and the
dis- tance between loci is expressed by the
number of base pairs of DNA.
Currently, 34 blood group systems are rec-
ognized by the ISBT.6 The genes for all of
them have been cloned and assigned to their
respec-
278 ■ AABB T EC HNIC AL MANUAL
tive chromosomes (see Table 11-1).
Tradition- ally, genes were mapped to
chromosomes ac- cording to the metaphase
banding patterns produced through staining
with Giemsa (G banding) or quinacrine (Q
banding). Other methods used in
chromosome mapping have included
deletion mapping (partial or total loss of a
chromosome related to the presence or
absence of a gene), somatic cell hybridiza-
tion (based on the fact that human-rodent
hy- brid cell lines randomly shed human
chromo- somes, permitting the association
of a trait with the presence or absence of a
chromo- some), in-situ hybridization (use of
fluores- cent DNA probes with a preparation
of intact chromosomes), and chromosome
walking (a technique to clone a gene from
its known clos- est markers). Details on
gene mapping proce- dures are beyond the
scope of this chapter, but reviews are
available.7
Advances in genetic technology and in-
formation generated through the Human Ge-
nome Project make it feasible to construct a
physical gene map of the absolute positions of
gene loci in which the distance between loci is
expressed by the number of base pairs of
DNA.
CHIMERISM
The observation that a sample gives mixed-
field agglutination is not unusual in a
labora- tory. Often this is the result of
artificially in- duced chimerism through the
transfusion of donor red cells or a stem cell
transplant. On rare occasions, the
observation of mixed-field agglutination
identifies a true chimera, that is, a person
with a dual population of cells de- rived
from more than one zygote. Indeed, the first
example of a human chimera was a female
blood donor discovered through mixed-field
agglutination during antigen typing. Most
hu- man chimeras can be classified as either
twin chimeras or tetragametic (dispermic)
chime- ras. Chimerism is not a hereditary
condition.41
Twin chimerism occurs through the for-
mation of placental blood vessel anastomo-
ses, which results in the mixing of blood be-
tween two fetuses. This vascular bridge
allows hematopoietic stem cells to migrate
to the marrow of the opposite twin. Each
twin may
have two distinct populations of cells (red cells
and leukocytes), that of their true genetic type
and that of their twin. The percentage of the
two cell lines in each twin tends to vary; the
major cell line is not necessarily the autolo-
gous cell line, and the proportions of the two
cell lines may change throughout life. Chime-
ric twins have immune tolerance; they do not
make antibody against the A or B antigens that
are absent from their own red cells but are
present on the cells of the engrafted twin. This
tolerance extends beyond red cells to negative
mixed lymphocyte cultures and the mutual ac-
ceptance of skin grafts.
In twin chimeras, the dual cell popula-
tion is strictly confined to blood cells.
Tetraga- metic or dispermic chimeras
present chime- rism in all tissues and are
more frequently identified because of
infertility than because of mixed populations
of red cells. The mecha- nism(s) leading to
the development of tetraga- metic chimeras
are unknown, but they arise through the
fertilization of two maternal nu- clei by two
sperm followed by fusion of the two zygotes
and development into one person containing
two cell lineages.
More commonly, chimeras occur
through medical intervention and arise from
the trans- fer of actively dividing cells, such
as through hematopoietic transplantation.41
However, chimerism may be more prevalent
than once thought. DNA analysis to confirm
the Rh-neg- ative status of Northern
European blood do- nors identified a donor
with a dual red cell population of which
95% was Rh negative and 5% was Rh
positive. The donor was confirmed to be a
chimera. Chimerism was found to be the
cause of a discrepancy observed when ABO
was determined, and news headlines were
made by a case of disputed maternity when a
woman was falsely excluded as the mother
of her children because of chime- rism.42,43
BLOOD GROUP TERMINOLOGY
Antigens were originally named using an al-
phabetical (eg, A/B, C/c) notation or they
were named after the proband whose red
cells car- ried the antigen or who made the
first known
 CH A P T E R 1 1 Blood Group Genetics ■ 279
antigen = 002; D antigen = 001).

antibody (eg, Duclos). A symbol with a super-

script letter (eg, Lua, Lub; Jka, Jkb) was used,
and a numerical (eg, Fy3, Jk3, Rh32)
terminology was introduced. In blood group
systems, anti- gens are named using more than
one scheme (eg, the Kell blood group system:
K, k, Jsa, Jsb, K11, K17, TOU).
In 1980, the ISBT established its
Working Party on Terminology for Red Cell
Surface An- tigens. The working party was
charged to de- velop a uniform
nomenclature that would be “both eye and
machine readable” and “in keeping with the
genetic basis of blood groups.” A blood
group system consists of one or more
antigens under the control of a single gene
locus or of two or more homologous genes
that are so closely linked that virtually no
recombination occurs between them. Thus,
each blood group system is genetically
independent from every other blood group
system. The failure of an antibody to be
reac- tive with red cells of a particular null
pheno- type is not sufficient for assignment
of the cor- responding antigen to a system.
Some null phenotypes are the result of
inhibitor or modi- fying genes that may
suppress the expression of antigens from
more than one system [eg,
the Rhnull phenotype lacks not only Rh anti-
gens but also LW system antigens, Fy5
antigen
(Duffy system), and sometimes U (MNS
sys- tem) antigen]. Similarly, a blood group
antigen must be shown to be inherited
through family studies, or the expression of
the antigen must be demonstrated to be
associated with a varia- tion in the
nucleotide sequence of the gene controlling
the system, to be assigned antigen status by
the ISBT terminology working party. A
blood group antigen must be defined sero-
logically by an antibody; a polymorphism
that is detectable only by DNA analysis and
for which there is no corresponding
antibody can- not be called a blood group
antigen.
The working party established a
terminol- ogy consisting of uppercase letters
and Arabic numerals to represent blood
group systems and antigens.6,44 Each system
can also be iden- tified by a set of numbers
(eg, ABO system = 001; Rh system = 004).
Similarly, each antigen in the system is
assigned a number (eg, A anti- gen = 001; B
 resources.6 An exam- ple of the traditional
and current ISBT termi- nology as it applies
Thus, 001001 identifies the A to alleles, genotypes, phe- notypes, and
antigen and 004001 identifies antigens is shown in Table 11-3.
the D antigen. Alternatively,
the sinistral zeros may be BLOOD GROUP GENOMICS
omitted so that the A antigen
becomes 1.1 and the D antigen As discussed in earlier sections of this
be- comes 4.1. Each system chapter, the antigens expressed on red cells
also has an alphabeti- cal are the products of genes and can be
abbreviation (Table 11-1); thus detected directly by hemagglutination
KEL is the ISBT symbol for techniques (as long as relevant antisera are
the Kell system, the Rh ISBT available). Their detec- tion is an important
system symbol is RH, and an aspect of the practice of transfusion
alternative name for the D medicine because an antigen can, if it is
antigen is RH1. This introduced into the circulation of an
alphanumeric terminology,
which was designed primarily
for computer use, is not ideal
for everyday com- munication.
To achieve uniformity, a
recom- mended list of user-
friendly alternative names was
compiled.45
The ISBT working party
meets periodical- ly to assign
names and numbers to newly
discovered antigens. For
terminology criteria; tables
listing the systems, antigens,
and phe- notypes; and other
information see ISBT Red
Cell Immunogenetics and
Blood Group Termi- nology
web resources.6 A
comprehensive re- view of the
terminology and its usage is
found in Garratty et al.45
The working party is
charged to develop, maintain,
and monitor a terminology for
blood group genes and their
alleles.6 The ter- minology
takes into account the
guidelines for human gene
nomenclature published by
the Human Genome
Organization (HUGO), which
is responsible for naming
genes based on the
International System for
Human Gene No-
menclature.46 For information
regarding the current status of
gene and allele terminology
see ISBT Red Cell
Immunogenetics and Blood
Group Terminology web
280 ■ AABB T EC HNIC AL MANUAL

TABLE 11-3. Example of Allele, Genotype, Phenotype and Antigen Terminology

Duffy System Traditional ISBT

Allele Fya, Fy b, Fy FY*01 or FY*A, FY*02 or FY*B, FY*N or FY*01N or
FY*02N
Genotype/haplotype Fya/Fyb FY*A/FY*B or FY*01/FY*02
Phenotype Fy(a+b+) FY:1,2
Antigen Fy , Fy
a b
FY1, FY2
ISBT = International Society of Blood Transfusion; N denotes “null”; FY*01N or FY*02N indicates
that the null allele is on a FY*A or FY*B background, respectively.

individual who lacks that antigen, elicit an sulting proteins differ by one amino acid, me-
im- mune response. thionine at residue 48 for S and threonine for s
It is the antibody from such an immune (designated c.143T>C p.Met48Thr). As a re-
response that causes problems in clinical sult, assay design and interpretation are fairly
practice, such as patient/donor blood straightforward for the prediction of most phe-
transfu- sion incompatibility, maternal-fetal
notypes.
incompat- ibility, and the reason why
However, detailed serologic and molecu-
antigen-negative blood is required for safe
lar studies have shown that there are far more
transfusion in these patients.
alleles than phenotypes for some systems, es-
Hemagglutination is simple, quick, and
pecially ABO and Rh. More than 100 different
relatively inexpensive. When carried out
alleles encoding the glycosyltransferases re-
correctly, it has a specificity and sensitivity
sponsible for the four ABO types have been
that is appropriate for most testing.
identified, and a single nucleotide change in
However, hemagglutination has limitations;
an A or B allele can result in an inactive trans-
for exam- ple, it is difficult and often
ferase and a group O phenotype (see Chapter
impossible to ob- tain an accurate phenotype
12). Testing for the common Rh antigens D,
for a recently transfused patient or to type
C/c, and E/e is uncomplicated for most popu-
red cells that are coated with IgG, and some
lations, but antigen expression is more com-
typing reagents are in short supply or not
plex in some ethnic groups. There are more
available. Because the genes encoding the
than 200 RHD alleles encoding weak D or par-
34 known blood group sys- tems have been
tial D phenotypes, and more than 100 RHCE
cloned and sequenced and the molecular
alleles encoding altered, or novel, hybrid Rh
bases of most blood group antigens and
proteins, some of which result in weakened
phenotypes are known, DNA-based meth-
antigen expression (see Chapter 13). RH geno-
ods (genotyping) are increasingly being
typing, particularly in minority populations,
used as an indirect method to predict a blood
requires sampling of multiple regions of the
group phenotype. This approach has
introduced blood group genomics, often gene(s) and algorithms for interpretation.
The basis of DNA assays is the amplifica-
referred to as “molecular
tion of a target gene sequence through the
immunohematology,” into the practice of
polymerase chain reaction (PCR), followed by
transfusion medicine. Prediction of a blood
manual, semi-automated, or automated
group antigen by testing DNA is sim- ple
downstream analysis. Commonly used meth-
and reliable for the majority of antigens be-
ods are sequence-specific PCR (SSP-PCR)
cause most result from SNPs that are
and allele-specific PCR (AS-PCR). For manual
inherited in a straightforward Mendelian
methods, gel electrophoresis is used to sepa-
manner. For example, the antithetical
antigens S and s arise from GYPB alleles
that differ by one nucleo- tide—143T for S
and 143C for s—and the re-
 CH A P T E R 1 1 Blood Group Genetics ■ 281
are summarized in Table 11-4.

rate the PCR products for fragment size

deter- mination. As an alternative, the assay
may in- clude digestion of the PCR products
with a restriction fragment length
polymorphism (RFLP), followed by
electrophoresis and visu- alization of the
fragments. Semi-automated approaches
include real-time PCR using fluo- rescent
probes with quantitative and qualita- tive
automated read-out. Manual methods are
labor-intensive, and each assay is performed
separately on each sample. Automated DNA
arrays allow for higher throughput with a
larg- er number of target alleles in the PCR
reaction, which makes possible the
determination of numerous antigens in a
single assay. Most available platforms are
based on fluorescent bead technology or
mass spectrometry. Rou- tine ABO and RhD
testing is not currently available on most
automated platforms be- cause the
expression of these antigens is com- plex
and further development is required.
To resolve discrepancies and identify
new
alleles, specialty referral laboratories use
methods that are similar to those used for
high-resolution HLA typing, ie, gene-
specific amplification of coding exons
followed by se- quencing, or gene-specific
cDNA amplifica- tion and sequencing.
These methods are used to investigate new
alleles and resolve serologic and molecular
discrepancies. The application of these
methods has been reviewed by several
groups.47-49

Clinical Application of the

Prediction of Blood Groups by DNA
Analysis
A major use of DNA-based assays is to
predict the red cell phenotype of a fetus, or
of a patient who has been transfused, or
when red cells are coated with IgG.
Additional applications in- clude the
resolution of discrepancies in the ABO and
Rh systems and identification of the
molecular basis of unusual serologic results.
DNA analysis also affords the capability of
dis- tinguishing alloantibodies from
autoantibod- ies. This section gives an
overview of some of the major applications
of DNA-based analysis that are currently
employed in patient and do- nor testing.
These, and additional clinical ap- plications,
 most prob- able phenotype through DNA-
based assays makes it possible to match the
DNA-Based Assays to antigen profile of the absorbing red cells to
Predict the Red Cell that of the patient, thereby reducing the
Phenotype: Recently number of cell types re- quired for
Transfused Patients absorption. This approach also al- lows
matching of the antigen profile of the do-
In patients receiving chronic nor to that of the patient for the most
or massive trans- fusions, the clinically significant, common antigens (eg,
presence of donor red cells Jka, Jkb; S, s) when transfusion is required.
often makes typing by This matching avoids the use of “least
hemagglutination inaccurate. incompatible” blood for transfusion, and
Time-consuming and allows transfusion of units that are “antigen-
cumbersome cell sepa- ration matched for clinically signifi- cant blood
methods that are often group antigens” to prevent delayed
unsuccessful in isolating and transfusion reactions and circumvent addi-
typing the patient’s tional alloimmunization.
reticulocytes can be avoided
when DNA typing is used.
PCR- based assays mostly use
DNA extracted from WBCs
isolated from a sample of
peripheral blood. Interference
from donor-derived DNA is
avoided by targeting and
amplifying a region of the
gene that is common to all
alleles so that the minute
quantity of donor DNA is not
de- tected. This approach
makes possible reliable blood
group determination with
DNA pre- pared from a blood
sample collected after
transfusion. DNA isolated
from a buccal smear or urine
sediment is also suitable for
testing. In transfusion-
dependent patients who
produce alloantibodies, an
extended antigen profile is
important to determine
additional blood group
antigens to which the patient
can be- come sensitized.
In the past, when a
patient with autoim- mune
hemolytic anemia was
transfused be- fore the
patient’s red cell phenotype
for minor antigens was
established, time- and
resource- consuming
differential allogeneic
absorptions were required to
determine the presence or
absence of alloantibodies
underlying the auto- antibody.
Establishing the patient’s
282 ■ AABB T EC HNIC AL MANUAL

TABLE 11-4. Applications of DNA-Based Assays for Patient and Donor Testing

To predict a patient’s red cell phenotype:

■ After a recent transfusion.

– Aid in antibody identification and RBC unit selection.
– Select RBCs for adsorption.

■ When antibody is not available (eg, anti-Doa, -Dob, -Jsa, -V, -VS).

■ Distinguish an alloantibody from an autoantibody (eg, anti-e, anti-Kpb).

■ Help identify alloantibody when a patient’s type is antigen-positive and a variant phenotype is possible
(eg, anti-D in a D-positive patient, anti-e in an e-positive patient).

■ When the patient’s red cells are coated with immunoglobulin (DAT+).
– When direct agglutinating antibodies are not available.
– When the antigen is sensitive to the IgG removal treatment (eg, antigens in the Kell system are
denatured by EDTA-glycine-acid elution).
– When testing requires the indirect antiglobulin test and IgG removal techniques are not
effective at removing cell-bound immunoglobulin.
– When antisera are weakly reactive and reaction is difficult to interpret (eg, anti-Doa, anti-Dob, anti-Fyb).
■ After allogeneic stem cell transplantation.
– If an antibody problem arises, test stored DNA samples from the patient and the donor(s).

■ To detect weakly expressed antigens (eg, Fyb with the FyX phenotype); where the patient is unlikely to
make antibodies to transfused antigen-positive RBCs.

■ Identify molecular basis of unusual serological results, especially Rh variants.

■ Resolve discrepancies, eg, A, B, and Rh.

■ Aid in the resolution of complex serologic investigations, especially those involving high-prevalence anti-
gens when reagents are not available.

■ Identify if a fetus is or is not at risk for hemolytic disease of the fetus and newborn.
– Predict if the partner of a prospective mother with anti-D is homozygous or heterozygous for RHD.
To predict the donor’s red cell phenotype:

■ Screen for antigen-negative donors.

■ When antibody is weak or not available (eg, anti-Doa, -Dob; -Jsa, -Jsb; -V/VS).

■ Mass screening to increase antigen-negative inventory.

■ Find donors whose red cells lack a high-prevalence antigen.

■ Resolve blood group A, B, and Rh discrepancies.

■ Detect genes that encode weak antigens.

■ Type donors for reagent red cells for antibody screening cells and antibody identification panels (eg,
Doa, Dob, Jsa, V, VS).

■ Determine zygosity of donors on antibody detection/identification reagent panels, especially D, S, Fya,

and Fyb.
 CH A P T E R 1 1
DNA-Based Assays to Predict the
Red Cell Phenotype: When Red
Cells Are Coated with IgG
In patients with or without autoimmune he-
molytic anemia, the presence of
immunoglob- ulin bound to the red cells
[positive result on direct antiglobulin testing
(DAT)] often makes antigen typing results
by serologic methods in- valid. Certain
methods, such as treatment of the red cells
with chloroquine diphosphate or EDTA-
glycine acid (EGA) may be employed to
remove the red-cell-bound IgG. These meth-
ods are not always successful, the antigen of
interest may be denatured by the treatment
(eg, EGA destroys antigens of the Kell
blood group system), and direct
agglutinating anti- bodies for the antigen of
interest may not be available. DNA testing
allows determination of an extended antigen
profile to select antigen- negative red cell
units for transfusion.
DNA-Based Assays to
Distinguish Alloantibody from
Autoantibody
When an antibody specificity is found in a
pa- tient whose red cells express the
correspond- ing antigen, it is essential to
know whether the antibody is an allo- or
autoantibody and a DNA-based investigation
is helpful for transfu- sion management. If
DNA typing predicts the red cells to be
antigen positive, further investi- gation by
high-resolution gene sequencing should be
considered because the sample may have a
novel amino acid change in the protein
carrying the blood group antigen. These
novel amino acid changes result in new
epitopes and altered (weakened or partial)
expression of the conventional antigen.
This situation is especially relevant in
pa- tients with sickle cell disease (SCD) or
thalas- semia who require long-term
transfusion sup- port and are at risk of
alloimmunization that is often complicated
by the presence of autoanti- bodies. In
patients of African ancestry who have SCD,
partial expression of common Rh antigens
(D, C, c, and e) is prevalent. Such pa- tients
frequently present with a combination of
anti-D, -C, and -e and yet their red cells type
serologically as D+, C+, and e+. Although
such
Blood Group Genetics ■ 283
patients may make alloantibodies to these
an- tigens, autoantibody production with
Rh- related specificity is prevalent, and
distin- guishing between the two is critical
for safe transfusion practice to avoid
hemolytic trans- fusion reactions.50,51
Delayed hemolytic trans- fusion reaction in
particular, places patients with SCD at risk
for life-threatening anemia, pain crisis, acute
chest syndrome, and/or acute renal failure.
Patients may also experi- ence
hyperhemolysis, in which hemoglobin levels
drop below pretransfusion levels due to
bystander hemolysis of the patients’ own
anti- gen-negative red cells. RH genotyping
has re- vealed that many of these patients
have vari- ant RHD and/or RHCE alleles
that encode amino acid changes in Rh
proteins that result in altered or partial
antigens. For details on RHD and RHCE
alleles that encode partial an- tigen, refer to
Chapter 13.
Reports of autoantibodies to Jka and Jkb
are not uncommon. With the discovery of vari-
ant JK alleles that encode partial Jka and Jkb
an- tigens, it is probable that some previously
identified autoantibodies were alloantibodies
(see Chapter 14). DNA analysis for JK
variants is helpful to clarify the situation. As in
other blood group systems, Kidd system
genetic di- versity is higher in populations of
African an- cestry.
DNA-Based Testing in Prenatal
Practice
DNA-based testing has affected prenatal
prac- tice in the areas that are discussed
below. Hemagglutination, including
antibody titers, gives only an indirect
indication of the risk and severity of
hemolytic disease of the fetus and newborn
(HDFN). Antigen prediction by DNA- based
assays can be used to identify the fetus who
is not at risk of HDFN (ie, who is predicted
to be antigen-negative) so that the mother
need not be aggressively monitored. Testing
of fetal DNA should be considered when a
moth- er's serum contains an IgG
alloantibody that has been associated with
HDFN and the fa- ther's antigen status for
the corresponding an- tigen is heterozygous
or indeterminable, or he is not available for
testing.
284 ■ AABB T EC HNIC AL MANUAL
To Identify a Fetus at Risk for Anemia
of the Neonate
The first application of DNA-based assays for
the prediction of blood group phenotype oc-
curred in the prenatal setting and was report-
ed by Bennett et al52 who tested fetal DNA for
the presence of RHD. Because of the clinical
significance of anti-D, RHD is probably the
most frequent target gene, but DNA-based as-
says can be used to predict the antigen type of
the fetus for any antigen if the molecular basis
is known. When the implicated IgG antibody
in the maternal circulation is not anti-D, it is
prudent, when possible, to also test the fetal
DNA for RHD to preempt unnecessary re-
quests for D– blood for intrauterine transfu-
sion; this is particularly relevant to avoid the
use of rare r'r' or r''r'' blood when anti-c or
anti-e is the implicated antibody.
PCR analyses for the prediction of fetal
D presence of a fetal RHD gene to eliminate
phenotype are based on detecting the pres-
ence or absence of specific portions of RHD.
In populations of European ancestry, the
molec- ular basis of the D-negative
phenotype is usu- ally associated with
deletion of the entire RHD, but several other
molecular bases have been described. In
populations of Asian ancestry 15% to 30%
of D-negative people have an in- tact but
inactive RHD, while others with red cells
that are nonreactive with anti-D have the
Del phenotype. Approximately a quarter of D-
negative people of African ethnicity have an
RHD pseudogene (RHD), which does not en-
code the D antigen, and many others have a
hybrid RHD-CE-D gene (eg, the rS
phenotype). Predicting the D type by DNA
analysis requires probing for multiple
nucleotide changes. The
choice of assays depends upon the patient’s
ethnicity and the degree of discrimination
de- sired. Establishing the fetal KEL
genotype is also of great clinical value in
determining whether a fetus is at risk for
severe anemia, be- cause the strength of the
mother's anti-K anti- body often does not
correlate with the severity of the infant’s
anemia. The same is true for anti-Ge3.53
Amniocytes, harvested from amniotic flu-
id, are the most common source of fetal
DNA. Chorionic villus sampling and
cordocentesis
are not favored because of their more
invasive nature and associated risk to the
fetus. A non- invasive sample source is the
cell-free fetal DNA that is present in
maternal plasma as ear- ly as 5 weeks of
gestation; the amount of DNA increases
with gestational age and reliable re- sults in
DNA-based assays are obtained start- ing at
about 15 weeks of gestation (sometimes
earlier, depending on the gene of interest).54,55
These assays are particularly successful for
D typing because the D-negative phenotype
in the majority of samples is due to the
absence of the RHD gene.
Testing for the presence or absence of a
gene is less demanding than testing for a
sin- gle gene polymorphism or SNP to
predict, for example, the K/k antigen status.
Cell-free fetal DNA from the maternal
plasma is routinely tested in Europe for the
the unnecessary administration of
antepartum RhIG to the ap- proximately
40% of D-negative women who are carrying
a D-negative fetus. In the United States, due
to intellectual property ownership, testing of
cell-free fetal DNA is limited to wom- en
with active anti-D and is available only
commercially.
DNA-Based Testing for D in
Pregnant Women
Serologic typing for D cannot easily distin-
guish women whose red cells lack some epi-
topes of D (partial D) and are at risk for D
im- munization, from those with a weak D
phenotype who are not at risk for D immuni-
zation. Red cells with partial D type as D+,
some in direct tests and others by indirect
tests. These women might benefit from
receiv- ing RhIG prophylaxis if they deliver
a D+ fetus. RHD genotyping can distinguish
weak D from partial D to guide RhIG
prophylaxis and blood transfusion
recommendations.
DNA-Based Testing of Paternal Samples
The father’s red cells should be tested for the
antigen corresponding to the antibody in the
maternal plasma. If the red cells are
negative, the fetus is not at risk. If the father
is positive
 CH A P T E R 1 1 Blood Group Genetics ■ 285
format

for the antigen, zygosity testing can

determine whether the father is homozygous
or heterozy- gous for the gene encoding the
antigen, partic- ularly when there is no
allelic antigen or no an- tisera to detect the
allelic product.
Zygosity testing of paternal samples is
most often performed when testing for
possi- ble HDFN due to anti-D or anti-K. If
the pater- nal red cells are K+ and the
mother has anti-K, they can be tested
serologically for expression of the allelic k
antigen. However, many centers do not have
a licensed reagent available and genetic
counselors often request DNA testing. If the
paternal red cells are K–, the maternal anti-
K is mostly likely the result of immuniza-
tion through transfusion.
For maternal anti-D, DNA testing of
RHD
zygosity is the only method to determine the
paternal gene copy number. Several
different genetic events cause a D-negative
phenotype, and multiple assays must be
conducted to ac- curately determine RHD
zygosity, especially in minority ethnic
groups. If the father is RHD homozygous,
all of his children will be D+ and any
pregnancy in his partner needs to be mon-
itored. If the father is heterozygous, the
fetus has a 50% chance of being at risk. The
D type of the fetus should be determined to
prevent invasive and unnecessary testing so
the moth- er need not be aggressively
monitored or re- ceive immune-modulating
agents.

DNA-Based Testing for Antigen-

Negative Blood Donors
DNA-based typing to predict donor antigen
profiles in the search for antigen-negative
units is now a standard procedure for blood
centers, especially when suitable antibodies
are not available. Because red cell typing for
Dombrock antigens is notoriously difficult,
one of the most frequent approaches is to
type for Doa and Dob. Many other antibody
specific- ities are unavailable for mass donor
screening. These specificities include anti-
Hy, -Joa, -Jsa,
-Jsb, -CW, -V, and -VS. Even specificities
consid- ered to be common, such as anti-S and
anti- Fyb, are not always readily available.
DNA arrays can be used to screen for
mul- tiple minor antigens in a single assay
 Discrepancies between Serologic
(Phenotype) and DNA (Genotype)
and have the potential to be Testing
used for mass screening of
donors. This practice not only Differences between serologic and DNA
in- creases the antigen- test- ing results do occur and must be
negative inventory by ex- investigated. Often, these discrepancies lead
panding combinations of the to interesting discoveries such as the
minor antigens and some presence of a novel al- lele or genetic
high-prevalence antigens, but variant, particularly when peo- ple of
also allows consideration of diverse ethnicities are tested. Causes of
the possibility of pro- viding discrepancies include recent transfusions,
donor components that are stem cell transplantation, and natural chime-
DNA matched to the patient’s rism. Stem cell transplantation and natural
type. Although DNA methods
are not yet licensed by the
Food and Drug
Administration for the
labeling of donor units in the
United States, DNA testing is
a valuable screening tool and
only negative test results need
to be confirmed using a
licensed reagent when one is
available.

DNA-Based Testing to
Confirm D Type of
Donors
Donor centers must test
donors for weak D to avoid
labeling a product as D-
negative that might result in
anti-D in response to trans-
fused RBCs. Some donor red
cells with very weak D
expression (weak D type 2,
and espe- cially those with
the Del phenotype) are not
typed as D+ using current methods and are la-
beled as D–. The prevalence of
weak D red cells not detected
by serologic reagents is
approxi- mately 0.1% (but may
vary depending on the test
method and population).
Although the clinical
significance has not been
established, donor red cells
with weak D expression have
been associated with
alloimmunization. RHD
genotyping would improve
donor testing by confirming D–
phenotypes,56 but a high-
throughput and cost-effective
platform is not yet available.
286 ■ AABB T EC HNIC AL MANUAL

chimerism also may cause results of testing
DNA from somatic cells to differ from
results of testing DNA from extracted from
peripheral WBCs. Thus, when using DNA
testing, it is im- portant to obtain an accurate
medical history. Many genetic events can
cause apparent dis- crepant results between
hemagglutination and DNA test results and
the genotype does not al- ways predict the
phenotype (see Table 11-5
examples).3,5,27
Silenced or Nonexpressed Genes
DNA testing interrogates a single SNP or a
few SNPs associated with antigen
expression and cannot sample every
nucleotide in the gene. Although a gene may
be detected by DNA test- ing, there are
times when the gene product is
not expressed on the red cells because of a
mutation that silences the gene. Such
changes result in discrepancies in the typing
of patients and donors. Homozygosity (or
compound het- erozygosity) for a silenced
gene results in a null phenotype and most
null phenotypes have more than one
molecular basis.5
With donor typing, the presence of a
grossly normal gene whose product is not
for
ex- pressed on the red cell surface results in
the donor being falsely typed as antigen-
positive. Although this situation means loss
of an anti- gen-negative donor, it does not
jeopardize the safety of blood transfusion.
However, if a grossly normal gene is
detected in a patient but the gene is not
expressed, the patient could produce an
antibody if he or she re- ceives a transfusion
of antigen-positive blood.

TABLE 11-5. Examples of Some Molecular Events Where Analysis of Gene and Phenotype Do
Not Agree

Molecular Event Mechanism Observed Blood Group Phenotype

Transcription Nt change in GATA box Fy(b–)
Alternative splicing Nt change in splice site: Partial/ S–s–; Gy(a–)
complete skipping of exon
Deletion of nt(s) Dr(a–)
Premature stop codon Deletion of nt(s)  frameshift Fy(a–b–); D–; c−E−; Rhnull; Gy(a–);
GE:–2,–3,–4; K0; McLeod
Insertion of nt(s)  frameshift D–; Co(a–b–)
Nt change Fy(a–b–); r; Gy(a–); K0; McLeod
Amino acid change Missense nucleotide change D–; Rhnull; K0; McLeod
Reduced amount of Missense nucleotide change FyX; Co(a–b–)
protein
Hybrid genes Cross-over GP.Vw; GP.Hil; GP.TSEN
Har
Gene conversion GP.Mur; GP.Hop; D– –; R0
Interacting protein Absence of RhAG Rhnull
Absence of Kx Weak expression of Kell antigens
Absence of aas 75 to 99 of GPA Wr(b–)
Absence of protein 4.1 Weak expression of Ge antigens
Modifying gene In(Jk) Jk(a–b–)
Nt = nucleotide; aas = amino acids.
 CH A P T E R 1 1
To avoid misinterpretation, routine as-
says must include appropriate tests to detect
a change that silences gene expression if
preva- lent in the population tested. Silenced
alleles can be specific to a particular ethnic
group. For example, in the Duffy blood
group system, a single nucleotide change (–
67T>C) within the promoter region (GATA
box) of FY prevents transcription of FY*A
and/or FY*B in red cells but not in other
tissues. Although silencing of FY*A is rare,
silencing of FY*B is frequent in persons of
African ethnicity where homozy- gosity for
the –67T>C change in FY*B results in the
Fy(a–b–) phenotype, which has a preva-
lence of 60% or higher. To ensure accuracy,
testing for the GATA box mutation must be
in- cluded in typing for Duffy in persons of
African ethnicity.
When the assay is used to predict the
presence or absence of D antigen, particularly
in populations of African ancestry, it is essen-
tial to include a test for the complete but inac-
tive RHD pseudogene (RHD), which has a
37-bp sequence duplication. If the assay tests
for GYPB*S (S antigen), additional testing
should be performed to detect a C>T change at
nucleotide 230 in GYP*B exon 5 or a change
in intron 5 (+5g>t); both changes prevent
expres- sion of S antigen when testing persons
of Afri- can ancestry. Homozygosity, or
compound heterozygosity, for the 230T, or the
+5g>t change, results in the S–s–U+W
phenotype.
Other common causes of discrepancies
include the presence in the sample of an
altered FY*B allele that encodes an amino
acid change causing an FyX phenotype with
greatly reduced expression of Fyb antigen.
The red cells type as Fy(b–) with most
serologic re- agents. The prevalence of the
allele encoding
KEY POINTS
Genetics is the study of heredity, that is, the mechanisms by which a particular characteris- tic, such as a blood group, is pa
A gene is a segment of DNA and is the basic unit of inheritance; it occupies a specific loca-
tion on a chromosome (the gene locus). Alleles are alternative forms of a gene at the same gene locus (eg, alleles JK*A and
Blood Group Genetics ■ 287
the FyX phenotype in people of European an-
cestry is as high as 2%, and the allele has been
found in persons of African ancestry also. Si-
lencing mutations associated with the loss of
Kidd antigen expression occur more often in
people of Asian ancestry, whereas nucleotide
changes encoding amino acid changes that
weaken Kidd expression occur in people of
Af- rican ethnicity.
The routine detection of some blood
group polymorphisms by DNA analysis is
complex and not practical at this time. This
in- cludes situations where 1) a large
number of alleles encode one phenotype (eg,
ABO, Rh, and null phenotypes in many
blood group sys- tems), 2) a phenotype
results from alleles with a large deletion (eg,
GE:–2,3 and GE:–2,–3,–4), or 3) a phenotype
results from hybrid alleles (eg, in the Rh and
MNS systems). In addition, there is a high
probability that not all alleles in all ethnic
populations are known.
Blood group genomics has become an
es- sential component of the practice of
transfu- sion medicine.57 Genomics has
provided a greater understanding of genetic
blood group variants, including the
complexity of the molecular basis of Rh
variants, such as those that encode the Hr–
hrS– and hrB–HrB– phenotypes58,59 and the
associated partial Rh antigens that are a daily
challenge for the man- agement of patients
with SCD.50,51 RH genotyp- ing expands and
extends matching for Rh in this patient
population. High-throughput plat- forms
provide a means to test relatively large
numbers of donors, thereby opening up the
possibility of changing the way antigen-
nega- tive blood is provided to patients to
prevent immunization or to eliminate
transfusion re- actions for those who are
already immunized.
288 ■ AABB T EC HNIC AL MANUAL

3. A human somatic (body) cell is diploid, containing 46 chromosomes in 23 pairs: 22 pairs

are alike (homologous) in males and females and are termed autosomes. The remaining pair
is the sex chromosomes: X and Y for males, or two X chromosomes for females.
4. Somatic cells divide for growth and repair by mitosis. Mitosis replicates the
chromosomes and produces two identical nuclei in preparation for cell division. The
new cells are diploid, like the parent cell, and have all the genetic information of the
parent cell.
5. Meiosis is the process by which germ cells divide to become gametes (sperm and egg
cells); diploid cells undergo DNA replication and two divisions to form four gametes,
each of which is haploid and has half the chromosomal complement of the parent
somatic cell.
6. The term “genotype” traditionally refers to the complement of genes inherited by each
per- son from his or her parents; the term is also used to refer to the set of alleles at a
single gene locus. Whereas the genotype of a person is his or her genetic constitution,
the phenotype is the observable expression of the genes and reflects the biologic
activity of the gene(s). Thus, the presence or absence of antigens on the red cells, as
determined by serologic testing, rep- resents the phenotype.
7. When identical alleles for a given locus are present on both chromosomes, a person is
ho- mozygous for the particular allele, whereas when nonidentical alleles are present at
a par- ticular locus, the person is heterozygous. Antigens encoded by alleles at the same
locus are said to be antithetical. Thus, genes are allelic but not antithetical, whereas
antigens are anti- thetical but not allelic.
8. The expression of blood group antigens on the red cell may be modified or affected by gene
interaction. Homozygosity (or compound heterozygosity) for a silenced gene results in a
null phenotype and most null phenotypes have more than one molecular basis.
9. A blood group system consists of one or more antigens under the control of a single
gene lo- cus (eg, KEL encodes the Kell blood group antigens) or of two or more
homologous genes (eg, RHD and RHCE encode the Rh blood group antigens) so
closely linked that virtually no recombination occurs between them; thus, each blood
group system is genetically indepen- dent. Currently, 34 blood group systems are
recognized.
10. The genes encoding the 34 blood group systems have been sequenced and the
molecular bases of most antigens and phenotypes are known so that DNA-based
methods (genotyp- ing) can be used to predict a blood group phenotype.
11. DNA-based assays (blood group genotyping) have major applications in patient and
donor testing. They can be used to predict the red cell phenotype of a fetus, or of a
patient who was transfused, or when red cells are coated with IgG; they can be used to
resolve ABO and Rh discrepancies and to identify the molecular basis of unusual
serologic results. DNA analysis aids in distinguishing alloantibodies from
autoantibodies and is being applied to high- throughput screening of donors.

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 C h a p t e r 1 2

ABO, H, and Lewis Blood Groups

and Structurally Related Antigens

Laura Cooling, MD, MS

THE AN TIGENS OF the ABO, H, Lew-

is, I, and P blood group systems are de-
fined by small carbohydrate epitopes on glyco-
proteins and glycolipids. Because the epitopes
represent posttranslational modifications, the
synthesis of those antigens requires the action
of several enzymes known as “glycosyltrans-
ferases.” Glycosyltransferases reside in the
Golgi apparatus and are responsible for add-
ing specific sugars, in a specific sequence and
steric or anomeric linkage (-linked or -
linked), to growing oligosaccharide chains on
glycolipids and glycoproteins.
Transcriptional regulation coupled
with the enzymatic specificity of these
glycosyl- transferases for both sugar donors
[nucleotide sugars; eg, uridine diphosphate
(UDP)-galac- tose] and acceptor substrates
(eg, Type 1 chain vs Type 2 chain) is
responsible for the tissue- specific
distribution of many blood group an-
tigens.1,2 Because they are widely
distributed on many tissues, including
embryonic stem cells, such antigens are
considered to be histo- blood-group
antigens.2-5 Several studies have shown that
these antigens have a role in devel-
opment, cell adhesion, malignancy, and
infec- tious disease.1,6,7
ABO SYSTEM
The ABO system, initially described by Karl
Landsteiner in 1900, remains the most impor-
tant blood group system in transfusion and or-
gan transplantation medicine.7 In blood, ABO
antigens are found on red cells, platelets, and
many circulating proteins. As histo-blood-
group antigens, ABO antigens are also present
on many tissues, including those of the
endotheli- um, kidney, heart, bowel, pancreas,
and lung.2
Transfusion of ABO-incompatible
blood can be associated with acute
intravascular he- molysis, renal failure, and
death. Likewise, transplantation of ABO-
incompatible organs is associated with
acute humoral rejection. 7 Because of the
dire clinical consequences as- sociated with
ABO incompatibilities, ABO typ- ing and
ABO compatibility testing remain the
foundation of pretransfusion testing and an
important component of typing before trans-
plantation.

Laura Cooling, MD, MS, Associate Professor, Department of Pathology, and Associate Medical Director,
Trans- fusion Medicine, University of Michigan, Ann Arbor, Michigan
The author has disclosed no conflicts of interest.

291
292 ■ AABB T EC HNIC AL MANUAL
The ABO system contains four major
ABO phenotypes: A, B, O, and AB. The four
pheno- types are determined by the presence
or ab- sence of two antigens (A and B) on
red cells (see Table 12-1). The ABO system is
also char- acterized by the presence or absence
of natu- rally occurring antibodies, termed
“isohemag- glutinins,” directed against
missing A and B antigens. As shown in Table
12-1, an inverse reciprocal relationship
exists between the presence of A and/or B
antigens on red cells and the presence of anti-
A, anti-B, or both, in sera. For example, group
O individuals, who lack A and B antigens
on red cells, possess both anti-A and anti-B.
It is believed that the immunizing sources for
such naturally occur- ring antibodies are gut
and environmental bacteria, such as the
Enterobacteriaceae, which have been shown
to possess ABO-like struc- tures on their
lipo-polysaccharide coats.8,9
Biochemistry
The A and B antigens are defined by three
sug- ar terminal epitopes on glycolipids and
glyco- proteins.7 As shown in Fig 12-1, H
antigen is characterized by a terminal 12
fucose; it is the immediate biosynthetic
precursor for both A and B antigens and is
required for their ex- pression. In group A
individuals, an N-acetyl- galactosamine is
added, in an 13 linkage,
TABLE 12-1. Routine ABO Grouping
Reaction of Red Cells Reaction of Serum
with Antisera with Reagent Red
(Red Cell Grouping) Cells (Serum
Grouping)
Anti-A Anti-B A1 Cells B Cells
0 0 + +
+ 0 0 +
0 + + 0
+ + 0 0
0 0 + +
*H null phenotype (see section on H antigen).
+ = agglutination; 0 = no agglutination.
to the subterminal galactose of the H antigen
to form A antigen. In group B individuals, an
13 galactose is added to the same subter-
minal galactose to form B antigen. In group
AB individuals, both A and B structures are
syn- thesized. In group O individuals, neither
A nor B antigens are synthesized as a result of
a mu- tation in the ABO gene.7,10 As a
consequence, group O individuals express only
H antigen. A and B antigens are also absent in
the very rare Bombay phenotype because of
the absence of the H-antigen precursor (see
section below on the H system).
As terminal motifs, the A and B antigens
can be displayed on a number of oligosaccha-
ride scaffolds that differ in their size, composi-
tion, linkages, and tissue distribution. On red
cells, ABH sites are present on N-linked (65%-
75%) and O-linked (5%-15%) glycoproteins,
polyglycosylceramides (10%-15%), and sim-
pler glycosphingolipids (Fig 12-2). ABO anti-
gens are subclassified by the carbohydrate se-
quence immediately upstream of the ABO
motif. In humans, ABH is expressed predomi-
nantly on four different oligosaccharide back-
bones (see Table 12-2). On human red cells,
the majority of endogenous ABH antigen syn-
thesized is present on Type 2 chain structures.
The ability to synthesize and use
different carbohydrate chains is genetically
determined and can contribute to antigenic
differences in
Interpre- Prevalence (%) in
tation US Population
European African
O Cells ABO Group Ethnicity Ethnicity
0 O 45 49
0 A 40 27
0 B 11 20
0 AB 4 4
+ Bombay* Rare Rare
 CH APT E R 1 2
FIGURE 12-1. GalNAc added to the subterminal
Gal confers A activity to the sugar; Gal added
to the subterminal Gal confers B activity. Unless the
fucose moiety that determines H activity is
attached to the number 2 carbon, galactose does
not accept either sugar on the number 3
carbon.
weak ABO subgroups.10-12 For instance, Type 3
(repetitive A) and Type 4 (globo-A) A
antigens are present on A1, but not A2, red
cells. Differ- ences in cis carbohydrate
sequences upstream
of the terminal ABO motif can also influence
antibody reactivity.12 As an example, ABO
anti- gens on Type 1 chain substrates can be
recog- nized by antibodies directed against
both ABO and Leb antigen (see “Lewis
System” section below).10,13
ABO in Development and Aging
ABO antigens can be detected on red cells
of embryos as early as 5 to 6 weeks of
gestation.13 The quantity of ABO antigens on
umbilical cord red cells is less than that of
adults as the
ABO, H, and Lewis Blood Groups ■ 293
result of the immaturity of Type 2 chain pre-
cursors on cord red cells (see “I and i
Antigens” section below).14 With increasing
age, precur- sor chains become increasingly
branched, thereby allowing more A and B
antigen to be expressed. Adult levels of ABO
expression are generally present by age 2 to 4
years.13,14
Anti-A and anti-B are not present at
birth or, if present, are of maternal origin.
Endoge- nous synthesis of anti-A and anti-B
can devel- op as early as age 3 to 6 months,
with nearly all children displaying the
appropriate isohemag- glutinins in their sera
at 1 year of age.13,15 Titers of anti-A and anti-
B continue to increase dur- ing early
childhood and achieve adult levels within 5
to 10 years.
Among healthy adults, ABO titers can
nat- urally vary from 4 to 2048 or
higher.13,15,16 High- titer ABO antibodies can
be present in group O multiparous women
and in patients taking certain bacteria-
based nutritional supple- ments.7,9,13
Although older reports indicated a fall in
isoagglutinin titers in the elderly, more
recent studies have disputed these findings.15
In industrialized countries, isoagglutinin
ti- ters have generally decreased with
increasing consumption of processed
foods.16
Genetics
The ABO gene is located on chromosome
9q34 and is fairly large, consisting of seven
exons spread over 18 kb.7 The open reading
frame of the protein is located primarily in
exons 6 and
7. A study of the promoter region indicates
that ABO gene expression is transcriptionally
regulated by several mechanisms, including
methylation, tissue-specific transcription-
factor-binding proteins, antisense RNA, and,
possibly, a minisatellite enhancer region 4 kb
upstream of exon 1.7 ABO expression is also
regulated by the H gene, which is responsible
for the synthesis of H antigen substrate, the
precursor of A and B antigens. The H gene is
tightly regulated in a tissue-specific manner
through tissue-specific transcription factors
and promoters.17 In the absence of H, no A or
B antigen is expressed regardless of ABO
geno- type (Bombay or Oh phenotype).7,10
294 ■ AABB T EC HNIC AL MANUAL

FIGURE 12-2. Schematic representation of the red cell membrane showing antigen-bearing glycosylation of
proteins and lipids.
GPI = glycosylphosphatidylinositol. (Courtesy of ME Reid)

A series of elegant studies over the past

ids (A B; Gly235 S e r, Leu266Met, a
decade has identified the molecular basis for
n d Gly268Ala) are critical in determining
A, B, O, cis-AB, and weak ABO
whether the glycosyltransferase uses UDP-N-
subgroups.7,10,18 Fundamentally, three common
acetyl-D- galactosamine or UDP-D-galactose
ABO alleles are responsible for the A, B, and
donor sug- ars to synthesize A or B antigens,
O phenotypes. The A and B consensus alleles
respective- ly.7,10 The rare cis-AB phenotype
are autosomal codominant and differ by only
is a chimeric enzyme with a mix of A-specific
seven nucleo- tides and four amino acids.7,18
and B-specific amino acids at those or other
Three amino ac-
amino acid posi-

TABLE 12-2. Chain Variants of A Antigen in Humans

AntigenOligosaccharide Sequence*

A epitope GalNAc1-3(Fuc1-2)Gal1-R
Type 1 A GalNAc1-3(Fuc1-2)Gal1-3GlcNAc1-3-R
Type 2 A GalNAc1-3(Fuc1-2)Gal1-4GlcNAc1-3-R
Type 3 A (repetitive A)
GalNAc1-3(Fuc1-2)Gal1-3GalNAc1-3(Fuc1-2)Gal1-4GlcNAc1-3-R

Type 4 GalNAc1-3(Fuc1-2)Gal1-3GalNAc1-4Gal1-4Gal1-4Glc-Cer
A (globo-
A)
*Underlined sequences denote the critical differences between Type 1, 2, and 4 chains. Linkages and anomery (
or  linked) of the A antigen galactose are in bold. Bracketed sequences denote repetitive A antigen characteristic of
Type 3 chain A antigen.
Cer = ceramide; Fuc = fucose; Gal = galactose; GalNAc = N-acetyl-galactosamine; Glc = glucose; GlcNAc = N-acetyl-glucosa-
mine; R = upstream oligosaccharide.
 CH APT E R 1 2 ABO, H, and Lewis Blood Groups
tions.18 A plethora of mutations associated
with weak A and B subgroups has been de-
scribed. As an example, group A 2 (a weak A
subgroup) is commonly the result of a nucleo-
tide deletion and frameshift, resulting in an
enzyme with an additional 21 amino acids at
the C-terminus of the molecule.10,18
The O allele is an amorph, encoding a
nonfunctional enzyme. The group O pheno-
type, therefore, is an autosomal recessive trait,
representing inheritance of two nonfunction-
al ABO genes. Overall, more than 50 O alleles
have been identified. 7,18 The two most com-
mon O alleles (O01 and O02) contain a
nucleo- tide deletion and frameshift, leading to
a trun- cated, 117-amino-acid protein.
Another common O allele is O03 (or O 2 ), a
group of nondeletional O alleles that contains
a muta- tion at amino acid 268 (Gly268Arg),
which is a critical residue for donor binding
(UDP-galac- tose or UDP-N-
acetylgalactosamine). One German study
found that O03 and a related al- lele (Aw08)
were responsible for 25% of all ABO typing
discrepancies caused by reverse-group- ing
problems in healthy donors.19 It was specu-
lated that weak anti-A and anti-B could reflect
weak residual glycosyltransferase activity;
however, a later study was unable to demon-
strate A antigen or enzyme activity in
individu- als with the O03 allele.20
ABO Subgroups
ABO subgroups are phenotypes that differ in
the amount of A and B antigen carried on red
cells and present in secretions. In general, A
subgroups are more common than B sub-
groups. Clinically, the two most common sub-
groups encountered are A1 and A2. A1 repre-
sents the majority of group A donors (80%)
and is characterized by approximately 1 mil-
lion A antigen epitopes per red cell. A2 is the
second most common subgroup (20%) and
possesses only one fifth (2.2 × 105) the number
of A antigen sites as A 1. Both A1 and A2 are
strongly agglutinated by reagent anti-A in rou-
tine direct testing. A1 can be distinguished
from A2 by the lectin Dolichos biflorus, which
agglutinates A1 red cells but not A2 red cells. In
addition, 1% to 8% of individuals with A2 and
■ 295
22% to 35% of individuals with A2B possess
an alloanti-A1 in their sera. Because the A2
pheno- type reflects the inefficient conversion
of HA antigen, A2 red cells have increased
reactivity with the anti-H lectin Ulex
europaeus. Enzyme studies comparing A1 and
A2 glycosyltransfer- ase activity show that the
A1 enzyme is 5 to 10 times more active than
the A2 enzyme, result- ing in quantitative and
qualitative differences in A antigen
expression.7,10 The latter includes
the synthesis of unusual Type 3 and Type 4
chain A antigen on A1 red cells that is not pres-
ent on A2 or weaker A subgroups.10,11
In addition to A2, several weaker A sub-
groups have been described (eg, A3, Ax, Am,
and Ael). The extremely weak A (and B)
subgroups are infrequently encountered and
are usually
recognized by apparent discrepancies be-
tween red cell (forward) and serum
(reverse) grouping results. Most weak A
and B sub- groups were originally
described before the advent of
monoclonal typing reagents and were
based on reactivity with human poly-
clonal anti-A, anti-B, and anti-A,B
reagents. Weak A subgroups are frequently
nonreactive with human polyclonal anti-A
(see Table 12-3) and can show variable
reactivity with human polyclonal anti-A1,
anti-A,B, and murine monoclonal
antibodies (not shown).10,13,18 The degree of
reactivity with commercial murine
monoclonal reagents is clone dependent;
however, most commercially available anti-
A agglutinates A3 red cells. Because of the
recip-
rocal relationship between H and synthesis of
A and B antigens, nearly all weak A and B
sub- groups have higher H expression. 7 In
clinical practice, it is seldom necessary to
identify a patient’s specific A or B subgroup.
When performed, classification of weak
A subgroups is typically based on the
following:
1. Degree of red cell agglutination by anti-A
and anti-A1.
2. Degree of red cell agglutination by
human and some monoclonal anti-A,B.
3. Degree of H antigen (anti-H lectin and
Ulex europaeus) expression.
4. Presence or absence of anti-A1 (Method 2-
9).
5. Presence of A and H in saliva.
296 ■ AABB T EC HNIC AL MANUAL

TABLE 12-3. Serologic Reactions Observed in A and B Subgroups

Red Cell Reactions with Antisera orSerum

LectinsReactions with Reagent Red Cells

Red Cell Phenotype

Saliva Anti-AAnti-BAnti-A,BAnti-HA1 CellsB CellsO Cells(Secretors)

A1 4+ 0 4+ 0 0 4+ 0 A&H
A2 4+ 0 4+ 2+ 0/2+* 4+ 0 A&H
A3 2+ mf
0 2+ mf
3+ 0/2+* 4+ 0 A&H
Ax 0/ 0 1-2+ 4+ 0/2+ 4+ 0 H
Ael 0 0 0 4+ 0/2+ 4+ 0 H
B 0 4+ 4+ 0 4+ 0 0 B&H
B3 0 1+ mf
2+ mf
4+ 4+ 0 0 B&H
Bx 0 0/ 0/2+ 4+ 4+ 0 0 H
B(A) /2+ †
4+ 4+ 0 4+ 0 0 B&H
*The occurrence of anti-A1 is variable in these phenotypes.
†
Most often detected with anti-A clones containing the MHO4 clone.
1+ to 4+ = agglutination of increasing strength;  = weak agglutination; mf = mixed-field agglutination; 0 = no agglutination.

6. Adsorption and elution studies. addition to UDP-galactose, resulting in detect-

7. Family (pedigree) studies.
B(A) and A(B) Phenotypes
The B(A) phenotype is an autosomal dominant
phenotype characterized by weak A expression
on group B red cells.13,21 Serologically, red
cells from B(A)-phenotype individuals are
strongly reactive with anti-B and weakly
reactive with monoclonal anti-A (<2+), and
they possess a strong anti-A that is reactive
with both A1 and
A2 red cells in their sera. B(A) red cells can
show varying reactivity with monoclonal anti-
A reagents; however, most cases are detectable
with monoclonal typing reagents containing
the MHO4 clone. In general, the agglutination
is weak with fragile, easily dispersed aggluti-
nates. Testing the sample with polyclonal anti-
A or a different monoclonal anti-A should
resolve the discrepancy. Amino acid polymor-
phisms have been identified in some B(A) in-
dividuals near (Pro234Ala) or at (Ser235Gly)
critical amino acids.10,18 The B glycosyltransfer-
ase in these individuals has an increased ca-
pacity to use UDP-N-acetylgalactosamine in
able A antigen synthesis.
An A(B) phenotype has also been de-
scribed with monoclonal anti-B. The A(B)
phe- notype was associated with elevated H
antigen and plasma H-transferase activity.13 It
is hy- pothesized that the increased H
precursor on these cells may permit the
synthesis of some B antigen by the A
glycosyltransferase.
Acquired B Phenotype
The acquired B phenotype phenomenon is a
transient serologic discrepancy in group A
in- dividuals that is a cause of red cell
grouping discrepancies.22 Acquired B should
be suspect- ed when a patient or donor who
has historical- ly typed as group A now
presents with weak B expression on forward
or red cell grouping. Se- rologically, the
acquired B phenotype shows strong
agglutination with anti-A, shows weak
agglutination (2+ or less) with monoclonal
anti-B, and contains a strong anti-B in
serum. Despite reactivity of the patient’s red
cells with reagent anti-B, the patient’s serum
is not reac- tive with autologous red cells.
 CH APT E R 1 2 ABO, H, and Lewis Blood Groups
Chemically, acquired B is the result
of deacetylation of the A antigen’s N-
acetyl- galactosamine, yielding a B-like
galactosamine sugar.23,24 In patients’
samples, acquired B is often present in the
setting of infection by gas- trointestinal
bacteria. Many enteric bacteria possess a
deacetylase enzyme capable of con- verting
A antigen to a B-like analog.24 Identifi-
cation of the acquired B phenotype can also
be influenced by reagent pH and specific
mono- clonal anti-B typing reagents. 22 In
the past, anti-B reagents containing the ES-4
clone were associated with an increased
incidence of ac- quired B.
To resolve a patient’s true red cell
type and confirm the presence of acquired
B, red cells should be retyped using a
different monoclonal anti-B or acidified
(pH 6.0) hu- man anti-B. Acidified human
anti-B does not react with acquired B
antigen. The ability of monoclonal anti-B
to recognize acquired anti- B should be
noted in the manufacturer’s insert.
ABO Antibodies
Anti-A and Anti-B
Immune globulin M (IgM) is the predominant
isotype found in group A and group B individ-
uals, although small quantities of IgG antibody
can be detected. In group O serum, IgG is the
major isotype for anti-A and anti-B. As a con-
sequence, hemolytic disease of the fetus and
newborn (HDFN) is more common among the
offspring of group O mothers than of mothers
with other blood types because IgG can cross
the placenta but IgM cannot.
Both IgM and IgG anti-A and anti-B
pref- erentially agglutinate red cells at
room tem- perature (20 to 24 C) or cooler,
and both can efficiently activate
complement at 37 C. The complement-
mediated lytic capability of these
antibodies becomes apparent if serum
testing includes an incubation phase at 37
C. Hemolysis caused by ABO antibodies
should be suspected when either the
supernatant se- rum is pink to red or the cell
button is smaller or absent. Hemolysis must
be interpreted as a positive result. The use of
plasma for testing or of reagent red cells
suspended in solutions
■ 297
that contain EDTA prevents complement ac-
tivation and hemolysis.
Anti-A,B
Sera from group O individuals contain an anti-
body known as “anti-A,B” because it is
reactive with both A and B red cells. Such
anti-A and anti-B reactivity cannot be
separated by differ- ential adsorption,
suggesting that the anti- body recognizes a
common epitope shared by the A and B
antigens.7 Saliva containing secret- ed A or B
substance can inhibit the activity of anti-A,B
against both A and B red cells.
Anti-A1
Anti-A1 is present as an alloantibody in the se-
rum of 1% to 8% of A 2 individuals and 22% to
35% of A2B individuals. Anti-A1 is sometimes
present in the sera of individuals with other
weak A subgroups. Group O serum contains a
mixture of anti-A and anti-A1.24 Anti-A1 can
cause ABO discrepancies during routine test-
ing and lead to incompatible crossmatches
with A1 and A1B red cells. Anti-A1 is usually
of IgM isotype, reacting best at room tempera-
ture or below, and is usually considered clini-
cally insignificant. Anti-A1 is considered
clini-
cally significant if reactivity is observed at
37 C.24 Group A2 patients with an anti-A1 that
is reactive at 37 C testing should be transfused
with group O or A2 red cells only; group A2B
pa- tients should receive group O, A2, A2B, or
B red cells.
Routine Testing for ABO
Donor blood samples are routinely typed for
ABO at the time of donation and on receipt of
Red Blood Cell (RBC) units in the hospital
transfusion service (confirmatory typing). Re-
cipient samples are typed before transfusion.
ABO grouping requires both antigen typing of
red cells for A and B antigen (red cell
grouping or forward type) and screening of
serum or plasma for the presence of anti-A and
anti-B isoagglutinins (serum grouping or
reverse type). Both red cell and serum or
plasma grouping are required for donors and
patients because each grouping serves as a
check on
298 ■ AABB T EC HNIC AL MANUAL
the other. Reverse or serum grouping is not
re- quired in two circumstances: 1) for
confirma- tion testing of labeled, previously
typed donor red cells and 2) in infants
younger than 4 months of age. As
previously discussed, isoag- glutinins are
not present at birth and develop only after 3
to 6 months of age.
Commercially available anti-A and anti-B
for red cell typing are extremely potent and ag-
glutinate most antigen-positive red cells di-
rectly, even without centrifugation. Most
monoclonal typing reagents have been formu-
lated to detect many weak ABO subgroups
(see manufacturers’ inserts for specific
reagent characteristics). Additional reagents
(anti-A1 and anti-A,B) and special techniques
to detect weak ABO subgroups are not
necessary for routine testing but are helpful
for resolving ABO-typing discrepancies.
In contrast to commercial ABO typing
re- agents, human anti-A and anti-B in the
sera of patients and donors can be relatively
weak, re- quiring incubation and
centrifugation. Tests for serum grouping,
therefore, should be per- formed using a
method that adequately de- tects human
anti-A and anti-B. Several meth- ods are
available for determining ABO group,
including slide, tube, microplate, and
column agglutination techniques.
ABO Discrepancies
Table 12-1 shows the results and interpreta-
tions of routine red cell and serum tests for
ABO. A discrepancy exists when the results of
red cell tests do not agree with those of serum
tests, usually due to unexpected negative or
positive results in either the forward or reverse
typing (see Table 12-3). ABO discrepancies
may arise from intrinsic problems with either
red cells or serum or from technical errors in
performing the test (see Table 12-4 and
section on Resolving ABO Discrepancies).
When a discrepancy is encountered,
the discrepant results must be recorded, but
inter- pretation of the ABO group must be
delayed until the discrepancy has been
resolved. If the specimen is from a donor
unit, the unit must be quarantined and
cannot be released for transfusion. When
an ABO discrepancy is
identified in a patient, it may be necessary
to transfuse group O red cells pending an
investi- gation. It is important to obtain a
sufficient pretransfusion blood sample from
the patient to complete any additional
studies that may be required.
Red Cell Testing Problems
ABO testing of red cells may give unexpected
results for many reasons, including the follow-
ing:
1. Weak ABO expression that results from
inheritance of a weak ABO subgroup.
Some patients with leukemia and other
malignancies can also show weakened
ABO expression.25
2. Mixed-field agglutination with circulating
red cells of more than one ABO group
fol- lowing out-of-group red cell
transfusion or hematopoietic progenitor
cell (HPC) transplantation (eg, group O
to group A). Mixed-field agglutination
is also present in some ABO subgroups
(A3), blood group chimerism in
fraternal twins, and very rare cases of
mosaicism arising from dispermy.
3. Neutralization of anti-A and anti-B typing
reagents by high concentrations of A or
B blood group substance in serum,
result- ing in unexpected negative
reactions with serum- or plasma-
suspended red cells.
4. Spontaneous agglutination or autoagglu-
tination of serum- or plasma-suspended
red cells caused by heavy coating of red
cells by potent autoagglutinins.
5. Nonspecific aggregation of serum- or
plasma-suspended red cells caused by
abnormal concentrations of serum pro-
teins or infused macromolecular solu-
tions.
6. False-positive reactions caused by a pH-
dependent autoantibody, a reagent-
dependent antibody (eg, EDTA or para-
ben), or rouleaux.
7. Anomalous red cell grouping resulting
from acquired B, B(A), or A(B) pheno-
types.
 CH APT E R 1 2 ABO, H, and Lewis Blood Groups ■ 299

TABLE 12-4. Possible Causes of ABO Typing Discrepancies

CategoryCauses

Weak/missing red cell reactivity ABO subgroup

Leukemia/malignancy

Transfusion

Intrauterine fetal transfusion

Transplantation

Excessive soluble blood group substance

Extra red cell reactivity Autoagglutinins/excess protein coating red cells

Unwashed red cells: plasma proteins

Unwashed red cells: antibody in patient’s serum to reagent constituent

Transplantation

Acquired B antigen

B(A) phenomenon

Out-of-group transfusion

Mixed-field red cell reactivity Recent transfusion

Transplantation

Fetomaternal hemorrhage

Twin or dispermic (tetragametic) chimerism

Weak/missing serum reactivity Age related (<4-6 months old, elderly)

ABO subgroup

Hypogammaglobulinemia

Transplantation

Extra serum reactivity Cold

autoantibody

Cold alloantibody

Serum antibody to reagent constituent

Excess serum protein

Transfusion of plasma components

Transplantation

Infusion of intravenous immune globulin

300 ■ AABB T EC HNIC AL MANUAL
8. Polyagglutination (eg, T activation)
resulting from inherited or acquired
abnormalities of the red cell membrane,
with exposure of “cryptic autoantigens.”24
Because all human sera contain naturally
occurring antibodies to such cryptic anti-
gens, those abnormal red cells are agglu-
tinated by sera from group A, B, and AB
individuals. Monoclonal anti-A and anti-
B reagents do not detect polyagglutina-
tion.
Problems with Serum or Plasma Testing
Problems may arise during ABO testing of se-
rum or plasma, including the following:
1. Small fibrin clots in plasma or incom-
pletely clotted serum that can be mis-
taken for red cell agglutinates.
2. Lack of detectable isoagglutinins in
infants younger than 4 to 6 months. Chil-
dren do not develop isoagglutinins until 3
to 6 months of age. Isoagglutinins present
at birth are passively acquired from the
mother.
3. Unexpected absence of ABO agglutinins
caused by a weak A or B subgroup (see
Table 12-3).
4. Unexpected absence in children of anti-
B 1. Specimen mix up.
agglutinins that are sterile, free of
bacte- ria, and result from long-term
parenteral and enteral nutrition.26
5. Unexpected absence of anti-A agglutinins
in patients receiving equine-derived
immunoglobulins.27
6. ABO-incompatible HPC transplantation
with induction of tolerance. For example,
a group A patient receiving a group O
marrow transplant will have circulating
group O red cells but will produce only
anti-B in serum. (Refer to Chapter 25 for
more information on ABO-mismatched
transplants.)
7. Severe hypogammaglobulinemia second-
ary to inherited immunodeficiency or
dis- ease therapy.
Hypogammaglobulinemia with dilution
of isoagglutinins can also
occur after several courses of plasma
exchange with albumin replacement.
8. Cold alloantibodies (eg, anti-M) or auto-
antibodies (eg, anti-I) reactive with
reverse-grouping cells, leading to unex-
pected positive reactions.
9. Antibodies directed against constituents
in the diluents used to preserve reagent
A1 and B red cells.24
10. Nonspecific aggregation or
agglutination caused by high-
molecular-weight plasma expanders,
rouleaux, high serum-protein
concentrations, or altered serum-protein
ratios.
11. Recent transfusion of out-of-group
plasma-containing components (eg, a
group A patient transfused with
platelets from a group O donor, causing
unex- pected anti-A in the patient’s
plasma).
12. Recent infusion of intravenous
immuno- globulin, which can contain
ABO isoag- glutinins.
Technical Errors
Technical problems with a sample or during
testing can also lead to problems in ABO
grouping, including:
2. Too heavy or too light red cell suspen-
sions.
3. Failure to add reagents.
4. Missed observation of hemolysis.
5. Failure to follow the manufacturer’s
instructions.
6. Under- or overcentrifugation of tests.
7. Incorrect interpretation or recording of
test results.
Resolving ABO Discrepancies
The first step in resolving an apparent
serolog- ic testing discrepancy should be to
repeat the test with the same sample to
exclude the pos- sibility of a technical error
during testing. Ad- ditional studies may
include testing washed red cells; testing a
new sample; testing for un- expected red
cell alloantibodies; and reviewing
 CH APT E R 1 2 ABO, H, and Lewis Blood Groups ■ 301
false-positive reac- tions in tests with anti-A and anti-B.
Usually,
the patient’s medical record for conditions,
medications, or recent transfusions that may
have contributed to the conflicting test
results (Method 2-4). Samples with apparent
weak or missing ABO antigens and/or
antibodies may require tests using methods
that enhance antigen-antibody binding,
including incubat- ing red cells at 4 C
(Method 2-5), using enzyme-treated red
cells (Method 2-6), and conducting
adsorption and elution studies (Method 2-7).
In some instances, it may be necessary to
test for the secretion of ABO anti- gens in
saliva (Method 2-8). Patients with sus-
pected B(A), acquired B, or A(B)
phenotypes should be retested using
different monoclonal and human polyclonal
reagents.
ABO discrepancies caused by
unexpected serum reactions are not
uncommon. Com- monly encountered
causes of serum-group- ing discrepancies
include cold autoantibodies, rouleaux, cold-
reacting alloantibodies (eg, anti-M), and
weak A subgroups with an anti- A1. To
resolve an ABO discrepancy caused by an
anti-A1 in a group A individual, red cells
should be tested with Dolichos biflorus
lectin, which agglutinates group A1 but not
A2 and
weaker A subgroups. The presence of an
anti-
A1 should be confirmed by testing serum
against A1, A2, and O red cells (Method 2-9).
Reverse-grouping problems resulting from
either a cold alloantibody (Method 2-10) or
autoantibody can be identified with a
room- temperature antibody detection test
and a di- rect antiglobulin test. Techniques
to identify ABO antibodies in the presence
of cold auto- antibodies include testing at 37
C without cen- trifugation (Method 2-11)
and cold autoad- sorption (Method 4-5).
Serum or plasma properties can induce
rouleaux formation that resembles
agglutination with A1 and B red cells.
Saline replacement or saline dilution
(Method 3-7) can be used to distinguish
rou- leaux from agglutination and identify
ABO an- tibodies.
Cold autoantibodies can cause autoag-
glutination of red cells and unexpected
reac- tions during red cell typing. Red cells
heavily coated with autoantibodies can
spontaneous- ly agglutinate and cause
 enzymes are capable of synthesizing H
antigen: FUT1 (H gene) and FUT2 (secretor
false-positive reactions gene). FUT1 specifically fu- cosylates Type 2
caused by autoanti- bodies chain oligosaccharides on red cell
can be eliminated by washing glycoproteins and glycolipids to form Type 2
red cells with warm saline chain H. In contrast, FUT2 or secretor recog-
(Method 2-17). Autoaggluti- nizes Type 1 chain precursors to form Type 1
nation caused by IgM can also chain H and Leb antigens in secretions (Fig 12-
be inhibited or dispersed by 3).10 Secretion of Type 1 chain ABH antigens
incubating red cells in the in saliva and other fluids requires a functional
pres- ence of either FUT2 gene. FUT2 is not expressed in red cells
dithiothreitol or 2-aminoethyl- but is expressed in salivary glands, gastrointes-
isothiouronium bromide tinal tissues, and genitourinary tissues.1,7,10
(Method 3-16). These
reagents reduce the disulfide
bonds on IgM molecules,
decreasing their polyvalency
and ability to directly
agglutinate red cells.

THE H SYSTEM
H antigen is ubiquitously
expressed on all red cells
except the rare Bombay
phenotype. Be- cause H antigen
serves as the precursor to both
A and B antigens, the amount
of H anti- gen on red cells
depends on an individual’s
ABO type. H antigen is highly
expressed on group O red cells
because group O individuals
lack a functional ABO gene. In
group A and B individuals, the
amount of H antigen is con-
siderably less because H is
converted to the A and B
antigens, respectively. The
amount of H antigen on red
cells, based on agglutination
with the anti-H lectin Ulex
europaeus, is repre- sented
thus: O>A2>B>A1>A1B. H
antigen is
present on HPCs, red cells, megakaryocytes,
and other tissues.2,3,28 H
antigen has been im- plicated
in cell adhesion, normal
hematopoi- etic
differentiation, and several
malignan- cies.6,7,29

Biochemistry and Genetics

H antigen is defined by the
terminal disaccha- ride fucose
12 galactose. Two different
fu- cosyltransferase (FUT)
 302
■
AABB T ECHNICAL M A NUAL

FIGURE 12-3. Synthesis of Type 1 chain ABH and Lewis antigens by Secretor (FUT2), Lewis (FUT3), and ABO enzymes. Type 2 chain ABH antigen synthesis is also
shown for comparison.
Fuc = fucose; Gal = galactose; GalNAc = N-acetylgalactosamine; GlcNAc = N-acetylglucosamine; R = other upstream carbohydrate sequence. Reproduced with
permission from Cooling.30
 CH APT E R 1 2 ABO, H, and Lewis Blood Groups
Type 1 chain ABH antigen present on red cells
is passively adsorbed from circulating glyco-
lipid antigen present in plasma (see “Lewis
System” section).24
Null Phenotypes
Bombay (Oh) Phenotype
Originally described in Bombay, India, the
Oh or Bombay phenotype is a rare,
autosomal re- cessive phenotype
characterized by the ab- sence of H, A, and
B antigens on red cells and
in secretions. Genetically, Oh individuals are
homozygous for nonfunctional H (hh) and
se- cretor (sese) genes, resulting in a
complete ab- sence of Type 1 and Type 2
chain H, A, and B.
Oh red cells type as H negative with the anti-H
lectin Ulex europaeus and monoclonal anti-H.
Because these individuals lack a functional
secretor gene necessary for Leb synthesis, Oh
individuals also type as Le(b–) (see “Lewis
System” section). Genotyping studies have de-
scribed a wide range of inactivating mutations
in both the H and secretor genes in Oh
individ- uals.10,18 The Oh phenotype is also
present in leukocyte adhesion deficiency 2
(LAD2) due to a mutation in the GDP-fucose
transporter gene.25
Because they lack all ABH antigens, Oh
in- dividuals possess natural
isohemagglutinins to A, B, and H (see
Table 12-1). In routine ABO typing, these
individuals initially type as group
O. The Oh phenotype becomes apparent dur-
ing antibody-detection tests with group O
red cells, which are rich in H antigen. The
anti-H present in Oh individuals strongly
agglutinates all group O red cells and
sometimes demon- strates in-vivo
hemolysis. The Oh phenotype can be
confirmed by demonstrating an ab- sence
of H antigen on red cells and the pres- ence
of an anti-H in serum that is reactive with
group O red cells but not with Oh red cells
from other individuals.
Para-Bombay Phenotype
Individuals with the para-Bombay
phenotype are H-deficient secretors.7,10
Genetically, these individuals are
homozygous for a nonfunc- tional H gene
(hh), but they have inherited at
■ 303
least one functional secretor gene (Se). The red
cells from H-deficient secretors lack serologi-
cally detectable H antigen but can carry small
amounts of A and/or B antigen because, unlike
persons with classic Bombay phenotype, para-
Bombay persons express Type 1 chain ABH
an- tigens in their secretions and plasma
(Method 2-8).24 Type 1 chain A or B antigen in
plasma is then passively adsorbed onto red
cells, result- ing in weak A or B antigen
expression. Para- Bombay can also occur in
group O individuals, as evidenced by trace
Type 1 chain H on their red cells and in their
secretions. Red cells from
para-Bombay individuals are designated “A h,”
“Bh,” and “ABh.”
In laboratory testing, red cells from para-
Bombay individuals may (or may not) have
weak reactions with anti-A and anti-B re-
agents. In some cases, A and B antigens may
be detected only after adsorption and elution.
Para-Bombay red cells are nonreactive with
anti-H lectin, monoclonal anti-H, and human
anti-H from Oh individuals. The sera of para-
Bombay individuals contain anti-H, anti-HI,
or both and, depending on their ABO type,
anti-A and anti-B.13,24
Anti-H
Alloanti-H (Bombay and Para-Bombay)
The anti-H in Bombay and para-Bombay
phe- notypes is clinically significant and
associated with acute hemolytic transfusion
reactions. The antibody is predominantly of
IgM isotype and exhibits a broad thermal
range (4 to 37 C)
with all red cells except O h red cells. As with
anti-A and anti-B, alloanti-H is capable of
acti- vating complement and causing red cell
he- molysis.
Autoanti-H and Autoanti-HI
Autoantibodies to H and HI antigens can be
encountered in healthy individuals. When
present, these autoantibodies are most com-
mon in A1 individuals, who have very little
H
antigen on their red cells. Autoanti-H and
autoanti-HI are usually of IgM isotype and
are reactive at room temperature.
304 ■ AABB T EC HNIC AL MANUAL
Transfusion Practice
Alloanti-H is highly clinically significant and
is capable of fixing complement and causing
he- molytic transfusion reactions. As a result,
pa- tients with alloanti-H caused by a Bombay
or para-Bombay phenotype must be transfused
with H-negative (Oh) RBCs.
In contrast, autoantibodies against H
and
HI are generally clinically insignificant. In
most patients, transfused group-specific or
group O RBCs should have normal in-vivo
sur- vival. Occasionally, autoanti-HI can
result in decreased red cell survival and
hemolytic transfusion reactions after
transfusion of group O RBCs.13,24 In most
instances, hemoly- sis follows transfusion of
group O RBCs to a
group A1 or B patient with an unusually potent
high-titer anti-HI that is reactive at 37 C. 24 In
such patients, it may be advisable to transfuse
group-specific (A1, B, or AB) RBCs.
THE LEWIS SYSTEM
The Lewis blood group system consists of two
main antigens, Lea (LE1) and Leb (LE2), and
three common phenotypes, Le(a+b–), Le(a–
b+), and Le(a–b–). Four additional Lewis anti-
gens represent composite reactivity between
Lea, Leb, and ABO antigens: Leab (LE3), LebH
(LE4), ALeb (LE5), and BLeb (LE6).18,25 In
addi-
tion to being present on red cells, Lewis
anti- gens are widely expressed on platelets,
endothelium, and the kidney as well as on
genitourinary and gastrointestinal
epithelium. Lewis antigens are not
synthesized by red cells but are passively
adsorbed onto red cell membranes from a
pool of soluble Lewis gly- colipid present in
plasma.25 The gastrointesti- nal tract, which
is rich in Lewis-active glyco- lipid and
glycoprotein, is thought to be the primary
source of Lewis glycolipid in plasma.
Because Lewis antigens are passively adsorbed
onto red cell membranes, they can be
eluted from red cells after transfusion or by
increases in plasma volume and increased
circulating li- poproteins, which also adsorb
Lewis glycolip- id. For example, Lewis
antigen is often de- creased on red cells
during pregnancy, with some women
transiently typing as Le(a–b–).
The latter is attributed to an increase in
circu- lating plasma volume and a fourfold
increase in lipoprotein.24 Leb expression and
immuno- reactivity are also influenced by
ABH type as a result of the synthesis of
hybrid structures with both Lewis and ABH
activity (Fig 12-3).2,10,28
Biochemistry and Synthesis
Lewis antigen synthesis depends on the inter-
action of two distinct fucosyltransferases (Fig
12-3): Lewis (FUT3) and secretor (FUT2).25,30
Unlike H or FUT1, which is relatively specific
for Type 2 chain substrates, Lewis and secretor
preferentially fucosylate Type 1 chain sub-
strates. Secretor (FUT2) can add a terminal
12 fucose to a Type 1 chain precursor to
form Type 1 chain H antigen. The Lewis gene
encodes an 13/4 fucosyltransferase that
transfers a fucose, in an 14 linkage, to the
penultimate N-acetyl-glucosamine of Type 1
chain precursor (“Lewis c”) to form Le a anti-
gen. Lewis (FUT3) can also add a second fu-
cose to Type 1 H antigen to form Le b antigen.
Note that Leb cannot be formed from Lea be-
cause the presence of a subterminal fucose on
Lea sterically inhibits binding by the secretor
enzyme.1
In individuals with both Lewis and secre-
tor enzymes, Type 1 chain H is favored over
Lea synthesis. As a result, most of the Lewis
anti- gen synthesized is Leb [Le(a–b+)
phenotype]. In group A1 and B individuals,
Leb and Type 1 chain H can be further
modified by ABO glyco- syltransferases to
form LE5, LE6, Type 1 A, and Type 1 B
antigens.2,30 In group A1 individuals, the
majority of Lewis antigen in plasma is ac-
tually ALeb.31
Genetics and Lewis Phenotypes
The three Lewis phenotypes commonly en-
countered represent the presence or absence
of Lewis and secretor enzymes (see Table
12- 5). Le(a+b–) individuals have inherited
at least one functional Lewis gene (Le) but
are homo- zygous for nonfunctional secretor
alleles (se- se). As a result, such individuals
synthesize and secrete Lea antigen but lack
Leb and Type 1 chain ABH antigens.
 CH APT E R 1 2 ABO, H, and Lewis Blood Groups ■ 305

The Le(a–b+) phenotype reflects Lewis Expression in Children

inheri- tance of both Le and Se alleles,
leading to the synthesis of Lea, Leb, and
Type 1 chain ABH. Because most Type 1
chain precursor is con- verted to Leb in
those individuals, they appear to type as
Le(a–). An Le(a+b+) phenotype is
transiently observed in infants because
secre- tor activity increases with
developmental age. An Le(a+b+) phenotype
is also present in 16% of Japanese
individuals as a result of inheri- tance of a
weak secretor gene (Sew).18 In the ab- sence
of a functional Lewis gene (lele), neither Lea
nor Leb is synthesized, leading to an Le(a–
b–) or null phenotype. Type 1 chain ABH
anti- gens may still be synthesized and
secreted in individuals who have inherited
at least one functional Se allele (Method 2-
8). The Le(a– b–) phenotype is significantly
more common in persons of African
ethnicity. Although rare, an Le(a-b-)
phenotype is also present in indi- viduals
with LAD2 due to defects in fucose
transport.25
Several inactivating mutations have
been
identified in both Lewis and secretor genes. 18
Many of the mutations are geographically and
racially distributed, with many populations
displaying a few predominant alleles. Of the
nonfunctional Le alleles described, most have
more than one mutation.10,18
Table 12-5 shows the distribution of Lewis
types in adults. In contrast, most newborns
type as Le(a–b–) with human anti-Lewis
typing reagents. Approximately 50% of
newborns subsequently type as Le(a+) after
ficin treat- ment. The prevalence of Le b
antigen, however, is low in newborns
compared to in adults be- cause of
developmental delays in secretor (FUT2)
activity. An Le(a+b+) phenotype can be
transiently present in children as the level of
secretor activity approaches adult levels. A
val- id Lewis phenotype is not developed until
age 5 or 6.13
Lewis Antibodies
Antibodies against Lewis antigens are general-
ly of IgM isotype and occur naturally. Clinical-
ly, Lewis antibodies are most often encoun-
tered in the sera of Le(a–b–) individuals and
may contain a mixture of anti-Le a, anti-Leb,
and anti-Leab, an antibody capable of recog-
nizing both Le(a+) and Le(b+) red cells. Be-
cause small amounts of Lea are synthesized in
the Le(a–b+) phenotype, Le(a–b+) individuals
do not make anti-Lea. Anti-Leb is present infre-
quently in the Le(a+b–) phenotype. Lewis anti-
bodies, accompanied by a transient Le(a–b–)

TABLE 12-5. Adult Phenotypes and Prevalence in the Lewis System

Red Cell Reactions Prevalence (%) Genotype*

Anti-Lea Anti-Leb Phenotype Whites Blacks Lewis Secretor Saliva†

+ 0 Le(a+b–) 22 23 Le sese Lea

0 + Le(a–b+) 72 55 Le Se Lea, Leb, ABH
0 0 Le(a–b–) 6 22 lele sese Type 1 precursor
lele Se Type 1 ABH
+ + Le(a+b+)‡ Rare Rare Le Sew Lea, Leb
*Probable genotype at the Lewis (FUT3 ) and Secretor (FUT2 ) loci.
†
Type 1 chain antigens present in saliva and other secretions.
‡
Le(a+b+) is present in 16% of Japanese individuals and is also transiently observed in infants.
Le = gene encoding functional Lewis enzyme; lele = homozygous for gene encoding an inactive enzyme; Se = gene
encoding sactive secretor enzyme; sese = homozygous for gene encoding an inactive enzyme; Se w = gene encoding
weak secretor enzyme.
306 ■ AABB T EC HNIC AL MANUAL
phenotype, are present during pregnancy. Fi-
nally, anti-Leb can demonstrate ABO specifici-
ty (anti-LebH, anti-ALeb, and anti-BLeb), and is
preferentially reactive with Le(b+) red cells of
a specific ABO group.24,28 Anti-LebH, the most
common reactivity, is more strongly reactive
with Le(b+) group O and A2 red cells than with
group A1 and B red cells, which have low H
an-
tigen levels. Anti-LebL is strongly reactive with
all Le(b+) red cells, regardless of ABO group.
Most examples of Lewis antibodies are
sa- line agglutinins that are reactive at room
tem- perature. Unlike ABO, the
agglutination is rela- tively fragile and
easily dispersed, requiring gentle
resuspension after centrifugation. Ag-
glutination is sometimes observed after 37
C incubation, but the reaction is typically
weaker than that at room temperature. On
occasion, Lewis antibodies can be detected
in the anti- human globulin (AHG) phase.
Such detection may reflect either IgG or
bound complement (if polyspecific AHG
reagent is used). Very rarely, Lewis
antibodies can cause in-vitro he- molysis.
Hemolysis occurs more often when fresh
serum containing anti-Lea or anti-Leab is
tested, particularly against enzyme-treated
red cells.
Transfusion Practice
In general, Lewis antibodies are not consid-
ered clinically significant. Red cells that are
compatible in tests at 37 C, regardless of
Lewis phenotype, are expected to have
normal in- vivo survival. It is not necessary
to transfuse antigen-negative RBCs in most
patients. Un- like ABO antigens, Lewis
antigens are extrinsic glycolipid antigens that
are readily eluted and shed from transfused
red cells within a few days of transfusion.25
Furthermore, Lewis anti- gens in transfused
plasma can neutralize Lew- is antibodies in
the recipient. For these rea- sons, hemolysis
is rare following transfusion of either Le(a+)
or Le(b+) red cells.
Lewis antibodies are not a cause of
HD- FN.13 Lewis antibodies are
predominantly of IgM isotype and do not
cross the placenta. In addition, Lewis
antigens are poorly expressed on neonatal
red cells, with many newborns
typing as Le(a–b–) with human Lewis antibod-
ies (see above).
Disease Associations
The Leb and H antigens are receptors for
Heli- cobacter pylori, a gram-negative
bacterium implicated in gastritis, peptic
ulcer disease, gastric carcinoma, mucosa-
associated lym- phoma, and idiopathic
thrombocytopenia. Leb and Type 1 H
antigens are also receptors for noroviruses,
common causes of acute gastro- enteritis.
An Le(a–b–) phenotype is associated with
increased susceptibility to infections by
Candida and uropathogenic Escherichia
coli, cardiovascular disease, and possibly
de- creased graft survival after renal
transplanta- tion.6,25
I AND i ANTIGENS
The I and i antigens are ubiquitous, structural-
ly related antigens present on all cell mem-
branes. The minimum epitope common to
both i and I is a terminal repeating lactos-
amine (Gal14GlcNAc) or Type 2 chain
pre- cursor. The minimum i antigen is a linear,
nonbranched structure containing at least two
successive lactosamine structures.14 The I anti-
gen is a polyvalent, branched glycan derived
from the i antigen (Fig 12-4). Both i and I
serve as substrate and scaffold for the
synthesis of ABO, Lewis X [Gal1-4(Fuc1-
3) GlcNAc], and other Type 2 chain
antigens.1,2,14 On red cells, i and I antigens are
present on N-linked glycoproteins and
glycolipids.
Phenotypes
Two phenotypes are recognized according to
the presence or absence of I antigen: I and i
(I–). The i phenotype is characteristic of
neonatal red cells, whereas I+ is the common
pheno- type in adults. With increasing age,
there is a gradual increase in I antigen
accompanied by a reciprocal decrease in i
antigen; most chil- dren develop an adult I+
phenotype by age 2.14 An increase in i antigen
can occur in people with chronic hemolytic
disorders and is a sign of stressed
erythropoiesis.32
 CH APT E R 1 2 ABO, H, and Lewis Blood Groups ■ 307

FIGURE 12-4. Structure of I gene (GCNT2) (above) and synthesis of I antigen from i antigen
(below). Gal = galactose; GlcNAc = N-acetylglucosamine; R = other upstream carbohydrate
sequence.

Two genetic disorders are associated ed with abnormal glycosylation, chronic he-
with an increase in i antigen.14 The iadult molysis, splenomegaly, and erythroid multi-
phenotype (I–i+) is an autosomal recessive nuclearity.
phenotype caused by mutations in the I
gene (GCNT2 or Genetics
IGnT). In populations of Asian ancestry,
the iadult phenotype can be associated with The I gene (GCNT2) encodes a 16 N-
acetyl- glucosaminyltransferase that converts
con- genital cataracts. Increased i antigen
the lin- ear i antigen into the branched I
levels are also present in people with
antigen.14,18 The gene resides on chromosome
congenital dys-
6p24 and contains five exons, including three
erythropoietic anemia Type 2 (also known
tissue- specific exons (exons 1A, 1B, and 1C).
as hereditary erythroblastic multinuclearity
As a re- sult, three slightly different mRNA
with positive acidified serum lysis test). The
transcripts
latter is a genetic defect in Golgi transport
associat-
308 ■ AABB T EC HNIC AL MANUAL
can be synthesized, depending on which
exon 1 is used.
In the iadult phenotype without cataracts,
there is a mutation in exon 1C that is
specific for I antigen synthesis in red cells.
As a conse-
quence, I antigen is missing on red cells but
is still synthesized in other tissues that use
either exon 1A or exon 1B. In the iadult
phenotype with cataracts, there is a loss of I
antigen synthesis in all tissues caused by
either gene deletion or mutations in exons 2
and 3.
Antibodies
Anti-I
Anti-I is common in the serum of healthy
indi- viduals. Anti-I is usually of IgM
isotype and is strongly reactive at 4 C with
titers of <64. Sam- ples with higher titers
may also be detectable at room temperature.
Anti-I is identified by strong reactions with
adult red cells but weak or no agglutination
with cord red cells (see Ta- ble 12-6). Anti-I
can be enhanced by 4 C incu- bation, the
presence of albumin, or use of enzyme-
treated red cells. An alloanti-I can be seen in
the iadult phenotype.
Some examples of anti-I can demon-
strate complex reactivity and are more strong-
ly reactive with red cells of specific ABO, P1,
or Lewis phenotypes. Many of those
antibodies appear to recognize branched
oligosaccha-
rides that have been further modified to ex-
press additional blood group antigens. Anti-
IH is commonly present in the serum of A1
indi- viduals. Anti-IH is more strongly
reactive with
TABLE 12-6. Comparative Serologic Behavior of the I/i Blood Group Antibodies with Saline Red Cell
Suspensions
Temperature Cell Type
4C I adult
i cord
i adult
22 C I adult
i cord
i adult
group O and group A2 red cells, which are rich
in H antigen, than with group A1 red cells.
Anti- IH is suspected when serum from a
group A in- dividual directly agglutinates all
group O red cells but is compatible with
most group A donor blood tested. Other
examples of com- plex reactivity include anti-
IA, -IP1, -IBH, and
-ILebH.24
Anti-i
Autoanti-i is a relatively uncommon cold ag-
glutinin in sera from healthy individuals. Like
anti-I, anti-i is primarily of IgM isotype but is
weakly reactive at 4 to 10 C. Anti-i is most
strongly reactive with cord and iadult red cells
and more weakly reactive with I+ adult red
cells (Table 12-6). Patients with infectious
mononucleosis often have transient but po-
tent anti-i. As with anti-I, complex reactivity
can sometimes occur (eg, anti-iH).
Cold Agglutinin Syndrome
Autoanti-I and autoanti-i are pathologically
significant in cold agglutinin syndrome
(CAS) and mixed-type autoimmune
hemolytic ane- mia. In those disorders,
autoanti-I (or anti-i) behaves as a
complement-binding antibody with a high
titer and wide thermal range. Pri- mary CAS
occurs with lymphoproliferative dis- orders
(eg, Waldenström macroglobulinemia,
lymphoma, and chronic lymphocytic leuke-
mia). A potent autoanti-I can also occur in
the setting of infection. Mycoplasma
pneumoniae infections are a common cause
of autoanti-I and can be accompanied by a
transient hemo-
Anti-I Anti-i
4+ 0-1+
0-2+ 3+
0-1+ 4+
2+ 0
0 2-3+
0 3+
 CH APT E R 1 2 ABO, H, and Lewis Blood Groups ■ 309

lysis. (See Chapter 17 for additional informa- P BLOOD GROUPS/GLOB

tion on CAS.)
The specificity of the autoantibody in
CAS may not be apparent when undiluted
samples are tested. Titration and thermal
amplitude studies may be required to
discern the speci- ficity of the
autoantibodies and their potential clinical
significance. Table 12-6 illustrates the
serologic behavior of anti-I and anti-i at 4
C and 22 C. (See Chapter 17 and Method 4-
7 for additional information regarding
titration and thermal amplitude studies.)
Transfusion Practice
Autoanti-I can interfere with ABO typing,
anti- body screening, and compatibility testing.
In laboratory testing, these antibodies can be
re- active in the antiglobulin phase of testing,
par- ticularly when polyspecific AHG is used.
Such
reactions rarely indicate antibody activity at 37
C
but are the consequence of antibody binding,
followed by complement binding, at low tem-
peratures. Usually, avoiding room-tempera-
ture testing and using anti-IgG-specific AHG
prevents detection of nuisance cold autoanti-
bodies. For stronger antibody samples, auto-
antibody can be removed from serum by
cold autoadsorption techniques (see Method
4-5). Cold autoadsorbed serum can also be
used for ABO testing.
COLLECTION
The first antigen of the P blood group sys-
tem was discovered by Landsteiner and
Levine in 1927 in a series of experiments
that also led to the discovery of M and N an-
tigens. Several related glycosphingolipid an-
tigens belong to P1PK (P1, Pk, NOR), GLOB
(P, PX2), and 209 collection (LKE).18 Pk, P,
and LKE are high-incidence antigens
expressed on nearly all red cells except rare
null phe- notypes, which lack P (Pk
phenotype) or both P and Pk antigens (p
phenotype) (see Table 12-7). Red cells are
particularly rich in P antigen, which makes
up nearly 6% of to- tal red cell lipid. P k and
P antigens are also widely expressed on
nonerythroid cells, in- cluding lymphocytes;
platelets; and plasma, kidney, lung, heart,
endothelium, placenta,
and synovium cells.33 In contrast, antigen
P1 is uniquely expressed on red
cells.33
Phenotypes
More than 99% of donors have the P 1 (P1+) or
P2 (P1–) phenotype (see Table 12-7). Both phe-
notypes synthesize Pk and P antigens and dif-
fer only in the expression of the P 1 antigen.
Three rare, autosomal recessive phenotypes
have been identified (p, 1P k, P k) as well as
weak

TABLE 12-7. Phenotypes and Prevalence in the P1PK and GLOB Group

Red Cell Reactions with Antisera Prevalence (%)

Antibodies European African

Anti-P1 Anti-P Anti-Pk Anti-PP1Pk in Serum Phenotype Ethnicity Ethnicity

+ + 0 + None P1 79 94
0 + 0 + P1* P2 21 6
0 0† 0 0 PP1Pk (Tja) p Rare Rare
k
+ 0 + + P P1 Rare Rare
k
0 0 + + P P2 Rare Rare

*An anti-P1 is detected in approximately 25% of P2 individuals.

†
Usually negative. Some examples may be weakly positive as a result of crossreactivity of anti-P with X2 and sialosyl-X2
glycolipid on p red cells.
310 ■ AABB T EC HNIC AL MANUAL

variants.34 Analogous to the ABO system, the
rare p and Pk phenotypes are associated with
the presence of naturally occurring antibodies
against missing antigens (anti-P1, anti-P, and
anti-PP1Pk).
Biochemistry
The synthesis of the Pk, P, and P1 antigens
proceeds through the stepwise addition of
sugars to lactosylceramide, a ceramide dihex-
ose (CDH). (See Fig 12-5 and Table 12-8.)
The
first step in this process is the synthesis of
the
Pk antigen, the ultimate precursor of all globo-
type glycosphingolipids. To make the Pk anti-
gen, 1,4 galactosyltransferase 1 (A4GALT1)
adds a terminal galactose, in an 14 linkage,
to CDH. The Pk antigen can then serve as a
substrate for 1,3 N-acetylgalactosaminyl-
transferase I (B3GALNACT1), which adds a
13 N-acetylgalactosamine to the terminal
galactose of Pk (Gb3) to form P antigen. In
some cells, including red cells, the P antigen is
further elongated to form additional, globo-
family antigens, such as Luke (LKE), Type 4

GlcCer

Neolacto Family LacCer Globo Family

(CDH)
A4GALT1

Lc3 Pk
(Gb3)

B3GalNAcT1
A4GALT1
Paragloboside P NOR
(PG, nLc4) (Gb4)
A4GalT1
B3GalT5

X2 SPG P1 Gb5
ST3GAL2 FUT1/2

Sialosyl-X2 LKE Globo-H

FIGURE 12-5. Synthesis of P1, Pk, P, and related glycosphingolipid antigens. Carbohydrate structures are
shown in Table 12-8.
 CH APT E R 1 2 ABO, H, and Lewis Blood Groups ■ 311

TABLE 12-8. Structures of P1, and Related Glycosphingolipids

GLOB,
Family* Name Oligosaccharide Structure

CDH Gal1-4Glc1-1Cer
Globo (Gb) Gb3, Pk Gal1-4Gal1-4Glc1-1Cer
Gb4, P GalNAc1-3Gal1-4Gal1-4Glc1-1Cer
Gb5 Gal1-3GalNAc1-3Gal1-4Gal1-4Glc1-1Cer
NOR1 Gal1-4GalNAc1-3Gal1-4Gal1-4Glc1-1Cer
Globo-H Fuc1-2Gal1-3GalNAc1-3Gal1-4Gal1-4Glc1-1Cer
LKE NeuAc2-3Gal1-3GalNAc1-3Gal1-4Gal1-4Glc1-1Cer
NOR2 Gal1-4GalNAc1-3Gal1-4GalNAc1-3Gal1-4Gal1-4Glc1-1Cer
Neolacto (nLc) Lc3 GlcNAc1-3Gal1-4Glc1-1Cer
nLc4, PG Gal1-4GlcNAc1-3Gal1-4Glc1-1Cer
P1 Gal1-4Gal1-4GlcNAc1-3Gal1-4Glc1-1Cer
SPG NeuAc2-3Gal1-4GlcNAc1-3Gal1-4Glc1-1Cer
X2 GalNAc1-3Gal1-4GlcNAc1-3Gal1-4Glc1-1Cer
Sialosyl-X2 NeuAc2-3GalNAc1-3Gal1-4GlcNAc1-3Gal1-4Glc1-1Cer
*Glycosphingolipid family. Note: Neolacto are type 2 chain glycosphingolipids.
CDH = ceramide dihexose or lactosylceramide; Cer = ceramide; Gal = galactose; GalNAc = N-acetylgalactosamine; Glc =
glu- cose, GlcNAc = N-acetylglucosamine; NeuAc = N-acetylneuramanic acid (sialic acid); PG = paragloboside; SPG =
sialosyl- paragloboside.

chain ABH antigens (globo-ABH), and NOR.

NOR, a rare polyagglutinable red cell pheno-
type, is the result of unusual globo-family anti-
gens characterized by the addition of an 14
galactose to the terminus of P and related
long- chain globo-glycolipids (see Table 12-
8).35
Unlike Pk and P antigens, the P1 antigen is
not a globo-glycosphingolipid but is a member
of the neolacto family ( Type 2 chain glyco-
sphingolipids). In P1 individuals, A4GALT1
adds an 14 galactose to the terminus of
paragloboside. The P1 antigen is not expressed
on red cell glycoproteins.36 The weak P-like
ac- tivity on p red cells is believed to be X2
(PX2), a related Type 2 chain glycolipid.
Recent studies have shown that
B3GALNACT1 is capable of
synthesizing PX2-active structures.37
Molecular Biology
Several inactivating mutations have been
identi- fied in both 4GALT1 and
3GALNACT1.18,38 The p phenotype is the
consequence of muta- tions in the protein-
coding sequence of A4GALT1. In the absence
of A4GALT1 activity, there is a loss of all
globo-family and P1 anti- gens. These patients
have a compensatory increase in Type 2 chain
glycolipid synthesis, as evidenced by increased
paragloboside, sialoparagloboside, and PX2.37
Mutations in B3GalNACT1 give rise to the P k
phenotype, which is characterized by a loss of
P and LKE antigens and by increased Pk
expression. The
P2 phenotype arises from a point mutation
that introduces an alternate start codon. It is
hypothesized that this alternate A4GALT1
312 ■ AABB T EC HNIC AL MANUAL
transcript or peptide may downregulate
A4GAT1 transcription with low enzyme levels
and decreased affinity for paragloboside. In-
terestingly, individuals with weak P 1 expres-
sion are heterozygous for P1P2 alleles.34
P Blood Group Antibodies
Anti-P1
Anti-P1
is a present in the
24
sera of one-quarter
to two-thirds P2 donors. Anti-P1 is a naturally
occurring antibody of IgM isotype and is
often detected as a weak, room-temperature
aggluti- nin. In rare cases, anti-P1 is reactive
at 37 C or shows in-vitro hemolysis.
Because anti-P1 is nearly always IgM, anti-
P1 does not cross the placenta and has not
been reported to cause HDFN. Anti-P 1 has
only rarely been reported to cause in-vivo
hemolysis. Anti-P1 titers are often elevated
in patients with hydatid cyst disease or
fascioliasis (liver fluke) and in bird
handlers. It is believed that P1-like substance
in bird excrement can stimulate anti-P 1 lev-
els.25 Some people with anti-P1 also have I
blood group specificity (anti-IP1).24
P1 expression varies in strength among
in- dividuals and has been reported to
decrease during in-vitro storage. 24 As a
consequence, anti-P 1 may not be reactive
with all P1+ red cells tested. Anti-P1 can be
enhanced by incu- bation at low
temperatures (eg, 4 C) or by test- ing serum
against enzyme-treated red cells. Anti-P1
reactivity can be inhibited in the pres- ence
of hydatid cyst fluid or P1 substance de-
rived from pigeon eggs. Inhibiting P1
activity may be helpful when testing sera
containing multiple antibodies.
Alloanti-PP1Pk and Alloanti-P
Anti-PP1Pk (historically known as anti-Tja) is a
separable mixture of anti-P, anti-P1, and anti-
Pk in the sera of p individuals. Alloanti-P is
present in the sera of P k and P k individuals,
1 2
occurs naturally, and is predominantly of
IgM isotype or a mixture of IgM and IgG
(see Table 12-7). The antibodies are potent
hemolysins and are associated with
hemolytic transfusion reactions and,
spontaneous abortion. The placenta, which is
of fetal origin, is rich in P k and P antigen and
is a target for maternal cytotoxic IgG
antibodies.
Autoanti-P (Donath-Landsteiner)
An autoantibody with P specificity is
present in patients with paroxysmal cold
hemoglobin- uria (PCH), a clinical
syndrome that most
commonly occurs in children following viral
infection. In PCH, autoanti-P is an IgG bipha-
sic hemolysin capable of binding red cells at
colder temperatures, followed by
intravascular hemolysis at body temperature.
This charac- teristic can be demonstrated in
vitro in the Donath-Landsteiner test (see
Chapter 17 and Method 4-11 for further
details).
Transfusion Practice
Alloanti-PP1Pk and alloanti-P are clinically
sig- nificant antibodies associated with acute
he- molytic transfusion reactions and
spontane-
ous abortion. Rare individuals of p and Pk
phenotypes should be provided with
antigen- negative, crossmatch-compatible
RBCs for transfusion.
In general, anti-P1 is a clinically
insignifi- cant, room-temperature
agglutinin. Patients with anti-P 1, which is
reactive only at room temperature or
below, can be safely trans- fused with P1+
RBCs, which results in normal red cell
survival. It is not necessary to provide
antigen-negative units to these patients. Very
rarely, anti-P1 can cause decreased red cell
sur- vival and hemolytic transfusion
reactions.
Anti-P1 that is capable of fixing comple-
ment at 37 C and is strongly reactive in the
AHG phase of testing is considered potentially
clinically significant. In such rare
instances, units selected for transfusion
should be nonre- active at 37 C and in an
indirect antiglobulin test with either
polyspecific AHG or anti-C3.24
Disease Associations
occasionally, HDFN. There is an association between anti-PP1Pk
and early
The Pk antigen is a
receptor for Shiga toxins,
the causative agent of
shigella dysentery, and
Escherichia coli-
associated hemolytic
uremic syndrome.33 The
Pk antigen is also a
receptor for
Streptococcus suis, a
species of bacteria that
 CH APT E R 1 2 ABO, H, and Lewis Blood Groups ■ 313

triggers zoonotic illness capable of causing

bacterial meningitis.33 Pk expression may also
modulate host resistance to human immuno-
deficiency virus.39 The P antigen is a receptor
for parvovirus B19, the etiologic agent of ery-
thema infectiosum (fifth disease). In some pa-
tients, B19 can cause a transient anemia or
aplastic crisis. P1, Pk, P, and LKE antigens
can all serve as receptors for P-fimbriated
uro- pathogenic E. coli, a cause of chronic
urinary
tract infections.33 LKE is a common
oncofetal marker present on tumors and
pluripotent embryonic, mesenchymal, and
very small em- bryonic-like stem cells.4,34

KEY POINTS

The antigens of the ABO, H, Lewis, I, and P blood group systems are defined by small car- bohydrate epitopes on glycopro
The ABO system contains four major ABO phenotypes: A, B, O, and AB. The four phenotypes are determined by the prese
ABO grouping requires both antigen typing of red cells for A and B antigen (red cell group- ing or forward type) and scree
H antigen is ubiquitously expressed on all red cells, except the rare Bombay phenotype.
H antigen is the precursor to both A and B antigens; thus, the amount of H antigen on red cells depends on the person’s AB
Lewis antigens are not synthesized by red cells but are passively adsorbed onto red cell membranes from a pool of soluble L
The three common Lewis phenotypes indicate the presence or absence of Lewis and secre- tor enzymes.
With increasing age, there is a gradual increase in I antigen accompanied by a reciprocal de- crease in i antigen. Most child
Autoanti-I and autoanti-i are pathologically significant in cold agglutinin syndrome and mixed-type autoimmune hemolytic
More than 99% of donors have the P1 (P1+) or P2 (P1–) phenotype. Both phenotypes synthe- size Pk and P antigens and diffe

REFERENCES

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 C h a p t e r 1 3

The Rh System

Gregory A. Denomme, PhD, FCSMLS(D), and

Connie M. Westhoff, PhD, SBB

RH IS TH E most complex of the 34 hu- antigens—antithetical C and c, and E and e—

man blood group systems. The clinical
importance of Rh is underscored by the D
anti- gen, which is considered to be the most
immu- nogenic of all antigens. Anti-D is
present in more than 50% of Rh-negative
recipients transfused with Rh-positive
blood, and the prevalence of
alloimmunization in Rh-nega- tive females
with an Rh-positive fetus was a significant
risk prior to the availability of Rh Immune
Globulin (RhIG) prophylaxis. A his- torical
account of the Rh system and its confu- sion
with the LW system has been described by
Rosenfeld.1 It was in 1939, when Levine and
Stetson made key observations that the
serum of a pregnant woman agglutinated
80% of samples within her ABO blood
group, that a new blood group antigen was
suspected to ex- ist. These authors also
proposed that “prod- ucts of the
disintegrating fetus” and an adverse
transfusion reaction in the mother to a blood
transfusion from her husband were related.2
Today, the terms “Rh positive” and “Rh
negative” refer to the presence or absence,
re- spectively, of the D antigen. Four
additional Rh
are the system’s principal antigens. The anti-
gens were named by Fisher using the next
available letters of the alphabet. Thus, the five
principal Rh antigens—D, C, c, E, and e—are
responsible for the majority of clinically
signif- icant Rh antibodies among the 61 Rh
antigens that have been characterized (Table
13-1).
A true success story in transfusion
medi- cine therapy, the development of
RhIG pro- phylaxis arose from the
observation that ABO incompatibility
between a mother and fetus had a partial
protective effect against immuni- zation to
D.4 The administration of IgG anti-D
obtained from human plasma was effective
in the prevention of hemolytic disease of the
fe- tus and newborn (HDFN).5 Beginning in
the mid-1960s, alloimmunization to D in
pregnan- cy was reduced to about 1 in 2000
live births.
CHARACTERIZATION OF RH
Rh proteins, unlike most membrane
proteins, are neither glycosylated nor
phosphorylated.6,7 The use of
immunoprecipitation followed by

Gregory A. Denomme, PhD, FCSMLS(D), Director of Immunohematology and Transfusion Services,

Diagnos- tic Laboratories, BloodCenter of Wisconsin, Milwaukee, Wisconsin, and Connie M. Westhoff, PhD,
SBB, Director of Immunohematology, Genomics, and Rare Blood, New York Blood Center, New York, New
York
G. Denomme disclosed no conflicts of interest. C. Westhoff has disclosed financial relationships with
Novar- tis, BioArray, and Grifols.
317
318 ■ AABB T EC HNIC AL MANUAL

TABLE 13-1. Terminology for Rh Antigens

ISBT Antigen or Numerical Antigen or

Designation Symbol(s) Prevalence Designation Symbol(s) Prevalence
RH1 D 85% Whites RH32 #
1% Blacks RN
92% Blacks with DBT
RH2 C 68% Whites RH33 R0 Har, DHAR 0.01% Germans
27% Blacks
RH3 E 29% Whites RH34** HrB High
22% Blacks
RH4 c 80% Whites RH35 Low
96% Blacks
RH5 e 98% RH36 Bea Low
RH6 ce or f 65% Whites RH37 Evans Low (several D/CE or
92% Blacks CE/D hybrids)
RH7 Ce or rhi 68% Whites RH39 C-like High
27% Blacks
RH8 CW Low, 2% Whites RH40 Tar Low (DVII)
RH9 CX Low, 1.8% Finns RH41 Ce-like 70% Whites
RH10 V 30% Blacks RH42 Ce S
2% Blacks
RH11 EW Low RH43 Crawford 0.1% Blacks
RH12* G 84% Whites RH44 Nou High
92% Blacks
RH17† Hr0 High RH45 Riv Low
RH18‡ Hr High RH46 Sec High
RH19 §
hr s
98% RH47 Dav High
RH20 VS 32% Blacks RH48 JAL Low
RH21 CG 68% Whites RH49 ††
STEM 6% Blacks
RH22 CE <1% (DCE, CE) RH50 FPTT Low (DFR, R Har)
0

RH23|| D W Low (DVa) RH51 MAR High

RH26 c-like High (most c+) RH52|| BARC Low (DVI)
RH27 cE 28% Whites RH53 JAHK Low
22% Blacks
RH28 hrH Low RH54|| DAK Low (DIIIa, DOL, RN)
RH29¶ total Rh 100% RH55 LOCR Low
RH30 ||
Go a
Low RH56 CENR Low
RH31 §
hr B
High RH57 CEST High (antithetical to
JAL)
 CH A PT E R 1 3 The Rh System ■ 319

TABLE 13-1. Terminology for Rh Antigens (Continued)

ISBT Antigen or Numerical Antigen or

Designation Symbol(s) Prevalence Designation Symbol(s) Prevalence
RH58 CELO High (antithetical to RH60 PARG Low
Crawford)
RH59 CEAG High RH61 CEVF
Note: RH13 through RH16, RH24, RH25, and RH38 are obsolete.
*Present on red cells expressing C or D antigen.
†
Antibody made by individuals with D-deletion phenotypes D– –, Dc–, and DCw–.
‡
Antibody made by individuals with altered e and/or D phenotypes prevalent in groups of African ethnicity.
§
Absent from red cells with DcE/DcE (R2R2) phenotype or variant e found in groups of African ethnicity.
||
Low-prevalence antigen associated with the partial D indicated.
¶
Antibody made by individuals with Rhnull red cells.
#
Low-prevalence antigen expressed by red cells with RN or the partial DBT antigen.3
**Antibody made by individuals with altered C, E, and/or D phenotypes prevalent in groups of African ethnicity.
††
Associated with 65% of hrS– Hr– and 30% of hrB– HrB– red cells.

sodium dodecylsulfate polyacrylamide gel

electrophoresis led to the discovery that Rh
proteins have a molecular weight of 30,000
to 32,000 kDa.8-10 N-terminal amino acid se-
quencing of Rh was accomplished in the late
1980s.11 The findings led to the cloning of
the RHCE gene by Cartron12 and colleagues
in 1990 and of RHD in 1992.13,14 The
different RHCE alleles responsible for the C
or c and E or e antigens were determined in
1994.15
TERMINOLOGY
Early Rh nomenclature reflects the differences
in opinion concerning the number of genes
that encode DCE antigens. The Fisher-Race
terminology was based on the premise that
three closely linked genes, C/c, E/e, and D,
were responsible. In contrast, the Wiener
nomen- clature (Rh-Hr) was based on the
belief that a single gene encoded several blood
group fac- tors. There are actually two genes,
RHD and RHCE, as proposed by Tippett.16
The Fisher-Race CDE terminology is
often preferred for written communication,
but a modified version of Wiener’s
nomenclature makes it possible to identify
the Rh antigens present on one
chromosome, that is, a haplo-
type, using a single term (Table 13-2). In the
modified Wiener’s nomenclature, “R” indi-
cates that D is present, and a subscripted
number or letter indicates the C/c and E/e
an- tigens: R1 for Ce, R2 for cE, Ro for ce,
and RZ for CE. In addition, “r” indicates
haplotypes that
lack D, and the C/c and E/e antigens are
indi- cated using superscripted symbols: r
for Ce, r for cE, and ry for CE (Table 13-2).
The International Society of Blood Trans-
fusion (ISBT) Working Party on Terminology
for Red Cell Surface Antigens adopted six-
digit numbers to indicate red cell antigens. The
first three numbers represent the system, and
the remaining three digits refer to the antigenic
specificity; the Rh system was assigned Num-
ber 004. In 2008, the ISBT committee recog-
nized the antigens of the Rh-associated glyco-
protein (RHAG) as a new blood group system
(Number 30).17
RH GENES AND Rh PROTEINS
Two genes (RHD and RHCE) are closely
linked on chromosome 1 and encode 416
amino ac- ids. One gene encodes the D
antigen, and the other encodes CE antigens in
four combina- tions (ce, cE, Ce, or CE) [Fig
13-1(A)]. Each
320 ■ AABB T EC HNIC AL MANUAL

TABLE 13-2. Prevalence of the Principal Rh Haplotypes

Modified Prevalence (%)

Fisher-Race Wiener
Haplotype Haplotype White Black Asian

Rh positive
DCe R1 42 17 70
DcE R2 14 11 21
Dce R0 4 44 3
DCE RZ <0.01 <0.01 1
Rh negative
ce r 37 26 3
Ce r 2 2 2
cE r 1 <0.01 <0.01
CE ry <0.01 <0.01 <0.01

FIGURE 13-1. (A) RHD and RHCE genes. The inverted gene orientation, the antigens they encode, and
the deletion of RHD that results in the D-negative red cell phenotype are shown. (B) Predicted 12-
transmembrane domain model of the RhD and RhCE proteins. The amino acid differences between RhD and
RhCE are shown as grey circles. The zigzag lines represent the location of possible palmitoylation sites.
Positions 103 and 226 in RhCE critical for C or c and E or e expression are indicated as open
circles.18
 CH A PT E R 1 3
gene has 10 exons, and the two genes share
97% identity. The two proteins created by
these genes differ by 32 to 35 amino acids
[Fig 13-1(B), shown as circles on RhD],
depending on whether D is compared to C
or c.
The last decade has witnessed the
devel- opment of an abundance of
information on the genetic diversity of the
RH locus, and the antigens identified by
molecular testing have far exceeded the
number of antigens identified by serology.
More than 275 RHD and 50 RHCE alleles
have been documented. A directory of RHD
alleles is maintained on a Rhesus-data-
base,19 and RHCE and RHD alleles are listed
on the National Center for Biotechnology
Infor- mation human blood group mutation
web- site.19,20 The ISBT Working Party on
Red Cell Immunogenetics and Blood Group
Terminolo- gy maintains, names, and
catalogs new al- leles.21
Most D-negative (Rh-negative) pheno-
types are the result of complete deletion of
the RHD gene [Fig 13-1(A)]. These
phenotypes provide the immunological
rationale for why the transfusion of Rh-
positive blood to a D- negative individual
often results in produc- tion of anti-D. The
immunogenicity of a pro- tein correlates
with the degree of foreignness to the host,
and the large number of amino acid
differences in D explains why exposure can
result in a potent immune response.
RHCE [found in all but rare D– –
individu- als, where the dashes represent
missing anti- gens] encodes both C/c and E/e
antigens on a single protein. C and c antigens
differ by four amino acids, but only the serine
to proline at position 103 is predicted to be
extracellular [Fig 13-1(B)]. The E and e
antigens differ by one amino acid, a proline to
alanine at amino acid position 226, located on
the fourth extra- cellular loop of the protein.
The RH genes and proteins shown in Fig 13-
1(B) are typical of the majority of individuals.
Commercial antibody reagents are available to
detect the expression of the principal Rh
antigens—D, C, c, E, and e (Table 13-3).
The five principal antigens are responsi-
ble for the majority of Rh incompatibilities, al-
though the Rh system is more complex, with
61 antigens identified to date (Table 13-1).
The Rh System ■ 321
New antigens may result from single
nucleo- tide polymorphisms (SNPs) to
major gene re- arrangements. For example,
the genetic ex- changes between RHD and
RHCE can create hybrid proteins that
express an RhD protein with a portion of
RhCE, or vice versa. Rear- rangements are
common and thought to be fa- cilitated by
the inverted orientation of the RH genes [Fig
13-1(A)].18 The structure promotes hairpin
loop formation and subsequent genet- ic
exchange via template conversion; one
member acts as a donor template during
repli- cation but remains unchanged in the
process. The donated region can span
several base pairs, single exons, or multiple
exons.
ANTIGENS
Routine donor and patient typing tests only for
D. Testing for other common Rh antigens is
performed primarily to resolve or confirm an-
tibody identification. Exceptions include pa-
tients receiving chronic transfusion, such as in
some sickle cell disease (SCD) programs, to
provide antigen-matched donor units to mini-
mize alloimmunization.
Rh Phenotypes
Testing of red cells with the five principal
Rh antisera has been used to predict the RH
geno- type (Table 13-3). Some haplotypes
are more common in certain ethnic groups.
The fre- quencies of the predicted RH
genotypes are uncertain (eg, the frequencies
of RoRo vs Ror in
persons of African ancestry), and frequencies
are more uncertain in people of mixed ethnici-
ty. Serologic testing cannot determine whether
red cells are from a homozygous (D/D) or het-
erozygous (D/–) person because anti-D sel-
dom shows any difference in reactivity be-
tween red cells with a single or a double dose
of D antigen. RHD zygosity can be determined
by DNA molecular testing for the presence of
a RHD deletion or an inactive RHD.18
The Rh haplotype influences the level of
D antigen expression. Less D is expressed in
the presence of C, a phenomenon called the
“Ceppellini effect.”22 Red cells from a
DCe/DCe (R1R1) individual express
significantly fewer D
322 ■ AABB T EC HNIC AL MANUAL

TABLE 13-3. Results of Tests with Five Principal Rh Antisera with Phenotype and Predicted RH
Genotype

Antisera
Predicted Alternative
Anti-D Anti-C Anti-E Anti-c Anti-e Phenotype Genotype Genotype
Rh positive*
+ + 0 + + D, C, c, e R1 r R1 R0
DCe/ce DCe/Dce
R0 r 
Dce/Ce
+ + 0 0 + D, C, e R1 R1 R1 r 
DCe/DCe DCe/Ce
+ + + + + D, C, c, E, e R1 R2 R1 r
DCe/DcE DCe/cE
R2 r 
DcE/Ce
RZ r
DCE/ce
R0 R Z
Dce/DCE
+ 0 0 + + D, c, e R0 r R0 R 0
Dce/ce Dce/Dce
+ 0 + + + D, c, E, e R2 r R2 R 0
DcE/ce DcE/Dce
R0 r
Dce/cE
+ 0 + + 0 D, c, E R2 R 2 R2 r
DcE/DcE DcE/cE
+ + + 0 + D, C, E, e R1 R Z RZ r 
DCe/DCE DCE/Ce
+ + + + 0 D, C, c, E R2 R Z RZ r
DcE/DCE DCE/cE
+ + + 0 0 D, C, E RZ R Z RZ r y
DCE/DCE DCE/CE
 CH A PT E R 1 3 The Rh System ■ 323

TABLE 13-3. Results of Tests with Five Principal Rh Antisera with Phenotype and Predicted RH
Genotype (Continued)

Antisera

Predicted Alternative
Anti-D Anti-C Anti-E Anti-c Anti-e Phenotype Genotype Genotype
Rh negative†
0 0 0 + + c, e rr
ce/ce
0 + 0 + + C, c, e rr
Ce/ce
0 0 + + + c, E, e r r
cE/ce
0 + + + + C, c, E, e r r
Ce/cE
*Rare genotypes (R r , R r , and R r ) not shown (prevalence of <0.01%).
0 y 1 y 2 y

†
Rare genotypes (rry, rry, rry, and ryry) not shown (prevalence of <0.01%).

antigen sites than red cells from a DcE/DcE
(R2R2) individual. Therefore, it is important to
choose a consistent haplotype when titrating
anti-D in the antenatal setting because signifi-
cantly different titers can be obtained.
D Antigen
The D antigen is composed of numerous
epit- opes (designated by “epD”) that were
original- ly defined by antibodies from D-
positive peo- ple who made anti-D.
Subsequent studies with monoclonal
antibodies defined 30 or more epitopes
designated as “epD1” to “epD9.”23 Each
epitope has additional subdivisions (eg,
epD6.1). D epitopes are highly
conformational and consist of more than
simple linear amino acid residues. Amino
acid changes in intracel- lular regions of the
protein may alter D epit- opes.
D-Positive (Rh-Positive) Phenotypes
Most D-positive red cell phenotypes have a
conventional RhD protein [Fig 13-1(B)]. How-
ever, more than 275 different RHD alleles
that
encode proteins with amino acid changes
have been reported. These alleles can cause
numerous variations in the expression of D,
and red cells with some form of altered D ex-
pression are encountered in routine transfu-
sion practice. An estimated 1% to 2% of indi-
viduals of European ethnicity carry RHD
alleles that encode altered D antigens, and the
incidence in individuals of African ethnicity is
higher. Altered D is organized into four
groups: weak D, partial D (including category
D), Del,
and nonfunctional RHD.24-27
WEA K D. Traditionally, weak D red cells
were defined as having a reduced amount of
D anti- gen (formerly called “Du”) that
required an in- direct antiglobulin test (IAT)
for detection. However, the number of
samples identified as being weak D
depended on the typing reagent and method
used, which have changed over the years. In
1999, Wagner and Flegel proposed a system
to classify altered D red cells on the basis of
their nucleotide substitutions (re- viewed in
Flegel and Denomme28). Weak D types are
the result of an SNP that encodes a single
amino acid change predicted to be
324 ■ AABB T EC HNIC AL MANUAL

located in the intracellular or protein resulting from regions of RhD joined

transmembrane region of the protein, rather
than on the outer surface of the red cell (Fig
13-2). The amino acid changes may affect
the insertion of the protein in the membrane
and, thus, reflect the reduced number of D
antigen sites on the red cells.
Uniquely different SNPs cause weak D
Types 1-84.19 The most common is weak D
Type 1, which has a valine-to-glycine amino
acid substitution at position 270. Types 1, 2,
and 3 represent approximately 90% of the
to RhCE can result in the loss of D epitopes
and also generate new antigens. For example,
DVI red cells carry the BARC antigen. A few
partial D phenotypes are the result of multiple
nucle- otide changes. Some partial D types are
de- tected only by the IAT. In contrast to weak
D types, partial changes are predicted to be lo-
cated on the exterior membrane surface (Fig
13-2).29
D EL . Red cells that express extremely low

weak D types in persons of European ethnici-
ty.25 Weak D types can be further weakened
when C is present in trans to a weak D type
(eg, r in trans with weak D Type 2).
PA RT I A L D. Red cells with “category D”
have historically been classified by epitope
studies. Category D individuals type as D
positive, but can make anti-D when they are
exposed to conventional D antigen.
Category D pheno- types are now classified
as partial D. The ma- jority of partial D
phenotypes are due to hy- brid genes in
which portions of RHD are replaced by
corresponding portions of RHCE (Fig 13-3).
The novel sequences of the hybrid
lev- els of D antigen that cannot be detected
by routine serologic methods are designated
as “D-elution” or Del. Red cells have enough
D an- tigens to adsorb and elute anti-D. Del
cells are found in 10% to 30% of D-negative
people of Asian ancestry and result from
several differ- ent RHD mutations that
severely reduce RhD expression in the
membrane. Del cells are much less common
in individuals of European ancestry
(0.027%) and carry different nucleotide
substitutions than those of Asian
ancestry.26,27 Del red cells can usually be char-
acterized by RHD genotyping and careful
ad- sorption-elution studies.

FIGURE 13-2. Structural models of weak D and partial D. The locations of amino acid changes are shown
as solid circles in the plasma membrane or on the interior. Weak D Types 1 and 2 (shown by the arrows)
are found in approximately 80% of people with weak D phenotypes. Partial D types are encoded by
single amino acid changes that are generally present on the exterior of the cell.
 CH A PT E R 1 3 The Rh System ■ 325

FIGURE 13-3. RHD and RHCE genes. The 10 exons of RHD and RHCE are depicted as white and grey
boxes, re- spectively. Also shown are some examples of RHD encoding partial D and of RHCE with mutations
often found in people of African ethnicity. These mutations complicate transfusions in patients with sickle
cell disease.

NONF U N CTI O NAL RHD. RHD genes that

cannot produce a full-length polypeptide are
deemed nonfunctional and have been given
allele designations.21
D EPITOPES ON RHCE (RHCE) . Expression
of D epitopes by the protein product of the
RHCE gene, in the absence of RHD,
further complicates serologic determination
of D sta- tus. Several Rhce (RHCE) proteins
have D-spe- cific amino acids result in
epitopes that are re- active with some
monoclonal anti-D. They are more often
found in a specific population. Ex- amples
include DHAR, which is found in indi-
viduals of German ancestry, and Crawford
(ce- CF), found in individuals of African
ancestry. These two examples are notable
because the red cells show strong
reactivity with some monoclonal reagents
but are nonreactive with others, including
polyclonal anti-D reagents
(Table 13-4). DHAR and Crawford
phenotypes can cause D typing
discrepancies. Less dra- matic are changes
in Rhce (RHCE) proteins that mimic a D-
epitope structure encoded by alleles
designated as “ceRT ” and “ceSL.”30,31 The
red cells are weakly reactive with some,
but not all, monoclonal anti-D. Most impor-
tantly, individuals with DHAR and Crawford
lack RhD and can be sensitized to D.32,33
EL EVAT ED D. Several rare deletion pheno-
types, designated as “D– –,” “Dc–,” and
“DCw–,” have an enhanced expression of D
antigen and no, weak, or altered C/c and E/e
antigens.34 These variants are the converse of
partial D (discussed above) and result from the
replace- ment of portions of RHCE by RHD.
The addi- tional RHD sequences in RHCE
result in the additional expression of (hybrid)
D along with
TABLE 13-4. Reactivity of FDA-Licensed Anti-D Reagents with Some D Variant Red
326

Reagent
Cells
■

Gammaclone GAMA401 F8D8 Neg/Pos Pos Pos Pos†

monoclonal
MS26
Immucor Series MS201 Neg/Pos Pos Pos Neg Weakly Neg
4 monoclonal pos
MS26
Immucor Series Th28 Neg/Pos Pos Pos Neg Weakly Weakly
5 monoclonal pos pos
Polyclonal
Ortho BioClone MAD2 Neg/Pos Neg/Pos Neg/Neg Neg

IgM Monoclonal
Pos
Ortho Gel (ID- MS201 Neg Pos Neg Weakly Neg
pos
MTS) Biotest RH1 BS226 Neg Pos Neg
Biotest RH1 Blend BS221 Neg/Pos Pos† Neg

IgG
BS232
H4111B7
AABB T ECHNICAL M A NUAL

Alba Bioscience alpha LDM1 Neg Pos Neg

Alba Bioscience beta LDM3 Neg Pos Neg
Alba Bioscience delta LDM1 Neg Pos Neg
ESD1-M
LDM3
Alba ESD1 Neg/Pos Pos Neg
blend Neg/Pos Neg/Neg Neg/Neg
Neg/Pos Weakly pos‡ Neg
Polyclonal
*Result following slash denotes anti-D test result by the indirect antiglobulin test (IAT).
†
Test results will be negative if an IAT is performed.
‡
Enzyme-treated cells.
FDA = Food and Drug Administration; IgM = immune globulin M; IS = immediate spin; AHG = antihuman globulin; pos = positive; neg =
negative.
IS/AHG*IS/ AHG*
DVI IS/AHG* DBTDHAR (Whites)
Crawford (Blacks)

ceR
 CH A PT E R 1 3
normal D, which explains the enhanced D
ex- pression and reduced or missing C/c and
E/e antigens.
D-Negative (Rh-Negative) Phenotype
The D-negative phenotype is most common
in people of European ancestry (15-17%), is
less common in people of African ethnicity
(3-5%), and is rare in people of Asian
ancestry (<0.1%).35 The D-negative phenotype
has arisen multiple times, as evidenced by the
different nonfunctional alleles responsible
for the lack of D expression in various
ethnic groups.
In most people of European ancestry,
the D-negative phenotype results from a
deletion of the entire RHD gene.36 There are
exceptions, however, and red cell samples
with uncom- mon haplotypes [r (Ce) or r
(cE)] are more likely to carry a
nonfunctional RHD. In other ethnic groups,
D-negative phenotypes are pri- marily
caused by inactivating changes in RHD. In
individuals of African ancestry, 66% have a
37-bp insertion in RHD,37 which results in a
premature stop codon. D-negative pheno-
types in people of Asian ancestry result from
mutations in RHD that are most often
associ- ated with a Ce (r) haplotype,
although 10% to 30% of people of Asian
ancestry who type as D negative are actually
Del.26,27
Testing for D
Monoclonal antibody technology introduced
in the 1980s freed manufacturers from
reliance on human source material to
manufacture anti-D reagents. However,
these antibodies are specific for a single D
epitope and do not de- tect all D-positive red
cells. Consequently, most Food and Drug
Administration (FDA)- approved anti-D
reagents combine a mono- clonal IgM,
which causes direct agglutination at room
temperature, with a monoclonal or
polyclonal IgG that is reactive by IAT for
the determination of weak D. Anti-D for
column agglutination testing is an exception
and con- tains only a monoclonal IgM.
Three of the five FDA-licensed reagents
contain unique IgM clones, and these may
exhibit different reactiv- ity with red cells
that have weak D, partial D, or D-like
epitopes (Table 13-4).
The Rh System ■ 327
Typing Donors for D
The goal of D typing of Red Blood Cell
(RBC) donors, including the identification
of units with weak D or partial D types, is to
prevent anti-D immunization of recipients.
Therefore, the AABB Standards for Blood
Banks and Transfusion Services requires
donor blood to be tested using a method that
is designed to detect weak expression of D.
There is no re- quirement that the typing be
done using an IAT. If the test results are
positive, the unit must be labeled as “Rh
positive.”38(p31) Most weak D or partial D
antigen units are detected but infrequently,
some very weak D red cells are not detected,
and Del red cells appear non- reactive with
anti-D. Red cells with weak D an- tigen are
less immunogenic than normal D- positive
red cells, and Del donor units can stim- ulate
anti-D in some D-negative recipients.39-43 A
unit labeled Rh negative must be confirmed
by testing an integrally attached segment be-
fore transfusion. Weak D testing is not re-
quired. The RhD type of the units labeled
Rh
positive do not require confirmatory test-
ing.38(p35)
Typing Patients for D
When the D type of a patient is determined,
a weak D test is not necessary except to
assess the red cells of an infant whose
mother is at risk of D immunization. Today,
monoclonal IgM reagents type many
samples as being D positive in direct testing
that would have pre- viously been detected
only by IAT. As a result, some of the
concerns regarding the unneces- sary use of
Rh-negative blood and RhIG have been
addressed.
DVI is the most common partial D found
in people of European ancestry, and anti-D
produced by women with partial DVI has re-
sulted in fatal hemolytic disease.44 Current
FDA-licensed monoclonal IgM reagents are
selected to be nonreactive with RBCs from
partial DVI in direct tests (Table 13-4). There-
fore, performing only the direct test on red
cells from female children and women of
childbearing potential avoids the risk of sensi-
tization by classifying DVI as D negative for
transfusion and RhIG prophylaxis. However,
328 ■ AABB T EC HNIC AL MANUAL
because no IAT is performed, the results of
positive rosetting tests (to detect fetomaternal
hemorrhage) must be carefully evaluated; ma-
ternal weak D types that are reactive only in
the IAT phase have a false-positive rosette test
result.
D Typing Discrepancies
D typing discrepancies should always be in-
vestigated and resolved (see “Resolving D
Typ- ing Discrepancies” below). An Rh-
negative blood transfusion is an appropriate
option for patients needing immediate
transfusion, but a thorough clerical and
serologic investigation should be performed.
RHD genotyping is also useful to resolve D
typing discrepancies.45
Because donor centers test for weak D,
a donor who is correctly classified as Rh
positive may be classified as Rh negative as
a recipient. This discrepancy should not be
considered problematic but, rather, should
be communi- cated to the patient and health-
care staff and be noted in the patient’s
medical record.
Clinical Considerations
The long history of transfusing patients who
have weak D red cells with D-positive
RBCs suggests that weak D Types 1, 2, and
3, which are present in the majority of
people of Euro- pean ancestry with weak D,
are unlikely to make anti-D. People with
these weak D types can therefore safely
receive D-positive blood. The less common
weak D Types 11 and 15 have been reported
to make anti-D, suggesting that they have
altered D epitopes.3 Empirical data are
needed to determine the risk of pro- duction
of anti-D in people with other weak D types.
Unfortunately, serologic reagents
cannot typically be used to distinguish
people with partial D that is reactive only
with enhanced methods and techniques from
D-positive indi- viduals. Many partial D red
cells (eg, DIIIa, the most common partial D
type in people of Afri- can ancestry) type as
strongly D positive in di- rect tests and are
recognized only after the pa- tients produce
anti-D.
Policies regarding D typing procedures
and selection of blood components for trans-
fusion should be based on the patient
popula- tion, risk of immunization to D,
and limited supply of D-negative blood
components. These policies should address
what should be done when a partial D type
is found before the patient makes anti-D.
Anti-D is a clinically sig- nificant antibody,
and preventing immuniza- tion in females
of childbearing potential is im- portant to
avoid the complications of HDFN. For other
patients, the complications of anti-D are less
serious, and the decision to transfuse Rh-
positive or Rh-negative blood should take
into consideration dependence on D-
negative blood transfusions for future
bleeding epi- sodes and a possible increased
risk of multiple blood group antibodies in
addition to anti-D.46 Not all D-negative
patients make anti-D when they are exposed
to D-positive RBCs. The incidence in D-
negative hospitalized pa- tients switched to
D-positive blood compo- nents is much
lower, than anticipated.47 AABB Standards
requires that transfusion services must have
policies that address the adminis- tration of
D-positive red cells to D-negative patients
and the use of RhIG, which is a hu- man
blood product that is not entirely without
risk.38(p37)
G Antigen
The G antigen is found on red cells
possessing C or D and maps to the 103Ser
residue on RhD, RhCe, and RhCE (Fig 13-1).
Antibodies to G ap- pear as anti-D plus anti-
C and cannot be sepa- rated. However, the
antibody can be adsorbed by either D–C+ or
D+C– red cells. The presence of anti-G can
explain why a D-negative person who was
transfused with D– (C+) blood or a D-
negative woman who delivered a D– (C+)
child and subsequently appeared to have
made anti-D. Anti-D, -C, and -G can be
distinguished by adsorption and elution
studies.48 These analyses are not usually
necessary in the pre- transfusion setting.
However, it is important to provide RhIG
prophylaxis to pregnant women who have
anti-G only.
C/c and E/e Antigens
The common RHCE alleles encode the
princi- pal C or c and E or e antigens.
However, more
 CH A PT E R 1 3
than 50 different RHCE alleles are known,
and many are associated with altered or
weak ex- pression of the principal antigens
and, in some cases, loss of high-prevalence
antigens.37 Par- tial C and many partial e
antigens are well rec- ognized, and the
majority are reported in indi- viduals of
African ethnicity.
Compound Antigens (ce, Ce, cE, and
CE)
Compound antigens define epitopes that de-
pend on conformational changes that result
from amino acids associated with both C/c
and E/e. These antigens were previously
referred to as “cis products” to indicate that
the antigens were expressed from the same
haplo- type. However, it is now known that
these anti- gens are expressed on a single
protein. These antigens are shown in Table
13-5 and include
ce or f, Ce or rhi, cE or Rh27, and the
uncom- mon CE or Rh22.
Altered or Variant C and e Antigens
Nucleotide changes in RHCE result in quanti-
tative and qualitative changes in C/c or E/e
antigen expression; altered C and altered e are
encountered most frequently. In persons of
European ancestry, altered C is associated
with amino acid changes on the first
extracellular loop of RhCe and the expression
of CW (Gln41Arg) or CX (Ala36Thr) antigens.
Altered C is also associated with changes that
result in the expression of the novel antigens
JAHK (Ser122Leu) and JAL (Arg114Trp).
These red
TABLE 13-5. Compound Rh Antigens on Rh
Proteins
Compound Present on Red
Antigen Cells with These
Designation Rh Protein Haplotypes
ce or f Rhce Dce (R0) or ce (r)
Ce or rhi, RhCe DCe (R1) or Ce (r)
Rh7
cE or Rh27 RhcE DcE (R2) or cE (r)
CE or Rh22 RhCE DCE (Rz) or CE (ry)
The Rh System ■ 329
cells type as C positive, but these patients
can make apparent anti-C or anti-Ce (rh i)
when they are stimulated.
In individuals of African ancestry, altered
C expression most often results from the in-
heritance of a RHDIIIa-CE(4-7)-D hybrid and
less often of a RHD-CE(4-7)-D hybrid.37 These
hybrids do not encode D antigen, but they do
encode C antigen on a hybrid background that
differs from the normal background (Fig 13-
3). The gene has an incidence of
approximately 20% in people of African
ancestry. It is inherit- ed with an RHCE allele
designated as “RHCE*ceS” that encodes
altered e antigen and a V–VS+ phenotype.50
The expressed product of the hybrid RHDIIIa-
CE(4-7)-D linked to RHCE*ceS is referred to
as the “(C)ceS” or “rS” haplotype. Red cells
with the rS haplotype of- ten type as strongly
C-positive with monoclo- nal reagents, and the
presence of the altered C is undetected.
Patients with these types of red cells
fre- quently make alloantibodies with C-
like, and occasionally e-like, specificities
that may appear to be autoantibodies. The
red cells may also lack the high-prevalence
hrB antigen (hrB–).51,52 Altered or partial e
expression is as- sociated with other RHCE*ce
alleles.37,53 These alleles are found primarily
in people of African ethnicity; some
examples are shown in Fig 13-
3. Some people lack the high-prevalence hrS
antigen (hrS–). Anti-hrS is a clinically signifi-
cant antibody and has caused transfusion fa-
talities.53 Not all red cells designated as hr B– or
hrS– by serologic testing are compatible with
antibodies produced by patients with altered
or partial e expression.
An additional complication is that altered
RHCE*ce is often inherited with a partial RHD
(eg, DIII, DAU, or DAR).54 As discussed
above, patients with partial D red cells are at
risk of producing anti-D.
Clinical Considerations
It has long been recognized that alloimmuni-
zation represents a significant problem in
pa- tients with SCD because 25% to 30% or
more of those who are chronically
transfused devel- op red cell antibodies. To
address the problem,
330 ■ AABB T EC HNIC AL MANUAL
many programs determine the pretransfusion
red cell phenotype in patients with SCD and
transfuse RBCs that are antigen matched for
D, C, E, and K because these antigens are
consid- ered to be the most immunogenic. In
addition, some programs attempt to supply
RBCs from donors of African ancestry
whenever possible. Although there is no
consensus on the need to perform red cell
phenotype matching for all patients with
SCD,55 transfusion programs that transfuse
RBCs that are antigen matched for D, C, E,
and K have reduced the incidence of allo-
antibody production.56 Unfortunately, despite
matching for D, C/c, and E/e, some patients
become sensitized because they express Rh
variants.57
RH GENOTYPING
RH genotyping is a powerful adjunct to
sero- logic testing for the typing of
transfused pa- tients, RHD zygosity
determination, noninva- sive fetal D typing,
determination of D status, and identification
of antigen-matched blood for patients with
SCD.
Typing Multitransfused Patients
In patients receiving chronic or massive
trans- fusions, the presence of donor red
cells in the peripheral blood often makes red
cell pheno- typing by agglutination difficult
or inaccurate. Genotyping overcomes this
limitation because blood grouping can be
determined with DNA prepared from a
blood sample, even if the sample was
collected after transfusion.58
RHD Zygosity Testing
RHD zygosity can be determined by
assaying RHD dosage or confirming the
presence of a hybrid rhesus box.18,59 In
prenatal practice, pa- ternal RHD zygosity
testing is important to predict fetal D status
when the mother has anti-D. Care must be
taken in the interpreta- tion of testing results
using either approach. Several different
genetic events cause an ap- parent D-
negative haplotype, and multiple targets
must be tested to accurately determine
zygosity.59,60
Fetal RHD Typing
To determine the D antigen status of a fetus,
fetal DNA can be isolated from cells
obtained by amniocentesis or chorionic
villus sampling. An alternative, noninvasive
approach is to test the maternal plasma,
which contains cell-free, fetal-derived DNA
beyond 5 weeks’ gesta- tion.61,62 In the
future, determination of fetal RHD status
using this noninvasive procedure could
become routine in clinical practice to
eliminate the unnecessary administration of
antepartum RhIG to women who are
carrying a D-negative fetus.62
Confirming D Status
RHD genotyping is useful to distinguish
partial D from weak D or to resolve serologic
D typing discrepancies. Although patients with
an un- certain D status can be treated as D
negative for transfusion and RhIG
administration, this approach may be
unsatisfactory for females of childbearing
potential who face unnecessary RhIG
injections and puts a strain on the limit- ed Rh-
negative blood supply. RHD genotyping
allows informed decisions to be made in this
setting (see “Weak D Types” above).
For donors, D typing discrepancies
must be resolved because errors in
determining their D status may be reportable
to the FDA and result in the recall of blood
components. D-negative, first-time donors
are screened for RHD to detect red cells
with very weak D in some European
centers.3,63,64
RH Genotyping for Patients with SCD
Currently, extensive RH genotyping is time
consuming and is used to find compatible do-
nors in the American Rare Donor Program for
patients with antibodies to high-prevalence
Rh antigens.52 The future availability of high-
throughput RH genotyping platforms enables
donors to be identified by genotyping. Howev-
er, RH genotype matching patients with SCD
who have rare Rh variant types may not be
possible for chronic prophylactic transfu-
sions.65 These patients may be candidates for
stem cell transplantation.66
 CH A PT E R 1 3
RHNULL SYNDROME AND RHAG
BLOOD GROUP SYSTEM
Erythrocytes express a third Rh-protein,
RhAG. RhAG shares 38% of its identity with
RhD/ RhCE, has the same topology in the
mem- brane, and is encoded by a single gene
on chromosome 6. RhAG associates with the
Rh blood group proteins in the membrane to
form an Rh-core complex. Three red cell anti-
gens resulting from single amino acid substi-
tutions form the RHAG blood group system:
Duclos is RHAG1, Ol(a) is RHAG2, and
DSLK is RHAG3 and RHAG4.67
Red cells lacking all Rh antigens are
desig- nated as “Rhnull.” Although
uncommon, the phenotype most often
results from nucleotide
changes in RHAG, known as a “regulator”
type Rhnull, indicating that the RhAG protein
plays a critical role in trafficking RhCE and
RhD to the membrane. Less often, Rhnull
individuals have RHCE nucleotide changes
along with the com- mon deletion of RHD,
and these individuals are called “amorph.”49
Rhnull red cells are stomatocytic and
asso- ciated with mild anemia, suggesting
that the Rh proteins have an important
structural role in the erythrocyte membrane.
The Rh complex
is linked to the membrane skeleton through
CD47-protein 4.2 interactions and
Rh/RhAG- ankryin interactions.68,69
RH ANTIBODIES
Most Rh antibodies are IgG, although some
sera may have an IgM component.
Typically, Rh antibodies do not activate
complement, al- though rare exceptions
have been reported. As a result, in
transfusion reactions involving Rh
antibodies, hemolysis is primarily
extravascu- lar rather than intravascular.
Most Rh antibodies have the potential to
cause clinically significant HDFN and transfu-
sion reactions. Anti-c, which is clinically the
most important Rh antibody after anti-D, may
cause severe HDFN. Anti-C, -E, and -e do not
often cause HDFN, and when they do, it is
usu- ally mild. The reactivity of Rh antibodies
is en- hanced by enzyme treatment of red cells,
and
The Rh System ■ 331
most Rh antibodies are optimally reactive at
37 C.
Concomitant Rh Antibodies
Some Rh antibodies are often found
together. For example, a DCe/DCe (R1R1)
patient with anti-E most certainly has been
exposed to the c antigen as well. Anti-c may
be present in ad-
dition to anti-E but the anti-c may be weak
and undetectable at the time of testing.
When seemingly compatible E-negative
blood is transfused, it is most likely to be c
positive and may elicit an immediate or
delayed transfu- sion reaction. Therefore,
some experts advo- cate for avoiding the
transfusion of c-positive blood in this
situation. In contrast, testing for anti-E in
serum containing anti-c is not war- ranted
because the patient has probably been
exposed to c without being exposed to E. In
addition, the vast majority of c-negative
donor blood is E negative (see Table 13-3).
Antibodies to High-Prevalence Rh
Antigens
Alloantibodies to high-prevalence Rh
antigens include anti-Rh29, made by some
Rhnull indi- viduals who lack Rh antigens,
and others (anti- hrS, -hrB, -HrB, and -Hr)
that are most often en- countered in
transfused patients with SCD.
TECHNICAL CONSIDERATIONS
FOR RH TYPING
High-Protein Reagents and Controls
Some Rh reagents for use in slide, rapid
tube, or microplate tests contain high
concentra- tions of protein (20% to 24%)
and other mac- romolecular additives. These
reagents are pre- pared from pools of human
sera and give reliable results; however, high-
protein levels and macromolecular additives
may cause false-positive reactions (see
“Causes of False- Positive and False-
Negative Rh Typing Results” below). These
reagents must be used accord- ing to the
manufacturers’ instructions and with the
appropriate controls. False-positive results
could cause a D-negative patient to receive
D- positive blood and become immunized.
If red
332 ■ AABB T EC HNIC AL MANUAL
cells exhibit aggregation in the control test, the
results of the test are not valid.
Low-Protein Reagents and Controls
Most Rh reagents in routine use are low-
pro- tein reagents formulated predominantly
with IgM monoclonal antibodies.
Spontaneous ag- glutination causing a false-
positive result can occur, although this
happens much less fre- quently than with
high-protein reagents. A negative result
from a test that was performed concurrently
with a similar reagent serves as a control.
For example, for ABO and Rh typing, the
absence of agglutination by anti-A or anti-B
serves as a negative control for spontaneous
aggregation. For red cells that show
agglutina- tion with all reagents (eg, type
AB or D+), a control performed as described
by the reagent manufacturer is required
(with the exception of donor retyping). In
most cases, a suitable control is a suspension
of the patient’s red cells with autologous
serum or 6% to 10% albumin. Indirect
antiglobulin testing is not valid for red cells
with a positive direct antiglobulin test
(DAT) result unless a method is used to re-
move the IgG antibody. Positive and
negative controls should be tested, and the
positive control cells should have a single
dose of the antigen or be known to show
weak reactivity.
Rh Testing Considerations in HDFN
Red cells from an infant with HDFN are
coated with immunoglobulin, and a low-
protein re- agent is usually necessary to test
these cells. Occasionally, red cells with a
strongly positive DAT result may be so
heavily coated that they are not agglutinated
by a reagent with the same specificity as the
bound antibody. This “blocking”
phenomenon probably results from steric
hindrance, and occupied antigen sites show
false-negative results. Heat elution of the
antibody performed at 45 C permits red cell
typing, but elution must be performed with
caution to avoid denaturing the antigen.
Although detection of the antibody in an
elu- ate confirms the presence of the antigen
on the eluted red cells, RHD genotyping can
be used for confirmation of D typing.
Causes of False-Positive and False-
Negative Rh Typing Results
False-positive typing results can be caused by:
1. Immunoglobulin coating of the cells as a
result of warm or cold autoagglutinins.
Wash the red cells several times and
retest them with low-protein reagents by
direct methods. If an IAT is required, IgG
coating on the red cells can be removed
by treating the cells with glycine/EDTA
(Method 2-21) or chloroquine (Method 2-
20) and retest- ing.
2. Induction of rouleaux by serum factors that
can be eliminated by thoroughly washing
the red cells and retesting.
3. Use of the wrong reagent.
4. Contamination with reagent from another
vial.
5. Nonspecific aggregation of the red cells due
to some component of the reagent other
than the antibody (ie, a preservative, antibi-
otic, or dye).
6. Testing of polyagglutinable red cells agglu-
tinated with reagents that contain human
serum.
False-negative typing results can be caused by:
1. Failure to add the reagent. It is good prac-
tice to add typing reagent to all test tubes
or wells before adding the red cells.
2. Use of the wrong reagent.
3. A red cell suspension that is too heavy for a
tube test or too weak for a slide test.
4. Failure to detect a weak D reaction with
direct testing (immediate centrifugation).
5. Nonreactivity of a reagent with a weak or
partial form of the antigen.
6. Aggressive resuspension of the red cell but-
ton dispensing the agglutination.
7. Contamination, improper storage, or out-
dating of the reagent.
8. Red cells with a strongly positive DAT result
and antigen sites blocked because of a
large amount of bound antibody (most
common in severe HDFN caused by anti-
D).
 CH A PT E R 1 3 The Rh System ■ 333

Resolving D Typing Discrepancies to specify the reactivity of their reagents with

To investigate D typing discrepancies, errors
in sample identification or of a clerical
nature should be eliminated by obtaining
and testing a new sample. Beyond clerical
errors, multiple variables contribute to D
typing discrepancies. These variables
include the use of different methods (ie,
slide, tube, microplate, gel, and automated
analyzers using enzyme-treated red cells),
phase of testing (direct or IAT), dif- ferent
IgM clones in manufacturers’ reagents, and
large number of RHD gene variations that
affect the level of expression and epitopes of
the D antigen.
It is important to know the
characteristics of the D typing reagent used
and to always consult and follow the
manufacturer’s instruc- tions during D
typing. The FDA has drafted
recommendations that require manufacturers
partial DIV, DV, and DVI red cells.70
The IgM anti-D in all of the tube reagents
currently licensed by the FDA is reactive by
di- rect testing (initial spin) with DIV and
DV red cells but has been selected to be
nonreactive with partial DVI red cells in
direct testing. Lim- ited studies have been
performed to charac- terize the reactivity of
anti-D reagents with other partial D and
weak D red cells. These studies have shown
that the anti-D reagents cannot reliably
predict whether a D variant is a weak or
partial D antigen.71,72 Table 13-5 shows the
reactivity of D variant red cells that have
predictable patterns among the different
anti- D reagents. In general, an individual
with par- tial D should be considered to be a
D-positive blood donor but a D-negative
transfusion re- cipient.

KEY POINTS

The Rh system is highly immunogenic, complex, and polymorphic. More than 50 Rh anti- gens have been characterized, al
“Rh positive” and “Rh negative” refer to the presence or absence, respectively, of the D anti- gen.
Contemporary Rh terminology distinguishes between antigens (such as D and C), genes (such as RHD and RHCE), alleles
Most D-negative (Rh-negative) phenotypes result from complete deletion of the RHD gene. Exposure of D-negative indivi
RHCE encodes both C/c and E/e antigens on a single protein. C and c differ by four amino acids, whereas E and e differ by
Routine donor and patient Rh typing procedures test only for D. Testing for other common Rh antigens is used to resolve o
Weak D phenotypes are defined as having a reduced amount of D antigen and are detected by IAT. Weak D usually results
RHD genotyping can identify those pregnant females and blood transfusion recipients with a serologic weak D phenotype w
Most anti-D reagents approved by the FDA combine a monoclonal IgM (that is reactive at room temperature for routine tes
334 ■ AABB T EC HNIC AL MANUAL

10. When determining the D type of a patient, an IAT for weak expression of D is not
necessary except when testing the red cells of an infant born to a mother at risk of D
immunization. Rh-negative donors must be tested by a method that detects weak D.
11. Most Rh antibodies are IgG, although some may have an IgM component. With rare
excep- tions, Rh antibodies do not activate complement and, thus, cause primarily
extravascular rather than intravascular hemolysis. Antibodies almost always result from
red cell immuni- zation through pregnancy or transfusion.

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 C h a p t e r 1 4
Other Blood Group
Systems and Antigens
Jill R. Storry, PhD, FIBMS
THE I N TER N A T I O NAL S O CIET Y of
Blood Transfusion (ISBT) currently rec-
ognizes 339 antigen specificities, of which 297
belong to 1 of 34 blood group systems. 1-3 Each
system represents either a single gene or two
or three closely linked homologous genes. The
ABO and Rh systems are the best known and
clinically most important systems and are de-
scribed in detail in Chapters 12 and 13. The
antigens of the H, Lewis, I, P1PK, and Globo-
side systems are carbohydrate structures that
are biochemically closely related to ABO anti-
gens and are discussed in Chapter 12. The re-
maining systems are described in this chapter;
some, generally the most important in transfu-
sion medicine, are described in some detail,
others in a few lines. They are listed in ISBT
or- der, as in Table 14-1.
In addition to the 34 systems, some
groups of antigens that are serologically,
bio- chemically, or genetically related but
not eligi- ble to join a system are classified
together as collections. Other antigens that
are not eligible to join a system or collection
are of either low or high prevalence in most
major populations
Jill R. Storry, PhD, FIBMS, Associate Professor, Region Skane Office of Medical Services, Department of
Clini- cal Immunology and Transfusion Medicine, Lund, Sweden.
The author has disclosed no conflicts of interest.
and make up the 700 and 901 series, respec-
tively.1 They are all discussed at the end of
this chapter.
The full ISBT classification can be found
on the ISBT website and in Appendix 6. 4
Many more references to blood group systems
and antigens than can be provided here are
avail- able in various textbooks and reviews.5-7
The most important aspect of blood
group antigens in transfusion medicine is
whether their corresponding antibodies are
hemolytic and therefore have the potential to
cause hemolytic transfusion reactions
(HTRs) and hemolytic disease of the fetus
and new- born (HDFN).5 A guide to the
potential clinical significance of blood group
antibodies is pro- vided in Table 14-1.
THE MNS SYSTEM
MNS is a highly complex blood group
system consisting of 46 antigens. As with
the Rh sys- tem, much of its complexity
arises from re- combination between closely
linked homolo- gous genes.
337
TABLE 14-1. Clinical Significance of Antibodies to Blood Group Antigens
338

No.Name
ISBTSystem
■

001 ABO 4 See Chapter 12. See Chapter 12.

002 MNS 46 Rare examples of anti-M and -N active at 37 C cause Anti-S, -s, -U, and some other antibodies cause
AHTRs and DHTRs; anti-S, -s, -U, and some other severe HDFN; anti-M rarely causes severe
antibodies may cause AHTRs and DHTRs. HDFN.

Only very rare examples active at 37 C cause AHTRs

003 P1PK 3 No.
and DHTRs.

No. of Antigens
Rh antibodies can cause severe AHTRs and
004 Rh 55 DHTRs (see Chapter 13). Anti-D can cause severe HDFN (see Chapter
22).
Anti-Lua and -Lub have caused mild DHTRs; anti-Lu8 has
005 Lutheran 20 caused AHTRs. No.

Kell antibodies can cause severe AHTRs and DHTRs.

006 Kell 35 Anti-K can cause severe
007 Lewis 6 Anti-Lea and -Leb are not generally considered to be clinically HDFN.
AABB T ECHNICAL M A NUAL

significant. No.

Anti-Fya, -Fyb, and -Fy3 cause AHTRs and DHTRs; anti-

008 Duffy 5 Fy5 causes DHTRs. Anti-Fya and -Fyb cause
HDFN.
Anti-Jka is a common cause of DHTRs; anti-Jka and -Jk3
009 Kidd 3 also cause AHTRs. No. Anti-Jka does not usually cause
HDFN.
One anti-Dia caused DHTR, but there is little evidence; anti-
010 Diego 22 Dib, DHTRs, and anti-Wra cause HTRs. Anti-Dia, -Dib, -Wra, and -Wrb plus some others have
caused severe HDFN.
Anti-Yta has very rarely caused HTR. No.
011 Yt 2
Hemolytic Transfusion Reaction (HTR), Acute (AHTR), or
(HDFN)
Hemolytic
Delayed (DHTR)
Disease of the Fetus and
012 Xg 2 No. No.
013 7 No. No.
Scianna
014 8 Anti-Doa and -Dob cause AHTRs and DHTRs. No.
Dombrock
015 Colton 4 Anti-Coa causes AHTRs and DHTRs; anti-Cob and -Co3 Anti-Coa has caused severe HDFN, and -Co3 has caused
have caused mild HTRs. mild HDFN.

No. No.
016 LW 3
017 9 No. No.
Ch/Rg
018 1 Anti-H in Bombay phenotype can cause severe Anti-H in Bombay phenotype has the potential to cause
H
intravascular HTRs; anti-HI in para-Bombay is not usually severe HDFN (see Chapter 12).
clinically signifi- cant (see Chapter 12).

Anti-Kx and -Km in McLeod syndrome has caused severe

019 Kx 1 HTRs. Antibodies found only in
males.
No.
020 Gerbich 11 Three examples have been reported of anti-Ge3
CHA P TER 1 4

causing HDFN.
No.
021 Cromer 22 No.
022 9 No. No.
Knops
023 4 There is one example of anti-Inb causing an HTR. No.
Indian
024 3 Anti-Oka is very rare and no cases of HTR have been No.
Ok
Other Blood Groups

025 1 reported. No. No.

Raph
026 JMH 6 One example has been reported of anti-JMH causing No.
AHTR.
■

(Continued)
339
 340

No.Name
ISBTSystem
■

TABLE 14-1. Clinical Significance of Antibodies to Blood Group Antigens

(Continued)

027 I 1 Anti-I in adult i phenotype has caused increased No.

destruction of I+ red cells.
Globoside has caused intravascular HTRs.

No. of Antigens
028 Globoside 1 No, but anti-PP1Pk is associated with a high rate of
spontane- ous abortion.
No.
029 Gill 1 No.
030 3 No. No.
RHAG
031 1 No. No.
FORS
032 JR 1 Mild DHTRs and one case of AHTR have been reported to Two examples of severe HDFN due to anti-Jra have
AABB T ECHNICAL M A NUAL

be caused by anti-Jra. been reported.

Mild to severe HTR due to anti-Lan has been Anti-Lan is generally not a cause of HDFN, although cases
033 Lan 1 reported. of mild HDFN have been reported.

Anti-Vel is generally not a cause of HDFN, although cases

034* Vel 1 Severe AHTR and mild to severe DHTR due to anti-Vel of severe HDFN have been reported.
have been reported.

*Provisionally assigned blood group systems.

ISBT = International Society of Blood
Transfusion.
Hemolytic Transfusion Reaction (HTR), Acute (AHTR), or
(HDFN)
Hemolytic
Delayed (DHTR)
Disease of the Fetus and
 CHA P TER 1 4
The MNS Glycoproteins and the
Genes that Encode Them
The antigens of the MNS system are located
on one or both of two glycoproteins:
glycophorin A (GPA, CD235A) and
glycophorin B (GPB, CD235B). Each crosses
the membrane once and has an external N-
terminal domain and a C-terminal cytosolic
domain. The extracellular domains of both
molecules have many sialic acid-rich O-
glycans; GPA is N-glycosylated at asparagine-
45 (26 in the mature protein), whereas GPB is
not N-glycosylated. The long cytosolic tail of
GPA interacts with the cyto- skeleton. GPA is
abundant, with about 106 cop- ies per red cell,
whereas GPB has only about 200,000 copies
per cell. GPA forms an associa-
tion in the membrane with Band 3 (Diego
blood group system), and both GPA and GPB
appear to be part of the Band 3/Rh ankyrin
complex (Fig 14-1).8,9
GYPA and GYPB, the genes encoding
GPA and GPB, comprise seven and five exons,
re- spectively. A region of intron 3 of GYPB is
ho- mologous to exon 3 of GYPA but is not ex-
pressed because of a defective splice site (Fig
14-2).10 Exon 1 of each gene encodes a 19-ami-
no-acid signal peptide that is not present in
the mature protein. A third gene, GYPE,
proba- bly produces a third glycoprotein,
glycopho- rin E, but this plays little or no part
in MNS an- tigen expression.
GPA is restricted to blood cells of ery-
throid origin and is often used as an
erythroid marker. Both GPA and GPB are
exploited by the malarial parasite
Plasmodium falciparum as receptors for
binding to red cells and may be critical to
the invasion process.11,12 A GPA- like
molecule has been detected on renal en-
dothelium.
M ( MNS1 ), N (MNS2), S ( MNS3
), AND s (MNS4)
M and N (as detected by most anti-N
reagents) are antithetical antigens and
polymorphic in all populations tested (Table
14-2). M and N are located at the N-
terminus of GPA. M-active GPA has serine
and glycine at the first and fifth
Other Blood Groups ■ 341
TABLE 14-2. Frequencies of Some Phenotypes
of the MNS System
Frequency (%)
Phenotype Whites Blacks
M+ N– 30 25
M+ N+ 49 49
M– N+ 21 26
S+ s– 10 6
S+ s+ 42 24
S– s+ 48 68
S– s– 0 2
positions of the mature protein (positions 20
and 25); N-active GPA has leucine and
glutam- ic acid at those positions.
S and s are another pair of polymorphic
antithetical antigens of the MNS system
(Table 14-2). Family studies show tight
linkage be- tween M/N and S/s. The S and s
antigens repre- sent a Met48Thr (29 in mature
protein) poly- morphism in GPB. The amino-
terminal 26 amino acids of GPB mature
protein are usually identical to those of the N
form of GPA. Conse- quently, in almost all
people of European an- cestry and most people
of other ethnicities, GPB expresses ‘N.’
However, because GPB is much less abundant
than GPA, most anti-N re- agents do not detect
the ‘N’ antigen on GPB. The N-terminal
region of GPA is cleaved from intact red cells
by trypsin, whereas that of GPB is not.
Consequently, M and N antigens on GPA are
trypsin sensitive, and S, s, and ‘N’ on GPB are
trypsin resistant. In contrast, with -
chymotrypsin treatment of red cells, M and N
activity is only partially reduced, whereas S, s,
and ‘N’ expression is completely destroyed.
M, N, S, s, and ‘N’ are all destroyed by
treatment of the red cells with papain, ficin,
bromelin, or pronase, although this effect with
S and s is variable.
 342
■
AABB T ECHNICAL M A NUAL

FIGURE 14-1. Model of two proposed membrane complexes containing Band 3 and Rh proteins: 1) containing tetramers of Band 3 and heterotrimers of RhD,
RhCE, and RhAG, and linked to the spectrin matrix of the cytoskeleton through Band 3, protein 4.2, and ankyrin; and 2) containing Band 3, RhD, and RhCE,
and linked to the spectrin/actin junction through glycophorin C (GPC), p55, and protein 4.1 and through Band 3 and adducin.
 CHA P TER 1 4
FIGURE 14-2. GYPA, GYPB, and the hybrid GYPB–A–B gene responsible for GP.Mur, and a representation of
the proteins they encode, showing the regions of proteins encoded by the various exons.
 = pseudoexon not represented in the mRNA or the encoded protein.
S–s–U– Phenotype
The red cells of about 2% of Americans of
Afri- can ancestry and a higher proportion
of Africans are S–s– and lack the high-
prevalence antigen U (MNS5). The S–s–U–
phenotype of- ten results from
homozygosity for a deletion of the coding
region of GYPB, but other, more complex
molecular phenomena involving hy- brid
genes may also give rise to an S–s– pheno-
type with expression of a variant U antigen.
U is generally resistant to denaturation by
prote- ases—papain, ficin, trypsin, -
chymotryp- sin—although anti-U is not
reactive with pa- pain-treated red cells in
rare cases.
M, N, S, s, and U Antibodies and
Their Clinical Significance
Anti-M is a relatively common antibody,
whereas anti-N is quite rare. Most anti-M
and
-N are not active at 37 C and are not
clinically significant.13 They can generally
be ignored in transfusion practice. If room-
temperature in- cubation is eliminated
from compatibility
Other Blood Groups ■ 343
testing and screening for antibodies, these an-
tibodies are not detected. When M or N anti-
bodies active at 37 C are encountered, antigen-
negative or red cells that are compatible by an
indirect antiglobulin test (IAT) should be pro-
vided.5 Very occasionally, anti-M and -N have
been implicated as the cause of acute and de-
layed HTRs, and anti-M has very rarely been
responsible for severe HDFN. A few cases of
warm autoimmune hemolytic anemia (AIHA)
caused by autoanti-N have been described,
one of which had a fatal outcome. Autoanti-M
responsible for warm AIHA has not been re-
ported.
Anti-S and -s are usually immune globu-
lin G (IgG) antibodies that are active at 37 C.
They have been implicated in HTRs and have
caused severe and fatal HDFN. Autoanti-S has
caused AIHA. If immunized, individuals with
S–s–U– red cells may produce anti-U. Anti-U
has been responsible for severe and fatal HTRs
and HDFN. Autoanti-U has been implicated in
AIHA.
344 ■ AABB T EC HNIC AL MANUAL
Other MNS Antigens and Antibodies
The other MNS antigens are either of high
or low prevalence in most populations. The
simi- larity of sequence between certain
regions of GYPA and GYPB may
occasionally lead to GYPA pairing with
GYPB during meiosis. If re- combination
then occurs, either by crossing over or by a
less well-defined mechanism called “gene
conversion,” a hybrid gene can be formed
consisting partly of GYPA and partly of
GYPB. A large variety of these rare hybrid
genes exists, and they give rise to low-preva-
lence antigens and, in the homozygous state,
to phenotypes that lack high-prevalence
anti- gens.10 Red cells of some of the
phenotypes re- sulting from hybrid genes
react with an anti- body called “anti-Mia.”
The phenotypes were grouped together as
the Miltenberger series, but this
classification is obsolete because the hybrids
are now well defined.14
One example that is relatively common in
Southeast Asia is the hybrid gene that is re-
sponsible for the GP.Mur (previously Mi.III)
phenotype. The hybrid gene is mostly
GYPB, but a small region of GYPB
encompassing the 3 end of the pseudoexon
and the 5 end of the adjacent intron has
been replaced by the equivalent region from
GYPA. This means that the defective splice
site in GYPB is now re- placed by the
functional splice site from GYPA, and the
new, composite exon is expressed in the
mRNA and represented in the protein.10 This
provides an unusual amino acid se- quence
that is immunogenic and represents the
antigen Mur. The amino acid sequence that
results from the junction of exons B3 and
A3 gives rise to Hil and MINY (Fig 14-2).
Mur antigen is rare in people of
European and African ethnicity but has a
prevalence of about 7% in people of
Chinese and 10% in people of Thai ancestry.
Anti-Mur has the po- tential to cause severe
HTRs and HDFN. In Hong Kong and
Taiwan, anti-Mur is the most common blood
group antibody after anti-A and -B. In
Southeast Asia, it is important that red cells
for antibody detection include a Mur+
sample.15
Antibodies to regions of GPA with the ge-
neric name Ena that may be made by very rare
individuals who lack all or part of GPA have
caused severe HTRs and HDFN.
THE LUTHERAN SYSTEM
Lutheran is a complex system consisting of 20
antigens, including four antithetical pairs: Lua/
Lub (His77Arg); Lu6/Lu9 (Ser275Phe); Lu8/
Lu14 (Met204Lys); and Aua/Aub
(Thr529Ala). The other Lutheran antigens are
16
highly preva- lent in all populations tested. Lua
(LU1) has a prevalence of about 8% in people
of European or African ethnicity but is rare
elsewhere; its antithetical antigen, Lub (LU2),
is common everywhere. In the other Lutheran
polymor- phism, Aua and Aub have a
prevalence of around 80% and 50%,
respectively, in people of European ancestry.
Lutheran antigens are destroyed by treat-
ment of the red cells with trypsin or -
chymo- trypsin, whereas papain and ficin
have little ef- fect. Most Lutheran antibodies
are not reactive with red cells treated with
the sulfhydryl re- agents 2-
aminoethylisothiouronium bromide (AET)
or dithiothreitol (DTT), which reduce the
disulfide bonds of the immunoglobulin su-
perfamily (IgSF) domains (Method 3-18).
The Lutheran antigens are located on a
pair of glycoproteins that span the
membrane once, have five extracellular IgSF
domains, and differ by the length of their
cytoplasmic do- mains as a result of
alternative RNA splicing. The isoform with
the longer cytoplasmic do- main interacts
with spectrin of the membrane skeleton.17
The location of the Lutheran anti- gens on
the IgSF domains is shown in Fig 14-3. The
Lutheran glycoproteins are adhesion mol-
ecules that bind isoforms of laminin that
con- tain -5 chains. Laminin is a
glycoprotein of the extracellular matrix, and
Lutheran-laminin interactions may play a
role in the migration of mature erythroid
cells from the marrow to the peripheral
blood at the latest stages of erythro- poiesis.
Upregulation of Lutheran glycopro- teins on
red cells of patients with sickle cell disease
could play a part in adhesion of these cells
to the vascular endothelium and the re-
sultant crises of vascular occlusion.17
 CHA P TER 1 4
FIGURE 14-3. Diagram of the two isoforms of
the Lutheran glycoprotein, showing the five
extracellular immunoglobulin superfamily
domains and the location of the Lutheran
antigens on these domains, the single
membrane-spanning domain, and the
cytoplasmic domains.
The extremely rare Lunull phenotype aris-
es from homozygosity for inactive LU gene.18
The red cells lack any expression of Lutheran
antigens, and individuals with this phenotype
Other Blood Groups ■ 345
may produce anti-Lu3, which is reactive
with all red cells except those from other
Lu(a–b–) individuals. Heterozygosity for
inactivating mutations in the erythroid
transcription fac- tor KLF1 is responsible
for In(Lu), a Lumod phe- notype with
extremely weak expression of Lu- theran
antigens that are detectable only by
adsorption/elution techniques. Mutations in
KLF1 also affect other blood group genes
and cause weakened expression of several
other antigens, including P1, Inb, and
AnWj.19 The In(Lu) phenotype has a
prevalence of around 0.03%. In one family,
hemizygosity for a muta-
tion in the X-linked gene for the major ery-
throid transcription factor GATA-1 resulted
in an Lumod phenotype with an X-linked
mode of inheritance.20
Lutheran antibodies are most often IgG
and demonstrate reactivity best by IAT, but
have generally been implicated only in mild
delayed HTRs and have not caused severe
HDFN. Anti-Lua may be “naturally occurring”
or immune, and it is often IgM but may also be
IgG and IgA. These antibodies are usually
reac- tive by direct agglutination of Lu(a+) red
cells but often also reactive by an IAT and
may show a mixed-field-like agglutination that
is charac- teristic of this and other antibodies
to Luther- an antigens.
THE KELL AND KX SYSTEMS
The antigen often referred to as “Kell,” but
cor- rectly named “K” or “KEL1,” is the
original an- tigen of the Kell system and the
first blood group antigen to be identified
following the discovery of the antiglobulin
test in 1946. Its antithetical antigen, k or
KEL2, was identified 3 years later. The Kell
system now consists of 35 antigens numbered
from KEL1 to KEL38, of which three are
obsolete.21,22 The Kell system includes six
pairs (K/k, Jsa/Jsb, K11/K17, K14/ K24,
VLAN/VONG, and KYO/KYOR) and one
triplet (Kpa/Kpb/Kpc) of Kell antithetical
anti- gens. Initially, most antigens joined the
Kell system through genetic associations
observed in family studies. These
associations have now been confirmed by
DNA sequencing of the KEL gene.
346 ■ AABB T EC HNIC AL MANUAL

The Kell Glycoprotein and the KEL TABLE 14-3. Frequencies of Some Kell
Gene Phenotypes

The Kell antigens are located on a red cell

membrane glycoprotein (CD238) with four Frequency (%)
or five N-glycans but no O-glycosylation. Phenotype Whites Blacks
Kell is unique as a blood group antigen
because it is a Type II membrane K– k+ 91.0 98
glycoprotein; it spans the membrane once
K+ k+ 8.8 2
and has a short N-terminal domain in the
cytosol and a large C-terminal domain K+ k– 0.2 Rare
outside the membrane (Fig 14-4).23 The Kp(a–b+) 97.7 100
extracellular domain has 15 cysteine resi-
dues and is extensively folded by disulfide Kp(a+b+) 2.3 Rare
bonding, although crystallographic studies Kp(a+b–) Rare 0
are required to determine the molecule’s
three- dimensional structure. Kell system Js(a–b+) 100.0 80
antigens depend on the conformation of the Js(a+b+) Rare 19
glycopro- tein and are sensitive to disulfide-
bond-reduc- ing agents, such as DTT and Js(a+b–) 0 1
AET. K, Kpa, and Jsa are extremely rare in populations
The Kell glycoprotein is linked through a of Asian ancestry.
single disulfide bond to the Xk protein (Fig 14-

4) , an integral membrane protein that express-

es the Kx blood group antigen (XK1).
Absence of Xk protein from the red cell results
in re- duced expression of the Kell
glycoprotein and weakened Kell antigens
(McLeod phenotype, see below).
The KEL gene is located on chromosome
7q34. It is organized into 19 exons: exon 1
en- codes the probable translation-initiating
me- thionine; exon 2, the cytosolic domain;
exon 3, the membrane-spanning domain;
and exons 4 through 19, the large
extracellular domain.

FIGURE 14-4. Diagram of the Kell and Xk
proteins linked by a single disulfide bond. The Xk
protein has cytoplasmic N- and C-terminal
domains and 10 membrane-spanning domains.
The Kell glycoprotein has a large, folded,
extracellular, C-terminal domain and an
intracellular N-terminal domain.
Kell Antigens
K has a prevalence of about 9% in people of
European ancestry and about 1.5% in people
of African ancestry. It is rare in East Asia
(Table 14-3). The k antigen is highly
prevalent in all populations. K and k result
from a single nu- cleotide polymorphism
(SNP) in exon 6, which encodes Met193 in
K and Thr193 in k. Asn191
 CHA P TER 1 4
is N-glycosylated in the product of the k but
not the K allele.
Kpa (KEL3) is found in about 2% of
people of European ancestry and is not present
in people of African or Japanese ancestry
(Table 14-3); Kpb (KEL4) has high prevalence
in all populations. Whereas 2.3% of people of
Euro- pean ancestry are Kp(a+) only 1.2% of
K+ per- sons of that same ancestry are Kp(a+).
Nine percent of people of European ancestry
are K+, but only 2.7% of Kp(a+) people from
the same population are K+. This strong allelic
associa- tion has been confirmed by family
studies. Only one example of the KKpa
haplotype has been found.24 Kpc (KEL21), an
antigen with very low prevalence, is the
product of another allele of Kpa and Kpb. KEL
alleles encoding the three Kp antigens differ by
single base substi- tutions within codon 281
(exon 8): Kpa, TGG, Trp281; Kpb, CGG,
Arg281; and Kpc, CAG, Gln281. The mutation
associated with Kpa ex- pression appears to
reduce the quantity of Kell glycoprotein in the
red cell membranes, giving rise to a slight
reduction in expression of Kell antigens in
Kpa/Kpa homozygotes but a more obvious
weakening of Kell antigens in individ- uals
who are heterozygous for Kpa and the null
allele K0.
Jsa (KEL6) is almost completely confined
to people of African ethnicity. The prevalence
of Jsa in African Americans is about 20%
(Table 14-3). Jsb (KEL7) is highly prevalent in
all popu- lations, and Js(a+b–) has not been
found in persons of non-African ethnicity. Jsa
represents Pro597; Jsb, Leu597.
K17 (Wka) (Ala302) has a prevalence of
0.3% in English donors; K11 (Val302), its
anti- thetical antigen, has very high
prevalence. K14 (Arg180) and K24 (Pro180)
are very high- and very low-prevalence
antigens, respectively. VLAN and VONG are
low-prevalence antigens associated with
Arg248Gln and Arg248Trp, re- spectively.
The presence of the low-prevalence anti-
gens Ula, KYO, and K23 and the absence of
the high-prevalence antigens K12, K13, K18,
K19, K22, TOU, RAZ, KALT, KTIM, KUCI,
KANT,
KASH, KELP, KETI, and KHUL result from
sin- gle amino acid substitutions in the Kell
glyco- protein.
Other Blood Groups ■ 347
Kell antigens are resistant to papain,
ficin, trypsin, and -chymotrypsin but are
de- stroyed by a mixture of trypsin and -
chymo- trypsin. They are also destroyed by
DTT and AET (see above) and by EDTA
glycine.
Kell Antibodies and Their
Clinical Significance
Kell antibodies are usually IgG, predominantly
IgG1.13 They should be considered potentially
clinically significant from the perspective of
causing severe HDFN and HTRs. Patients with
Kell antibodies should be transfused with anti-
gen-negative blood whenever possible.
Anti-K is the most common immune red
cell antibody outside the ABO and Rh
systems; one-third of all non-Rh red cell
immune anti- bodies are anti-K. An
antiglobulin test is usual- ly the method of
choice for detecting anti-K, although
occasional samples may agglutinate red cells
directly. Most anti-K appears to be in- duced
by blood transfusion. Because anti-K can
cause severe HDFN, it is usual practice in
some countries for girls and women of child-
bearing potential to receive only K– red cells.
Anti-K, -k, -Kpa, -Kpb, -Jsa, -Jsb, -Ku, -Ula, -
K11,
-K19, and -K22 are all reported to have
caused severe HDFN. Anti-K, -k, -Kpb, -Jsa,
-Jsb, -Ku, and -K19 have all been implicated
in acute or delayed HTRs.
The pathogenesis of HDFN caused by
anti-K differs from that resulting from anti-
D. Anti-K HDFN is associated with lower
concen- trations of amniotic fluid bilirubin
than anti-D HDFN of comparable severity.
Postnatal hy- perbilirubinemia is not
prominent in infants with anemia caused by
anti-K. There is also re- duced
reticulocytosis and erythroblastosis in the
anti-K disease compared with anti-D HDFN.
These symptoms suggest that anti-K HDFN
is associated with a lower degree of he-
molysis and that fetal anemia in anti-K
HDFN results predominantly from a
suppression of erythropoiesis. The Kell
glycoprotein appears on erythroid
progenitors at a much earlier stage of
erythropoiesis than do Rh antigens.
Consequently, anti-K probably facilitates
phagocytosis of K+ erythroid progenitors at
an early stage of development by
macrophages in
348 ■ AABB T EC HNIC AL MANUAL

en-
the fetal liver, before the erythroid cells pro-
duce hemoglobin.25
Antibodies mimicking Kell specificities
have been responsible for severe AIHA.
Pres- ence of the autoantibody is often
associated with apparent depression of all
Kell antigens. Although most examples of
anti-K are stimu- lated by pregnancy or
transfusion, a few cases of apparently non-
red-cell immune anti-K have been
described. In some cases, the anti- bodies
were found in untransfused, healthy, male
blood donors; in others, microbial infec-
tion was implicated as an immunizing agent.

Null (K0) and Mod Phenotypes

Like most blood group systems, Kell has a
null phenotype (K0) in which none of the Kell
anti- gens is expressed and the Kell
glycoprotein
cannot be detected in the membrane. Immu-
nized K0 individuals may produce anti-Ku
(anti-KEL5), an antibody that is reactive
with all cells except those of the K0
phenotype. Ho- mozygosity for a variety of
nonsense, mis- sense, and splice-site
mutations have been as- sociated with K0
phenotype.26
Kmod red cells have only very weak
expres-
sion of Kell antigens, and individuals with
this phenotype are homozygous (or doubly
hetero- zygous) for missense mutations,
resulting in single-amino-acid substitutions
within the Kell glycoprotein.27 Some Kmod
individuals
make an antibody that resembles anti-Ku but
differs in being nonreactive with K mod red
cells. Other phenotypes in which Kell antigens
have substantially depressed expression result
from Kpa/K0 heterozygosity (see above),
absence of Xk protein (see below), and
absence of the Gerbich antigens Ge2 and Ge3,
which are lo-
cated on the glycophorins C and D. The reason
for this phenotypic association between Kell
and Gerbich is not known, although Kell
glyco- proteins, Xk, and glycophorins C and D
could all be located within the same
membrane pro- tein complex (Fig 14-1).

Functional Aspects
The Kell protein has structural and sequence
homology with a family of zinc-dependent
 and psychiatric symptoms. It results from
dopeptidases that hemizygosity for inactivating mutations and
process a variety of deletions of the XK gene.29 McLeod
peptide hormones. syndrome is associated with McLeod
Although the function phenotype, in which Kell antigens are
of the Kell glycoprotein expressed weakly and Km (KEL20) as well
is not known, it is as Kx are absent. When transfused, people
enzymatically active with McLeod phenotype without chronic
and can cleave the granulomatous disease (CGD) produce anti-
biologically inactive Km only, which is compatible
peptide big-endothelin-3 with both McLeod and K0 phenotype red
to create the biologi- cells. The function of the Xk-Kell complex
cally active is not known, but Xk has structural
vasoconstrictor resemblance to a family of neurotransmitter
endothelin-3. Con- transporters.
sequently, Kell might Deletion of part of the X chromosome
play a role in regulating that includes XK may also include CYBB, ab-
vascular tone, but there sence of which is responsible for X-linked
is no direct evidence for CGD. When transfused, CGD patients with
this.28 No obvious McLeod syndrome usually produce anti-Kx
pathogenesis is associat- plus anti-Km, making it almost impossible to
ed with the K0 find compatible donors. It is recommended,
phenotype.
In addition to erythroid cells, Kell anti-
gens may be present on
myeloid progenitor cells,
and Kell glycoprotein has
been detected in testis,
lymphoid tissues, and
with Xk protein in
skeletal muscle.

Kx Antigen (XK1),
McLeod Syndrome,
and McLeod
Phenotype
Kx is the only antigen of
the Kx blood group
system. It is located on a
polytopic protein that
spans the red cell
membrane 10 times and
is linked to the Kell
glycoprotein by a single
di- sulfide bond (Fig 14-
4). Xk protein is encoded
by the XK gene on
chromosome Xp21.1.
McLeod syndrome
is a very rare X-linked
condition that develops
almost exclusively in
males and is associated
with acanthocytosis and
a variety of late-onset
muscular, neurolog- ic,
 CHA P TER 1 4 Other Blood Groups ■ 349

therefore, that transfusion of males with
CGD and McLeod syndrome be avoided.
THE DUFFY SYSTEM
The Duffy system officially includes five anti-
gens that reside on a glycoprotein known as
the Duffy antigen receptor for chemokines
(DARC). The DARC gene consists of two
exons, with exon 1 encoding only the first
seven ami- no acids of the Duffy
glycoprotein.21,22 DARC is on chromosome
1q23.2.
Fya (FY1) and Fyb (FY2)
In people of European and Asian ancestry, the
Duffy polymorphism consists of two antigens,
Fya and Fyb, giving rise to three phenotypes:
Fy(a+b–), Fy(a+b+), and Fy(a–b+) (Table 14-
4). The Fya and Fyb alleles represent an SNP in
exon 2 of DARC that encodes Gly42 and
Asp42, respectively, in the N-terminal
extracellular domain of the glycoprotein (Fig
14-5). Fya and Fyb are very sensitive to most
proteolytic en- zymes, including bromelin, -
chymotrypsin, ficin, papain, and pronase, but
are not de- stroyed by trypsin. People of
African ethnicity have a third allele, Fy, that is
more abundant than Fya and Fyb. Fy produces
no Duffy glyco- protein on red cells and,
hence, neither Fya nor Fyb. Individuals who are
homozygous for Fy have the red cell
phenotype Fy(a–b–), whose prevalence varies
from about 70% in African Americans to
100% in residents of Gambia (Ta- ble 14-4).
The coding region for the Fy allele in
peo- ple of African ancestry is identical to that
of the Fyb allele, which encodes Asp42. Fy
pro- duces no Duffy glycoprotein and no Fyb
anti- gen in red cells because of an SNP in the
pro- moter region of DARC. This mutation
disrupts the binding site for the erythroid-
specific GATA-1 transcription factor and
prevents ex- pression of the gene in erythroid
tissue.30 Duffy glycoprotein is present on many
cells through- out the body; thus, Fy(a–b–)
people of African ethnicity lack Duffy
glycoprotein on their red cells but not on cells
from other tissues. This explains why they do
not make anti-Fyb and only very rarely make
anti-Fy3 or anti-Fy5 (see below). Although the
GATA-1 binding site mu- tation in people of
African ancestry has been found only in Duffy
genes with the Fyb se- quence, the mutation has
been detected in silent Fya alleles in Papua
New Guinea and Brazil.
Fyx is a weak form of Fyb that results from
an Fyb allele with a missense mutation
encod- ing an Arg89Cys substitution in a
cytosolic do- main of the glycoprotein (Fig
14-5). Fyb anti- gen may be undetected in
some samples of anti-Fyb, although the
antigen can be detected by
adsorption/elution.
Fy3, Fy4, Fy5, and Fy6
Very rare people of non-African ancestry with
Fy(a–b–) red cells are homozygous for inacti-
vating mutations in their DARC genes. These
individuals have no Duffy glycoprotein on

TABLE 14-4. Duffy Phenotypes and Genotypes in Selected Populations

Genotype Frequency (%)

European or
Phenotype Asian Ethnicity African Ethnicity Whites Blacks Japanese

Fy(a+b–) Fya/Fya Fya/Fya or Fya/Fy 20 10 81

Fy(a+b+) Fya/Fyb Fya/Fyb 48 3 15

Fy(a–b+) Fyb/Fyb Fyb/Fyb or Fyb/Fy 32 20 4

Fy(a–b–) Fy/Fy Fy/Fy 0 67 0

350 ■ AABB T EC HNIC AL MANUAL
FIGURE 14-5. Diagram of the Duffy
glycoprotein (DARC), with a glycosylated
external N-terminal domain, seven membrane-
spanning domains, and cytoplasmic C-terminus.
The positions of the Fya/Fyb polymorphism and of
the amino acid substitution responsible for the
Fyx phenotype are shown.
their red cells and would not be expected to
have it in any other tissues. All were found
through the presence in their sera of anti-
Fy3, an antibody that is reactive with all red
cells except those of the Fy(a–b–)
phenotype. FY6, like Fya and Fyb, is
sensitive to protease treat- ment, whereas
FY3 and FY5 are resistant. Fy5 is also
absent from cells of the Fy(a–b–) pheno-
type, but unlike Fy3, Fy5 is also absent from
cells of the Rhnull phenotype. The Duffy
glyco- protein may belong to the junctional
mem- brane protein complex, which also
contains
Rh proteins (Fig 14-1).9 Anti-Fy5 has been
found only in multitransfused individuals of
African ethnicity. Anti-Fy6 was the
designation given to a monoclonal antibody
that produced serologic reactions very
similar to those of anti-Fy3. Anti-Fy4
appeared to be reactive with red cells of
individuals with the silent Fy allele.
However, the work could not be repeated,
the antibody is no longer available, and Fy4
is now obsolete.
Duffy Antibodies and Their
Clinical Significance
Anti-Fya is a relatively common antibody,
and anti-Fyb is about 20 times less
common.31 IgG1
usually predominates, and these antibodies
are generally detected by an antiglobulin
test. Naturally occurring examples are very
rare.
Anti-Fya and anti-Fyb may cause acute
or delayed HTRs. Although generally mild,
some have proved fatal. These antibodies
have also been responsible for from mild to
severe HDFN. Anti-Fy3 has been
responsible for acute and delayed HTRs and
anti-Fy5 for de- layed HTRs.
The Duffy Glycoprotein, a Receptor
for Chemokines
The Duffy glycoprotein is a red cell receptor
for a variety of chemokines, including
interleukin- 8, monocyte chemotactic protein-
1, and mela- noma growth stimulatory
activity.32 It traverses the membrane seven
times, and a 63-amino- acid extracellular N-
terminal domain contains two potential N-
glycosylation sites and a cyto- plasmic C-
terminal domain (Fig 14-5). This ar-
rangement is characteristic of the G protein-
coupled superfamily of receptors that includes
chemokine receptors.
The function of the DARC on red cells
is not known. It has been suggested that it
might act as a clearance receptor for
inflammatory mediators and that Duffy-
positive red cells function as a “sink,” or as
scavengers, for the removal of unwanted
chemokines.33 If so, this function must be of
limited importance be- cause Duffy is not
present on the red cells of most individuals
of African ancestry. It has been suggested
that DARC on red cells reduces
angiogenesis and, consequently, the progres-
sion of prostate cancer by clearing
angiogenic chemokines from the tumor
microenviron- ment. This potential effect of
erythroid DARC could provide an
explanation for the substan- tially higher
levels of prostate cancer in men of African
ancestry compared with those of Euro- pean
ancestry.34
DARC is present in many organs,
where it is expressed on endothelial cells
lining post- capillary venules.35 Duffy
glycoprotein on vas- cular endothelium may
be involved in the inhibition of cancer-cell
metastasis and induc- tion of cellular
senescence.34 DARC may also
 CHA P TER 1 4 Other Blood Groups ■ 351

facilitate movement of chemokines across

the endothelium.

The Duffy Glycoprotein and Malaria

The Duffy glycoprotein is a receptor for mero-
zoites of Plasmodium vivax, the parasite re-
sponsible for a form of malaria that is widely
distributed in Africa but is less severe than
ma- laria resulting from P. falciparum
infection. Red cells with the Fy(a–b–)
phenotype are re- sistant to invasion by P.
vivax merozoites. Con- sequently, the Fy allele
confers a selective ad- vantage in geographic
areas where P. vivax is endemic; this advantage
probably balances out any potential
disadvantage resulting from the absence of the
chemokine receptor on red cells.33

THE KIDD SYSTEM FIGURE 14-6. Diagram of the Kidd glycoprotein,

The Kidd system consists of three antigens lo-
cated on a glycoprotein with 10 membrane-
spanning domains, cytoplasmic N- and C-ter-
mini, and one extracellular N-glycosylation
site (Fig 14-4).22,36 The Kidd gene (SLC14A1)
contains 11 exons, 4 through 11 encoding the
mature protein, and is located on chromo-
some 18q12.3.
a b
Jk (JK1) and Jk (JK2)
Jka and Jkb are the products of antithetical al-
leles and represent Asp280 and Asn280 in the
fourth external loop of the Kidd glycoprotein
(Fig 14-6). They have similar prevalence in
populations of European and Asian ancestry,
but Jka is more common than Jkb in people of
African ancestry (Table 14-5). Kidd antigens
are resistant to proteolytic enzymes, such as
papain and ficin.
Jk(a–b–) and Jk3
The null phenotype, Jk(a–b–) Jk:–3, usually
re- sults from homozygosity for a silent gene at
the JK locus. Although very rare in most
popu- lations, the null phenotype is relatively
com- mon in people of Polynesian ethnicity,
with a prevalence of around 1 in 400, but as
high as
a urea transporter, with cytoplasmic N- and C-
terminal domains, 10 membrane-spanning
domains, and an N-glycan on the third
extracellular loop. The position of the Jka/Jkb
polymorphism is shown on the fourth external
loop.
sian null allele contains a splice site
mutation in intron 5 that results in the loss of
exon 6 from the mRNA. In people of
Finnish ancestry, where Jk(a–b–) is less rare
than in other popu- lations of European
ancestry, the mutation re- sponsible encodes
a Ser291Pro substitution. Immunized
individuals with the Jk(a–b–) phe- notype
may produce anti-Jk3. An extremely rare
form of Jk(a–b–) phenotype found in
TABLE 14-5. Kidd Phenotypes in Three
Populations
 Jk(a+b–) 26 52 23
Frequency (%)
Jk(a+b+) 50 40 50
Phenotype Whites Blacks Asians
Jk(a–b+) 24 8 27
1.4% in those of Niuean ancestry. The Polyne-
352 ■ AABB T EC HNIC AL MANUAL
people of Japanese ethnicity results from
het- erozygosity for a dominant inhibitor
gene, named In(Jk) in analogy with the
In(Lu) domi- nant inhibitor of Lutheran and
other antigens. Very weak expression of Jka
and/or Jkb can be detected on In(Jk) red cells
by adsorption/elu- tion tests.
Kidd Antibodies and Their
Clinical Significance
Anti-Jka and –Jkb are not common antibodies
and are generally found in antibody
mixtures. They are usually IgG1 and IgG3,
but some are partly IgG2, IgG4, or IgM.
About 50% of anti-Jka and –Jkb bind
complement.13
Kidd antibodies are often difficult to
de- tect. Some agglutinate antigen-positive
cells directly, but the reactions are usually
weak. Generally, an antiglobulin test is
required, and use of enzyme-treated cells
may be necessary to detect weaker
antibodies.
Kidd antibodies may cause severe acute
HTRs. They are also a very common cause
of delayed HTRs, probably because they are
of- ten not detected in pretransfusion testing
due to their tendency to drop to low or
undetect- able levels in plasma. Anti-Jk3
can also cause acute or delayed HTRs.
Despite their hemolyt- ic potential, Kidd
antibodies only very rarely cause severe
HDFN.
Kidd antibodies have been implicated
in
acute renal transplant rejection, suggesting
that Kidd antigens can behave as histocom-
patibility antigens.37
The Kidd Glycoprotein in
Urea Transportation
The Kidd antigens are located on a red cell
urea transporter, also known as “human urea
transporter 11” (HUT11 or UT-B1).38 When
red cells approach the renal medulla, which
con- tains a high concentration of urea, the
urea transporter permits rapid uptake of urea
and prevents the cells from shrinking in the
hyper- tonic environment. As the red cells
leave the renal medulla, urea is transported
rapidly out of the cells, preventing the cells
from swelling and carrying urea away from
the kidney.
HUT11 has been detected on endothelial
cells of the vasa recta, the vascular supply of
the re- nal medulla, but it is not present in
renal tu- bules.
Normal red cells are rapidly lysed by 2M
urea because urea transported into the cells
makes them hypertonic and they burst as a
re- sult of the osmotic influx of water.
Because of the absence of the urea
transporter, Jk(a–b–) cells are not
hemolyzed by 2M urea, and this can be used
as a method for screening for Jk(a– b–)
donors.39
The Jk(a–b–) phenotype is not associated
with any clinical defect, although two
unrelat- ed Jk(a–b–) individuals had a mild
urine- concentrating defect.40
THE DIEGO SYSTEM
Band 3, the Red Cell Anion Exchanger
The 22 antigens of the Diego system are locat-
ed on Band 3, the common name for the red
cell anion exchanger or solute carrier family
4 A1 (SLC4A1).41 Band 3 is a major red cell
membrane glycoprotein with approximately
106 copies per red cell. Band 3 has a
transmem- brane domain that traverses the
membrane about 14 times, with an N-glycan
on the fourth extracellular loop. Band 3 also
has a long cyto- plasmic N-terminal domain
that interacts with the membrane skeleton
proteins ankyrin, 4.1R, and protein 4.2 and
functions as a bind- ing site for hemoglobin
(Figs 14-1 and 14-7). The short cytoplasmic
C-terminal domain binds carbonic anhydrase
II.
Band 3 in red cells has at least two major
functions: the rapid exchange of HCO 3
–
and
Cl ions, which are important in CO2 transport,
–
and attachment of the red cell membrane to
the cytoskeleton.9 Tetramers of Band 3 form
the core of the Band 3/Rh ankyrin macrocom-
plex of red cell membrane proteins, which
could function as a gas channel for O2 and
CO2.8 Band 3 is also a component of the junc-
tional complex that links the red cell mem-
brane to the membrane skeleton via glycopho-
rin C (Fig 14-1).9 The Band 3 gene (SLC4A1)
 CHA P TER 1 4
FIGURE 14-7. Diagram of Band 3, the Diego
glycoprotein and anion exchanger, with cytoplasmic
N- and C-terminal domains, 14 membrane-spanning
domains, and an N-glycan on the fourth
extracellular loop (although the precise
conformation is still controversial). The locations
of 22 antigens of Diego system on the
extracellular loops are shown.
consists of 20 exons of coding sequence and
is on chromosome 17.
Dia (DI1) and Dib (DI2); Anti-Dia and -
Dib
Dia, the original Diego antigen, is very rare
in people of European or African ancestry
but has a prevalence of 5% in people of
Chinese or Japanese ancestry and an even
higher preva- lence in the indigenous
peoples of North and South America,
reaching 54% in the Kaingan- ges Indians of
Brazil. Dib is a high-prevalence antigen in
almost all populations. Dia and Dib represent
an amino acid substitution in the seventh
extracellular loop of Band 3—Leu854 in Dia
and Pro854 in Dib.
Anti-Dia and -Dib are usually IgG1 plus
IgG3 and typically require an antiglobulin
test for detection, although a few directly
aggluti- nating samples have been found.
Anti-Dia oc- casionally binds complement
and lyses un-
Other Blood Groups ■ 353
treated red cells. Anti-Dia, which is present
in 3.6% of multitransfused patients in
Brazil, can cause severe HDFN. Anti-Dib
has, very rarely, been responsible for serious
HDFN. Apart from one example of anti-Dia
causing a de- layed reaction, neither anti-Dia
nor anti-Dib has been reported to be
responsible for an HTR.13,21
Antigens of the Diego system are not de-
stroyed by proteolytic enzymes, such as papa-
in, ficin, or trypsin; however, the antigens car-
ried on the third extracellular loop (Rba, Tra,
WARR, Vga, Wda, BOW, NFLD, Wu, DISK,
Jna,
KREP, and Bpa) are sensitive to -chymotryp-
sin.
Wra (DI3) and Wrb (DI4); Anti-Wra and -Wrb
The low-prevalence antigen Wra and its anti-
thetical antigen of extremely high prevalence,
Wrb, represent an amino acid substitution in
the fourth loop of Band 3—Lys658 in Wra and
Glu658 in Wrb. Wrb expression, however, also
depends on the presence of GPA. Despite its
homozygosity for the Glu658 codon in the
Band 3 gene, Wrb is not expressed in the rare
phenotypes associated with a complete ab-
sence of the MN glycoprotein GPA or of the
part of GPA that is close to insertion into the
red cell membrane. This provides strong evi-
dence for an interaction between Band 3 and
GPA within the red cell membrane.
Anti-Wra is a relatively common antibody,
usually detected by an antiglobulin test but
sometimes by direct agglutination of red
cells. Wra antibodies are mostly IgG1 but
sometimes IgM or IgM plus IgG. Anti-Wr a
has been re- sponsible for severe HDFN and
HTRs. Alloan- ti-Wrb is rare and little is
known about its clini- cal significance, but
autoanti-Wrb is a relatively common
autoantibody and may be implicated in
AIHA.
Other Diego Antigens
Since 1996, 17 antigens of very low
prevalence have been shown to represent
amino acid sub- stitutions in Band 3 and have
joined the Diego system: Wda, Rba, WARR,
ELO, Wu, Bpa, Moa, Hga, Vga, Swa, BOW,
NFLD, Jna, Krep, Tra, Fra,
354 ■ AABB T EC HNIC AL MANUAL
and SW1. Anti-DISK detects a high-
prevalence antigen that is antithetical to Wu.
Anti-ELO and anti-BOW have caused
severe HDFN.
THE YT SYSTEM
Yta (YT1; His353) and Ytb (YT2; Asn353) are
an- tithetical antigens on acetylcholinesterase,
an enzyme that is important in neurotransmis-
sion but has unknown function on red cells.
Ytb has a prevalence of about 8% in people of
European or African ancestry but has not been
found in people of Japanese ancestry; Yta has
relatively high prevalence in all populations.
Yta is not affected by trypsin but is destroyed
by
-chymotrypsin treatment of the red cells;
pa- pain and ficin may also destroy the
antigen, but this ability appears to depend on
the anti- Yta used. Yta and Ytb are sensitive
to the disul- fide-bond-reducing agents AET
and DTT.
Yt antibodies are usually IgG and require
an IAT for detection. They are not generally
considered clinically significant, although
anti-Yta may cause accelerated destruction of
Yt(a+)-transfused red cells and has been impli-
cated in acute and delayed HTRs.21,41
THE XG SYSTEM
Xga (XG1) is the only polymorphic blood
group antigen encoded by an X-linked
gene.41 Xga has a prevalence of about 66% in
males and 89% in females. Part of the XG
gene is within the X chromosome
pseudoautosomal region, a sec- tion at the
tip of the short arm that pairs with the Y
chromosome. CD99 (XG2) is the second
antigen of the Xg system. The CD99 gene is
homologous to XG and is located on both X
and Y chromosomes, with pairing occurring
at meiosis. Both CD99 and Xga expression
appear to be controlled by a common
regulator gene, XGR. Although Xga
antibodies occasionally ag- glutinate red cells
directly, they are generally IgG and are
reactive by an IAT. They are not re- active
with red cells treated with proteolytic
enzymes. Anti-Xga is not clinically significant.
CD99 antibodies in common use are mostly
monoclonal and of mouse origin; a few
human alloanti-CD99 occur, although little
is known about their characteristics.
THE SCIANNA SYSTEM
The Scianna system consists of seven
antigens on erythrocyte membrane-
associated protein, a member of the IgSF
that has one IgSF do- main.41,42 Sc1 (Gly57)
and Sc2 (Arg57) are anti- thetical antigens of
high and low prevalence, respectively. Rd
(SC4) has very low prevalence; Sc3, STAR,
SCER, and SCAN all have very high
prevalence. Anti-Sc3 is produced by
individu- als with the very rare Scianna-null
phenotype.
No Scianna antibody has been implicat-
ed in an HTR or severe HDFN, although
evi- dence is limited because of the scarcity
of the antibodies. Although directly
agglutinating SC1 antibodies are known,
Scianna antibodies are generally reactive by
an IAT. Treatment of red cells by proteolytic
enzymes has little ef- fect on their reactivity
with Scianna antibod- ies, but disulfide-
bond-reducing agents (AET and DTT)
substantially reduce reactivity.
THE DOMBROCK SYSTEM
The Dombrock system consists of eight anti-
gens: the polymorphic antithetical antigens
Doa (DO1; Asn265) and Dob (DO2; Asp265)
and the high-prevalence antigens Gya, Hy, Joa,
DOYA, DOMR, and DOLG.3,43 Doa and Dob
have a prevalence of 66% and 82%,
respectively, in populations of European
ancestry (Table 14- 6). The prevalence of Doa
is somewhat lower in populations of African
ancestry and substan- tially lower in people of
East Asian ancestry. Anti-Gya is the antibody
that is characteristi- cally produced by
immunized individuals with the Dombrock-
null [Gy(a–)] phenotype that results from
various inactivating mutations. Two
uncommon phenotypes are present in in-
dividuals of African ethnicity: Hy– Jo(a–)
(Gly108Val) and Hy+w Jo(a–) (Thr117Ile),
which are usually associated with weak
expression of Dob and Doa, respectively (Table
14-6). The Dombrock glycoprotein (CD297)
has a struc- ture that is characteristic of an
adenosine di- phosphate ribosyltransferase,
and the Dom- brock gene has the designation
“ART4.”
Dombrock antigens are resistant to papa-
in and ficin treatment of the red cells but are
sensitive to trypsin, -chymotrypsin, and
pro-
 CHA P TER 1 4 Other Blood Groups ■ 355

TABLE 14-6. Phenotypes of the Dombrock System and Their Approximate Frequencies

Frequency (%)

Phenotype Doa Dob Gya Hy Joa Whites Blacks

Do(a+b–) + – + + + 18 11
Do(a+b+) + + + + + 49 44
Do(a–b+) – + + + + 33 45
Gy(a–) – – – – – Rare 0
Hy– – +w +w – – 0 Rare
Jo(a–) +w –/+w + +w – 0 Rare
DOYA– – – +w +w +w Rare Rare
DOMR– – + – +w +w Rare Rare
DOLG– + – +w + + Rare Rare
+ = weakened expression of antigen.
w

nase. They are also sensitive to the disulfide-

14-8).45 Colton antibodies are usually IgG and
bond-reducing agents AET and DTT.
reactive by an IAT, although agglutinating IgM
Dombrock antibodies are usually IgG
anti-Coa has been found. Colton antibodies
and reactive by an IAT. They are in short
have been implicated in severe HDFN and
supply and are often of poor quality, with
HTRs. Colton antigens are resistant to proteo-
very weak reac- tivity. Screening for
lytic enzymes.
Dombrock-compatible donors, therefore, is
best performed by molec- ular genetics
(SNP testing). THE L ANDSTEINER-WIENER
Anti-Doa and -Dob have been SYSTEM
responsible for acute and delayed HTRs.
LWa (LW5) and LWb (LW7; Gln100Arg) are
There is little in- formation regarding the
anti- thetical antigens of high and low
clinical significance of other Dombrock
prevalence, respectively.41 Anti-LWab is
antibodies. No Dombrock antibody has
reactive with all red cells except those of the
caused HDFN.
extremely rare LW-
null phenotype and Rhnull cells, which are
THE COLTON SYSTEM also LW(a–b–). LW antigens are expressed
more strongly on D+ than D– red cells and
Coa (CO1; Ala45) is a high-prevalence
more strongly on umbilical cord red cells
antigen; Cob (CO2; Val45), its antithetical
than on
antigen, has a prevalence of about 8% in
those of adults. LW antigens are unaffected by
people of European ancestry but is less
treatment of the red cells with papain, ficin,
common in other ethnic groups.41 Anti-Co3
trypsin, or -chymotrypsin but are destroyed
is reactive with all red cells except those of
by pronase. Disulfide-bond-reducing agents
the extremely rare Co(a–b–) phenotype that
(AET and DTT) either destroy or greatly
results from various inactivat- ing
reduce LWa or LWab (LW6) on red cells.
mutations. Co4 (Gln47) is a high-preva-
The LW glycoprotein is intercellular
lence antigen whose presence is required for
adhe- sion molecule-4 (ICAM-4), an IgSF
the expression of Coa due to the proximity
adhesion molecule. ICAM-4 binds integrins
of the polymorphism.44 The Colton antigens
on macro- phages and erythroblasts, and it is
are located on aquaporin-1, a water channel
probably
(Fig
356 ■ AABB T EC HNIC AL MANUAL

FIGURE 14-8. A three-dimensional model of aquaporin-1, showing the six membrane-spanning domains
as cylinders. The first extracellular loop is glycosylated and contains the Co a/Cob polymorphism. The
third extracellular loop and first intracellular loop contain alanine (A)-proline (P)-asparagine (N) motifs
and form a channel in the membrane through which water molecules pass.

involved in the stabilization of erythroblastic rary LW-negative phenotypes sometimes oc-

islands in the marrow during the later stages of
erythropoiesis.45 ICAM-4 is also part of the
Band 3/Rh ankyrin macrocomplex (Fig 14-1)
of red cell surface antigens and might main-
tain close contact between the red cell surface
and the vascular endothelium.8 Upregulation
of ICAM-4 on red cells of patients with sickle
cell disease could play a part in the adhesion
of these cells to the vascular endothelium and
the resultant crises of vascular occlusion.17,46
Most LW antibodies are reactive by an
IAT.
They are not generally considered to be
clini- cally significant and have not been
implicated in HTRs or HDFN. Acquired and
often tempo-
cur with production of anti-LWa or anti-
LWab, a phenomenon that is usually
associated with pregnancy or hematologic
malignancy. The transient antibodies behave
like alloantibod- ies but, strictly speaking,
should be considered autoantibodies.
THE CHIDO/RODGERS SYSTEM
The nine antigens of the Chido/Rodgers sys-
tem are not true blood group antigens
because they are not produced by erythroid
cells. They are located on a fragment of the
fourth com- ponent of complement (C4d)
that attaches to
 CHA P TER 1 4
the red cells from the plasma. Ch1 to Ch6,
Rg1, and Rg2 each have a prevalence
greater than 90%; WH has a prevalence of
about 15%. A complex relationship exists
between the nine determinants and SNPs in
C4A and C4B, the genes encoding the C4
chains. The expres- sion of Chido/Rodgers
on red cells is destroyed by treatment of the
cells with proteolytic en- zymes, such as
papain or ficin.
No Chido/Rodgers antibodies are known
to have caused HTRs or HDFN, and antigen-
negative blood is not required for transfusion.
Chido/Rodgers antibodies are generally IgG.
Detection of these antibodies with native red
cells usually requires an IAT, but they often
directly agglutinate red cells coated artificially
with C4d. Binding of Chido/Rodgers antibod-
ies to red cells is readily inhibited by plasma
from Ch/Rg+ individuals; this is a useful aid to
identification of these antibodies (Method 3-
17).
THE GERBICH SYSTEM
The Gerbich system consists of six highly
prev- alent antigens—Ge2, Ge3, Ge4, GEPL,
GEAT, and GETI—and five antigens with
very low prevalence—Wb, Lsa, Ana, Dha, and
GEIS. These antigens are located on the
sialoglyco- proteins glycophorin C (GPC),
glycophorin D (GPD), or both. These two
glycoproteins are produced by the same gene,
GYPC, by initia- tion of translation at two
different sites on the mRNA. GPD lacks the
N-terminal 21 amino ac- ids of GPC. GPC and
GPD are part of the junc- tional complex of
membrane proteins. Their C-terminal
cytoplasmic domains interact with the
membrane skeleton through 4.1R, p55, and
adducin and serve as an important link
between the membrane and its skeleton.9,45
GPC appears to be exploited as a receptor
by some strains of the malarial parasite P.
falci- parum. There are three types of
“Gerbich-neg- ative” phenotypes (Table 14-7).
The first of these phenotypes, Ge:–2,–3,–4, is
the true null
in which both GPC and GPD are absent from
the red cells, and the cells are elliptocytic. In
the other phenotypes, Ge:–2,3,4 and Ge:–
2,
–3,4, GPD is absent and an abnormal form
of
GPC is present. Ge2, Ge3, and Ge4 are de-
Other Blood Groups ■ 357
stroyed by trypsin treatment of red cells.
Whereas Ge2 and Ge4 are also sensitive to
pa- pain, Ge3 is resistant to papain
treatment. Consequently, papain-treated red
cells can be used for distinguishing anti-Ge2
from anti- Ge3 in the absence of the very
rare Ge:–2,3,4 phenotype red cells.
Gerbich antibodies may be IgM and di-
rectly agglutinating, but most are IgG and re-
quire an IAT for detection. They are not gener-
ally considered to be clinically significant and
have not caused HTRs. However, anti-Ge3 has
caused HDFN that tends to manifest 2 to 4
weeks after birth. Some autoantibodies with
specificities resembling anti-Ge2 or -Ge3 have
been responsible for AIHA.
THE CROMER SYSTEM
The 18 Cromer antigens are located on the
complement-regulatory glycoprotein, decay
accelerating factor (DAF or CD55). 3,47 They
in- clude the antithetical antigens Tc a (Arg52),
Tcb (Leu52), Tcc (Pro52), WESa (Arg82), and
WESb (Leu82). Tca and WESb have high
prevalence, and Tcb, Tcc, and WESa, have low
prevalence, although both Tcb and WESa are
present in ap- proximately 0.5% of people of
African ancestry and WESa is present in 0.6%
of people of Finn- ish ancestry. Other antigens
—Cra, Dra, Es, IFC, UMC, GUTI, SERF,
ZENA, CROV, CRAM, and
CROZ, CRUE, and CRAG—have high preva-
lence.
Anti-IFC is the antibody made by
individ- uals with the very rare Cromer-null
phenotype (Inab phenotype), and it is
reactive with all red cells, apart from those
of individuals with the
TABLE 14-7. Phenotypes Lacking High-
Prevalence Gerbich Antigens and the
Antibodies that May Be Produced
PhenotypeAntibodies
Ge:–2,3,4 (Yus type) Anti-Ge2
Ge:–2,–3,4 (Ge type) Anti-Ge2 or -Ge3
Ge:–2,–3,–4 (Leach Anti-Ge2, -Ge3, or -Ge4
type)
 358 ■ AABB T EC HNIC AL MANUAL

Inab phenotype. Cromer antigens are readily TABLE 14-8. Approximate Frequencies of
destroyed by -chymotrypsin treatment of Knops Antigens in Two Populations
red cells but not by papain, ficin, or trypsin
treat- ment. The disulfide-bond-reducing
agents AET and DTT reduce antigen Frequency (%)
expression only slightly. Antigen Whites Blacks
CD55 helps protect the red cells from
lysis resulting from autologous complement by Kna KN1 99 100
in- hibiting the action of C3-convertases. Inab- Kn b
KN2 6 0
phenotype red cells do not undergo undue he-
McC a
KN3 98 94
molysis, however, because of the activity of
another complement regulatory glycoprotein, Sl1 (Sla) KN4 98 60
CD59. CD55 and CD59 are both linked to the Yk a
KN5 92 98
red cell membrane by a glycosylphosphati-
McC b
KN6 0 45
dylinositol (GPI) anchor. Pathological levels
of Sl2 KN7 0 80
hemolysis occur in paroxysmal nocturnal he- Sl3 KN8 100 100
moglobinuria, which is associated with a clon-
al defect in GPI biosynthesis and the absence of KCAM KN9 98 20
both CD55 and CD59 in affected red cells.
Cromer antibodies are not usually con-
sidered to be clinically significant because tigens are generally resistant to papain and
there is no firm evidence that any of them fi- cin, although this may depend on the
has caused an HTR, and the evidence from antibod- ies used, and are destroyed by
func- tional cellular assays is equivocal. No trypsin or - chymotrypsin treatment. They
Cromer antibodies have been implicated in are also de- stroyed, or at least weakened, by
HDFN, and they are probably sequestered AET and DTT. CR1 appears to be involved
by high lev- els of CD55 in the placenta. in the roset-
Cromer antibodies are usually IgG and ting of red cells that is associated with severe
require an IAT for detec- tion. They are P. falciparum malaria. The McCb and Sl2
inhibited by serum or concen- trated urine alleles, present almost exclusively in
from antigen-positive individuals and are individuals of Af- rican ancestry, may confer a
removed from serum by platelet con- degree of protec- tion from the parasite. This
centrates. might explain the very strong difference in the
prevalence of some antigens, especially Sl1,
THE KNOPS SYSTEM McCb, Sl2, and KCAM, among populations of
European and African ethnicity (Table 14-8).
The nine antigens of the Knops system are Knops antibodies are not clinically
lo- cated on the complement-regulatory signif- icant and can be ignored when
glyco- protein complement receptor 1 (CR1 selecting blood for transfusion.5 They are
or CD35).48 All are polymorphic, although usually difficult to work with, often making
Kna, McCa, Sl1, and Yka have relatively high it difficult to distin- guish antigen-negative
preva- lence (Table 14-8). cells from those with weak expression. They
Kna/Knb represent Val 1561Met, McCa/ are generally IgG and reactive only by an
McC , Lys1590Glu, Sl1/Sl2, and Arg1601Gly.
b
IAT.
Sl3 requires the presence of Ser1610 and
Arg1601 (Sl2) for expression. Absence of
KCAM results from an Ile1615Val THE INDIAN SYSTEM
substitution. An apparent null phenotype, the The low-prevalence antigen Ina (Arg46) and
Helgeson phenotype, indi- cates very low its antithetical antigen Inb (Pro46) plus two
levels of red cell CR1 and very weak other high-prevalence antigens, INFI and
expression of Knops antigens. Knops an- INJA, are located on CD44, the predominant
cell surface
 CHA P TER 1 4
receptor for the glycosaminoglycan hyaluro-
nan, a component of the extracellular
matrix.41 AnWj (901009), an antigen with very
high prev- alence, may also be located on or
associated with CD44, but the evidence is
incomplete. In- dian antigens have reduced
expression on red cells with the In(Lu)
phenotype, and AnWj is virtually
undetectable on In(Lu) cells. Ina and Inb are
sensitive to treatment of red cells with
proteolytic enzymes—papain, ficin, trypsin, -
chymotrypsin—and are also destroyed by
the disulfide-bond-reducing agents AET and
DTT. AnWj, however, is resistant to all these
en- zymes but shows variable outcomes with
re- ducing agents.
Anti-Ina and -Inb often agglutinate red
cells directly, but the reaction is usually en-
hanced by an IAT. Indian antibodies are not
generally considered to be clinically signifi-
cant, although there is one report of anti-Inb
causing an HTR. AnWj, however, has
caused severe HTRs, and In(Lu) red cells
should be se- lected for transfusion.5
THE OK SYSTEM
Oka, OKGV, and OKVM have very high
preva- lence and are located on the IgSF
molecule CD147 or basigin, which has two
IgSF do- mains. Oka is resistant to
proteolytic enzymes and disulfide-bond-
reducing agents. Only two alloanti-Oka
antigens and a single example each of anti-
OKGV and -OKVM are known; all are
reactive by an IAT.49 In-vivo survival tests
and cellular functional assays with one anti-
Oka have suggested that it could be clinically
significant, but no clinical information
exists.
THE RAPH SYSTEM
MER2 (RAPH1), which is located on the
tet- raspanin CD151, was initially defined
by mouse monoclonal antibodies that
recognized a quantitative polymorphism, and
about 8% of the population has undetectable
levels of MER2 on their mature red cells.
Alloanti-MER2 was found in three Israeli
Jews originating from India who had a
RAPH-null phenotype resulting from a
single nucleotide deletion that led to a
premature stop codon. These three in-
Other Blood Groups ■ 359
dividuals were CD151 deficient and had
end- stage renal failure, sensorineural
deafness, and pretibial epidermolysis
bullosa, suggesting that CD151 is essential
for the proper assem- bly of basement
membranes in kidney, inner ear, and skin.50
MER2-negative individuals with anti-MER2
but only single amino acid substitutions in
CD151 do not have these symptoms.
MER2 antigen is resistant to treatment of
red cells with papain but is destroyed by tryp-
sin, -chymotrypsin, and pronase and by AET
and DTT. MER2 antibodies react in an IAT.
There is no evidence that anti-MER2 is clini-
cally significant.
THE JOHN MILTON HAGEN
SYSTEM
This system consists of six antigens with
very high prevalence—JMH, JMHK, JMHL,
JMHG, JMHM, and JMHQ—on the
semaphorin glyco- protein CD108 (Sema7A).
Anti-JMH is typically produced by
individuals with an acquired loss of CD108.
This most often occurs in elderly pa- tients
and is associated with a weakly positive
direct antiglobulin test result. The absence
of the other JMH antigens results from
different missense mutations in SEMA7A.51
JMH anti- gens are destroyed by proteolytic
enzymes and disulfide-bond-reducing
agents. They are not detected on cord red
cells.
JMH antibodies are usually reactive in an
IAT. They are not generally considered to be
clinically significant, although one example
was implicated in an acute HTR.
THE GILL SYSTEM
GIL antibodies detect a very high-
prevalence antigen, GIL, located on
aquaporin 3 (AQP3), a member of the
aquaporin superfamily of wa- ter and
glycerol channels (like the Colton anti-
gen).52 AQP3 enhances the permeability of
the red cell membrane by glycerol and water.
GIL antigen is resistant to proteolytic
en- zymes and disulfide-bond-reducing
agents. GIL antibodies are reactive by an IAT.
Anti-GIL has not been implicated in HTRs
or HDFN,
360 ■ AABB T EC HNIC AL MANUAL
although monocyte monolayer assays have
suggested a potential to cause accelerated
de- struction of GIL+ red cells.
THE RHAG SYSTEM
The four antigens of the RHAG system are
lo- cated on the Rh-associated glycoprotein
(RhAG), which is also described in Chapter
13.53 RhAG is closely associated with the Rh
protein in the membrane as part of the Band
3/Rh/ankyrin macrocomplex (Fig 14-1). Ola is
very rare, and homozygosity for the allele en-
coding Ola is associated with an Rhmod pheno-
type. Duclos and DSLK have high prevalence,
and absence of these antigens is associated
with an aberrant U (MNS5) antigen. RHAG4
is a low-prevalence antigen whose antibody
was associated with a single case of severe
HDFN.3
THE FORS SYSTEM
FORS is a new blood group system consisting
of a single antigen, Forssman glycosphingolip-
id antigen (FORS1). The presence of FORS1
on human erythrocytes is unusual and was
shown to be the result of an enzyme-activating
amino acid substitution arising from a
missense mu- tation in the human Forssman
synthase gene GBGT1. FORS1 was
demonstrated biochemi- cally on the red cells
of two blood donors from
different families with the A pae phenotype,
which was first described in 1987.54,55 Apae had
been previously thought to constitute a sub-
group of A in the ABO system but has now
been shown to be based on the presence of
FORS1 antigen on red cells. Forssman syn-
thase adds a terminal 3--N-acetylgalactos-
amine to its globoside acceptor. FORS1 is not
usually present on the red cells of primates but
is highly expressed on the red cells and
uroepi- thelia of lower mammals, such as
dogs and sheep. As with other carbohydrate
blood group antigens, naturally occurring
antibodies to FORS1 are present in all
human sera, with the exception of rare
FORS1+ individuals, and
have the potential to cause a positive cross-
match.
THE JR SYSTEM
The high-prevalence antigen Jra has been
pro- moted to a new blood group system, JR,
fol- lowing the independent findings of two
groups demonstrating that the Jr(a–)
phenotype was due to inactivating
nucleotide changes in ABCG2.56,57 The gene
encodes ABCG2, a multi- pass membrane
protein family member of the adenosine
triphosphate (ATP)-binding cas- sette
transporters that is broadly distributed
throughout the body. Jra has long been
associ- ated with drug resistance in cancer
and resis- tance to xenobiotics, and it might
be impor- tant for porphyrin homeostasis.58
The Jr(a–) phenotype is present predomi-
nantly in people of Japanese ancestry. Jra
anti- gen is resistant to proteolytic enzymes
and disulfide-bond-reducing agents. Anti-Jra
is re- active by an IAT and has caused HTR.
It is not usually implicated in HDFN,
although one case has been reported.
THE L AN SYSTEM
Lan, another high-prevalence antigen, was
also elevated to a new blood group system
fol- lowing the discovery that it was carried
on ABCB6, another ATP-binding cassette
trans- porter molecule on the erythrocyte
mem- brane.59 Unlike Jra, Lan is not
associated with any single geographic or
ethnic group, and this is mirrored by the
diversity of mutant alleles in the Lan–
individuals studied. ABCB6 is associ- ated
with porphyrin transport and was thought to
have an important role in heme synthesis.60
However, the existence of ABCB6- deleted
individuals indicates that there may be
compensation by other transporters in the
ab- sence of ABCB6.
Lan antigen is expressed variably on red
cells in different individuals but is resistant
to proteolytic enzymes and disulfide-bond-
reducing agents. Anti-Lan is reactive by an
IAT
 CHA P TER 1 4
and has been implicated in HTR but not
gen- erally in HDFN.
THE VEL SYSTEM
Vel is a high prevalence blood group antigen
that has been shown to depend on the pres-
ence of small integral protein 1 (SMIM1), a
protein of unknown function newly
discovered on the erythrocyte surface.60-62
Absence of Vel antigen in the vast majority
of individuals re- gardless of ethnic
background, is due to a 17- base pair
deletion in SMIM1, which results in the
absence of the protein at the cell mem-
brane.
Vel antigen expression is generally weak
on cord RBCs and differs substantially from
one individual to another. Patterns of expres-
sion are a consequence both of zygosity for the
17-bp deletion and also for a SNP in a GATA-
1 transcription factor site in intron 2.62
Serologi- cal expression is not affected by
protease treat- ment although sensitivity to
reducing agents such as 0.2 M DTT is
variable. Anti-Vel are of- ten a mixture of IgG
and IgM, readily activate complement and
have been implicated in mild to severe HTRs,
although HDFN is rare.
ANTIGENS THAT DO NOT
BELONG TO A BLOOD GROUP
SYSTEM
CD59—A Potential New Blood
Group System
An antibody to a high-prevalence antigen
de- tected in the plasma of a transfused
CD59- deficient child was shown to be
specific for CD59.63 The antibody was
readily inhibited with soluble protein.
Sequence analysis of samples from the
family revealed that the par- ents (first-
degree cousins) were heterozygous and the
child homozygous for a silencing mu- tation
in CD59. The antibody was an IgG anti-
body, and although the child’s RBCs had
been weakly DAT-positive following
transfusion, in- compatible blood was well-
tolerated. Thus, CD59 has been proposed as
a blood group sys- tem but is as yet,
unconfirmed.
Other Blood Groups ■ 361
Blood Group Collections
Although many antigens belong to blood
group systems, others have not been shown to
belong to a system. These are mostly antigens
with either very high or very low prevalence.
Some of them are included in blood group col-
lections that contain two or more antigens that
are related serologically, biochemically, or ge-
netically but do not fit the criteria for system
status.1
The high-prevalence antigen ABTI is
sero- logically related to Vel. However, it has
been excluded from SMIM1 by sequencing
analysis and thus remains in a collection. Like
Vel, ABTI expression differs substantially
and it is gener- ally expressed only weakly
on cord red cells. ABTI is resistant to
treatment of red cells with proteolytic
enzymes or disulfide-bond-reduc- ing
agents. Anti-ABTI has not caused HDFN
and clinical data are limited.
The Cost collection contains Csa and
Cs , antithetical antigens with relatively high
b
and low prevalence, respectively. These
antigens are serologically related to those of
the Knops system but do not appear to be
located on CR1. Cost antibodies are not
clinically signifi- cant.
Era and Erb are antithetical antigens with
very high and low prevalence, respectively.
Anti-Er3 is produced by individuals with Er(a–
b–) red cells. There is no evidence that Er anti-
bodies are clinically significant.
Carbohydrate antigens of the Ii and
GLOB collections are described in Chapter
12.
Carbohydrate antigens associated with
MNS antigens that are not encoded by
GYPA or GYPB are included in a seventh
collection, MNS CHO. These antigens have
been shown to be due to altered
glycosylation of the O-linked sugars on
GPA and GPB.2
High-Prevalence Antigens (901 Series)
The 901 series of the ISBT classification con-
tains six antigens (Table 14-9): five have a
prevalence well in excess of 99%, whereas Sd a
has a prevalence of about 91%. All are inherit-
ed, and none is eligible to join a system.1 All
six antigens are resistant to papain, trypsin, -
chymotrypsin, and AET treatment of the red
362 ■ AABB T EC HNIC AL MANUAL

TABLE 14-9. Antigens of the 901 Series of High-Frequency Antigens and Their Clinical Significance

Antigen Number Clinical Significance

At a
901003 Reported to have caused an HTR and mild HDFN
Emm 901008 No evidence of clinical significance
AnWj 901009 Severe AHTRs
Sd a
901011 No evidence of clinical significance
PEL 901014 No evidence of clinical significance
MAM 901016 Severe HDFN
HTR = hemolytic transfusion reaction; HDFN = hemolytic disease of the fetus and newborn; AHTR = acute HTR.

cells, and all except AnWj and Sda are ex-
pressed strongly on cord cells.
Sda represents carbohydrate structures on
red cells, and the product of the Sda gene is
probably a (1,4)N-acetylgalactosaminyltrans-
ferase. The strength of Sda on red cells is
highly variable, and Sda is not detected on cord
red cells. Agglutination of Sd(a+) red cells has
a characteristic “mixed-field” appearance of
ag- glutinates and free red cells; when viewed
mi- croscopically, the agglutinates are
refractile. Anti-Sda is inhibited by urine from
Sd(a+) individuals (Method 3-19), and by
guinea pig urine.
Low-Prevalence Antigens (700 Series)
Eighteen antigens of very low prevalence in all
of the populations tested constitute the 700 se-
ries of the ISBT classification: By, Chra, Bi,
Bxa, Toa, Pta, Rea, Jea, Lia, Milne, RASM, JFV,
Kg, JONES, HJK, HOFM, SARA, and REIT.
All are
inherited and do not fit any criteria for
joining or forming a system.1
Antibodies to low-prevalence antigens do
not present transfusion problems because
compatible blood is readily available. These
antibodies remain undetected if a serologic
crossmatch is not employed. Anti-JFV, -Kg,
-JONES, -HJK, and -REIT have all caused
HDFN.
HLA Antigens on Red Cells
“Bg” is the name given to HLA Class I
antigens expressed on mature red cells. Bga
represents
HLA-B7; Bgb, HLA-B17 (B57 or B58); and Bgc,
HLA-A28 (A68 or A69, which cross-reacts
with HLA-A2). Many individuals, however, do
not express Bg antigens on their red cells,
despite having the corresponding HLA
antigens on their lymphocytes.
There are a few reports of Bg antibodies
causing HTRs.64 These antibodies are some-
times present as contaminants in reagents.
HLA antigens on red cells are not destroyed
by papain, ficin, pronase, trypsin, -
chymotryp- sin, AET, or DTT. They can be
stripped from red cells with chloroquine
(Method 2-20) or EDTA/glycine-HCl (Method
2-21).
ERY THROID PHENOTYPES
CAUSED BY MUTATIONS IN
TRANSCRIPTION FACTOR
GENES
Mutations in genes encoding erythroid tran-
scription factors are emerging as important
modifiers of blood-group-antigen
expression. As described in the “Lutheran
System” section above, heterozygosity for
different mutations in KLF1 has been
identified in individuals with the In(Lu)
phenotype. In these individuals, ex- pression
of antigens carried on CD44 (Ina/Inb) and the
AnWj and P1 antigens is weak.19 How- ever,
KLF1 mutations have also been shown to
affect other genes, notably the -globin
gene, resulting in the hereditary persistence
of fetal hemoglobin syndrome.65 Affected
individuals have elevated HbF levels, some
30%, and demonstrate an In(Lu)
phenotype. Further-
 CHA P TER 1 4 Other Blood Groups ■ 363

more, discrete mutations in KLF1 appear to

give rise to different phenotypes; for example,
the change of Glu325Lys does not result in the
In(Lu) phenotype but is associated with severe
congenital dyserythropoietic anemia. These
red cells demonstrate weakened expression of
antigens in the Colton (AQP1), Cromer
(DAF), and LW (ICAM-4) blood group
systems.66
Although mentioned above in the “Lu-
theran System” section, it is worth repeating
that a mutation in GATA-1 resulted in the X-
linked Lu(a–b–) phenotype in one family.20 It
is likely that additional mutations in these and
other erythroid-specific transcription factors
will be identified as the causes of altered blood
group antigen expression.

KEY POINTS

1. Of 339 recognized antigen specificities, 297 belong to 1 of 34 blood group systems repre-
senting either a single gene or two or three closely linked homologous genes. Some groups
of antigens that are not eligible to join a system are classified together as collections. Anti-
gens not classified in a system or collection have either low or high prevalence and make up
the 700 and 901 series, respectively.
2. M and N are antithetical, polymorphic antigens. M, N, S, s, and ‘N’ are all destroyed by
treat- ment of the red cells with papain, ficin, bromelin, or pronase, although this effect with
S and s is variable. M and N—but not S, s, or ‘N’—are destroyed by trypsin treatment.
3. Anti-M is relatively common, and anti-N is quite rare. Most anti-M and -N are not
clinically significant. When M or N antibodies active at 37 C are encountered, antigen-
negative or compatible red cells should be provided. Anti-S, -s, and -U are generally
IgG antibodies that are active at 37C. They have been implicated in HTRs and severe
and fatal HDFN.
4. The antigen often referred to as “Kell” is correctly named “K” or “KEL1”; its
antithetical anti- gen is k or KEL2.
5. Because Kell antibodies can cause severe HDFN and HTRs, patients with Kell
antibodies should be transfused with antigen-negative blood whenever possible. Anti-K
is the most common immune red cell antibody not in the ABO and Rh systems.
6. In people of European or Asian ancestry, the Duffy polymorphism consists of two
antigens, Fya and Fyb, and three phenotypes, Fy(a+b–), Fy(a+b+), and Fy(a–b+). Fya
and Fyb are very sensitive to most proteolytic enzymes. In people of African ancestry, a
third allele, Fy, may result in neither Fya nor Fyb. Individuals who are homozygous for
Fy have the red cell pheno- type Fy(a–b–).
7. Anti-Fya (common) and anti-Fyb (rare) are generally detected by an IAT and may cause
acute or delayed HTRs that are usually mild, although some have been fatal.
8. Kidd antigens are resistant to proteolytic enzymes, such as papain and ficin.
9. Kidd antibodies anti-Jka and -Jkb are not common, are generally present in antibody
mix- tures, and are often difficult to detect. An IAT is usually required, and use of
enzyme-treated cells may be necessary to detect weaker antibodies. Kidd antibodies
may cause severe acute HTRs and are a common cause of delayed HTRs.
10. The 22 antigens of the Diego system are located on Band 3, the red cell anion exchanger.
Anti-Dia and -Wra can cause severe HDFN. Anti-Wra can also cause HTRs.

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Haploinsufficiency for the erythroid transcrip-
62. Cvejic A, Haer-Wigman L, Stephens JC, et al.
tion factor KLF1 causes hereditary persistence
SMIM1 underlies the Vel blood group and in-
of fetal hemoglobin. Nat Genet 2010;42:801-
fluences red blood cell traits. Nat Genet 2013;
5.
45:542-5.
66. Arnaud L, Saison C, Helias V, et al. A
63. Anliker M, von Zabern I, Höchsmann B, et dominant mutation in the gene encoding the
al. A new blood group antigen is defined
erythroid transcription factor KLF1 causes a
by anti- congenital dyserythropoietic anemia. Am J
Hum Genet 2010;87:721-7.
 C h a p t e r 1 6

Identification of Antibodies to
Red Cell Antigens

Phyllis S. Walker, MS, MT(ASCP)SBB, and

Janis R. Hamilton, MS, MT(ASCP)SBB

N A TUR A LLY OCCURR IN G ANT I -A

and anti-B are the only red cell
antibod- ies that are commonly found in
human serum or plasma. All other
antibodies are called “un- expected red cell
antibodies.” This chapter dis- cusses
methods for detecting and identifying
unexpected red cell antibodies.
There are two types of unexpected red
cell antibodies: alloantibodies and
autoantibodies. When someone produces
an antibody to an antigen that he or she
lacks, the antibody is called an
“alloantibody.” When someone pro- duces
an antibody to an antigen that he or she
possesses, the antibody is called an
“autoanti- body.” Therefore, by definition,
alloantibodies react only with allogeneic red
cells that express the corresponding antigens
—not with the an- tibody producer’s red
cells. Conversely, auto- antibodies are
reactive with the red cells of the antibody
producer. In fact, autoantibodies usually
are reactive with most reagent red cells as
well as with autologous red cells.
Immunization to red cell antigens
may result from pregnancy, transfusion,
transplan- tation, needle sharing, or
injections of immu- nogenic material. The
frequency of alloimmu- nization is
extremely variable depending on the
patients or donors being studied. Healthy
blood donors have very low
immunization rates. In chronically
transfused patient popu- lations, such as
those with sickle cell anemia or
thalassemia, as many as 14% to 50% of indi-
viduals are reported to be alloimmunized.1-3
In some instances, no specific immuniz-
ing event can be identified. In such cases,
naturally occurring antibodies have presum-
ably resulted from exposure to environmen-
tal, bacterial, or viral antigens that are
similar to blood group antigens. Also,
antibodies de- tected in serologic tests may
be passively ac- quired from injected
immunoglobulin, do- nor plasma,
passenger lymphocytes in transplanted
organs, or hematopoietic pro- genitor cells
(HPCs).

Phyllis S. Walker, MS, MT(ASCP)SBB, retired from Reference Laboratory, Blood Centers of the Pacific-
Irwin Center, San Francisco, California, and Janis R. Hamilton, MS, MT(ASCP)SBB, Manager, Reference
Laboratory, American Red Cross Blood Services, Detroit, Michigan
P. Walker has disclosed no conflict of interest. J. Hamilton has disclosed a financial relationship with Bio-Rad
Laboratories, Inc.

391
392 ■ AABB T EC HNIC AL MANUAL
SIGNIFICANCE OF
ALLOANTIBODIES
Alloantibodies to red cell antigens may be
de- tected initially with any test that uses
serum or plasma (eg, an ABO test, antibody
detection test, or compatibility test) or in an
eluate pre- pared from red cells coated with
alloantibody. Serum and plasma are
interchangeable for an- tibody testing unless
complement is required for antibody
detection. In such rare cases, only serum
provides complement. Throughout this
chapter, serum can be considered to be inter-
changeable with plasma unless the text indi-
cates otherwise.
After an antibody has been detected,
its specificity should be determined and
its clinical significance assessed. A
clinically significant red cell antibody is
defined as an antibody that is frequently
associated with he- molytic disease of the
fetus and newborn (HD- FN), hemolytic
transfusion reactions, or a no- table decrease
in transfused red cell survival. The degree
of clinical significance varies among
antibodies with the same specificity; some
cause destruction of incompatible red cells
within hours or even minutes, whereas
others decrease red cell survival by only a
few days, and still others do not shorten
red cell survival discernibly. Some
antibodies are known to cause HDFN,
whereas others may cause a positive direct
antiglobulin test (DAT) result in the fetus
without clinical evidence of HDFN.
PREANALYTICAL
CONSIDERATIONS
Before antibody identification testing is start-
ed, the patient’s medical history should be
considered if such information can be ob-
tained. Exposure to foreign red cells through
blood transfusion or pregnancy is the usual
cause of red cell immunization. It is uncom-
mon for patients who have never been trans-
fused or pregnant to produce clinically
significant alloantibodies, although “naturally
occurring” antibodies may be present. If the
patient has been transfused, it is critical to
know when the most recent transfusion was
given. If the patient was transfused during
the past 3 months, primary immunization to
red cell antigens may be a risk. In addition,
the presence of circulating donor red cells
may cause mixed-field results in antigen-
typing tests, and autologous adsorption
techniques should not be used because
alloantibodies could be adsorbed onto
transfused donor red cells. In general,
women who may have been sensitized by
pregnancy are more likely to have
alloantibodies than men. Infants who are
younger than 6 months usually do not pro-
duce alloantibodies, but newborns may have
passive antibody of maternal origin.
Certain diseases have been
associated with red cell antibodies;
depending on the methods used, such
antibodies may be detect- able in antibody
detection and identification tests. Cold
agglutinin syndrome, Raynaud
phenomenon, and infections with
Mycoplas- ma pneumoniae are often
associated with anti-I. Infectious
mononucleosis is sometimes associated with
anti-i. Patients with paroxys- mal cold
hemoglobinuria, which is associated with
syphilis in adults and viral infections in
children, may demonstrate autoantibodies
with anti-P specificity. Warm
autoantibodies often accompany diagnoses
such as warm au- toimmune hemolytic
anemia, systemic lupus erythematosus,
multiple myeloma, chronic lymphocytic
leukemia, or lymphoma. Patients who have
received solid-organ or HPC trans- plants
may demonstrate passive antibodies that
originate from donor passenger lympho-
cytes.
Drugs are known to cause antibody iden-
tification problems. (See Chapter 17 for a
dis- cussion of drug-related mechanisms
and drugs that are associated with serologic
prob- lems.) Administration of intravenous
immune globulin (IVIG) and Rh Immune
Globulin (RhIG) can interfere with
antibody-screening tests. Some lots of IVIG
have been reported to contain unexpected
antibodies, including anti-A and anti-B.
Intravenous RhIG, which is sometimes used
to treat thrombocytopenia, could explain
the presence of anti-D in an Rh- positive
patient.
Finally, when a patient is suspected of
having an antibody to a high-prevalence
anti-
 C H A P TE R 1 6 Identification of Antibodies to Red Cell Antigens
gen, the patient’s ethnic origin may
provide clues to the specificity of the
antibody. Table 16-1 lists some rare blood
types that are more commonly associated
with certain ethnic groups.4
ANALYTICAL PHASE OF
ANTIBODY IDENTIFICATION
Specimen Requirements
Either serum or plasma may be used for
anti- body detection and identification;
however, plasma may not be suitable for
detecting com- plement-activating
antibodies. Depending on the test methods
used, a 5-mL to 10-mL ali- quot of whole
blood usually contains enough serum or
plasma for identifying simple anti- body
specificities; more whole blood may be
required for complex studies. When autolo-
gous red cells are studied, the use of
samples anticoagulated with EDTA avoids
problems as- sociated with the in-vitro
uptake of comple- ment components by red
cells, which may oc- cur when clotted
samples are used.
Reagents and Test Methods
Antibody Detection Red Cells
Group O red cells suitable for pretransfusion
antibody screening are commercially available
and offered as sets of either two or three re-
agent single-donor red cells. Pooled red
cells for antibody detection are usually
obtained from two different donors and may
be used only when donor serum is tested.
Each labora- tory should decide whether to
use two or three reagent donor red cells for
antibody-detection testing. All reagent red
cells licensed by the Food and Drug
Administration (FDA) for this purpose must
express the following antigens: D, C, E, c, e,
M, N, S, s, P1, Lea, Leb, K, k, Fya, Fyb,
Jka, and Jkb. Three-cell antibody detection sets
usually offer red cells from presumed homozy-
gous donors with double-dose expression for
the following common antigens: D, C, E, c, e,
M, N, S, s, Fya, Fyb, Jka, and Jkb. Some weakly
re- active antibodies are reactive only with red
cells from donors who are homozygous for the
genes encoding the antigens; this serologic
■ 393
phenomenon is known as “dosage.”
Antibod- ies in the Rh, MNS, Duffy, and
Kidd systems most commonly demonstrate
dosage. Reagent red cells should be
refrigerated when not in use, and they
should not be used for antibody detection
after their expiration date.
Antibody Identification Panels
Identification of an antibody to red cell anti-
gen(s) requires testing the serum against a
panel of selected red cell samples (typically
8- 14 samples) with known antigenic
composi- tion for the major blood groups.
Usually, the red cell samples are obtained
from commer- cial suppliers, but institutions
may assemble their own panels using red
cells from local sources. Except in special
circumstances, pan- el cells are group O,
thereby allowing the se- rum of any ABO
group to be tested.
Each reagent red cell of the panel is
from a different donor. The reagent red
cells are se- lected so that if one takes all of
the examples of red cells into account, a
distinctive pattern of positive and negative
reactions exists for each of many antigens.
To be functional, a reagent red cell panel
must make it possible to identify with
confidence those clinically significant al-
loantibodies that are most commonly
encoun- tered, such as anti-D, -E, -K, and -
Fya. The phe- notypes of the reagent red
cells should be distributed so that single
specificities of the common alloantibodies
can be clearly identi- fied and most others
can be excluded. Ideally, patterns of
reactivity for most examples of sin- gle
alloantibodies should not overlap with any
other (eg, all of the K+ samples should not
be the only ones that are also E+). Also, it
is im- portant to include reagent red cells
with dou- ble-dose antigen expression to
detect com- mon antibodies that frequently
show dosage. Commercial panels are
accompanied by a sheet that lists the
phenotypes of the reagent red cells. The
combination of reagent red-cell samples
varies from lot to lot, so it is essential to use
the correct phenotype sheet when inter-
preting panel results. Commercial reagent
red cells for tube testing are diluted to a 2%
to 5% suspension in a preservative solution
that can be used directly from the bottle.
Washing the
394 ■ AABB T EC HNIC AL MANUAL

TABLE 16-1. High-Prevalence Antigens Absent In Certain Ethnic Populations

PhenotypePopulation

AnWj– Transient>Israeli Arabs (inherited)

At(a–) Blacks
Cr(a–) Blacks

Di(b–) South Americans>Native Americans>Japanese

DISK– Dutch>Europeans>any

Dr(a–) Jews from Bukhara>Japanese

En(a–) Finns>Canadians>English>Japanese

Es(a–) Mexicans, South Americans, Blacks

Fy(a–b–) Blacks>Arabs/Jews>others of Mediterranean ancestry>Whites

Ge:-2,-3 (Gerbich phenotype)
Ge:-2,3 (Yus phenotype)
Ge:-2,-3,-4 (Leach phenotype)
GUTI–
Gy(a–)
Papua New Guineans>Melanesians>Whites>any
Mexicans>Israelis>Mediterraneans>any
Any
Chileans
Eastern Europeans (Romany)>Japanese

hrB– Blacks
hr –
s
Blacks

Hy– Blacks
IFC (Crnull, Inab) Japanese>any
In(b–) Indians>Iranians>Arabs
Jk(a–b–) Polynesians>Finns>Japanese>any

Jo(a–) Blacks
Jr(a–) Japanese>Asians>Europeans>Bedouin Arabs>any

Js(b–) Blacks
k– Whites>any

Ko (Knull) Reunion Islanders>Finns>Japanese>any

K12– Whites

K14– French-Cajuns
K22– Israelis

KCAM– Blacks>any
Kn(a–) Whites>Blacks>any
 C H A P TE R 1 6 Identification of Antibodies to Red Cell Antigens ■ 395

TABLE 16-1. High-Prevalence Antigens Absent In Certain Ethnic Populations (Continued)

PhenotypePopulation

Kp(b–) Whites>Japanese

KUCI– Native Americans

Lan– Whites>Japanese>Blacks>any

Lu(a–b–) Any
Lu20– Israelis

Lu21– Israelis
LW(a–b–) Transient in any>inherited in Canadians
LW(a–) Those of Baltic Sea region
MAM– Arabs>any

MAR– Finns>any
McC(a–) Blacks>Whites>any

MER2– Indian Jews, Turks, Portuguese

MkMk Swiss>Japanese

Oh (Bombay) Indians>Japanese>any
Ok(a–) Japanese

P– Japanese>Finns>Israelis>any
Para-Bombay Reunion Islanders>Indians>any

PEL– French-Canadians
PP1Pk– Swedes>Amish>Israelis>Japanese>any

Sl(a–) Blacks>Whites>any
Tc(a–b+c-) Blacks

Tc(a–b–c+) Whites
SERF– Thais

U– and S–s–U+ var

Blacks
UMC– Japanese

Vel– Swedes>any
WES(b–) Finns>Blacks>any

Yk(a–) Whites>Blacks>any
Yt(a–) Arabs>Jews>any
Used with permission from Reid, Lomas-Francis, and Olsson. 4
Any = may be found in any population; > = more prevalent
than.
396 ■ AABB T EC HNIC AL MANUAL
red cells before use is usually unnecessary un-
less the preservative solution is suspected of
interfering with antibody identification.
Panel cells should not be used after the
expiration date; however, this restriction is
not always practical. Most serologists use
in-date reagent cells for initial antibody
identification panels and, if necessary, use
expired reagent red cells to exclude or
confirm specificities. Each laboratory
should establish a policy for using expired
reagent red cells and validate any
procedures associated with this prac-
tice.5(p21)
Antiglobulin Reagents
Most antibody detection and identification
studies include an indirect antiglobulin test
(IAT) phase. Antihuman globulin (AHG) can
be specific only for human immunoglobulin G
(IgG), or a polyspecific reagent that contains
anti-IgG and anti-complement may be used. A
polyspecific reagent may detect—or may de-
tect more readily—antibodies that bind com-
plement. To detect complement binding, se-
rum rather than plasma must be used because
the anticoagulant binds calcium, making it
unavailable for complement activation. Al-
though complement binding may be advanta-
geous in some instances, such as the detection
of certain JK system antibodies, many serolo-
gists prefer the routine use of IgG-specific
AHG reagents to avoid unwanted reactivity re-
sulting from in-vitro complement binding by
cold-reactive antibodies.6
Enhancement Media
Although the test system may consist solely
of serum and red cells (either reagent red
cells as provided by the manufacturer or
saline-sus- pended red cells), most
serologists use some type of enhancement
medium. Several differ- ent enhancement
media are available, includ- ing low-ionic
strength saline (LISS), polyethyl- ene glycol
(PEG), and 22% bovine albumin. Additional
enhancement techniques may be used for
complex studies. Enhancement tech- niques
are discussed in more detail later in this
chapter.
Test Methods
Hemagglutination testing performed in tubes
has been the gold standard of immunohema-
tology testing for decades, but testing by
gel- column agglutination and solid-phase
tech- nology are commonplace. These
methods offer stable and possibly less
subjective end- points, work-flow
standardization, and the ability to be
incorporated into semiautomated or
automated systems. They provide a sensi-
tive detection system for most blood-group
antibodies. Both gel and solid-phase
methods have also been shown to enhance
serologic re- activity that may not be
clinically significant in the selection of units
for transfusion, including the reactivity of
warm autoantibodies. Reactiv- ity that is
dependent on the diluent in com- mercially
prepared reagent cells can also be seen in
gel-column tests. Published studies have
compared the various methods for detec-
tion of wanted and unwanted red cell
alloanti- bodies.7-9
Recent reports have illustrated the
poten- tial effect of using red cell
membranes vs intact red cells to coat the
microwells in solid-phase tests. 10,11 In
selected samples, reactivity was found with
the disrupted red cell membranes, but the
samples were not reactive when the same
intact red cells were used. Laboratories
using these techniques for routine testing
and initial problem solving should be
familiar with the unique reactivity
characteristics of their chosen method.
Testing algorithms that in- volve both
nontube and tube techniques are frequently
developed to address anomalous reactivity
in either gel-column or solid-phase testing if
specificity cannot be assigned. When careful
review of initial and extended testing using
these methods does not allow identifica- tion
of alloantibody specificity(ies), a tube test
performed with LISS or PEG
enhancement media has been used to
further evaluate the sample for clinically
significant alloantibodies.
Basic Antibody Identification
Identification Panel
For initial antibody identification panels, it
is common to use the same methods and
test
 C H A P TE R 1 6 Identification of Antibodies to Red Cell Antigens ■ 397

phases as in the antibody-detection test or Selected Cell Panel

crossmatch. The gel and solid-phase
If a patient has antibodies that were
methods involve a single reading of the test
identified previously, the known antibodies
at the indi- rect antiglobulin phase. Tube-
should be considered when selecting panel
testing proto- cols have greater flexibility
cells to test. For example, if the patient has
for readings at dif- ferent test phases but
known anti-e, it will not be helpful to test
many serologists also utilize this single test
the patient’s serum with a routine panel, in
reading because the IAT detects the
which 9 of 10 cells are e-positive. Testing a
overwhelming majority of clinical- ly
selected panel of e-nega- tive red cells is a
significant alloantibodies.
better approach to find any newly formed
Some serologists using tube methods
antibodies. It is not necessary to test e-
may choose to include an immediate
positive cells to reconfirm the previously
centrifuga- tion reading, a room-
identified anti-e.
temperature incubation that is read before an
If the patient’s red cell phenotype is
enhancement medium is added, or both.
known, reagent red cells selected to detect
Such an approach may en- hance the
only those alloantibodies that are potentially
detection of certain antibodies (eg, anti-M, -
formed by the patient may be tested. For
N, -P1, -I, -Lea, or –Leb) and may help
example, if the patient’s Rh phenotype is C–
explain reactions detected in other phases.
E+c+e–, cells selected to exclude anti-E and
These steps are frequently omitted in
anti-c should not be necessary or can be limit-
initial antibody-identification studies
ed to a single selected cell because the patient
because most antibodies that are reactive
is not expected to form alloantibodies to these
only at lower tem- peratures have little or
antigens. Exceptions include patients with
no clinical signifi- cance.
weak or altered (partial) Rh antigens, which
Test observation after 37 C incubation
are usually found in minority ethnic groups,
in tube testing is influenced by the
and patients whose Rh phenotype was deter-
enhancement media used. Tests employing
mined by deoxyribonucleic acid (DNA) testing
PEG enhance- ment cannot be centrifuged
rather than serology and who could be carry-
and read because the reagent causes
ing a silenced allele. This approach can mini-
nonspecific aggregation of all red cells.
mize the amount of testing required.
LISS, albumin, and saline (no en-
hancement) tests do not have this restriction. IN TERPR E TIN G R E S U LTS. Antibody-detec-
A 37 C reading can detect some antibodies tion results are interpreted as positive or nega-
(eg, potent anti-D, -E, or -K) that may cause tive according to the presence or absence of
direct agglutination of red cells. Other reactivity (ie, agglutination or hemolysis), re-
antibodies (eg, anti-Lea or -Jka) may spectively. Interpretation of panel results can
occasionally be detected by the lysis of be a more complex process combining tech-
antigen-positive red cells during the 37 C nique, knowledge, and intuitive skills. Panel
incubation if serum is tested. Omit- ting results generally include both positive and
centrifugation and the reading at 37 C negative results, sometimes at different phases
should lessen the detection of unwanted of testing; each positive result should be ex-
posi- tive reactions caused by clinically plained by the final conclusion. The patient’s
insignifi- cant cold-reactive autoantibodies phenotype and the probability of the antibody
and alloan- ti bo di es. However, in som specificity are also taken into account in the
e i ns t a nces, potentially clinically final interpretation.
significant antibodies are detected only by
their 37 C reactivity. Exam- ples of 103 P O S I TI V E AN D NEG ATI V E RE ACT I ON S.
such antibodies (63-E, 27-K, 5-Jka , 4-D, 3- Both positive and negative reactions are im-
cE, and 1-C) were identified in 87,480 portant in antibody identification. The phase
samples.12 If the 37 C reading is desired in and strength of positive reactions can suggest
a specific antibody study, an alternative certain specificities. (See Method 1-9 for
strategy to avoid the uptake of cold
antibodies is to set up duplicate tests. One
test is read after the 37 C incubation, and the
other test is read only at the AHG phase.
398 ■ AABB T EC HNIC AL MANUAL
grading agglutination.) Positive reactions
are compared to the antigen patterns of the
panel cells to help assign specificity. A
single alloan- tibody usually produces a
clear pattern with antigen-positive and
antigen-negative re- agent red cells. For
example, if a serum sample is reactive only
with red cell samples 3 and 5 of the reagent
red cell panel shown in Table 16-2, anti-E is
very likely present. Both reactive red cells
express the antigen, and all nonreactive cells
lack it. When there is no discernible pat-
tern to explain the reactivity, possible
explana- tions include multiple antibodies,
dosage, and variations in antigen expression.
These factors are discussed in more detail
later in this chap- ter. Negative reactions are
important in anti- body identification
because they allow tenta- tive exclusion of
antibodies to antigens expressed on the
nonreactive red cells. Exclu- sion of
antibodies is an important step in the
interpretation process and must be
performed to ensure proper identification of
all of the an- tibodies present.
EXCLUSION, “ RU L E OUT,” OR “ CRO SS
OUT.” A widely used first approach to the
in- terpretation of panel results is to exclude
spec- ificities on the basis of nonreactivity
of the pa- tient’s serum with red cells that
express the antigen. Such a system is
sometimes referred to as a “cross-out” or
“rule-out” method. Once results have been
recorded on the work sheet, the antigen
profile of the first nonreactive red cell is
examined. If an antigen is present on the red
cell sample and the serum was not reactive
with it, the presence of the corresponding
an- tibody may tentatively be excluded.
Many technologists actually cross out such
antigens from the list at the top of the panel
sheet to fa- cilitate the process. After all of
the antigens on the list for that red cell have
been crossed out, the same process is
performed with the other nonreactive red
cells; additional specificities are then
excluded. In most cases, this process leaves
a group of antibodies that have not been
excluded.
Next, the red cells that are reactive
with
the serum are evaluated. The pattern of
reac- tivity for each specificity that was not
excluded is compared to the pattern of
reactivity ob-
tained with the test serum. If there is an anti-
gen pattern that matches the test serum pat-
tern exactly, this pattern most likely
identifies the specificity of the antibody in
the serum. If there are remaining
specificities that were not excluded,
additional testing is needed to elimi- nate
the possibilities and confirm the suspect- ed
specificity. This process requires testing of
the serum with additional selected red cells.
Although the exclusion (rule-out) ap-
proach often identifies simple antibodies, it
should be considered only as a provisional
step, particularly if the rule out was based on
the lack of reactivity with red cells that have
weaker expression of the antigen (eg, cells
from heterozygous donors).
SE LE CTE D CE LLS . Selected cells are red
cells that have been chosen because they
express some specific antigens and lack
others. Select- ed red cells with different
antigen combina- tions can be used to
confirm or rule out the presence of
antibodies. For example, if a pat- tern of
reactive red cells fits anti-Jka exactly, but
anti-K and anti-S were not excluded, the
serum should be tested with selected red cells.
Ideally, red cells with the following
phenotypes should be chosen: Jk(a–), K+, S–;
Jk(a–), K–, S+; and Jk(a+), K–, S–. The
reaction pattern with these red cells should
both confirm the pres- ence of anti-Jka and
include or exclude anti-K and anti-S.
Whenever possible, selected red cells should
have a strong expression of the antigen being
tested (ie, from homozygous do- nors or red
cells with double-dose expression). Such red
cells help ensure that nonreactivity with the
selected red cell indicates the absence of the
antibody and not that the antibody was too
weak to be reactive with a selected red cell
that had a weak expression of the antigen.
PROBABIL IT Y. To ensure that an
observed pattern of reactions is not the
result of chance alone, conclusive antibody
identification re- quires the serum to be
tested against a suffi- cient number of
reagent red cell samples that lack—and
express—the antigen that corre- sponds
with the antibody’s apparent specifici- ty. A
standard approach (which is based on
Fisher’s exact method) has been to require
that
TABLE 16-2. Example of a Reagent Red Cell Panel for Antibody
Identification
Cell Rh-hr MNS Kell P Lewis Duffy Kidd Others Cell Results
w a a a b a b a b
DCEcefC V MNSs KkKp Js P1 Le Le Fy Fy Jk Jk 37 C AHG

1 ++00+000 +00+ 0+00 + 0+ ++ 0+ Bg(a+) 1

CH A PT E R 1 6

2 ++00+0+0 +++0 0+00 + 00 00 +0 2

3 +0++0000 0+0+ 0+00 0 +0 0+ ++ 3

4 0+0+++00 +0++ 0+00 + 0+ +0 +0 4

5 00++++00 0+++ 0+00 + 0+ 0+ 0+ 5

6 000+++00 +0+0 ++00 + 0+ +0 0+ 6

7 000+++00 ++++ 0+00 + 0+ 0+ +0 7

8 +00+++0+ 0+00 0+00 + 00 00 0+ 8

9 000+++00 ++++ +000 0 +0 +0 ++ 9

10 000+++00 +00+ +++0 + 00 0+ ++ Yt(b+) 10

Identification of Antibodies to Red Cell Antigens

11 ++00+000 ++0+ 0+00 + 0+ 0+ 0+ 11

AC AC

+ indicates presence of antigen, 0 indicates absence of antigen, AC = autocontrol, AHG = antihuman

■

globulin.
399
400 ■ AABB T EC HNIC AL MANUAL

three antigen-positive red cell samples are
re- active and that three antigen-negative red
cell samples are not reactive for each
specificity identified.13 In some cases, the
use of two reac- tive and two nonreactive
red cell samples is also an acceptable
approach for antibody con- firmation.5,14
When that approach is not possi- ble, a more
liberal approach (which is derived from
calculations by Harris and Hochman15)
allows the minimum requirement for a
proba- bility (p) value of 0.05 to be met
with two re- active and three nonreactive
red cell samples or with one reactive and
seven nonreactive red cell samples (or the
reciprocal of either combi- nation).
Comparative p-value calculations are shown
in Table 16-3. Additional details on cal-
culating probability may be found in the
sug- gested readings list at the end of this
chapter. The possibility of false-negative
results with antigen-positive red cells must
be considered as well as that of unexpected
positive results (ie, caused by the presence
of an additional antibody or an error in the
presumptive anti- body identification).
Autologous Control
The autologous control (autocontrol), in
which serum and autologous red cells are
test-
ed under the same conditions as serum and
reagent red cells, is an important part of
anti- body identification. The autocontrol is
not the same as or equivalent to a DAT
(Method 3-14). Incubation and the presence
of enhancement reagents may cause
reactivity in the autocon- trol that is only an
in-vitro phenomenon. If the autocontrol is
positive in the antiglobulin phase, a DAT
should be performed. If the DAT result is
negative, antibodies to an enhance- ment
medium constituent or autoantibodies that
are reactive only in the enhancement me-
dium should be considered. Warm autoanti-
bodies and cold autoantibodies, such as anti-
I,
-IH, or -Pr, may be reactive in an IAT when
cer- tain enhancement media are used;
therefore, testing should be repeated in
another medi- um. If the DAT result is
positive, it must be in- terpreted with careful
attention to the transfu- sion history.
Autoantibodies or drugs could explain a
positive DAT result; however, if the patient
has an alloantibody and was recently
transfused with blood that expressed the
corresponding antigen, the circulating donor
red cells could be coated with alloantibody,
resulting in a positive DAT result associated
with a clinically significant delayed transfu-
sion reaction.

TABLE 16-3. Probability Values for Selected Antibody Identification Approaches

No. No. No.

Tested Positive Negative Fisher13 Harris-Hochman15

5 3 2 0.100 0.035
6 4 2 0.067 0.022
6 3 3 0.050 0.016
7 5 2 0.048 0.015
7 4 3 0.029 0.008
8 7 1 0.125 0.049
8 6 2 0.036 0.011
8 5 3 0.018 0.005
8 4 4 0.014 0.004
 C H A P TE R 1 6 Identification of Antibodies to Red Cell Antigens
Phenotyping Autologous Red Cells
Determining the phenotype of the
autologous red cells is an important part of
antibody iden- tification. When an antibody
is tentatively identified in the serum, the
corresponding an- tigen is expected to be
absent from the autolo- gous red cells. For
example, if serum from an untransfused
individual appears to contain anti-Fy a but
the autologous red cells have a negative
DAT result and type as Fy(a+), the re- sults
are clearly in conflict and further testing is
indicated. Thus, knowing the patient’s phe-
notype can help guide exclusion testing.
It may be difficult to determine the
pa- tient’s phenotype if he or she was
transfused in the past 3 months. A
pretransfusion speci- men, if available,
should be used to determine the phenotype.
If a pretransfusion sample is not available,
the patient’s newly formed autologous
red cells can be separated from the
transfused red cells and then typed (Method
2- 22). Separation of young red cells by
centrifu- gation is based on the difference in
the densi- ties of new and mature red cells.
Centrifuga- tion is most successful when 3
or more days have elapsed since the last
transfusion, which will provide time for
new autologous red cell production. New
autologous red cells must be isolated from
the sample while it is fresh. The technique is
ineffective if the sample is too old (>24
hours), the patient is not producing new red
cells, or the patient has sickle cell anemia.
Sickle cells are quite dense, and
centrifu-
gation is not an effective technique for
sepa- rating the autologous red cells from
the trans- fused donor red cells in a patient
with sickle cell disease. However,
autologous sickle cells may be separated
from donor red cells using washes with
hypotonic saline (Method 2-23). Sickle
cells containing hemoglobin SS are re-
sistant to lysis by hypotonic saline,
whereas donor red cells containing
hemoglobin AA are lysed.
The use of potent blood-typing
reagents, appropriate controls, and
observation for mixed-field reactions can
allow a specimen contaminated with donor
red cells to be phe- notyped. Phenotyping
results on posttransfu- sion samples can be
misleading, however, and
■ 401
should be interpreted with caution. 16 If the
specificity of the antibody(ies) in the
patient’s plasma is clear, extensive efforts
to separate and type the patient’s own red
cells are not necessary. A compatible AHG
crossmatch us- ing antigen-negative donor
units provides ad- ditional confirmation of
the antibody’s speci- ficity.17(p37) Definitive
typing can be performed on the patient’s red
cells 3 months after the last transfusion if
the patient is not transfusion de- pendent or
experiencing marrow aplasia or failure.
Cold and warm autoantibodies may
also complicate red cell typing. When red
cells are coated with cold autoantibodies,
it may be possible to remove the
autoantibodies with warm (37 C) saline
washes (Method 2-17). If the cold
autoantibodies are very potent, it may be
necessary to treat the red cells with dithio-
threitol (DTT ) to dissociate IgM
molecules that cause spontaneous
agglutination (Meth- od 2-18). When red
cells are coated with IgG autoantibodies, it
is not possible to perform typing that
requires an IAT without first re- moving
the bound IgG. However, it is often
possible to type antibody-coated red cells
with direct-agglutinating antisera, such
as IgM monoclonal reagents. With rare
exceptions, most direct-agglutinating
monoclonal re- agents give valid
phenotyping results despite a positive DAT
result.18 For antisera that require an IAT (eg,
anti-Fya and anti-Fyb), it is neces- sary to
remove the IgG from the test red cells
before typing. Common techniques for
remov- ing IgG antibodies include gentle
heat elution (Method 2-19), treatment with
chloroquine di- phosphate (Method 2-20),
and treatment with acid glycine/EDTA
(Method 2-21).
Molecular DNA genotyping offers an al-
ternative to serologic typing and is especially
useful when the patient has been recently
transfused or the patient’s red cells are heavily
coated with IgG. Molecular testing relies on
the extraction of DNA from white cells. Be-
cause of the short life-span of white cells in
vivo and the assay design, the presence of
transfused white cells from donors is not a lim-
iting factor in determining the patient’s geno-
type.
402 ■ AABB T EC HNIC AL MANUAL
There are situations, however, where
the genotype of a person may not predict
the red cell phenotype. Mutations that
inactivate gene expression or rare new
alleles may not be iden- tified by the
specific assay performed. The genotype
obtained from DNA isolated from
leukocytes and other hematopoietic cells
may differ from that of other tissues in
people with a history of transplantation.19
When DNA testing is used as a tool in an-
tibody identification, the predicted red cell
phenotype should be used judiciously. If a
sample appears to contain an antibody speci-
ficity when the predicted phenotype of the pa-
tient is antigen positive, the apparent antibody
should be further investigated. This discrepan-
cy may indicate that the patient’s red cells do
not actually express the antigen due to a gene
mutation not detected by the assay. Alterna-
tively, the patient might have an altered or par-
tial antigen due to an additional gene poly-
morphism. It is important to remember that the
antibody in the sample could be, in fact, an
alloantibody.
Complex Antibody Problems
Not all antibody identifications are simple.
The exclusion procedure does not always
lead directly to an answer, and additional
testing may be required. When an antibody
screen or incompatible crossmatch detects
an unex- pected antibody, the next step may
be to deter- mine whether the antibody is an
autoantibody or alloantibody. An
autocontrol, which may not have been
performed in the initial testing, can start the
identification process. Figure 16- 1 shows
some approaches to identifying anti- bodies
in a variety of situations when the auto-
control is negative, and Fig 16-2 shows
some approaches to identifying antibodies
when the autocontrol is positive.
Selected Serologic Procedures
Many techniques and methods are used in
complex antibody identification. Some of
the methods described in this chapter are
used routinely by many laboratories; others
are used selectively and may apply only in
special circumstances. It is important to
remember
that no single method is optimal for
detecting all antibodies. Any laboratory that
performs antibody detection or identification
should use routine methods and have access
to some alternative approaches.
When a pattern of weak reactions fails
to indicate specificity or the presence of an
anti- body is suspected but cannot be
confirmed, the use of enhancement
techniques or testing of panel cells treated
with enzymes or chemi- cals may be
helpful. An autocontrol should al- ways be
included with each technique.
LISS and PEG
LISS and PEG techniques (Methods 3-4 and 3-
5) are used to enhance reactivity and reduce
incubation time. LISS may be used to
suspend test red cells for use in tube or
column aggluti- nation tests or as an
additive medium for tube or solid-phase
tests. Care should be taken to follow the
instructions in the manufacturer’s product
insert closely to ensure that the ap- propriate
proportion of serum to LISS is achieved.
Commercially prepared LISS addi- tives or
PEG additives may contain additional
enhancing agents. Because LISS and PEG
en- hance autoantibodies, their use may
compli- cate alloantibody identification in
samples that also contain autoantibodies.20,21
Enzymes
Ficin and papain are the most frequently
used enzymes for complex antibody
identification. They destroy or weaken
antigens, such as M, N, Fya, Fyb, Xga, JMH,
Ch, and Rg (Table 16-4). Antibodies to these
antigens are nonreactive with treated red
cells. Conversely, ficin-treated and papain-
treated red cells show enhanced reactivity
with other antibodies (eg, Rh, P, I, Kidd, and
Lewis). Additional enzymes that are
commonly used in immunohematology
labo- ratories include trypsin, -
chymotrypsin, and pronase. Depending on
the enzyme and meth- od used, other
antigens may be altered or de- stroyed.
Antigens that are inactivated by one
proteolytic enzyme may not be inactivated
by other enzymes. The clinical significance
of antibodies that are reactive only with
enzyme-treated cells is questionable; such
 CH A PT E R 1 6
Identification of Antibodies to Red Cell Antigens
■

FIGURE 16-1. Antibody identification with negative

autocontrol. DTT = dithiothreitol.
403
 404
■
AABB T ECHNICAL M A NUAL

FIGURE 16-2. Antibody identification with positive autocontrol.

HPCs = hematopoietic progenitor cells; IVIG = intravenous immune globulin.
 C H A P TE R 1 6 Identification of Antibodies to Red Cell Antigens ■ 405

TABLE 16-4. Alterations of Antigens by Various Agents

AgentAntigens Usually Denatured or Altered*

Proteolytic enzymes† M, N, S, Fya, Fyb, Yta, Ch, Rg, Pr, Tn, Mg, Mia/Vw, Cla, Jea, Nya, JMH,
some Ge, and Inb
Dithiothreitol (DTT) or
Yta, JMH, Kna, McCa, Yka, LWa, LWb, all Kell, Lutheran, Dombrock,
2-aminoethylisothiouronium
and Cromer blood group antigens
bromide (AET)
*Some antigens listed may be weakened rather than completely denatured. Appropriate controls should be used with
modi- fied red cells.
†
Different proteolytic enzymes may have different effects on certain antigens.

“enzyme-only” antibodies may not have clini-
cal significance.22
In addition to enhancing the reactivity
of certain antibodies, enzyme techniques
may be used to separate mixtures of
antibodies. For example, if a serum sample
contains anti-Fya and anti-Jka, many of the
red cell samples on the initial panel would
be reactive. Then, if a panel of enzyme-
treated red cells were tested, the anti-Jk a
reactivity would be enhanced, whereas
the anti-Fya reactivity would be de-
stroyed. Procedures for the preparation and
use of proteolytic enzymes are given in
Methods 3-8 to 3-13.
Temperature Reduction
Some antibodies (eg, anti-M, -N, -P1, -Lea,
-Leb, and -A1) react better at room temper-
ature or below, and their specificity may be ap-
parent only at a temperature below 22 C. An
autocontrol is especially important for tests at
low temperatures because many sera also
contain anti-I or other cold-reactive autoanti-
bodies.
Increased Serum-to-Cell Ratio
Increasing the volume of serum incubated
with a standard volume of red cells may en-
hance the reactivity of antibodies that are
present in low concentrations. One
acceptable procedure involves mixing four
volumes (drops) of serum with one volume
of a 2% to
5% saline suspension of red cells and
incubat- ing the mixture for 60 minutes at 37
C. Periodic mixing during the incubation
promotes con- tact between the red cells and
the antibodies. It is helpful to remove the
serum before wash- ing the cells for an IAT
because the standard three to four washes
may be insufficient to re- move all of the
unbound immunoglobulin if increased
amounts of serum are used. More than four
washes are not recommended be- cause
bound antibody molecules may dissoci- ate.
Increasing the serum-to-red-cell ratio is not
appropriate for tests using LISS or com-
mercial PEG, which may contain LISS.
Tests performed in a low-ionic-strength
medium require specific proportions of
serum and additive.
Increased Incubation Time
For some antibodies, a 15-minute incubation
period may not be sufficient to achieve equi-
librium; therefore, the reactions may be
nega- tive or weak, particularly in saline or
albumin media. Extending the incubation
time to be- tween 30 and 60 minutes for
albumin or saline tests often improves the
reactivity and helps clarify the pattern of
reactions. Extended incu- bation may be
contraindicated when LISS or PEG is used.
If the incubation period exceeds the
recommended times for these methods, the
reactivity may be diminished or lost. Care
must be taken to use all reagents according
to the manufacturer’s directions.
406 ■ AABB T EC HNIC AL MANUAL
Alteration in pH
Altering the pH of the test system can
change the reactivity of certain antibodies,
enhancing the reactivity of some and
decreasing that of others.
Some examples of anti-M are
enhanced when the pH of the test system is
lowered to
6.5.23 If anti-M is suspected because the only
reactive red cells are M+N–, a definitive pat-
tern (ie, reactivity with M+N+ red cells also)
may be seen if the serum is acidified. The
addi- tion of one volume of 0.1 N HCl to
nine vol- umes of serum lowers the pH to
approximately
6.5. The acidified serum should be tested
with known M-negative cells to control for
nonspe- cific agglutination.
Lowering the pH, however,
significantly decreases the reactivity of
other antibodies.24 If unbuffered saline with
a pH <6.0 is used to prepare red cell
suspensions or for washing in an IAT,
antibodies in the Rh, Duffy, Kidd, and MNS
blood groups may lose reactivity. Phos-
phate-buffered saline (Method 1-8) can be
used to control the pH and enhance the
detec- tion of antibodies that are poorly
reactive at a lower pH.25
Inhibition Techniques
Soluble forms of some blood group antigens
exist in body fluids, such as saliva, urine,
and plasma. These substances are also
present in other natural sources, and they
can be pre- pared synthetically. Soluble
substance can be used to inhibit the
reactivity of the corre- sponding antibody
that could mask the pres- ence of underlying
nonneutralizable antibod- ies. Also, when an
antibody is suspected, inhibition of the
reactivity by a soluble sub- stance can help
with the identification of the antibody. For
example, if a suspected anti-P1 does not
produce a definitive pattern of agglu-
tination, the loss of reactivity after the
addition of soluble P1 substance strongly
suggests that the specificity is anti-P1 if a
parallel dilution control with saline.
Inhibition results can be interpreted only
when the test is nonreactive and the dilution
control that substitutes an equal volume of
saline for the soluble sub- stance is reactive.
The most commonly used substances for
inhibition include the following:
1. Lewis substances. Lea substances, Leb sub-
stances, or both are present in the saliva of
individuals who possess the Lewis gene
(FUT3). Lea substance is present in the
saliva of Le (a+b–) individuals, and both
Lea and Leb substances are present in the
saliva of Le(a–b+) individuals (Method 2-
8). Com- mercially prepared Lewis
substance is available.
2. P1 substance. Soluble P1 substance is pres-
ent in hydatid cyst fluid and the ovalbumin
of pigeon eggs. Commercially prepared P1
substance is available.
3. Sda substance. Soluble Sda blood group
substance is present in various body
fluids, but urine has the highest
concentration of Sda.26 To confirm the
presence of anti-Sda in a serum sample,
urine from a known Sd(a+) individual (or
a pool of urine specimens) can be used to
inhibit the antibody reactiv- ity (Method
3-19).
4. Chido and Rodgers substances. Ch and Rg
antigens are epitopes on the fourth compo-
nent of human complement (C4).27,28 Most
normal red cells have a trace amount of C4
on their surface. Anti-Ch and anti-Rg are
reactive with this C4 in an IAT. If red cells
are coated in vitro with excess C4, these
antibodies may cause direct agglutina-
tion.29 A useful test to identify anti-Ch and
anti-Rg is inhibition of the antibodies with
plasma from Ch+, Rg+ individuals (Method
3-17).
Inactivation of Blood Group Antigens
Certain blood group antigens can be
destroyed or weakened by chemical
treatment of the cells (Table 16-4). Modified
red cells can be useful for both confirming
the presence of sus- pected antibodies and
detecting additional antibodies. The use of
modified red cells can be especially helpful
if a sample contains an antibody to a high-
prevalence antigen because antigen-negative
red cells are rare. Proteolytic enzymes,
described above, are commonly used to alter
red cell antigens. Sulfhydryl re-
 C H A P TE R 1 6 Identification of Antibodies to Red Cell Antigens
agents, such as 2-aminoethylisothiouronium
bromide (AET), 2-mercaptoethanol (2-ME), or
DTT, can be used to weaken or destroy anti-
gens in the Kell system and some other anti-
gens (Method 3-18).30,31 ZZAP reagent,
which contains proteolytic enzyme and DTT,
dena- tures antigens that are sensitive to
DTT (eg, all Kell system antigens) as well
as antigens that are sensitive to enzymes
(Method 4-8).32 Gly- cine-HCl/EDTA
treatment of red cells destroys Bg and Kell
system antigens as well as the Era antigen
(Methods 2-21 and 4-2).33 Chloroquine
diphosphate can be used to weaken the ex-
pression of Class I HLA antigens (Bg
antigens) on red cells.34 Chloroquine
treatment also weakens some other antigens,
including Rh antigens (Method 2-20).
Sulfhydryl Reagents
Sulfhydryl reagents, such as DTT and 2-ME,
can be used to cleave the disulfide bonds that
join the monomeric subunits of the IgM pen-
tamer. Intact 19S IgM molecules are cleaved
into 7S Ig subunits, which have altered sero-
logic reactivity.35 The interchain bonds of 7S
Ig monomers are relatively resistant to such
cleavage. Sulfhydryl reagents (DTT and 2-ME
as well as AET) can also be used to cleave di-
sulfide bonds that are responsible for the con-
formation of certain blood group antigens
and, therefore, are used to destroy certain red
cell antigens.
Uses of sulfhydryl reagents include the
following:
1. Determining the Ig class of an antibody
(Method 3-16).
2. Identifying antibodies in a mixture of IgM
and IgG antibodies, particularly when an
agglutinating IgM antibody masks the pres-
ence of IgG antibodies.
3. Determining the relative amounts of IgG
and IgM components of a given specificity
(eg, anti-A or -B).
4. Dissociating red cell agglutinates caused by
IgM autoantibodies (Method 2-18).
5. Dissociating IgG antibodies from red cells
using a mixture of DTT and proteolytic
enzyme (ZZAP reagent) (Method 4-8).
■ 407
6. Destroying selected red cell antigens for
use in antibody investigations (eg, antigens
in the Kell, Dombrock, Cartwright, LW, and
Knops systems) (Method 3-18).
Adsorption
Antibody can be removed from a serum
sam- ple by adsorption onto red cells that
express the corresponding antigen. After the
antibody attaches to the membrane-bound
antigens and the serum and cells are
separated, the spe- cific antibody remains
attached to the red cells. It may be possible
to harvest the bound antibody by elution or
examine the adsorbed serum for
antibody(ies) remaining after the adsorption
process.
Adsorption techniques are useful for
the following purposes:
1. Separating multiple antibodies present in
a single serum.
2. Removing autoantibody to permit the
detection or identification of underlying
alloantibodies.
3. Removing unwanted antibody (often anti-
A, anti-B, or both) from serum that
contains an antibody that is suitable for
reagent use.
4. Confirming the presence of specific anti-
gens on red cells by their ability to
remove antibody of corresponding
specificity from previously characterized
serum.
5. Confirming the specificity of an antibody
by showing that it can be adsorbed onto
red cells of only a particular blood group
phe- notype.
Adsorption serves different purposes
in different situations; no single procedure is
sat- isfactory for all purposes (Methods 4-
5, 4-8, 4-9, and 4-10). A basic procedure for
antibody adsorption can be found in Method
3-20. The usual serum-to-cell ratio is one
volume of se- rum to an equal volume of
washed, packed red cells. To enhance
antibody removal, a larger volume of red
cells increases the proportion of antigen.
The incubation temperature should be that
at which the antibody is optimally re-
active. Pretreating red cells with a
proteolytic enzyme may enhance antibody
uptake and
408 ■ AABB T EC HNIC AL MANUAL
reduce the number of adsorptions required
to remove an antibody completely. Because
en- zymes destroy some antigens,
antibodies di- rected against those antigens
are not removed by enzyme-treated red
cells. To ensure that an adsorption process is
complete (ie, that no un- adsorbed antibody
remains), it is essential to confirm that the
adsorbed serum is nonreac- tive with a
reserved sample of the adsorbing red cells
that was not used for adsorption. Ad-
sorption requires a substantial volume of
red cells, and vials of reagent red cells are
usually not sufficient. Blood samples from
donor units or staff members are the most
convenient sources.
When separating mixtures of
antibodies, the selection of red cells of the
appropriate phenotype is extremely
important. If one or more antibodies have
been previously identi- fied, red cells that
express the corresponding antigens can be
used to remove the known an- tibodies and
leave the unknown antibody(ies) in the
adsorbed serum. For example, if a per- son
who types K+k–, Fy(a–b+) has produced
anti-k, it may be necessary to adsorb the
anti-k onto K–k+, Fy(a–b+) reagent red
cells to re- move the anti-k. Then, the
adsorbed serum can be tested with
common K–k+, Fy(a+b–) red cells to
detect possible anti-Fy a.
Elution
Elution dissociates antibodies from
sensitized red cells. Bound antibody may be
released by changing the thermodynamics of
antigen- antibody reactions, neutralizing or
reversing forces of attraction that hold
antigen-antibody complexes together, or
disturbing the struc- ture of the antigen-
antibody binding site. The usual objective is
to recover bound antibody in a usable form.
Various elution methods have been
de- scribed in the literature. Selected
procedures are given in Methods 4-1
through 4-4. No sin- gle method is best for
all situations. Heat or freeze-thaw elution
techniques are usually re- stricted to the
investigation of HDFN caused by ABO
incompatibility because these elution
procedures rarely work well for other
antibod- ies. Acid or organic solvent
methods are used
for eluting warm-reactive auto- and alloanti-
bodies. Commercial kits are also available for
performing elution. (See Table 17-2 for a list
of elution methods and their uses,
advantages, and disadvantages.)
Elution techniques are useful for the
fol- lowing:
1. Investigation of a positive DAT result
(Chapter 17).
2. Concentration and purification of
antibod- ies, detection of weakly
expressed antigens, and identification of
multiple antibody specificities. Such
studies are used in con- junction with an
appropriate adsorption technique, as
described below and in Method 2-7.
3. Preparation of antibody-free red cells for
phenotyping or autologous adsorption
studies. Procedures used to remove cold-
and warm-reactive autoantibodies from
red cells are discussed in Methods 4-5
and 4-8.
Technical factors that influence the
out- come of elution procedures include the
follow- ing:
1. Incomplete washing. Sensitized red cells
should be thoroughly washed before an
elution to prevent contamination of the
eluate with unbound residual antibody.
Six washes with saline are usually
adequate, but more washes may be
needed if the serum contains a high-titer
antibody (the considerations in Item 3
below should be kept in mind). To
confirm the efficacy of the washing
process, supernatant fluid from the final
wash should be tested for antibody
activity and found to be nonreactive.
2. Binding of protein to glass surfaces. If an
eluate is prepared in the test tube that was
used during the sensitization or washing
phases, antibody that nonspecifically
binds to the test tube surface may
dissociate dur- ing the elution. Similar
binding can also occur from a whole
blood sample when a patient has a
positive DAT result and has free antibody
in the serum. To avoid such
contamination, red cells used to prepare
an
 C H A P TE R 1 6 Identification of Antibodies to Red Cell Antigens
eluate should be transferred to a clean test
tube before washing and then to another
clean tube before the elution procedure is
initiated.
3. Dissociation of antibody before elution.
IgM antibodies, such as anti-A or anti-M,
or low-affinity IgG may spontaneously
disso- ciate from the red cells during the
wash phase. To minimize the loss of
bound anti- body, cold (4 C) saline or
wash solution pro- vided by the
manufacturer should be used for washing.
4. Incorrect technique. Such factors as incom-
plete removal of organic solvents or
failure to correct the tonicity or pH of an
eluate may cause the reagent red cells
used to test the eluate to hemolyze or
appear “sticky.” The presence of stromal
debris may inter- fere with the reading of
test results. Careful technique and strict
adherence to proce- dures should
eliminate such problems.
5. Instability of eluates. Dilute protein solu-
tions, such as those obtained by elution
into saline, are unstable. Eluates should
be tested as soon after preparation as
possible. Alternatively, bovine albumin
may be added to a final concentration of
6% w/v, and the preparation may be
frozen during storage. Eluates can also be
prepared in antibody-free plasma, 6%
albumin, or a similar protein medium.
When commercial elution kits are used,
the manufacturer’s instructions for
preparation and storage should be
followed.
Combined Adsorption-Elution
Combined adsorption-elution tests can be
used to separate a mixture of antibodies in a
single serum sample, detect weakly
expressed antigens on red cells, or help
identify weakly reactive antibodies. The
process consists of first incubating serum
with selected red cells and then eluting
antibody from the adsorbing red cells.
Care must be taken when selecting the
adsorbing cells to separate a mixture of anti-
bodies. The cells should express only one of
the antigens corresponding to an antibody in
the mixture so that the eluate from the cells
■ 409
will contain only that antibody. Both the
eluate and adsorbed serum can be used for
further testing. Unmodified red cells are
generally used for adsorptions when
subsequent elu- tions are being prepared.
Titration
The titer of an antibody is usually
determined by testing serial twofold
dilutions of the serum with selected red
cells. Results are expressed as the reciprocal
of the highest serum dilution that shows
macroscopic agglutination. Titra- tion values
can provide information about the relative
amount of antibody present in a se- rum
sample or the relative strength of antigen
expression on red cells.
Titration studies are useful for the
follow- ing purposes:
1. Prenatal studies. When the antibody has
a specificity that is known to cause
HDFN or the antibody’s clinical
significance is unknown, the results of
titration studies may contribute to the
decision about per- forming additional
procedures (eg, Doppler sonography or
amniocentesis). (See Chap- ter 22 and
Method 5-3.)
2. Antibody identification. Some antibodies
that agglutinate virtually all reagent red
cells may give an indication of specificity
by dem- onstrating reactivities of different
strengths with different red cell samples in
titration studies. For example, potent
undiluted auto- anti-I may be reactive with
both adult and umbilical cord blood red
cells, but titration studies may reveal
reactivity with adult I+ cells at a higher
dilution than with cord blood I– red cells.
The reactivity of most antibodies weakens
progressively with serial dilutions (ie, a 2+
reaction becomes 1+ in the next dilution),
and weak antibodies (<1+) may lose their
reactivity when diluted. How- ever, some
antibodies that have weak reac- tions when
undiluted continue to react at dilutions as
high as 1 in 2048. Such anti- bodies
include anti-Ch, -Rg, -Csa, -Yka, -Kna,
-McCa, and -JMH. When weak reactions are
observed in an IAT, titration studies may
be performed to determine whether the
410 ■ AABB T EC HNIC AL MANUAL
reactivity is consistent with the antibodies
in this group; however, not all examples of
these antibodies demonstrate such high
titer, low-avidity characteristics. Thus, the
serologic characteristics may suggest
certain specificities, but failure to do so
does not eliminate these possibilities. The
antibodies listed above are not expected to
cause short- ened red cell survival, although
there are examples of other antibodies (eg,
anti- Lub, -Hy, and –Yta) that may mimic
these serologic characteristics and cause
short- ened red cell survival. Details about
titration are given in Method 3-15.
3. Separating multiple antibodies. Titration
results may suggest that one antibody is
reactive at higher dilutions than another
antibody. That information can allow the
serum to be diluted before it is tested
with a red cell panel, which effectively
removes one antibody and allows the
other to be identified. For example, if a
serum contains anti-c that is reactive to a
titer of 2 and anti- Jka that is reactive to a
titer of 16, it may be possible to eliminate
the anti-c reactivity by diluting the serum
to a titer of 8.
Other Methods
Methods other than traditional tube, gel, or
solid-phase techniques may be used for anti-
body identification. Some methods are espe-
cially useful for testing small volumes of
serum or reagents. Such methods include
testing in capillary tubes, microplates, or
enzyme-linked immunosorbent assays.
Other methods that are useful in laboratories
with specialized equipment include
radioimmunoassay, im- munofluorescence,
flow cytometry, and im- munoblotting.
Factors Affecting Antibody
Identification
Variation in Antigen Expression
For a variety of reasons, antibodies are not
al- ways reactive with all red cells that
express the corresponding antigen. Basic
interpretation by exclusion, as described
previously, may result in an antibody being
excluded because the se-
rum is nonreactive with an antigen-positive
red cell sample, despite the presence of the
an- tibody. Technical error, weak antibody
reactiv- ity, and variant or weak antigenic
expression are all possible causes.
Therefore, whenever possible, antibody
exclusions should be based only on red cells
that strongly express the anti- gen.
Enhancement techniques often help re-
solve problems associated with variations in
antigen expression (Methods 3-3 through
3-
13).
ZYGOSIT Y. Reaction strength of some anti-
bodies may vary because of dosage, in which
antibodies are more strongly reactive (or only)
with red cells that possess a “double-dose” ex-
pression of the antigen. Double-dose antigen
expression occurs when an individual is ho-
mozygous for the gene that encodes the anti-
gen. Red cells from individuals who are het-
erozygous for the gene may express fewer
antigens and, therefore, may be weakly reac-
tive or nonreactive with a weak example of the
corresponding antibody. Alloantibodies vary in
their tendency to demonstrate dosage. Many
antibodies to antigens in the Rh, Duffy, MNS,
and Kidd blood groups demonstrate dosage.
VA RIAT ION IN ADU LTS AND INFA NT S .
Some antigens (eg, I, P1, Le a, and Sda) show
variable expression on red cells from different
adults. The antigenic differences can be dem-
onstrated serologically; however, the variabili-
ty is unrelated to zygosity. Certain antibodies
(eg, anti-I or –Lub) demonstrate weaker reac-
tivity with cord red cells than with red cells
from adults (Table 16-5).
CHANGES WITH STORAG E . Blood group an-
tibodies may be more weakly reactive with
stored red cells than with fresh red cells. Some
antigens (eg, Fya, Fyb, M, P1, Kna/McCa, and
Bg) deteriorate more rapidly than others dur-
ing storage, and the rate varies among red cells
from different individuals.36 Because red cells
from donors are often fresher than commer-
cial reagent red cells, some antibodies have
stronger reactions with donor red cells than
with reagent red cells. Similarly, storage of red
cells in a freezer may cause antigens to deteri-
 C H A P TE R 1 6 Identification of Antibodies to Red Cell Antigens
TABLE 16-5. Antigen Expression on Cord Red Blood Cells*
ExpressionAntigens
Negative Lea, Leb, Sda, Ch, Rg, and AnWj
Weak I, H, P1, Lua, Lub, Yta, Vel, Bg, KN and DO antigens, Yka, Csa,
and Fy3 Strong i, LWa, and LWb
*Modified with permission from Reid, Lomas-Francis and Olsson. 4
orate, thus producing misleading antibody
identification results.
The pH or other characteristics of the
storage medium can affect the rate of
antigen deterioration.36,37 For example, Fya
and Fyb an- tigens may weaken when the
red cells are stored in a medium with low
pH and low ionic strength. Thus, certain
antibodies may dem- onstrate differences in
reactivity with red cells from different
manufacturers if the suspend- ing media are
different.
The age and nature of the specimen
must be considered when red cells are
typed. Anti- gens on red cells from clotted
samples tend to deteriorate more quickly
than antigens on red cells from donor units
that are collected in ci- trate anticoagulants,
such as acid-citrate-dex- trose or citrate-
phosphate-dextrose. Red cells in donor units
collected in approved anticoag- ulants
usually retain their antigens throughout the
standard shelf life of the blood component.
EDTA samples up to 14 days old are
suitable for antigen typing.38 However, the
manufactur- er’s instructions should be
consulted when commercial typing
reagents are used.
No Discernible Specificity
Factors other than variation in antigen
expres- sion may contribute to difficulty in
interpret- ing results of antibody
identification tests. If the reactivity with the
serum is very weak, the pattern of reactivity
and cross-out process have excluded all
likely specificities, or both, alternative
approaches to interpretation should be used.
PR E S E N CE O F A N TIGE N S IN CO M M O N .
In -
stead of excluding antibodies to antigens
on nonreactive red cells, it might be helpful
to ob-
■ 411
serve which antigens the reactive red cells
have in common. For example, if all of the red
cells reactive at room temperature are P1+ but
the anti-P1 pattern is not complete, the anti-
body could be anti-P1 that is not reactive with
red cells with a weaker expression of the anti-
gen. (Sometimes, such red cells are designated
on the panel sheet as “+w.”) In this case, it
might be helpful to use a method that enhanc-
es anti-P1, such as testing at a colder tempera-
ture.
If all of the reactive red cells are Jk(b+)
but not all Jk(b+) red cells are reactive, the
reactive red cells might be Jk(a–b+) with a
double-dose expression of the antigen. In
this case, en- hancement techniques,
such as enzymes, LISS, or PEG, might
help demonstrate reactivi- ty with all of the
Jk(b+) red cells. Typing the pa- tient’s red
cells to confirm that they lack the
corresponding antigen is also very helpful.
Finally, the presence of some
antigens in common may suppress the
expression of certain antigens. This
suppression can cause weak antibodies to
be missed or certain cells to be
unexpectedly nonreactive when a sus-
pected antibody fails to show reactivity with
all antigen-positive cells. For example,
In(Lu) is known to suppress the expression
of Lutheran antigens, P1, Inb, and AnWj.
Similarly, Kpa is known to weaken the
expression of Kell anti- gens. (See Chapter
14 for a more detailed dis- cussion.)
INHERENT VA RI ABILIT Y. Ne b u l o u s re
a c - tion patterns that do not appear to fit
any par- ticular specificity are characteristic
of certain antibodies, such as anti-Bga, -Kna,
-McCa, -Sla,
-Yka, -Csa, and -JMH. Antigens
corresponding to these antibodies vary
markedly in their
412 ■ AABB T EC HNIC AL MANUAL
expression on red cells from different
individ- uals. For example, the expression
of Knops blood group antigens shows
marked differenc- es between individuals of
either African or Eu- ropean ethnicity, and
this difference in expres- sion is caused by
variations in the CR1 copy numbers on the
red cells.39 Rarely, a pattern of reactive and
nonreactive red cells cannot be interpreted
because the typing result for a re- agent red
cell is incorrect or the reagent red cell has
a positive DAT result. If the red cell
sample is from a commercial source, the
man- ufacturer should be notified
immediately of the discrepancy.
UN LISTE D A N TIGE NS . Sometimes, a
serum reacts with an antigen that is not
routinely list- ed on the antigen profile
supplied by the re- agent manufacturer—
Ytb is an example. Even though serum
studies yield clearly reactive and
nonreactive test results, anti-Ytb may not be
suspected. In such circumstances, it is use-
ful to ask the manufacturer for additional
phe- notype information. If only one cell is
unex- pectedly reactive, this reaction is
most likely caused by an antibody to a low-
prevalence an- tigen. These antibodies are
discussed in more detail later in this chapter.
ABO T Y PE OF R E D CELLS TESTED. A serum
sample may be reactive with many or all of
the group O reagent red cells but not with
red cells of the same ABO group as the
autologous red cells. Such a reaction occurs
most frequently with anti-H, anti-IH, or
anti-LebH. Group O and A2 red cells have
more H antigen than A1 and
A1B red cells, which express very little H.
(See
Chapter 12 for more information.) Thus,
sera containing anti-H or anti-IH are
strongly reac- tive with group O reagent
red cells, whereas autologous A1 or A1B
red cells or donor red
cells used for crossmatching may be weakly
re-
active or nonreactive. Anti-LebH is strongly re-
active with group O, Le(b+) red cells but
weak- ly reactive or nonreactive with Le(b+)
red cells
from A or A B individuals.
1 1
Multiple Antibodies
When a serum sample contains two or more
the results of testing performed with a single
panel of reagent red cells. Perhaps the
easiest way to identify multiple antibodies is
to deter- mine the phenotype of the patient’s
pretrans- fusion autologous red cells and
then use a se- lected cell panel to identify or
exclude all common antibodies to the red
cell antigens that the patient lacks. (See the
discussion on selected cells earlier in this
chapter.) The presence of multiple
antibodies may be sug- gested by a variety
of test results, such as the following:
1. The observed pattern of reactive and
nonre- active red cells does not fit a
single antibody. When the exclusion
approach fails to indi- cate a specific
pattern, it is helpful to deter- mine
whether the pattern matches two
combined specificities. For example, if
the reactive red cells on the panel in
Table 16-2 are numbers 3, 5, 6, 9, and 10,
none of the specificities remaining after
crossing out fits a pattern exactly.
However, if both E and K are considered
together, a pattern is dis- cerned, with
cells 3 and 5 showing reactivity because
of anti-E, and cells 6, 9, and 10 because
of anti-K. If the reaction pattern does not
fit two combined specificities, the
possibility that more than two antibodies
are present must be considered. The more
antibodies a serum sample contains, the
more complex identification and
exclusion become, but the basic process
remains the same.
2. Reactivity occurs at different test phases.
When reactivity occurs at several phases,
each phase should be analyzed separately.
The pattern at room temperature may
indi- cate a different specificity from the
pattern at the AHG phase. It is also
helpful to look for variations in the
strength of the reac- tions at each phase
of testing. Table 16-6 provides
information about the character- istic
reactivity of several antibodies.
3. Unexpected reactions occur when attempts
are made to confirm the specificity of a sus-
alloantibodies, it may be difficult to interpret
pected single antibody.
If a serum suspected to
contain anti-e is reactive
with some e- negative
cells, another antibody
may be present or the
suspected antibody may
not
 C H A P TE R 1 6 Identification of Antibodies to Red Cell Antigens ■ 413

TABLE 16-6. Serologic Reactivity of Some Common Blood Group Antibodies

Immuno- Reactivity Associated with

globulin Papain/ DTT
Antibody Class 4C 22 C 37 C AHG Ficin (200 mM) HDFN HTR
Anti-M IgG > IgM Most Most Rare Sensitive Resistant Rare Rare
Anti-N IgM > IgG Most Most Rare Sensitive Resistant No Rare
Anti-S IgG > IgM Most Most Variable Resistant Yes Yes
Anti-s IgG > IgM Most Variable Resistant Yes Yes
Anti-U IgG Most Resistant Resistant Yes Yes
Anti-P1 IgM Most Most Resistant Resistant No Rare
(IgG rare)
Anti-D IgG > IgM Some Some Most Resistant Resistant Yes Yes
(IgA rare)
Anti-C IgG > IgM Some Some Most Resistant Resistant Yes Yes
Anti-E IgG > IgM Some Some Most Resistant Resistant Yes Yes
Anti-c IgG > IgM Some Some Most Resistant Resistant Yes Yes
Anti-e IgG > IgM Some Some Most Resistant Resistant Yes Yes
Anti-Lu a
IgM > IgG Most Most Resistant or Variable Rare No
weakened
Anti-Lub IgG > IgM Some Most Resistant or Variable Mild Yes
weakened
Anti-K IgG > IgM Some Most Resistant Sensitive Yes Yes
Anti-k IgG > IgM Most Resistant Sensitive Yes Yes
Anti-Kpa IgG Most Resistant Sensitive Yes Yes
Anti-Kpb IgG > IgM Most Resistant Sensitive Yes Yes
Anti-Js a
IgG > IgM Most Resistant Sensitive Yes Yes
Anti-Js b
IgG Most Resistant Sensitive Yes Yes
Anti-Le a
IgM > IgG Most Most Most Most Resistant Resistant No Rare
Anti-Leb IgM > IgG Most Most Most Most Resistant Resistant No No
Anti-Fya IgG > IgM Most Sensitive Resistant Yes Yes
Anti-Fy b
IgG > IgM Most Sensitive Resistant Yes Yes
Anti-Jk a
IgG > IgM Most Resistant Resistant Yes Yes
Anti-Jk b
IgG > IgM Most Resistant Resistant Yes Yes
Anti-Di a
IgG Most Resistant Resistant Yes Yes
Anti-Dib IgG Most Resistant Resistant Yes Yes
414 ■ AABB T EC HNIC AL MANUAL

TABLE 16-6. Serologic Reactivity of Some Common Blood Group Antibodies (Continued)

Immuno- Reactivity Associated with

globulin Papain/ DTT
Antibody Class 4C 22 C 37 C AHG Ficin (200 mM) HDFN HTR
Anti-Yta IgG Most Variable Sensitive No Yes
or weak-
ened
Anti-Ytb IgG Most Variable Sensitive No No
or weak-
ened
Anti-Xga IgG > IgM Some Most Sensitive Resistant No No

Anti-Sc1 IgG Most Resistant Variable No No

Anti-Sc2 IgG Most Resistant Variable No No

Anti-Doa IgG Most Resistant Variable No Yes

Anti-Dob IgG Most Resistant Variable No Yes

Anti-Coa IgG > IgM Most Resistant Resistant Yes Yes

Anti-Cob IgG Most Resistant Resistant Yes Yes

AHG = antiglobulin reagent; DTT = dithiothreitol; HDFN = hemolytic disease of the fetus and newborn; HTR =
hemolytic transfusion reaction; Ig = immunoglobulin.

be anti-e. Testing a panel of selected e-neg-
ative red cells may help identify an addi-
tional specificity.
4. No discernible pattern emerges. When vari-
able reaction strengths are observed and
dosage or other variations in antigen
strength do not provide an explanation,
additional approaches and methods of
test- ing are needed. Some helpful steps
include the following:
a. If strongly positive results are
obtained, use the exclusion method
with nonreactive cells to eliminate
some specificities from initial consid-
eration.
b. If weak or questionable positive
results are obtained, test the serum
against cells with a strong expression
of the antigen that corresponds with
any sus- pected specificity, and
combine this approach with methods
that enhance the reactivity.
c. Type the patient’s red cells for com-
mon red cell antigens, and eliminate
from consideration specificities that
correspond to antigens on the
patient’s autologous red cells. This
step may not be possible if the
patient has been transfused recently
or has had a posi- tive DAT result.
d. Use a method to inactivate certain
antigens on the reagent cells.
Enzyme treatment renders cells
negative for such antigens as Fya and
Fyb (see Table 16-4).
e. Use adsorption or elution methods to
separate antibodies (Methods 3-20, 4-1,
and 4-2).
f. Enhance antibody reactivity by using
a more sensitive method (eg, PEG,
enzymes, increased incubation time,
or increased serum-to-cell ratio; see
Methods 3-5 and 3-8 through 3-13).
 C H A P TE R 1 6 Identification of Antibodies to Red Cell Antigens
Antibodies to High-Prevalence Antigens
If all reagent red cells are reactive but the
auto- control is nonreactive, an alloantibody
to a high-prevalence antigen should be
consid- ered, especially if the strength and
test phase of reactions are uniform for all of
the red cells tested. Antibodies to high-
prevalence antigens can be identified by
testing selected red cells of rare phenotypes
and by typing the patient’s autologous red
cells with antisera to high- prevalence
antigens. Knowing the race or eth- nic origin
of the antibody producer can be helpful
when selecting additional tests to per- form
(Table 16-1). Red cells that lack all of the
antigens in a blood group system (eg, Rhnull
or
Ko) or chemically modified red cells (eg, DTT-
treated red cells) can help limit possible speci-
ficities (Table 16-4).
If red cells negative for a particular high-
prevalence antigen are not available, red cells
that are positive for the lower-prevalence anti-
thetical antigen can sometimes be helpful. For
example, if a serum contains anti-Co a, which
reacts with a high-prevalence antigen, weaker
reactions may be observed with Co(a+b+) red
cells than with Co(a+b–) red cells because of a
dosage effect.
Antibodies to high-prevalence antigens
may be accompanied by antibodies to
com- mon antigens, which can make
identification much more difficult. In such
cases, it may be necessary to determine the
patient’s pheno- type for common antigens,
choose a pheno- typically similar red cell
sample (ie, one that lacks the patient’s
common antigens) that is incompatible
with the patient’s plasma, and adsorb the
antibody to the high-prevalence antigen
onto that red cell sample. This ap- proach
leaves antibodies to common red cell
antigens in the adsorbed plasma, where they
can be identified with a routine selected
red cell panel. Because the identification of
anti- bodies to high-prevalence antigens is
compli- cated, it may be necessary to refer
such speci- mens to a reference laboratory.
SE RO LO GIC CLUES. Knowing the serologic
characteristics of particular antibodies to
high-prevalence antigens and/or the preva-
■ 415
lence of such antigens in different popula-
tions may help with identification.
1. Reactivity in tests at room temperature
suggests anti-H, -I, -IH, -P, -PP1Pk (-
Tja),
-Ena, -LW (some), -Ge (some), -Sda, or -Vel.
2. Lysis of reagent red cells during testing
with fresh serum is characteristic of anti-
Vel, -P,
-PP1Pk (-Tja), -Jk3, and some examples of
anti-H and -I.
3. Reduced or absent reactivity with
enzyme- treated red cells occurs with
anti-Ch,
-Rg, -Ena, -Inb, -JMH, -Ge2, and some
examples of anti-Yta. Weak nebulous
reac- tions in the AHG phase are often
associated with anti-Kna, -McCa, -Yka,
and -Csa.
4. Complement-binding autoantibodies,
such as anti-I and -IH, or alloantibodies,
such as anti-Lub, -Vel, and -Yta, may give
similar results when polyspecific AHG
reagent is used.
ETHNIC CLUES . Antibodies, such as anti-U,
-McCa, -Sla, -Jsb, -Hy, -Joa, -Tca, -Cra, and –
Ata, should be considered if the serum is
from an individual of African ethnicity
because the antigen-negative phenotypes
occur almost ex- clusively in persons of
African ethnicity. Indi- viduals with anti-
Kpb are almost always of Eu- ropean
ethnicity. Anti-Di b is usually found among
populations of Asian, South American
Indian, and Native American ethnicity
(Table 16-1).
IN TERPR E TIN G A POSITIV E D AT R E SULT.
When a patient produces an antibody to a
high-prevalence antigen after transfusion,
the patient’s posttransfusion red cells may
have a positive DAT result, and both serum
and elu- ate may be reactive with all red
cells tested. Be- cause this pattern of
reactivity is identical to that of many warm-
reacting autoantibodies that appear after
transfusion, the two scenari- os can be very
difficult to differentiate. A post- transfusion
alloantibody to a high-prevalence antigen
would be expected to produce a DAT result
of mixed-field appearance (ie, some red
cells agglutinated among many
unagglutinated red cells) because only the
transfused red cells would be coated with
antibody. In practice,
416 ■ AABB T EC HNIC AL MANUAL
however, weak sensitization and mixed-field
agglutination can be difficult to
differentiate. If a pretransfusion specimen is
not available, it may be helpful to use red
cell separation pro- cedures to isolate
autologous red cells for test- ing or perform
a DNA-based genotype, as de- scribed
earlier in this chapter. Performing a DAT on
autologous red cells, testing the post-
transfusion serum with DAT-negative
autolo- gous red cells, or both may help
distinguish an autoantibody from an
alloantibody. If a DAT result from
autologous red cells is negative, the
reactivity is consistent with an alloantibody.
If the posttransfusion serum is reactive with
DAT-negative autologous red cells, the
reactiv- ity is consistent with an
autoantibody (Chap- ter 17 and Fig 16-2).
Antibodies to Low-Prevalence Antigens
If a serum sample is reactive only with a single
donor or reagent red cell sample, an antibody
to a low-prevalence antigen should be sus-
pected. To identify such an antibody, one can
test the serum with a panel of reagent red cells
that express low-prevalence antigens. Alterna-
tively, the one reactive red cell sample can be
tested with known antibodies to low-preva-
lence antigens. Unfortunately, sera that con-
tain antibodies to low-prevalence antigens
often contain multiple antibodies to low-prev-
alence antigens. Although low-prevalence an-
tigens are rare by definition, antibodies that
recognize some of them are much less rare.
Many antibodies to low-prevalence antigens
are reactive only at temperatures below 37 C
and therefore have doubtful clinical signifi-
cance. To confirm the suspected specificities,
one may need the expertise and resources of a
reference laboratory.
If an antibody to a low-prevalence anti-
gen is suspected, transfusion should not
be delayed while identification studies are
per- formed. Because antisera to type donor
units for low-prevalence antigens are
rarely avail- able, it is usually necessary to
rely on the cross- match to avoid transfusion
of antigen-positive units. When the serum
is reactive with only one donor unit or
reagent red cell, the most likely cause is an
antibody to a low-prevalence
antigen; however, other possible explanations
include that the red cells may be ABO incom-
patible, have a positive DAT result, or be poly-
agglutinable.
If an antibody in the serum of a pregnant
woman is suspected of reacting with a low-
prevalence antigen, testing the father’s red
cells with maternal plasma (if the plasma is
ABO compatible) can predict the possibility
that the fetal red cells also carry the paternal
antigen and are incompatible with the mater-
nal antibody, even if the antibody specificity is
unknown. If a newborn has a positive DAT re-
sult, testing the mother’s serum or an eluate
from the infant’s red cells against the father’s
red cells can implicate an antibody to a low-
prevalence antigen as the probable cause.
That test can be performed only if the mother’s
plasma is ABO compatible with the father’s
red cells or if the eluate from the infant’s red
cells does not contain anti-A or -B that would
be re- active with the father’s red cells. Some
refer- ence laboratories do not attempt to
identify antibodies to low-prevalence
antigens be- cause the antibodies are not
clinically mean- ingful. Antibodies may be
identified when time permits and suitable
reagents are avail- able.
Antibodies to Reagent Components and
Other Anomalous Serologic Reactions
Antibodies to a variety of drugs and
additives can cause positive results in
antibody detec- tion and identification tests.
The mechanisms are probably similar to
those discussed in Chapter 17. Most of these
anomalous reac- tions are in-vitro
phenomena and have no clinical significance
in transfusion therapy, other than causing
laboratory problems that delay transfusions.
The reactions rarely cause erroneous
interpretations of ABO typing that could
endanger the patient. For a more de- tailed
discussion, see the suggested reading by
Garratty.
RO UL E AU X . Rouleaux are aggregates of
red cells that often look like a stack of coins
when viewed microscopically. Rouleaux
formation is an in-vitro phenomenon produced
by abnor-
 C H A P TE R 1 6 Identification of Antibodies to Red Cell Antigens
mal serum protein concentrations. It may be
difficult to detect antibodies by direct
aggluti- nation in a test serum that contains
rouleaux- producing proteins; however, it is
not a prob- lem in an IAT, where the
serum is washed away. The saline
replacement technique can be used to detect
direct-agglutinating antibod- ies in the
presence of rouleaux (Method 3-7).
PRES ERVATIVE S O LUTIONS . Antibodies
that are reactive with an ingredient in the
solu- tion used to preserve reagent red
cells (eg, chloramphenicol, neomycin,
tetracycline, hy- drocortisone, EDTA, sodium
caprylate, or vari- ous sugars) may
agglutinate red cells suspend- ed in that
solution. The autologous control is often
nonreactive unless a suspension of au-
tologous red cells is prepared with the
manu- facturer’s red cell diluent or a similar
preserva- tive. Such reactions can often be
avoided by washing the reagent red cells
with saline be- fore testing. Adding the
medium to the autolo- gous control and
converting a nonreactive test to a reactive
test can often confirm the role of the
preservative. In some cases, however,
washing the reagent red cells does not
circum- vent the reactivity, and the
resolution may be more complex.
ENHA NCEME N T MEDIA . Antibodies that
are reactive with ingredients in other
reagents, such as commercially prepared
LISS additives or albumin, can cause
agglutination in tests that use reagent red
cells, donor red cells, au- tologous red cells,
or all three. Ingredients that have been
implicated include parabens (in some
LISS additives), sodium caprylate (in
some albumins), and thimerosal (in some
LISS/saline preparations). Antibody to
ingre- dients in enhancement media may be
suspect- ed if the autologous control is
positive but the DAT result is negative.
Omitting the enhance- ment medium
usually circumvents the reac- tivity.
In some cases, antibodies to reagent
in- gredients show blood group specificity
(eg, paraben-dependent anti-Jka, paraben-
depen- dent antibody to Rh protein, or
caprylate-de- pendent autoanti-e).40-42 The
autocontrol may be reactive if the patient’s
own red cells express
■ 417
the antigen, but the DAT result should be
neg- ative.
RE D-CEL L - REL ATE D ANOMA L IE S. The age
of red cells can cause anomalous serologic
re- actions. Antibodies exist that are reactive
only with stored red cells. Such
antibodies can cause agglutination of
reagent red cells by all techniques, and the
reactivity may be en- hanced with
enzyme-treated red cells. Such re- activity is
not affected by washing the red cells, and
the autocontrol is usually nonreactive. No
reactivity is seen when freshly collected
red cells (ie, from freshly drawn donor or
autolo- gous blood samples) are tested.
Patients with a Positive Autocontrol
Reactivity of a patient’s serum with the
autolo- gous cells may indicate the presence
of warm or cold autoantibodies, antibodies
to certain drugs, alloantibodies to transfused
red cells if the patient was recently
transfused, or antibod- ies to a constituent of
the test medium. When the autocontrol is
positive, a DAT should be performed
(Chapter 17 and Fig 16-2).
NO R E CENT T R AN SF US IONS . If the reactivi-
ty in the serum occurs at room temperature or
below, the cause is often anti-I or another cold
autoagglutinin. If the reactivity occurs in the
AHG phase, the reactivity is usually associated
with a positive DAT result and possible auto-
antibodies. If, in addition, the serum is reac-
tive with all cells tested, autoadsorption or
other special procedures may be necessary to
determine whether there are underlying allo-
antibodies that are masked by the autoanti-
bodies. If the serum is nonreactive or shows
only weak reactivity, an eluate may demon-
strate more potent autoantibody reactivity.
(See “Cold Autoantibodies” and “Warm Auto-
antibodies” sections below and Chapter 17 for
a more detailed discussion.)
RE CENT TR ANS F US IONS . If the
autocontrol is positive in the AHG phase,
there may be an- tibody-coated donor red
cells in the patient’s circulation, resulting in
a positive DAT result that may show
mixed-field reactivity. An elu- tion should
be performed, especially when
418 ■ AABB T EC HNIC AL MANUAL
tests on serum are inconclusive. For
example, a recently transfused patient may
have a posi- tive autocontrol and serum that
is weakly reac- tive with most but not all
Fy(a+) red cells. It may be possible to
confirm anti-Fya specificity in an eluate
because more antibody is often bound to
donor red cells than is free in the plasma.
Furthermore the preparation of an el- uate
concentrates the antibody. It is rare for
transfused red cells to make the autocontrol
positive at other test phases, but this can
oc- cur, especially with a newly
developing or cold-reacting alloantibody.
Some patients may form warm autoanti-
bodies following red cell transfusion;
there- fore, if the positive DAT test does
not have a mixed-field appearance—and
especially if the serum is reactive with all
red cells—then the possibility of an
autoantibody should be con- sidered.
Detection of alloantibodies in sam- ples
with autoantibody may require allogeneic
adsorptions (Method 3-20). If the
patient’s phenotype is known for the
common red cell antigens, an allogeneic
adsorption may re- quire only one
adsorption using a reagent cell that is
matched for the antigens that the pa- tient
lacks. If the patient’s phenotype is un-
known, it is necessary to perform multiple
al- logeneic adsorptions using
combination of reagent cells, each of which
lacks some of the common red cell
antigens. Cells that are ho- mozygous for
the common red cell antigens, such as R1
(DCe), R2 (DcE), and r (ce), are fre-
quently used. The adsorbed serum is tested
with reagent cells to rule out the common an-
tibodies that correspond to the antigens that
are missing from the adsorbing cell. For exam-
ple, if the adsorbing cell types R1 (DCe), K–,
Fy(a+b–), Jk(a–b+), and S–s+, the adsorbed
plasma could be tested for anti-c, anti-E, anti-
K, anti-Fyb, anti-Jka, and anti-S. It is never
possible, however, to rule out antibodies to
high-prevalence antigens when allogeneic ad-
sorption techniques are used.
If the DAT result does have a mixed-
field appearance and the serum is reactive
with all cells tested, a transfusion reaction
caused by an alloantibody to a high-
prevalence antigen should be considered
(Fig 16-2).
CO LD AUTOA N T I BO D I E S . Potent cold auto-
antibodies that are reactive with all red cells,
including the patient’s own, can create special
problems—especially when the reactivity per-
sists at temperatures above room temperature.
Cold autoantibodies may be benign or patho-
logic. (See Chapter 17 for a more detailed dis-
cussion.)
There are different approaches to
testing sera with potent cold agglutinins.
To detect underlying and potentially
clinically signifi- cant antibodies, methods
that circumvent the cold autoantibody can be
used. Procedures for the detection of
alloantibodies in the presence of cold-
reactive autoantibodies include the
following:
1. Prewarming techniques in which red cells
and serum are prewarmed to 37 C sepa-
rately before they are combined (Method 3-
6).
2. The use of anti-IgG rather than polyspecific
AHG reagent.
3. Cold autoadsorption of the patient’s
serum with autologous red cells to
remove autoan- tibodies but not
alloantibodies.
4. Adsorption with rabbit erythrocytes or
rab- bit erythrocyte stroma.
WA R M AU TOAN TIB O D I E S . P a t i en ts
a wi th warm-reactive autoantibodies in
their sera create a special challenge
because the anti- body is reactive with
virtually all red cells test- ed. The majority
of warm autoantibodies are IgG, although
some warm autoantibodies are IgM. IgM
warm autoantibodies are unusual, but they
have caused severe (often fatal) auto-
immune hemolytic anemia.43 If a patient
with warm autoantibodies requires a
transfusion, it is important to detect any
underlying clinically significant
alloantibodies that the autoanti- bodies may
mask. (For more information, see Chapter
17 and Methods 4-8 through 4-10.) The
reactivity of most warm-reactive autoanti-
bodies is greatly enhanced by certain
methods such as PEG and enzymes and, to a
lesser ex- tent, by LISS. It is often helpful to
omit the en- hancement media when testing
sera that con- tain warm autoantibodies. If
such tests are nonreactive, the same
procedure can be used
 C H A P TE R 1 6 Identification of Antibodies to Red Cell Antigens
for compatibility testing without the need
for adsorptions.
Frequency of Antibody Testing
After a clinically significant antibody has
been identified, antigen-negative Red Blood
Cell (RBC) units must be selected for all
future transfusions, even if the antibodies
are no lon- ger detectable.17(p37) In addition,
an AHG cross- match must be performed. It
is rarely neces- sary to repeat the
identification of known antibodies. AABB
Standards for Blood Banks and Transfusion
Services states that in patients with
previously identified antibodies, testing
methods should be used that identify addi-
tional clinically significant antibodies.17(p36)
Each laboratory should define and validate
methods for the detection of additional anti-
bodies in these patients.
Immunohematology Reference
Laboratories
When antibody problems cannot be resolved
or rare blood is needed, a reference
laboratory can provide consultation and
assistance through its access to the
American Rare Donor Program (ARDP).
(See Method 3-21.)
POSTANALY TICAL
CONSIDERATIONS: SELECTING
BLOOD FOR TRANSFUSION
After an antibody has been identified, it is im-
portant to determine its clinical significance.
Antibodies that are reactive at 37 C, in an IAT,
or both are potentially clinically significant.
Antibodies that are reactive at room tempera-
ture and below are usually not clinically
signif- icant; however, there are many
exceptions. For example, anti-Vel, -P, and -
PP1P k (-Tja) may be reactive only at cold
temperatures yet may cause red cell
destruction in vivo. Anti-Ch, anti-Rg, and
many of the Knops and Cost anti- bodies have
little or no clinical significance despite their
reactivity in an IAT. Reported experience with
examples of antibodies with the same
specificity can be used in assessing the
■ 419
clinical significance. Table 16-6 summarizes
the expected reactivity and clinical signifi-
cance of commonly encountered
alloantibod- ies. For some antibodies, little
or no data exist, and the decision about
clinical significance must be based on the
premise that clinically significant antibodies
are those that are active at 37 C, in an IAT,
or both.
Certain laboratory tests have been used to
predict the clinical significance of
antibodies. The monocyte monolayer assay,
which quanti- fies phagocytosis, adherence
of antibody-coat- ed red cells, or both, can
be used to predict the in-vivo clinical
significance of some antibod- ies. The test
for antibody-dependent cellular
cytotoxicity, which measures lysis of
antibody- coated red cells, and the
chemiluminescence assay, which measures
the respiratory release of oxygen radicals
after phagocytosis of anti- body-coated red
cells, have been helpful in predicting in-
vivo antibody reactivity—partic- ularly for
predicting the severity of HDFN. For cold-
reactive antibodies, in-vitro thermal am-
plitude studies may predict the likelihood
of in-vivo hemolysis.44
In-vivo tests may also be used to evaluate
the significance of an antibody. The most
common technique is a red cell survival
study in which radiolabeled, antigen-
positive red cells (usually labeled with 51Cr)
are infused into the patient. After a
specified period has elapsed, a sample of
blood from the patient is tested for
radioactivity. With this technique, it is
possible to measure the survival of 1 mL or
less of infused cells. Another in-vivo
tech- nique, flow cytometry, can also be
used to measure the survival of infused red
cells, but a larger aliquot of red cells (about
10 mL) is usu- ally required. Interpretation
of in-vivo survival test results is
complicated by the fact that small
aliquots of incompatible red cells may have
a faster rate of destruction than an entire
transfused RBC unit. Comparison with
docu- mented cases in the literature and
consulta- tion with a reference laboratory
should pro- vide guidance about previous
examples of similar specificities.
420 ■ AABB T EC HNIC AL MANUAL

with anti-
Phenotyping Donor Units
Whenever possible, RBC units selected for
transfusion to a patient with potentially
clini- cally significant antibodies should be
tested and found negative for the
corresponding an- tigen(s). Even if the
antibodies are no longer detectable, all
subsequent RBC transfusions to that patient
should lack the antigen to prevent a
secondary immune response. The transfu-
sion service must maintain records of all pa-
tients in whom clinically significant
antibodies have been previously identified,
and an AHG crossmatch procedure is
required if the serum contains—or has
previously contained—a clinically
significant antibody.17(pp37-38,75)
A potent example of the antibody
should be used to identify antigen-negative
blood. Of- ten, the antibody is a commercial
antiserum, but to save expensive or rare
reagents, units can be tested first for
compatibility with the patient’s serum.
Then, the absence of the anti- gen in
compatible units can be confirmed with
commercial reagents. If the antibody is
unusu- al and commercial antiserum is not
available, a stored sample from the
sensitized patient can be used to select
units for transfusion at a later time,
especially if the patient’s later sam- ples lose
reactivity. If a patient’s serum is used as a
typing reagent, the antibody should be
well characterized and retain its reactivity
after storage. Appropriate negative and
weak-posi- tive controls (eg, from
heterozygous donors) should be used at the
time of the testing. The following criteria,
established by the FDA for licensing some
reagents, should be used as guidelines for
human-source reagents used in lieu of
commercial reagents45,46:

1. Anti-K, -k, -Jka, -Fya, and –Cw: dilution of

1:8 must produce at least a 1+ reaction.
2. Anti-S, -s, -P1, -M, -I, -c (saline), -e
(saline), and -A1: dilution of 1:4 must
produce at least a 1+ reaction.
3. Most other specificities: undiluted must
produce at least a 2+ reaction.

When selecting units for patients with

clinically significant antibodies, some
serolo- gists recommend typing the units
 and the patient’s serum can be used to select
bodies from two different serologically compatible RBC units. This is
sources, but others especially true for antibod- ies that
consider this step characteristically are reactive below 37 C (eg,
unnecessary—especially anti-M, -N, -P1, -Lea, -Leb, and -A1) and that
when potent reagents are do not ordinarily produce a second- ary
available and an AHG immune response following the transfu- sion
crossmatch will be of antigen-positive RBC units.
performed. Different lots It can sometimes be a best practice to
of antibody from the provide phenotypically matched, antigen-
same manufacturer and neg- ative RBC units as a prophylactic
even different reagents measure when the patient has no detectable
from different antibody.
manufacturers may have When a patient of the R1R1 phenotype
been prepared from the produc- es anti-E, some serologists suggest
same source material that RBC units should be negative for both
because manufac- turers the E and c antigens. This recommendation
often share these is based on
resources. the assumption that the stimulus to produce
If a donor unit is anti-E may also have stimulated anti-c or
tested for selected anti- anti- cE that remains undetected by routine
gens and labeled by the tests.48 Similarly, for an R2R2 patient with
blood center, the use of demonstra-
licensed (commercial)
reagents, if available, is
required. If no licensed
reagent is available, the
unit may be labeled
with appropriate
wording (eg, “Tested and
found negative for XX
antigen using
unlicensed typing re-
agents”).47 Except for
results of ABO and D
typ- ing, there is no
requirement that the
hospital repeat testing of
donor minor antigen
typing if the results are
attached to the units on
the la- bel or by a tag.
Minor antigen typing
results on packing slips or
not physically attached to
the donor unit should be
confirmed by the hospi-
tal if the unit is used for
clinical care.

Antigen-Negative
Blood vs Crossmatch
for Compatibility
For certain antibodies,
typing the donor units
may not be necessary,
 C H A P TE R 1 6 Identification of Antibodies to Red Cell Antigens
ble anti-C, the use of e-negative donor blood
may be considered.
When a patient has potent warm autoan-
tibody and compatibility cannot be demon-
strated by routine testing or when an antibody
has not been specifically demonstrated but
cannot be conclusively excluded, it may be
prudent to select RBC units that are phenotyp-
ically matched for clinically significant anti-
gens.
When Rare Blood Is Needed
Rare blood includes units that are negative
for high-prevalence antigens or are negative
for a combination of common antigens.
When a pa- tient has multiple antibodies, it
can be helpful to determine the prevalence
of compatible do- nors. To calculate this
prevalence, one must multiply the
prevalence of donors who are negative for
one antigen by the prevalence of donors who
are negative for each of the other antigens.
For example, if a serum contains anti-c, anti-
Fya, and anti-S and if the preva- lence of
antigen-negatives are c-negative = 18%,
Fy(a–) = 34%, and S-negative = 45%, then the
prevalence of compatible units is 0.18 ×
0.34 × 0.45 = 0.028, or 2.8%. If the patient is
group O, the prevalence of group O donors
(45%) is factored into the calculation as fol-
lows: 0.028 × 0.45 = 0.013, or 1.3%.
If any of these antibodies is present
alone, finding compatible blood is not very
difficult; but the combination requires a
large number of units to provide one
compatible unit. The calculation above uses
the prevalence in pop- ulations of European
ethnicity, and prevalence may be different
in populations of non- European
ethnicity. In calculating the proba- bility of
compatible donors, one should use the
prevalence that corresponds with the eth-
KEY POINTS
It is important to consider the patient’s medical history (transfusions, pregnancies, transplantations, diagnoses, drugs, and e
An antibody may be tentatively excluded or ruled out if an antigen is present on a reagent cell and the patient’s serum does
■ 421
nic composition of the donor population, if
available.
When units of rare (<1 in 5000) or
uncom- mon (<1 in 1000) phenotypes are
needed, the ARDP can be very helpful.
This program, which is accessible only to
personnel from an accredited reference
laboratory, can identify blood suppliers that
are known to have poten- tially compatible
units (usually frozen RBC units) and donors
who may be eligible to do- nate (Method 3-
21).
Family members are another potential
source of rare blood donors. The absence
of high-prevalence antigens is usually
associated with the inheritance of the same
rare recessive blood group gene from each
heterozygous parent. Children from the
same parents have one chance in four of
inheriting the same two rare genetic
mutations, making siblings much more
likely than the general population to have
the rare blood type. In most cases, blood
from the patient’s parents, children, and half
of the patient’s siblings express only one
rare gene. If transfusion is essential and
there is no alternative to transfusing
incompatible blood, these heterozygous
(single-dose) donors are preferable to
random donors. For infants with HDFN
resulting from multiple antibodies or an
antibody to a high-prevalence antigen, the
mother (if she is ABO compatible) is often
the logical donor.
If the clinical situation allows, autologous
RBC transfusions should be considered for pa-
tients with rare phenotypes who are expected
to need rare blood in the future. For some
patients with multiple antibodies who are not
able to donate autologous units, it may be
necessary to determine whether any of the
antibodies is less likely to cause red cell
destruction and, in a crit- ical situation, to give
blood that is incompatible for that particular
antigen.
422 ■ AABB T EC HNIC AL MANUAL

3. Common clinically significant alloantibodies that should be excluded during antibody

identification testing are anti-D, -C, -E, -c, -e, -K, -Fya, -Fyb, -Jka, -Jkb, -S, and -s.
4. Based on probability, the use of two reactive and two nonreactive red cell samples is an
ac-
ceptable approach for antibody confirmation.
5. The autologous control, in which serum and autologous red cells are tested under the
same conditions as the serum and reagent red cells, is an important part of antibody
identifica- tion. The autologous control is not the same as a DAT.
6. The phenotype of the autologous red cells is an important part of antibody identification.
When an antibody has been tentatively identified in the serum, the corresponding
antigen is expected to be absent from the autologous red cells, although exceptions can
occur.
7. An antibody can be removed from a serum sample by adsorption onto red cells that
express
the corresponding antigen.
8. Elution dissociates antibodies from sensitized red cells. Bound antibody may be
released by changing the thermodynamics of an antigen-antibody reaction, neutralizing
or reversing forces of attraction that hold antigen-antibody complexes together, or
disturbing the struc- tures of the antigen-antibody binding site.
9. A clinically significant red cell antibody is an antibody that is frequently associated with
HDFN, hemolytic transfusion reactions, or a notable decrease in the survival of
transfused red cells.
10. Whenever possible, RBC units selected for transfusion to a patient with a potentially clin-
ically significant antibody should be tested and found negative for the corresponding

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Levitt J, ed. Standards for blood banks and transfu-
sion services. 29th ed. Bethesda, MD: AABB,
2014.
Lomas-Frances C, DePalma H. 2007 Rock Øyen
Symposium. DNA-based assays for patient
testing: Their application, interpretation, and
correlation of results. Immunohematology
2008;24:180-90.
Menitove JE. The Hardy-Weinberger principle:
Selection of compatible blood based on math-
ematic principles. In: Fridey JL, Kasprisin CA,
Chambers LA, Rudmann SV, eds. Numbers
for blood bankers. Bethesda, MD: AABB,
1995: 111.
Reid ME, Lomas-Francis C, Olsson M. The blood
group antigen factsbook. 3rd ed. London:
Elsevier Academic Press, 2012.
Rolih S. A review: Antibodies with high-titer, low-
avidity characteristics. Immunohematology
1990;6:59-67.
Rudmann SV, ed. Serologic problem-solving: A
sys- tematic approach for improved practice.
Bethesda, MD: AABB Press, 2005.
Weisbach V, Kohnhauser T, Zimmermann R, et al.
Comparison of the performance of microtube
column systems and solid-phase systems and
the tube low-ionic-strength solution additive
indirect antiglobulin test in the detection of
red cell alloantibodies. Transfus Med 2006;16:
276-84.
Westhoff C. 2007 Rock Øyen Symposium. Potential
of blood group genotyping for transfusion
medicine practice. Immunohematology 2008;
24:190-5.
 C h a p t e r 1 7

The Positive Direct Antiglobulin

Test and Immune-Mediated
Hemolysis

Regina M. Leger, MSQA, MT(ASCP)SBB, CMQ/OE(ASQ)

THE D I R E CT ANTIGLOB ULIN test

I (DAT) is a simple test used to determine

if red cells have been coated in vivo with im-
munoglobulin (Ig), complement, or both. The
DAT is used primarily for the investigation of
hemolytic transfusion reactions, hemolytic
disease of the fetus and newborn (HDFN), au-
toimmune hemolytic anemia (AIHA), and
drug-induced immune hemolysis. A positive
DAT result may or may not be associated with
immune-mediated hemolysis. As shown in Ta-
ble 17-1, there are many causes of a positive
DAT result.
THE DAT
The DAT should be performed on every pa-
tient in whom the presence of hemolysis has
been established to distinguish immune from
nonimmune hemolytic anemia. The DAT
should also be performed when a positive au-
tocontrol is found in antibody identification
studies (see Chapter 16), but there is no bene-
fit to performing a DAT (or autocontrol) as
part of routine pretransfusion testing. The
predic- tive value of a positive DAT result is
83% in a patient with hemolytic anemia, but
only 1.4% in a patient without, hemolytic
anemia.1
Small amounts of IgG and complement
that are lower than the detection limit of
rou- tine testing techniques appear to be
present on all red cells. Using sensitive
testing tech- niques, 5 to 90 IgG
molecules/red cell2 and 5 to 40 C3d
molecules/red cell3 have been detected in
healthy individuals. Depending on the tech-
nique and reagents used, the DAT can detect
100 to 500 molecules of IgG/red cell and
400 to 1100 molecules of C3d/red cell.
Positive DAT results are reported in 1:1000
to 1:14,000 blood donors and 1% to 15% of
hospital patients.4 These large differences in
incidence are proba- bly related to the
different DAT techniques used.
Most blood donors with a positive DAT
re- sult appear to be perfectly healthy, and
most patients with positive DAT results have
no

Regina M. Leger, MSQA, MT(ASCP)SBB, CMQ/OE(ASQ), Research Associate, American Red Cross Blood
Ser- vices, Southern California Region, Pomona, California
The author has disclosed no conflicts of interest.

425
426 ■ AABB T EC HNIC AL MANUAL
TABLE 17-1. Some Causes of a Positive DAT Result
Autoantibodies to intrinsic red cell antigens
Hemolytic transfusion reactions
Hemolytic disease of the fetus and newborn
Drug-induced antibodies
Passively acquired alloantibodies (eg, from donor plasma, derivatives, or immunoglobulin)
Nonspecifically adsorbed proteins (eg, hypergammaglobulinemia, high-dose intravenous immune globulin, or
modification of red cell membrane by some drugs)
Complement activation due to bacterial infection, autoantibodies, or alloantibodies
Antibodies produced by passenger lymphocytes (eg, in transplanted organs or hematopoietic components)
DAT = direct antiglobulin test
obvious signs of hemolytic anemia. However,
a careful evaluation may show evidence of in-
creased red cell destruction. A recent study
suggests that a positive DAT result in a
healthy blood donor may be a marker of risk
of future development of malignancy.5,6
A positive DAT result in a patient with
he- molytic anemia indicates that the most
likely diagnosis is one of the immune
hemolytic ane- mias. However, the DAT result
can be positive, coincidentally, in patients with
hemolytic ane- mia that is not immune
mediated. Conversely, some patients with
immune hemolytic anemia have a negative
DAT result (see “DAT-Negative AIHA”
section below).
The DAT can also be positive for IgG
or complement without a clear correlation
with anemia in patients with sickle cell
disease, beta-thalassemia, renal disease,
multiple my- eloma, autoimmune disorders,
AIDS, or other diseases associated with
elevated serum glob- ulin or blood urea
nitrogen levels.7-9 The inter- pretation of a
positive DAT result should take into
consideration the patient’s history, clini- cal
data, and results of other laboratory tests.
Initial transfusion reaction
investigations include a DAT on a
posttransfusion specimen. In the presence of
immune-mediated hemoly- sis, the DAT
result may be positive if sensitized red cells
have not been destroyed or negative if
hemolysis and rapid clearance have
occurred. Preparation and testing of an
eluate from DAT-
positive posttransfusion-reaction red cells is
indicated. Even if the DAT result is only
weakly positive or negative, testing of an
eluate may be informative. If the DAT result
is positive on the postreaction specimen, a
DAT should also be performed on the
pretransfusion specimen for comparison and
appropriate interpreta- tion.
The Principles of the DAT
The DAT is based on the test developed by
Coombs, Mourant, and Race10 for the detec-
tion of antibodies attached to red cells that
do not produce direct agglutination. This
test, an indirect antiglobulin test, was
initially used to demonstrate antibody in
serum, but it was lat- er applied to
demonstrate the in-vivo coating of red cells
with antibody or complement components
(the DAT).
Most of the antiglobulin reactivity is di-
rected at the heavy chains (eg, Fc portion of
the sensitizing antibody) or the complement
component, thus bridging the gap between
adjacent red cells to produce visible
agglutina- tion. The strength of the observed
agglutina- tion is usually proportional to the
amount of bound protein.
The DAT is performed by testing freshly
washed red cells directly with antiglobulin re-
agents containing anti-IgG and anti-C3d. In
the United States, only polyspecific anti-IgG,
-C3d and monospecific anti-IgG, anti-C3d,
 CH A PT E R 1 7
and anti-C3b,-C3d reagents are currently li-
censed. The red cells need to be washed to
re- move free plasma globulins and
complement; otherwise, the antiglobulin
reagent can be neutralized, leading to a
false-negative result. The saline used for
washing the red cells should be at room
temperature; washing red cells with warm
(eg, 37 C) saline can result in the loss of
red-cell-bound, low-affinity IgG. The red
cells should be tested immediately af- ter
washing to prevent false-negative results
due to the elution of IgG.
Although any red cells may be tested,
EDTA-anticoagulated blood samples are pre-
ferred. The EDTA prevents in-vitro fixation of
complement by chelating the calcium that is
needed for C1 activation. If red cells from a
clotted blood sample have a positive DAT re-
sult due to complement, the results should be
confirmed on red cells from freshly collected
blood kept at 37 C or an EDTA-anticoagulated
specimen if these results are to be used for di-
agnostic purposes.
The DAT can be initially performed
with a polyspecific antihuman globulin (AHG)
reagent that is capable of detecting both IgG
and C3d (see Method 3-14). If the results
are positive, tests with monospecific
reagents (anti-IgG and anticomplement)
need to be performed to ap- propriately
characterize the immune process involved
and determine the diagnosis. Be- cause
polyspecific reagents are usually blend- ed
and testing conditions for optimally detect-
ing IgG and C3d on red cells may differ,
some laboratories perform the DAT initially
with anti-IgG and anti-C3d reagents
separately. If the polyspecific reagent is
polyclonal, pro- teins other than IgG or C3d
(eg, IgM, IgA, or other complement
components) can occa- sionally be detected;
however, specific re- agents to distinguish
these other proteins by serologic techniques
are not readily available. If umbilical cord
blood samples are to be test- ed, it is
appropriate to use anti-IgG only be- cause
HDFN results from fetal red cell sensiti-
zation with maternally derived IgG antibody
and complement activation rarely occurs.4
It is important to follow the reagent
man-
ufacturer’s instructions and recognize any
product limitations. False-negative or
weaker
DAT/Immune Hemolysis ■ 427
results can be obtained if the washed red
cells are allowed to sit before they are tested
with anti-IgG or if the reading of the results
is de- layed. Some anticomplement reagents,
in con- trast, demonstrate stronger reactivity
if cen- trifugation is delayed for a short time
after the reagent has been added. When the
DAT result is positive with both anti-IgG
and anti-C3, the red cells should be tested
with an inert control reagent (eg, 6%
albumin or saline). Lack of ag- glutination
of the red cells in the control re- agent
provides some assurance that the test results
are accurately interpreted. If the con- trol is
reactive, the DAT result is invalid [see
sections below on warm AIHA (WAIHA)
and cold agglutinin disease (CAD)].
Reactivity with this control reagent can
indicate spontaneous agglutination caused
by heavy coating of IgG or rare warm-
reactive IgM, or it can indicate IgM cold
autoagglutinins that were not disso- ciated
during routine washing.
Evaluation of a Positive DAT Result
A positive DAT result alone is not
diagnostic of hemolytic anemia.
Understanding the signifi- cance of this
positive result requires knowl- edge of the
patient’s diagnosis; recent drug, pregnancy,
and transfusion history; and the presence of
acquired or unexplained hemolyt- ic anemia.
Dialogue with the attending physi- cian is
important. Clinical considerations to- gether
with laboratory data should dictate the
extent to which a positive DAT result is
evalu- ated.
Patient History
The following situations may warrant further
investigation of a positive DAT result.
1. Evidence of in-vivo hemolysis (ie, red cell
destruction). If a patient with anemia who
has a positive DAT result shows evidence
of hemolysis, testing to evaluate a possible
immune etiology is appropriate.
Reticulocy- tosis; spherocytes observed on
the peripheral blood film; hemoglobinemia;
hemoglobin- uria; decreased serum
haptoglobin; and elevated levels of serum
unconjugated (indirect) bilirubin or lactate
dehydroge-
428 ■ AABB T EC HNIC AL MANUAL
nase (LDH), especially LDH1, may be
asso- ciated with increased red cell
destruction. These factors are indicative
of hemolytic anemia but not specifically
immune hemo- lytic anemia. If there is
no evidence of hemolytic anemia, no
further studies are necessary unless the
patient requires a red cell transfusion and
the serum contains incompletely
identified antibodies to red cell antigens.
Testing an eluate may be helpful for
antibody identification (see “Elution”
section below and Chapter 16).
2. Recent transfusion. When a patient has
recently been transfused, a positive DAT
result may be the first indication of a
devel- oping immune response. The
developing antibody sensitizes the
transfused red cells that have the
corresponding antigen, and the DAT
result becomes positive. The anti- body
may not be present in sufficient quan- tity
to be detected in the serum. Antibody
may appear as early as 7 to 10 days after
transfusion in a primary immunization or
as early as 1 to 2 days in a secondary
response.4,11 These alloantibodies could
shorten the survival of red cells that have
already been transfused or administered
in subsequent transfusions. A mixed-field
appearance in the posttransfusion DAT
result (ie, agglutination of donor red cells
and no agglutination of the patient’s red
cells) may or may not be observed.
3. Administration of drugs associated with
immune-mediated hemolysis. Many drugs
have been reported to cause a positive
DAT result and/or immune-mediated
hemoly- sis, but this occurrence is not
common.12 (See “Drug-Induced Immune
Hemolytic Anemia” section below.)
4. History of hematopoietic progenitor cell or
organ transplantation. Passenger
lympho- cytes of donor origin produce
antibodies directed against ABO or other
blood group antigens on the recipient’s
red cells, causing
a positive DAT result.4
5. Administration of intravenous Ig (IVIG)
or IV anti-D. IVIG may contain ABO
antibod- ies, anti-D, or, sometimes, other
antibod- ies.13 Intravenous anti-D used to
treat
immune thrombocytopenia (previously
known as “immune thrombocytopenic
purpura”) causes Rh-positive patients to
develop a positive DAT result. IV anti-D
may also contain other antibodies, includ-
ing ABO antibodies.14
Serologic Investigation
Three investigative approaches are helpful in
the evaluation of a positive DAT result.
1. Test the DAT-positive red cells with anti-
IgG and anti-C3d reagents to characterize
the type of protein(s) coating the red
cells. This will help classify an immune-
mediated hemolytic anemia.
2. Test the serum/plasma to detect and iden-
tify clinically significant antibodies to red
cell antigens. Additional tests that are
use- ful in classifying the immune
hemolytic anemias and procedures for
detecting allo- antibodies in the presence
of autoantibod- ies are described later in
this chapter.
3. Test an eluate prepared from the DAT-
posi- tive red cells with reagent red cells
to deter- mine whether the coating
protein has red cell antibody specificity.
When the only coating protein is
complement, the eluate is likely to be
nonreactive. However, an elu- ate from
the patient’s red cells coated only with
complement should be tested if there is
clinical evidence of antibody-mediated
hemolysis, for example, after transfusion.
The eluate preparation can concentrate
small amounts of IgG that may not be
detectable in routine testing of the
patient’s plasma.
Results of these tests combined with the
patient’s history and clinical data should assist
in classification of the problem involved.
Elution
Elution can be informative in the following sit-
uations:
■ Clinical signs and symptoms of immune
hemolysis are present.
 CH A PT E R 1 7 DAT/Immune Hemolysis ■ 429

■ Serum test results are negative or
inconclu- sive for a patient who has been
recently transfused.
■ HDFN is suspected but no alloantibodies
were detected in the maternal plasma.
Performing an elution routinely on the
red cells of all patients who have a positive
DAT re- sult is not recommended. The
majority of pre- transfusion patients with a
positive DAT result have a nonreactive
eluate that is often associat- ed with an
elevated serum globulin level.7-9
Elution frees antibody from sensitized red
cells and recovers antibody in a usable form.
Multiple elution methods have been described
and reviewed.15 Many laboratories use com-
mercial acid elution kits, primarily for ease of
use and decreased exposure to potentially
harmful chemicals; these kits are suitable to
recover antibody in most cases. False-positive
eluate results associated with high-titer anti-
bodies have been reported when the low ionic
wash solution supplied with the commercial
acid eluates was used.16 Because no single elu-
tion method is ideal in all situations, an alter-
native elution method (eg, an organic solvent)
may be used in some high-complexity refer-
ence laboratories when a nonreactive acid elu-
ate result is not in agreement with clinical da-
ta.17
Table 17-2 lists the uses of some common
elution methods. Typically, eluates are tested
only at the antiglobulin phase. If an IgM
anti- body is being investigated or
suspected, how- ever, centrifugation and
reading after the 37 C incubation should be
performed. Technical considerations for
elution are discussed in Chapter 16.
In cases of hemolytic transfusion reac-
tions or HDFN, specific antibody (or
antibod- ies) is usually detected in the eluate
that may or may not be detectable in the
serum. For transfusion reactions, newly
developed anti- bodies that are initially
detectable only in the eluate are usually
detectable in the serum after about 14 to 21
days.18 If the eluate is nonreac- tive and a
non-group-O patient has received plasma
containing anti-A or anti-B (as a result of
the transfusion of group O platelets, for ex-
ample) and the recipient appears to have im-
mune hemolysis, the eluate should be tested
against A1 and/or B cells. It may be
appropriate to test the eluate against red
cells from recently transfused donor units,
which could have caused immunization to a
rare antigen. For cases of HDFN when no
maternal anti- body has been detected and
paternal red cells are ABO incompatible
with maternal plasma, testing an eluate
prepared from the infant’s red cells with the
paternal red cells may detect a maternally
derived antibody to a low-preva- lence
antigen,
When the eluate reacts with all cells
test- ed, autoantibody is the most likely
explana-

TABLE 17-2. Antibody Elution Methods

Method Use Comments

Lui freeze-thaw
Heat (56 C)
Acid elution kits (commercial)
Chemical/organic solvent
HDFN = hemolytic disease of the fetus and newborn; IgM = immunoglobulin M.
ABO HDFN
ABO HDFN, IgM agglutinating
antibodies
Warm auto- and alloantibodies
Warm auto- and alloantibodies
Quick, small volume of red
cells needed, poor recovery
of other antibodies
Easy, poor recovery of IgG allo-
and autoantibodies
Easy, possible false-positive elu-
ate results when high-titer anti-
body is present16
Chemical hazards, eg, flammabil-
ity, toxicity, or carcinogenicity
430 ■ AABB T EC HNIC AL MANUAL
tion, especially if the patient has not been
transfused recently. However, if the patient
has been recently transfused, an antibody to
a high-prevalence antigen should be consid-
ered. When no unexpected antibodies are
present in the serum and the patient has not
been transfused recently, no further serologic
testing of an autoantibody detected only in
the eluate is necessary.
The patient’s complete history,
including the presence of potential passive
antibodies, needs to be reviewed when the
serologic test results are evaluated. If both
the serum and el- uate are nonreactive, there
is evidence of im- mune hemolysis, and the
patient has received a drug reported to have
caused immune-me- diated hemolysis,
testing to demonstrate drug- related
antibodies should be considered (see
“Laboratory Investigation of Drug-Induced
Immune Hemolysis” section below).
AUTOIMMUNE HEMOLYTIC
ANEMIA
Immune-mediated hemolysis is the shorten-
ing of red cell survival as a result of an
immune response. If the marrow is able to
adequately compensate, the reduced red cell
survival may not result in anemia. Immune-
mediated he- molysis is only one cause of
hemolytic anemia, and many causes of
hemolysis are unrelated to immune
reactions.
The serologic investigations carried out
in the blood bank do not determine whether
a patient has a “hemolytic” anemia. The
diagno- sis of hemolytic anemia rests on
clinical find- ings and laboratory data, such
as hemoglobin or hematocrit values;
reticulocyte count; red cell morphology;
bilirubin, haptoglobin, and
LDH levels; and, sometimes, red cell survival
studies. The serologic findings help
determine whether the hemolysis has an
immune basis
and, if so, what type of immune hemolytic
anemia is present. This is important because
the treatment for each type is different.
In some cases, the destruction of red cells
takes place in the intravascular space with
the release of free hemoglobin into the
plasma. The red cells are ruptured following
activation
of the classical complement cascade. The
characteristic features of this rare type of he-
molysis are hemoglobinemia, and, when the
plasma hemoglobin level exceeds the renal
threshold, hemoglobinuria. Conversely and
more commonly, extravascular hemolysis re-
sults when macrophages in the spleen and
liv- er phagocytose red cells completely or
partial- ly (producing spherocytes) or
destroy red cells by cytotoxic events,
resulting in an increase in serum bilirubin.
This distinction is a simplifi- cation,
however, because hemoglobin can also be
released into the plasma following extravas-
cular destruction if hemolysis is brisk.
Immune hemolytic anemias can be clas-
sified in various ways. One classification
sys- tem is shown in Table 17-3. The AIHAs
are sub- divided into the major types:
WAIHA, CAD, mixed- or combined-type
AIHA, and paroxys- mal cold
hemoglobinuria (PCH). Not all cases fit
neatly into these categories. Table 17-4
shows the typical serologic characteristics of
the AIHAs. Drugs (discussed in the “Drug-
In- duced Immune Hemolytic Anemia”
section below) may also induce immune
hemolysis; the effects of drug-induced
autoantibodies are serologically
indistinguishable from WAIHA.
Warm Autoimmune Hemolytic
Anemia
The majority of AIHA cases are caused by
warm-reactive autoantibodies that are opti-
TABLE 17-3. Classification of Immune
Hemolytic Anemias
Autoimmune hemolytic anemia (AIHA)
Warm AIHA
Cold agglutinin disease
Mixed-type AIHA
Paroxysmal cold hemoglobinuria
Alloimmune hemolytic anemia
Hemolytic transfusion reaction
Hemolytic disease of the fetus and newborn
Drug-induced immune hemolytic anemia
 CH A PT E R 1 7 DAT/Immune Hemolysis ■ 431

TABLE 17-4. Typical Serologic Findings in AIHA

WAIHA CAD Mixed-Type AIHA PCH

DAT IgG C3 only IgG + C3 C3 only
(routine) IgG + C3 C3
C3

Ig type IgG IgM IgG, IgM IgG

Eluate IgG antibody Nonreactive IgG antibody Nonreactive

Serum IAT, 35% aggluti- IgM agglutinating IgG IAT-reactive Negative routine
nate untreated red antibody, titer antibody plus IgM IAT result, IgG
cells at 20 C 1000 (60%) at agglutinating anti- biphasic hemolysin
4 C, reactive at body reactive at in Donath-Land-
30 C 30 C steiner test

Specificity Broadly reactive, Usually anti-I Usually unclear Anti-P

multiple specifici-
ties reported
AIHA = autoimmune hemolytic anemia; WAIHA = warm AIHA; CAD = cold agglutinin disease; PCH = paroxysmal
cold hemoglobinuria; DAT = direct antiglobulin test; IgG = immunoglobulin G; IAT = indirect antiglobulin test.

mally reactive with red cells at 37 C. The auto-
antibody is usually IgG, but it can be IgM or
IgA.
Serologic Characteristics
The DAT result may be positive due to IgG
plus complement (67% of cases), IgG
without com- plement (20%), or
complement without IgG (13%).4
Performing an elution at initial diagno- sis
and/or during pretransfusion testing is use-
ful to demonstrate that the IgG coating the
pa- tient’s red cells is autoantibody.
Typically in WAIHA, the eluate is
reactive with virtually all red cells tested,
and reactivity is enhanced in tests against
enzyme-treated red cells, with polyethylene
glycol (PEG) en- hancement, or in column
agglutination and solid-phase tests. The
eluate usually has no se- rologic activity if
the only protein coating the red cells is
complement.
If the autoantibody has been adsorbed
by the patient’s red cells in vivo, the serum
may not contain detectable free antibody.
The se- rum contains free antibody when the
amount
of autoantibody exceeds the available
binding sites on the patient’s red cells. The
DAT result in such cases is usually strongly
positive.
Autoantibody in the serum typically is
re- active against all cells by an IAT.
Approximately 60% of patients with WAIHA
have serum anti- bodies that react with
untreated saline- suspended red cells. When
tested with PEG, enzyme-treated red cells,
column agglutina- tion, or solid-phase
methods, more than 90% of these sera can
be shown to contain autoan- tibody.
Agglutination at room temperature is
present in about one-third of patients with
WAIHA, but these cold agglutinins have
nor- mal titers at 4 C and are nonreactive at
30 C and 37 C. Thus, these cold agglutinins
are non- pathogenic and the patient does not
have CAD in addition to WAIHA.4
An unusual subcategory of WAIHA is
as- sociated with IgM agglutinins in the
plasma that are reactive at 37 C.4,19 This type
of WAIHA is characterized by severe
hemolysis, and the prognosis for these
patients can be poor. The red cells are
typically spontaneously aggluti- nated in the
DAT; that is, the washed red cells
432 ■ AABB T EC HNIC AL MANUAL
are reactive with all reagents tested,
including a control, such as 6% albumin
(see “Serologic Problems” section below).
Complement is usually detected on the red
cells; IgG or IgM may or may not be
detected. IgM agglutinins are often detected
in an eluate (eg, acid) when it is inspected
for agglutination after the 37 C incubation
and before the antiglobulin test is
conducted. Some serum IgM warm
autoagglu- tinins may be difficult to detect;
some are en- hanced in the presence of
albumin or at low pH. Optimal reactivity of
the agglutinin some- times occurs between
20 C and 30 C rather than at 37 C. These
antibodies have low titers at 4 C, usually
<64, which easily differentiates this IgM
warm antibody from those in CAD. To
prevent misinterpretation of titration results,
titrations at different temperatures (eg, 37 C,
30 C, room temperature, and 4 C) need to be
carried out with separate sets of tubes to
avoid carry-over agglutination.4,19
Serologic Problems
Warm autoantibodies can cause technical
dif- ficulties during red cell testing.
Spontaneous agglutination can occur if the
red cells are heavily coated with IgG and the
reagent con- tains a potentiator, such as
albumin. This has been observed when high-
protein Rh typing sera are used. If the
control reagent provided by the
manufacturer for these antisera is reac- tive,
the typing is invalid. IgG can less com-
monly cause spontaneous agglutination in
lower protein reagents (eg, monoclonal
typing sera); this reactivity is often weaker
or more fragile than true agglutination and
may not be detected by a 6% albumin
control.20
Warm-reactive IgM agglutinins can
also cause spontaneous agglutination,
resulting in ABO and Rh typing problems
and/or reactivi- ty with the negative control
reagent for the DAT.19 In these cases,
treatment with dithio- threitol (DTT) or 2-
mercaptoethanol (2-ME) (Method 2-18) to
disrupt the IgM agglutinin is required to
accurately interpret typing and DAT results.
When the spontaneous agglutina- tion is
disrupted, the control reagent is nonre-
active.
When the DAT result is positive due to
IgG, antiglobulin-reactive typing reagents can-
not be used unless the red-cell-bound IgG is
first removed (see Methods 2-20 and 2-21).
An alternative is to use low-protein antisera
(eg, monoclonal reagents) that do not require
an antiglobulin test (refer to the
manufacturer’s instructions for the detection of
spontaneous agglutination). It is helpful to
know which of the common red cell antigens
are lacking on the patient’s red cells to predict
which clinical- ly significant alloantibodies the
patient may have produced or may produce in
the future. Antigens absent from autologous
cells could well be the target of present or
future alloanti- bodies.
The presence of autoantibody in the se-
rum increases the complexity of the
serologic evaluation and the time needed to
complete pretransfusion testing. If a patient
who has warm-reactive autoantibodies in the
serum needs a transfusion, it is important to
deter- mine whether alloantibodies are also
present. Some alloantibodies may make
their presence known by reacting more
strongly or at differ- ent phases than the
autoantibody, but quite often, routine testing
may not suggest the exis- tence of masked
alloantibodies.21,22
Methods to detect alloantibodies in the
presence of warm-reactive autoantibodies
are used to attempt to remove, reduce, or
circum- vent the autoantibody. Antibody
detection methods that use PEG, enzymes,
column ag- glutination, or solid-phase red
cell adherence usually enhance
autoantibodies. Antibody de- tection tests
using low-ionic-strength saline (LISS) or
saline tube methods may not detect
autoantibodies but they do detect most clini-
cally significant alloantibodies. Other proce-
dures involve adsorption; two widely used
ad- sorption approaches are discussed below.
Adsorption with Autologous Red Cells
In a patient who has not been transfused re-
cently, adsorption with autologous red cells
(autologous adsorption; see Method 4-8) is
the best way to detect alloantibodies in the
pres- ence of warm-reactive autoantibodies.
Only
 CH A PT E R 1 7
autoantibodies are removed, and
alloantibod- ies, if present, remain in the
serum.
Autologous adsorption typically
requires some initial preparation of the
patient’s red cells. At 37 C, in-vivo
adsorption has occurred, and all antigen
sites on the patient’s own red cells may be
blocked. A gentle heat elution at 56 C for 3
to 5 minutes can dissociate some of the
bound IgG. This can be followed by treat-
ment of the autologous red cells with
proteo- lytic enzymes to increase their
capacity to ad- sorb autoantibody. Treatment
of the red cells with ZZAP, a mixture of
papain or ficin and DTT, accomplishes both
of these actions in one step. It is proposed
that the sulfhydryl component makes the
IgG molecules more susceptible to the
protease and dissociates the antibody
molecules from the cell.23 Multiple
sequential autologous adsorptions with new
aliquots of red cells may be necessary if the
se- rum contains high levels of
autoantibody. Once autoantibody has been
removed, the ad- sorbed serum is tested for
alloantibody reac- tivity.
Autologous adsorption is not recom-
mended for patients who have been trans-
fused within the last 3 months because a
blood sample may contain some transfused
red cells that might adsorb alloantibody.
Red cells nor- mally survive for about 110
to 120 days. In pa- tients with AIHA,
autologous and transfused red cells can be
expected to have shortened survival.
However, determining how long transfused
red cells remain in circulation in patients
who need repeated transfusions is not
feasible. It has been demonstrated that very
small amounts (<10%) of antigen-positive
red cells are capable of removing
alloantibody re- activity in in-vitro studies.24
Therefore, it is recommended to wait for 3
months after transfusion before performing
autologous ad- sorptions.
Adsorption with Allogeneic Red Cells
The use of allogeneic red cells for
adsorption (allogeneic adsorption) may be
helpful when the patient has been recently
transfused or in- sufficient autologous red
cells are available. The goal is to remove
autoantibody and leave
DAT/Immune Hemolysis ■ 433
the alloantibody in the adsorbed serum. The
adsorbing red cells must not have the
antigens against which the alloantibodies are
reactive. Because alloantibody specificity is
unknown, red cells of different phenotypes
are usually used to adsorb several aliquots
of the patient’s serum.
Given the number of potential alloanti-
bodies, the task of selecting the red cells
may appear formidable. However, red cell
selection is based only on those few
antigens for which alloantibodies of clinical
significance are like- ly to be present. These
include the common Rh antigens (D, C, E, c,
and e), K, Fya and Fyb, Jka and Jkb, and S and
s. Red cell selection is made easier by the
fact that some of these an- tigens can be
destroyed by appropriate pre- treatment (eg,
with enzymes or ZZAP) before use in
adsorption procedures (see Table 16-4).
Antibodies to high-prevalence antigens can-
not be excluded by allogeneic adsorptions
be- cause the adsorbing red cells are
expected to express the antigen and adsorb
the alloanti- body along with autoantibody.
When the patient’s phenotype is not
known, group O red cell samples of three
dif- ferent Rh phenotypes (R1R1, R2R2, and
rr) should be selected (see Method 4-9). One
sam-
ple should lack Jka and another, Jkb. As
shown in Table 17-5, ZZAP or enzyme
pretreatment of the adsorbing red cells
reduces the phenotype requirements.
Untreated red cells may be used, but the
adsorbing red cells must include at least one
sample that is negative for the S, s, Fya, Fyb,
and K antigens in addition to the Rh and
Kidd requirements stated above.
If the patient’s phenotype is known or
can be determined, adsorption with a single
sample of red cells may be possible. Red
cells can be selected that match the patient’s
phenotype or at least match the Rh and Kidd
phenotypes if ZZAP treatment is used. For
example, if a pa- tient’s phenotype is E– K–
S– Fy(a–) Jk(a–), untreated adsorbing red
cells need to lack all five antigens, but
enzyme-treated red cells only need to be E–
K– Jk(a–) and ZZAP-treated red cells only
need to be E– Jk(a–). Adsorption using
untreated red cells in the presence of PEG
(Method 4-10) or LISS25,26 are modifica-
tions that have been used to decrease the
434 ■ AABB T EC HNIC AL MANUAL
TABLE 17-5. Selection of Red Cells for Allogeneic Adsorption
Step 1. Select red cells for each Rh phenotype.
R1 R1
R 2R 2
rr
Step 2. On the basis of the red cell treatment, or lack of treatment (below), at least one of the Rh-
phenotyped cells should be negative for the antigens listed below.
ZZAP-Treated Red Cells Enzyme-Treated Red Cells
Jk(a–)
Jk(b–)
incubation time for adsorptions and increase
efficiency.
Testing of Adsorbed Serum
In some cases, each aliquot of serum may
need to be adsorbed two or three times to re-
move the autoantibody. The fully adsorbed
ali- quots are then tested against reagent red
cells known to either lack or carry common
anti- gens of the Rh, MNS, Kell, Duffy, and
Kidd blood group systems (eg, antibody
detection cells). If an adsorbed aliquot is
reactive, the ali- quot should be tested to
identify the antibody. Adsorbing several
aliquots with different red cell samples
provides a battery of potentially informative
specimens. For example, if the ali- quot
adsorbed with Jk(a–) red cells subse-
quently is reactive only with Jk(a+) red
cells, the presence of alloanti-Jk a can be
inferred confidently.
Occasionally, autoantibody is not re-
moved by three sequential adsorptions.
Addi- tional adsorptions can be performed,
but the performance of multiple adsorptions
has the potential to dilute the serum. If the
adsorbing cells do not appear to remove the
antibody, the
Untreated Red Cells
Jk(a–) Jk(a–)
Jk(b–) Jk(b–)
K– K–
Fy(a–)
Fy(b–)
S–
s–
autoantibody may have an unusual
specificity that is not reactive with the red
cells used for adsorption. For example,
autoantibodies with Kell, LW, or EnaFS
specificity are not removed by ZZAP-treated
red cells (see Table 16-4 for a list of
antigens altered by various agents). The
possibility that the sample contains an auto-
or alloantibody to a high-prevalence antigen
should always be considered when
adsorption fails to remove the reactivity.
Autoantibodies sometimes have patterns
of reactivity that suggest the presence of allo-
antibody. For example, the serum of a D– pa-
tient may have apparent anti-C reactivity. The
anti-C reactivity may reflect warm-reactive au-
toantibody even if the patient’s red cells lack
C. The apparent alloanti-C would, in this case,
be adsorbed by C– red cells, both autologous
and allogeneic. This is unlike the behavior of a
true alloanti-C, which would be adsorbed only
by C+ red cells. In one study, the serum
adsorbed with autologous red cells often
retained auto- antibodies that mimicked
alloantibodies in addition to the true
alloantibody(ies) present, whereas serum
adsorbed with allogeneic red cells most often
contained only alloantibod- ies.27 This reflects
an inefficiency of autologous
 CH A PT E R 1 7
adsorption that is primarily caused by
limited volumes of autologous red cells
available for removing all of the
autoantibody reactivity from the serum.
Specificity of Autoantibody
In many cases of WAIHA, no autoantibody
specificity is apparent. The patient’s serum
re- acts with all of the red cell samples
tested. If testing is performed with cells of
rare Rh phe-
notypes, such as D– – or Rhnull, some
autoanti- bodies are weakly reactive or are
nonreactive, and the autoantibody appears to
have broad specificity in the Rh system.
Apparent specific-
ity for simple Rh antigens (D, C, E, c, and e) is
occasionally seen, especially in saline or LISS
indirect antiglobulin tests. A “relative” speci-
ficity based on stronger reactivity with cells of
certain phenotypes may also be seen; relative
specificity may also be apparent after adsorp-
tion. Autoantibody specificities are clearer in
the serum than in the eluate.
Apart from Rh specificity, warm autoanti-
bodies with many other specificities have been
reported (eg, specificities in the LW, Kell,
Kidd, Duffy, and Diego systems).28,29 Patients
with autoantibodies of Kell, Rh, LW, Ge, Sc,
Lu, and Lan specificities may have transiently
de- pressed expression of the respective
antigen, and the DAT result may be negative
or very weakly positive.29 In these cases, the
autoanti- body may initially appear to be
alloantibody. Proof that it is truly autoantibody
is demon- stration that after the antibody and
hemolytic anemia subside, the antigen
strength returns to normal and stored serum
containing anti- body is reactive with the
patient’s red cells.
Tests against red cells of a rare
phenotype and by special techniques to
determine auto- antibody specificity have
limited clinical or practical application. If
the autoantibody re- acts with all red cells
except those of a rare Rh
phenotype (eg, Rhnull), compatible donor
blood is unlikely to be available. Such
blood, if available, should be reserved for
alloimmu- nized patients of that uncommon
phenotype.
DAT/Immune Hemolysis ■ 435
Selection of Blood for Transfusion
The most important consideration is to ex-
clude the presence of potentially clinically
sig- nificant alloantibodies before selecting
Red Blood Cell (RBC) units for transfusion.
There are multiple reports in the literature
demon- strating that patients who have
warm autoan- tibodies in their sera have a
higher rate of allo- immunization (eg, 12%
to 40%, with a mean of 32%).21,30-33
Although these patients present a serologic
challenge, they deserve the same protection
from hemolytic transfusion reac- tions as
any other patient. Autoantibodies that react
with all reagent red cells, even weakly, are
capable of masking alloantibody reactivity
(ie, reactivity of red cells with both
alloantibody and autoantibody may not be
any stronger than with autoantibody
alone).21,22
It is the exclusion of newly formed alloan-
tibodies that is of concern. Because of the
presence of autoantibodies, all crossmatches
are incompatible. This is unlike the case of
clinically significant alloantibodies without
autoantibodies, where a compatible cross-
match with antigen-negative red cells is
possi- ble. Monitoring for evidence of red
cell de- struction caused by alloantibodies is
difficult in patients who already have AIHA;
these pa- tients’ own red cells and transfused
red cells have shortened survival.
If no alloantibodies are detected in ad-
sorbed serum, random units of the appropri-
ate ABO group and Rh type may be selected
for transfusion. If clinically significant
alloanti- bodies are present, the transfused
cells should lack the corresponding
antigen(s). For patients facing long-term
transfusion support, it is pru- dent to obtain
an extended phenotype or gen- otype.
Consideration can then be given to
transfusion with donor units that are antigen
matched for clinically significant blood
group antigens to avoid additional
alloimmunization and potentially decrease
the number of adsorptions required and the
complexity of pretransfusion workup.
If the autoantibody has clear-cut specific-
ity for a single antigen (eg, anti-e) and active
hemolysis is ongoing, blood lacking that anti-
gen should be selected. There is evidence that
436 ■ AABB T EC HNIC AL MANUAL
such red cells survive longer than the patient’s
own red cells.4 In the absence of hemolysis or
evidence of compromised survival of trans-
fused cells, autoantibody specificity is not im-
portant. However, donor units that are nega-
tive for the antigen may be chosen because
this is a simple way to circumvent the autoan-
tibody and detect potential alloantibodies.
If the autoantibody shows broader reac-
tivity—reacting with all cells but showing
some relative specificity (eg, preferentially re-
acting with e+ red cells)—whether to transfuse
blood lacking the corresponding antigen is de-
batable. It may be undesirable to expose the
patient to Rh antigens absent from autologous
cells, especially D and especially in females of
childbearing potential, merely to improve se-
rologic compatibility testing results with the
autoantibody. (For example, when a D– pa-
tient has autoanti-e, available e– units are like-
ly to be D+; D–e– units are extremely rare.)
Referral of the sample for molecular investiga-
tion to determine the risk for allo- or autoanti-
body production will aid in decision making in
these complex cases and potentially improve
patient care.
Some laboratories use the adsorbed se-
rum to screen and select nonreactive units
(units that are antigen-negative for clinically
significant alloantibodies, if detected) for
transfusion. Other laboratories do not
perform a crossmatch with the adsorbed
serum be- cause all units will be
incompatible in vivo due to the
autoantibody. Issuing a unit that is sero-
logically compatible with adsorbed serum
may provide some assurance that the correct
unit has been selected and avoid incompati-
bility because of additional antibodies (eg,
anti-Wra), but this practice can also provide
a false sense of security about the safety of
the transfusion for these patients.
A transfusion management protocol us-
ing prophylactic antigen-matched units for
patients with warm autoantibodies, where
fea- sible, in combination with streamlined
ad- sorption procedures has been
described.34 The same antigens for the
commonly occurring, clinically significant
antibodies (D, C, E, c, e, K, Fya, Fyb, Jka, Jkb,
and S and s) are taken into ac- count, as
discussed in the previous section on
adsorptions. The ability to implement such a
protocol depends on the ability of the
transfu- sion service, and more often the
blood suppli- er, to maintain an adequate
inventory of phe- notyped units to meet the
antigen-matching needs.
In recent years, molecular technologies
have been applied to red cell genotyping for
patients with warm autoantibodies to deter-
mine which alloantibodies the patient can
make. DNA tests are attractive for
determining the predicted phenotype of
patients with a positive DAT (IgG) result
because IgG is not al- ways successfully
removed and some red cell antigens are
sensitive to IgG removal treat- ment.35-37
Recent transfusions do not interfere with
molecular testing. It must be remem- bered
that genotyping may not accurately pre- dict
the phenotype if uncommon or rare si-
lencing mutations are present or the patient
has received a stem cell transplant.
Some experts propose that an electronic
crossmatch can be safely used for patients
with autoantibodies when the presence of
common, clinically significant alloantibodies
has been excluded.38,39 This approach circum-
vents the need to issue units that are labeled
“incompatible”; however, as discussed above,
this practice can also lead to a false sense of
se- curity.
Although resolving serologic problems
for these patients is important, delaying
transfu- sion in the hope of finding
serologically com- patible blood may, in
some cases, cause greater danger to the
patient. Only clinical judgment can resolve
this dilemma; therefore, dialogue with the
patient’s physician is impor- tant.
Transfusion of Patients with
Warm- Reactive Autoantibodies
Patients with warm-reactive autoantibodies
may have no apparent hemolysis or may
have life-threatening anemia. Patients with
little or no evidence of significant hemolysis
tolerate transfusion quite well. The risk of
transfusion is somewhat increased in these
patients be- cause of the difficulties with
pretransfusion testing. The duration of
survival of the trans-
 CH A PT E R 1 7
fused red cells is about the same as that of
the patient’s own red cells.
In patients with active hemolysis,
transfu- sion may increase hemolysis, and
the trans- fused red cells may be destroyed
more rapidly than the patient’s own red
cells. This is related to the increased red cell
mass available from the transfusion and the
kinetics of red cell de- struction.4
Destruction of transfused cells may increase
hemoglobinemia and hemoglobin- uria.
Disseminated intravascular coagulation can
develop in patients with severe posttrans-
fusion hemolysis.
The transfusion of patients with AIHA is
a clinical decision that should be based on the
balance between the risks and clinical need.
Transfusion should not be withheld solely be-
cause of serologic incompatibility. The vol-
ume transfused should usually be the smallest
amount required to maintain adequate oxygen
delivery and not necessarily the amount re-
quired to reach an arbitrary hemoglobin level.4
The patient should be carefully monitored
throughout the transfusion.
DAT-Negative AIHA
Clinical and hematologic evidence of
WAIHA is present in some patients whose
DAT result is negative. The most common
causes of AIHA associated with a negative
DAT result are red- cell bound IgG below
the detection threshold of the antiglobulin
test, red-cell-bound IgM and IgA that are
not detectable by routine AHG reagents, and
low-affinity IgG that is washed off the red
cells during the washing phase for the
DAT.4,40
Nonroutine tests can be applied in these
situations. Unfortunately, these assays
require standardization and many have a low
predic- tive value. One of the easier tests is
for low- affinity antibodies. Washing with
ice-cold (eg, 4 C) saline or LISS may help
retain antibody on the cells; a control (eg,
6% albumin) is neces- sary to confirm that
cold autoagglutinins are not causing the
positive results.4,40 Comple- ment fixation
antibody consumption assay, enzyme-linked
antiglobulin test, radiolabeled anti-IgG, flow
cytometry, solid-phase, direct PEG test,
direct Polybrene test, column agglu-
DAT/Immune Hemolysis ■ 437
tination, and concentrated eluates are all
methods that have been used to detect lower
levels of red cell-bound IgG.40
Anti-IgG, anti-C3d, and the combined
anti-C3b,-C3d reagents are the only licensed
products available in the United States for
use with human red cells. AHG reagents that
react with IgA or IgM are available
commercially but probably have not been
standardized for use with red cells in
agglutination tests. They must be used
cautiously, and their hemagglutina- tion
reactivity must be carefully standardized by
the user.4 In countries outside the United
States, AHG reagents for the detection of
IgM and IgA in tube tests or column
agglutination tests may be available.
Cold Agglutinin Disease
CAD, which is less common than WAIHA,
is the hemolytic anemia that is most
commonly associated with autoantibodies
that react pref- erentially in the cold. CAD
occurs as an acute or chronic condition. The
acute form is often secondary to
Mycoplasma pneumoniae infec- tion. The
chronic form is often seen in elderly patients
and is sometimes associated with
lymphoma, chronic lymphocytic leukemia,
or Waldenström macroglobulinemia.
Acrocyano- sis and hemoglobinuria may
occur in cold weather. CAD is often
characterized by aggluti- nation, at room
temperature, of red cells in an EDTA
specimen, sometimes to the degree that the
red cells appear to be clotted.
Serologic Characteristics
Complement is the only protein detected on
red cells in almost all cases of CAD. If the
red cells have been collected properly and
washed at 37 C, there will be no
immunoglobulin on the cells and no
reactivity in the eluate. If other proteins are
detected, a negative control for the DAT (eg,
6% albumin or saline) should be tested to
ensure that the cold autoagglutinin is not
causing a false-positive result. The cold-re-
active autoagglutinin is usually IgM, which
binds to red cells in the lower temperature of
the peripheral circulation and causes
comple- ment components to attach to the
red cells. As
438 ■ AABB T EC HNIC AL MANUAL
the red cells circulate to warmer areas, the
IgM dissociates but the complement
remains.
IgM cold-reactive autoagglutinins associ-
ated with immune hemolysis usually react at
30 C, and 60% have a titer of 1000 when
test- ed at 4 C.4 If 22% to 30% bovine
albumin is in- cluded in the test system,
pathologic cold ag- glutinins will react at 30
C or 37 C.4 On occasion, pathologic cold
agglutinins have a lower titer (ie, <1000),
but they have a high thermal amplitude (ie,
reactive at 30 C with or without the addition
of albumin). The thermal amplitude of the
antibody has greater signifi- cance than the
titer. Hemolytic activity against untreated
red cells can sometimes be demon- strated at
20 C to 25 C. Except in rare cases with Pr
specificity, enzyme-treated red cells are
hemolyzed in the presence of adequate
complement.
To determine the true thermal amplitude
or titer of the cold autoagglutinin, the speci-
men is collected and maintained strictly at
37 C until the serum and red cells are
separated to avoid in-vitro autoadsorption.
Alternatively, plasma can be used from an
EDTA-anticoagu- lated specimen that has
been warmed for 10 to 15 minutes at 37 C
(with repeated mixing) and then separated
from the cells, ideally at 37 C. This process
should release autoadsorbed an- tibody back
into the plasma.
In chronic CAD, the IgM
autoagglutinin is usually a monoclonal
protein with kappa light chains. In the acute
form induced by Myco- plasma or viral
infections, the antibody is polyclonal IgM
with normal kappa and lamb- da light-chain
distribution. Rare examples of IgA and IgG
cold-reactive autoagglutinins have also been
described.4
Serologic Problems
Problems with ABO and Rh typing and other
tests are not uncommon. Often, it is only nec-
essary to maintain the blood sample at 37 C
immediately after collection and to wash the
red cells with warm (37 C) saline before test-
ing. Alternatively, an EDTA sample can be
warmed to 37 C for about 10 minutes, after
which the red cells are washed with warm sa-
line. It is helpful to perform a parallel control
test with 6% bovine albumin to determine
whether autoagglutination persists. If the
con- trol test result is nonreactive, the results
ob- tained with anti-A and anti-B are usually
valid. If autoagglutination still occurs, it
may be nec- essary to treat the red cells with
sulfhydryl re- agents. Because cold-reactive
autoagglutinins are almost always IgM and
sulfhydryl reagents denature IgM molecules,
reagents (such as 2- ME or DTT) can be
used to abolish autoagglu- tination (see
Method 2-18). Treating the red cells with
ZZAP reagent, as in the preparation for
adsorptions, can also be used (see Method 4-
8).
When the serum agglutinates group O re-
agent red cells, ABO serum tests are invalid.
Repeating the tests using prewarmed serum
and group A1, B, and O red cells and allowing
the red cells to “settle” after incubation at 37 C
for 1 hour (instead of centrifuging the
sample) often resolves any discrepancy (see
Method 2- 11). By eliminating the
centrifugation step, in- terference by cold-
reactive autoantibodies might be avoided.
Weak anti-A and/or -B in some patients’
sera may not react at 37 C. Al- ternatively,
adsorbed serum (either autoad- sorbed or
adsorbed with allogeneic group O red cells)
can be used. Rabbit erythrocyte stro- ma
should not be used for ABO serum tests be-
cause anti-B and anti-A1 may be
removed.41,42
Detection of Alloantibodies in the
Presence of Cold-Reactive Autoantibodies
Cold-reactive autoagglutinins rarely mask
clinically significant alloantibodies if serum
tests are conducted at 37 C and IgG-specific
reagents are used for the antiglobulin phase.
The use of potentiators (eg, albumin or
PEG) is not recommended because they may
increase the reactivity of the autoantibodies.
In rare in- stances, it may be necessary to
perform autol- ogous adsorption at 4 C (see
Method 4-5). Achieving the complete
removal of potent cold-reactive
autoagglutinins is very time con- suming
and usually unnecessary. Removal of
sufficient cold autoagglutinins may be facili-
tated by treating the patient’s cells with en-
zymes or ZZAP before adsorption. One or
two cold autologous adsorptions should
remove
 CH A PT E R 1 7
enough autoantibody to make it possible to
detect alloantibodies at 37 C that were other-
wise masked by the cold-reactive autoanti-
body. As an alternative, the allogeneic adsorp-
tion process used for WAIHA can be
performed at 4 C. Rabbit erythrocyte stroma,
which removes autoanti-I and -IH from sera,
should be used with caution because this
method can remove clinically significant allo-
antibodies—notably anti-D, -E, -Vel, and IgM
antibodies regardless of blood group specifici-
ty.43,44
Specificity of Autoantibody
The autoantibody specificity in CAD is most
often anti-I but is usually of academic interest
only. Anti-i is found less commonly, and it is
usually associated with infectious mononucle-
osis. On rare occasions, other specificities are
seen.
Autoantibody specificity is not
diagnostic
for CAD. Autoanti-I may be seen in healthy
in- dividuals as well as in patients with
CAD. The nonpathologic forms of autoanti-
I, however, rarely react at titers above 64 at
4 C and are usually nonreactive with I–
(cord i and adult i) red cells at room
temperature. In contrast, the autoanti-I of
CAD may react quite strongly with I– red
cells in tests at room temperature, and equal
or even stronger reactions occur with I+ red
cells. Autoanti-i reacts in the oppo- site
manner, demonstrating stronger reac- tions
with I– red cells than with red cells that are
I+. Anti-IT, originally thought to recognize a
transition state of i to I (explaining the
desig- nation “IT”), reacts strongly with cord
red cells, weakly with normal adult I red
cells, and most weakly with the rare adult i
red cells. In rare cases, the cold agglutinin
specificity may be anti-Pr, which reacts
equally well with untreat- ed red cells of I or
i phenotypes but does not react with
enzyme-treated red cells. Proce- dures to
determine the titer and specificity of cold-
reactive autoantibodies are given in Methods
4-6 and 4-7. Typical reactivity pat- terns of
cold autoantibodies are shown in the table in
Method 4-6.
DAT/Immune Hemolysis ■ 439
Mixed-Type AIHA
Although about one-third of patients with
WAIHA have nonpathologic IgM antibodies
that agglutinate at room temperature,
another group of patients with WAIHA have
cold agglu- tinins that react at or above 30 C.
This latter group is referred to as having
“mixed” or “com- bined warm and cold”
AIHA and can be subdi- vided into patients
with high-titer, high-ther- mal-amplitude
IgM cold antibodies (the rare WAIHA plus
classic CAD) and patients with normal-titer
(<64 at 4 C), high-thermal-ampli- tude cold
antibodies.45-47 Patients with mixed- type
AIHA often present with hemolysis and
complex serum reactivity in all phases of
testing.
Serologic Characteristics
In mixed-type AIHA, both IgG and C3 are
usu- ally detectable on patients’ red cells;
however, C3, IgG, or IgA alone may be
detectable on the red cells.4 An eluate contains
a warm-reactive IgG autoantibody. Both warm-
reactive IgG au- toantibodies and cold-
reactive, agglutinating IgM autoantibodies are
present in the serum. These autoantibodies
usually result in reactivi- ty at all phases of
testing and with virtually all cells tested. The
IgM agglutinating autoanti- body reacts at 30
C or above. If adsorptions are performed to
detect alloantibodies, it may be necessary to
perform them at both 37 C and 4 C.
Specificity of Autoantibodies
The unusual cold-reactive IgM agglutinating
autoantibody can have specificities that are
typical of CAD (ie, anti-I or -i) but often has
no apparent specificity.45,46 The warm-
reactive IgG autoantibody often appears to
be serologi- cally indistinguishable from
autoantibodies encountered in typical
WAIHA.
Transfusion of Patients with Mixed-Type
AIHA
If blood transfusions are necessary, the con-
siderations for the exclusion of
alloantibodies and the selection of blood for
transfusion are identical to those described
for patients with
440 ■ AABB T EC HNIC AL MANUAL
acute hemolysis caused by WAIHA and CAD
(see above).
Paroxysmal Cold Hemoglobinuria
PCH is the rarest form of DAT-positive
AIHA. Historically, PCH was associated with
syphilis, but this association is now
unusual.48 More commonly, PCH presents as
an acute transient condition that is
secondary to a viral infection, particularly in
young children. In such cases, the biphasic
hemolysin may be only transient- ly
detectable. PCH can also occur as an idio-
pathic chronic disease in older people.
Serologic Characteristics
PCH is caused by a cold-reactive IgG
comple- ment-binding antibody. As with
IgM cold-re- active autoagglutinins,
reactivity occurs with red cells in colder
areas of the body (usually the extremities)
and causes C3 to bind irre- versibly to red
cells. The antibody then dissoci- ates from
the red cells as the blood circulates to
warmer parts of the body. Red cells washed
in a routine manner for the DAT are usually
coated only with complement, but IgG may
be detectable on cells that have been washed
with cold saline and tested with cold anti-
IgG reagent.4 Keeping the test system close
to its optimal binding temperature allows the
cold- reactive IgG autoantibody to remain
attached to its antigen. Because complement
compo- nents are usually the only globulins
present on circulating red cells, eluates
prepared from the red cells of patients with
PCH are almost al- ways nonreactive.
The IgG autoantibody in PCH is
classical- ly described as a biphasic
hemolysin because binding to red cells
occurs at low temperatures but hemolysis
does not occur until the com- plement-
coated red cells are warmed to 37 C. This is
the basis of the diagnostic test for the
disease, the Donath-Landsteiner test (see
Method 4-11). The autoantibody may
aggluti- nate normal red cells at 4 C but
rarely to titers greater than 64. Because the
antibody rarely reacts above 4 C,
pretransfusion antibody de- tection tests are
usually nonreactive and the serum is usually
compatible with random do- nor cells by
routine crossmatch procedures.
Specificity of Autoantibody
The autoantibody of PCH has most
frequently been shown to have P specificity.
The autoan- tibody reacts with all red cells
by the Donath- Landsteiner test (including
the patient’s own red cells), except those of
the very rare p or Pk phenotypes.
Transfusion of Patients with PCH
Transfusion is rarely necessary for adult pa-
tients with PCH, unless their hemolysis is
se- vere. In young children, the thermal
amplitude of the antibody tends to be much
wider than in adults and hemolysis is often
more brisk, so transfusion may be required
as a lifesaving measure. Although there is
some evidence that p red cells survive
longer than P+ (P1+ or P1–) red cells, the
prevalence of p blood is approxi- mately 1
in 200,000, and the urgent need for
transfusion usually precludes attempts to ob-
tain this rare blood. Transfusion of donor
blood should not be withheld from patients
with PCH whose need is urgent. Transfusion
of red cells that are negative for the P
antigen should be considered only for those
patients who do not respond adequately to
randomly selected units of donor blood.4
DRUG-INDUCED IMMUNE
HEMOLY TIC ANEMIA
Drugs rarely cause immune hemolytic anemia;
the estimated incidence is 1 in 1 million peo-
ple.49 Many drugs have been implicated in he-
molytic anemia over the years, as can be seen
in the list provided in Appendix 17-1 and re-
viewed elsewhere.12
Drugs sometimes induce the formation
of antibodies—against the drug, red cell
mem- brane components, or an antigen
formed by the drug and the red cell
membrane. These antibodies may cause a
positive DAT result, immune red cell
destruction, or both.12,40 In some instances, a
positive DAT result can be caused by
nonimmunologic protein adsorp- tion
(NIPA) onto the red cell, which is caused by
the drug.12
 CH A PT E R 1 7 DAT/Immune Hemolysis ■ 441

Theoretical Mechanisms of
Drug- Induced Antibodies
Numerous theories have been suggested to ex-
plain how drugs induce immune responses
and what relation such responses may have to
the positive DAT result and immune-mediated
cell destruction observed in some patients. 4
For many years, drug-associated positive DAT
results were classified by four mechanisms:
drug adsorption (penicillin-type), immune
complex formation, autoantibody produc-
tion, and NIPA. This classification has been
useful serologically, but many aspects lack de-
finitive proof. In addition, some drugs demon-
strate serologic reactivity that appears to in-
volve more than one mechanism. A more
comprehensive approach, termed a “unifying
hypothesis,” is shown in Fig 17-1. One or
more populations of antibodies may be
present. In addition, NIPA, which is
independent of anti- body production, appears
to play a role in drug-induced immune
hemolytic anemia.12
Serologic Classification
Drug-induced antibodies can be classified
into two groups: drug dependent (those that
re- quire the presence of the drug in the test
sys- tem to be detected) and drug
independent (those that do not require the
in-vitro addition of the drug for detection).4
Drug-dependent antibodies are subdivided
into those that react with drug-treated red
cells (eg, antibodies to penicillin and some
cephalosporins) and those that react with
untreated red cells in the presence of a
solution of the drug (eg, antibod- ies to
quinine and ceftriaxone). Drug-indepen-
dent antibodies (eg, autoantibodies induced
by methyldopa and fludarabine) have
serolog- ic reactivity that is independent of
the drug de- spite the fact that the drug
originally induced the immune response.
Because the drug does not need to be added
to the test system, drug- independent
antibodies behave like autoanti- bodies that
are serologically indistinguishable from
idiopathic warm autoantibodies.

FIGURE 17-1. Proposed unifying theory of drug-induced antibody reactions (based on a cartoon by Habibi
as cited by Garratty28). The thicker lines represent antigen-binding sites for the Fab region of the drug-
induced antibody. Drugs (haptens) bind loosely or firmly to cell membranes, and antibodies may be made
to 1) the drug [producing in-vitro reactions typical of a drug adsorption (penicillin-type) reaction]; 2)
membrane components or mainly membrane components (producing in-vitro reactions typical of
autoantibody); or 3) part-drug, part- membrane components (producing an in-vitro reaction typical of
the so-called immune complex mechanism).28(p55)
442 ■ AABB T EC HNIC AL MANUAL
If a patient is suspected of having drug-
induced immune hemolytic anemia
(DIIHA), then the suspected drug should be
stopped. Laboratory testing to detect drug-
dependent antibodies can be performed, but
DIIHA caused by drug-independent
antibodies or NIPA can only be suggested
by showing a tem- poral association of the
drug administration and hemolysis.
The historical details of penicillin- and
methyldopa-induced antibodies are not de-
scribed in this chapter but have been exten-
sively reviewed elsewhere.4,49 DIIHA caused
by high-dose intravenous penicillin therapy
is no longer seen, and methyldopa, the
prototype for drug-independent antibodies, is
not used as frequently as in the past.
Currently, the drugs most commonly
associated with DIIHA are piperacillin,
ceftriaxone, and cefotetan.50
Drug-Dependent Antibodies that Are
Reactive with Drug-Treated Red
Cells
Some drugs (eg, penicillin, ampicillin, and
many cephalosporins) covalently bind to red
cells, thus making it possible to coat red
cells in the laboratory with the drug.
Antibodies di- rected to these drugs will
react with the drug- treated red cells but not
with untreated red cells (unless the patient
also has alloantibod- ies to red cell antigens
present on these cells).
Penicillin and the cephalosporins are
beta-lactam antibiotics. It was thought for
some time that antibodies to any drug in the
penicillin and cephalosporin families could
be detected by testing red cells with drug-
treated cells using methods previously
described for penicillin and cephalothin. It is
now known that this is not the case.
Synthetic penicillins and newer
cephalosporins cannot be as- sumed to have
the same red cell binding char- acteristics as
penicillin and cephalothin (a first-generation
cephalosporin). Cefotetan (a second-
generation cephalosporin) binds very well to
red cells, and antibodies caused by ce-
fotetan typically react to very high titers
with cefotetan-treated red cells. However,
ceftriax- one (a third-generation
cephalosporin) does not bind well to red
cells; therefore, antibodies to ceftriaxone
cannot be tested by this meth-
od.50 Piperacillin, a semisynthetic penicillin,
binds to red cells at high pH. However, a
large percentage of plasma from healthy
blood do- nors and patients react with
piperacillin-treat- ed red cells, so this
method is not recommend- ed for testing for
piperacillin antibodies.51 For drug antibodies
detected using drug-treated red cells, the
following are expected:
■ The DAT result is usually positive for
IgG, but complement may also be
present.
■ The serum contains an antibody that
reacts with the drug-treated red cells but
not the untreated red cells.
■ Antibody eluted from the patient’s red
cells reacts with drug-treated red cells
but not untreated red cells.
Hemolysis develops gradually but may be
life threatening if the etiology is unrecognized
and drug administration is continued. The pa-
tient may or may not have been previously ex-
posed to the drug and, in the case of cefotetan,
only a single dose given prophylactically can
result in severe hemolysis. Normal plasma has
been shown to react with some drug-treated
red cells (eg, red cells treated with cefotetan, 4
piperacillin,51 or oxaliplatin52), suggesting prior
exposure to these drugs through environmen-
tal routes.
Drug-Dependent Antibodies that Are
Reactive with Untreated Red Cells in the
Presence of Drug
Antibodies to many drugs that have been re-
ported to cause immune hemolytic anemia
are detected by testing untreated red cells in
the presence of the drug. Piperacillin and
some of the second- and third-generation
cephalospo- rins react by this method; anti-
ceftriaxone has been detected only by testing
red cells in the presence of drug.49 The
following observations are characteristic:
■ Complement may be the only protein
easily detected on the red cells, but IgG
may be present.
■ The serum antibody can be IgM, IgG, or
IgM with IgG.
 CH A PT E R 1 7
■ A drug (or metabolite) must be present in
vitro for the antibody in the patient’s
serum to be detected. Antibodies may
cause he- molysis, agglutination, and/or
sensitization of red cells in the presence
of the drug.
■ The patient need only take a small
amount of the drug (eg, a single dose).
■ Acute intravascular hemolysis with
hemo- globinemia and hemoglobinuria is
the usu- al presentation. Renal failure is
quite com- mon.
■ Once antibody has been formed, severe
he- molytic episodes may recur after
exposure to very small quantities of the
drug.
On occasion, it appears that a patient’s
se- rum contains an “autoantibody” in
addition to a drug antibody reacting in the
presence of the drug. Rather than a true
autoantibody, it is be- lieved that this
reactivity is due to the presence of
circulating drug or drug plus antibody com-
plexes.50,53 In these cases, an eluate is usually
nonreactive when the drug is not present in
the system. However, in some cases
involving piperacillin, the eluate reacts
while the patient is still taking the drug. A
sample collected sev- eral days after the
drug has been discontinued will be
nonreactive. A true warm autoantibody is
expected to be reactive in an eluate prepared
from the patient’s red cells, and
autoantibody in the serum persists.
Consequently, DIIHA caused by piperacillin
can be misdiagnosed as WAIHA, especially
if the eluate reacts. Differ- entiation of
warm-reactive autoantibody from drug-
induced immune hemolytic anemia is
important for clinical management.53
Drug-Independent Antibodies:
Autoantibody Production
Some drugs induce autoantibodies that ap-
pear serologically indistinguishable from
those of WAIHA. Red cells are coated with
IgG, and the eluate as well as the serum
react with virtually all cells tested in the
absence of the drug. The antibody has no
direct or indirect in- vitro interaction with
the drug. The prototype drug for such cases
is methyldopa, which is now used much less
frequently than in the past. Currently,
fludarabine, used to treat
DAT/Immune Hemolysis ■ 443
chronic lymphocytic leukemia, is the most
commonly used drug to produce drug-inde-
pendent antibodies and AIHA.49
Non-Immunologic Protein Adsorption
The positive DAT result associated with some
drugs is caused by modification of the red cell
membrane by the drug and is independent of
antibody production. Hemolytic anemia asso-
ciated with this mechanism is rare.
Cephalosporins (primarily cephalothin)
are the drugs with which positive DAT results
and NIPA were originally associated. In vitro,
red cells coated with cephalothin in pH 9.8
buffer and incubated with normal plasma ad-
sorb albumin, IgA, IgG, IgM, C3, and other
proteins in a nonimmunologic manner. For
this reason, the indirect antiglobulin test result
with virtually all plasma will be positive.
Other drugs that cause NIPA and a positive
DAT re- sult include diglycoaldehyde,
cisplatin, oxali- platin, and beta-lactamase
inhibitors (clavu- lanic acid, sulbactam, and
tazobactam).12
NIPA should be suspected when a pa-
tient’s plasma/serum and most normal plas-
ma/sera are reactive in an indirect antiglobu-
lin test with drug-treated red cells but the
eluate from the patient’s red cells is nonreac-
tive with the drug-treated cells.
Laboratory Investigation of Drug-
Induced Immune Hemolysis
The drug-related problems that are most
com- monly encountered in the blood bank
are those associated with a positive DAT
result and a nonreactive eluate. Recent red
cell transfu- sions and/or dramatic
hemolysis may result in a weak DAT result
by the time hemolysis is sus- pected. When
other, more common causes of immune-
mediated hemolysis have been ex- cluded
and a temporal relationship exists be- tween
the administration of a drug and the
hemolytic anemia, a drug antibody
investiga- tion should be pursued.
The patient’s serum should be tested for
unexpected antibodies by routine
procedures. If the serum does not react with
untreated red cells, the tests should be
repeated with the
444 ■ AABB T EC HNIC AL MANUAL
drug(s) suspected of causing the problem.
Some drug formulations contain inert
ingredi- ents (eg, pill or capsule forms), and
other drugs are combinations of two drugs
(eg, piperacillin plus tazobactam). Although
it would seem logical to test the patient’s
serum with the actual drug that the patient
received, inert ingredients or drug
combinations can make preparation of drug-
treated red cells dif- ficult or make the
results confusing. It is pref- erable to test
serum using pure drug formula- tions as
well as separate components of combination
drugs.
If the drug has already been reported to
cause hemolytic anemia, testing methods
may be described in the case reports. Far
more drug antibodies are detected by testing
serum in the presence of drug; therefore,
when a previous report of antibodies to a
drug is not available, an initial screening test
can be performed with a solution of the drug
at a concentration of ap- proximately 1
mg/mL in phosphate-buffered saline (see
Method 4-13). Serum, rather than plasma, is
the preferred specimen for testing for
hemolysis to be observed; this also allows
for the addition of fresh normal serum (as a
source of complement) to the test system.
The addition of the fresh complement
increases the sensitivity of the test for the
detection of in-vitro hemolysis resulting
from complement activation.
If these tests are not informative, at-
tempts can be made to coat normal red cells
with the drug. The patient’s serum and an
elu- ate from the patient’s red cells can be
tested against the drug-treated red cells (see
Method 4-12). This is the method of choice
when ceph- alosporins (except for
ceftriaxone) are thought to be implicated.
Results that are definitive for a drug-
induced positive DAT result are reactiv- ity
of the eluate with drug-treated red cells and
absence of reactivity with untreated red
cells.
KEY POINTS
The DAT is used to determine whether red cells have been coated in vivo with immunoglob- ulin, complement, or both. The D
The DAT should be used to determine whether a hemolytic anemia has an immune etiology.
Drug-treated red cells should always be
tested with saline and normal serum (or
plas- ma) as negative controls. This
approach en- sures that the observed
reactivity with the patient’s serum/plasma is
appropriately inter- preted. Antibodies
reactive with red cells treat- ed with some
drugs (eg, beta-lactams and plat- inums)
have been detected in the plasma from blood
donors and patients without hemolytic
anemia thought to be due to environmental
exposure. Therefore, misinterpretation of re-
activity in a patient’s serum is possible.51-52
Whenever possible, a positive control
should be tested with drug-treated red cells.
Negative results of a patient’s serum and elu-
ate without a positive control can only be in-
terpreted as showing that antibodies to that
drug were not detected. The drug may or
may not be bound to the test red cells.
If the drug in question is known to
cause NIPA, the patient’s serum and the
controls (negative and positive) should also
be tested at a dilution of 1 in 20. Normal
sera at this dilu- tion do not usually contain
enough protein for NIPA to be detected.
An immune response may be caused by
a metabolite of a drug rather than the drug
itself. If the clinical picture is consistent
with im- mune-mediated hemolysis and the
above tests are noninformative, it may be
helpful to test metabolites of the parent drug
that are present in the serum or urine of an
individual who is taking that drug.54
Antibodies to some nonste- roidal
antiinflammatory drugs have required
testing in the presence of metabolite.55 The
metabolism and half-life of the drug deter-
mines when the drug metabolite should be
collected. Pharmacology information for the
metabolite(s) detectable in serum or urine
and to previous reports for the drug under
investi- gation should be consulted.
 CH A PT E R 1 7 DAT/Immune Hemolysis ■ 445

3. A positive DAT result may or may not be associated with hemolysis.

4. Performance of the DAT on postreaction specimens is part of the initial investigation of a
transfusion reaction. The DAT result may be positive if sensitized red cells have not been
de- stroyed, or may be negative if hemolysis and rapid clearance have occurred.
5. The DAT is performed by testing freshly washed red cells directly with antiglobulin
reagents containing anti-IgG and anti-C3d. False-negative or weaker results can be obtained
if the washed red cells are allowed to sit before testing with anti-IgG or if the reading is
delayed.
6. When the DAT result is positive with both anti-IgG and anti-C3, the red cells should be test-
ed with an inert control reagent (eg, 6% albumin or saline). If the control is reactive, the
DAT result is invalid, possibly indicating spontaneous agglutination from heavy coating of
IgG or rare warm-reactive IgM. The invalid DAT result could also be due to IgM cold
autoaggluti- nins that were not dissociated during routine washing.
7. A positive DAT result alone is not diagnostic of hemolytic anemia. The interpretation
of the significance of this positive result requires additional patient-specific
information. Dia- logue with the attending physician is important. Clinical
considerations together with labo- ratory data should dictate the extent to which a
positive DAT result is evaluated.
8. The following situations may warrant further investigation of a positive DAT:
a. Evidence of in-vivo red cell destruction.
b. Recent transfusion.
c. Administration of drugs that have previously been associated with immune-mediated
hemolysis.
d. History of hematopoietic progenitor cell or organ transplantation.
e. Administration of IVIG or intravenous anti-D.
9. Elution frees antibody from sensitized red cells and recovers antibody in a usable form.
Elu- tion is useful in certain situations for implicating an autoantibody, detecting
specific anti- bodies that may not be detectable in the serum, and deciding to test
patient’s serum for drug-related antibodies.
10. AIHAs are subdivided into the major types: WAIHA, CAD, mixed- or combined-type
AIHA, and PCH. Drugs may also induce immune hemolysis.

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49. Garratty G. Immune hemolytic anemia associ-
ated with drug therapy. Blood Rev 2010;24:
143-50.
50. Arndt PA, Leger RM, Garratty G. Serologic
characteristics of ceftriaxone antibodies in 25
patients with drug-induced immune hemolyt-
ic anemia. Transfusion 2012;52:602-12.
51. Leger RM, Arndt PA, Garratty G. Serological
studies of piperacillin antibodies. Transfusion
2008;48:2429-34.
52. Leger RM, Garratty G. Antibodies to
oxaliplat- in, a chemotherapeutic, are found
in plasma of healthy blood donors.
Transfusion 2011;51: 1740-4.
53. Bandara M, Seder DB, Garratty G, et al. Piper-
acillin-induced immune hemolytic anemia in
an adult with cystic fibrosis (article 10
161454). Case Report Med 2010.
54. Salama A, Mueller-Eckhardt C, Kissel K, et al.
Ex vivo antigen preparation for the serological
detection of drug-dependent antibodies in
immune haemolytic anaemias. Br J Haemat
1984;58:525-31.
55. Johnson ST, Fueger JT, Gottschall JL. One
cen- ter’s experience: The serology and drugs
asso- ciated with drug-induced immune
hemolytic anemia—a new paradigm.
Transfusion 2007; 47:697-702.
448 ■ AABB T EC HNIC AL MANUAL

■ APPENDIX 17-1
Drugs Associated with Immune Hemolytic Anemia
Drug Method of Detection
Aceclofenac +Drug
Acetaminophen + Drug
Acyclovir DT
Aminopyrine DT
Amoxicillin DT
Amphotericin B + Drug
Ampicillin DT + Drug
Antazoline + Drug
Azapropazone AA DT
Butizide + Drug
Carbimazole AA DT + Drug
Carboplatin AA DT + Drug NIPA
Carbromal DT
Cefamandole DT
Cefazolin DT
Cefixime DT + Drug
Cefotaxime DT + Drug
Cefotetan AA DT + Drug NIPA
Cefoxitin AA DT + Drug
Ceftazidime AA DT + Drug
Ceftizoxime DT + Drug
Ceftriaxone + Drug
Cefuroxime DT
Cephalexin DT
Cephalothin DT + Drug NIPA
Chloramphenicol AA DT
Chlorinated hydrocarbons AA DT + Drug
Chlorpromazine AA + Drug
Chlorpropamide + Drug
Cimetidine DT +Drug
 CH A PT E R 1 7 DAT/Immune Hemolysis ■ 449

■ APPENDIX 17-1
Drugs Associated with Immune Hemolytic Anemia (Continued)
Drug Method of Detection
Ciprofloxacin +Drug
Cisplatin DT +Drug NIPA
Cladribine AA
Clavulanate NIPA
Cyanidanol AA DT + Drug
Cyclofenil AA + Drug
Cyclosporin DT
Diclofenac AA DT + Drug
Diethylstilbestrol + Drug
Diglycoaldehyde NIPA
Dipyrone DT + Drug
Erythromycin DT
Etodolac + Drug
Fenoprofen AA + Drug
Fluconazole DT + Drug
Fludarabine AA
Fluorescein DT + Drug
Fluorouracil + Drug
Furosemide + Drug
Hydralizine DT
Hydrochlorothiazide DT + Drug
Hydrocortisone DT + Drug
9-Hydroxy-methyl-ellipticinium + Drug
Ibuprofen +Drug
Imatinib mesylate DT
Insulin DT
Isoniazid DT + Drug
Levodopa AA
Levofloxacin DT +Drug
Mefenamic acid AA
(Continued)
450 ■ AABB T EC HNIC AL MANUAL

■ APPENDIX 17-1
Drugs Associated with Immune Hemolytic Anemia (Continued)
Drug Method of Detection
Mefloquine DT + Drug
Melphalan + Drug
6-Mercaptopurine DT
Methadone DT
Methotrexate AA DT + Drug
Methyldopa AA
Nabumetone +Drug
Nafcillin DT
Naproxen + Drug
Oxaliplatin DT + Drug NIPA
p-Aminosalicylic acid + Drug
Penicillin G DT
Phenacetin + Drug
Phenytoin DT
Piperacillin DT + Drug
Probenicid + Drug
Procainamide AA
Propyphenazone + Drug
Pyrazinamide DT + Drug
Pyrimethamine DT
Quinidine DT + Drug
Quinine + Drug
Ranitidine DT + Drug
Rifabutin + Drug
Rifampicin DT + Drug
Sodium pentothal/thiopental + Drug
Stibophen + Drug
Streptomycin AA DT + Drug
Sulbactam NIPA
Sulfamethoxazole + Drug
 CH A PT E R 1 7 DAT/Immune Hemolysis ■ 451

■ APPENDIX 17-1
Drugs Associated with Immune Hemolytic Anemia (Continued)
Drug Method of Detection
Sulfasalazine + Drug
Sulfisoxazole +Drug
Sulindac AA DT + Drug
Suprofen AA + Drug
Tazobactam NIPA
Teicoplanin AA + Drug
Teniposide AA + Drug
Tetracycline DT
Ticarcillin AA DT
Tolbutamide DT
Tolmetin AA + Drug
Triamterene DT + Drug
Trimethoprim + Drug
Vancomycin + Drug
Zomepirac AA + Drug
AA = drug-independent autoantibody; DT = testing with drug-treated red cells; + Drug = testing in the presence of
drug; NIPA = nonimmunologic protein adsorption.
 C h a p t e r 1 8

Platelet and Granulocyte Antigens

and Antibodies

Brian R. Curtis, PhD, D(ABMLI), MT(ASCP)SBB

THIS CHAP TER DIS C USSES anti-

gens expressed on platelets and
granulo- cytes together with antibodies that
arise when individuals are sensitized to
these markers. These antigens and the
immune responses to them are of importance
in alloimmune, auto- immune, and drug-
induced immune syn- dromes involving
platelets and granulocytes.
PLATELET ANTIGENS AND
ANTIBODIES
Platelets express a variety of antigenic markers
on their surface. Some of these antigens are
shared with other cells, as with ABO and
HLA, whereas others are essentially platelet
specific, like human platelet alloantigens
(HPAs).
HPAs
Platelets reportedly play roles in inflamma-
tion; innate, adaptive, and autoimmunity;
car- diovascular disease; and even cancer. 1,2
How- ever, they are most well known for
their function in forming clots to stem
bleeding. Platelets perform these functions
through
multiple ligand-receptor interactions involv-
ing glycoproteins (GPs) expressed on their
cell-surface membranes.
Platelet membrane GPs are expressed in
different forms due to single nucleotide poly-
morphisms (SNPs) in the genes that encode
them. The amino acid changes resulting from
these SNPs induce changes in the glycoprotein
structure to form antigens that can elicit anti-
bodies through exposure from pregnancy or
platelet transfusions. Currently, 33 different
HPAs expressed on six different platelet mem-
brane glycoproteins—GPIIb, GPIIIa, GPIb,
GPIb, GPIa, and CD109—have been identi-
fied (Table 18-1).3 These antigens are often re-
ferred to as “platelet specific,” but some are
found on cells other than platelets (especially
leukocytes and endothelial cells), although
their chief clinical importance appears to be
linked to their presence on platelets.
Twelve antigens are clustered into six bi-
allelic groups (HPA-1, HPA-2, HPA-3, HPA-
4, HPA-5, and HPA-15). The nomenclature for
HPAs consists of numbering the antigens in
their order of discovery, with the higher-

Brian R. Curtis PhD, D(ABMLI), MT(ASCP)SBB, Director, Platelet and Neutrophil Immunology Lab, Blood-
Center of Wisconsin, Milwaukee, Wisconsin
B. Curtis has disclosed a financial relationship with Gen-Probe Incorporated.

453
454 ■ AABB T EC HNIC AL MANUAL

TABLE 18-1. Human Platelet Alloantigens

Phenotypic Amino Acid Encoding Nucleotide

Antigen Frequency* Glycoprotein (GP) Change Gene Change
HPA-1a 72% a/a GPIIIa Leu33Pro ITGB3 176T>C
HPA-1b 26% a/b
2% b/b
HPA-2a 85% a/a GPIb Thr145Met GPIBA 482C>T
HPA-2b 14% a/b
1% b/b
HPA-3a 37% a/a GPIIb Ile847Ser ITGA2B 2621T>G
HPA-3b 48% a/b
15% b/b
HPA-4a >99.9% a/a GPIIIa Arg143Gln ITGB3 506G>A
HPA-4b < 0.1% a/b
< 0.1% b/b
HPA-5a 88% a/a GPIa Glu505Lys ITGA2 1600G>A
HPA-5b 20% a/b
1% b/b
HPA-6bw < 1% b/b GPIIIa Arg489Gln ITGB3 1544G>A
HPA-7bw < 1% b/b GPIIIa Pro407Ala ITGB3 1297C>G
HPA-8bw < 1% b/b GPIIIa Arg636Cys ITGB3 1984C>T
HPA-9bw < 1% b/b GPIIb Val837Met ITGA2B 2602G>A
HPA-10bw < 1% b/b GPIIIa Arg62Gln ITGB3 263G>A
HPA-11bw < 1% b/b GPIIIa Arg633His ITGB3 1976G>A
HPA-12bw < 1% b/b GPIb Gly15Glu GPIBB 119G>A
HPA-13bw < 1% b/b GPIa Met799Thr ITGA2 2483C>T
HPA-14bw < 1% b/b GPIIIa Lys611del ITGB3 1909_1911delAAG
HPA-15bw 35% a/a CD109 Ser682Tyr CD109 2108C>A
42% a/b
23% b/b
HPA-16bw < 1% b/b GPIIIa Thr140Ile ITGB3 497C>T
HPA-17bw < 1% b/b GPIIIa Thr195Met ITGB3 662C>T
HPA-18bw < 1% b/b GPIa Gln716His ITGA2 2235G>T
HPA-19bw < 1% b/b GPIIIa Lys137Gln ITGB3 487A>C
HPA-20bw < 1% b/b GPIIb Thr619Met ITGA2B 1949C>T
 C H A P T E R 1 8 Platelet and Granulocyte Antigens and Antibodies ■ 455

TABLE 18-1. Human Platelet Alloantigens (Continued)

Phenotypic Amino Acid Encoding Nucleotide

Antigen Frequency* Glycoprotein (GP) Change Gene Change
HPA-21bw < 1% b/b GPIIIa Glu628Lys ITGB3 1960G>A
HPA-22bw < 1% b/b GPIIb Lys164Thr ITGA2B 584A>C
HPA-23bw < 1% b/b GPIIIa Arg622Trp ITGB3 1942C>T
HPA-24bw < 1% b/b GPIIb Ser472Asn ITGA2B 1508G>A
HPA-25bw < 1% b/b GPIa Thr1087Met ITGA2 3347C>T
HPA-26bw < 1% b/b GPIIIa Lys580Asn ITGB3 1818G>T
HPA-27bw < 1% b/b GPIIb Leu841Met ITGA2B 2614C>A
*Phenotypic frequencies are for people of European ancestry who live in North America. Human platelet alloantigen (HPA)
frequencies in other races and ethnic groups can be found in the IPD-Immuno Polymorphism Database. 3

frequency antigens designated “a” and the Glanzmann thrombasthenia, in which

lower-frequency antigens designated “b.”4
HPAs for which antibodies against only one of
the two antithetical antigens have been de-
tected are labeled with a “w” for “workshop,”
such as HPA-6bw.
Platelet Alloantigens on GPIIb/IIIa
HPA-1a is the platelet alloantigen that was dis-
covered first and is most familiar.5 Originally
named “Zwa” and more commonly referred to
as “PlA1,” it is expressed on GPIIIa, the -sub-
unit of the integrin GPIIb/IIIa (2b/3) com-
plex.
Integrins are a broadly distributed
family of adhesion molecules consisting of
an  and a
 chain held together by divalent cations in
a heterodimeric complex.6 Integrins are
essen- tial for platelet adhesion and
aggregation be- cause they serve as
receptors for ligands, such as fibrinogen,
collagen, fibronectin, von Wille- brand
factor (vWF), and other extracellular matrix
proteins.
Binding of fibrinogen by GPIIb/IIIa re-
sults in platelet aggregation, which leads to
the formation of the “platelet plug” to stop
bleed- ing. The importance of GPIIb/IIIa in
hemosta- sis is demonstrated by the serious
bleeding that occurs in patients with the rare
disorder
platelet GPIIb/IIIa is absent or dysfunctional
due to in- herited mutations in ITGA2B
and/or ITGB3.7 Patients with Glanzmann
thrombasthenia who are exposed to normal
platelets by trans- fusion or pregnancy can
make isoantibodies against GPIIb/IIIa.
GPIIb/IIIa is the most abundantly ex-
pressed (approximately 80,000 molecules/
platelet) glycoprotein complex on the platelet
membrane, making it highly immunogenic.
Antibodies against HPA-1a account for the
vast majority (>80%) of the HPA-specific
plate- let antibodies detected in the sera of
alloim- munized people of European ancestry.
HPA-1a antibodies are produced by the 2% of
individu- als with the platelet type HPA-1b/1b.
Antibod- ies specific for HPA-1b are
commonly detected in patients with
posttransfusion purpura (PTP).
Twenty of the 33 HPAs are carried by GPI-
Ib (6) and GPIIIa (14). Like HPA-1a/1b, the
HPA-4a/4b antigens are also expressed on
GPIIIa and have been implicated in fetal and
neonatal alloimmune thrombocytopenia
(FNAIT), PTP, and platelet transfusion
refrac- toriness. The low-frequency HPA-4b
antigen is more common in populations of
Japanese and Chinese ancestry.
456 ■ AABB T EC HNIC AL MANUAL
The HPA-3a/3b antigens are expressed on
GPIIb, but despite the high rate of incompati-
bility for both antigens in the general popula-
tion, detection of HPA-3 antibodies is uncom-
mon. Some HPA-3 antibodies are difficult to
detect in monoclonal antigen capture assays,
such as the modified antigen capture enzyme-
linked immunosorbent assay (MACE) and
monoclonal antibody immobilization of plate-
let antigens (MAIPA), in which GPIIb is ex-
tracted from platelets with detergents that can
denature the antigenic epitopes recognized by
HPA-3 antibodies.8,9 X-ray crystallography
studies could not determine the portion of the
domain of GPIIb where the HPA-3 antigens
are located, which might explain the
difficulties with detection of HPA-3
antibodies.10
An additional 17 different low-frequency
platelet antigens are expressed on either GPIIb
or GPIIIa (Table 18-1). These antigens were
all discovered in cases of FNAIT when
specific an- tibodies in maternal sera were
detected that were reactive only with the
fathers’ GPIIb/IIIa. The vast majority of these
antigens are private antigens restricted to the
single families in which they were discovered.
HPA-6bw and HPA-21bw are exceptions,
having antigen fre- quencies of 1% and 2%,
respectively, in people of Japanese ancestry,
and HPA-9bw has been implicated in several
cases of FNAIT.11-14
Platelet Alloantigens on GPIb/V/IX
The GPIb/V/IX complex forms the vWF
recep- tor on platelets, and platelets express
approxi- mately 25,000 copies of the
complex. Follow- ing vascular injury,
binding of the GPIb/V/IX complex to vWF
facilitates platelet adhesion to vascular
subendothelium and initiates signal- ing
events within adherent platelets that lead to
platelet activation, aggregation, and hemo-
stasis. GPIb is composed of  (GPIb) and
 (GPIb) subunits that form noncovalent
asso- ciations with GPIX and GPV. GPIb
carries HPA-2a/2b, and GPIb carries HPA-
12bw. An- tibodies against HPA-2a, -2b, and
-12bw have all been implicated in causing
FNAIT.
Deficiency of the entire GPIb/V/IX com-
plex can occur due to mutations in the encod-
ing genes GPIBA, GPIBB, or GP9, and is the
cause of Bernard Soulier syndrome (BSS).
BSS is a disorder characterized by prolonged
bleeding time, thrombocytopenia, and the
presence of “giant platelets” that affects ap-
proximately 1 in 1 million people.7,15 BSS
pa- tients whose platelets are devoid of
GPIb/V/IX can produce antibodies when
they are ex- posed to the protein complex on
normal plate- lets through transfusions or
pregnancy.
Platelet Alloantigens on GPIa/IIa
The integrin GPIa/IIa, also known as integrin
21 , is a major collagen receptor on platelets.
The GPIa protein carries the HPA-5a/5b
anti- gens. Antibodies against HPA-5
antigens are the second most frequently
detected, after anti-HPA-1a, in patients with
FNAIT and are also frequently detected in
patients with PTP. About 3000 to 5000
molecules of the GPIa/IIa heterodimeric
complex are expressed on platelets, and
higher expression correlates with the
presence of both HPA-5b and threo- nine at
amino acid 807 in GPIa.16 HPA-13bw,
-18bw, and -25bw are low-frequency
antigens that are also expressed on GPIa and
have all been implicated in FNAIT.
Interestingly, the HPA-13bw polymorphism
has been reported to cause functional defects
that reduce platelet responses to collagen-
induced aggregation and spreading on
collagen-coated surfaces.4
Platelet Alloantigens on CD109
CD109 is a glycosylphosphatidylinositol
(GPI)- linked protein and a member of the 2-
macro- globulin/complement superfamily. Its
func- tion is still not completely understood,
but CD109 has been reported to bind to and
nega- tively regulate the signaling of
transforming growth factor beta. CD109 is
also expressed on activated T lymphocytes,
CD34+ hematopoiet- ic cells, and endothelial
cells, and it carries the HPA-15 antigens.
The clinical significance of HPA-15 anti-
bodies is uncertain because platelets express
only about 1000 molecules of CD109. Studies
show the presence of HPA-15 antibodies in
0.22% to 4% of maternal sera in patients with
suspected FNAIT, and one report suggests that
HPA-15 antibodies are more frequently detect-
 C H A P T E R 1 8 Platelet and Granulocyte Antigens and Antibodies ■ 457

ed in sera from patients with immune
platelet refractoriness.17-19
Other Antigens on Platelets
ABO and Other Blood Groups
Most of the ABO antigen on platelets is
carried on saccharides attached to the major
platelet membrane glycoproteins (Table 18-2).
GPIIb and platelet endothelial cell adhesion
mole- cule 1 (PECAM-1/CD31) carry the
largest amounts of A and B antigens.20 Platelet
A and B antigen levels are quite variable from
individu- al to individual, with 5% to 10% of
non-group- O individuals expressing
extremely high levels
of A1 or B on their platelets.20,21 These “high
ex- pressers” have highly active
glycosyltransfer- ases, which are much more
efficient at attach- ing A or B antigens.20
Interestingly, although individuals with
the subgroup A2 red cell phenotype express
lower levels of A on their red cells than A1
indi-
viduals, they do not express detectable A anti-
gens on their platelets. As a result, A 2 platelets
have been successfully transfused to group O
patients, even those with high-titer anti-A.22
Although platelets are often transfused
without regard to ABO compatibility, the
use of mismatched platelets frequently
results in lower posttransfusion recovery
rates.23,24 In some cases, high-titer
immunoglobulin G (Ig
G) A,B antibodies in blood group O recipients
are reactive with transfused platelets carrying
large amounts of A or B antigens, resulting in
platelet transfusion refractoriness. 21 Recovery
of transfused platelets can also be influenced
by the transfusion of group O platelets to
group A recipients. Anti-A and/or anti-B in the
donor plasma might be reactive with soluble A
or B in the recipient plasma to form immune
complexes that bind to transfused (and autol-
ogous) platelets via FcRIIa, thereby influenc-
ing the survival of the transfused platelets.25
Clinical trials comparing ABO-identical to
-unmatched platelets in patients with cancer
who require multiple platelet transfusions
have suggested that rates of refractoriness
are significantly higher when unmatched
compo- nents are used.22,25 Although other
red cell an- tigens (eg, Lea, Leb, I, i, P, Pk,
and Cromer) are also present on platelets,
there is no evidence that these antigens
significantly reduce plate- let survival in
vivo.26,27

TABLE 18- Other Platelet Antigens

2.
Phenotypic Glycoprotein Amino Acid Encoding Nucleotide
Antigen Frequency (GP)* Change† Gene Change‡
ABO Same as for red cells GPIIb, IIIa, IV, Ia/ Multiple ABO Multiple
IIa, GPIb/V/IX,
CD31
HLA-A, B, Same as for leukocytes Class I HLA Multiple MHC Multiple
and C
GPIV 90-97% (Africans) CD36 Tyr325Thr* CD36 1264T>G*
90-97% (Asians) Pro90Ser* 478C>T*
99.9% (Caucasians) Exons 1-3 del
GPVI N/A GPVI N/A GP6/N/A N/A
*ABO saccharides are attached to platelet GPs during their glycosylation j.
†
Only the most common changes are shown.
‡
Only the most common mutations are
shown. N/A = not applicable.
458 ■ AABB T EC HNIC AL MANUAL
GPIV/CD36
Platelets, monocytes/macrophages, and nu-
cleated erythrocytes are the only blood cells
that express GPIV/CD36 (Table 18-2). GPIV
be- longs to the Class B scavenger receptor
family and binds a number of different
ligands, in- cluding low-density lipoprotein
cholesterol, thrombospondin, Types I and IV
collagen, and malaria-infected red cells. A
number of muta- tions in CD36 have been
described that result in a complete lack of
protein expression on both platelets and
monocytes in populations of Asian and
African ancestry.28-30 CD36-defi- cient
individuals exposed to normal platelets can
produce antibodies to CD36 that have been
reported to cause FNAIT, PTP, and plate- let
refractoriness.29,31,32
GPVI
GPVI is a major collagen receptor on platelets
and a member of the Ig superfamily. GPVI in-
teractions with collagen exposed on the extra-
cellular matrix result in platelet activation and
aggregation. To date, no HPAs have been iden-
tified on GPVI, but platelet autoantibodies
formed against GPVI have been reported to
cause a mild form of autoimmune thrombocy-
topenia.33,34 Interestingly, GPVI autoantibod-
ies induce shedding of GPVI from platelets,
resulting in reduced collagen binding and clin-
ically significant bleeding.
HLA
HLA is present on all nucleated cells of the
body (see Chapter 19). HLA associated with
platelets is the main source of Class I HLA in
whole blood.35 Most Class I HLA on platelets
is expressed as integral membrane proteins,
whereas smaller amounts may be adsorbed
from surrounding plasma. HLA-A and -B
locus antigens are significantly represented,
but there appears to be only minimal platelet
ex- pression of HLA-C.36 With rare exceptions,
Class II HLA is not present on the platelet
membrane.
Several factors influence the likelihood
that HLA antibodies will develop after
transfu- sions, and sensitization may be
clinically im-
portant in patients receiving multiple platelet
transfusions. Transfusion-associated HLA
allo- immunization appears to be influenced
by the underlying disease,
immunosuppressive ef- fects of treatment
regimens, and whether the blood
components contain a significant amount of
leukocytes. With widespread use of
leukocyte-reduced (LR) blood components
for transfusion, associated HLA
alloimmunization has been reduced
considerably. HLA antibod- ies also
commonly develop following pregnan- cy
and are present in the sera of more than 32%
of women who have had four or more
pregnancies.37 HLA antibodies have also
been identified in 1.7% of never-pregnant or
-trans- fused women and men with no
previous trans- fusions.37 Sensitization to
HLA antigens be- comes important in
managing patients receiving platelet
transfusions when HLA anti- bodies cause
destruction of transfused plate- lets and,
especially, when patients develop platelet
transfusion refractoriness.
Immune Platelet Disorders
Platelet Transfusion Refractoriness
A less-than-expected increase in platelet
count occurs in about 20% to 70% of
multitransfused patients with
thrombocytopenia.38 Those treated for
malignant hematopoietic disorders are
particularly likely to become refractory to
platelet transfusions. Responses to platelet
transfusions are often determined 10 to 60
minutes after transfusion by calculating
either a corrected platelet count increment
(CCI) or a posttransfusion platelet recovery
(PPR), both of which normalize transfusion
responses for patient blood volume and
platelet dose (see Chapter 20, Table 5). Most
experts would agree that a 1-hour
posttransfusion CCI of less than 5000 to
7500 after two consecutive transfu- sions
adequately defines the refractory state.
The CCI and PPR measurements are
com- monly used to assess the success of
platelet transfusions. However, these
calculations may be misleading, particularly
when smaller dos- es of platelets are
administered, as can occur during LR
transfusions. Therefore, absolute
posttransfusion platelet count increments are
more accurate than CCI or PPR.23
 C H A P T E R 1 8 Platelet and Granulocyte Antigens and Antibodies
HLA sensitization is the most common
immune cause of refractoriness and can be di-
agnosed by demonstration of significant lev-
els of antibodies to Class I HLA in the
refracto- ry patient’s serum (see Chapter 19 for
more information on detecting HLA
antibodies). Other immune causes to be
considered in- clude antibodies to HPA, ABO
incompatibility, and antibodies in the sera of
patients with congenital platelet glycoprotein
deficiencies (eg, Glanzmann’s
thrombasthenia).
Although platelet alloimmunization is
one cause of refractoriness, there are
multiple nonimmune-related reasons why
transfused platelets may not yield the
expected increase in platelet count, such as
sepsis, disseminated intravascular
coagulation, and the administra- tion of
certain drugs. It cannot be stressed enough
that these nonimmune factors are more often
implicated in transfusion refracto- riness
than alloimmunization.39 Some of the most
commonly cited nonimmune factors as-
sociated with refractoriness are listed in
Table 18-3. Even when possible immune
causes of refractoriness are identified,
nonimmune fac- tors are often
simultaneously present.
TABLE 18-3. Some Nonimmune Causes of
Platelet Refractoriness
■ Massive bleeding
■ Fever
■ Sepsis
■ Splenomegaly (splenic sequestration)
■ Disseminated intravascular coagulation
■ Allogeneic transplantation
■ Poor storage of platelets before transfusion
■ Effects of drugs (may include immune
mecha- nisms)
■ Intravenous amphotericin B
■ Thrombotic thrombocytopenic purpura
■ Treatment regimen (ie, total body irradiation
or chemotherapy)
■ Liver dysfunction
■ 459
Selection of Platelets for Transfusion in
Patients with Alloimmune
Refractoriness
Several strategies may be considered when se-
lecting platelets for transfusion to patients
with alloimmune refractoriness. When HLA
antibodies are present, a widely used ap-
proach is to supply apheresis platelets from
donors whose Class I HLA closely matches
those of the patient. This approach is primarily
performed using molecular methods. A disad-
vantage of relying on HLA-matched platelets
is that a pool of 1000 to 3000 or more random,
HLA-typed, potential apheresis donors is gen-
erally necessary to find sufficient HLA-com-
patible donors to support a typical patient.40
Moreover, donor selection on the basis of HLA
type can lead to the exclusion of donors whose
HLA types, although different from that of the
recipient, may still be suitable for transfusion
to the patient.
An additional concern when using HLA-
selected platelets is that requesting “HLA-
matched” platelets does not necessarily lead
to receiving HLA-identical or even well-
matched platelets. It is important to under-
stand the degree of matching that may be
provided (Table 18-4). Platelets received fol-
lowing an HLA-matched request are typically
the closest match obtainable within the con-
straints of time and donor availability. In one
study, 43% of platelets provided as HLA
matched were relatively poor Grade B or C
matches.41 In alloimmune refractory patients,
the best increases in CCI occur with the subset
of Grade A and B1U or B2U HLA-matched
platelets, but platelets mismatched for some
antigens (eg, B44, 45) that are poorly
expressed on platelets can also be
successfully trans- fused.
According to AABB Standards for
Blood Banks and Transfusion Services, HLA-
matched platelets should be irradiated to
prevent transfusion-associated graft-vs-host
disease (GVHD).42(p40) HLA-matched platelets
are select- ed to minimize incompatible
antigens. There- fore, these components are
more likely to cause GVHD than random
platelets because the re-
cipient’s immune system may fail to recognize
460 ■ AABB T EC HNIC AL MANUAL
TABLE 18-4. Degree of Matching for HLA-Matched Platelets
Match Grade
Description
A 4-antigen match
B1U 1 antigen unknown or blank
B1X 1 cross-reactive group
B2UX 1 antigen blank and 1 cross-reactive
C 1 mismatched antigen present
D 2 or more mismatched antigens present
R Random
the donor T lymphocytes as foreign. Treating
the component with gamma irradiation effec-
tively eliminates this risk by inactivating the
donor lymphocytes so that they cannot prolif-
erate.
An alternative approach for supplying
HLA-compatible transfusions is to determine
the specificity of the patient’s HLA antibodies
and select donors whose platelets lack the cor-
responding antigens. This antibody specificity
prediction (ASP) method is as effective as se-
lection by HLA matching or platelet cross-
matching and is superior to random selection
of platelets.43 Furthermore, many more poten-
tial HLA-typed donors are identified by the
ASP method than are available using tradition-
al HLA-matching criteria. A variation of the
ASP method involves analysis of antibody
specificities detected in sensitive flow cytome-
try or Luminex-based assays, where single
HLAs are represented on discrete and identifi-
able populations of beads.44 Reactivity of the
patient’s serum with the specific bead popula-
tions yields both the specificity and the rela-
tive strength of the antibodies. Bead popula-
tions that lack reactivity with patient serum
are used to identify antigens that could be
used in donor selection, even though they may
not be matched to the patient’s HLA type
using classic criteria for HLA matching.
In the absence of information concern-
ing which specific platelet donor HLA to
avoid, permissive HLA-mismatched antigens
can also be identified using an HLA
Matchmaker
Examples of Donor Phenotypes for a Recipient Who Is A1, 3; B8,
A1, 3; B8, 27
A1, –; B8, 27
A1, 3; B8, 7
A1, –; B8, 7
A1, 3; B8, 35
A1, 32; B8, 35
A2, 28; B7, 35
algorithm that is based on knowledge of
shared HLA epitopes.45 The identification of
so-called permissive HLA epitopes is
modeled on a strategy used for identifying
potentially compatible deceased donor
kidney grafts for HLA-sensitized recipients.46
This is yet another way to expand the donor
pool to provide plate- lets that are
compatible with the patient’s HLA
antibodies.
Pretransfusion crossmatching of the pa-
tient’s serum against platelets from potential
donors is an additional approach to provide
effective platelet transfusions to patients who
are alloimmune refractory. Each potential
platelet product is tested in the crossmatch as-
say with a current sample of the patient’s se-
rum. The solid-phase red cell adherence
(SPRCA) test is the most widely used method.
Good correlation between test results and
posttransfusion platelet counts have been
achieved.47 Compared with HLA matching,
crossmatching can be more convenient and
cost effective. It avoids exclusion of HLA-
mis- matched but compatible donors and has
the added advantage of facilitating the
selection of platelets when platelet-specific
antibodies are present.
Platelet crossmatching, however, will not
always be successful, particularly when pa-
tients are highly alloimmunized, which can
make finding sufficient amounts of compati-
ble platelets problematic. Although the inci-
dence of platelet-specific antibodies causing
patients to be refractory to most or all platelet
 C H A P T E R 1 8 Platelet and Granulocyte Antigens and Antibodies
transfusions is very small, this possibility
should be investigated when most of the
crossmatches are positive or HLA-matched
transfusions fail. If platelet-specific antibodies
are present, donors of known platelet antigen
phenotype or family members, who may be
more likely to share the patient’s phenotype,
should be tested. Platelet crossmatching or
HPA genotyping should be considered for pa-
tients who do not respond to ABO- and HLA-
compatible platelets.
Fetal and Neonatal
Alloimmune
Thrombocytopenia
FNAIT (also known as neonatal alloimmune
thrombocytopenia and abbreviated as
“NATP,” “NAT,” “NAIT,” or “NIT”) is a
syndrome involv- ing immune destruction of
fetal platelets by maternal antibody that is
analogous to red cell destruction in
hemolytic disease of the fetus and newborn.
During pregnancy, a mother may become
sensitized to an incompatible fe- tal platelet
antigen inherited from the father of the
fetus. IgG specific for the platelet antigen
crosses the placenta, causing thrombocyto-
penia.
FNAIT is the most common cause of se-
vere fetal/neonatal thrombocytopenia, and
af- fected infants are at risk of major
bleeding complications, especially
intracranial hemor- rhage. The most
commonly implicated plate- let antigen
incompatibility in FNAIT is for HPA-1a,
but all HPAs identified to date have been
implicated.48 A serologic diagnosis of
FNAIT may be made by 1) testing maternal
se- rum for platelet antibodies using assays
that can differentiate platelet-specific from
non- platelet-specific reactivity and 2)
performing platelet genotyping on parental
deoxyribonu- cleic acid (DNA).49
Demonstration of both a platelet-specific
(HPA) antibody in the mater- nal serum and
the corresponding incompati- bility for the
antigen in the parental platelet types
confirms the diagnosis.
Treatment of acutely thrombocytopenic
newborns includes the administration of in-
travenous immune globulin (IVIG) with or
without antigen-compatible platelet transfu-
sions that are sometimes provided by the
■ 461
mother as washed platelet products.50 Once
the diagnosis of FNAIT has been made in a
family, subsequent fetuses are at risk.
Antena- tal treatment with IVIG with or
without ste- roids has proven to be an
effective means of reducing fetal
thrombocytopenia and prevent- ing
intracranial hemorrhage.51 (For a more in-
depth discussion of FNAIT, see Chapter 22.)
Posttransfusion Purpura
PTP is a rare syndrome characterized by the
development of dramatic, sudden, and self-
limiting thrombocytopenia 5 to 10 days after
a blood transfusion in patients with a
previous history of sensitization by
pregnancy or trans- fusion.52 Coincident
with the thrombocytope- nia is the
development of a potent platelet- specific
alloantibody, usually anti-HPA-1a, in the
patient’s serum. Other specificities have
been implicated; these are almost always
asso- ciated with antigens on GPIIb/IIIa.
PTP differs from transfusion reactions
caused by red cell antibodies in that the
patient’s own antigen- negative platelets as
well as any transfused antigen-positive
platelets are destroyed. The pathogenesis of
autologous platelet destruc- tion in PTP is
not fully understood; however, mounting
evidence suggests that distinct platelet
autoantibodies transiently arise, along with
the alloantibodies, and cause both autol-
ogous and transfused antigen-negative plate-
let destruction.52 These panreactive autoanti-
bodies often target the same glycoprotein
that expresses the HPA against which
alloantibod- ies were made.
Platelet antibody assays usually reveal an
antibody in the serum with HPA-1a specificity.
Genotyping documents the absence of HPA-1a
or other platelet-specific antigens. Plasma ex-
change—once the treatment of choice for PTP
—has now largely been supplanted by IVIG as
first-line therapy. Transfusion of anti- gen-
negative platelets may be of value during the
acute phase of PTP; however, such plate- lets
have reduced in-vivo survival due to de-
struction by the aforementioned platelet auto-
antibodies.53
Following recovery, future transfusions
should be from HPA-la-negative donors if
462 ■ AABB T EC HNIC AL MANUAL
possible. Washed red cells may offer some
pro- tection against recurrence; however, this
is controversial and there is at least one
report of PTP being precipitated by a frozen
deglycero- lized red cell transfusion.54
Moreover, accord- ing to recent data reported
from the Serious Hazards of Transfusion
surveillance program, the frequency of PTP
has rather dramatically decreased
coincidently with the introduction of
leukocyte-reduced blood products. 55 Al-
though no data have been reported to explain
this trend, use of leukocyte-reduced products
may also be of benefit in reducing the risk
of PTP recurrence.
Drug-Induced Thrombocytopenia
Thrombocytopenia caused by drug-induced
platelet antibodies is a recognized complica-
tion of drug therapy. Drugs commonly
impli- cated include quinine, sulfa drugs,
vancomy- cin, GPIIb/IIIa antagonists, and
heparin.56,57 Both drug-dependent and non-
drug-depen- dent antibodies may be
produced. Non-drug- dependent antibodies,
although stimulated by drugs, do not require
the continued presence of the drug to be
reactive with platelets and are serologically
indistinguishable from other platelet
autoantibodies.
Although several mechanisms for drug-
induced antibody formation have been de-
scribed, most clinically relevant drug-depen-
dent platelet antibodies are thought to result
when a drug interacts with platelet membrane
glycoproteins, inducing conformational chang-
es recognized by drug-dependent antibod-
ies.58,59 These antibodies can cause a thrombo-
cytopenia of sudden and rapid onset that
usually resolves within 3 to 4 days after the
drug is discontinued.
Among the drug-induced immune re-
sponses to platelets, those triggered by expo-
sure to heparin have particular clinical
impor- tance because of both the widespread
use of this anticoagulant and the devastating
throm- botic complications associated with
the hepa- rin-induced thrombocytopenia
(HIT) syn- drome.60 The incidence of HIT is
unknown, but
it may develop in up to 5% of patients treated
with unfractionated heparin. Low-molecular-
weight heparin is less likely to be associated
with HIT than unfractionated heparin.
A reduction in baseline platelet count by
30% to 50% occurs generally within 5 to 14
days after primary exposure to heparin and
sooner if the patient has been exposed to hep-
arin within the last 3 months. The platelet
count is often less than 100,000/µL but usually
recovers within 5 to 7 days upon discontinua-
tion of heparin. More than 50% of patients
with HIT develop thrombosis, which can occur
in the arterial, venous, or both systems. 61 Pa-
tients may develop stroke, myocardial infarc-
tion, limb ischemia, deep venous thrombosis,
or ischemia of other organs. The thrombotic
complications may lead to limb amputation or
prove fatal. Because the rate of HIT-associated
thrombosis is so substantial, it is of critical im-
portance to discontinue heparin therapy when
a diagnosis of HIT is suspected. Moreover,
strong consideration should be given to using
an alternative (nonheparin) anticoagulant (eg,
a direct thrombin inhibitor) to prevent throm-
bosis.61
The mechanism of HIT includes forma-
tion of a complex between heparin and
plate- let factor 4 (PF4), a tetrameric protein
released from platelet  granules. Antibodies
(IgG, IgA, and some IgM) are produced to
the complex, and IgG in the complex
attaches secondarily to platelet receptor
FcRIIa via its Fc, resulting in platelet
activation with subsequent thrombin
generation. The antibody may also bind to
complexes formed at other sites, notably on
endothelial cells and monocytes. Thus, HIT
might involve activation and damage not
only of platelets but also of endothelium and
monocytes/macrophages, causing increased
susceptibility to thrombosis. This
understand- ing of the mechanism of HIT
antibodies is ex- ploited by enzyme-linked
immunosorbent as- say (ELISA) tests in
which microtiter wells are coated with the
complexes of PF4 and heparin (or heparin-
like molecules) rather than with the platelets
themselves.62
 C H A P T E R 1 8 Platelet and Granulocyte Antigens and Antibodies
Autoimmune or Immune
Thrombocytopenic Purpura
Immune thrombocytopenic purpura (ITP) is
an immune platelet disorder in which
autoan- tibodies are directed against platelet
antigens, resulting in platelet destruction.
Chronic ITP, which is most common in
adults, is character- ized by an insidious
onset and moderate thrombocytopenia that
may exist for months to years before
diagnosis. Females are twice as likely to be
affected as males.
Spontaneous remissions are rare, and
treatment is usually required to raise the
plate- let count. First-line therapy consists of
steroids or IVIG followed by more potent
immunosup- pressive agents or splenectomy
in nonre- sponders. Many other therapies
have been used in patients who do not
respond to sple- nectomy, with variable
results.
Chronic autoimmune thrombocytopenia
may be idiopathic or associated with other
conditions, such as human
immunodeficiency virus infection,
malignancy, or other autoim- mune diseases.
Acute ITP is mainly a child- hood disease
characterized by the abrupt on- set of severe
thrombocytopenia and bleeding symptoms,
often after a viral infection. The majority of
cases resolve spontaneously over a 2- to 6-
month period. If treatment is required, IVIG
or anti-D immunoglobulin infusions giv- en
to D-positive patients are usually effective
in raising platelet counts. Steroids are used
less often because of their serious side
effects in children. Splenectomy, if used, is
reserved for children whose disease is
severe and lasts longer than 6 months; this
condition is similar to chronic ITP in adults.
Rituximab and vari- ous thrombopoietin
receptor agonists have been used as second-
line therapies for acute ITP.63
Studies of both sera and washed
platelets
from patients with ITP have identified
autoan- tibodies of IgG, IgM, and IgA that
are reactive with a number of platelet
surface-membrane structures, most often
including GP complexes IIb/IIIa, Ia/IIa, and
Ib/IX but that can also in- clude GPIV, GPV,
and GPVI.64 In the majority of cases, platelet-
associated autoantibodies are reactive with
two or more platelet glycopro-
■ 463
teins.65 There is no compelling evidence to
date suggesting that a patient’s profile of
auto- antibody specificities correlates with
the se- verity of the disease or predicts that
patient’s response to therapy.
Testing for Platelet Antigens and
Antibodies
Detection of platelet antibodies in the labora-
tory provides important results to aid in mak-
ing a clinical diagnosis of an immune platelet
disorder. The leaders of the International Soci-
ety of Blood Transfusion (ISBT) platelet
immu- nology workshops, designed to
establish best practices in platelet antibody and
antigen test- ing, have determined that a
comprehensive work-up for platelet antibodies
requires the use of multiple test methods,
including a gly- coprotein-specific assay, a test
employing in- tact/whole platelets, and HPA
genotyping.66 Glycoprotein-specific assays are
the most sen- sitive and specific for identifying
the HPA specificity of serum antibodies (Fig
18-1). The inclusion of assays that use intact
platelet tar- gets is critical for detection of
antibodies that can be missed by glycoprotein-
specific tests because the process of platelet
lysis with detergent and capture of GP with a
specific monoclonal antibody can disrupt HPA
epit- opes recognized by some antibodies.
HPA ge- notyping by DNA methods is helpful
to con- firm the HPA specificity of the
antibodies and for prenatal typing of a fetus in
suspected cas- es of FNAIT. The test methods
that follow are examples of the current state-
of-the-art meth- ods used by reference
laboratories. For in- depth descriptions of
platelet antibody and antigen testing, readers
should consult recent reviews.49,67,68
Assays Using Intact Platelets
An assay that is widely used for the detection
of platelet-specific antibodies for platelet
crossmatching is the SPRCA.47 Intact platelets
are immobilized in round-bottomed wells of a
microtiter plate and are then incubated with
the patient’s serum. After washing, detector
red cells coated with antihuman IgG are
464 ■ AABB T EC HNIC AL MANUAL
MACE MAIPA
Enz-AHG
HPA-Aby
GP
MoAb
G-AMIgG
Microtiter Plate Well
FIGURE 18-1. Antigen capture enzyme-linked
immunosorbent assay (ELISA) testing. Modified
antigen capture ELISA (MACE) involves incubation
of a patient’s serum with target platelets, washing
and solubilization of the platelets in nonionic
detergent, and addition of lysate to micrototer
plate well for capture of platelet glycoprotein
(GP) by a specific mouse immunoglobulin G (IgG)
monoclonal antibody (MoAb). Platelet-specific
antibodies [human platelet alloantigen (HPA)-Aby]
in the patient’s serum bound to the GP are
detected by the addition of an enzyme- labeled
goat antihuman IgG (Enz-AHG). The
monoclonal antibody immobilization of platelet
antigens (MAIPA) assay is very similar to the
MACE, but the patient’s serum and MoAb are
incubated with platelets before washing and
platelet solubilization, and GP/HPA-Aby
complex is captured by goat antimouse IgG (G-
AMIgG) adhered to the bottom of the well.
added, centrifuged, and examined visually.
The method’s main limitations are its subjec-
tive endpoint and failure to distinguish
plate- let-specific from non-platelet-specific
anti- bodies.
Flow cytometry is commonly used for
im- munofluorescent detection of platelet
anti- bodies utilizing intact platelets.49
Following in- cubation of platelets with the
patient’s serum, platelet-bound antibodies
are detected with a fluorescent-labeled
antiglobulin reagent that is specific for
human IgG or IgM. The results can be
expressed as a ratio of the mean or me- dian
channel fluorescence of platelets sensi- tized
with patient serum to that of platelets in-
cubated with negative control serum.
Platelet autoantibodies coating the patient’s
platelets can also be detected in a direct
flow-cytometry assay.69
Flow cytometry has proven to be a very
sensitive method for detection of antibodies
to platelets. Alloantibodies that are specific
for labile epitopes and unreliably detected
by an- tigen capture assays (ACAs) can be
detected with intact platelets using flow
cytometry.8 Flow cytometry does not
differentiate between platelet-specific (ie,
platelet-glycoprotein-di- rected/HPA) and
non-platelet-specific anti- bodies (ie, HLAs
or autoantibodies). This is a drawback when
suspected FNAIT or PTP is in- vestigated
because the more relevant platelet- specific
antibodies that are characteristic of these
syndromes can be obscured by non- platelet-
specific reactivity.
Antigen Capture Assays
Platelet glycoprotein ACAs are used to
deter- mine the HPA that is recognized by
platelet an- tibodies in a patient’s serum. The
assays devel- oped for this purpose include
MACE and MAIPA (Fig 18-1).49,70 The
assays require the use of monoclonal
antibodies that recognize the target antigens
of interest but do not com- pete with the
patient’s antibody. These assays capture
specific platelet glycoproteins on plas- tic
wells of a microplate after sensitization with
patient’s serum. The patient’s bound anti-
body is detected with an enzyme-labeled
anti- human Ig. Because only the
glycoproteins of interest are immobilized,
interference by reac- tions from non-
platelet-specific antibodies, especially anti-
HLA, is eliminated.
 C H A P T E R 1 8 Platelet and Granulocyte Antigens and Antibodies
Platelet Genotyping
Genotyping for the SNPs in the genes
encod- ing HPA can be performed by any of
the myri- ad molecular methods available.
Polymerase chain reaction (PCR) followed
by restriction fragment length
polymorphism analysis and allele-specific
PCR are two methods that have been used
successfully.67 These techniques are reliable,
but they are also laborious and time
consuming. Higher-throughput methods
have been developed, such as real-time PCR
and al- lele-specific fluorescent probes.67
Testing for Platelet Autoantibodies
Numerous assays have been developed to
de- tect platelet autoantibodies in patients
with ITP. Although many tests are quite
sensitive, particularly in detecting cell-
surface, platelet- associated Igs, none has
been sufficiently spe- cific to be particularly
useful in either the diag- nosis or
management of ITP. The American Society
of Hematology practice guidelines for ITP
state that serologic testing is unnecessary,
assuming that the clinical findings are com-
patible with the diagnosis.71 However,
platelet antibody tests may be helpful in the
evaluation of patients suspected of having
ITP when non- immune causes may be
present. The goal of serologic testing in ITP
is to detect autoanti- body bound to the
patient’s own platelets with or without
demonstration of similar reactivity in the
patient’s plasma.
Newer assays are designed to detect im-
munoglobulin binding to platelet-specific
epi- topes found on platelet GPIIb/IIIa,
GPIa/GPIIa, and/or GPIb/IX complexes.
These solid-phase, GP-specific assays appear
to have improved specificity in
distinguishing ITP from nonim- mune
thrombocytopenia, but this benefit is often
balanced by a decrease in sensitivity.65,72 One
commercially available test uses eluates
prepared from the patient’s washed
platelets.65 The eluates are tested against a
panel of monoclonal-antibody-immobilized
platelet glycoprotein complexes, and platelet
antibod- ies are detected using an enzyme-
linked anti- human Ig. In the indirect phase
of the assay, patient plasma is tested against
the same gly- coprotein panel. Although
autoantibodies are
■ 465
most often detected in the eluates, they are in-
frequently detected (in approximately 17% of
cases) in the plasma. Patients with ITP may
have antibodies that are reactive with one or
several GP targets.61
Testing for Drug-Dependent
Platelet Antibodies
Any platelet serology test that is used to
detect platelet-bound Ig can be modified to
detect platelet drug-dependent antibodies.
Each pa- tient serum or plasma sample must
be tested against normal platelets in the
presence and absence of the drug. Moreover,
at least one normal control serum sample
should be tested with and without the drug
to control for the nonspecific antibody
binding that might be induced by the drug’s
presence. A positive con- trol serum sample
that is known to be reactive with the drug
being assayed should be tested with and
without the drug to complete the evaluation.
A positive result shows that the se- rum is
positive (or more positive) against nor- mal
platelets in the presence of the drug vs
without the drug and that the drug did not
nonspecifically cause a positive result with
normal serum controls. Flow cytometry is
the most sensitive and most commonly used
method to detect both IgG and IgM drug-
dependent antibodies.49,73 Limitations to
detection of drug-dependent platelet
antibod- ies include that 1) for many drugs,
the optimal concentration for antibody
detection has not been determined and
hydrophobic drugs are difficult to solubilize;
2) the presence of non- drug antibodies can
mask the antibodies; and
3) a patient may be sensitized to a drug metab-
olite and not the native drug.
Assays for heparin-dependent
antibodies include the PF4 ELISA, which
involves the ad- dition of the patient’s serum
diluted at a 1:50 ratio alone and in the
presence of high-dose (100 U/mL) heparin
to plastic microplate wells to which bound
complexes of PF4 and heparin or heparin-
like molecules (eg, polyvinyl sulfo- nate)
are affixed.62 Heparin-dependent anti- bodies
bind to the complexes and are detected via
enzyme-conjugated antihuman Ig. An op-
tical density value, generally above 0.4, in
the
466 ■ AABB T EC HNIC AL MANUAL
PF4-heparin well that can be inhibited by
high-dose heparin confirms the presence of
heparin-dependent antibodies. Although IgG
antibodies are the most clinically relevant
an- tibodies, a few patients with HIT appear
to have only IgM or IgA antibodies. PF4
ELISAs are available in two forms: those
that detect but do not differentiate IgG, IgM,
and IgA hep- arin-dependent antibodies, and
those that de- tect only IgG.
The 14C-serotonin release assay (SRA)
is a functional assay that uses washed
platelets for detection of heparin-dependent
antibodies.74 Normal, fresh platelets are
incubated with 14C-serotonin, which is
taken up into their dense granules. The
platelets are then ex- posed to the patient’s
serum in the presence of low (0.1 U/mL)
and high (100 U/mL) concen- trations of
heparin. Release of at least 20% of the
radioactivity at the low dose of heparin and
inhibition of this release below 20% at the
high dose confirms the presence of heparin-
depen- dent antibodies.
Other functional tests used to detect
hep- arin-dependent antibodies include the
hepa- rin-induced platelet-aggregation test
and the heparin-induced platelet-activation
test. The PF4 ELISA and the SRA are both
more sensitive and specific than the platelet-
aggregation test for the detection of heparin-
dependent plate- let antibodies in patients
for whom there is a clinical suspicion that
these antibodies are present. However, in
asymptomatic patients receiving heparin or
who have not yet received the drug, neither
test is sufficiently predictive of HIT to
warrant its use in screening.75
GRANULOCYTE ANTIGENS AND
ANTIBODIES
Antibodies against granulocyte (neutrophil)
antigens are implicated in the following
clini- cal syndromes: neonatal alloimmune
neutro- penia (NAN), transfusion-related
acute lung injury (TRALI), immune
neutropenia after HPC transplantation,
refractoriness to granu- locyte transfusion,
and chronic benign auto- immune
neutropenia of infancy (AIN). To date, nine
neutrophil antigens carried on five different
glycoproteins have been character-
ized and given human neutrophil alloantigen
(HNA) designations by the Granulocyte Anti-
gen Working Party of the ISBT (Table 18-5).76
This nomenclature system follows a similar
convention to that used for HPA nomencla-
ture. Several of the antigens on granulocytes
are shared with other cells and are not granu-
locyte specific.
HNA
Antigens on FcRIIIb
The first granulocyte-specific antigen detected
was NA1, later named “HNA-1a.” Three
alleles of HNA-1 have now been identified—
HNA-1a, HNA-1b, and HNA-1c—and are
located on the protein FcRIIIb (CD16b).77
FcRIIIb is a GPI- linked protein receptor for
the Fc of IgG and is present only on the
surfaces of neutrophils. Neutrophils express
100,000 to 200,000 mole- cules of FcRIIIb,
but there are rare individuals (approximately
0.1%) whose neutrophils ex- press no
FcRIIIb (CD16 null) and who can produce
antibodies that are reactive with FcRIIIb
when they are exposed to it through
transfusion or pregnancy.78,79 Antibodies to
HNA-1a and -1b have been implicated in
TRALI, NAN, and AIN.77
Antigens on CD177
HNA-2 (previously known as “NB1”) is not an
alloantigen because HNA-2 antibodies are iso-
antibodies that recognize common epitopes
on CD177 protein, which is missing from the
neutrophils of immunized individuals. Neu-
trophils from approximately 3% to 5% of peo-
ple lack expression of CD177 on neutrophils.77
Lack of CD177 is thought to be caused by a
messenger ribonucleic acid splicing defect
that results in a truncated protein that cannot
be expressed.80 Interestingly, CD177 is ex-
pressed only on a subpopulation of neutro-
phils in CD177-positive individuals.81 The pro-
portion of the CD177-positive neutrophil
population ranges from 0% to 100%.82 CD177
is expressed only on neutrophils and belongs
to the Ly-6/uPAR/snake-toxin family of
proteins.81
 C H A P T E R 1 8 Platelet and Granulocyte Antigens and Antibodies ■ 467

TABLE 18-5. Human Neutrophil Antigens

Phenotypic Amino Acid Nucleotide

Antigen Frequency* Glycoprotein Change Encoding Gene Change
HNA-1a 12% a/a CD16 Multiple† FCGR3B Multiple†
HNA-1b 54% a/b
46% b/b
HNA-1c 5%
HNA-2 97% CD177+ CD177 N/A CD177 N/A
3% CD177-
HNA-3a 56%-59% a/a CTL2 Arg152Gln SLC44A2 455G>A
HNA-3b 34%-40% a/b
3%-6% b/b
HNA-4a 78.6% a/a CD11b Arg61His ITGAM 230G>A
HNA-4b 19.3% a/b
2.1% b/b
HNA-5a 54.3% a/a CD11a Arg766Thr ITGAL 2466G>C
HNA-5b 38.6% a/b
7.1% b/b

Nucleotide Changes Amino Acid Changes

141 147 227 266 277 349 36 38 65 78 82 106

HNA-1a G C A C G G Arg Leu Asn Ala Asp Val
HNA-1b C T G C A A Ser Leu Ser Ala Asn Ile
HNA-1c C T G A A A Ser Leu Ser Asp Asn Ile
*Phenotypic frequencies are for people of European ancestry who live in North America.
†
HNA-1 amino acid and nucleotide changes are shown in a separate section of the
table. HNA = human neutrophil antigen; N/A = not applicable.

A recent report documented cation-de- Antigens on CTL2

pendent heterophilic interactions between
CD177 and the endothelial cell membrane
protein PECAM-1(CD31), suggesting a role
for CD177 in neutrophil transendothelial
migra- tion to sites of infection. 83 Antibodies
against HNA-2 have been implicated in NAN,
TRALI, and neutropenia in marrow
transplant recipi- ents.84-86
HNA-3a and HNA-3b are carried on the
cho- line transporter-like protein 2 (CTL2),
and a SNP in the gene (SLC44A2) accounts
for the polymorphism (Table 18-5).87,88
CTL2 is also expressed on both T and B
lymphocytes, and small amounts are present
on platelets. HNA- 3a antibodies are
usually agglutinins. They
468 ■ AABB T EC HNIC AL MANUAL
occasionally develop in women after pregnan-
cy, and HNA-3a antibodies are the most fre-
quent cause of fatal TRALI.89 HNA-3b
antibod- ies are rarely detected, but several
have been found during screening of the serum
of mul- tiparous blood donors.
Antigens on CD11a and CD11b
HNA-4a and HNA-5a, both high-prevalence
antigens, are present on monocytes and lym-
phocytes as well as granulocytes. HNA-4a is
carried on the CD11b/18 (Mac-1, CR3,
m2) glycoprotein.77 CD11b/18 plays a role
in neu- trophil adhesion to endothelial cells
and
phagocytosis of C3bi opsonized microbes.
There is some evidence showing that
alloanti- bodies against HNA-4a interfere
with CD11b/ 18-dependent neutrophil
adhesion and en- hance neutrophil
respiratory burst.90 Antibod- ies against
HNA-4a have been implicated in NAN, and
autoantibodies against CD11b/18 have also
been described.91,92
HNA-5a is carried on CD11a/18 (LFA-1,
L2) glycoprotein.93 CD11a/18, like CD11b/
18, plays a role in neutrophil adhesion to en-
dothelial cells. Antibodies that are reactive
with HNA-5a have been found in a chronically
transfused patient with aplastic anemia and
have also been reported to be associated
with NAN.94 The patient who made the
original HNA-5a antibody experienced
prolonged sur- vival of an HLA nonidentical
skin graft that was attributed to the HNA-5a
antibody.93
Other Neutrophil Antigens
Neutrophils do not express ABH or other
red cell group antigens, but they do express
mod- est amounts of Class I and II HLA
only upon activation.
Immune Neutrophil Disorders
Neonatal Alloimmune Neutropenia
NAN is caused by maternal antibodies against
antigens on fetal neutrophils; the most fre-
quent specificities are against HNA-1a, HNA-
1b, and HNA-2 antigens. NAN may also occur
in the children of women who lack the
FcRIIIb protein. Neutropenia in NAN can
oc- casionally be life-threatening because of
in- creased susceptibility to infection.95
Manage- ment with antibiotics, IVIG,
granulocyte colony-stimulating growth
factor, and/or plas- ma exchange may be
helpful.
TRALI
TRALI is an acute, often life-threatening
reac- tion characterized by respiratory
distress, hypo- or hypertension, and
noncardiogenic pulmonary edema that
occurs within 6 hours of a blood component
transfusion.96 TRALI has been the most
common cause of transfusion- related death
for more than 10 years.97 In TRALI, the
causative antibodies are most often found in
the plasma of the blood donor. When these
antibodies are transfused, they cause ac-
tivation of primed neutrophils that are
seques- tered in the lungs of certain patients.
The acti- vated neutrophils undergo
oxidative burst, releasing toxic substances
that damage pul- monary endothelium and
resulting in capillary leak and pulmonary
edema. Class I and II HLA and HNA
antibodies have all been implicated in
TRALI. However, in a recent large clinical
study of TRALI, HNA and Class II HLA
anti- bodies, but not Class I HLA antibodies,
were significantly associated with TRALI. 98
(For a more in-depth discussion of TRALI,
see Chap- ter 27.)
Autoimmune Neutropenia
Autoimmune neutropenia may occur in
adults or in infants. When present in adults,
it may be idiopathic or be secondary to such
diseases as rheumatoid arthritis or systemic
lupus erythe- matosus or to bacterial
infections.99 In autoim- mune neutropenia of
infancy, usually occur- ring in children
between the ages of 6 months and 2 years,
the autoantibody has neutrophil antigen
specificity (usually HNA-1a or -1b) in about
30% of the patients. The condition is
generally self-limiting, with recovery
usually occurring in 7 to 24 months, and is
relatively benign and manageable with
antibiotics.87
 C H A P T E R 1 8 Platelet and Granulocyte Antigens and Antibodies ■ 469

Testing for Granulocyte Antibodies
and Antigens
Granulocyte antibody testing is technically
complex and labor intensive. The inability
to maintain the integrity of granulocytes for
test- ing that are stored at room temperature,
in re- frigerated conditions, or by
cryopreservation requires that cells be
isolated from fresh blood on each day of
testing. This demands that readily available
blood donors typed for the various
granulocyte antigens be available. Class I
HLA antibodies that are often present in
patient sera complicate detection and iden-
tification of granulocyte antibodies. For
these reasons, it is critical that granulocyte
antibody and antigen testing be performed
by an expe- rienced laboratory using
appropriate controls.
GR ANULOCYTE AGGLUTINATION TE ST . This
was one of the first tests developed for the
de- tection of granulocyte antibodies. It is
typically performed by overnight incubation
of small volumes of isolated fresh
neutrophils with the patient’s serum in a
microplate. The wells are viewed under an
inverted phase microscope for neutrophil
agglutination or aggregation.
GR A N UL OCY T E IMMUN O FL UORE S CEN
CE
TE S T . This test also requires fresh target
cells that are incubated, usually at room
tempera-
ture for 30 minutes, and washed in EDTA
and phosphate-buffered saline. Neutrophil-
bound antibodies are then detected with
fluorescein isothiocyanate-labeled
antihuman IgG or IgM with either a
fluorescence microscope or a flow
cytometer.89,100 A combination of aggluti-
nation and immunofluorescence tests is
bene- ficial.79,88 Other methods include
chemilumi- nescence, SPRCA, and the
monoclonal antibody immobilization of
granulocyte anti- gens (MAIGA) assay,
which is similar to the MAIPA assay, but
uses monoclonal antibodies to capture the
various glycoproteins that ex- press HNA.
The MAIGA assay is used to differ- entiate
between HLA- and HNA-specific anti-
bodies.
HNA TY PIN G. As with HPA, typing for
HNA is largely performed using molecular
methods to detect the allelic variants that
determine the antigens. Any methods used
in HPA typing can be applied to HNA
typing with simple modifi- cations to the
primer and probe sequences. Readers are
referred to several publications on this
subject.101,102 Because the splicing defect that
results in CD177 deficiency is not known,
typing for HPA-2/CD177 requires testing
for CD177 on freshly isolated neutrophils
using specific monoclonal antibodies and
the granu- locyte immuno fluorescence test.

KEY POINTS

Platelets express a variety of antigenic markers on their surfaces. Some of these antigens, such as ABH and HLA, are share
HLA sensitization is the most common immune cause of platelet refractoriness and can be diagnosed by the demonstration
Compared with HLA matching for platelet-refractory patients, crossmatching can be both more convenient and cost effecti
Although blood group antibodies (anti-A and -B) and platelet-specific antibodies can be re- sponsible for poor responses to
470 ■ AABB T EC HNIC AL MANUAL

5. Sensitization to HPA is the most common cause of FNAIT, a syndrome involving

immune destruction of fetal platelets by maternal antibody. Platelet-specific antibodies
are also in- volved in PTP, a rare syndrome characterized by severe thrombocytopenia
that occurs 5 to 10 days after a blood transfusion. The most commonly implicated
antibody in both condi- tions is anti-HPA-1a. Serologic testing using intact platelets
and antigen capture assays to- gether with HPA genotyping is used to confirm both of
these diagnoses.
6. Autoantibodies directed against platelet antigens may result in ITP. Chronic ITP, which
is most common in adults, is characterized by an insidious onset and moderate
thrombocyto- penia that may be present for months to years before diagnosis. Females
are twice as likely to be affected as males. The goal of serologic testing in ITP is to
detect autoantibody bound to the patient’s own platelets.
7. Granulocyte (neutrophil) antigens are implicated in the clinical syndromes NAN,
TRALI, immune neutropenia after hematopoietic progenitor cell transplantation,
refractoriness to granulocyte transfusion, and chronic benign autoimmune neutropenia
of infancy.
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in transfusion-related acute lung injury and re-
sults of leucocyte antibody screening of blood
donors. Vox Sang 2008;95:313-17.
90. Sachs UJ, Chavakis T, Fung L, et al. Human al-
loantibody anti-Mart interferes with Mac-1-
dependent leukocyte adhesion. Blood 2004;
104:727-34.
91. Fung YL, Pitcher LA, Willett JE, et al.
Alloim- mune neonatal neutropenia linked to
anti- HNA-4a. Transfus Med 2003;13:49-52.
92. Hartman KR, Wright DG. Identification of au-
toantibodies specific for the neutrophil adhe-
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with autoimmune neutropenia. Blood 1991;
78:1096-104.
474 ■ AABB T EC HNIC AL MANUAL

93. Simsek S, van der Schoot CE, Daams M, et

gov/BiologicsBloodVaccines/SafetyAvailabili
al. Molecular characterization of antigenic
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leukocyte alloan- tisera. Blood 2, 2013).]
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94. Porcelijn L, Abbink F, Terraneo L, et al. related acute lung injury: Incidence and risk
Neona- tal alloimmune neutropenia due to factors. Blood 2012;119:1757-67.
immuno- globulin G antibodies against human 99. Akhtari M, Curtis B, Waller EK. Autoimmune
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95. Davoren A, Saving K, McFarland JG, et al. 100. Curtis BR, Reno C, Aster RH. Neonatal alloim-
Neo- natal neutropenia and bacterial sepsis mune neutropenia attributed to maternal im-
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 C h a p t e r 1 9

The HLA System

Robert A. Bray, PhD; Marilyn S. Pollack, PhD;

and Howard M. Gebel, PhD

T H E H L A SY ST E M is composed of a found on the surface of platelets and, with a

complex array of genes located within
the human major histocompatibility complex
(MHC) on the short arm of chromosome 6.
Their protein products, the HLA antigens, con-
tribute to the recognition of self and nonself,
the immune responses to antigenic stimuli,
and the coordination of cellular and humoral
immunity.
HLA antigens are cell-surface glycopro-
teins that are divided into two groups (Class I
and Class II) according to their coding gene
lo- cus, function, tissue distribution, and bio-
chemistry. HLA Class I molecules contain one
copy each of two polypeptides: a heavy chain
that is an integral cell-membrane protein and
a noncovalently associated light chain called
“2-microglobulin” (the 2-microglobulin gene
resides on chromosome 15). HLA Class II
mol-
ecules are composed of one copy each of an
- chain and a -chain, both of which are
integral membrane proteins. Class I
molecules are
few exceptions, on all nucleated cells of the
body. Mature red cells usually lack HLA anti-
gens that are identifiable by conventional
methods, whereas nucleated immature ery-
throid cells do express HLA antigens. MHC
Class II antigen expression is typically restrict-
ed to a few cell types, including B lympho-
cytes, monocytes, macrophages, dendritic
cells, and activated T lymphocytes.
HLA molecules play a key role in antigen
presentation and the initiation of the immune
response. The HLA system is generally
viewed as second in importance only to the
ABO anti- gens in influencing the survival of
transplant- ed solid organs. In hematopoietic
progenitor cell (HPC) transplantation, the
HLA system is paramount with regard to graft
rejection and graft-vs-host disease (GVHD).
HLA antigens and antibodies are also
important in compli- cations of transfusion
therapy, such as platelet refractoriness, febrile
nonhemolytic transfu-

Robert A. Bray, PhD, Professor of Pathology, and Co-director, Histocompatibility and Molecular Immunoge-
netics Laboratory, Department of Pathology, Emory University, Atlanta, Georgia; Marilyn S. Pollack, PhD,
Pro- fessor of Pathology, University of Texas Health Science Center, and Director, University Health System
Histocompatibility and Immunogenetics Laboratory, San Antonio, Texas; and Howard M. Gebel, PhD, Profes-
sor of Pathology, and Co-director, Histocompatibility and Molecular Immunogenetics Laboratory, Depart-
ment of Pathology, Emory University, Atlanta, Georgia
The authors have disclosed no conflicts of interest.

475
476 ■ AABB T EC HNIC AL MANUAL
sion reactions (FNHTRs), transfusion-related
acute lung injury (TRALI), and transfusion-
associated GVHD (TA-GVHD).
Interest in HLA polymorphisms has ex-
tended beyond their role as transplantation
antigens. Studies correlating them with dis-
ease susceptibility and disease resistance be-
gan soon after serologic techniques for HLA
Class I typing were developed. Historically,
HLA antigen typing had been of value in
rela- tionship assessments and forensic
investiga- tions. However, with the
development of de- oxyribonucleic acid
(DNA) typing, molecular methods replaced
the mixed leukocyte culture (MLC) as the
preferred method to select matched donor-
recipient pairs for HPC trans- plantation.
Molecular HLA testing also fos- tered a
resurgence in the investigation of dis- ease
associations that permitted the analysis of
peptide-binding restriction parameters
needed for effective vaccine development
and provided a more accurate tool for
anthropo- logic population studies. Because
of the poly- morphic nature of the HLA
genes, a complex nomenclature was
developed (and is continu- ally evolving) to
define unique allele sequenc- es based on
the relationship of each allele’s protein
sequence to the serologic specificity of the
corresponding antigen.1,2
GENETICS OF THE MHC
Class I and II HLA antigens are cell-surface
gly- coproteins that are products of closely
linked genes mapped to the p21.3 band on
the short arm of chromosome 6 (Fig 19-1).
That genom- ic region is called the “MHC”
and is usually in- herited en bloc as a
haplotype. Each of the many loci has
multiple alleles with codomi- nant
expression of the products from each
chromosome. With the exception of
immuno- globulin (Ig) idiotypes, the HLA
system is the most polymorphic genetic
system described in humans.
The genes HLA-A, HLA-B, and HLA-C
encode the corresponding Class I A, B, and C
antigens. The genes HL A-DRB1, -DRB3,
-DRB4, -DRB5; HLA-DQA1, -DQB1; and
HLA-
DPA1, -DPB1 encode the corresponding
Class II antigens. Located between the Class
I and
Class II genes is a group of non-HLA genes
that code for molecules that include the
comple- ment proteins C2, Bf, C4A, and C4B;
a steroid enzyme (21-hydroxylase); and a
cytokine (tu- mor necrosis factor). This non-
HLA region is often referred to as “MHC
Class III” even though it does not contain any
HLA genes.
Organization of HLA Genetic Regions
The HLA Class I region contains (in addition
to the classical genes HLA-A, HLA-B, and
HLA-C) other gene loci designated HLA-E,
HLA-F, HLA-G, HLA-H, HFE, HLA-J, HLA-K,
HLA-L,
MICA, and MICB. The latter genes encode
nonclassical, or Class Ib, HLA proteins,
which have limited polymorphism and low
levels of expression.4 Some Class Ib genes
express non- functional proteins or no
proteins whatsoever. Genes that are unable
to express a functional protein product are
termed “pseudogenes” and presumably
represent an evolutionary dead end. In
contrast, other nonclassical HLA proteins
that are expressed have been associ- ated
with a variety of functions. For example,
HLA-E is associated with the surveillance
sys- tem of one subset of natural killer cells.
HLA-G is expressed by the trophoblast and
may be in- volved in the development of
maternal im- mune tolerance of the fetus.
Hereditary hemo- chromatosis (HH), an iron
overload disorder with a 10% carrier
frequency in people of Northern European
ancestry, is associated with two missense
mutations in a Class I-like gene.5 The gene
that causes HH was initially named “HLA-
H”; however, the HLA-H desig- nation had
already been assigned to an HLA Class I
pseudogene by the World Health Orga-
nization (WHO) Nomenclature Committee.6
The gene that is responsible for HH is now
called “HFE.” Additional Class I-like genes
that code for molecules, such as CD1, are
also lo- cated outside the MHC. These
molecules pres- ent nonprotein antigens
(such as lipids) to T cells.
The genomic organization of the MHC
Class II (HLA-D) region is more complex. An
MHC Class II molecule consists of a noncova-
lent complex of two structurally similar
chains: the -chain and the -chain. Both of
 C H A P T E R 1 9 The HLA System
■

FIGURE 19-1. The HLA complex is located on the short arm of chromosome 6. The centromere is to the top left of the figure, the telomere to the bottom right.
The organi- zation of the Class I, II, and III regions is shown.3
477
478 ■ AABB T EC HNIC AL MANUAL
these chains are encoded within the MHC.
The polymorphism of HLA Class II
molecules re- sults from differences in both
the -chain and the -chain; this
polymorphism depends on the Class II
isoform. For example, with HLA- DR, the
-chain is essentially monomorphic, but the
-chain is very polymorphic. Multiple loci
code for the - and -chains of the Class II
MHC proteins.
Different haplotypes have different num-
bers of Class II genes and pseudogenes. The
proteins coded by DRA1 and DRB1 result in
HLA-DR1 through HLA-DR18 antigens. The
products of DRA1 and DRB3 (if present) ex-
press HLA-DR52; those of DRA1 and DRB4
(if present) express HLA-DR53; and those of
DRA1 and DRB5 (if present) express HLA-
DR51. The HLA-DQ1 through DQ9 antigens
are expressed on the glycoproteins coded by
DQA1 and DQB1 in the DQ cluster. Many of
the other genes of the DQ cluster are probably
pseudogenes. A similar organization is found
in the HLA-DP gene cluster.
Although not generally considered part
of the HLA system, the MHC Class III
region con- tains four complement genes
with alleles that are typically inherited
together as a unit, termed a “complotype.”
More than 10 different complotypes are
inherited in humans. Two of the Class III
genes, C4A and C4B, encode for variants of
the C4 molecule and antigens of the
Chido/Rodgers blood group system. These
variants have distinct protein structures and
functions; the C4A molecule (if present)
car- ries the Rg antigen, and the C4B
molecule (if present) carries the Ch antigen.
Both of these antigens are adsorbed onto the
red cells of in- dividuals who possess the
gene(s).
Patterns of Inheritance
Although MHC organization is complicated,
its inheritance follows the established princi-
ples of Mendelian genetics. Every person has
two different copies of chromosome 6 and
possesses two HLA haplotypes, one from each
parent. The expressed gene products consti-
tute the phenotype, which can be determined
by typing for HLA antigens or alleles. Because
HLA genes are autosomal and codominant,
the phenotype represents the combined ex-
pression of both haplotypes. However, to de-
fine haplotypes, parents (and possibly other
family members) must also be phenotyped
to determine which alleles are inherited
together. Figure 19-2 illustrates inheritance
of haplo- types.
Finding HLA-Identical Siblings
A child inherits one copy of chromosome 6
from each parent; hence, one MHC
haplotype is inherited from each parent.
Because each parent has two different
copies of chromo- some 6, four different
combinations of haplo- types are possible in
the offspring (assuming that no
recombination occurs). The inheri- tance
pattern is important in predicting whether
family members will be compatible donors
for transplantation. The chance that two
siblings will be genotypically HLA identi-
cal is 25%. The chance that any one patient
with “n” siblings will have at least one
HLA- identical sibling is 1 – (3/4)n. Having
two sib- lings provides a 44% chance, and
having three siblings provides a 58%
chance, that one sib- ling will be HLA
identical. Moreover, each time a new sibling
is tested, that new sibling has (only) a 25%
chance of being a match, no mat- ter how
many siblings have previously been tested.
Absence of Antigens
Before the advent of molecular-based HLA
typing, the absence of an antigen in
serologic phenotyping results was attributed
to homo- zygosity at a locus (eg, inheritance
of A1 from both parents, which in reality
represented only an apparent absence of the
antigen as a result of limitations of
phenotyping methods) or to a null
(nonexpressed) allele. With DNA sequenc- ing
and other molecular HLA typing methods,
homozygosity can now be presumed with a
higher degree of confidence. However,
homo- zygosity still can be proven only
through fami- ly studies or methods
permitting hemizygous typing (ie, typing of
an individual haplotype). A null allele is
characterized by one or more substitutions
that are within or outside the gene’s coding
region and that prevent expres-
 C H A P TE R 1 9 The HLA System
FIGURE 19-2. The designations a/b and c/d represent paternal and maternal HLA haplotypes,
respectively. Except for crossovers, the HLA complex is transmitted en bloc from parent to
offspring.
sion of a functional protein at the cell
surface. Such inactivation of a gene may be
caused by nucleotide substitutions,
deletions, or inser- tions that lead to a
premature cessation in the antigen’s
synthesis. In the absence of a family study, a
phenotyping study revealing a single allele
at any locus offers only presumptive evi-
dence for homozygosity. In this situation,
the allele should be listed only once because
it is unknown whether that allele is present
twice (a true homozygote) or there is
another allele not detected by the available
method.
Crossovers
The genes of the HLA region occasionally
demonstrate chromosome crossover, in which
segments containing linked genetic material
are exchanged between the two chromosomes
during meiosis or gametogenesis. The recom-
binant chromosomes are then transmitted as
new haplotypes to the offspring. Crossover fre-
quency is related partly to the physical dis-
tance between the genes and partly to the re-
sistance or susceptibility of specific A, B, and
■ 479
DR antigens to recombination (see below).
The HLA-A, HLA-B, and HLA-DR loci are
close together, with 0.8% crossover between
the A and B loci and 0.5% between the B
and DR loci. Crossovers between the HLA-B
and HLA-C loci or between the HLA-DR and
the HLA-DQ loci are extremely rare,
whereas crossovers be- tween the DQ and
DP loci are relatively com- mon.7,8 In family
studies and relationship eval- uations, the
possibility of recombination should always
be considered.
Linkage Disequilibrium
The MHC system is so polymorphic that the
number of possible unique HLA phenotypes
is theoretically greater than that of the
global hu- man population. Moreover, new
HLA alleles are constantly being discovered
and character- ized. As of March 2014, 2579
HLA-A alleles, 3285 HLA-B alleles, 1512
DRB1 alleles, and 509 DQB1 alleles had
been identified.9 However, because HLA
genes are inherited as an entire
chromosome, in reality, many HLA
haplotypes are overrepresented compared
with what
480 ■ AABB T EC HNIC AL MANUAL

would be expected if the distribution of HLA BIOCHEMISTRY, TISSUE

genes were random. The phenomenon of link- DISTRIBUTION, AND
age disequilibrium accounts for the discrepan- STRUCTURE
cy between expected and observed HLA hap-
lotype frequencies. Characteristics of Class I and Class II
Expected frequencies for HLA haplotypes Antigens
are derived by multiplication of the frequen-
Class I antigens (HLA-A, -B, and -C) have a
cies of each allele. For example, in
molecular weight of approximately 57,000
individuals of European ancestry, the
Dal- tons and consist of two protein chains: a
overall frequency of the gene coding for
glyco- protein heavy chain (45,000 Daltons)
HLA-A1 is 0.15 and that for HLA-B8 is
encoded on the short arm of chromosome 6
0.10; therefore, 1.5% (0.15 × 0.10) of all HLA
and, as a light chain, the 2-microglobulin
haplotypes in people of European eth- nicity
would be expected to contain genes coding molecule
(12,000 Daltons) encoded by a gene on chro-
for both HLA-A1 and HLA-B8 if the
mosome 15. The heavy chain penetrates the
haplotypes were randomly distributed. The
cell membrane, whereas 2microglobulin does
actual haplotype frequency of the A1 and
not. Rather, 2microglobulin associates (non-
B8 combination, however, is 7% to 8% in
covalently) with the heavy chain through the
that population.
latter’s nonvariable (3) domain (see Fig 19-
Certain allelic combinations occur with 3).
increased frequency in different racial The external portion of the heavy chain con-
groups and constitute common haplotypes in sists of three amino acid domains (1, 2, and
those populations. These common
3), of which the outermost 1 and 2 do-
haplotypes are called “ancestral haplotypes”
mains contain the majority of polymorphic
because they ap- pear to be inherited from a
re- gions conferring serologic HLA antigen
single common an- cestor or to be conserved
speci- ficity.
within the popula- tion because of resistance
Class I molecules are present on
to recombination or survival advantage. The
platelets and most nucleated cells in the
most common ances- tral haplotype in
body, with some exceptions that include
people of Northern European ancestry—A1,
neurons, corneal epithelial cells,
B8, DR17 (DRB1*03:01), DQ2—
trophoblasts, and germinal cells. Only
includes both Class I and Class II regions.
vestigial amounts remain on ma- ture red
Some haplotypes in apparent linkage
cells, with certain allotypes better ex-
dis- equilibrium may represent relatively
pressed than others. These Class I types
young haplotypes that have not had
were independently recognized as red cell
sufficient time to undergo recombination,
antigens by serologists and designated as
whereas some old haplotypes are resistant to
“Bennett- Goodspeed” (Bg) antigens. The
recombination be- cause of selection or
specificities called “Bga,” “Bgb,” and “Bgc”
physical limitations. For example, the A1,
are actually HLA- B7, HLA-B17 (B57 or
B8, DRB1*03:01 haplotype appears to be
B58), and HLA-A28 (A68
resistant to recombination be- cause that
or A69), respectively. Platelets express
sequence of DNA has a different length than
primari- ly HLA-A and HLA-B antigens.
most other comparable sequenc- es due to
HLA-C antigens are present at very low levels,
the deletion of the complement gene C4A.
and Class II anti- gens are generally not
Linkage disequilibrium in the HLA sys- tem
present.
is important in relationship studies be- cause
Class II antigens (HLA-DR, -DQ, and -
haplotype frequencies in the relevant
DP) have a molecular weight of
population make the transmission of certain
approximately 63,000 Daltons and consist of
gene combinations more likely than others.
two structurally similar glycoprotein chains
Linkage disequilibrium also affects the
( and ), both of which traverse the
likeli- hood of finding suitable unrelated
membrane (see Fig 19-3). The
donors for HLA-matched platelet
extramembranous portion of each chain has
transfusions and HPC transplantation.
two amino acid domains, of which the out-
ermost one contains the variable regions of
the Class II alleles. The expression of Class
II
 C H A P TE R 1 9 The HLA System
FIGURE 19-3. Stylized diagram of Class I and Class II major histocompatibility complex molecules showing 
and  polypeptide chains, their structural domains, and attached carbohydrate units.
antigens is more restricted than that of Class
I antigens. Class II antigens are expressed
con- stitutively on B lymphocytes,
monocytes, mac- rophages, dendritic cells,
intestinal epitheli- um, and early
hematopoietic cells. There is also
constitutive expression of Class II anti- gens
on some endothelial cells, especially those
lining the microvasculature. However, in
general, endothelium, particularly that of
larg- er blood vessels, is negative for Class
II antigen expression, although Class II
antigen expres- sion can be readily induced
(for instance, by interferon- during immune
activation). Rest- ing T lymphocytes are
negative for Class II an- tigen expression
but can become positive when activated.
Soluble HLA Class I and Class II
antigens shed from cells are present in blood
and body fluids and may play a role in
modulating im- mune reactivity.10 Levels of
soluble HLA in- crease with infection
[including with human immunodeficiency
virus (HIV)], inflammato- ry disease, and
transplant rejection, but HLA levels decline
with progression of malignancy. Levels of
soluble HLA in blood components are
proportional to the number of residual do-
■ 481
nor leukocytes and the duration of storage. 11
Soluble HLA in blood components may be
in- volved in the immunomodulatory effect
of blood transfusion.
Configuration
A representative three-dimensional structure
of Class I and Class II molecules can be ob-
tained by x-ray crystallographic analysis of
pu- rified HLA antigens (see Fig 19-4). The
outer domains, which contain the regions of
amino acid variability and the antigenic
epitopes of the molecules, form a structure
known as the “peptide-binding groove.”
Alleles that are de- fined by polymorphisms
in the HLA gene se- quences encode unique
amino acid sequences and therefore form
unique binding grooves, each of which is
able to bind peptides of differ- ent
sequences. The peptide-binding groove is
critical for the functional aspects of HLA
mole- cules (see “Biologic Function” section
below).
Nomenclature for HLA Antigens
An international committee sponsored by the
WHO establishes the nomenclature of the
HLA
482 ■ AABB T EC HNIC AL MANUAL
FIGURE 19-4. Ribbon diagram of HLA Class I and Class II molecule. Note the peptide in the groove of each
molecule.
system. This nomenclature is updated regular-
ly to incorporate new HLA alleles.2 HLA anti-
gens are designated by a number following the
letter that denotes the HLA series (eg, HLA-
A1 or HLA-B8). Previously, antigenic
specificities that were not fully confirmed
carried the prefix “w” (eg, HLA-Aw33) for
“workshop.” When the antigen’s identification
became definitive, the WHO Nomenclature
Committee dropped the “w” from the
designation. (The Committee meets regularly
to update nomenclature by recognizing new
specificities or genetic loci.) The “w” prefix is
no longer applied in this manner and is now
used only for the following:
1) Bw4 and Bw6, to distinguish such “public”
antigens (see “‘Public’ Antigens” section be-
low) from other B locus alleles; 2) all serologi-
cally defined C locus specificities, to avoid
confusion with components of the comple-
ment system; and 3) Dw specificities that were
defined by mixed leukocyte reactions but are
now known to be caused by HLA-DR, HLA-
DQ, and HLA-DP polymorphisms. The
numeric designations for the HLA-A and
HLA-B speci- ficities were assigned according
to the order of their discovery.
Splits and Cross-Reactive Groups
Refinement of serologic methods permitted
antigens that were previously believed to rep-
resent a single specificity to be “split” into
specificities that were serologically (and,
later, genetically) distinct. The designation
for an in- dividual antigen that was split
from an earlier recognized antigen often
includes the number of the parent antigen in
parentheses [eg, HLA- B44 (12)].
In addition to “splits,” certain apparently
distinct HLA antigens may have other epitopes
in common. Antibodies that are reactive with
these shared determinants often cause cross-
reactions in serologic testing. The collective
term for a group of HLA antigens that exhibit
such cross-reactivity is “cross-reactive group”
(CREG).
“Public” Antigens
In addition to splits and CREGs, HLA proteins
have reactivity that is common to many differ-
ent HLA specificities. Called “public”
antigens, these common amino acid sequences
appear to represent the less variable portion of
the HLA molecule. Two well-characterized
public antigens, HLA-Bw4 and HLA-Bw6, are
present in almost all HLA-B molecules. 12 The
HLA-A locus molecules A23, A24, A25, and
A32 also have a Bw4-like epitope.
Public antigens are clinically important
because patients exposed to them through
pregnancy, transfusion, or transplantation
can
 C H A P TE R 1 9 The HLA System
make antibodies to these antigens if the pa-
tients do not express the epitopes
themselves. A single antibody, when
directed against a public antigen, can
resemble multiple discrete alloantibodies,
and this has significant conse- quences for
identifying compatible donors for
transplantation and platelet transfusion.
Nomenclature for HLA Alleles
Because nucleotide sequencing is used to in-
vestigate the HLA system, increasing numbers
of HLA alleles are being identified, many of
which share a common serologic phenotype.
The minimum requirement for designation of
a new allele is the sequence of exons 2 and 3
for HLA Class I and exon 2 for HLA Class II
(DRB1). These exons encode the variable ami-
no acids that confer HLA antigen specificity.
A uniform nomenclature has been adopt-
ed that takes into account the locus, major se-
rologic specificity, and allele group deter-
mined by molecular typing techniques. For
example, although many alleles have been se-
quenced only for exon 2, nucleotide sequenc-
ing has identified at least 165 unique amino
acid sequence variants (alleles) of HLA-DR4
as of March 2014. The first HLA-DR4 variant
is designated “DRB1*04:01,” indicating the
locus (DR), protein (1 chain), major
serologic spec- ificity (04 for HLA-DR4), and
sequence 2 varia- tion allele number (variant
01). The asterisk indicates that an allele name
follows (and that
TABLE 19-1. HLA Nomenclature 2013
Antigen
Species Locus Equivalent Allele
HLA DRB1 * 04 : 01 :
Examples:
DR4 - Serology
DRB1*04:xx - Serologic
Equivalent
DRB1*04:02 - Allele
DRB1*04:01:01; DRB1*04:01:02 - Silent Mutations
A*02:15N; DRB4*01:03:01:02N - Null Alleles (exon,
intron) A*24:02:01:02L
B*44:02:01:02 - - Expression Modifiers
B*32:11Q
■ 483
the typing was determined by molecular
tech- niques).
A similar system is used for naming
Class I alleles. The name of the locus—for
example, “HLA-B”—is followed by an
asterisk and then by several digits separated
by colons. The first two digits correspond to
the antigen’s serologic specificity in most
cases. The next group of digits makes up the
code for a unique amino acid sequence in
exons 2 and 3, with numbers being assigned
in the order in which the DNA sequences
were determined. Therefore, B*27:04
represents the HLA-B locus, has a se-
rologic specificity of B27, and was the
fourth unique sequence 2 and 3 allele
described in this family (see Table 19-1). A
third “place” in the allele name is added for
alleles that differ only by synonymous
(“silent”) nucleotide sub- stitutions in exons
2 and 3 for Class I or in exon 2 for Class II.
For example, A*01:01:02 differs from
A*01:01:01 only in that the codon for iso-
leucine in position 142 is ATT instead of ATC.
A fourth “place” in the allele name can be
added for alleles that differ only in
sequences within introns or in 3 or 5
untranslated regions. Fi- nally, the
nomenclature accommodates al- leles with
null or low expression or other char-
acteristics by the addition of an “N” or “L,”
respectively, or another letter as appropriate
to the end of the allele name. The other
official expression modifiers are as follows:
S (secret- ed, not on cell surface), Q
(expression level
Silent Outside Expression
Mutation Exon Modifier
01 : 01:02 N,L,S,Q
484 ■ AABB T EC HNIC AL MANUAL
questionable), A (unknown but aberrant ex-
pression, perhaps null), and C (cytoplasmic
expression only). The last two have not been
used to date.
Biologic Function
The essential function of the HLA system is
self/nonself discrimination, which is accom-
plished by the interaction of T lymphocytes
with peptide antigens presented by HLA
pro- teins. T lymphocytes interact with
peptide an- tigens only when the T-cell
receptor (TCR) for antigen engages both an
HLA molecule and the antigenic peptide
contained within the TCR’s peptide-binding
groove. This limitation is referred to as
“MHC restriction.”13
In the thymus, T lymphocytes with TCRs
that bind to a self HLA molecule are selected
(positive selection), with the exception of
those with TCRs that also bind to a peptide de-
rived from a self-antigen, in which case the T
lymphocytes are deleted (negative selection).
Some self-reactive T cells escape negative se-
lection. If not functionally inactivated (for in-
stance, by the mechanism of anergy), such
self-reactive T cells may become involved in
an autoimmune process.
Role of Class I Molecules
Class I molecules are synthesized, and
peptide antigens are inserted into the
peptide-binding groove, in the endoplasmic
reticulum. Peptide antigens that fit into the
Class I peptide-bind- ing groove are
typically eight or nine amino ac- ids in
length and are derived from proteins made
by the cell (endogenous proteins). Such
endogenous proteins—which may be normal
self-proteins; altered self-proteins, such as
those in cancer cells; or viral proteins, such
as those in virus-infected cells—are
degraded in the cytosol by a large
multifunctional protease (LMP) and are
transported to the endoplasmic reticulum by
a transporter associated with an- tigen
processing (TAP). The LMP and TAP genes
are both localized to the MHC.
Class I molecules are transported to the
cell surface, where the molecules are available
to interact with CD8-positive T lymphocytes.
If the TCR of a CD8 cell can bind the
antigenic
peptide in the context of the specific Class I
molecule displaying it, then TCR binding acti-
vates the cytotoxic properties of the T cell,
which attacks the cell, characteristically elicit-
ing an inflammatory response. The presenta-
tion of antigen by Class I molecules is
especial- ly important in host defense against
viral pathogens and malignant transformation.
Tu- mor cells that do not express Class I
antigens escape this immune surveillance.
Role of Class II Molecules
Like Class I molecules, Class II molecules
are synthesized in the endoplasmic
reticulum, but peptide antigens are not
inserted into the pep- tide-binding groove
there. Instead, an invari- ant chain (Ii) is
inserted as a place holder. The Class II-
invariant chain complex is transport- ed to
an endosome, where the invariant chain is
removed by a specialized Class II molecule
called “DM.” The DM locus is also localized
to the MHC. A Class II antigenic peptide is
then inserted into the peptide-binding
groove.
Peptide antigens that fit into the Class II
peptide-binding groove are typically 12 to
25 amino acids in length and are derived
from proteins that are taken up by the cell
through endocytosis (of exogenous
proteins). Exoge- nous proteins, which may
be normal self-pro- teins or proteins derived
from pathogens, such as bacteria, are
degraded to peptides by en- zymes in the
endosomal pathway. Class II mol- ecules are
then transported to the cell surface, where
the molecules are available to interact with
CD4-positive T lymphocytes, which se-
crete immunostimulatory cytokines in re-
sponse. That mechanism is especially
impor- tant for the production of antibodies.
IDENTIFICATION OF HLA
ANTIGENS AND ALLELES
Methods for the identification of HLA
antigens and alleles fall into two categories: 1)
molecu- lar (DNA based) and 2) serologic
(antibody based). Historically, cell-based
assays were also used.
Detailed procedures for commonly used
assays are provided in the ASHI Laboratory
 C H A P TE R 1 9 The HLA System ■ 485

Manual from the American Society for
Histo- compatibility and Immunogenetics.14
De- pending on the clinical situation, a
particular HLA antigen/allele detection or
typing meth- od may be preferable (see
Table 19-2).
DNA-Based Assays
DNA-based typing has several advantages
over serologic assays: 1) high sensitivity and
speci- ficity, 2) use of small sample volumes,
and 3) no need for cell-surface-antigen
expression or cell viability. Although serologic
methods can distinguish among a limited
number of HLA specificities, high-resolution
DNA-based methods have the capability to
identify all known alleles.
Polymerase Chain Reaction Testing
Polymerase chain reaction (PCR)
technology allows amplification of large
quantities of a particular target segment of
genomic DNA. Low- to intermediate-
resolution typing de- tects the HLA
serologic equivalents with great accuracy
(eg, it distinguishes DR15 from
DR16), whereas high-resolution typing distin-
guishes individual alleles (eg, DRB1*01:01:01
from DRB1*01:02:01). Several PCR-based
methods have been developed; three general
approaches are described below.
OLIGON UCLEOT IDE PROBE S . This method
for establishing HLA genotypes features ar-
rays of oligonucleotide probes that have
been individually attached to a solid-phase
matrix— for example, each probe is attached
to a differ- ent microbead or well of an
enzyme-linked immunosorbent assay
(ELISA) plate. DNA for an entire locus is
then amplified and the bind- ing to different
probes is evaluated. The micro- bead array
assay is a sequence-specific oligo-
nucleotide probe method for HLA Class I
and Class II low-to-high-resolution, DNA-
based, tissue typing. This method allows
high throughput with a reduction in sample
pro- cessing time.15
SEQUENCE-SPE CIFIC PRI M ERS. A second
major technique uses sequence-specific
prim- er (SSP) pairs that target and amplify
a particu-

TABLE 19-2. HLA Typing Methods and Appropriate Applications

Method Clinical Application Resolution

Microlymphocytotoxicity
SSP (PCR)
Forward SSOP Hybridization
Reverse SSOP Hybridization
DNA Sequencing
SSP = sequence-specific primer; PCR = polymerase chain reaction; HPC = hematopoietic progenitor cell; SSOP =
sequence- specific oligonucleotide probe.
Solid-organ transplantation, evaluation Serologic specificity
of platelet refractoriness, HLA
typing
(Class I only) of platelet recipients
and
platelet donors
Solid-organ, related and unrelated Serologic to allele level,
higher
HPC transplantation resolution with large number
of
primers
Solid-organ and HPC transplantation Serologic to allele level
(can accommodate high-volume
testing)
Solid-organ, related and unrelated Serologic, higher resolution
with
HPC transplantation larger number of probes
Unrelated HPC transplantation, resolu- Allele level
tion of typing problems with other
methods, characterization of new alleles
486 ■ AABB T EC HNIC AL MANUAL
lar DNA sequence.16 The sequence-specific
method requires the performance of multiple
PCR assays in which each reaction is
specific for a particular allele or group of
alleles. The amplified alleles are directly
visualized after agarose gel electrophoresis.
Because SSPs have such specific targets, the
amplified material indicates the presence of
the allele or alleles that have that sequence.
The pattern of positive and negative PCR
amplifications is examined to determine the
actual HLA allele(s) present. Primer pair
sets are commer- cially available that can
determine a complete HLA-A, -B, -C, -DR, -
DQA1, -DQB1, and -DPB1
allele-level phenotype.
S E QU EN CE- B A S ED T Y PI NG. High-
resolution nucleic acid sequencing of HLA
alleles gener- ates allele-level sequences.
Sequence-based typing (SBT) can be used to
characterize new allele(s). Although SBT is
considered the “gold standard” for HLA
typing, ambiguities often occur when two
different base pairs at the same position can
result in two different possi- ble
combinations of alleles. These ambiguities
occur because SBT evaluates both maternal
and paternal HLA genes (haplotypes)
simulta- neously, and which haplotype to
assign each base pair to is not always
certain. New tech- niques for high-
resolution SBT allow separa- tion of HLA
haplotypes before sequencing, thereby
avoiding such ambiguities.
Serologic (Lymphocytotoxicity) Assays
The microlymphocytotoxicity test can be used
to detect HLA-A, -B, -C, -DR, and -DQ anti-
gens. Lymphocytes are used for testing be-
cause they are readily obtained from anticoag-
ulated peripheral blood and are a more
reliable target than granulocytes. Lymphocytes
obtained from lymph nodes or spleen may
also be used. HLA-typing sera are obtained
primarily from multiparous women. Some
mouse antihuman HLA monoclonal antisera
are also available.
HLA sera of known specificities are
placed
in different wells of a microdroplet test plate.
A suspension of lymphocytes is added to each
well. Rabbit complement is then added; if suf-
ficient antibody has bound to the lymphocyte
membranes, the complement cascade is acti-
vated through the membrane attack complex,
leading to lymphocytotoxicity. Damage to the
cell membrane can be detected by the addi-
tion of dye. Cells that have no attached anti-
body, activated complement, or damage to the
membrane keep the vital dyes from penetrat-
ing; however, cells with damaged membranes
allow the dye to enter. The cells are examined
for dye exclusion or uptake under phase con-
trast microscopy. If a fluorescent microscope is
available, fluorescent vital dyes can also be
used.
Because HLA-Class II antigens (DR, DQ,
and DP) are expressed on B cells and not on
resting T cells, typing for these antigens usual-
ly requires manipulation of the initial lympho-
cyte preparation to yield an enriched B-cell
population before testing.
The interpretation of serologic reactions
requires skill and experience. The extreme
polymorphic nature of the HLA system; varia-
tion in antigen frequencies among different
racial groups; reliance on biologic antisera and
living target cells; and complexities introduced
by splits, CREGs, and “public” antigens all
con- tribute to difficulties in accurate serologic
HLA typing. Given these problems, most US
labora- tories now rely primarily on DNA-
based meth- ods for HLA typing. However,
although the more common “null alleles,” such
as DRB4*01: 03:01:02N and C*04:09N, can
now be identified by routine molecular typing
methods, rare null alleles may not be as easily
identified. It is im- portant to stay current on
the ever-changing array of expressed and
nonexpressed HLA polymorphisms. A very
useful resource is the International
Immunogenetics Project’s IMGT/ HLA
Database.2,9
Cellular Assays
Historically, the MLC [also called “mixed leu-
kocyte reaction” (MLR)] and primed lympho-
cyte typing (PLT) assays were used to detect
genetic differences in the Class II region. In
the MLC, mononuclear cells from different
indi- viduals are cultured together; the ability
of one population (the responder) to recognize
the
 C H A P TE R 1 9 The HLA System
HLA-D (combined DR, DQ, and DP)
antigens/ alleles of the other (the target) as
foreign can be detected by measuring the
proliferation of the responders. In PLT, reagent
cells previously stimulated by specific Class II
mismatched types allow the identification of
those types by their accelerated proliferative
responses to stimulator cells sharing the
original mis- matches.
These classical cellular assays have
been largely replaced by molecular typing
methods for HLA antigens. However, these
assays are still used in some laboratories to
monitor im- mune function, assess relative
functional compatibility, or select a partially
mismatched unrelated donor for HPC
transplantation.
CROSSMATCHING AND
DETECTION OF HLA
ANTIBODIES
Serologic Assays
The same microlymphocytotoxicity testing
used for serologic HLA typing can be used to
test serum specimens for antibodies against
selected target cells. This type of testing is
referred to as “lymphocyte crossmatching.”
Crossmatching consists of incubating serum
from a potential recipient with lymphocytes
(unfractioned or separated into T and B lym-
phocytes) from prospective donors. A varia-
tion of the microlymphocytotoxicity test uses
an antiglobulin reagent, which increases assay
sensitivity. Flow cytometry is yet another
crossmatch method with even greater sensitiv-
ity than the antiglobulin-enhanced cross-
match.
Testing the patient’s serum against a
pan- el of 30 to 60 (or more) different target
cells was historically used to assess the
extent of HLA alloimmunization. A positive
result was target cell death. The percentage
of panel cells that died after reacting with
the cytotoxic anti- bodies in patient serum
was referred to as the “panel-reactive
antibody” (PRA) level in that patient.
Determination of cytotoxic PRA was (and
may still be) useful for following patients
awaiting deceased-donor solid-organ trans-
plantation, conducting the work-up of pa-
■ 487
tients for platelet refractoriness, and investi-
gating FNHTR or TRALI cases. The PRA
“HLA antibody screen” not only detects the
presence of cytotoxic HLA antibodies but can
often also identify the specificity of those
antibodies.
Although they have long been the gold
standard for antibody identification, comple-
ment-dependent cytotoxic assays are not opti-
mal tests. Their sensitivity and specificity are
marginal, but more importantly, most cytotox-
ic assays cannot identify all of the possible
HLA antibody specificities in highly sensitized
patients whose cells are reactive with 80% or
more of test panel cells or in patients with
Class II antibodies.
Solid-Phase Assays
The current approach to identify HLA antibod-
ies (especially for highly sensitized solid-
organ transplant candidates) relies on the use
of beads or microparticles (ie, solid-phase
meth- odology) coated either with clusters of
HLA Class I or Class II antigens (ie, an HLA
pheno- type) or with recombinantly expressed,
indi- vidually purified HLA antigens (single
antigen beads).17 Antibody binding is detected
by staining with a fluorescently labeled antihu-
man globulin (AHG). Flow cytometry and
microarray methods are more sensitive than
lymphocytotoxicity or ELISA testing and
focus on the detection of IgG antibodies. The
use of single-antigen bead assays are of
particular importance for highly sensitized
patients where multiple HLA antibody
specificities cannot be reliably distinguished
and identified with either cell-based cytotoxic
assays or sol- id-phase assays using clusters of
HLA mole- cules.
The clinical significance of the low-level
antibodies that are detectable by the more
sensitive solid-phase assays and that may
not cause a positive cytotoxicity or flow-
cytomet- ric crossmatch is controversial.
Newer adapta- tions of the solid-phase
technology can deter- mine whether these
antibodies do or do not fix complement.
However, use of this method is also
controversial because the significance of
noncomplement-fixing antibodies is also un-
known.
488 ■ AABB T EC HNIC AL MANUAL
THE HLA SYSTEM AND
TRANSFUSION
HLA system antigens and antibodies play
im- portant roles in a number of transfusion-
relat- ed events, including platelet
refractoriness, FNHTRs, TRALI, and TA-
GVHD. HLA antigens are highly
immunogenic. In response to preg- nancy,
transfusion, or transplantation, immu-
nologically competent individuals are more
likely to form antibodies to HLA antigens
than to any other antigens.
Platelet Refractoriness
The incidence of HLA alloimmunization
and platelet refractoriness among patients
receiv- ing repeated transfusions of cellular
blood components ranges from 20% to
71%.18 The re- fractory state exists when a
transfusion of suit- ably preserved platelets
fails to increase the re- cipient’s platelet
count. Platelet refractoriness may be caused
by clinical factors, such as sepsis, high fever,
disseminated intravascular coagulopathy,
medications, hypersplenism, complement-
mediated destruction, or a com- bination of
these factors; alternatively, it may have an
immune basis.
Antibody Development
Antibodies against HLA Class I antigens are
a common cause of immune-mediated
platelet refractoriness, but antibodies to
platelet-spe- cific or ABH antigens may also
be involved. HLA alloimmunization can
follow pregnancy, transfusion, or organ
transplantation because the foreign antigens
are the donor MHC anti- gens themselves. A
common example is the development of
HLA antibodies directed against Class I and
Class II antigens that oc- curs with platelet
transfusion, even though platelets express
only Class I antigens. It is likely that the
presence of donor leukocytes (bearing Class
I and II antigens) elicits alloim- munization.
The likelihood of HLA immuniza- tion can be
lessened (but not eliminated) by transfusion
of leukocyte-reduced blood com- ponents.
The threshold level of leukocytes required
to provoke a primary HLA alloimmune re-
sponse is unclear and probably varies among
different recipients. Some studies have sug-
gested that 5 × 106 leukocytes per
transfusion may represent an immunizing
dose. In pa- tients who have been sensitized
by prior trans- plantation, pregnancy, or
transfusion, expo- sure to even lower
numbers of allogeneic cells is likely to
provoke an anamnestic antibody re- sponse.
Identifying Compatible Donors
The HLA antibody response of transfused
indi- viduals may be directed against
individual specificities or public alloantigens.
Precise characterization may be difficult and is
best accomplished using a single-antigen,
solid- phase assay as described in the “Solid-
Phase Assays” section above. Platelet-
refractory pa- tients with broadly reactive
antibodies may be difficult to support with
platelet transfusions. HLA-matched platelets,
obtained by platelet- pheresis, benefit some,
but not all, of those re- fractory patients.
Donors who are phenotypi- cally matched with
the immunized recipient for four Class I
antigens (two alleles each at the HLA-A and
HLA-B loci) are difficult to find, and strategies
to obtain HLA-compatible platelets vary.
Although selection of partially mismatched
donors (based on serologic CREGs) has been
emphasized, such donations may fail to
provide an adequate transfusion re- sponse in
vivo.
An alternative approach to the selection
of donors is based on matching for immuno-
genic epitopes as described by the “HLA
Matchmaker” program developed at the Uni-
versity of Pittsburgh. HLA Matchmaker is
used to consider epitopes on all the patient’s
HLA Class I (and if indicated, Class II)
molecules re- gardless of which allele encodes
them. HLA Matchmaker is an excellent tool to
predict compatibility between a specific
donor-recipi- ent pair, but, unfortunately, the
tool does not generate accurate predictions
100% of the time. Use of single-antigen beads
or microar- rays to identify HLA antibody
specificities can also help improve the
selection of donors with acceptable
mismatched antigens.19
 C H A P TE R 1 9 The HLA System
Approaches such as HLA Matchmaker
can be referred to as “pull” approaches,
where- in donors are selected (or pulled
into) the pro- cess. In contrast, antibody
specificity identifi- cation strategies can be
considered “push” approaches that exclude
(push away) inappro- priate donors. Either
approach has a similar net effect:
identification of donors who have the
greatest likelihood of optimizing post-
transfusion platelet counts.
As an alternative approach, HLA-
alloim- munized patients often respond to
cross- match-compatible platelets selected
by using patient serum and samples of
apheresis plate- lets in a platelet antibody
assay. Crossmatch- ing techniques may
assess compatibility for both HLA and
platelet-specific antibodies.20
Histocompatible platelet components are
dis- cussed further in Chapter 18.
Febrile Nonhemolytic Transfusion
Reactions
HLA, granulocyte, and platelet-specific anti-
bodies have been implicated in the pathogen-
esis of FNHTRs. The recipient’s antibodies, re-
acting with transfused antigens, elicit the
release of cytokines (eg, interleukin-1) that are
capable of causing fever. Serologic investiga-
tion, if undertaken, may require multiple tech-
niques and target cells from a number of dif-
ferent donors (see Chapter 27).
TRALI
In TRALI (a potentially fatal transfusion
reac- tion that is recognized with increasing
fre- quency), acute noncardiogenic
pulmonary edema develops in response to
transfusion (see Chapter 27). The
pathogenesis of TRALI appears to reflect
the presence of HLA or neu- trophil
antibodies in donor blood. Studies have
shown that up to one-third of blood com-
ponents can contain detectable amounts of
HLA antibodies.21 If present, such antibodies
can be reactive with and fix complement to
the recipient’s granulocytes, leading to
severe cap- illary leakage and pulmonary
edema. Rarely, the recipient’s HLA
antibodies are reactive with transfused
leukocytes from the donor.
■ 489
Cases of TRALI have been reported
that appear to be caused by donor antibodies
against Class I or Class II antigens in recipi-
ents. Because Class II antigens are not ex-
pressed on resting neutrophils, an alternate
explanation for activation of neutrophils is
re- quired in these instances. One hypothesis
is that Class II antigens on the recipient’s
pulmo- nary macrophages are targeted by
the comple- ment-activating antibodies. The
subsequent release of cytokines and
chemokines results in the recruitment and
activation of neutrophils in the lungs. 22 Yet
another explanation is that activated
neutrophils, which can express HLA Class
II antigens in response to a patient’s in-
flammatory state, could be further activated
with the binding of donor anti HLA Class II
an- tibodies, resulting in TRALI.
Chimerism and TA-GVHD
“Chimerism” refers to the presence of two
cell populations, such as transfused or
transplant- ed donor cells in a recipient, in
an individual. Persistent chimerism after
blood transfusion may lead to the
development of TA-GVHD in the recipient.
The development of TA-GVHD depends on
the following factors: 1) the degree to which
the recipient is immunocompro- mised, 2)
the number and viability of lympho- cytes in
the transfused component, and 3) the degree
of HLA similarity between the donor and
recipient. The development of TA-GVHD
with the use of fresh blood components
from blood relatives has highlighted the
pathogenic role of the HLA system.
Figure 19-5 illustrates the conditions
for increased risk of TA-GVHD. The parents
have one HLA haplotype in common. Each
child, therefore, has one chance in four of
inheriting the same haplotype from each
parent, and child 1 is homozygous for the
shared parental HLA haplotype. Transfusion
of blood from child 1 to an unrelated
recipient with different haplotypes would
have no untoward conse- quences. If,
however, child 1 were a directed donor for a
relative who was heterozygous for that
haplotype (eg, one of the parents or child 3),
the recipient’s body would fail to recognize
the antigens on the transfused lymphocytes
as
490 ■ AABB T EC HNIC AL MANUAL

FIGURE 19-5. HLA haplotypes in a family at risk for transfusion-associated graft-vs-host disease (GVHD).
In contrast to the family shown in Fig 19-2, each parent shares a common HLA haplotype, HLA-A1,B8,DR17.
Child 1 is homozygous for the haplotype shared by the parents and by child 3. The lymphocytes of child
1 are capable of producing posttransfusion GVHD if they are transfused to either parent or to child
3.

foreign and would not eliminate them. The
donor cells would recognize the recipient’s
other haplotype as foreign and would
become activated, proliferate, and attack the
host.
To avoid this situation, it is
recommended
that all cellular components from blood rela-
tives be irradiated before transfusion. Other
specially chosen donor units, including
HLA- matched platelets, may also present
an in- creased risk of TA-GVHD. Rarely, TA-
GVHD has occurred after the transfusion of
blood from an unrelated donor, usually
within popu- lations with relatively limited
genetic diversi- ty, such as in Japan.
Chimerism is proposed to be
responsible
for the maintenance of tolerance in some or-
gan transplant recipients as well as for the
maintenance of HLA sensitization.23,24 It has
been postulated that scleroderma is a form
of GVHD resulting from chimeric cells
derived from fetal cells transferred across
the placenta during pregnancy.25
Furthermore, the persis- tence of donor
lymphocytes originally present in and
transplanted with a solid-organ al- lograft
has been documented to cause fatal GVHD
in recipients of these organs.26
Hemolytic Transfusion Reactions
HLA incompatibility has rarely been implicat-
ed in shortened red cell survival in patients
with antibodies to HLA antigens, such as Bg a
(B7), Bgb (B17-B57 or B58), and Bgc (A28-A68
or
A69). These antigens are expressed, although
weakly, on red cells. Such incompatibility may
not be detected by conventional pretransfu-
sion testing.
 C H A P TE R 1 9 The HLA System
HLA TESTING AND
TRANSPLANTATION
HLA testing is an integral part of solid-organ
and HPC transplantation. The extent of
testing differs depending on the type of
transplanta- tion (see Chapters 29 and 30).
Hematopoietic Progenitor Cell
Transplants
It has long been recognized that disparity
within the HLA system is an important
barrier to successful HPC transplantation.27
HLA simi- larity and compatibility between
the donor and the recipient are required for
engraftment and to help prevent GVHD.
However, some de- gree of rejection or
GVHD is a common prob- lem for
recipients of allogeneic HPCs, despite
immunosuppressive conditioning.
Candidate donors and recipients are
typed for their HLA-A, -B, -C, -DR, and -DQ
al- leles and, in some cases, for their HLA-DP
al- leles. The goal of HLA typing is to match
the al- leles of the prospective donor and
recipient at the HLA-A, -B, -C, and -DRB1
loci.28 Some transplant programs also attempt
to match donors and recipients for HLA-DQ
alleles, HLA-DP alleles, or both.
Although HLA-identical sibling donors
remain the best choice for HPC transplanta-
tion, there is increasing use of unrelated do-
nors identified by searching the files of more
than 10 million HPC donors listed in the
National Marrow Donor Program’s registry
of volunteer donors or other international
regis- tries. The use of umbilical cord HPCs
and HPC grafts from mismatched donors
that have undergone T-cell depletion may
allow an increased number of donor-
recipient mis- matches.29,30
Kidney Transplants
ABO compatibility is the most important fac-
tor in determining the immediate outcomes of
kidney transplants. Because ABH antigens are
expressed in varying amounts on all of the
body’s cells, transplanted ABO-incompatible
tissue comes into continuous contact with the
recipient’s ABO antibodies. Of particular im-
■ 491
portance is the expression of ABH antigens
on vascular endothelial cells because the
vascular supply in the transplant is a
common site for rejection. Currently, the use
of non-A1 A blood
group organs for group B and group O recipi-
ents with low anti-A blood group titers has
be- come acceptable.31,32 In fact, several
transplan- tation programs have used ABO-
incompatible donors with higher anti-A
titers following pro- tocols that include
various combinations of rituximab,
splenectomy, plasmapheresis with
intravenous Ig, and other treatments to re-
move preexisting antibodies and promote
ac- commodation of the transplanted organ.
Both recipients and donors are routinely
typed for ABO and HLA-A, -B, and -DR anti-
gens. HLA-C and -DQ typing is usually also
performed. Before surgery, a crossmatch be-
tween recipient serum and donor lymphocytes
is required. The ASHI Standards for
Accredited Laboratories requires that the
crossmatch be performed using a method that
is more sensi- tive than routine
microlymphocytotoxicity testing, such as
prolonged incubation, wash- ing,
augmentation with AHG reagents, or flow
cytometry.33 Flow cytometry is the most sensi-
tive method and has been credited with pre-
dicting early acute rejection and delayed graft
function, both of which are strong predictors
of chronic rejection (if results are positive) and
long-term allograft survival (if results are
nega- tive).34
In a study of patients undergoing de-
ceased-donor kidney retransplantation, the 7-
year graft survival rate using a negative T-cell
flow crossmatch to select the donor kidney
was comparable to that of patients undergoing
primary deceased-donor transplantation (68%
vs 72%) and was significantly better than that
of regraft patients for whom only the antiglob-
ulin lymphocytotoxicity crossmatch was used
(45%).35
Because HLA antibody responses are
dy- namic, the serum used for the
crossmatch is often obtained within 48 hours
of surgery for sensitized potential recipients
and is retained in the frozen state for any
required subsequent testing. An
incompatible crossmatch with un-
fractionated or T lymphocytes is typically a
contraindication to kidney transplantation. A
492 ■ AABB T EC HNIC AL MANUAL
positive B-cell crossmatch is significant when
caused by donor-specific HLA Class I or Class
II antibodies.
Serum from a patient awaiting
deceased- donor kidney transplant surgery is
tested at regular intervals for the degree of
alloimmuni- zation by determining the
percent PRA as well as the specificities of
the detected antibodies. If an antibody with a
defined HLA specificity is identified in a
recipient, a common practice is to avoid
donors who express the correspond- ing
HLA antigen(s). Such antigens are deemed
“unacceptable.” A more recent approach is
the use of solid-phase assays to identify
HLA anti- bodies and unacceptable antigens
and then calculate the patient’s PRA (cPRA)
using a da- tabase of more than 12,000
HLA-typed do- nors.36 Frozen serum
samples used for period- ic PRA testing are
often stored so that “historic” samples with
the highest PRA can be used in addition to
the preoperative sample for
pretransplantation crossmatching.
Prospective crossmatching is often not
performed for recipients who are
conclusively devoid of HLA antibodies (ie,
cPRA = 0%). Prompt transplantation with
reduced cold- ischemia time for the renal
allograft may pro- vide greater benefit to the
patient than pro- spective crossmatching,
provided that 1) a very sensitive method for
antibody detection, such as flow cytometry
or microarrays, has been used, and 2) it is
certain that the patient has had no additional
sensitizing event (ie, im- munizations or
transfusions in the 2 weeks be- fore or at
any time after that serum was screened). 37
The approach to kidney transplants with
living donors is different. In the past, when
several prospective living donors were being
considered, MLC testing of recipients and do-
nors was sometimes performed, but this is
rarely (if ever) the case today. HLA matching
of recipients with kidney donors (both living
and deceased) contributes to long-term
allograft survival by decreasing the likelihood
of chron- ic rejection. According to the
Scientific Regis- try for Transplant Recipients,
recent 1-year graft survival rates from living
and deceased renal donors were 96.5% and
91.7%, respec- tively, and the half-lives of
living-donor and
deceased-donor renal allografts were 21.6
years and 13.8 years, respectively.38
The significantly better graft survival rate
for recipients of living- vs deceased-donor re-
nal allografts, even when donors and recipi-
ents are completely unrelated, coupled with
inadequate numbers of deceased-organ do-
nors has led to a relatively new practice, kid-
ney paired donation (KPD).39 Recently, KPDs
have been facilitated through local and na-
tional registries, which permit patients with
ABO- or HLA-incompatible potential living
donors to exchange these donors for the do-
nors of other patients in the same situation. As
a simple example, a blood group A transplant
candidate with an incompatible blood group B
potential living kidney donor could exchange
that donor for the incompatible blood group A
living kidney donor of a blood group B trans-
plant candidate. Patients with HLA-incompat-
ible potential donors have similar possibilities
for donor exchange, and multiple “pairs” can
be involved in one continuous exchange
(chain) process.
The introduction of altruistic donors (ie,
individuals who choose to donate a kidney
without having a specific intended recipient)
can significantly expand KPD options.
Briefly, the altruistic donor donates a kidney
to a pa- tient with an incompatible potential
living do- nor who then donates to a
different recipient with an incompatible
donor, starting a “chain” with the possibility
of a large number of living- donor
transplants. One chain of 10 trans- plants
was recently reported.40 Currently, there is a
movement to establish a national KPD pro-
gram.
Other Solid-Organ Transplants
For liver, heart, lung, and heart/lung trans-
plants, ABO compatibility remains the prima-
ry immunologic concern for donor selection,
and determining pretransplant ABO compati-
bility between the donor and recipient is re-
quired. However, it has been shown that young
pediatric heart or liver transplant recipients,
who have low levels of ABO isoagglutinins,
have successful outcomes with ABO-incom-
patible hearts or livers.41,42 Although it is not a
 C H A P TE R 1 9 The HLA System ■ 493

requirement, HLA typing of potential recipi- TABLE 19-3. HLA-Associated Diseases

ents of the above organs is recommended.
Furthermore, a crossmatch should be avail- Disease HLA RR43-46
able before transplantation when the Celiac disease DQ2 >250
recipient has demonstrated presensitization,
except for emergency situations. Although a Ankylosing spondylitis B27 >150
degree of HLA compatibility correlates with Narcolepsy DQ6 >38
graft surviv- al after heart, lung, small-
Subacute thyroiditis B35 14
intestine, and liver transplantations,
prospective HLA matching is generally not Type 1 diabetes DQ8 14
performed for these proce- dures.43 Pancreas Multiple sclerosis DR15, DQ6 12
transplantation generally follows the same
Rheumatoid arthritis DR4 9
guidelines as kidney trans- plantation.
Juvenile rheumatoid DR8 8
Relationship and Other Forensic arthritis
Testing Grave disease DR17 4
HLA typing (particularly DNA-based HLA RR = Relative Risk
typ- ing) has been useful in forensic testing.
Al- though rarely used now for relationship
test- ing, DNA-based HLA typing alone can susceptibilities have several features in com-
exclude more than 90% of falsely accused mon. The susceptibilities are known or sus-
males. Hap- lotype frequencies, rather than pected to be inherited, display a clinical
gene frequen- cies, are used in such course with acute exacerbations and remis-
calculations because linkage disequilibrium is sions, and usually have characteristics of
very common in the HLA system. It is auto- immune disorders. Furthermore, their
important, however, to con- sider the racial exact cause is often unknown.
differences that exist in HLA haplotype Evidence has been accumulating that im-
frequencies in the calculations used; the plicates the HLA molecules themselves, rather
possibility of recombination events should than linked genes, in most cases of disease
also be considered. susceptibility. One mechanism that could lead
More commonly used DNA-based assays to the association of HLA phenotype and dis-
for relationship testing and forensic detection ease is the presence of a Class I or Class II het-
of polymorphisms between individuals are erodimer encoded by a specific allele that pref-
employed to measure variations in the num- erentially presents autoantigens to the TCR.
ber of short tandem repeats, which are used to The ancestral haplotype HLA-A1, B8, DR17
assess other polymorphic, non-HLA genetic (DRB1*03:01), DQ2, discussed in the
regions, and single nucleotide polymorphisms “Linkage Disequilibrium” section above, is
(SNPs). DNA-based assays, including HLA associated with susceptibility to Type I
typ- ing, allow identification of individuals on diabetes, systemic lupus erythematosus, celiac
the basis of extremely small samples of fluid disease, common variable immunodeficiency,
or tis- sue, such as hair, epithelial cells, or IgA deficiency, and myasthenia gravis. 47 This
semen. haplotype is also associated with an
accelerated course of HIV infection that is
OTHER CLINICALLY likely caused by the presence of multiple
SIGNIFICANT ASPECTS OF HLA genes.
However, HLA typing has only limited
For some conditions, especially those believed val- ue in assessing the risk of most diseases
to have an autoimmune etiology, an associa- be- cause the association is incomplete. The
tion exists between HLA phenotype and the asso- ciation of HLA-B27 and ankylosing
occurrence of, or resistance to, clinical disease spondylitis
(see Table 19-3).43-46 HLA-associated disease
494 ■ AABB T EC HNIC AL MANUAL
in patients of European ancestry is instructive.
The test is highly sensitive; more than 90% of
patients with ankylosing spondylitis possess
the HLA-B27 antigen. Conversely, the test’s
specificity is low; only 20% of individuals
with the B27 antigen develop ankylosing
spondyli- tis. A second condition, narcolepsy,
is strongly associated with the HLA allele
DQB1*06:02.48 As with HLA-B27 and
ankylosing spondylitis, more than 90% of
individuals with narcolepsy are positive for
HLA-DQB1*06:02, but only a minority of
individuals with that HLA allele develop the
disease. For some autoimmune diseases, the
specific peptide that might trig- ger the
autoimmune response has been at least
tentatively identified: a gluten peptide, gliadin,
for celiac disease; cyclic citrullinated peptides
for rheumatoid arthritis; and a pep- tide from
glutamic acid decarboxylase for type I
diabetes.49-51 Resistance to cerebral malaria
seems to result from a strong cytotoxic T-cell
response to particular malarial peptides that
are restricted by (ie, fit into the peptide-bind-
ing grooves of ) two specific HLA
molecules.52
A similar peptide-binding specificity is
important to consider in the development of
vaccines. For example, a vaccine to enhance
immune responses to melanoma using a
mel- anoma-specific peptide that binds only
to the cells of individuals with the HLA type
HLA- A*0201 was selected for development
because A*02:01 is the most common allele
in virtually all populations.53
HLA typing is also important in pharma-
cogenomic applications. For example, the
presence of certain HLA alleles confers in-
creased risk for several hypersensitivity reac-
KEY POINTS
Genes encoded by the major histocompatibility complex (or HLA complex in humans) are critical components of the immune
HLA genes are located within multiple loci on chromosome 6. Each locus is extremely poly- morphic.
HLA genes encode multiple Class I (eg, HLA-A, -B, and -C) and Class II (eg, HLA-DR, -DQ, and -DP) cell-surface proteins.
Class I proteins are expressed ubiquitously, but Class II proteins have restricted tissue distri- bution.
tions to certain drugs: HLA-B*57:01 confers
sensitivity to abacavir, HLA-B*15:02 to carba-
mazepine, and HLA-B*58:01 to
allopurinol.54.55 The degree of association
between a given HLA type and a disease is
often described in terms of relative risk
(RR), which is a measure of how much more
frequently a disease occurs in individuals
with a specific HLA type than in individuals
not having that HLA type. Calcula- tion of
RR is usually based on the cross-prod- uct
ratio of a 2 × 2 contingency table. However,
because the HLA system is so polymorphic,
there is an increased possibility of finding
an association between an HLA antigen and
a dis- ease by chance alone. Therefore,
calculating RRs for HLA disease associations
is more com- plex and is typically
accomplished by use of Haldane’s
modification of Woolf’s formula.56,57 The RR
values for some diseases associated
with HLA types are shown in Table 19-3.
SUMMARY
In conclusion, the HLA system is a complex
and highly polymorphic set of genes that are
collectively involved in all aspects of the
im- mune response. The recent development
of molecular tools to explore this genetic
oasis is providing additional information,
such as the elucidation of unrecognized
polymorphisms within the HLA complex
(ie, SNPs). In the fu- ture, the translation of
this basic information will undoubtedly lead
to new clinical applica- tions in
transplantation, autoimmune diseas- es,
vaccine development, pharmacogenetics,
and infectious diseases.
 C H A P TE R 1 9 The HLA System ■ 495

5. Everyone inherits a set of HLA genes from his or her mother and father, referred to as a
“ma- ternal haplotype” and “paternal haplotype,” respectively.
6. Together, the maternal and paternal haplotypes are referred to as a “genotype.” The cell-sur-
face expression of proteins encoded by these HLA genes is referred to as a “phenotype.”
7. Class I and Class II HLA proteins are strongly immunogenic and can induce an
immune re- sponse, eg, formation of HLA antibodies.
8. Donor-directed HLA antibodies are associated with graft dysfunction and/or loss.
9. Solid-phase assays (eg, flow cytometry and Luminex) have become the gold standard for
de- tecting and identifying HLA antibodies.
10. Identification of donor-directed HLA antibodies can be used to perform a virtual (in-silico)
crossmatch.

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 C h a p t e r 2 0

Hemotherapy Decisions
and Their Outcomes

Theresa Nester, MD; Shweta Jain, MD; and Jessica Poisson, MD

AS WITH OTHER medical interven-

tions, transfusion offers both benefits
and risks that must be balanced for each pa-
tient. Although the myriad situations in which
transfusion might be considered prevent the
promulgation of “one-size-fits-all” transfu-
sion guidelines, the accumulating data in the
medical literature can certainly help clinicians
make hemotherapy decisions based on in-
creasingly sound evidence.
This chapter provides a review of the
cur- rent literature on the indications for, and
out- comes of, transfusion of blood
components. When health-care providers
use this chapter in combination with
information presented else- where in the
Technical Manual on the content of blood
components and the risks they pose, they
should have sufficient data to form evi-
dence-based decisions about hemotherapy.
RED CELL TRANSFUSION
At first glance, the decision to transfuse a
Red Blood Cell (RBC) unit appears to be
easy; for
example, a patient with acute anemia
requires an RBC transfusion to restore the
body to its baseline state. For the trauma
patient with massive injuries, death will
ensue without a transfusion. For the
critically ill patient with anemia stemming
from a variety of causes, it would seem
reasonable to maintain adequate oxygen
stores in the tissues through transfu- sion.
Yet the decision to transfuse a stored RBC
unit has become more complex in the last de-
cade, with substantial evidence suggesting
that RBC transfusion may be detrimental to
some patients. For example, a sentinel 1999
study randomly assigned critically ill patients
(who are among the most frequent recipients
of RBC products) to a restrictive or liberal
transfusion strategy (thresholds of hemoglo-
bin = 7 vs 10 g/dL). 1 The lower threshold re-
duced morbidity and mortality significantly
and was not associated with increased length
of stay or morbidity in patients with heart dis-
ease or with prolonged ventilation for those re-
quiring a respirator. The authors concluded

Theresa Nester, MD, Associate Medical Director, Puget Sound Blood Center, and Associate Professor of
Labo- ratory Medicine, University of Washington, Seattle, Washington; Shweta Jain, MD, Transfusion
Medicine Fel- low, Puget Sound Blood Center, Seattle, Washington; and Jessica Poisson, MD, Director of
Transfusion Services, USC Medical Center, Los Angeles, California
The authors have disclosed no conflicts of interest.

499
500 ■ AABB T EC HNIC AL MANUAL
that a hemoglobin threshold of 7 g/dL was
ap- propriate for all critically ill patients,
except perhaps those with unstable cardiac
condi- tions. Similarly, a 2008 systematic
review of the critical care literature
determined that in most cohort studies in
272,586 patients, RBC trans- fusions were
an independent predictor of death, infection,
multiorgan dysfunction, and acute
respiratory distress syndrome.2
Most randomized, controlled trials
(RCTs) conducted to date have demonstrated
that a restrictive RBC transfusion strategy is
not infe- rior to a liberal transfusion strategy.
A recent RCT that evaluated transfusion
strategies in patients with severe upper
gastrointestinal bleeding showed a superior
outcome with a restrictive strategy for
patients with cirrhosis or Child-Pugh Class A
or B disease.3 Because of such evidence,
many studies now compare the risk of
anemia to that of transfusion and the degree
to which each risk affects clinical out-
comes.
The appropriate RBC transfusion
thresh- old has become a central point of
discussion. The hemoglobin level in any
individual patient only tells a portion of the
story and, by itself, does not reflect the
compensatory mecha- nisms used to respond
to the anemia. Stable tissue oxygenation
may be maintained by increased cardiac
output (depending on the coronary arteries’
ability to dilate) and in- creased oxygen
extraction, which result in low- er venous
oxygen content.4 Clinicians are ap-
propriately looking for ways to monitor a
patient’s response to anemia before deciding
to administer a transfusion. At the same
time, hospital-utilization and blood-
management committees look for guidance
on the levels of hemoglobin, below which to
consider transfu- sion in a given patient
population.
Parallel to the RCTs performed thus far is
the observation that patients without evidence
of cardiovascular disease who refuse transfu-
sion on religious grounds and undergo elective
surgery tolerate hemoglobin reductions of 6 to
7 g/dL with only a minimal rise in periopera-
tive mortality risk.5
Although many otherwise-healthy
adults can tolerate significant anemia
equivalent to a halving of their red cell
mass, comorbidities in
many patients may limit their reserve. So what
should the indication be for RBC transfusion
in the setting of acute anemia? A recent meta-
analysis of 19 RCTs evaluated the effects of
dif- ferent RBC transfusion thresholds on
clinical outcomes between 1956 to 2011, a
period during which the definitions of liberal
vs re- strictive RBC transfusion strategies
varied.6,7 Of the 6264 patients in these studies,
most were in surgical or intensive care units.
The lower transfusion threshold (hemoglobin
= 7.0-10.0 g/dL, and most commonly 7.0-8.0
g/dL) was not inferior to the higher
hemoglobin level (9.0-13.3 g/dL, and most
commonly 9.5-10.0 g/dL). In the lower
threshold groups, fewer RBC units were
transfused without an adverse impact on
mortality, morbidity, or time to functional
recovery. There were also no signifi- cant
differences in the incidence of major
complications, such as stroke, pulmonary ede-
ma, or infection. Caution must be used in ex-
tending these findings to all patient popula-
tions because some high-risk groups (eg,
patients with acute brain injury or renal fail-
ure) were not adequately represented.
Although the findings of this meta-analy-
sis suggest that a restrictive transfusion strate-
gy is appropriate for many hospitalized, stable
patients, many clinicians still pause at the
thought of withholding transfusion from a pa-
tient with coronary artery disease. In the past,
large observational studies gave conflicting re-
sults regarding whether a higher or lower red
cell transfusion threshold was beneficial.8-10
More recently, an RCT known as “FOCUS” in-
cluded 2016 patients (average age = 82 years)
with risk factors for, or a history of, cardiovas-
cular disease who required hip surgery. 11 The
results showed that the restrictive strategy (he-
moglobin <8 g/dL) was not inferior to a liberal
transfusion strategy (hemoglobin <10 g/dL)
with respect to mortality, morbidity, or func-
tion. The primary outcome of death or inabili-
ty to walk independently across the room at 60
days was similar in the two groups (34.7% in
the restrictive vs 35.2% in the liberal groups).
Rates of in-hospital cardiac events or death as
well as other complications were also similar.
The 1999 multicenter Transfusion Re-
quirements in Critical Care (TRICC) trial in 838
 CH A P T E R 20
critically ill patients, including 326 with ath-
erosclerotic disease but not acute, unstable
myocardial conditions showed that mortality
at 30 days was no different in the subgroup
with a primary or secondary diagnosis of car-
diac disease between the two strategies
(hemoglobin <7 g/dL vs <10 g/dL).1 Cardiac
events (pulmonary edema and myocardial in-
farction) were more frequent in the liberal
strategy group, although there were no signifi-
cant differences in rates of these events during
the 48 hours prior to death in the patients who
died. The subgroup analysis showed a trend
toward increased mortality in patients with
ischemic heart disease in the restrictive arm,
leading the investigators to suggest that pa-
tients with active coronary ischemic syn-
dromes may need a higher transfusion thresh-
old than 7 g/dL.
The FOCUS and TRICC trials account for
60% of the available data on the risk of myo-
cardial infarction following restrictive vs liber-
al RBC transfusion strategies in patients who
have coronary artery disease. An analysis of
combined data from these trials and six small-
er trials did not find an elevated risk [risk ratio
(RR) = 0.88; 95% confidence interval (CI) =
0.38, 2.04] for myocardial infarction using a
re- strictive strategy.12 The statistical power of
the combined data was sufficient to detect
moder- ate to large differences in risk of
myocardial in- farction, but the analysis could
have missed a twofold higher risk of
myocardial infarction with a restrictive
transfusion strategy. In light of the available
data, the Clinical Transfusion
Medicine Committee of the AABB published
guidelines suggesting that transfusion
should be considered for hospitalized
patients with preexisting cardiovascular
disease who have
symptoms or a hemoglobin level of 8 g/dL or
less.12
For patients with acute coronary syn-
drome, no clinical trials have compared re-
strictive and liberal transfusion strategies.
For this reason, the development of a
guideline based on hemoglobin levels is
difficult for pa- tients with this diagnosis.
The most appropriate hemoglobin
threshold for transfusion is a patient-
specific,
and even situation-specific, parameter. A he-
Hemotherapy Decisions ■ 501
moglobin level <6 g/dL almost always
requires a transfusion, whereas a level >10
g/dL rarely does so. Of course, most patients
have a hemo- globin level between these
limits, and many have one or more
comorbidities that affect their tolerance of
anemia. The assemblage of experience and
knowledge must be blended into the art of
medicine to address this need. In addition to
symptoms, research continues to identify
consistent signs, such as venous ox- ygen
saturation, that reflect how an individual
patient is tolerating anemia.13
Transfusion in Patients with
Hemorrhagic Shock
A brief history of the changes in transfusion
support over the last 40 years for patients ex-
periencing hemorrhagic shock can be re-
viewed elsewhere.14 Currently, in the civilian
setting, such patients represent 2% to 3% of all
trauma admissions. Signs of acute blood loss
in such patients include tachycardia, hypoten-
sion, increased respiratory rate, and mental
status changes (Table 20-1). When these
symptoms are not addressed quickly, the mor-
tality rate in patients experiencing hemorrhag-
ic shock ranges from 40% to 70%.
Before 1995, aggressive resuscitation
us- ing crystalloid and RBCs was the
standard of
TABLE 20-1. Signs and Symptoms of
Acute Blood Loss
Tachycardia
Palpitations
Cooling of extremities
Pallor
Hypotension
Reduced arterial pressure
Reduced central venous (jugular) pressure
Acidosis
Increased respiratory rate
Decline in urinary output
Mental status changes
502 ■ AABB T EC HNIC AL MANUAL
care in civilian hospitals. In the late 1990s,
trauma surgeons started to recognize the po-
tential deleterious effects of too much
crystal- loid, including increased risk of
acute respira- tory distress syndrome,
multiple-organ failure, and abdominal
compartment syndrome. Gradually, and after
new data became avail- able from the
military experience supporting combat
casualties in the Iraq and Afghanistan wars,
a new approach to hemorrhagic shock,
damage control resuscitation, was adopted.
This approach included the early transfusion
of plasma and platelets in addition to RBCs
while minimizing crystalloid use. More em-
phasis was also placed on addressing the
lethal triad of acidosis, hypothermia, and
coagulopa- thy early in patient resuscitation.
Since publication in 2007 of a seminal
study involving 252 military personnel with
combat casualties and using almost a 1:1 ratio
of plasma to RBCs, many publications from
ci- vilian hospitals have echoed the idea that
us- ing an increased ratio of plasma or
platelets to RBCs can improve outcomes. 15 In
2012, the Prospective, Observational, Multi-
Center Mas- sive Transfusion Study evaluated
905 patients in 10 Level I US civilian trauma
centers who survived for 30 minutes after
admission and received at least 1 RBC unit
within the first 6 hours and at least three other
blood products within 24 hours of admission. 16
An increased ratio of plasma to RBCs or
platelets to RBCs was independently
associated with decreased 6-hour mortality.
Furthermore, patients with plasma to red cell
ratios less than 1:2 during the first 6 hours
were three to four times more likely to die
than patients with ratios of 1:1 or higher.
The Army Surgeon General has estab-
lished a clinical policy requiring
transfusions of Fresh Frozen Plasma (FFP),
platelets, and RBCs in a 1:1:1 ratio for
patients injured in combat who require a
massive transfusion of 6 whole blood
platelet units or 1 apheresis plate- let unit
following transfusion of 6 RBC units. This
emphasis on addressing coagulopathy early
in a massive resuscitation pushes labora-
torians to develop faster ways of evaluating
co- agulation results to provide optimal
transfu- sion support.17 The benefit of
cryoprecipitate
infusion for low fibrinogen levels, although
not widely emphasized in these protocols,
has also been established.18
Transfusion in Patients with
Chronic Anemia
Transfusion is much less commonly indicated
when anemia has persisted for weeks or
months because compensatory mechanisms
have had time to work than in patients with
anemia of more recent onset. These longer-
term anemias are usually best treated by ad-
dressing their etiologies, such as providing
supplementation to treat a nutritional defi-
ciency (eg, of iron) or reducing the rate of au-
toimmune hemolysis. Congenital hemoglo-
binopathies, such as sickle cell disease, are
treated according to disease-related protocols
for purposes that are not necessarily related to
oxygen delivery. Hypoproliferative anemias
secondary to chemotherapy or end-stage renal
disease are often treated with marrow stimu-
lants, such as recombinant erythropoietin.
Any of these diseases could conceivably re-
quire a transfusion if patient symptomatology
requires more rapid reversal than would be
possible by treating the underlying mecha-
nisms, but transfusion is usually considered
only as a last resort in these patients.
Some patients become transfusion de-
pendent because of their inability to create
and maintain an adequate red cell mass.
These patients often “declare” the
hemoglobin at which their symptoms are
best controlled. The symptomatology
reported by patients with chronic anemia
does not generally correlate well with
laboratory values in different pa- tients but
often corresponds well with these values in
an individual over time.
Selected Clinical Issues
Use of Whole Blood
Whole blood provides oxygen-carrying
capaci- ty, stable coagulation factors
(concentrations of Factors V and VIII decrease
during storage), and blood volume expansion.
Thus, it is po- tentially useful for patients with
concomitant red cell and volume deficits,
such as actively
 CH A P T E R 20
bleeding patients, and helps support
coagula- tion if coagulation factor
consumption is the primary cause of
coagulopathy.19 However, whole blood is
rarely available for allogeneic transfusion.
Component therapy has evolved as an
improved way to expand the blood com-
ponent supply to meet the demand. Whole
blood is mostly commonly used in the
United States today for autologous
transfusion. The ABO type of transfused
whole blood must be identical to that of the
recipient.
Duration of Storage
With the rapid disappearance of 2,3-diphos-
phoglycerate (DPG) from stored red cells and
the concomitant increase in hemoglobin’s af-
finity for O2, a concern is that RBC units
stored for more than 1 to 2 weeks may not
provide the intended increased availability
of O2 at the tis- sue level, at least for the first
12 to 24 hours af- ter transfusion.20,21
Beyond 7 to 10 days of stor- age, the P50 of
hemoglobin decreases from 27 to 16 mmHg,
markedly shifting the dissocia- tion curve to
the left.
There are other known changes to red
cells that occur with storage over time, some-
times referred to collectively as the “storage
le- sion.” These changes include shape
changes, such as decreased deformability of
the red cell, and decreased adenosine
triphosphate. In ad- dition, a loss of nitric
oxide occurs within a few hours after
collection.22 Whether these chang- es are
clinically important is unclear.
The corollary question regarding whether
the age of blood matters for clinical outcomes
is an important and controversial one. A re-
cent meta-analysis that evaluated both obser-
vational studies and RCTs in which death was
the primary outcome showed that transfu-
sions of older RBC units (>21 days old) were
associated with a significantly increased risk
of death [odds ratio (OR) = 1.16, 95% CI =
1.07, 1.24]. The majority of studies in this
analysis were observational, and the three
RCTs ana- lyzed had only small numbers of
patients. Therefore, the conclusion that
transfusions of older blood components caused
the increased mortality is weakened by the
confounding and unintentional bias that arises
with observa-
Hemotherapy Decisions ■ 503
tional studies.23,24 At least one of the studies
was difficult to interpret because of clinical
differences in the two groups of patients.25
The authors of an earlier meta-analysis that
fo- cused on homogeneous subgroups
concluded that the available data were
inadequate to prove causality between older
stored blood components and increased
morbidity and mortality.26
Three larger RCTs have been undertaken
to clarify the issue. The Age of Red Blood
Cells in Premature Infants trial included 377
very low-birthweight premature infants
requiring transfusion. Transfusions of fresh
RBC units (average age = 5.1 days) vs
standard-issue RBC units (average age = 14.6
days) were compared, with rates of major
neonatal morbidities as the primary outcome
measure and the rate of no- socomial infection
as a secondary outcome. In this study, there
were no clinically meaningful or statistically
significant differences in either outcome.27 The
Red Cell Storage Duration Study randomly
assigned patients aged at least 12 years who
required complex cardiac sur- gery to receive
RBCs less than 11 days old or greater than 20
days old throughout their peri- operative
course.28 The primary outcome mea- sure is an
assessment of clinical outcomes us- ing the
Multiple Organ Dysfunction Score. One
ongoing RCT, the Age of Blood Evaluation
study, involves critically ill patients who re-
quire transfusion; this prospective random-
ized trial will compare all-cause mortality at
90 days in patients receiving RBCs less than 8
days old vs standard-issue RBC units.29
Certainly, if the storage lesion is detri-
mental to a patient’s clinical course, research
must determine whether and, if so, why cer-
tain patient populations are more affected
than others. With the tenuous balance that
al- ready exists between the safety and
supply of this human-derived resource,
having fewer blood components available to
support pa- tients may prove more
detrimental than hav- ing stored blood
components available. Deter- mining a
better way to store RBCs—one that prevents
the storage lesion—seems to be a prudent
approach. Opportunities to improve RBC
storage, prevent cell damage, and
504 ■ AABB T EC HNIC AL MANUAL
improve the preservation of 2,3-DPG
continue to be pursued.30
ABO Matching
Although matching patient and donor ABO
types ensures compatibility in that blood
group system, inventory considerations may
suggest that an ABO-compatible rather than
an ABO-identical RBC unit should be trans-
fused (Table 20-2). Although a compatible but
nonidentical RBC unit introduces some isoag-
glutinins directed against the recipient’s A
and/or B antigens, the small amount of plas-
ma in an additive system or packed RBC unit
(or even multiple units) is insufficient to cause
hemolysis.
Components containing larger amounts
of plasma, such as platelets (see below) or
whole blood units, raise the potential for iso-
agglutinins to cause hemolysis. Whole blood,
when used, would be transfused to an ABO-
identical recipient because of this issue. In the
setting of ABO-incompatible heart transplan-
tation in an infant, the transfusion service may
be asked to plasma-reduce or wash an RBC
unit, largely because the original protocol
called for this modification rather than be-
cause solid evidence indicates that incompati-
ble passive ABO isoagglutinins can impair a
new graft.31
Leukocyte Reduction
The most appropriate use of leukocyte
reduc- tion remains controversial. Ardent
proponents on both sides argue about
whether all cellular
TABLE 20-2. Selection of ABO-Compatible
Red Blood Cell Units
Recipient Blood GroupCompatible Red Blood excellent
A A, O
B B, O
AB AB, A, B, O TRIM, but the results have been
O O
*Red Blood Cells prepared as additive system or leu- kocyte-reduced blood supply, and the
“packed” units.
components should have their white cells
reduced.32 Although initially introduced in
several European countries to decrease the
transmission of prions, universal leukocyte
re- duction is most often used to reduce the
risk of postoperative infection and improve
post- transfusion survival by reducing
transfusion- related immunomodulation
(TRIM).
The benefit of removing leukocytes for
patients who will be multitransfused and/or
transplanted is clearly evident in terms of re-
duced rates of alloimmunization, episodes of
refractoriness to platelet transfusion, and fe-
brile reactions.33-36 However, universal imple-
mentation of leukocyte reduction may not
cause a measureable drop in febrile reaction
rates, given the proportion of patients who
are susceptible to these reactions.37 A
positive clinical impact of universal
leukocyte reduc- tion has been difficult to
identify and was not seen in the only large-
scale, prospective RCT of its
implementation.38 Even in more defined
situations, such as cardiac surgery,
prospective trials have reached contradictory
conclusions, including when they were
performed by the same research team.39-42
The many retrospec- tive analyses ascribing
benefits to leukocyte reduction have often
been limited by con- founders.43,44 However,
some support strong arguments that
removing leukocytes does lead to reduced
lengths of stay, reduced infection rates, and
improved outcomes.45,46 Other po- tential
concerns associated with the presence of
leukocytes in blood components, such as
increased red cell adhesive properties, raise
new questions about their impact.47 For a
thor- ough examination of this complex
subject, the reader is referred to several
Cell Units* meta- analyses and other
publications.48-51
Many studies have addressed the possi-
bility that leukocyte reduction can reduce
the incidence of clinical outcomes due to
contradictory.50 One hypothesis was that the
savings resulting from reduced
immunomodulation could offset the costs of
leukocyte reduction, but this was not
demonstrable in a large prospective RCT.42
Nonetheless, several countries maintain a
need for leukocyte reduction remains
controversial.43
 CH A P T E R 20
The value of leukocyte reduction as a
means of preventing cytomegalovirus (CMV)
transmission has been well documented.52,53
Studies using polymerase chain reaction
(PCR) indicate that the highest risk of CMV
transmis- sion through blood transfusion may
be from recently seroconverted donors rather
than do- nors who have been seropositive for
more than a year.54,55 This information is
interesting but difficult to translate into an
operational policy. Because of a 1% to 2% risk
of “break- through” transmission with either
seronega- tive or leukoreduced components,
patients at high risk of disseminated CMV
infection should be actively monitored for
evidence of CMV using antigenemia assays or
reverse- transcription PCR techniques.56
The leukocyte content of units varies by
component type (Table 20-3).57,58 Current
fed- eral guidelines and AABB Standards for
Blood Banks and Transfusion Services define
a leuko- cyte-reduced component as one
with <5 × 106 residual donor leukocytes per
final product (including RBCs; apheresis
platelets, and pooled platelets).59,60(p26) The
AABB Standards calls for <8.3 × 105 residual
leukocytes in plate- lets prepared from a
single unit of whole blood, and 5 × 106 in
Pooled Platelets Leuko- cytes Reduced.60(p29)
The Food and Drug Ad-
TABLE 20-3. Approximate Leukocyte Content of Blood Components (Per Unit)
Whole blood
Red Blood Cells
Red Blood Cells, washed
Red Blood Cells, deglycerolized
Red Blood Cells, leukocyte reduced (by filtration)*
Apheresis Platelets
Apheresis Platelets, leukocyte reduced
Platelets†
Platelets, leukocyte reduced
Platelets, pooled, leukocyte reduced
Fresh Frozen Plasma, thawed
*Leukocyte reduction with third-generation leukocyte adsorption filter.
†
Derived from 1 unit of whole blood via platelet-rich plasma process.
Hemotherapy Decisions ■ 505
ministration (FDA) recommends quality-
con- trol steps to indicate with 95%
confidence that at least 95% of units meet
these criteria.59 By comparison, European
guidelines define leu- kocyte-reduced
components as those with
<1 × 106 residual leukocytes per unit and re-
quire no more than a 10% failure rate in the
leukoreduction process.61
Matching Units to Patient Hemoglobin
Targets
For adult patients, the discussion of when or
whether to transfuse RBCs has not usually
pro- ceeded to a consideration of how much
to transfuse. Although pediatricians
commonly prescribe blood components
based on the pa- tient’s size, the typical
order for adult RBC transfusion until
recently had been 2 units, re- gardless of
patient size and usually without re- gard to a
desired target hemoglobin level. In addition,
the widely variable hemoglobin con- tent of
RBC units usually is not considered.62
A more physiologic approach would be
to identify the target hemoglobin desired
after transfusion and then base the dose (ie,
num- ber of units or g of hemoglobin to be
trans- fused) on the patient’s current
hemoglobin, any ongoing blood loss, and
the calculated blood volume. A pilot study,
however, showed
109
108
107
106-107
<5 × 106
106-108
<5 × 106
107
<8.3 × 105
<5 × 106
0.6 × 106 - 1.5 × 107
506 ■ AABB T EC HNIC AL MANUAL
that the number of units transfused using
this approach to a group of patients could be
re- duced, thus reaping benefits for
inventory management as well as donor
exposure rates.63 Issues that remain to be
resolved for this ap- proach include the
following:
■ Whether the approach could be imple-
mented successfully for all patients in an
institution.
■ Whether blood collection establishments
would be able to provide the needed
hemo- globin content for all RBC units.
■ Whether the approach could be sustained
in an era when most units are produced
by apheresis, with a standardized
hemoglobin content.
Emergency Transfusion
The unexpected need to transfuse possibly a
large amount of RBCs may require
application of alternative procedures.
Release of group O RBCs or antigen-
negative, uncrossmatched units may be
necessary if waiting to complete standard
pretransfusion testing routines could induce
anemic morbidity (see Chapter 15).
An emergency-release protocol might
be integrated into one that allows rapid
provision of a large number of RBC units to
accommo- date the needs of patients who
are bleeding rapidly. Elements of such a
block-release sys- tem might include
completing the paper work for multiple
units in advance and prepackag- ing these
units to facilitate immediate delivery to the
patient. The need for and components of
such a system vary according to the types of
patients served by the institution and the lo-
gistics of blood delivery. The expectations of
clinicians (particularly surgeons,
anesthesiol- ogists, and emergency
physicians) for rapid delivery of large
volumes of RBCs should be discussed with
them so that appropriate re- quirements can
be met reliably. In evaluating these clinical
needs, attention should be given not just to
the time required to issue RBCs from
inventory to the laboratory but also for the
units to reach the patient.
Incompatible RBC Transfusion
Occasionally, transfusion of RBCs may be
nec- essary when no serologically
compatible units are available for a patient.
This most often oc- curs in patients with
autoantibodies that typi- cally are reactive
with red cells from all donors; however, as
long as the presence of alloanti- bodies can
be ruled out, the transfused cells should
survive as long as autologous cells.
The key point in ensuring a safe and
suc- cessful transfusion in such a situation is
the exclusion of the presence of
alloantibodies. Because this may be difficult
and the benefi- cial effects of the transfusion
may be tempo- rally limited, a conservative
transfusion strate- gy is usually
recommended for patients with autoimmune
hemolytic anemia. Determina- tion of the
patient’s phenotype before transfu- sion
simplifies subsequent investigations of the
presence of alloantibodies. (Chapters 15- 17
contain a more complete discussion of this
issue.)
Other situations in which all units
appear to be incompatible include the
presence of al- loantibodies to high-
frequency antigens and/ or multiple antibody
specificities. If serologic testing fails to
resolve the problem or the prob- lem is
identified but sufficient time is not available
to acquire compatible units, consul- tation
between the transfusion service physi- cian
and the patient’s clinician is advised to
weigh the risks and benefits of transfusion
and to consider which alternative therapies
are suitable. If the need is sufficiently
urgent, ABO-compatible but crossmatch-
incompati- ble RBCs may need to be used.
Depending on the alloantibody’s
specific- ity or possible specificities that
have not been ruled out, incompatible RBC
transfusion does not always result in
immediate hemolysis, and the incompatible
cells may remain in the cir- culation long
enough to provide therapeutic benefit.64
If time permits and equipment is avail-
able, the survival of a radiolabeled aliquot of
the incompatible RBCs can be determined.
However, this is beyond the capability of
most laboratories and is rarely needed. An
in-vivo crossmatch can be performed by
cautiously
 CH A P T E R 20
transfusing 25 to 50 mL of the incompatible
cells, watching the patient’s clinical
response, and checking a 30-minute
posttransfusion specimen for hemoglobin-
tinged serum. Such an assessment does not
guarantee normal red cell survival, but it can
indicate whether an acute reaction might
occur. If no adverse symptoms or hemolysis
are observed, the re- mainder of the unit can
be transfused slowly and with careful
clinical monitoring. If the condition
requiring a transfusion intervention is life
threatening, RBC units may sometimes be
given without special testing, but clinical
staff should be prepared to treat any reaction
that may result.
RBC Substitutes
A variety of means have been explored to
pro- vide the O2-carrying capacity of
hemoglobin without utilizing red cells.
Development has proceeded furthest using
Hb solutions derived
from red cell lysates in which the
hemoglobin molecules have undergone one
or more chem- ical modifications to
optimize their O2 dissoci- ation and prevent
renal damage. Two of the
greatest challenges with cell-free hemoglobin
are its vasoconstrictive properties and its short
half-life within the intravascular circulation.
The goal of creating a hemoglobin substitute
that the FDA considers to be as safe as donor
red cells continues to be an elusive one.65,66
PLATELET TRANSFUSION
Prophylactic vs Therapeutic
Transfusion
Before the advent of platelet therapy,
bleeding exceeded infection as the most
common cause of death in patients with
acute leukemia. The availability of platelet
concentrates led to the natural consideration
that prophylactic trans- fusion to prevent or
limit the most severe thrombocytopenia
would prevent the onset of severe
hemorrhage. Today, approximately 80% of all
platelet transfusions are administered to
patients with hypoproliferative
thrombocyto- penia.
Hemotherapy Decisions ■ 507
However, the value of this approach has
never been definitively documented. Early
studies yielded equivocal results or utilized
statistical approaches that were less rigorous
than would be expected today.67,68 In fact, an
RCT in children with leukemia prior to the
era of leukocyte reduction indicated that the
use of prophylactic platelet transfusion from
the outset of chemotherapy increased the
likeli- hood of bleeding due to the
development of platelet refractoriness.67
The alternative strategy of directing
plate- let resources to those patients who are
bleed- ing may be a wiser and more
effective use of a limited resource.69 In fact,
a recently published Cochrane review
showed that overall, a thera- peutic platelet
transfusion strategy might not be inferior to
a prophylactic strategy, but the evidence
supporting this approach is not ro- bust. 67
The majority of studies reviewed were
conducted in the 1970s, and the conclusions
did not take into account practice changes
since that time.
Overall, the role of prophylactic platelet
transfusions for the prevention and control
of thrombocytopenic bleeding is not yet
clear. When bleeding risk is identified in
patients with thrombocytopenia, several
factors in ad- dition to platelet count should
be considered. These include altered platelet
function due to intrinsic or acquired defects
and hemostatic defects involving the
coagulation system. The disease process
itself must also be considered. For example,
in acute leukemia, intracranial hemorrhage
is more often associated with high
circulating blast counts than with low
platelet counts.70 The leukemic blasts may
cause sludging of cells in capillaries and
possi- ble hemorrhagic infarction,
particularly in the presence of a concomitant
coagulopathy.
Transfusion Thresholds
If prophylactic platelet transfusions are to be
given to patients with hypoproliferative
thrombocytopenia, what is the most
appropri- ate threshold for transfusion? The
first study to address this question sought to
define a clini- cally useful threshold by
observing and cate- gorizing bleeding and
associating the risks of
508 ■ AABB T EC HNIC AL MANUAL
hemorrhage with patients’ platelet counts.71
The researchers were unable to identify a
platelet count threshold below which the risk
of hemorrhage increased rapidly. However,
this paper was often cited as the source of
the dictum that patients’ platelet counts
should be kept above 20,000/µL. In the
1960s, when this study was conducted,
aspirin was commonly used to treat fever,
pain, and transfusion reac- tions among
patients with neutropenia. But with current
knowledge about the platelet dys- function
induced by aspirin and associated changes in
hematology practice, the study re- sults are
less applicable today (Table 20-4).73
Over the last decade, several
prospective
studies using either historical or randomized
control groups have documented the
success- ful application of 10,000/µL as a
prophylactic transfusion threshold in
inpatients.67,74-76 These results parallel the
finding that stool blood loss does not
accelerate until a platelet count of
approximately 5000/µL is reached.77 Many
institutions have now adopted 10,000/
µL as their standard prophylactic platelet
transfusion threshold, but others have opted
to combine laboratory data with the patient’s
clinical status to determine the most
appropri- ate transfusion point 72 (Table 20-
4). Further- more, because platelets adsorb
circulating thrombopoietin (TPO) and
higher platelet counts are associated with
lower levels of free TPO,78 maintaining
patients at a lower platelet count has been
suggested to lead to shorter in- tervals of
thrombocytopenia, which is a poten- tial
additional benefit.
TABLE 20-4. Current Prophylactic Platelet Transfusion Thresholds
All patients
- or –
Stable patients
Patients with fever or recent hemorrhage
Patients with coagulopathy, on heparin, or with anatomic lesion
likely to bleed72
Note: These thresholds are most commonly applied to inpatients. Adjustment of the transfusion threshold may be necessi-
tated by unusual clinical situations.
For patients who are already bleeding or
are about to undergo a hemostatic challenge,
such as a surgical procedure, attempts are
made to keep the platelet count higher,
usually in the vicinity of 50,000/µL.79
Although there are no trials to document that
this is the most appropriate level, it has
become generally ac- cepted as adequate.80
An even higher target, up to 100,000/µL, is
often used for intracerebral, pulmonary, and
ophthalmic hemorrhage. This is to provide a
greater “cushion” to ensure ade- quate
hemostasis in these vital and suscepti- ble
organs even if the platelet count should
drop. Higher cut points might also be used in
massive transfusion or disseminated
intravas- cular coagulation (DIC), where the
platelet count may drop rapidly.
The role of anemia should also be consid-
ered in the prevention or treatment of hemor-
rhage. In a 2001 study in healthy volunteers, a
reduction in hematocrit from 41% to 35% re-
sulted in almost a doubling of the bleeding
time, whereas a decrease in the platelet count
by one-third had no effect.81 Traditionally, this
result has been attributed to the rheologic
properties of flowing blood. Given their larger
size and higher density, red cells tend to occu-
py the central (axial) portion of the blood flow,
pushing platelets to the periphery in proximity
with the vessel wall. This makes teleologic
sense because it is only along the vessel wall
that a rent in the endothelium can occur
where the platelets would then perform their
hemostatic functions.
Recent research has focused on
elucidat- ing the mechanisms of red cell
and platelet
10,000/L
5,000/L
10,000/L
20,000/L
 CH A P T E R 20
communication, which might also explain
the hemostatic effect of a higher hematocrit.
These newer studies have shown, for
example, that the red cell membrane
augments throm- bin generation,82 adenosine
diphosphate re- leased from red cells may be
a chemical mes- senger for platelet
activation,83 and red cell phosphatidyl serine
expression might be an- other pathway for
thrombin generation. Re- gardless of the
postulated mechanism, correc- tion of
anemia may be an additional tool to use for
bleeding prevention, particularly in pa-
tients with thrombocytopenia. In patients
with uremia, RBC transfusion or the
administra- tion of erythropoietin to increase
the hemato- crit improves hemostasis
similarly.84 Thus, al- though the patient’s
cardiovascular system might be tolerating
the anemia associated with chemotherapy
(or hemorrhage), the he- mostatic system
may not.85
Transfusion to Correct
Thrombocytopathy
Patients whose platelets are not able to com-
plete all of the complex metabolic steps
neces- sary for activation, granular release,
and aggre- gation may have an increased
likelihood of bleeding and/or an inability to
respond to hemorrhage appropriately. These
abnormali- ties may be congenital (eg,
Glanzmann throm- basthenia) or acquired as
the result of disease (eg, myelodysplasia) or
drug treatment (eg, with aspirin or
glycoprotein IIb/IIIa antago- nists). In
addition, patients who have recently
undergone extracorporeal circulation (eg,
dur- ing cardiac bypass surgery) may have
platelet counts that appear to be adequate for
hemo- stasis but, in fact, are dysfunctional
due to pro- longed exposure to roller pumps
and foreign surfaces, leading to partial
activation and de- granulation.86
In all of these circumstances, the
decision
whether to transfuse platelets probably
needs to be based on the patient’s clinical
status rather than his or her platelet count.
Patients undergoing surgery while still under
the effect of previously ingested aspirin do
not necessar- ily bleed more than other
patients, although clinician knowledge that
the patient has been
Hemotherapy Decisions ■ 509
taking aspirin has been associated with in-
creased blood component usage.87-92 The pro-
phylactic use of platelets (and plasma) after
cardiac surgery has been shown to be
neither necessary nor beneficial, but a
patient experi- encing excessive blood loss
postoperatively whose heparin level has
been reversed may benefit from a dose of
platelets even before his or her postoperative
platelet count is known. Patients treated
with irreversible platelet an- tagonists (eg,
clopidogrel) during cardiac cath- eterization
and who proceed directly to cardi- ac
surgery may need one or more doses of
platelets (as well as increased RBC transfu-
sions) because their own platelets are no
lon- ger functional.93,94 Antiplatelet therapies
con- tinue to be used in catheterization
because they appear to improve outcomes
even if the patient requires immediate
surgery.95 The ef- fect of these drugs can be
antagonized through pharmacologic
intervention, including the use of
desmopressin acetate.96
Given the high proportion of patients
treated with aspirin for a variety of reasons,
patients who experience bleeding while tak-
ing aspirin may be encountered frequently.
Al- though the daily consumption of 81 mg
aspi- rin for cardiac prophylaxis is unlikely
to contribute to hemostatic difficulties, some
pa- tients are hyperresponders to aspirin and
their bleeding times may be dramatically
extended. Platelet transfusions are
occasionally request- ed to correct the effect
of aspirin. In these cas- es, only a small
proportion—approximately 20%—of
circulating platelets need to be func- tional
to correct the deficiency.97 Therefore, neither
achievement of donor platelet levels of
50,000/µL nor a full therapeutic dose of
plate- lets is required in most patients.
Patients with significant renal disease (ie,
a creatinine level exceeding 3 mg/dL) may
also have dysfunctional platelets due to the
uremic environment. Transfusion of platelets
to these patients is of little value, however,
because they, too, rapidly succumb to the same
meta- bolic derangement. Desmopressin
acetate (1- deamino-8-D-arginine vasopressin,
or DDAVP) treatment is usually recommended
to aug- ment the responsiveness of these
platelets.98 Alternatively, cryoprecipitate may
provide the
510 ■ AABB T EC HNIC AL MANUAL

increased amount of von Willebrand factor smallest number of platelets that would need
(vWF) that is believed to be helpful in activat- to be transfused if patients received just
ing these platelets if tachyphylaxis to multiple enough platelets to achieve the desired
doses of desmopressin acetate precludes fur- target level when they reached the
ther treatment.99 Dialysis to decrease the ure- transfusion threshold.101 This analysis
mia is often indicated in clinical scenarios argued for the use of small therapeutic doses
where optimal platelet function is desired. administered more frequently. The use of
larger units in a clinical study resulted in
Dosage longer intertransfusion inter- vals, although
this temporal increase was smaller than the
The amount of platelets considered a thera-
increase in the number of platelets
peutic dose remains undecided, but recent
transfused.102 In a paired study of marrow
re- search is elucidating this issue. 100 Much
transplant patients, larger units pro- vided
early research was performed using platelet
the expected lengthening of intertrans-
units with significantly lower platelet counts
fusion intervals and, unexpectedly, resulted
than units currently being produced. Initial
in both count increments and corrected
platelet separation efficiencies were
count increments (CCIs) that were higher
significantly lower than those achieved
than those achieved with smaller units.103
through evolutionary im- provements in
The Strategies for Transfusion of
both component production processes and
Platelets study104 was a multicenter,
larger whole-blood collections. Accordingly,
prospective RCT designed to show that a
many blood centers now report mean
lower platelet dose for prophylactic
contents that are 20% to 40% above the
transfusions was not inferior to the standard
required minimum of 5.5 × 10 10 platelets per
dose; the primary outcome was incidence of
unit derived from whole blood. As a result,
WHO Grade 2 (or higher) bleed- ing. The
fewer units need to be pooled to obtain the
trial enrolled patients undergoing he-
same platelet dose. At the same time, accep-
matopoietic stem cell transplantation and
tance of lower platelet counts in patients has
nontransplant patients with chemotherapy-
facilitated the progressive reduction in the
induced thrombocytopenia. The experimen-
standard dose from 10 to 8, 6, or even 4
tal arm received low-dose prophylactic
units per pool (or transfusion).
plate- let transfusions (1.5-2.9 × 10 11
The amount of platelets that can be col-
platelets per transfusion, defined as half the
lected by apheresis also merits
standard dose) and the control arm received
consideration. The standard of 3.0 × 10 11
a standard dose (3.0-6.0 × 1011 platelets per
apheresis platelets per unit is the amount that
transfusion). Al- though sample size
could be practically collected with early
calculations indicated that approximately
instruments and may also have accounted
270 patients would be neces- sary in each
for the maximum collection time that donors
treatment arm, the study was stopped after
would tolerate. These apher- esis units
enrolling 130 patients based on a
yielded an increment similar to the
preestablished safety threshold because the
transfusion of a pool of 6 to 8 units of
cumulative incidence of Grade 4 bleeding
whole- blood-derived platelet components.
ex- ceeded 5% between the two study arms.
Today, the increased efficiency of apheresis
The main trends of this study did not
instruments allows the collection of two or
support the hypotheses that patients in the
even three times the standard quantity.
low-dose arm would require fewer products
Although blood centers derive a significant
and have a shorter duration of
economic boost from split- ting platelet
thrombocytopenia.
apheresis units, whether patients are better
Another recent multicenter, prospective,
served by receiving larger platelet units RCT, the platelet dose (PLADO) study,105 did
remains to be determined. proceed to completion. Patients with hypo-
The question of what the optimal proliferative thrombocytopenia secondary to
platelet dose is has been approached in chemotherapy for hematologic malignancies
several ways. A mathematical model was or undergoing either autologous or
used to calculate the allogeneic stem cell transplantation were
randomly
 CH A P T E R 20 Hemotherapy Decisions ■ 511

assigned to a prophylactic platelet transfusion based on content and blood volume,108 can
dose of 1.1 (low dose), 2.2 (medium dose), or provide a yardstick to determine whether
4.4 × 1011/m2 platelets (high dose). The pa- spe- cial efforts, such as selection of
tients were transfused with their assigned dose antigen- negative units to combat
prophylactically for morning platelet counts of alloimmunization, may be helpful.
10,000/µL. Additional platelet transfusions
could be given for active bleeding or Unit Type and Age
planned invasive procedures. The primary
The types of platelet units used for routine
endpoint was the percentage of patients in
transfusion varies by institution. Currently,
each group with at least one episode of
about 90% of platelet transfusions in the
WHO Grade 2 or higher bleeding. Of the
Unit- ed States are derived from apheresis
1272 patients who re- ceived at least one
collec- tions, and the usage of these platelets
platelet transfusion, the pri- mary endpoint
has in- creased in a steady, linear manner for
was achieved in 71%, 69%, and 70% in the
the past two decades.109 Local pricing
low-, medium-, and high-dose groups,
policies and the preference of transfusion
respectively. These differences were not
services to avoid the extra processing steps
statistically significant. Those in the low-
associated with whole- blood-derived units
dose arm did receive an average of one more
are probably significant factors in this
platelet transfusion, however.
choice (Table 20-6). Some in- vitro
The lifespan of transfused platelets ap-
measures have identified a larger platelet
pears to be abnormally short in patients with
storage lesion in platelets produced via the
thrombocytopenia. Although autologous
platelet-rich plasma (PRP) method than by
platelets stored for 5 or 7 days and then re-
apheresis,110 and one paired reinfusion study
infused to healthy people usually survive for
has confirmed these findings.111 However,
more than 5 days, the lifespan of platelets in
the radiolabeled autologous recovery and
patients with severe thrombocytopenia may
survival rates of the two types of platelets do
be only 2 days. This shortened lifespan may
not ap- pear dissimilar even after 7 days of
be attributable to the fixed loss of platelets
stor- age.112,113 Platelets derived from whole-
of ap- proximately 7100/µL per day from
blood buffy coats may have the advantages
circulation for maintenance of normal
of re- duced activation during centrifugal
hemostasis.106 When the patient’s platelet
prepara- tion steps, but these are currently
count is low (and, of course, still subject to
not available in the United States, although
daily reductions due to senescence), this
they are proba- bly the most widely used
obligatory loss represents a large proportion
platelet component around the world. From
of circulating platelets and leads to the need
a clinical efficacy standpoint, all three
for transfusions every 2 to 3 days even in
platelet preparations yield good clinical
patients achieving good incre- ments from
results.
transfusions.107 More RCTs similar to the
All platelet unit types accumulate a stor-
PLADO study are needed to clarify which
age lesion over time that leaves them less re-
dosage provides optimal hemostasis.
sponsive to physiologic agonists. Typically,
As with adult RBC transfusions, the size
of these platelets also bear markers of platelet
the patients (and their spleens) are usually ac- tivation, although none of these markers
not considered in selecting the platelet unit has proven to be a useful predictor of
dose to be transfused. One of the dose recovery or survival after transfusion.114-117
studies men- tioned above attempted to set Studies in which radiolabeled platelets at the
the dose based on patient weight, and the limit of the storage period are reinfused are
outcomes of the two studies may be helpful commonly re- quired for FDA licensure of
in determining the clinical importance of new techniques of collecting or storing
such a strategy. The pa- tient’s size and the platelets. Both recovery and survival appear
content of the unit are rou- tinely assessed to become shorter with in- creased storage
through the CCI (Table 20-5). This measure, time in such radiolabeling studies.
or a similar approach of calcu- lating the However, not all series of clinical
recovery rate of transfused platelets
512 ■ AABB T EC HNIC AL MANUAL

TABLE 20-5. Determination of Platelet Response

CCI

CI (per L) × BSA

CCI = unit content
Platelet Recovery
CI × patient mass (kg) × blood volume (estimated at 75 mL/kg) × 100
Platelet recovery (%)
= unit content

Sample Calculations
Patient mass: 80 kg blood volume = 80 kg × 75 mL/kg = 6000 mL
Patient BSA: 2.0 m2 (determined from a table or nomogram, available in many textbooks)
Pretransfusion platelet count: 5000/L
Posttransfusion platelet count: 25,000/L  CI = 20,000/L
Platelet count in unit: 1.5 × 106/L
Volume of unit: 267  unit content = 4.0 × 1011 platelets
mL
CCI = (20,000/L × 2.0 m2)/4.0 = 10,000
Successful transfusion: 7500
Refractory patient: Two or more transfusions with CCI <7500
Recovery = (20,000/L × 1000 L/mL × 6000 mL × 100%)/(4.0 × 1011) = 30%
Maximum achievable if the patient has a spleen: 65% to 70%
CCI = corrected count increment; CI = count increment; BSA = body surface area.

TABLE 20-6 Characteristics of Platelet Unit Types Available in the United States

Whole-Blood Derived

Prestorage
Platelet Unit Characteristic Individual Unit Pooled* Apheresis

Cost of preparation Lower Higher

Ease of bacterial testing Lower Higher Higher
Ease of leukoreduction Lower Higher Higher
Hospital preparation required More Less Less
Donor exposures Greater Fewer
HLA selection possible No Yes
Platelet content known No Yes
*Storage of pools of platelets for longer than 4 hours requires bacteria detection by a culture technique
approved by the Food and Drug Administration.
 CH A P T E R 20
observations have shown a decrement in
CCI with increased storage time, perhaps
due to limited study sizes and the inherent
variability between patients and outcomes of
transfu- sions at different points during the
course of illness.118,119 Therefore, platelet-
transfusion policies in most institutions call
for the most efficient use of the short-lived
and scarce re- source by transfusing the most
appropriate units that are closest to their
outdate. Some patients may appear to be
more sensitive to the storage lesion than
others and may benefit from receipt of
platelets that have been stored for less time;
this can only be determined em- pirically.
Out-of-Group Transfusions
The importance of providing platelet transfu-
sions using units of the same ABO group as
that of the recipient is an unresolved issue.
There are several points to consider: 1) the
presence of ABH antigens on platelets that
could be targeted by the recipient’s isoaggluti-
nins,120 2) the presence of plasma in the unit
that could lead to hemolysis of the recipient’s
red cells, and 3) the potential that the incom-
patibility could have immunologic effects that
could affect patient outcomes. Typically, the
presence of ABO-incompatible donor red cells
in the platelet product is not a significant con-
cern because of their very low levels.
A recently published systematic review121
showed that ABO-identical and ABO-
compati- ble platelet transfusions (eg, group A
platelets in an AB recipient) result in a higher
CCI than ABO-incompatible platelet
transfusions (eg, group O platelets in a non-
group O recipient).
Whether the benefits of transfusing ABO-
identical or -compatible platelets also trans-
lates into improved clinical outcomes in terms
of decreased bleeding severity or need for
transfusions is not clear from the existing data.
These data do indicate that a subset of recipi-
ents with a higher isoagglutinin titer, particu-
larly anti-A, have poorer recovery after trans-
fusion with a high-ABH-antigen-expression
unit, especially if the platelets comes from an
A1 donor.122-126 Any diminution in response ap-
pears to be more pronounced with repeated
Hemotherapy Decisions ■ 513
out-of-group transfusions, possibly due to the
stimulation of higher isoagglutinin titers in the
recipients.127 Failure to achieve expected plate-
let increments with an out-of-group transfu-
sion should prompt a trial of ABO-identical
transfusions, particularly if the patient is not
alloimmunized to platelet-specific or HLA an-
tigens (Fig 20-1).128
An out-of-group platelet transfusion,
such as of a group O unit to a group A
recipi- ent, results in the transfusion of about
300 mL of plasma containing isoagglutinins
that are directed against antigens present on
the recip- ient’s red cells. Although it is not
standard practice to transfuse a group O
plasma unit to a group A recipient, this
practice is common for platelet
transfusions.80 Many patients show no signs
or symptoms of the mismatch, al- though the
majority may have a transiently positive
direct antiglobulin test result that, at least,
causes some immunohematologic con-
fusion regarding its cause.129
A high titer of isoagglutinins in a
platelet unit can cause hemolysis of
recipient red cells that may be clinically
significant and even fa- tal. This outcome
may be more likely in situa- tions where the
recipient is smaller (and, thus, the volume
transfused represents a greater proportion of
the recipient’s plasma volume), when
apheresis units are used (and all the plasma
comes from the same donor and is not
“diluted” by plasma of lower isoagglutinin
ti- ters from other units in the pool or
neutralized by the presence of soluble ABO
antigens in the other units), and in patients
undergoing multi- ple transfusions.130 The
risk of hemolysis with apheresis platelet
units is in the range of 1:3000 to
1:10,000.131,132 Whether ABO-compat- ible but
nonidentical plasma poses a risk through
circulating immune complexes has been
considered.133
Different approaches may be used to pre-
vent acute hemolysis when incompatible
plasma must be transfused as part of a
platelet transfusion. One approach is to limit
the amount of plasma being transfused by
centri- fuging the unit and expressing off the
majority of the plasma shortly before
transfusion (see Method 6-13). This reduces
the potential se- verity of any hemolysis but
may not prevent it
514 ■ AABB T EC HNIC AL MANUAL
FIGURE 20-1. One approach to management of patients with thrombocytopenia.
and, in any case, does result in the loss of
some proportion of the unit’s platelet content.
An approach that achieves a similar result is to
use platelet additive solutions, which allow
platelets to be stored in electrolyte solutions
that replace up to 65% of plasma, reducing the
amount of incompatible isoagglutinins. An-
other approach is to avoid out-of-group trans-
fusions of units having a dangerously high
titer of isoagglutinins. This approach usually
in- volves identifying units with amounts of
anti-A above a particular threshold, often a
titer of
200. This approach lacks a solid evidence
base, its outcomes vary by method,134 and it
does not identify all potentially harmful
units. 135 Avoid- ance of out-of-group plasma
or amelioration of the situation through
volume reduction is especially important for
pediatric recipients, in whom the volume
transfused is relatively greater, and in
neonates, where hyperbilirubi- nemia may
have particularly adverse conse- quences.
The possibility exists that the
transfusion of incompatible plasma may also
have other, less-immediate but still
untoward effects on recipients’ immune
systems. It is postulated
that these effects might be mediated by the
formation of circulating immune complex-
es.133 For example, minimization of exposure
to incompatible plasma was associated with
improved survival probability in marrow
transplant recipients in one retrospective
analysis,135 and another retrospective study
suggested that such a strategy reduced in-
hos- pital mortality after cardiac surgery by
two- thirds.136 This finding was not
replicated in a subsequent, larger study,
however.137 There is also some suggestion
that transfusion with ABO-incompatible
platelets may hasten the development of
alloimmunization and plate- let
refractoriness138 and may reduce survival
after marrow transplantation.139
Any delayed immunologic impact of
the transfusion of mismatched plasma with
plate- lets remains to be fully elucidated,
although avoidance of out-of-group
transfusion when possible would obviate
this concern. Weighed against the practical
issue of a human-derived therapeutic
product that expires in 4 to 5 days issuing a
platelet unit with incompatible plas- ma
generally carries a lower risk than with-
holding transfusion.
 CH A P T E R 20 Hemotherapy Decisions ■ 515
multiple transfusions over a 2- to 4-week peri-

Rh Matching
Platelets themselves do not express or carry
Rh antigens. Despite improvements in
apheresis- collection and whole-blood-
processing tech- niques, a small but
immunogenic dose of red cells can be
contained in a platelet unit. This is more
likely in whole-blood-derived than apheresis
platelet units. In one study, an inter- nal
quality control check of platelet units using
flow cytometry found that mean red cell
con- tent of platelet concentrates from
whole-blood donations and from apheresis
were 0.036 mL and 0.00043 mL,
respectively.138
The risk of developing
alloimmunization to the D antigen from a
platelet transfusion is also dependent on a
multitude of other plate- let unit factors (eg,
ABO compatibility and whether the unit
was leukoreduced) and recip- ient factors
(eg, gender, immunologic status, whether
the patient needs a massive transfu- sion,
and whether a concurrent febrile transfu-
sion reaction occurs). The development of
anti-D has been studied in many
observation- al and retrospective studies.140-
142

The clinical magnitude of this issue is

far less than one might expect. Most patients
re- ceiving platelet transfusions are severely
im- munosuppressed, and a primary
response to the D or other red cell antigens
is very uncom- mon.140,142 In addition, for
most patients, the formation of anti-D has
minimal impact on their subsequent
hemotherapy support. How- ever, if the
patient is a female of childbearing potential,
the formation of anti-D could have a
significant impact on her future pregnancies.
Therefore, attempts are usually made to pro-
vide Rh-negative recipients with Rh-
negative platelet units even though platelets
do not ex- press or carry Rh antigens. When
an Rh-nega- tive patient must receive an Rh-
positive unit of platelets, a dose of Rh
Immune Globulin (RhIG) may be
administered to prevent Rh(D)
alloimmunization.
Rather than provide RhIG to all Rh-
nega-
tive recipients of Rh-positive platelets, many
centers supply RhIG only to premenopausal
females. Given the 3-week half-life of IgG,
a single dose should provide prophylaxis for
 alloimmunization status may help blood
services prepare to provide special- ly
od and certainly for the period selected units. Usually, however, the hunt for
during which anti-D is alloantibodies begins when
detectable serologically. alloimmunization is suspected from poor
Because the recipient had, responses to platelet transfusions that cannot
and probably still has, be explained by oth- er, nonimmunologic
thrombo- cytopenia, an factors, such as spleno- megaly or sepsis.
intravenous form of RhIG The availability of kits to screen for HLA
may be administered to avoid and other platelet-directed an- tibodies by
a hematoma, partic- ularly if enzyme immunoassay or other simple
the platelet count remains approaches has expanded the number of
below 50,000/µL.143 laboratories able to perform this testing and
provide clinically useful information about
Alloimmunization: the results with a short turnaround time.
Prevention and Identifi- cation of the specificities of
Response multiple HLA anti-
Although patients who are
receiving multiple platelet
transfusions are usually
immunosup- pressed by virtue
of their underlying disease
and/or therapy, they are still
able to mount an immune
response against antigens on
plate- lets. This response is
usually in the form of an-
tibodies to HLA Class I
antigens, but some pa- tients
may make antibodies to
platelet-specific antigens. The
former require presentation of
the antigens via donor
lymphocytes. Leuko- cyte
reduction of both platelet and
RBC units has proven to be
highly effective in reducing
the risk of primary HLA
alloimmunization.33 However,
if a patient has previously
been sen- sitized, such as
through pregnancy or a prior,
nonleukoreduced transfusion,
transfusion of the Class I
antigens on the platelets may
be sufficient to provoke an
anamnestic response and the
appearance of platelet
refractoriness.
For patients who can be
expected to re- quire multiple
platelet transfusions, such as
those who will undergo
hematopoietic stem cell
transplantation, advance
knowledge of their (current)
516 ■ AABB T EC HNIC AL MANUAL

the presence of DDPAs.

bodies, however, may still require
lymphocyto- toxicity testing in some
circumstances.
Before the ready availability of such
test- ing, the detection or supposition of
immuno- logic platelet refractoriness led to
a call for HLA-matched platelets.144,145 Even
a large do- nor registry may not be able to
provide a com- pletely matched unit for a
given patient; many “HLA-matched” units
actually carry antigens against which the
patient may have an alloan- tibody. (The
presence of serologic cross-reac- tive groups
prevents recognition of some anti- gens as
foreign but, at the same time, reduces the
range of donors whose platelets are truly
compatible.) Platelet crossmatching is
another approach that can be utilized, with
the lack of in-vitro reactivity used as a
predictor of good in-vivo compatibility. This
technique is often still used when
alloimmunization is directed at a platelet
antigen because few blood-collec- tion
agencies have phenotyped their donors for
these antigens. However, neither approach
can guarantee a good result more than 50%
to 80% of the time, although a “good” HLA
match (A or BU grade) provides the best
prediction of transfusion success.146 An
alternative, simpler approach that parallels
the practice with pa- tients who are
alloimmunized against red cell antigens is
the selection of platelet units that lack the
antigen(s) against which the recipient is
immunized and those from cross-reacting
groups (Fig 20-1).147
Antibodies provoked by or directed at a
wide variety of drugs can lead to
thrombocyto- penia.148 These drug-
dependent platelet anti- bodies (DDPAs) can
lead to mild or profound thrombocytopenia
and to refractoriness to transfused
platelets.149 Although often thought of as
rare, DDPAs can be caused by many drugs
and are present in many patients. For
example, in one study, approximately 10%
of patients receiving gentamicin had a drop
in their platelet counts, and almost half of
these patients (5.9% overall) had an
antibody that interacted with platelets in the
presence of gentamicin. 150 When
refractoriness to platelet transfusion cannot
be explained by the pa- tient’s condition or
alloimmunization to HLA or platelet
antigens, consideration should be given to

Dealing with Platelet Refractoriness
Responses to platelet
transfusion are most of-
ten quantitated using the
CCI 1 hour after
transfusion (Table 20-
5).151 However, a sample
collected 10 minutes
after transfusion yields
similar information and
may be easier to ob- tain
routinely.152 The CCI
calculation is based on
the count increment
(count increment =
posttransfusion count –
pretransfusion count),
platelet content of the
unit (expressed as × 10-
11
), and size of the
patient [expressed as
body sur- face area
(BSA) in m2]. For an
adult patient with a
BSA of 2.0 m2 whose
platelet count rose from
5,000/µL to 25,000/µL
after a platelet
transfusion containing
4.0 × 1011 platelets, the
CCI would be:
transfuse to a patient with platelet
refractoriness. Alter- native measures can be
tried to stem or fore- stall hemorrhage, but
these are of unpredict- able benefit.
Administration of antifibrinolytic agents,
such as -aminocaproic acid (Amicar),
either intravenously or, in the case of
gingival bleeding, as a mouthwash may
allow the clot that is formed to be
sustained.108
Curiously, administration of intravenous
Ig (IVIG), an effective therapy for
autoimmune thrombocytopenia, appears to
be of little ben- efit in alloimmune
refractoriness. IVIG can yield increased
increments shortly after trans- fusion, but
the durability of the response is limited and,
by 24 hours after transfusion, the patient
usually returns to his or her baseline level. 108
The claim that platelets are accumu- lating at
sites of bleeding even though they are not
detected in circulation via a platelet count

(count increment 
BSA)/unit content
= (20,000/µL ×
2.0 m2)/4.0 =
10,000

Units (m2/µL) are

usually omitted when
reporting the result. A
CCI above 7500 is con-
sidered evidence of a
successful transfusion;
two transfusions with
CCIs below 7500 within
an hour after transfusion
are evidence of re-
fractoriness. A similar
approach is used to
compare the recovery
rate of platelets to the
expected rate.108
Occasionally,
despite diligent efforts,
no compatible platelets
can be found to
 CH A P T E R 20
increment has not been substantiated and is
unlikely to be true.108,153-155 Some experts have
advocated administration of platelets by
slow drip, perhaps after making aliquots of a
thera- peutic dose and administering it over
4 to 12 hours. This approach has the benefit
of allow- ing the clinician to feel that
“something” is be- ing done while
minimizing the demand on the transfusion
service inventory; however, the predicted
benefit to the patient is based pri- marily on
anecdotal experience.
Contraindications to Platelet
Transfusion
Idiopathic (autoimmune) thrombocytopenia
(ITP) can cause profoundly low platelet
counts, but patients with autoimmune ITP
(particular- ly children) rarely suffer
hemorrhagic conse- quences.156,157 The
transfusion of platelets in the stable,
nonbleeding patient with ITP offers no
benefit because the platelets are rapidly
cleared by the circulating antibodies. In
situa- tions where the patient is bleeding,
clinicians may feel compelled to take some
action, including using antifibrinolytic
agents and platelet transfusion. Success in
stemming hemorrhage with transfusion is
not universal, but bleeding may slow down
or cease after transfusion in perhaps half or
two-thirds of at- tempts; this proportion is
high enough to war- rant trying transfusion,
particularly when the bleeding is serious.158
Thrombotic thrombocytopenic purpura
(TTP) has traditionally been regarded as a
contraindication to platelet transfusion.159,160
Thrombocytopenia occurs in TTP as
platelets are activated and consumed in
thromboses triggered by abnormally large
multimers of vWF that are capable of
inciting inappropriate platelet activation
without other cofactors.161 The resulting
thrombocytopenia may be pro- tective, then,
in slowing the formation of addi- tional
pathologic thromboses. Case reports
describing the development of coma in close
temporal relationship to platelet transfusion
led to the idea that platelets may “add fuel to
the fire” and prompt further thromboses in
critical sites. A recent review of the
literature accompanied by a prospective
observational
Hemotherapy Decisions ■ 517
study of 54 patients with TTP found no in-
creased frequency of neurologic events or
death in patients who received platelet trans-
fusions.154 Other, well-designed trials are
need- ed to refute or confirm the belief that
platelet transfusion is contraindicated in
TTP.
Platelet transfusion is also usually avoid-
ed in patients with heparin-induced thrombo-
cytopenia (HIT), especially the immunologic
(Type II) form, to forestall the development of
limb- and life-threatening thromboses.162
Other Uses of Platelets
Autologous or allogeneic platelets may also
be applied topically to an area of surgical
recon- struction. The presence of platelet-
derived growth factor through applications
of PRP or platelet gels is thought to
stimulate angiogene- sis and promote more
rapid tissue repair.163,164 A recent systematic
review of RCTs on PRP concluded that,
although the use of PRP may reduce the
volume of blood components transfused for
an average savings of 247 mL per patient,
the use of PRP in adult elective surgery
cannot be justified due to the risk of adverse
events, such as hypotension, and the fact that
other blood-sparing techniques (eg, use of a
lower transfusion threshold or antifi-
brinolytic agents) are supported by stronger
evidence.165
PLASMA TRANSFUSION
Indications for Plasma
The current data needed to support
evidence- based guidelines for plasma
transfusion are surprisingly weak. A
systematic review of the available data for
subsequent development of guidelines by an
expert panel found the data to be sparse and
of low quality.166 The panel developed a
guidance document after taking into account
the potential benefits and harms in the
available data.167 Indications for plasma
transfusion with reasonable support included
massive transfusion and reversal of warfarin
anticoagulation in patients with intracranial
hemorrhage. In other clinical scenarios, such
as surgery without massive transfusion or
518 ■ AABB T EC HNIC AL MANUAL
warfarin reversal in the absence of
intracranial hemorrhage, the panel did not
develop recom- mendations due to
insufficient data. The panel recommended
against plasma infusion in situ- ations that
might result in prophylactic plasma infusion,
such as acute pancreatitis or critical- ly ill
nonsurgical noncardiac patients. In these
types of patients where coagulopathy or
bleeding is absent, the risk of lung injury
and mortality outweigh any perceived
benefit.165 A more recent review echoes the
conclusion that prophylactic plasma infusion
may not provide benefit.168
Common Clinical Scenarios and the
Limitations of Laboratory Tests to
Support Transfusion Decisions
Many studies have shown that abnormal
coag- ulation test results do not predict an
increased risk of hemorrhage. 169 Standard
coagulation screening tests are poor
predictors of hemor- rhage risk, in part
because they are inordinate- ly sensitive to
mild deficiencies of multiple
procoagulants.170 As a result, the test results
become abnormal at factor levels that are
not clinically associated with bleeding.
Additional confusion arises from the fact
that the critical thresholds in these studies,
1.3 times the up- per limit of normal or 1.5
times the midpoint of the reference range
(usually almost the same number), usually
fall at about an inter- national normalized
ratio (INR) of 1.5 to 2.0.
Because the usual target for oral anticoag-
ulation therapy is an INR of 2.0 to 3.0, some
cli- nicians assume that patients with an INR
close to 2.0 require correction before surgery.
How- ever, although reducing coagulation in
pa- tients with an INR of 2.0 prevents their
con- genital risk factor for hypercoagulability
(eg, Factor V Leiden) or acquired abnormality
(eg, prosthetic cardiac valve) from triggering
un- wanted clotting, this does not mean that
nor- mal, physiologic triggers of the clotting
sys- tem will be unable to cause an appropriate
thrombotic sequence. This distinction is im-
portant to make when deciding whether a pa-
tient requires correction of a slightly abnor-
mal coagulation level. The guidelines for
plasma administration of several professional
associations, including the American Society
of Anesthesiologists and the College of Ameri-
can Pathologists, recommend correction of the
overcoagulation by plasma transfusion prior
to surgery or to facilitate thrombosis only for
an INR of approximately 1.5 to 2.0.171,172
The British Committee for Standards in
Haematology noted, similarly, the limited
utility of attempting to correct mild
overcoag- ulation.173 Their guidelines for
correcting excessive anticoagulation and
those of the American College of Chest
Physicians174 (Ta- ble 20-7) notably avoid
calling for the use of plasma in lieu of
administration of vitamin K until very
abnormal INRs and bleeding are en-
countered. Administration of vitamin K can
re- verse the effects of warfarin promptly
(within 6-24 hours) without complicating
the reestab- lishment of healthy levels of
anticoagula- tion.175
Prothrombin complex concentrates
(PCCs) can also be used to rapidly correct
the effects of warfarin.176 Success has been
reported from combining the transfusion of 2
units of plasma with three-factor PCCs, or
by using a four-fac- tor PCC.177 This is
discussed more fully in the “PCC” section
below.
With the approval of new
anticoagulants that act at different points of
the coagulation system, questions about
reversal have arisen when emergent surgery
is required or bleeding occurs. The direct
thrombin inhibitors and Factor Xa inhibitors
function as their names indicate and offer
the benefits of short half- lives and a return
to baseline coagulation lev- els in
approximately 24 hours with adequate renal
function upon cessation of the drug.
However, there is currently no approved
rever- sal agent. Case reports indicate that
attempted reversal with plasma in bleeding
patients was not successful, and more
evidence on the utili- ty of PCCs and
prohemostatic therapies is nec- essary.178
Laboratory tests to determine the amount of
drug circulating are still in develop- ment.
Patients with cirrhosis commonly have
coagulation abnormalities due to their syn-
thetic difficulties. They also often
experience gastrointestinal bleeding due to
increased por- tal vein pressure. However,
much more fre-
 CH A P T E R 20 Hemotherapy Decisions ■ 519

TABLE 20-7. Guidelines for Correction of Excessive Oral Anticoagulation

Clinical SituationGuideline

INR > therapeutic but < 5, no

significant bleeding

Lower anticoagulant dosage.

INR > 5 but < 9, no significant bleeding
INR is in therapeutic range or, if patient is at increased
risk of hemorrhage, omit a dose and give 1-2.5 mg
INR > 9, no significant bleeding
Serious bleeding at any
elevation of INR
Life-threatening hemorrhage
Temporarily discontinue drug if necessary.
Omit 1-2 doses; monitor INR. Resume oral anticoagulation when
vitamin K1 orally.
For rapid reversal before urgent surgery: 2-4 mg vitamin K1
orally; repeat dose with 1-2 mg at 24 hours if INR remains
elevated.
Omit warfarin; give 5-10 mg vitamin K1 orally.
Closely monitor INR; give additional vitamin K1 if necessary.
Resume warfarin at lower dose when INR is within therapeutic
range.
Omit warfarin.
Give 10 mg vitamin K1 by slow intravenous infusion.
Supplement with plasma or prothrombin complex concentrate
depending on urgency of correction.
Vitamin K1 infusions can be repeated every 12 hours.
Omit warfarin.
Give prothrombin complex concentrate with 10 mg vitamin
K1 by slow intravenous infusion.

Repeat as necessary, depending on INR.

INR = international normalized ratio.
Adapted from guidelines developed by the American College of Chest Physicians. 174

quent hemorrhage and an inability to clot protein C.

might be expected in these patients based on
their prothrombin times (PTs) only. The
reason why people with cirrhosis do not
bleed abnor- mally—and why intricate pro-
and antithrom- botic balances are inherent in
their clotting system—was recently
elucidated by demon- stration that thrombin
generated in vitro in plasma from healthy
individuals and people with cirrhosis was
the same, but only after the addition of
thrombomodulin, which activates protein C,
to the test system.179
As the liver’s synthetic production de-
clines, the amount of procoagulants circulat-
ing in plasma decreases, but so does the
level of certain anticoagulants, such as
 Less activity may be expected
in the procoagu- lant system
of a patient with cirrhosis,
but, at the same time, there
will be less of an opposing
force from the coagulation-
control system.179 The PT and
partial thromboplastin time do
not reflect thrombin
generation in these patients in
a way that predicts bleeding.
Until a more accurate
predictive test becomes
available, one role of the
transfusion medicine
physician will continue to be
to help other cli- nicians
understand why patients with
severe liver disease often do
not require heroic efforts (or
heroic amounts of plasma) to
completely normalize their
coagulation system.
What happens if an attempt is
made to
correct the patient’s PT before a
procedure?
520 ■ AABB T EC HNIC AL MANUAL
Often, the answer is “surprisingly little.”
The authors of one study noted that the
mean re- duction in INR was only 0.03 per
unit of plas- ma transfused.180 Another study
showed that the PT decreased to the normal
range in less than 1% of plasma recipients
who had mildly abnormal PTs before
transfusion, and the dif- ference in the upper
limit of normal was re- duced by half in only
14.5%.181 The mean de- crease in PT was
only 0.2 second, and there was no
correlation between PT abnormality and
subsequent RBC transfusion. This study also
revealed that only 1 in 10 patients receiv-
ing plasma had their PT rechecked within 8
hours—despite the fact that the ordering
clini- cian thought that the correction was
clinically important and the expected
shortening of PT occurred infrequently. The
small corrections in PT/INR may reflect the
fact that these re- ductions have an
exponential relationship, rather than a linear
relationship, to the pro- portion of
coagulation factors in circulation (Fig 20-2).
In cases of rapid bleeding, a more
proac- tive approach may be beneficial. The
altera- tions that occur in a massive
transfusion situation are a combination of
dilutional coag- ulopathy, injury-driven
factor consumption, and activation of
fibrinolysis.182 Correction of a true
coagulopathy after it becomes clinically
7
6
5
4
INR
3
2
1
0
0 20 40
% Factor Levels
FIGURE 20-2. Exponential relationship of international normalized ratio (INR) to % factor
levels. (Used with permission from Wayne L. Chandler, MD, Houston Methodist
Hospital.)
manifest can be much more difficult, and in-
creased transfusion of other components
may be needed. Although the early use of
non-RBC components reduces mortality
risk,16 this ap- proach fails to take into
consideration each patient’s situation; direct
involvement of a transfusion medicine
specialist can best guide hemotherapy in
these complex, rapidly evolv- ing situations.
Such consultation also takes into account
other important factors, such as reduced
patient temperature and the presence of
acidosis that decrease the in-vivo activity of
the coagulation system significantly.183
Rapid whole-blood testing techniques
have become integrated into many centers’
transfusion protocols for massively bleeding
patients. Common viscoelastic coagulation
testing (VCT) platforms include
thromboelas- tography and rotational
thromboelastometry. These tests measure
the speed and strength of clot formation.
The benefits of these tests include that they
generate initial results within 15-20 minutes
and can be used to assess fibri- nolysis.
Current challenges, however, involve lack of
standardization of results, correlation with
standard coagulation tests, or training in
interpreting the results.184
A recent meta-analysis of RCTs on
TEG- based treatment in massive transfusion
in- cluded nine studies of cardiac surgery
and one
60 80 100 120
 CH A P T E R 20 Hemotherapy Decisions ■ 521

liver transplant study in 776 patients. 185 The
authors found no difference in mortality be-
tween TEG-guided therapy and
conventional practice. There were moderate
decreases in bleeding and numbers of
patients transfused with both FFP and
platelets in patients under- going TEG-
guided treatment; however, the
heterogeneity of the studies limited the
ability to draw definitive conclusions. VCT
use in trauma appears to predict the need for
mas- sive transfusion and risk of mortality;
however, most data come from observational
studies, and this issue would benefit from
additional RCT evidence.186
Dose and Timing of Plasma
Transfusion
Although the level of coagulation factors nor-
mally varies widely between 50% and 150%
of the activity of circulating clotting factors,
nu- merous publications show that people
have the ability to form clots with significantly
low- er levels of clotting factors. 187 For single
factor deficiencies, such as deficiencies of
Factors VIII or IX, 30% activity is often
needed for he- mostasis. In patients with
multiple factor defi- ciencies, such as after
trauma, factor levels closer to 40% may be
needed for hemostasis (see Fig 20-2 and Table
20-8). In addition, the further the patient’s
procoagulant activity is from the normal
range, the easier it is to effect
a change in the PT because the relationship
between factor activity and PT is more
expo- nential than linear.179,188
A usual dose of plasma is 10-20 mL/kg.
This dose is expected to increase the level of
coagulation factors by 20% immediately
after infusion. Precise prediction of the
amount of plasma needed to be transfused to
correct a particular coagulopathy is not
currently possi- ble. Thus, posttransfusion
repetition of the co- agulation test that
prompted the transfusion is warranted.179,181
When attempting to correct a coagulopa-
thy with plasma transfusion, the biological
half-life of procoagulants must also be consid-
ered. Factor VII has the shortest half-life in
vivo (approximately 5 hours). If a transfusion
raises the patient’s activity of this factor from
30% to 45% 5 hours later, for example, the
activity level will be halfway back to the
steady-state level for that patient (ie, to 37%).
Additional correction attempts must now
change the factor concentration in an en-
larged plasma volume, and multiple plasma
transfusions can produce pulmonary edema
through fluid overload. In addition, if correc-
tion is truly required before a hemostatic chal-
lenge, such as major surgery, the plasma
should be infused shortly before the procedure
for the benefit to be incurred at the time of the
hemostatic challenge.

TABLE 20-8. Coagulation Factor Half-Lives

Factor In-Vivo Half-Life % Needed for Hemostasis

I 3-6 days 12-50
II 2-5 days 10-25
V 5-36 hours 10-30
VII 2-5 hours >10
VIII 8-12 hours 30-40
IX 18-24 hours 15-40
X 20-42 hours 10-40
XI 40-80 hours 20-30
XIII 12 days <5
522 ■ AABB T EC HNIC AL MANUAL
Types of Plasma
Several types of plasma may be available for
transfusion, and, for most situations, they
can be used interchangeably. To be called
“FFP,” the unit must be frozen within 8
hours of col- lection and transfused within
24 hours of thawing, thus optimizing the
levels of the most labile procoagulants,
Factors V and VIII. How- ever, congenital
Factor V deficiency is rare. Most patients
requiring plasma transfusion al- ready have
an elevated Factor VIII level be- cause it is
an acute phase reactant. Therefore, Plasma
Frozen within 24 Hours After Phlebot- omy
(PF24) has procoagulant contents that are as
capable as FFP of correcting most clinical
coagulopathies.189,190
Thawed Plasma made from PF24 or FFP
and kept refrigerated after thawing can be
ef- fectively used throughout its 5-day shelf
life because most coagulation factors remain
at hemostatic levels.191,192 Thawed Plasma
can be part of an effective strategy aimed at
reducing wastage of plasma in combination
with the implementation of appropriate
ordering prac- tices, as described above.
Transfusion of Plasma for Other
Indications
There are several other clinical situations
where the transfusion of plasma is indicated
based on slightly different rationales. Thera-
peutic plasmapheresis may call for partial or
complete replacement of the removed
volume of patient plasma. This may
occasionally be necessitated by frequent (ie,
daily) procedures that can deplete
procoagulant levels more quickly than they
can be replenished. Howev- er, most patients
do not require such an ag- gressive
plasmapheresis schedule, and the need to
use plasma for factor replenishment is very
unusual. TTP presents a different situa- tion,
however, because the plasma’s content of
ADAMTS13 is therapeutic, replenishing the
patient’s supply that has been reduced by au-
toantibody or congenital deficiency, for
exam- ple.161
Congenital deficiencies of procoagulants
may require prophylactic or therapeutic re-
plenishment via plasma transfusion, but pa-
tients with these deficiencies are rare. C1 es-
terase inhibitor deficiency causes hereditary
angioedema due to inappropriate activation
of complement. Its management may
include administration of a C1 inhibitor
derived from human plasma.191 Factor XI
deficiency, al- though not a cause of
spontaneous bleeding, often results in
excessive postoperative bleed- ing.191 In
emergent situations where specific
concentrates are not available, transfused
plasma can be used as a source of the
deficient factor. 193,194
CRYOPRECIPITATE
TRANSFUSION
The discovery that certain proteins critical to
the coagulation system could be
concentrated by thawing FFP at 4 C and
centrifugally sepa- rating the plasma from
the supernatant pro- vided the first truly
effective treatment for pa- tients with
hemophilia A, the congenital deficiency of
Factor VIII. Cryoprecipitated an- tihemophilic
factor, known as “cryoprecipi- tate,” is
rarely if ever used today for its original
purpose, having been supplanted by other
simpler and safer means of providing the
defi- cient factor. Nevertheless,
cryoprecipitate is an essential component in
modern hemotherapy.
Although the US regulatory
requirements for the content of
cryoprecipitate are based on Factor VIII
content (minimum = 80 U/unit), it is for its
fibrinogen content that cryoprecipi- tate is
most commonly used. When fibrinogen
consumption (eg, in DIC) and/or loss (eg,
due to massive hemorrhage) occur,
exogenous re- placement may be necessary
to maintain plas- ma’s coagulation potential.
The fibrinogen concentration necessary to
maintain hemo- stasis is in the range of 50-
100 mg/dL. Howev- er, coagulation test
results that are used to fol- low patients with
such conditions usually become abnormal
due to hypofibrinogen- emia when the
fibrinogen concentration drops below 100
mg/dL. Therefore, maintaining the
fibrinogen concentration above this level
aids both the patient and those attempting to
un- derstand the situation through laboratory
test- ing. When fibrinogen levels drop as a
result of
 CH A P T E R 20 Hemotherapy Decisions ■ 523
to stop bleeding

DIC or ongoing, high-volume hemorrhage,

it may be wise to initiate cryoprecipitate
transfu- sion (or at least prepare the
component) as the critical point of 100
mg/dL is approached (eg, at approximately
120 mg/dL).
Although the dosage of cryoprecipitate
is often stated in tens of units (eg, 10, 20, or
30 units), the dosage required to achieve the
de- sired effect is readily calculated. This
calcula- tion is based on the difference
between the current and desired (usually 200
mg/dL) con- centration of fibrinogen, a
projection of the patient’s plasma volume [as
(1 – hematocrit) ×
0.7 dL/kg × body mass, or 30 dL if the
patient’s weight is unknown], and the usual
fibrinogen content of cryoprecipitate (250
mg/unit):

Dose (units) = [desired fibrinogen

increment (mg/dL) × plasma
volume]/250 mg/unit

Although the small volume (5-15 mL)

of each cryoprecipitate unit facilitates rapid
thawing, the pooling of units (usually
includ- ing rinsing the bags to maximize
recovery) can be time consuming. Planning
ahead for rapid use later in case a patient
needs a massive transfusion, for example,
can certainly facili- tate patient care. Some
facilities have chosen to produce pools of
cryoprecipitate using ster- ile techniques
before freezing the component. This product
has a higher fibrinogen content and a shorter
time to issue when needed.
Dysfibrinogenemias are rare congenital
abnormalities that result in dysfunctional fi-
brinogen molecules. These molecules can
lead to an increased risk of thrombosis,
bleeding, or both, although they can also
have no clinical manifestations.195 Patients
may require admin- istration of
cryoprecipitate to correct the deficiency.
Some degree of dysfunctional fi- brinogen is
also produced in damaged or re- generating
livers and in neonates, but this rarely results
in the need to transfuse normal fibrinogen.196
States of localized fibrinolysis can be
in- duced during surgeries that disrupt the
pros- tatic bed, urothelium, or salivary gland
tissue because plasminogen activators are
produced by these tissues. In these cases,
cryoprecipitate infusions may be warranted
 rent practice because products that have
both lyophilized plasma and thrombin are
with or without the use of avail- able commercially.200 Similarly, virus-
antifibrinolytic ther- apy. inactivat- ed fibrinogen concentrates are
Note that bleeding from the available that are more readily stored
urinary tract is a relative (lyophilized) and pack- aged for simple use
contraindication to (discussed below).201
antifibrinolytic therapy. States
of systemic fibrinolysis can GRANULOCYTE TRANSFUSION
oc- cur with medical
conditions such as wide- The development of apheresis technology
spread amyloidosis or as a sev- eral decades ago allowed the collection
result of chemo- therapy with of granulocytes (neutrophils) to transfuse to
L-asparaginase, typically used pa- tients with neutropenia and serious
to treat acute lymphoblastic infections. Most of these patients were not
leukemia. able to mount an appropriate response
Although no longer because of marrow hypoplasia (eg, due to
considered for rou- tine use chemotherapy). The
due to the availability of
concentrated plasma
derivatives of copurified
Factor VIII and vWF,
cryoprecipitate can be used as
a source of vWF for patients
with von Wille- brand disease
(vWD). The most common
form of vWD is Type 1,
which results in a deficiency
of a normal protein. Type 1
vWD most often can be
managed with administration
of des- mopressin. Patients
with Types 2 and 3 vWD need
to have their bleeding
managed with an exogenous
source of vWF. See Table 20-
9 for general factor
replacement guidelines for the
treatment of vWD.
Cryoprecipitate can also be applied topi-
cally on surfaces where
suturing is ineffective to
achieve hemostasis and that
are slow to generate clot, such
as the raw surface of trau-
matically injured liver.199
When applied simul-
taneously with calcium and
thrombin (usually using two
syringes), a thick, gelatinous
mass quickly forms over the
area and quells the bleeding.
This approach can also be
used to bind together
structures, such as in surgery
in the inner ear.
Cryoprecipitate is not
typically requested from the
transfusion service in cur-
524 ■ AABB T EC HNIC AL MANUAL

TABLE 20-9. General Factor Replacement Guidelines for Treatment of Hemophilia A and von
Willebrand Disease

Minimum Desired Factor VIII Dose Factor IX Duration

Indication Factor Level (%) (IU/kg) Dose (IU/kg) (days)

Hemophilia*
Severe epistaxis, oral mucosal 20-30 10-15 20-30 1-2
bleeding†
Hemarthrosis, hematoma, persistent 30-50 15-25 30-50 1-3
hematuria,‡ gastrointestinal bleed-
ing, retroperitoneal bleeding
Trauma without signs of bleeding, 40-50 20-25 40-50 2-4
tongue/retropharyngeal bleeding†
Trauma with bleeding, surgery, 100 50 100 10-14
intracranial bleeding§
von Willebrand Disease¶
Major surgery 50 40-60 daily
Minor surgery 30 30-50 daily or every other day
Dental extractions 30 20-30, single dose 12 hours
Spontaneous bleeding 30 20-30, single dose
*Data from US Pharmacopeia. Dosing intervals based on a half-life of Factor VIII over 8-12 hours (2-3
197

doses/day) and half-life of Factor IX over 18-24 hours (1-2 doses/day). Maintenance doses of one-half the initial
dose (as shown) may be given at these intervals. The dosing frequency depends on the severity of bleeding, with more
frequent dosing used for seri- ous bleeding.
†
In addition to antifibrinolytics.
‡
Painless spontaneous hematuria usually requires no treatment. Increased oral or intravenous fluids are necessary to
main- tain renal output.
§
Factor may be administered continuously. Following the initial loading dose, a continuous infusion at a dose of 3
IU/kg per hour is given. Subsequent doses are adjusted according to measured plasma factor levels.
¶
Concentrates labeled in terms of the ratio of von Willebrand factor to ristocetin cofactor. The recommended doses
for adults, number of infusions, and target plasma levels are the same as those for Factor VIII.198

therapy was also administered to patients ery of granulocyte production, differentiation,

with congenital neutrophil dysfunction [eg,
chronic granulomatous disease (CGD)]. Not
all pa- tients benefited from this approach,
however, and only modest improvements in
outcome were noted in a majority of—but
not all— trials.202-206 The published literature
offers re- views of this and other topics
related to granu- locyte transfusion
therapy.207,208
With the development of more potent
an-
timicrobial drugs and the availability of
cyto- kines that promote more rapid marrow
recov-
and function [such as granulocyte colony-
stimulating factor (G-CSF) and granulocyte/
macrophage colony-stimulating factor], the
frequency of granulocyte transfusion fell to
very low levels.209,210 This low utilization was
also prompted by the recognition that the
amount of granulocytes that could be collect-
ed from a donor, even one mobilized with pri-
or administration of a corticosteroid, on the
order of 1 × 1010, was only 1% to 10% of what
a healthy marrow would produce each day in
re-
 CH A P T E R 20
sponse to a serious infection. Trials whose au-
thors reported success in using granulocyte
transfusion therapy generally transfused high-
er doses of granulocytes, but these higher
yields were very difficult to obtain.
More recently, the concept of
granulocyte transfusion therapy using
components with higher, “more therapeutic”
content has gener- ated renewed attention to
this component. Despite the availability of
newer antimicrobial agents, infection
remains a common problem in patients
undergoing rigorous chemothera- py
regimens, especially those culminating in
hematopoietic stem cell transplantation, and
more rigorous T-cell depletion schemes in
al- logeneic hematopoietic transplantation
that lead to an increased frequency of
serious and fatal infections, usually due to
yeasts and fungi.211,212 Case reports of
success in treating patients with CGD with
fungal infections dur- ing transplantation
have also rekindled inter- est in granulocyte
transfusion.213,214
With the administration to donors of 8
mg dexamethasone orally and 5 µg/kg G-
CSF sub- cutaneously 8 to 16 hours before
apheresis, yields of up to 1011 granulocytes
(or more) have been reported.215 A clinical
trial to document the effectiveness of these
more potent compo- nents is under way
through the Transfusion Medicine
Hemostasis Clinical Trials Network of the
National Heart, Lung and Blood Insti- tute.
This use of G-CSF is not approved by the
FDA, and donor informed consent should be
obtained before using this approach. In addi-
tion, the suggestion that corticosteroid
admin- istration may lead to posterior
subcapsular cataracts has prompted some
clinicians to consider avoiding the
dexamethasone admin- istration to donors
even though this decreases yield by one-
quarter.216 The potential utility of
prophylactic granulocyte transfusions
remains unclear at present, again owing to
dosage con- siderations.217
Neonates born preterm or after pro-
longed premature rupture of membranes are
at increased risk of bacterial sepsis. Their
neu- tropenia is related to a temporary
limitation in the production of neutrophils
from a marrow primed to produce red cells.
Some of these pa- tients have been treated
with transfusions of
Hemotherapy Decisions ■ 525
granulocytes generated either through an
apheresis procedure or from whole-blood-
derived buffy coats. The latter can provide
only a limited number of cells and has not
been found to be as clinically efficacious as
apheresis.218 Today, potent antibiotics are
used much more commonly than granulocyte
transfusions in neonates. IVIG may also be
giv- en because premature infants may have
hypo- gammaglobulinemia,219 although IVIG
may in- crease these infants’ susceptibility to
other potentially fatal infections.220
Patients with severe neutropenia (abso-
lute neutrophil count <500/µL) are consid-
ered for granulocyte therapy if the infection
cannot be controlled with appropriate
bacteri- cidal antimicrobials and the
neutropenia is temporary, meaning that
recovery of endoge- nous production in a
few days is likely. Patients with CGD are
usually considered for granulo- cyte
transfusions if they have deep-seated ab-
scesses and/or fungal infections that are not
responding to antimicrobial therapy.
Components must be ABO compatible
if the granulocyte collection contains 2 mL
or more of red cells, as is often the case. If
the pa- tient requires CMV-reduced-risk
transfusions, the donor should be CMV
seronegative be- cause leukocyte reduction
cannot be used on these components. If the
recipient is alloim- munized to HLA
antigens, HLA-compatible donors need to
be found or the transfused cells could be
cleared rapidly and possibly cause untoward
reactions without achieving their intended
purpose. If patients are at risk of
transfusion-associated graft-vs-host dis-
ease, as is usually the case for patients with
neutropenia, units may be gamma irradiated.
Donors often undergo infectious disease
testing when they present for predonation
stimulation to facilitate rapid release of the
collected component following documenta-
tion of ABO/Rh type. Granulocyte units
should be stored for the shortest period
possible at room temperature without
agitation and ad- ministered through regular
blood component filters (eg, 170 microns).
Drugs known to inter- act with granulocytes,
such as amphotericin, are best given at times
remote from the granu- locyte transfusions
(ie, 12 hours before or
526 ■ AABB T EC HNIC AL MANUAL
after). Transfusions are usually given daily,
and sometimes more frequently if yields are
low, until the patient recovers from the
infection and/or neutrophil production returns.
PLASMA-DERIVATIVE
TRANSFUSION
Plasma proteins are derived from both source
plasma, collected for the purpose of manufac-
turing plasma derivatives, and recovered plas-
ma that has been separated from whole-blood
donations. In the United States, approximately
6 million liters of source plasma and 2.5 mil-
lion units of recovered plasma are produced
each year. The final products are subjected to
one or more virus-inactivation techniques.
Additional testing (eg, for hepatitis A virus or
parvovirus) may be performed by nucleic acid
amplification techniques to add an additional
margin of safety.
The prions associated with variant
Creutzfeldt-Jakob disease (vCJD) appear to be
successively removed through multiple steps
in the separation process to levels that are be-
yond the limit of detection. Neither classical
CJD nor vCJD cases have reportedly been at-
tributed to the transfusion of plasma deriva-
tives.221
The pathogen-inactivation techniques
described above have an excellent record of
not transmitting infectious diseases.
However, most patients with hemophilia in
the United States prefer transfusions of
Factor VIII or Fac- tor IX concentrates
produced via recombinant DNA technology
and, whenever possible, that have not been
manufactured using any human proteins.
Immunoglobin products have often
been regarded as free of infectious risks
because of their production method and/or
neutralizing antibodies. However, multiple
examples of hepatitis C transmission have
raised doubts regarding this belief, although
the units in- volved in these cases were
usually prepared through nonstandard
donor-selection or pro- duction methods.222-
225
Albumin
Albumin was the first of the modern plasma
proteins to be developed for transfusion. It
serves as a colloid, expanding plasma
volume by approximately the volume
administered at the 5% concentration with a
half-life of ap- proximately 16 hours.226
Unless the container has been damaged,
albumin’s pasteurization at 60 C for 10 hours
prevents the transmission of viruses or
bacteria.
There are several obvious and accepted
applications for albumin, including volume
replacement in patients who are
unresponsive to crystalloids,
postparacentesis volume man- agement in
patients who are unresponsive to colloids,
volume replacement in patients with severe
necrotizing pancreatitis, patients with
diarrhea (>2 L/d) or hypoalbuminemia (<2.0
d/dL) on enteral feedings who do not
respond to short-chain peptide
supplementation, and plasmapheresis.227 Use
of albumin infusions simply to raise the
albumin concentration in patients with
cachexia or other chronic hypo-
albuminemia is ineffective and inappropriate
but may be helpful in treating the complica-
tion of acute hypoalbuminemia if a
concentra- tion of at least 3.0 g/dL can be
achieved.228
In most cases of hypovolemia, crystal-
loids are used as the primary means of
volume replacement because of their
effectiveness, availability, and cost. A meta-
analysis of recent RCTs found no decrease in
mortality associat- ed with the use of
colloids compared to crys- talloids in
critically ill patients, with no benefit from
albumin vs crystalloid specifically.229 When
colloid support is required, other, non-
human sources, such as hydroxyethyl starch
and dextran, may also be useful for both
clini- cal use and apheresis, although the
volume that can be administered in a day
without af- fecting coagulation function may
be limited.230 Synthetic substitutes may
prompt an anaphy- lactic reaction in rare
cases and have been as- sociated with a
syndrome of pruritus begin- ning several
weeks after administration.231
Clotting Factor Concentrates
A complete discussion of the treatment of he-
mophilia A (Factor VIII deficiency) and
hemo-
 CH A P T E R 20
philia B (Factor IX) deficiency is beyond
the scope of this text. Although the first
effective treatment for hemophilia A was
cryoprecipi- tate transfusion, patients with
moderate (1%- 5% Factor VIII activity) or
severe (<1% activity) disease are now treated
exclusively with a lyophilized concentrate.
The same treatment has evolved for patients
deficient in Factor IX, also an X-linked
recessive condition.
For both conditions, the dose of the
factor to be administered can be easily
calculated based on the patient’s plasma
volume [body mass  70 mL/kg  (1 –
hematocrit)] multi- plied by the desired
increase in activity level. Thus, a 70-kg
male (hematocrit = 40%) with 1% activity
who needs his activity level raised by 99%
(0.99 IU/mL) to 100% (1 IU/mL) would re-
quire approximately 2900 IU of Factor VIII.
Be- cause the half-life of Factor VIII is 8 to
12 hours, repeat doses will be required to
ensure hemostasis (ie, >30% activity)
through surgery or a bleeding episode (often
a period of at least 10 days); see Table 20-9.
Because recovery of activity may vary by
product and patient, close follow-up of pa-
tients using repetitive assays of factor activity
is advised. Patients may also be maintained on
regular prophylactic doses of Factor VIII to re-
duce the risk of spontaneous hemorrhage and
preserve joint function, although the value of
this approach has been difficult to prove be-
cause of the small sizes of most trials. 232,233
Nevertheless, prophylaxis is a widely recom-
mended course of action.
About one-third of patients with severe
hemophilia A develop antibodies against
Fac- tor VIII because they do not produce it
or they produce a defective form. These
patients can be very difficult to support
because it is im- practical to overwhelm the
inhibitor (anti- body) with higher and higher
doses of Factor VIII to stem hemorrhage. A
variety of treat- ments have been tried,
including a high-dose desensitization
program, immunosuppres- sion, and, when
rapid reduction in inhibitor ti- ters is needed,
plasmapheresis. The advent of recombinant
Factor VIIa to bypass the inhibi- tion of
Factor VIII activity has improved the
management of bleeding in this dreaded
com- plication of hemophilia therapy.
Hemotherapy Decisions ■ 527
Prothrombin Complex Concentrates
Procoagulants that require vitamin K for ap-
propriate carboxylation and normal activity
— Factors II (prothrombin), VII, IX and X
—are called the “vitamin K-dependent
factors.” They have long been known to be
separable from plasma by simple chemical
means and have been available as Factor IX
complex con- centrate or PCC. This product
should not be confused with a Factor IX
concentrate used to treat hemophilia B. The
latter, now also avail- able as a recombinant
product, contains only Factor IX.
The first technique to produce PCC re-
sulted in activation of a substantial propor-
tion of some procoagulants and sometimes
led to unwanted thromboses. This
complication precluded its routine use, but
the product was beneficial in bypassing the
effect of Factor VIII inhibitors in some
patients with hemophilia A. This activated
form of PCC concentrate was known for its
Factor VIII inhibitor bypassing activity.
Subsequent development of PCC with
minimal amounts of activated factors has al-
lowed its reconsideration for broader use,
par- ticularly as an antidote to warfarin
overdos- age.175
Four-factor PCCs have been available
in Europe for several years and have
demonstrat- ed rapid correction of
coagulation status. In 2013, a four-factor
PCC was approved by the FDA for reversal
of acute major bleeding for patients taking
vitamin K antagonists.234 Be- cause the
product (Kcentra, CSL Behring, Kankakee,
IL) contains adequate concentra- tions of
coagulation Factors II, VII, IX, and X,
plasma infusion should not be necessary.
Experience with this product is limited at the
time of this writing and its value for reversal
of direct thrombin inhibitors or anti-Xa
inhibi- tors is not established.
The administration of PCCs in other off-
label settings (eg, massive hemorrhage in the
absence of warfarin or intracranial pressure
monitor placement in fulminant liver failure)
should be weighed against the risk of throm-
bosis from giving, in the case of PCCs, a non-
activated but supraphysiologic concentration
of clotting factors. The clinical trials preceding
528 ■ AABB T EC HNIC AL MANUAL
FDA approval did not include patients who
had experienced acute thromboembolic
events, myocardial infarction, DIC, stroke,
un- stable angina, or severe peripheral
vascular disease within 3 months before
administra- tion. For this reason, the
package insert indi- cates that the product
may not be suitable in patients who have
experienced thromboem- bolic events in the
prior 3 months. In patients with a history of
HIT who are subsequently shown to be
negative for HIT on enzyme- linked
immunosorbent assay, exposure is like- ly to
be safe because the heparin will be gone by
the time the HIT antibody is recalled. How-
ever, use is contraindicated in patients with
known HIT. The reduced capacity of a
cirrhotic liver to clear the circulating
byproducts of the coagulation cascade, such
as D-dimers, may lead to DIC or other
derangements of the co- agulation system.
Therefore, the use of PCC in patients with
liver disease remains risky.173
Recombinant Factor VIIa
The production of the activated form of
coagu- lation Factor VII via recombinant
Factor VIIa (rFVIIa) has led to the
exploration of this drug as a means of
addressing a wide variety of hemorrhagic
conditions. The drug was devel- oped and is
approved for use in bypassing an- tibodies
against Factor VIII in patients with he-
mophilia A and achieving hemostasis
without needing to achieve hemostatic
levels of Factor VIII in the face of potent
antibodies. It can also be used to address
congenital deficiency of Factor VII.235
Beyond these applications, rFVIIa been
considered for use in a wide variety of
condi- tions that are not related to
hemophilia or Fac- tor VII deficiency in
which bleeding is difficult to control or is
threatening the patient’s life. For example,
rFVIIa has been used to counter- act the
effect of warfarin by replacing the inac- tive
factor (produced in the presence of the vi-
tamin K antagonist) with active rFVIIa that
can immediately stimulate the downstream
proco- agulants and produce fibrin. 236 The
use of rFVIIa has also received much
attention in trauma treatment, liver
transplantation, and massive transfusion,
where correction of pro-
coagulant deficiency and achievement of he-
mostasis can be challenging. 237,238 However,
rFVIIa is less effective in patients with signifi-
cant acidosis or hypothermia.
In a registry that collected reports of ad-
verse events after use of rFVIIa, the drug
was listed as a contributing cause of death
for a large proportion of patients who died
after its administration.239 However, in a
review of 35 placebo-control trials involving
4468 people, a significant increase in
arterial thrombosis, but not venous
thrombosis, was attributed to rFVIIa use.240
A recent Cochrane analysis iden- tified 29
RCTs with 4290 patients who were treated
off label with rFVIIa in a variety of clin-
ical situations. The meta-analysis included
separate analyses of 16 studies of
prophylactic use and 13 of therapeutic use in
addition to an analysis of all of the trials.
The authors found no effect on mortality in
the prophylactic group and a trend toward
decreased mortality in the therapeutic group.
Bleeding and trans- fusion rate outcomes
favored rFVIIa use, but these data were only
reported in the smaller studies, so these
findings are of uncertain val- ue. Of concern
was a trend toward increased
thromboembolic events in the therapeutic
group. When all studies were analyzed
togeth- er, a statistically significant increase
in arterial thromboembolic events was found
(RR 1.45; 95% CI 1.02 to 2.05).241 Given the
open ques- tion of whether any adverse
events are related to unwanted clotting and
the exclusion of pa- tients with a propensity
to clot, including those with atherosclerotic
disease, from many of the trials, it may be
prudent to refrain from employing rFVIIa in
patients who might be at increased risk of
unwanted thrombosis.
A substantial stumbling block in the use
of rFVIIa has been its cost. If it is effective in
substantially reducing hemorrhage or morbid-
ity, then its use might be cost-effective. Anoth-
er approach is to adopt a standardized rather
than weight-based dosing practice, which re-
duces consumption of the drug and appears to
provide similar clinical results.242 Clinical con-
sultation by a transfusion medicine specialist
may be very helpful in guiding the use of this
product.243
 CH A P T E R 20
Fibrinogen Concentrate
Originally, fibrinogen concentrate, which de-
livers a small volume of purified plasma-de-
rived factor, was approved by the FDA for
treatment of dysfibrinogenemia or deficiency.
As the pathogenesis of coagulopathy of trau-
ma and surgery and the importance of fibrino-
lysis in these situations is better understood,
the use of fibrinogen concentrate in these situ-
ations is being explored. 182 Although cryopre-
cipitate can also replace fibrinogen, fibrinogen
concentrate can be prepared in advance with-
out the storage and thawing issues that arise
with cryoprecipitate.
A systematic review of the use of fibrino-
gen concentrate and plasma in patients at risk
of bleeding found 1) decreased bleeding, 2) re-
duced transfusion requirements, and 3) a low
rate of adverse events in patients treated with
fibrinogen concentrate. Results for bleeding
and transfusion requirements were inconsis-
tent in plasma-treated patients.201 The hetero-
geneity among the studies precluded any true
meta-analysis, but the overall trend favored
use of fibrinogen concentrate early in care.
Intravenous Immune Globulin
Concentrates of plasma immune globulins
were developed to treat congenital immuno-
deficiencies and treat or provide prophylaxis
against certain viral exposures. Because im-
mune globulins tend to polymerize during
Cohn fractionation and purification, initial
preparations were given intramuscularly to
avoid severe hypotensive and/or anaphylac-
toid reactions. Subsequent development of a
variety of chemical modifications has
allowed the development of IVIG
preparations that have low proportions of
aggregates. These products allow the
administration of larger quantities over
shorter intervals to achieve more
pronounced clinical effects.
Although the initial intended use of
IVIG was to reduce infections in
immunodeficient patients, the optimal dose
for this indication remains under discussion.
The clinical appli- cation of IVIG has
extended far beyond this in- dication (Table
20-10).244 For a thorough re- view of the
indications for IVIG, see the report
Hemotherapy Decisions ■ 529
by Durabi and colleagues.245 When used for
the treatment of hypogammaglobulinemia,
an IVIG dose of 600 mg/kg in adults or 800
mg/kg in children every 4 weeks rather than
the usual 300 mg/kg in adults or 400 mg/kg
in children slightly reduces the frequency
and duration of infections in patients with
primary immune deficiencies.246 However,
the cost-effective- ness of increased doses
may be poor, and these doses accelerate
usage rates.
The ever-increasing off-label use of IVIG
poses an additional challenge to the adequacy
of the supply for treating the 50,000 patients
with congenital hypogammaglobulinemia in
the United States. The FDA-approved indica-
tions for IVIG account for only 30% of its us-
age. The other situations in which IVIG is
administered may have greater or lesser scien-
tific support, but the usage rate in developed
countries appears to be increasing at a rate of
approximately 10% per year. The wide vari-
ability of doses used (from < 0.03 g per person
in Australia to more than 0.06 g per person in
the United States and Canada) without con-
comitant variations in outcomes, however,
raises the question of the true utility of some
of these applications.
The administration of IVIG can be
associ- ated with a wide variety of adverse
events (Ta- ble 20-11). Some of these effects
appear to result from the use of idiosyncratic
combina- tions of a particular product with a
particular patient. Therefore, advantage
should be taken of the wide variety of IVIG
products in the marketplace to find the form
of the medica- tion that is well tolerated by a
given patient.247 Slow rates of infusion and
pretreatment with antihistamines and/or
steroids usually pre- vent reactions.
Clinically dramatic reactions usually occur
only in patients with agamma- globulinemia
who have not previously been treated and
who have an infection. IgA may be present
in sufficient concentration in some
preparations to prompt an anti-IgA reaction
in sensitized patients.
Although the antibody specificities in
IVIG products represent the range and
relative concentrations of the donors’
immune globu- lin responses and, thus, may
convey varying degrees of protection
against particular patho-
530 ■ AABB T EC HNIC AL MANUAL

TABLE 20-10. Applications of Intravenous Immune Globulin

Primary Immune Deficiencies

Hypo/agammaglobulinemia
Selective antibody
deficiency
Class/subclass deficiency with recurrent
infection Secondary (Acquired) Immune
Deficiencies
Chronic lymphocytic leukemia
Multiple myeloma
Prevention of cytomegalovirus pneumonitis after hematopoietic stem cell transplantation
Reduction of bacterial infection in children with acquired immunodeficiency syndrome
Immune Cytopenias
(Auto)immune thrombocytopenic purpura [idiopathic thrombocytopenia (ITP)]*
Pure red cell aplasia
Other cytopenias: neonatal alloimmune thrombocytopenia, posttransfusion purpura, and warm autoimmune
hemolytic anemia
Human immunodeficiency virus-related idiopathic thrombocytopenia
(Presumed) Autoimmune Disorders
Kawasaki disease
Guillain-Barré syndrome
Multiple sclerosis
Myasthenia GRAVis
Dermatomyositis
Systemic vasculitides
Factor VIII inhibitor deficiency (congenital/acquired hemophilia)
Prevention/treatment of infections
Post-hematopoietic stem cell transplantation

Post-solid-organ transplantation and immunosuppression

Parvovirus infection
Neonatal sepsis

Other (Presumed) Immunologic Conditions

Recurrent spontaneous abortion
Graft-vs-host disease
Asthma
Myocarditis
 CH A P T E R 20 Hemotherapy Decisions ■ 531

TABLE 20-10. Applications of Intravenous Immune Globulin (Continued)

Inflammatory bowel disease

Stevens-Johnson syndrome
Other Conditions
Alzheimer’s disease
Predisposition to atherosclerosis
Autism
Chronic fatigue syndrome
Multifocal motor neuropathy
Rh prophylaxis (if patient cannot receive intramuscular product)
Approved indications shown in bold. Commonly accepted indications shown in italics.
*Usually used only in chronic ITP because most acute ITP cases are self-limited. (Many other agents potentially effective in
ITP are being developed.244

gens, the large pool sizes used to manufacture
these products reduces this variability. Hyper-
immune globulins are manufactured from
samples provided by donors chosen for their
higher titers of activity against selected agents,
such as hepatitis A or B virus, tetanus, rabies,
varicella-zoster virus, or CMV, but are usually
available only in the intramuscular form.
The products may, on occasion, convey
sufficient isoagglutinins or red cell alloanti-
bodies of a particular specificity (usually anti-
D) to cause a positive direct antiglobulin test
result in the recipient, but clinically
detectable or significant hemolysis
following use of these products is rare.248
Administration of hyperim- mune anti-D to
an Rh-positive patient for treatment of ITP
may induce a life-threatening or fatal acute
hemolysis, and the product car- ries a black-
box warning to remind physicians of this
potential outcome.
Antithrombin
Antithrombin circulates in plasma as a
serine protease inhibitor that inactivates the
serine proteases of the coagulation system,
including thrombin and Factors IXa, Xa, XIa,
and XIIa, by covalent binding at their serine
active site.249 The ability of antithrombin to
accomplish this
inhibition is greatly accelerated by heparin,
which induces a change in the polypeptide’s
conformation; this is why antithrombin is
known as “heparin cofactor.”
Patients who are congenitally deficient
in antithrombin have an increased risk of
devel- oping thrombosis.250 Acquired
deficiency of antithrombin can also occur
due to reduced antithrombin synthesis (as in
hepatic dis- ease), increased antithrombin
loss (as in a ne- phrotic syndrome),
increased antithrombin breakdown (eg, due
to L-asparaginase treat- ment), or increased
antithrombin consump- tion (eg, in DIC,
trauma, or surgery). The ad- ministration of
heparin also accelerates the metabolism of
antithrombin and can lead to a relative
resistance to heparin.250
Although antithrombin is stable in
frozen or thawed plasma, antithrombin
deficiencies are usually addressed through
the infusion of antithrombin concentrate.
Congenital anti- thrombin deficiency is rare.
However, anti- thrombin has been used in
the treatment of sepsis and DIC to stem
thrombotic complica- tions and in patients
being heparinized for extracorporeal
circulation who do not experience the
expected effects of heparin treatment.
532 ■ AABB T EC HNIC AL MANUAL

TABLE 20-11. Adverse Events Associated with Activated Protein C

Intravenous Immune Globulin Use
Protein C is a serine protease that acts with
Infusion Associated protein S to hydrolyze and thus inactivate
Fac-
tors Va and VIIIa. It therefore serves an anti-
Fever Chest tightness thrombotic regulatory function. Protein C is
Chills Dyspnea normally activated along the prothrombotic
cascade, which leads to an innate regulation of
Facial flushing Wheezing the clotting system. When given as an
Tachycardia Hypotension infusion over 96 hours to patients with
sepsis, recombi-
nant activated protein C concentrate reduced
Palpitations Anxiety mortality by 19%, although the risk of
Abdominal pain Nausea serious bleeding almost doubled.251

Headache Vomiting 1-Antitrypsin

Lumbar pain Urticarial rash -Antitrypsin (also known as 1-proteinase
Other Possible Adverse inhibitor) is a natural inhibitor of the
Events elastase elaborated by activated
polymorphonuclear leukocytes. If the
Volume overload activity of elastase is left un- checked, as in
Arterial thrombosis congenital deficiencies of 1- antitrypsin,
(myocardial infarction, excessive damage to pulmonary parenchyma
stroke) occurs after even minor infec- tions, leading
to development of fatal emphy-
Venous thromboembolism sema at an early age in the 100,000 patients in
Disseminated intravascular the United States with this deficiency. Hepatic
coagulation cirrhosis can also occur.
Until the development of several
Hemolysis (due to plasma- derived replacements,252 congenitally
alloanti- deficient patients could stem the advance of
bodies/isoagglutinins) tissue de- struction only by attempting to
Nephrotoxicity avoid and rap- idly treat respiratory
infections. Periodic (usu- ally weekly)
Neutropenia infusions of 1-antitrypsin can be
administered prophylactically to prevent addi-
tional lung damage in these patients.

KEY POINTS

Transfusion in the presence of an autoantibody that precludes a negative crossmatch is safe and could be life-saving, provided th
The data needed to support evidence-based guidelines for RBC transfusion have become stronger over the past several years. Th
Compared to amounts of circulating platelets and coagulation factors in healthy people, the amount required to achieve hemosta
The laboratory test results that are currently available to aid in component therapy deci- sions do not correlate with an individual
 CH A P T E R 20 Hemotherapy Decisions ■ 533

5. Data on using blood components to reverse the effects of newer antiplatelet agents and
an- ticoagulants are very limited, as are the data on using clotting factor concentrates.
6. In the setting of hemorrhagic shock, the current data support the transfusion of
platelets, plasma, and cryoprecipitate early in the resuscitation effort. An increased ratio
of these products to RBCs, often referred to as a 1:1:1 protocol, has been shown to
decrease mortality in one large prospective cohort study and a series of retrospective
studies.
7. For patients without underlying cardiac disease, the data support using an RBC
transfusion threshold of 7 to 8 g/dL of hemoglobin. For patients with underlying
cardiac disease, cur- rent guidelines recommend a threshold of 8 g/dL of hemoglobin or
less. Data are lacking on the appropriate transfusion threshold in patients with acute
coronary syndrome; therefore, a transfusion trigger cannot be established in this setting
at this time. The decision to trans- fuse RBCs should also take into account the patient’s
clinical situation and response to anemia.
8. Although prophylactic platelet transfusions during aplasia are common practice, it is
uncer- tain whether a prophylactic vs a therapeutic transfusion approach is optimal.
9. The most common prophylactic transfusion threshold is 10,000 platelets/µL. The current
standard prophylactic dose of platelets of 3 to 4 × 1011 for an adult may be halved without
risk of bleeding.
10. When platelets bearing ABO antigens that are foreign to the recipient are transfused,
the ef- fect on the platelet count may be blunted. When plasma in platelet units contains
antibod- ies against A and/or B antigens expressed on the recipient red cells, hemolysis
may occur, with a 1:3000 to 1:10,000 risk from apheresis platelets. Steps are often
taken to limit the amount of incompatible plasma transfused or to avoid high-titer units,
especially for pedi- atric patients, when time permits.
11. Alloimmunized platelet refractory patients may be supported by transfusion of platelets
from donors lacking the targeted epitopes by either phenotyping the donor (unit) or
provid- ing HLA-matched units to patients with HLA alloimmunization.
12. There are very limited data to suggest a benefit in transfusing plasma in settings other
than intracranial hemorrhage after anticoagulation with vitamin K antagonists or
massive trans- fusion.

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 C h a p t e r 2 1
Administration of Blood
Components
Kim Maynard, BSN, RN, OCN
THE SAFE ADMINISTR A T I ON of
blood and its components requires
mul- tidisciplinary collaboration among
clinical and ancillary services and clinicians.
Policies and procedures should be
developed with in- put from transfusionists,
the transfusion ser- vice, surgeons,
anesthesiology care providers, primary-care
physicians, and transport per- sonnel. The
transfusionist typically provides the last line
of defense in the detection of er- rors before
the transfusion commences. All personnel
involved in the chain of preparing,
delivering, and administering a transfusion
should be given appropriate training to
ensure the provision of the safest transfusion
possible for patients.
EVENTS AND CONSIDERATIONS
BEFORE DISPENSING
COMPONENTS
Before a transfusion begins, thoughtful con-
sideration, planning, and preparation are re-
quired.
Kim Maynard, BSN, RN, OCN, Breast Coordinator (former Transfusion Safety Officer, Transfusion Medicine
Service), Dartmouth-Hitchcock Medical Center, Lebanon, New Hampshire
The author has disclosed no conflicts of interest.
Recipient Consent
The AABB Standards for Blood Banks and
Transfusion Services states, “The blood bank
or transfusion service medical director shall
par- ticipate in the development of policies,
pro- cesses, and procedures regarding
recipient consent for transfusion.”1(p44)
Recipient in- formed consent should address
indications for; risks, benefits, and possible
side effects of; and alternatives to
transfusions of allogeneic blood
components. Some state laws require certain
additional elements in the patient con- sent.
The patient has the right to choose or
re- fuse a transfusion and must have an
opportu- nity to ask questions of a learned
professional before providing consent.
Documentation of the consent process must
be entered into the patient’s medical record.
Some facilities re- quire an institution-
approved signed consent form to document
that the consent process has occurred and
the risks/benefits of transfu- sion were
discussed with the patient or legal
545
546 ■ AABB T EC HNIC AL MANUAL
representative. Each institution needs to
have a process for recording a patient’s
refusal to re- ceive blood or blood
components in the pa- tient’s medical record.
Institutional policies should identify health-
care providers who are allowed to obtain
consent and the length of time for which that
consent remains valid.
Consent for transfusion must be ob-
tained from patients who have the requisite
capacity to make such decisions. If a patient
is unable to give consent, a legally
authorized representative or surrogate may
do so (de- pending on local and state laws).
If no one is available to provide consent and
the need for transfusion is considered a
medical emergen- cy, the blood component
may be administered based on the doctrine
of implied consent. In- dividual state and
local laws governing re- quirements for
implied consent may vary, but the emergent
need for transfusion should be carefully
documented in the medical record.2
Patient Education and History
The transfusionist should educate the patient
about reporting any symptoms that may be
in- dicative of a reaction and how long the
trans- fusion will take. The patient’s
questions should be answered before the
transfusion is started.
It is important to collect a history from
the patient before the component is ordered
to assess whether the patient is at increased
risk of a transfusion reaction. This history
includes previous transfusions and any
adverse reac- tions. If the patient has had
previous reactions to transfusions, the
medical team should de- termine whether
the patient needs to receive medications
before the transfusion or wheth- er special
processing of the component is indi- cated to
mitigate the risk of an adverse reac- tion.
Baseline Assessment of Recipient
A baseline physical assessment should
include vital signs. Many institutions also
routinely measure oxygen saturation using
oximetry. The assessment should include
pretransfusion symptoms, such as shortness
of breath, rash, pruritus, wheezing, and
chills, as a basis for comparison after the
transfusion starts. A pa-
tient with renal or cardiopulmonary disease
may require a slower infusion rate to
prevent fluid overload, for example.
A patient with an elevated temperature
may destroy cellular components at an in-
creased rate.3 Moreover, if the patient
presents with an elevated temperature before
the trans- fusion, it may be difficult to
determine later whether this was caused by a
transfusion reac- tion. Administration of an
antipyretic should be considered in such
cases.
Medical Order
Blood Component
A licensed provider writes two orders for the
components to be administered. The first or-
der requests that compatible crossmatch (if
appropriate) unit(s) be prepared for the pa-
tient and notes special processing require-
ments. The second order explains to the
trans- fusionist how to administer the
component, including the transfusion rate.
Both of these orders should specify:
■ Patient name and other independent iden-
tifier (eg, date of birth or medical record
number).
■ Component [eg, Red Blood Cells (RBCs)
or apheresis platelets] to prepare or
adminis- ter.
■ Special processing required (eg,
leukocyte reduction, irradiation, or
washing).
■ Number of units or volume to administer.
■ Date and time for the infusion.
■ Flow rate or period for administering the
component.
Note: It is appropriate for the rate and
du- ration of the transfusion (eg, not to
exceed 4 hours from the time that the
container is en- tered until completion)4 to
be described in a policy approved by the
hospital’s medical staff.
Laboratory Testing
After receiving an order from a licensed pro-
vider, the transfusion service ensures that
compatible components are provided. To
issue blood components for a specific
patient (ex-
 C H A P TE R 2 1 Administration of Blood Components
cept in the case of an emergency
transfusion), the laboratory must collect a
pretransfusion blood sample. Typically, the
sample is ob- tained within 3 days of
transfusion, with the draw date considered
to be day 0.1(p36) Institu- tional policies may
vary regarding the sample outdate. If the
patient has not had a transfu- sion or been
pregnant in the prior 3 months, the sample
may be acceptable for longer than 3 days for
testing purposes.
According to The Joint Commission,
con- tainers used for blood specimens must
be la- beled in the presence of the patient.5
The sam- ple must be labeled at the patient’s
side with at least two unique identifiers (eg,
the patient’s name, date of birth, or
identification number). The identification of
the person collecting the sample and the
date on which the sample was collected
must be traceable.1(p35)
Pretransfusion testing of the recipient’s
blood sample, including for unexpected anti-
bodies to red cell antigens, is described in
de- tail in Chapters 15 and 16. The time
interval from sample receipt to availability
of the re- quested component can vary
greatly. One reason is that availability
depends on the in- ventory of components
maintained by the transfusing facility, and
some components may need to be obtained
from an external sup- plier. Furthermore, if
the test results from the pretransfusion
sample show that the patient has clinically
significant red cell antibodies, obtaining
corresponding antigen-negative or
crossmatch-compatible units may require
ad- ditional time. Moreover, some
components re- quire thawing, pooling,
relabeling, or other preparation before
release. All of these factors necessitate
timely communication between laboratory
and transfusing personnel. For ex- ample,
components that are pooled or require
thawing may have a shortened shelf life
after being prepared (4-24 hours);
transfusionists should be aware of the
decreased time avail- able to complete a
transfusion of such compo- nents.1(pp51-59)
Venous Access
The transfusionist must determine whether
the patient’s venous access [with a central
line,
■ 547
peripheral intravenous line, implanted port, or
peripherally inserted central catheter (PICC)]
is acceptable for the infusion of blood compo-
nents.
Acceptable intravenous catheter sizes
for use in transfusing cellular blood
components range from 22 to 14 gauge.6 A
20- to 18-gauge intravenous catheter is
suitable for the general adult population and
provides adequate flow rates without
excessive discomfort to the pa- tient. When
an infant or a toddler is trans- fused, a 24- to
22-gauge intravenous catheter may be
suitable but requires infusion through a
syringe (see Chapter 23).7 The flow rate
may need to be adjusted depending on the
catheter size.
There is an increased possibility of hemo-
lysis when red cells are transfused rapidly
us- ing handheld syringes through 23-gauge
or smaller needles.8 With the use of smaller
cath- eters, blood dilution and a pump are
helpful to administer the unit to prevent
slow flow rates that lead to clogging of the
intravenous cathe- ter. Units suspended in a
preservative solution, such as Adsol, usually
do not require addition- al dilution. It is
important that the infusion line be patent
before the component is re- ceived at the
patient’s bedside.
Prophylactic Medications Given
Before Transfusion
Although antipyretics (eg, acetaminophen)
were commonly ordered in the past to
reduce the risk of febrile nonhemolytic
transfusion re- actions, indications for their
use are contro- versial.9 Some providers use
antipyretics in a prophylactic manner, some
wait to use them until the patient has
experienced at least one febrile transfusion
reaction, and others believe that
prophylactic use of antipyretics may mask
the elevated temperature that results from a
transfusion reaction. Evidence supporting
any of these approaches is limited.9-12 Some
experts recommend that “in the absence of
definitive evidence-based studies,
pretransfusion medi- cation to prevent
transfusion reactions should not be
encouraged.”13
In a Cochrane review14 of studies on pre-
medication to prevent allergic reactions and
548 ■ AABB T EC HNIC AL MANUAL
febrile nonhemolytic transfusion reactions
(FNHTRs), the authors stated that current
evi- dence from three randomized controlled
trials (RCTs) involving 462 patients indicate
that no pretransfusion medication regimen
reduces the risk of allergic reaction or
FNHTR. They further state that no evidence
shows that pre- transfusion medications
prevent NHTR. How- ever, the conclusion is
based on the evidence from a review of
three trials with a moderate risk of bias and
low to moderate quality. A bet- ter-powered
RCT is necessary to evaluate the role of
pretransfusion medication in the pre-
vention of NHTR.
Antihistamines (diphenhydramine and/
or H2 blockers) may be ordered as
premedica- tion for individuals who have
had allergic reac- tions to transfusions in the
past. Meperidine or corticosteroids are
occasionally ordered for patients who have
experienced severe rigors during
transfusion.15 Corticosteroids have also been
used to premedicate patients who have had
prior anaphylactoid reactions or recurrent
febrile nonhemolytic transfusion reactions.
The onset of corticosteroid immunosuppres-
sion takes a long time. The efficacy of
premed- ication with corticosteroids has not
been ade- quately assessed, and the optimal
timing and efficacy of corticosteroids have
not been es- tablished in the transfusion
setting. However, corticosteroids have been
used to prevent ana- phylactoid reactions to
iodinated contrast me- dia in the radiology
setting in patients who have had prior
reactions, a situation that is somewhat
analogous to that of transfusion. The authors
of a number of publications rec- ommend
repeat corticosteroid dosing in the radiology
setting, often in combination with H1 and
H2 blockers over 13 to 24 hours before the
procedure.16,17
If premedication is required, it should be
administered in advance of the component’s
arrival. If the premedication is given orally, the
transfusionist should wait 30 minutes before
initiating the transfusion. If the premedica-
tion is given intravenously, a 10-minute wait-
ing period before initiating the transfusion is
suggested.
Equipment
Blood Warmers
Infusions of cold components can cause
hypo- thermia and cardiac complications,
increasing morbidity and mortality.18 The
likelihood of clinically important
hypothermia is increased when blood is
transfused through a central ve- nous device
directly into the right atrium.
Blood warmers are rarely needed during
routine transfusions. However, they are used
when rapid transfusion of components is re-
quired, especially in trauma or surgery set-
tings. Blood warmers are also advantageous
during transfusions to neonates, where
hypo- thermia can cause serious adverse
effects. Opinions vary on the utility of blood
warmers in patients with cold
agglutinins.19,20
AABB Standards1(p6) states that warming
devices shall be equipped with a
temperature- sensing device and a warning
system to detect malfunctions and prevent
hemolysis or other damage to blood or blood
components. Warming blood to
temperatures >42 C may cause hemolysis.21
The transfusion service should work with
departments that use blood warmers to make
sure that the devices are ap- proved by the
Food and Drug Administration (FDA) for
infusion of components. Warming devices
should be validated and maintenance and
testing of alarms should be performed ac-
cording to the manufacturer’s suggestions.
Blood components should not be warmed by
placing them in a microwave, on a heat
source, or in hot water, or by using devices
that are not approved by the FDA
specifically for blood warming.
Infusion Systems
Infusion pumps or systems are used to
admin- ister fluids, medications, blood, and
blood components through clinically
accepted routes of administration. These
devices allow for a controlled infusion rate
and, thus, controlled administration over a
desired period; they also provide an alarm
system to notify clinicians of problems with
the infusion. Consequently, the use of
infusion pumps or systems may be pre-
ferred over simple gravity administration.
 C H A P TE R 2 1 Administration of Blood Components
However, there is a potential for hemolysis
of the cellular components infused through
these pumps. The manufacturer of the pump
should be consulted to determine whether
the pump is approved for the infusion of
blood compo- nents. If it is not approved,
the institution should establish a validation
plan to confirm that the pump will not
damage components before their use. Many
of the electromechani- cal pump devices that
are approved for blood administration
require use of administration sets with
standard in-line filters.
Using infusion pumps to deliver
transfu- sions via PICC lines may lead to
pressure in the catheter that exceeds the
allowed pounds per square inch. The
institution should ensure that the infusion
pump used does not generate pressure that
exceeds the recommended limits of the
catheter.22
Syringe Infusion Pumps
A syringe infusion pump may be used for
small-volume transfusions to neonatal or pe-
diatric patients. Using this pump requires
drawing the blood component through a filter
into the syringe. For more details, see Chapter
23.
Pressure Devices
The use of an externally applied pneumatic
pressure device may achieve flow rates of 70
to 300 mL per minute, depending on the
pressure applied. The device should have a
gauge to monitor the pressure, which should
be applied evenly over the entire bag. Pressure
in excess of 300 mmHg may cause the seams
of the blood component bag to leak or
rupture. When a pressure device is used, a
large-gauge cannula should be employed to
prevent he- molysis.
The application of an external pressure
device to the blood bag to expedite the
trans- fusion of RBC units causes minimal
damage to the red cells and is a safe practice
in the major- ity of patients.23 However, the
use of pressure devices has been reported to
provide only a small increase in component
flow rates. When rapid infusion is desired,
an increase in cannu- la size typically
provides better results.
■ 549
Availability of Emergency Equipment
The transfusionist should be prepared to ob-
tain and administer emergency interventions
when needed. Items used to respond to a
transfusion reaction include the following:
■ A 0.9% sodium chloride intravenous (IV)
solution and administration set to keep an
IV line open.
■ Medications to treat a reaction along with
a mechanism to obtain emergency
medica- tions prescribed to treat the
sequelae of transfusion reactions.
■ A mechanism to activate emergency resus-
citation measures in the event of a severe
reaction.
■ Ventilatory assistance and an oxygen
source.
BLOOD COMPONENT
TRANSPORTATION AND
DISPENSING
Delivery of Components to the Patient
Area
Each institution must have policies and
proce- dures for issuing components and
ensuring that they are delivered in a timely
manner to the receiving transfusionist.
Components should not leave the controlled
environment until the patient has been
properly prepared, including insertion of a
patent intravenous catheter, and the
transfusionist is ready to be- gin the
infusion.
Transfusion service personnel should
in- spect the unit before issuing it for
abnormal appearance (significant color
change, cloudi- ness, clots, clumps, or loss
of bag integrity). The component must not
be used if any of these are noted. 4 There
must be a mechanism to correctly identify
the intended recipient and component at the
time of the request to issue the component.
Institutions should train staff to request
components so that they arrive in an
expedited manner and are not left outside a
controlled environment.
550 ■ AABB T EC HNIC AL MANUAL

Institutions may use dedicated Delays in Starting Transfusion

personnel or automated delivery systems
If an unexpected occurrence does not allow
(eg, pneumatic tube systems, automated
for the transfusion to start immediately, the
blood delivery ro- bots, or blood dispensing
blood component unit should be promptly
kiosks in remote sites) to facilitate the
returned to the transfusion service for proper
delivery of components to their final
storage.
destination. Provision of RBCs via
The transfusion service may set limits,
automated blood vending machines [remote,
based on validation, on the amount of time
automated, computerized controlled blood
that a unit may be out of controlled storage
storage and dispensing refrigerators, such as
be- fore it is considered unsuitable for
BloodTrack HemoSafe (Neoteric
reissue. Components may be suitable for
Technology, Vancouver, BC, Canada)] at the
reissue if the appropriate temperature has
point of care may help circumvent delays in
been main- tained.1(p40) They should never be
transportation. This solution employs an
electronic issuance process, requiring stored or held in a patient care unit unless
there is a controlled and monitored
infrastructure and bidirec- tional informatics
environment for storing components.
connectivity with the cen- tral blood bank to
Trauma areas and surgi- cal units may have
safely issue and return blood units to/from
arrangements with the transfusion service to
remote sites.24 Staff educa- tion in the
provide controlled stor- age of units. If the
electronic issuance process em- ployed by
temperature of a transport- ed component
vending machines is required to use these
falls outside of 1-10 C, reissue is not
devices. The use of automated and re- mote
permissible.1(p47)
delivery systems requires validation pri- or
to implementation to ensure a safe and ef- If a unit has been entered (spiked for
fective system for blood delivery and remote transfusion), it may not be returned to the
blood issue.25 transfusion service for reissue. It must either
Transfusion services generally allow the be infused within 4 hours of the time it was
issue of only 1 unit at a time unless it is an spiked, or it must be discarded.
emergent or large-volume transfusion.
EVENTS AND CONSIDERATIONS
1. At the time a unit is issued, AABB BEFORE COMPONENT
Standards requires a final clerical check ADMINISTRATION
of transfusion service records with each
unit or compo- nent. Verification must Identification of the Recipient
include1(p42) the intended recipient’s two and Correct Component
independent iden- tifiers (name, date of
Once the unit is received, a qualified
birth, or patient iden- tification number
transfu- sionist who will administer the
and/or unique identifier given at the time
blood or blood component to the patient
the crossmatch sample is drawn), ABO
verifies the compo- nent at the patient’s side
group, and Rh type.
with another health- care provider who is
2. The donation identification number,
qualified in performing identification
donor ABO group, and, if required, donor
verification, as determined by the hospital.5
Rh type.
Alternatively, a one-person veri- fication
3. The interpretation of results of
process using automated identifica- tion
crossmatch tests, if performed.
4. Special transfusion requirements. technology, such as bar coding, may be used.
5. The expiration date and, if applicable, time. The following items should be checked
6. The date and time of issue. immediately before transfusion:

AABB Standards requires that there be a
process to confirm agreement among identify-
ing information, records, the blood or blood
component, and the request. Discrepancies
must be resolved before a unit is issued.1(p42)
■ Appearance of the unit. Units should be
re- turned to the transfusion service if there
is discoloration, abnormal cloudiness, pres-
 C H A P TE R 2 1 Administration of Blood Components
ence of clots or clumps, or loss of bag
integ- rity.
■ Identification of patient and unit. The pa-
tient’s two independent identifiers (eg,
name and identification number) must
match the information on both the label
on the unit or attached tag and the
medical or- der. The requirements of the
institution for patient identification must
be satisfied. In- stitutions often require,
for example, an ad- ditional check to
verify that the unit was se- lected for the
patient whose sample was collected for
crossmatch.
■ Medical order. The transfusionist should
verify that the component matches the
unit called for in the medical order and
that any special processing in the order
was per- formed. In particular, the
transfusionist should ensure that
leukocyte reduction or irradiation was
performed if ordered.
■ Blood type. The patient’s ABO group (and
Rh type if required) should be compatible
with that of the unit. Interpretation of
crossmatch tests (if performed) is also veri-
fied.
■ Donation identification number.
■ Expiration date (and time, if applicable).
The unit should not be transfused if the ex-
piration date or time has passed.
The transfusion should be withheld if
any discrepancy or abnormality is found.
Proper bedside identification of the
recip- ient is the final step to prevent the
administra- tion of an incorrect blood
component to a pa- tient. Although
individuals often worry about the possibility
of exposure to infectious agents from
transfusion, equal concern should focus on
the inadvertent transfusion of incompati- ble
blood. Approximately 1 in every 19,000
units of RBCs is transfused to the wrong pa-
tient each year; 1 in 76,000 transfusions
results in an acute hemolytic transfusion
reaction, and 1 in 1.8 million units of
transfused RBCs results in death from an
acute hemolytic trans- fusion reaction.26
To prevent the potentially fatal conse-
quences of misidentification, specific
systems have been developed and marketed,
including identification bracelets with bar
codes and/or
■ 551
radiofrequency identification devices, biomet-
ric scanning, mechanical or electronic locks
that prevent access to bags assigned to other
patients, and handheld computers suitable
for transferring blood request and
administration data from the patient’s
bedside to the transfu- sion service
information system in real time. Each
system provides a method to bring staff
toward self-correction during the proce-
dure.27,28 Studies show that rates of positive
re- cipient identification can be increased by
such systems. However, none of these
systems ne- gates the need for good quality
management, such as adequate standard
operating proce- dures, regular training,
periodic competency assessment, and
system monitoring.
Infusion Sets
Components must be administered through
special IV tubing with a filter designed to re-
move blood clots and particles that are poten-
tially harmful to the patient. Standard blood
administration tubing typically has a 170- to
260-micron (macroaggregate) filter, but this
micron size is not mandated or required. The
tubing can be primed with either 0.9% sodium
chloride or the component itself. The manu-
facturer’s instructions should be reviewed for
proper use. The IV setup should have a mecha-
nism that allows bypass of the blood IV
admin- istration tubing to start 0.9% sodium
chloride in the event of a reaction. A suggested
mecha- nism is to have a “Y” port or three-
way stop- cock close to the infusion site that
allows for the administration of 0.9% sodium
chloride.
Microaggregate Filters
Microaggregate filters are not used for
routine blood administration. These second-
genera- tion filters were originally
developed to re- move leukocytes and to
complement or replace the clot screen in the
1970s.29 They have since been replaced by
more efficient leukocyte reduction filters.30
Microaggregate filters have a screen filter
depth of 20 to 40 microns and retain fibrin
strands and clumps of dead cells. Red cells,
which are 8 microns in diameter, can flow
through the filters. Micro- aggregate filters
are typically used for the
552 ■ AABB T EC HNIC AL MANUAL
reinfusion of shed autologous blood
collected during or after surgery.
Leukocyte Reduction Filters
Leukocyte reduction filters are designed to
re- duce the number of leukocytes to less
than 5 × 106 per RBC unit (so that more than
99.9% of the leukocytes are removed from
the unit). Leukocyte reduction decreases the
incidence of febrile transfusion reactions,
risk of HLA al- loimmunization, and
transmission of cyto- megalovirus by
cellular blood components (see Chapter
6).29,30 These filters are provided by various
manufacturers for prestorage use shortly
after collection of the units or for
poststorage use at the patient’s bedside.
Prestorage leukocyte reduction is
usually preferred for a number of reasons,
one of which is that it allows monitoring of
leukocyte reduction efficacy. Furthermore,
the use of bedside leukocyte reduction filters
has been associated with dramatic
hypotension in some individuals, often in
the absence of other symptoms. This
happens more frequently with patients
taking angiotensin-converting en- zyme
inhibitors. The use of components that were
filtered in the blood center or transfu- sion
service before storage decreases the inci-
dence of such reactions.31 If a precipitous
drop in blood pressure is noted, the
transfusion should be stopped immediately.
It is important to verify that the filter is
in- tended for use with the component being
transfused (RBCs or platelets) and to note
the maximum number of units that can be
admin- istered through one filter. Filters
designed for RBCs or platelets may not be
used inter- changeably. The manufacturer’s
instructions should be followed for priming
and adminis- tering blood components
through the filter. Otherwise, leukocyte
removal may be ineffec- tive or an air lock
may develop, preventing passage of the
component through the filter. Leukocyte
filters should never be used to ad- minister
granulocytes or hematopoietic pro- genitor
cells.
Compatible IV Solutions
No medications or solutions other than 0.9%
sodium chloride injection, USP, should be
ad- ministered with blood components
through the same tubing. Solutions
containing dex- trose alone may cause red
cells to swell and lyse. Lactated Ringer’s
solution or other solu- tions containing high
levels of calcium may overcome the
buffering capacity of the citrate
anticoagulant in the blood preservative solu-
tion and cause clotting of the component.32
AABB Standards allows exceptions to
the above restrictions when 1) the drug or
solution has been approved by the FDA for
use with blood administration, or 2) there is
documen- tation available to show that the
addition is safe and does not adversely
affect the blood or component.1(p45)
Acceptable solutions accord- ing to these
criteria include ABO-compatible plasma,
5% albumin, or plasma protein frac- tion.
Certain solutions are compatible with blood
or blood components as noted in the
package inserts reviewed by the FDA,
includ- ing Normosol-R pH 7.4 (Hospira,
Lake Forest, IL), Plasma-Lyte-A injection
pH 7.4 (Baxter Healthcare, Deerfield, IL),
and Plasma-Lyte 148 injection (Multiple
Electrolytes Injection, Type 1, USP, Baxter
Healthcare). There are sev- eral
formulations of Plasma-Lyte that are not
isotonic or that contain calcium; package in-
serts must be checked to confirm their com-
patibility with components.
MANUAL ADMINISTRATION
Starting the Transfusion
Once the identification of the unit and the
re- cipient is verified, the unit is spiked
using an aseptic technique. At institutions
that use Joint Commission hospital
accreditation, Joint Commission
requirements for the transfusion- ist
(HR.01.02.01) apply: “If blood transfusions
and intravenous medications are adminis-
tered by staff other than doctors of medicine
or osteopathy, the staff members must have
special training for this duty.”33
 C H A P TE R 2 1 Administration of Blood Components
The blood administration tubing should
be filled with either 0.9% sodium chloride
or the contents of the blood component. If
any solution or medication other than 0.9%
sodi- um chloride is infused before
component ad- ministration, the tubing
should be flushed with 0.9% sodium chloride
immediately before the blood infusion.
The infusion should start slowly, at ap-
proximately 2 mL per minute, for the first 15
minutes while the transfusionist remains near
the patient. Severe reactions may occur after
as little as 10 mL has been transfused. Poten-
tially life-threatening reactions most com-
monly occur within 10 to 15 minutes of the
start of a transfusion.34
The rate of transfusion should be in-
creased after 15 minutes to ensure the unit is
administered within the 4-hour window. The
advantages of using relatively rapid transfu-
sion rates (eg, 240 mL/hour) include correc-
tion of deficiency as rapidly as possible as
well as reduced patient and nursing time
dedicated to transfusion. Disadvantages
include the po- tential to cause reactions (eg,
volume over- load) or to make reactions
more severe (eg, FNHTR, septic reactions,
or allergic reac- tions). More rapid infusion
of leukocytes (with use of nonleukoreduced
cellular components) can result in a more
rapid rise in body temper- ature, a FNHTR,
or the transmission of bacte- ria and/or
allergenic substances.35 Many FNHTRs as
well as septic, allergic, and even some
hemolytic reactions may not be evident
within the first 15 minutes.
If there is no sign of a reaction after the
first 15 minutes, the flow rate can be
increased to the designated infusion rate,
taking into consideration the patient’s size,
blood vol- ume, and hemodynamic
condition in deter- mining the flow rate (see
Table 21-1). Careful attention should be paid
to avoid transfusing the unit too rapidly in
relation to the patient’s cardiac and/or and
respiratory status.
Monitoring the Transfusion
The unit identifiers should never be removed
during the transfusion.
■ 553
The transfusionist should continue to
monitor the patient throughout the infusion
and check the IV site and flow rate. If the IV
rate has slowed down, the transfusionist
should take one or more of the following ac-
tions: 1) check to make sure that the IV is
pat- ent and there is no swelling at the site;
2) at- tempt to administer the component
through an infusion pump; 3) raise or
elevate the unit;
4) examine the filter for air, excessive
debris, or clots; or 5) consider the addition
of 0.9% sodi- um chloride as a diluent if the
unit is too vis- cous. Frequent patient
monitoring during the infusion helps alert
the transfusionist to a pos- sible transfusion
reaction and allows for early interdiction.
Vital signs should be taken within 5 to
15 minutes of beginning the transfusion and
then according to institutional policy. There
is little evidence to support a best practice
related to the frequency of vital-sign
monitoring other than at baseline, soon after
the start of the transfusion, and after
transfusion.36 AABB Standards requires that
the medical record must include pre- and
posttransfusion vital signs.1(p45) Vital signs
should be taken at once if there is a
suspected transfusion reaction.
The transfusionist should be
knowledge- able about signs and symptoms
indicative of an adverse reaction and able to
act quickly (see Chapter 27). Visual
observation and pa- tient reporting of any
changes should be uti- lized to determine
that a reaction has occurred because the
patient may experience symp- toms before
changes occur in vital signs. If a transfusion
reaction is suspected, the transfu- sion
should be stopped and 0.9% sodium chlo-
ride administered. The 0.9% sodium
chloride should be infused near the IV
insertion site to avoid flushing the tubing
with the residual component. The unit
identification informa- tion should be
rechecked. The transfusionist should notify
the patient’s care provider of any suspected
transfusion reaction and obtain emergency
medication orders as needed to treat the
suspected reaction. It is helpful for in-
stitutions to summarize and have readily
avail- able to the transfusionist descriptions
of com- mon reactions and immediate steps
to be taken in the event of certain
symptoms.
 554

TABLE 21-1. Blood Component Transfusions in Nonemergency Settings

Component
■

Red Blood 1-2 mL/min As rapidly as tolerated; Infusion duration should Whole blood: ABO In-line (170-260
Cells (RBCs) (60-120 mL/hour) approximately 4 mL/ not exceed 4 hours. identical micron)
minute or 240 For patients at risk of RBCs: ABO compatible with Leukocyte reduction if
mL/hour fluid overload, may recipient’s plasma indicated
adjust flow rate to as low Crossmatch required
as 1 mL/kg/ hour.
Usually given

First 15 Minutes
Platelets 2-5 mL/min 300 mL/hour or as Crossmatch not required In-line (170-260
over 1-2 hours micron)
(120-300 mL/hour) toler- ated ABO/Rh compatibility pref-
For patients at risk of erable but not required Leukocyte reduction if
fluid overload, use indicated
May be HLA matched
slower flow rate (see
under RBCs)
Plasma 2-5 mL/min As rapidly as Crossmatch not required In-line (170-260
AABB T ECHNICAL M A NUAL

Thaw time may be

micron)
Suggested Adult Flow Rate
(120-300 mL/hour) tolerated; ABO compatibility with
needed before issue
approximately 300 mL/ recipient red cells
For patients at risk of
After 15 Minutes

hour
fluid overload, use
slower flow rate (see
Granulocytes 1-2 mL/min 120-150 mL/hour or Crossmatch required In-line (170-260 micron)
under RBCs)
(60-120 mL/hour) as tolerated ABO/Rh compatibility No leukocyte reduction fil-
Over approximately 2
required ter or depth-type micro
hours
May be HLA aggregate filters
Infuse as soon as matched
possi- ble after
Cryoprecipitated AHF As rapidly as collection/release of Crossmatch and ABO In-line (170-260
tolerated product; irradiate com- patibility not micron)
Special Considerations

required
Infuse as soon as
ABO Compatibility
 C H A P TE R 2 1 Administration of Blood Components
Once the patient is stable, the
transfusion service should be notified of a
suspected transfusion reaction, and
institutional policy should be followed for
returning the compo- nent bag and/or to
order the laboratory stud- ies needed to
evaluate the reaction.
Completing the Transfusion
The patient is assessed at the completion of
the transfusion, and his or her vital signs are
obtained. The bag and tubing are discarded
in a biohazard container if the transfusion
was uneventful.
Because patients can experience
transfu- sion reactions several hours to days
after the transfusion is complete, clinical
staff should continue to monitor the patient
periodically for 4 to 6 hours after the end of
the transfusion to detect febrile or
pulmonary reactions that may be associated
with blood administration. If the patient is
not under direct clinical super- vision after a
transfusion, clinical staff should provide
written instructions to the patient and
caregiver regarding signs and symptoms to
re- port and a phone number to call or a
person to contact should a reaction occur
later.
The transfusion should be documented in
the patient’s medical record. At a minimum,
AABB Standards requires documentation of
the following1(p45):
1. Transfusion order.
2. Recipient consent.
3. Component name.
4. Donation identification number.
5. Date and time of transfusion.
6. Pre- and posttransfusion vital signs.
7. Volume transfused.
8. Identification of the transfusionist.
9. Transfusion-related adverse events.
If additional units are transfused, the in-
stitution’s guidelines and/or manufacturer’s
recommendations should be consulted to de-
termine whether the same blood administra-
tion tubing may be used for subsequent
units. If there are no contraindications from
the manufacturer, institutions frequently
allow the tubing to be reused as long as
subsequent
■ 555
units are transfused within 4 hours of the
start of the initial transfusion . Therefore, if
more than 1 unit can be infused in 4 hours,
blood
administration tubing sets may be used for
more than one component.
UNIQUE TRANSFUSION
SET TINGS
See Chapter 23 for information about
transfu- sion in pediatric and neonatal
patients.
Operating Room and Trauma:
Rapid Infusions
If components need to be administered rapid-
ly, the use of pressure infusion, large-bore ad-
ministration tubing, and 8-Fr intravenous
catheters can decrease the infusion time with-
out inducing hemolysis.37,38 Specific tubing
sets are designed for rapid blood administra-
tion with appropriate filters. Flow rates as fast
as 10 to 25 mL/second (600-1500 mL/minute)
have been reported with such tubing. Howev-
er, at such rapid infusion rates, steps should be
taken to avoid hypothermia. Furthermore,
when multiple units are infused through the
same tubing, the flow rate may decrease ap-
preciably.
Hypocalcemia has been noted with
rapid transfusions. This is usually transient
and de- pendent on the amount and rate of
citrate in- fused. Calcium replacement may
be adminis- tered based on the patient’s
ionized serum calcium level and the rate of
citrate adminis- tration.39
Transfusion-associated hyperkalemic car-
diac arrest has been reported with rapid ad-
ministration of RBCs. It may develop with
rap- id RBC administration even with a
modest transfusion volume of between 1 (in a
neo- nate) and 54 units. Contributing factors
are ac- idosis, hypoglycemia, hypocalcemia,
and hy- pothermia at the time of cardiac
arrest.40
If components are urgently needed and
a delay in transfusion could be detrimental
to the patient, the transfusion service should
have a process to provide components
before all pretransfusion compatibility
testing is completed. In such cases,
uncrossmatched
556 ■ AABB T EC HNIC AL MANUAL

units are released with a signed statement
from the requesting physician indicating that
the clinical situation requires urgent release
before the completion of testing. If compo-
nents in the transfusion service inventory are
not immediately accessible to a trauma unit
or operating room, a supply of group O red
cells may be maintained at these sites. The
transfu- sion service must ensure proper
storage of components at these satellite
storage sites.
Out-of-Hospital Transfusion
Transfusing blood in a nonhospital setting
re- quires a well-planned program that
incorpo- rates all the relevant aspects of the
hospital setting and emphasizes safety
consider- ations.41,42 An outpatient surgery
center, oncol- ogy clinic, or dialysis center is
likely to be able to provide medical
assistance in a timely man- ner in the event
of an adverse reaction. Medi- cal staff
availability, medications, and equip- ment to
handle adverse reactions must be arranged
for in advance. Staff should be com- petent
in performing blood administration
procedures and patient monitoring. Blood
ad- ministration outside the hospital should
be performed by personnel with substantial
ex- perience in blood administration in this
set- ting.
Transfusion in the home generally
allows close monitoring of the transfusion
event be- cause the personnel-to-patient ratio
is one to
one. The disadvantage is that there is no
trained assistant available in the event of a
se- vere adverse reaction. Issues to consider
when preparing for a transfusion in the
home in- clude the following:
■ Availability of a competent adult in the
home to assist in patient identification
and to summon medical assistance if
needed.
■ A mechanism to obtain immediate physi-
cian consultation.
■ A telephone to contact emergency person-
nel and easy access for emergency vehicles.
■ Documentation of prior transfusions
unac- companied by adverse reactions.
■ The ability to properly dispose of medical
waste.
CONCLUSIONS
Transfusion of blood components and the
cre- ation of blood administration
procedures and policies should be patient
centric. Following these policies helps the
transfusionist quickly recognize and report
suspected transfusion re- actions. Close
monitoring and early interven- tion can
make a critical difference in patient
outcomes. The transfusion service should
pe- riodically audit the blood administration
pro- cess to identify instances of
nonconformance, analyze their causes, and
institute corrective actions.

KEY POINTS

Blood administration involves the process of informed consent, preparation of the recipi- ent, administration of the appropriate
A licensed care provider should initiate requests for blood administration with an order for the appropriate blood component a
The recipient should be informed of and educated about the upcoming administration of the blood component so that he or she
A baseline assessment of the recipient should be performed for subsequent comparison.
Just before the planned administration, the transfusionist must verify appropriate venous access, administration of any prophyl
 C H A P TE R 2 1 Administration of Blood Components ■ 557

6. Institutions should identify appropriate blood and blood component issue and delivery
mechanisms to ensure that the transfusionist receives the components in a timely
manner.
7. Transfusion services should ensure that other departments are aware of the requirement for
returning components if a transfusion is delayed.
8. Before a transfusion is initiated, verification of recipient and component identification
should be performed. The following items should be verified: 1) the appearance of the
unit,
2) identity of the recipient and unit, 3) medical order, 4) blood type, and 5) expiration
date/ time of the component.
9. Components must be administered through the appropriate infusion sets and filter if
indi- cated. No solution other than 0.9% sodium chloride injection, USP, should be
administered through the same tubing unless the tubing has been flushed with 0.9%
sodium chloride, USP, immediately before and after the transfusion.
10. The infusion should start slowly at approximately 2 mL per minute for the first 15
minutes. During this time, the transfusionist should remain near the patient. If no sign
of reaction appears, the infusion rate can be increased. The transfusionist monitors the
patient throughout the infusion and stops the infusion in the event of an adverse
reaction.
11. Infusions must be completed within 4 hours. After completion, the transfusionist takes
the patient’s vital signs. If the patient will not be under direct clinical supervision after
the transfusion, the patient and caregiver should receive instructions regarding signs
and symptoms to report and whom to report these reactions to.
12. The following information, at a minimum, about the transfusion must be documented in
the patient’s medical record: 1) the transfusion order, 2) patient consent for transfusion,
3) name of component, 4) donation identification number, 5) date and time of infusion,
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Indi- cators of erythrocyte damage after
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29. Wortham ST, Ortolano GA, Wenz B. A brief
his- tory of blood filtration: Clot screens,
microag- gregate removal, and leukocyte
reduction. Transfus Med Rev 2003;17:216-22.
30. Lane TA. Leukocyte reduction of cellular
blood components: Effectiveness, benefits,
quality control, and costs. Arch Pathol Lab
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31. Zoon KC, Jacobson ED, Woodcock J.
Hypoten- sion and bedside leukocyte
reduction filters. Int J Trauma Nurs
1999;5:121-2.
32. Dickson DN, Gregory MA. Compatibility of
blood with solutions containing calcium. S Afr
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33. The Joint Commission. Comprehensive ac-
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34. Bradbury M, Cruickshank JP. Blood transfu-
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35. Perkins HA, Payne R, Ferguson J, Wood M.
Nonhemolytic febrile transfusion reactions:
Quantitative effects of blood components with
emphasis on isoantigenic incompatibility of
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37. Iserson KV, Knauf MA. Confirmation of high
blood flow rates through 150 micron filter/
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38. Floccare DJ, Kelen GD, Altman RS, et al.
Rapid infusion of additive red blood cells:
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39. Denlinger JK, Nahrwold ML, Gibbs PS,
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40. Smith HM, Farrow SJ, Ackerman JD, et al.
Car- diac arrests associated with hyperkalemia
dur- ing red blood cell transfusion: A case
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41. Fridey JL. Practical aspects of out-of-hospital
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42. Evans CS. Out-of-hospital transfusion. Trans-
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 C h a p t e r 2 2

Perinatal Issues in Transfusion

Practice

Melanie S. Kennedy, MD; Meghan Delaney, DO,

MPH; and Scott Scrape, MD

HE MOLYTIC DISEASE OF the fetus and
newborn (HDFN), fetal/neonatal al-
loimmune thrombocytopenia (FNAIT), and
immune thrombocytopenia (ITP; previously
known as “immune thrombocytopenic purpu-
ra”) affect pregnant women and their fetuses
and newborns. The blood bank and transfu-
sion service play critical roles in supporting
the diagnosis and treatment of these condi-
tions, including the appropriate provision of
Rh Immune Globulin (RhIG).
HDFN
HDFN is the destruction of fetal and
newborn red cells by maternal red cell
alloantibodies that are specific for inherited
paternal red cell alloantigen(s). The
maternal IgG antibody is transported across
the placenta into the fetal circulation, where
it binds to the correspond-
ing red cell antigen, causing the antibody-
coated red cells to be destroyed by macro-
phages in the fetal spleen. The fetal hemato-
poietic tissue initially responds by increasing
erythropoiesis and releasing many of the new-
ly produced red cells into the circulation pre-
maturely as nucleated precursors, a condition
known as “erythroblastosis fetalis.” With
wors- ening anemia, excess erythropoiesis
occurs in the liver and spleen, causing organ
enlarge- ment and portal hypertension. A
resulting de- crease in liver production of
albumin leads to reduced plasma colloid
osmotic pressure, gen- eralized edema, ascites,
and effusions known as “hydrops fetalis.”
Untreated, hydrops feta- lis, with its associated
high-output cardiovas- cular failure, can lead
to fetal death. Severe disease can occur as
early as 18 to 20 weeks’ gestation; severity
usually increases in subse- quent pregnancies.

Melanie S. Kennedy, MD, Clinical Associate Professor Emeritus, Attending Physician, Transfusion Medicine,
Wexner Medical Center, Department of Pathology, The Ohio State University, Columbus, Ohio; Meghan Del-
aney, DO, MPH, Medical Director, Red Cell Genomics, Puget Sound Blood Center, Medical Director, Blood
Bank, Seattle Children’s Hospital, Assistant Professor, University of Washington, Seattle, Washington; Scott
Scrape, MD, Assistant Professor, Director, Transfusion Medicine, Wexner Medical Center, Department of
Pathology, The Ohio State University, Columbus, Ohio
The authors have disclosed no conflicts of interest.

561
562 ■ AABB T EC HNIC AL MANUAL
Maternal Alloimmunization
Females can be alloimmunized to red cell anti-
gens by previous transfusion or transplanta-
tion and/or previous or current pregnancy.
Fetomaternal hemorrhage (FMH) occurs
spontaneously during pregnancy and its likeli-
hood increases with gestational age (from 3%
in trimester I to 12% and 45% in trimesters II
and III, respectively).1,2 Because exposure to
fetal red cells and resulting maternal alloim-
munization typically occur late during preg-
nancy and at delivery, the fetus and newborn
of the first pregnancy are rarely affected. The
risk of FMH is higher in women who have ex-
perienced trauma or undergone amniocente-
sis, cordocentesis, abortion, or other proce-
dures.2
Complex factors influence the ability of
individuals to immunologically respond to red
cell antigens. Rh(D) is the most potent immu-
nogen, in that a 200-mL transfusion of Red
Blood Cells (RBCs) stimulates anti-D in about
85% of D-negative individuals, except those
who are immunosuppressed. About half of the
remaining 15% will never become immunized,
even after repeated challenges with D-positive
red cells. Although as little as 0.1 to 1 mL of
D- positive red cells can stimulate antibody
pro- duction, the volumes of FMH are
generally small, which contributes to relatively
low alloimmunization rates in pregnancy. 3
Before Rh(D) immunoprophylaxis, 16% of
ABO-com- patible, D-negative mothers with
D-positive infants became immunized; the rate
was 2% in ABO-incompatible, D-negative
mothers.3-5
Antibodies targeted to blood group anti-
gens in addition to RhD can cause HDFN. The
most common of these antigens are K and c. 6
Unlike HDFN caused by anti-D, HDFN
caused by anti-K uniquely results in
destruction of fe- tal erythropoietic precursors
in addition to he- molysis.7,8(p527) Other
antibodies that have been less commonly
reported to cause moderate or severe disease
include E, k, Kpa, Kpb, Ku, Jsa, Jsb, Jka, Fya,
Fyb, S, s, and U.8 In rare cases, anti-Ge and
anti-M have been reported to cause de-
struction of fetal erythropoietic precursors.
Pathophysiology of HDFN
Hemolysis occurs when the maternal
antibody binds to a fetal red cell antigen,
causing at- tachment to the Fc receptor of
macrophages in the spleen of the fetus. The
rate of hemolysis and severity of disease are
determined by the IgG subclass, amount of
antibody, and number of antigenic sites on
the red cells.9 The sub- classes IgG1 and
IgG3 are more efficient in causing
hemolysis than IgG2 or IgG4. Trans-
portation of IgG1 and IgG3 across the
placenta is mediated by the Fc receptor
beginning in the second trimester and
continuing until birth.10 Because IgG1 is
transported across the placenta earlier and in
larger amounts than IgG3, IgG1 is
associated with more severe dis- ease. After
delivery, antibody persistence can cause
continuing destruction of red cells, pro-
gressive anemia, and hyperbilirubinemia.
However, the amount of maternal antibody
present continues to decrease over the next
12 weeks, with a half-life of about 25 days.
During gestation, fetal red cell destruc-
tion releases hemoglobin. The hemoglobin
is broken down into bilirubin, which passes
into the maternal circulation and is
conjugated by the maternal liver, preventing
the bilirubin’s return to the fetus. Birth
severs the connection with the mother,
causing the bilirubin (result- ing from red
cell destruction) to remain in the neonatal
circulation. Because of the infant’s immature
liver enzymatic pathways, the amount of
unconjugated bilirubin can in- crease to
dangerous levels and cause perma- nent
damage to the brain, known as “kernic-
terus.”11
Diagnosis
The diagnosis and management of HDFN
in- volve the close cooperation of the
patient, ob- stetrician, and blood
bank/laboratory person- nel. During the first
trimester, the patient’s obstetric and
transfusion history should be obtained. A
previously affected pregnancy alerts the
obstetrician to possible problems in the
current pregnancy.12
 CH APT E R 2 2 Perinatal Issues in Transfusion Practice
Laboratory Testing
During the first prenatal visit, maternal ABO
and Rh should be typed and an antibody
screen should be performed. If an RhD-nega-
tive woman has an initial negative antibody
screening result, she is a candidate for RhIG
administration (see “RhIG” section below).
The antibody screen should be conducted us-
ing techniques that detect IgG antibodies that
are reactive at 37 C and in which separate
screening cells represent all clinically impor-
tant specificities. A positive antibody screen-
ing result requires antibody identification. An-
tibodies such as anti-I, -P1, -Lea, and -Leb,
whether IgM or IgG, may be ignored because
these antigens are poorly developed at birth.
Treatment of the mother’s plasma with dithio-
threitol (DTT) can help distinguish IgG from
IgM antibodies.13
After identifying an antibody known to
cause HDFN, the next step for fetal risk
stratifi- cation is to test the father for the
presence of the corresponding red cell antigen.
If the fa- ther is homozygous for the
corresponding an- tigen, 100% of the fathers’
offspring will be at risk of HDFN. If the father
is heterozygous, 50% of the father’s offspring
will be at risk.
For pregnancies sensitized with anti-D,
no serologic methods can determine paternal
zygosity. In these situations, paternal DNA
testing is indicated.14 Using fetal amniocytes,
direct testing of the fetal red cell genotype can
predict the red cell phenotype. The reported
sensitivity and specificity of DNA testing by
polymerase chain reaction are 98.7% and
100%, respectively, with a low false-negative
rate (1-3%).14 Molecular typing of fetal DNA
can also be performed on maternal plasma
early in the second trimester.15
During a sensitized pregnancy, antibody
titers can assist with management decisions.
The AABB-recommended method is the use of
saline antihuman globulin (AHG) incubated
for 60-minutes at 37 C (Method 5-3). Other
methods, such as using albumin AHG or gel,
may result in higher titers than the recom-
mended method and should be validated with
clinical findings and laboratory data to ensure
appropriate interpretation by the obstetrician
■ 563
and avoid inappropriate referral of patients
for high-risk obstetric care. The critical titer
for anti-D (the level below which HDFN
and hy- drops fetalis are unlikely and no
invasive pro- cedures are needed) is 16 in
the AHG phase. As long as the titer is 8 or
lower, except in the case of anti-K, the
pregnancy can be followed by titers.
Kell-system antigens are present on early
red cell precursors, so even a maternal anti-K
titer that is relatively low can cause erythropoi-
etic failure and severe anemia. A critical titer
of 8 is generally accepted for Kell system
antibod- ies.7 Due to the poor reproducibility
of titers, individual blood banks should
validate their testing internally and keep
previous speci- mens for subsequent
comparison. Flow cytometric quantitation of
antibody mass has proven to be more precise
than antibody titers.16
Pregnancy Monitoring
Obstetric care usually involves monitoring
af- fected fetuses with a combination of
maternal antibody titers and fetal ultrasound
of the middle cerebral artery to assess fetal
anemia.12 When a critical antibody titer is
reached at 16 weeks of gestation,
ultrasound and color Dop- pler
ultrasonography are performed to estab- lish
disease severity. Decisions about how and
when to treat an affected fetus are based on
the degree of fetal anemia and gestational
age.
Treatment
In cases of severe fetal anemia from hemolytic
disease, fetal transfusion is indicated to treat
the anemia and suppress fetal erythropoiesis.
Intrauterine transfusion (IUT) is performed by
inserting a needle into the umbilical vein using
color Doppler enhancement of high-resolu-
tion sonography to guide the procedure. A pre-
transfusion sample of fetal blood is obtained
at the same time to determine the fetus’s blood
type, direct antiglobulin test (DAT) result,
anti- gen type, hemoglobin level, hematocrit,
plate- let count, and bilirubin level. DNA
typing may be performed as well.
Cordocentesis is associ- ated with a 1% to 2%
risk of fetal infection, bleeding, bradycardia,
and/or premature
564 ■ AABB T EC HNIC AL MANUAL
membrane rupture. Because cordocentesis
may cause FMH, RhIG immunoprophylaxis
should be administered after the procedure
to D-negative women who do not have anti-
D.
Blood Product Selection
and Administration
For IUT, the blood should be 1) irradiated to
prevent transfusion-associated graft-vs-host
disease in the immunologically immature fe-
tus, 2) cytomegalovirus (CMV) reduced-risk
(through leukocyte reduction and/or by
being CMV seronegative), and 3) known to
lack he- moglobin S to prevent sickling
under low oxy- gen tension. Donor group O
RBCs that are an- tigen negative for the
mother’s corresponding antibodies and
crossmatch compatible with maternal
plasma are selected. Generally, RBC units
that were collected within 7 days before
transfusion are preferred if they are
available. Centers have reported that using
RBCs that are antigen matched to the
mother decreases the risk of further maternal
sensitization from IUT.17
In rare cases, the mother’s antibody is
di-
rected at a high-prevalence antigen and no
compatible blood is available. In these situa-
tions, the mother’s washed or frozen and de-
glycerolized RBCs can be used for fetal
IUT. Testing the mother’s siblings or
searching rare donor registries may provide
additional sourc- es of compatible units.
The volume of blood to be transfused
can
be calculated, as shown in the following
exam- ple, by 1) determining the fetal and
placental total blood volume by multiplying
the ultra- sound-estimated fetal weight in
grams (eg, 1000 g) by 0.14 mL/g, 2)
multiplying this amount (eg, 140 mL) by the
difference in post- transfusion (desired) and
pretransfusion he- matocrit (eg, 0.40 – 0.15
= 0.25), and 3) dividing the resulting
amount by the hematocrit of the RBC unit
(eg, 0.85). In this example, the result is 41.2
mL.
Example:
[(1000 g) × (0.14 mL/g) × (0.40 – 0.15)]/85
=
41.2 mL
The blood volume transfused by cordo-
centesis and the rate of transfusion should
be adjusted to accommodate the clinical
status of the fetus.18 Transfusion is repeated
if needed according to the severity of the
disease or based on an estimated decline in
hematocrit of 1% per day to maintain the
fetal hematocrit at about 30%.
Other Treatments
Maternal plasma exchange and the adminis-
tration of intravenous immune globulin
(IVIG) have been used early in gestation
before IUT can be accomplished or as
alternatives to in- trauterine transfusion.4
Plasma exchange can temporarily reduce
antibody levels by as much as 75%. Because
its efficacy was established before the use of
IUT was widespread, its use today is
reserved for treatment failures on a case-by-
case basis. The American Society for
Apheresis classifies plasma exchange as a
Cat- egory II treatment based on weak
(Grade 2C) evidence for this indication.19
IVIG infusion has been shown to stabilize
anti-D titers, and results were best when the
procedure was started before 28 weeks’
gesta- tion in a case series of 24 patients.20
Neonatal Management
During the first days after birth, close
monitor- ing of the bilirubin level is
necessary because of the threat of
kernicterus, especially in pre- mature
neonates.21 The infant may require blue-
green light therapy, which oxidizes the el-
evated unconjugated bilirubin, allowing the
oxidation products to be excreted in the
urine. In addition, IVIG may be given to the
infant to help control hemolysis and, thus,
elevated bili- rubin. In neonates who are
unresponsive to phototherapy and IVIG, a
double-volume- exchange transfusion
removes approximately 90% of the fetal red
cells and 50% of the biliru- bin. Exchange
transfusion is generally unnec- essary if the
infant received IUTs. See Chapter 23 for a
discussion of exchange transfusion in the
newborn.
 CH APT E R 2 2 Perinatal Issues in Transfusion Practice
RhIG
RhIG is available in 300-µg and 50-µg
doses to prevent alloimmunization to the D
antigen. The risk of a D-negative mother
becoming im- munized by a D-positive fetus
can be reduced from about 16% to less than
0.1% by the ap- propriate administration of
RhIG.3,4
Screening and Dosing for RhIG
Antepartum Administration
When a pregnant mother is D negative and
the father is D positive, the fetus may be D
positive and the mother may be at risk of D
alloimmu- nization. Such women are
candidates for RhIG prophylaxis to prevent
alloimmunization. D- negative females
whose infants are D negative, D-negative
females who have been previously
immunized to D, and D-positive females are
not candidates for RhIG. Whether women
with variants of D should be considered to
be D positive is controversial. Maternal red
cell ge- notyping can assist with RhIG
treatment deci- sions because some D
variants have been found to make anti-D.22,23
The American College of Obstetricians
and Gynecologists (ACOG) recommends
RhIG administration at 28 weeks’ gestation
because 92% of women who develop anti-D
during pregnancy do so at or after 28
weeks.3,24 Ante- natal RhIG administration
reduces alloimmu- nization to 0.1%
compared to 1.5% with post- partum
administration only. In addition, RhIG is
indicated after amniocentesis, cordocente-
sis, version, abortion, or abdominal trauma.
Postpartum Administration
D-negative mothers without anti-D should
receive RhIG after delivery of a D-positive in-
fant. A postpartum blood sample should be
screened for FMH to determine the RhIG
dose. The rosette test is 99.5% sensitive to an
FMH of 10 mL or more. After incubation with
anti-D, indicator D-positive red cells form
aggluti- nates (rosettes) with the fetal D-
positive red cells. Weak D phenotypes in the
mother or fe- tus can cause false-positive or -
negative test results. Therefore, D-negative
mothers with
■ 565
positive results on a test for fetal blood in
the maternal circulation should undergo a
sero- logic weak D test or molecular RHD
testing. If the rosette test result is negative, a
dose of 300
µg RhIG (100 µg in the United Kingdom) is
giv- en, which is sufficient to prevent
immuniza- tion by 15 mL of red cells (5 mL in
the United Kingdom) or 30 mL of whole
blood. The pres- ence of residual anti-D from
antepartum RhIG does not indicate ongoing
protection.
A positive rosette test result indicates a
large FMH. About 0.3% of deliveries have
an FMH larger than 30 mL. When a rosette
test re- sult is positive, a quantitative test,
such as the Kleihauer-Betke (acid/elution)
test, or flow cy- tometry is needed to
calculate the dose of RhIG. Flow cytometry
can precisely measure fetal hemoglobin and/
or D-positive red cells. The use of both
markers avoids false-positive results from
maternal red cells containing fetal
hemoglobin.25,26
The Kleihauer-Betke test has been shown
to be far less precise than flow cytometry be-
cause of its technical difficulty. The
Kleihauer- Betke test is based on the resistance
of fetal he- moglobin to acid treatment
(Method 5-2). A thin smear of maternal blood
is placed on a slide, treated with acid, rinsed,
counter- stained, and read microscopically by
counting 2000 cells. The maternal cells appear
as ghosts, and the fetal cells are pink. The
formula below is used to calculate the fetal
bleeding:
(Fetal cells/total cell counted) × maternal
blood volume (mL) = fetal hemorrhage
(mL)
Example:
(6 cells/2000 cells) × 5000 mL
= 15 mL fetal whole blood
According to the results for this
example, a 300-µg vial of RhIG will
suppress alloimmu- nization by 30 mL of
fetal whole blood. Fetal hemorrhage is 15
mL, so the number of RhIG vials is 15
mL/30 mL/vial = 0.5 vial. Because of the
inherently wide estimate generated by the
test, if the calculated dose to the right of the
decimal point is 0.5 vial, it should be
rounded up to the next whole number plus
one vial; if
566 ■ AABB T EC HNIC AL MANUAL

the calculated dose to the right of the
decimal point is <0.5 of a vial, it should be
rounded down to the next whole number
plus one vial (Table 22-1). In the above
example, the dose to be given is two vials.
Additional examples are as follows:
Examples:
1.6 vials calculated =
2 (round up) + 1 (add 1) = 3
1.4 vials calculated =
1 (round down) + 1 (add 1) = 2
Postpartum RhIG should be given to the
mother within 72 hours of delivery. If prophy-
laxis is delayed, the ACOG recommends that
treatment still be administered. If the D type of
the newborn is unknown or undetermined (eg,
for a stillborn infant), RhIG should be admin-
istered.
Depending on the preparation, RhIG
can be given by intramuscular (IM) or
intravenous (IV) injection. If IM
preparations are used, multiple doses are
given in different sites or at different times
within 72 hours. Multiple doses of the IV
preparation may be administered ac- cording
to the instructions in the package in- sert.
Serology and Mechanism
Administration of RhIG during pregnancy
may produce a positive antibody screening
result in
the mother, but the titer is rarely greater than
4 and thus poses no risk to the fetus.
Occasion- ally, the DAT result may be
positive in a new- born with no evidence of
hemolysis. About 10% of the 28-week
gestation dose will be pres- ent at delivery
(the half-life of IgG is 25 days). This anti-D
is not active immunization, so postpartum
RhIG should be given if the new- born is D
positive.
RhIG is entirely IgG, whereas active im-
munization has an IgM component. Thus, new
anti-D produced by the mother can often be
detected in the saline phase and can be com-
pletely or partially inactivated by 2-mercapto-
ethanol or DTT treatment, whereas RhIG can-
not. In addition, passively acquired anti-D
rarely achieves a titer above 4. Antibody titers
do not correlate with the effectiveness of the
RhIG or the amount of FMH.
The mechanism of action of RhIG has
not been completely elucidated. Current
evidence shows that D-positive red cells are
opsonized by RhIG and removed by
macrophages, which release cytokines that
result in immunomodu- lation.27 The number
of IgG molecules known to prevent
immunization is much smaller than the D
antigen sites on red cells.
ABO HEMOLY TIC DISEASE
Because of the use of RhIG, ABO
incompatibil- ity is now the most common
cause of HDFN. HDFN is triggered when
naturally occurring

TABLE 22-1. Amount of RhIG to Administer Based on Amount of Fetomaternal Hemorrhage

Dose

% Fetal Cells Vials to Inject g (mcg) IU

0.3-0.8 2 600 3000
0.9-1.4 3 900 4500
1.5-2.0 4 1200 6000
2.1-2.6 5 1500 7500

Notes:
1. Based on a maternal blood volume of 5000 mL.
2. 1 vial of 300 g (1500 IU) is needed for each 15 mL of fetal red cells or 30 mL of fetal whole blood.
 CH APT E R 2 2 Perinatal Issues in Transfusion Practice
IgG anti-A,B in a group O mother is
transport- ed across the placenta and bound
to fetal red cells expressing A or B antigens.
Destruction of fetal red cells rarely leads to
severe anemia be- cause fetal ABO antigens
are poorly developed and antibody is
neutralized by tissue and solu- ble antigens.
Also, ABO HDFN has no comple- ment-
mediated hemolytic mechanism. If an
umbilical cord DAT result is negative, ABO
HDFN is unlikely even if the mother has the
corresponding ABO antibody(ies). After birth,
hyperbilirubinemia can be successfully
treat- ed with phototherapy in most cases; in
rare sit- uations, exchange transfusions may
be re-
quired.
Group A and B infants of group O
mothers are more severely affected by ABO
HDFN. In populations of European or Asian
ancestry, group A infants are most commonly
affected; in populations of African ancestry,
group B in- fants are most likely to be
affected. The overall incidence of ABO HDFN
is higher in people of African than European
ancestry.8(p529) In pa- tients with severe disease,
the DAT result is nearly always positive.28
If ABO HDFN is ruled out, antibodies
against low-prevalence red-cell antigens in-
herited from the father should be suspected.
Testing the eluate from cord blood or maternal
serum (if ABO compatible) against the father’s
red cells with an antiglobulin technique is of-
ten diagnostic.
IMMUNE THROMBOCYTOPENIA
Maternal IgG antibodies to platelets can
cross the placenta and cause severe
thrombocyto- penia. Two categories of
immune thrombocy- topenia are recognized:
FNAIT and ITP. The diagnostic distinction
between them is impor- tant for therapy
selection.
FNAIT
FNAIT is caused by antibodies that are
specific for platelet antigens inherited from
the father that are are absent in the mother.
Platelet anti- gens represent specific
polymorphisms in platelet membrane
glycoproteins. Approxi- mately 80% of
FNAIT cases are caused by hu-
■ 567
man platelet antigen HPA-1a, which is present
in about 98% of the US population.29 About
10% of cases are caused by anti-HPA-5b, 4%
by anti-HPA-1b, 2% by anti-HPA-3a, and 6%
by other antibodies. The incidence of affected
pregnancies is approximately 1 per 1500 to
2000.30
In about 25% of FNAIT cases, the platelet
antibody develops during the first pregnancy
and that fetus is affected. The maternal anti-
body has been detected as early as 17 weeks’
gestation and the fetus may develop thrombo-
cytopenia as early as 20 weeks’ gestation.
How- ever, the disease is often not discovered
until birth, when the newborn presents with
pete- chiae, ecchymoses, or intracranial
hemor- rhage. Intracranial hemorrhage occurs
in 10% to 30% of infants and 50% of fetuses
with FNAIT.29 The greatest risk of hemorrhage
oc- curs when the fetal platelet count is less
than 50,000/µL. The response of the fetal
hemato- poietic system to FNAIT is variable
and may include compensatory extramedullary
hema- topoiesis. In rare cases, hydrops fetalis
devel- ops. Fetal anemia without red cell
incompati- bility can also occur.
A history of giving birth to a newborn
with thrombocytopenia can alert the
obstetrician to a potentially affected
pregnancy. The preg- nant woman and the
father should be typed for platelet antigens,
and the woman should be screened for the
alloantibody. DNA testing of the father can
determine the zygosity of the antigen
involved.31
The DNA genotype of the fetus can be
de- termined as early as 11 to 13 weeks’
gestation. Assessment of the fetus should
begin at or be- fore 20 weeks’ gestation, when
severe throm- bocytopenia and hemorrhage
can occur. When cordocentesis is used to
determine the platelet count, irradiated, CMV-
reduced-risk, antigen-negative platelets should
be used.
If needed, platelet transfusion should be
given to the fetus to treat thrombocytopenia
and avoid hemorrhage. Many blood
suppliers have identified donors who are
negative for human platelet alloantigen 1a,
the most widely implicated platelet antibody,
and can prepare suitable platelets for IUT.
The mother is negative for the implicated
alloantigen and
568 ■ AABB T EC HNIC AL MANUAL
can be a donor if her platelets are washed.
When platelet transfusion is needed urgently
and maternal platelets are unavailable, un-
selected platelets may be used.32
The mother is given IVIG after the
fetus is determined to be affected. The dose
is usually 1 g/kg each week. In a
retrospective review, IVIG treatment alone
appeared to be as safe and effective as
cordocentesis with platelet transfusion.33
However, IVIG treatment failures may occur
and can necessitate fetal platelet count
monitoring by cordocentesis and re- quire
platelet transfusions.34 The goal of both
transfusion and IVIG treatment is to avoid
hemorrhage. Ultrasound monitoring of the
fe- tus to detect hemorrhage is not
recommend- ed because intracranial
hemorrhage usually indicates permanent
brain damage. Before vaginal delivery, the
fetal platelet count should be >50,000/µL.
After birth, the infant’s platelet count
may decrease in the first few hours to days.
In 2 to 3 weeks, when the antibody has been
cleared, the infant’s platelet count returns to
normal. The presence or absence of severe
thrombocy- topenia and intracranial
hemorrhage in the firstborn correlates with
the outcomes of sub- sequent pregnancies.35
ITP
Autoantibodies against platelets, which are
re- active with the mother’s own platelets as
well as donor or fetal platelets, are another
cause of fetal and neonatal
thrombocytopenia. Throm-
KEY POINTS
HDFN is caused by maternal antibodies that are specific to a paternal red cell antigen. The maternal IgG antibody is transporte
Some antibodies, such as anti-I, -P1, -Lea and -Leb, can be ignored. The most common clini- cally significant antibodies are a
Molecular typing of fetal DNA can be performed on maternal plasma early in the second tri- mester.
The recommended titer method is saline AHG with 60-minute incubation at 37 C. Other methods, such as those using albumin
For IUT, the blood should be irradiated, CMV reduced-risk, hemoglobin S negative, group O (in most cases), and less than 7 d
bocytopenia in a pregnant woman is
common and is rarely associated with ITP.
However, ITP may cause thrombocytopenia
in both the mother and fetus. Fortunately,
only about 10% of newborns with ITP
have platelet counts
<50,000/µL, and only 1% to 2% have a high
risk of hemorrhage.36
A pregnant woman with
thrombocytope- nia or a preexisting
diagnosis of ITP should be tested for serum
platelet autoantibody. A neg- ative result
generally indicates that the throm-
bocytopenia was caused by other conditions
and suggests that the fetus or neonate is not
at risk. In contrast, a pregnant woman with
sig- nificant thrombocytopenia and
petechiae or other evidence of hemorrhage
caused by auto- antibody should undergo
similar treatment to that used in nonpregnant
women with ITP.
Prednisone at a dose of 1 to 2 mg/kg is
usually effective. However, with persistent
ma- ternal thrombocytopenia, IVIG 1
g/kg/day for 2 to 5 days is indicated.
Although the maternal platelet count is often
monitored in these cas- es, it does not
correlate with the newborn’s platelet count.36
Fetal blood sampling to deter- mine platelet
count is not usually recom- mended because
the risk of morbidity and mortality from
cordocentesis is greater than or equal to the
risk of severe bleeding in utero or at
delivery. However, if fetal sampling is per-
formed, a platelet transfusion should be ad-
ministered at the same time. Platelet transfu-
sions may be needed in about 15% of
newborns.36
 CH APT E R 2 2 Perinatal Issues in Transfusion Practice ■ 569

6. The rosette test is a sensitive method for detecting fetomaternal hemorrhage of ap-
proximately 10 mL or more. Flow cytometry can precisely measure hemoglobin F and/
or D-positive red cells.
7. The calculated RhIG dose should be rounded up if the number to the right of the
decimal point is 0.5 or rounded down if the number is <0.5. In either case, a vial
should be added to the result.
8. Despite the prevalence of ABO HDFN, severe anemia rarely occurs. After birth,
hyperbiliru- binemia can usually be treated with phototherapy alone.
9. In fetal/neonatal alloimmune thrombocytopenia, the platelet antibody may develop at
around 17 weeks of gestation in the first pregnancy and fetal thrombocytopenia may
devel- op as early as 20 weeks. Irradiated, CMV-reduced-risk, antigen-negative
platelets should be given to treat thrombocytopenia and avoid hemorrhage.

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J. Flow-cytometric quantitation of anti-D
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17. Schonewille H, Klumper FJCM, Watering
LMG, et al. High additional maternal red
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20. Margulies M, Voto LS, Mathet E. High dose

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24. Prevention of Rh D alloimmunization. ACOG
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34. Silver RM, Porter TF, Branch DW, et al.
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Neona- tal alloimmune thrombocytopenia:
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27. Branch DR, Shabani F, Lund N, Denomme
35. Birchall JE, Murphy MF, Kroll H. European
GA. Antenatal administration of Rh-immune
col- laborative study of the antenatal
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immu- nomodulary cytokines transforming
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Transfusion 2006;48:1316-22. 36. Webert KE, Mittal R, Sigouin C, et al. A retro-
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 C h a p t e r 2 3

Neonatal and Pediatric

Transfusion Practice

Cassandra D. Josephson, MD, and Erin Meyer, DO, MPH

TR AN SFU S IO N PR ACTICE IN neo-

natal and pediatric patients differs
from that in adults.1 The differences are
related to physiologic changes occurring
during the transition from fetus to
adolescent. Blood volume, hematologic
values, immune system maturity, and
physiologic responses to hypo- volemia and
hypoxia are variable in this het- erogeneous
population, contributing to the complexity
and intricacies of pediatric trans- fusion
practice.
This chapter discusses neonatal and
pedi- atric transfusion practice during two
distinct periods: infancy from birth to 4
months, and infancy after 4 months and
childhood. The pe- diatric practices
addressed include 1) small- volume
component preparation, 2) transfu- sion
indications for blood components, 3)
transfusion administration and exchange
transfusion, 4) transfusion support in
specific diseases, 5) rationale for special
processing of blood components, and 6)
pediatric massive transfusion protocols
(MTPs).
TRANSFUSION IN INFANTS
YOUNGER THAN 4 MONTHS
Considerations for Component
Preparation and Therapy
The fact that patients younger than 4 months
have small blood/plasma volumes and imma-
ture organ system functions necessitates spe-
cial approaches to component therapy. This is
especially important for very-low-birthweight
(VLBW) infants (<1500 g) and extremely low-
birthweight infants (<1000 g).
Fetal and Neonatal Physiology
Affecting Transfusion Practice
Healthy full-term neonates have a mean
cord blood hemoglobin level of 16.9 ± 1.6
g/dL, whereas that of preterm neonates is
15.9 ±
2.4 g/dL. The hemoglobin concentration
nor- mally declines during the first few
weeks of life, resulting in the physiologic
anemia of in- fancy in newborns and
physiologic anemia of

Cassandra D. Josephson, MD, Director of Transfusion, Tissue, and Apheresis Services, Children’s Healthcare
of Atlanta, and Associate Professor, Pathology and Pediatrics, Emory University School of Medicine, and
Erin Meyer, DO, MPH, Associate Director of Transfusion, Tissue, and Apheresis Services, Children’s
Healthcare of Atlanta, and Assistant Professor of Pathology and Laboratory Medicine, Emory University
School of Medicine, Atlanta, Georgia
C. Josephson has disclosed financial relationships with Immucor and Octapharma. E. Meyer has
disclosed no conflicts of interest.

571
572 ■ AABB T EC HNIC AL MANUAL

full-term neo-
prematurity in preterm infants.2 Both
anemias are considered self-limited and are
usually tol- erated without harmful effects.
The rate of decline in hemoglobin
levels is a function of gestational age at
birth. At 4 to 8 weeks after birth,
hemoglobin decreases to as low as 8.0 g/dL
in preterm infants weighing 1000 to 1500 g
and 7.0 g/dL in neonates weigh- ing less
than 1000 g at birth.3 The physiologic
decrease in hemoglobin concentration is due
to several factors: 1) a decrease in
erythropoie- tin (EPO) resulting in
diminished red cell pro- duction, 2) a
decrease in survival of fetal red cells, and 3)
an increasing blood volume due to rapid
growth. Reduced EPO production re- sults
from increased oxygen delivery to tissues
because of increased pulmonary blood flow,
elevated arterial pO2 levels, and increased
red
cell 2,3-diphosphoglycerate (2,3 DPG) and he-
moglobin A levels.

Clinical Considerations Related

to Infant Size and Blood Volume
Blood volumes of pediatric patients vary with
body weight. A full-term newborn has a blood
volume of approximately 85 mL/kg compared
to 100 mL/kg in a preterm newborn. Due to
the small total blood volumes of preterm in-
fants (100 mL or less), blood banks must be
ca- pable of providing appropriately sized
blood components.
Many factors, including iatrogenic
blood loss from repeated phlebotomy, lead
to fre- quent transfusions. Hypovolemia is
not toler- ated well in newborns because
their left ven- tricular stroke volume
decreases without an increase in heart rate
when >10% of their blood volume is lost.
Thus, newborns must physiologically
increase their peripheral vas- cular
resistance with decreasing cardiac out- put
to maintain systemic blood pressure. Ulti-
mately, poor tissue perfusion and
oxygenation occur, resulting in metabolic
acidosis.4
Although transfusion may be required,
it
is neither necessary nor acceptable to
replace the amount of blood lost mL for mL;
rather, transfusions can be administered to
maintain a target hemoglobin level in certain
clinical sit- uations.5 When ill preterm and
 same goal as EPO therapy (ie, decreases the
nates receive multiple use of transfusions and the number of donor
transfusions, they have exposures) without expensive
proportionately lower pharmacotherapy and its risk of ad- verse
levels of circulating fetal effects.
hemoglobin and an
increase in adult hemo- Cold Stress
globin levels.
Hypothermia in the neonate can trigger or
ex- aggerate a number of responses,
Erythropoietic Response
including 1) an increase in metabolic rate; 2)
The EPO response in hypoglyce- mia; 3) metabolic acidosis; and
newborns differs from 4) potential apneic events that may lead to
that in adults and older hypoxia, hypo- tension, and cardiac arrest.
children. In the latter In-line blood warmers are required for all
groups, oxygen sensors Red Blood Cell (RBC) exchange
in the kidney recog- nize transfusions to combat the ef- fects of
decreases in oxygen hypothermia. A radiant heater should never
delivery, resulting in the be used to warm the blood being trans-
release of EPO into the fused due to the risk of hemolysis. Further-
circulation. In con- trast, more, to prevent hemolysis in neonates
in the fetus, this sensor is under-
located in the liver and is
less sensitive, resulting in
reduced EPO production
in the face of hypoxia
(hypo- responsiveness).
This response likely
occurs to prevent
polycythemia of the fetus
in the hy- poxic
intrauterine environment.
Although EPO
production eventually
shifts from the liver to
the kidney, most prema-
ture infants produce the
smallest amount of EPO
for any degree of
anemia.6 As an alterna-
tive to transfusion, the
use of recombinant hu-
man EPO has been
shown to reduce donor
ex- posures in
premature infants and
minimize the severity of
anemia.7 As compliance
with strict transfusion
threshold criteria has in-
creased, clinicians have
decreased their phle-
botomy rates in VLBW
infants, reducing the
rates of iatrogenically
induced anemia and
numbers of
transfusions. Thus, in
most cases, this
approach achieves the
 C H AP T E R 23 Neonatal and Pediatric Transfusion Practice
going phototherapy, the blood-
administration tubing should be positioned
to minimize ex- posure to phototherapy
light.8
Immunologic Status
Their immature immune system predisposes
infants to infectious and noninfectious haz-
ards of transfusion. In fact, much of the
special processing of products for preterm
infants and neonates is directly related to
their underde- veloped immune function.
Most of their hu- moral immunity (antibody
protection) is pro- vided by the mother
starting early in pregnancy (approximately
12 weeks) through placental transfer of
immunoglobulins. Be- tween 20 and 33
weeks of gestation, fetal IgG levels rise
significantly because of selective transport
system maturation in the placenta. The
breakdown of IgG occurs at a slower rate in
the fetus than in the mother, enabling con-
servation of the transplacental maternal anti-
body during the neonatal period. Unexpected
red cell alloantibodies of either IgM or IgG
class are rarely produced by the infant
during the neonatal period. This lack of red
cell allo- antibody production is not well
understood but has been postulated to be
due to deficient T-helper-cell function,
enhanced T-suppres- sor-cell activity, and
poor antigen-presenting- cell function.9
Cellular immune responses are also in-
completely developed during this period and
may make infants susceptible to transfusion-
associated graft-vs-host disease (TA-GVHD).
TA-GVHD has been reported most frequently
in newborns with confirmed or suspected
congenital immunodeficiency. The majority of
TA-GVHD cases reported in nonimmunocom-
promised infants have occurred after intra-
uterine transfusion and subsequent postnatal
exchange transfusion.10,11 There have also been
rare cases of TA-GVHD associated with ex-
treme prematurity, neonatal alloimmune
thrombocytopenia, or extracorporeal mem-
brane oxygenation (ECMO).10,12 Once an in-
fant develops TA-GVHD, the chance of
associ- ated mortality is higher than 90%. TA-
GVHD can be prevented by pretransfusion
irradiation of cellular blood components.12,13
■ 573
Metabolic Problems
In infants younger than 4 months, large-vol-
ume transfusions of reconstituted whole
blood or plasma may result in acidosis
and/or hypo- calcemia because of the
immature liver’s in- ability to effectively
metabolize citrate. The immature kidneys
also contribute to these complications
because they have lower glo- merular
filtration rates and concentrating abil- ity
than older infants and children, leading to
difficulties in excreting excess potassium,
acid, and/or calcium.
POTA SSIUM. Small-volume, simple transfu-
sions administered slowly have been shown to
have little effect on serum potassium concen-
trations in infants younger than 4 months de-
spite the high potassium levels in the plasma
of stored RBCs. In calculating levels of
infused potassium, Strauss13,14 determined that
an RBC unit (80% hematocrit) stored in an
extended storage medium for 42 days would
deliver 2 mL of plasma containing only 0.1
mmol/L of potassium when transfused at 10
mL/kg. This amount of potassium is much less
than the daily requirement of 2 to 3 mmol/L
for a pa- tient weighing 1 kg. It must be
stressed that this calculation does not apply to
the transfu- sion of large volumes of RBCs
(>20 mL/kg). Se- rum potassium can rise
rapidly in these small patients—particularly
during surgery, exchange transfusion, or
ECMO—and is dependent on the plasma
potassium levels in the blood and ma-
nipulation of the blood components.15,16
The type of anticoagulant-preservative
solution used to store RBCs at collection
deter- mines the amount of potassium
leakage. For instance, a unit of RBCs
preserved in an addi- tive solution (AS), such
as AS-1, AS-3, or AS-5, delivers less
extracellular potassium than RBCs stored in
citrate-phosphate-dextrose- adenine
(CPDA)-1. 13,17
In addition, special
component processing, such as irradiation,
can potentiate potassium leaks. If such com-
ponents are stored for more than 24 hours,
washing may be required to remove the
excess potassium before transfusion.12 This
washing practice is supported by several
reports of infants who received via central
line or intra-
574 ■ AABB T EC HNIC AL MANUAL
cardiac line either older RBC units or units
that had been irradiated (>1 day before
transfu- sion) and had severe adverse effects,
including cardiac arrest and death.18,19 This
washing practice is controversial, however, and
several institutions accept AS units for low-
volume neonatal transfusions without washing
as long as these units do not exceed a certain
storage age or time after irradiation.20
2 ,3-D P G. Levels of 2,3-DPG in red cells are
known to decline rapidly after 1 to 2 weeks of
storage. This deficit does not affect older chil-
dren and adult recipients negatively because
of their ability to replenish the missing 2,3-
DPG in vivo and to compensate for hypoxia
by increasing their heart rate. Infants younger
than 4 months are not able to do this as effec-
tively as a result of their low levels of intracel-
lular 2,3-DPG that reach even lower levels
with respiratory distress syndrome or septic
shock. Thus, if a large proportion of the
neonate’s blood volume is composed of
transfused 2,3- DPG-depleted blood, the
resulting shift in the hemoglobin oxygen
dissociation curve further increases oxygen
affinity for hemoglobin and reduces oxygen
availability to the tissues.
Therefore, the recommended therapy
for
newborns is an exchange transfusion with
RBCs that are usually less than 14 days old,
al- though this practice is variable and depen-
dent on institutional standard operating pro-
cedures. However, the medical need for fresh
RBC units for small-volume transfusions has
not been established and these transfusions
have even been characterized as unneces-
sary.5,13,16 A prospective randomized controlled
trial to assess the outcomes of longer vs short-
er storage times of RBCs is necessary in this
population (see “RBC Age” section below).21
RBC Transfusion Support
Ill neonates are more likely to receive RBC
transfusions than any other patient age group,
and RBCs are the component most often
transfused during the neonatal period.5 RBC
replacement is considered for sick neonates
when approximately 10% of blood volume has
been lost or they have symptomatic anemia.
Indications
Several guidelines have been published over
the past 15 years regarding the indications
for RBC transfusions in neonates.22-26 Most
of the recommendations are based on
experience ac- quired in clinical practice
rather than pub- lished evidence. To this
end, a critical need ex- ists for clinical
studies in this area.27 Table 23-1 lists the
most recently published guide- lines.23,26
Compatibility Testing
AABB Standards allows limited
pretransfusion serologic testing for infants
younger than 4 months.28(p39) Initial patient
testing must in- clude ABO and D typing of
the patients’ red cells and screening for
unexpected red cell an- tibodies using either
plasma or serum from the infant or mother.
During any hospitaliza- tion, crossmatch-
compatibility testing and re- peat ABO and
D typing need not be conducted as long as
all of the following criteria are met:
1) the antibody screening result is negative; 2)
transfused RBCs are group O, ABO identical,
or ABO compatible; and 3) transfused cells are
either D negative or the same D type as the pa-
tient. Testing the infant’s reverse type for anti-
A and/or anti-B is not necessary. However, be-
fore non-group O RBCs can be issued, testing
of the infant’s plasma or serum is required to
detect passively acquired maternal anti-A or
anti-B and should include antiglobulin phase.
If the antibody is present, ABO-compatible
RBCs must be transfused until the acquired
antibody is no longer detected.
If an unexpected non-ABO alloantibody
is detected in the infant’s or mother’s
specimen, the infant must be transfused with
RBC units lacking the corresponding
antigen(s) or units that are compatible by
antiglobulin cross- match. This regimen
should continue until the maternal antibody
is no longer detected in the infant’s plasma
or serum. The policy of the hospital
transfusion service determines the frequency
for reevaluating the patient’s anti- bodies.
Once a negative antibody screening result is
obtained, crossmatches and use of an- tigen-
negative blood are no longer required in
 C H AP T E R 23 Neonatal and Pediatric Transfusion Practice ■ 575

TABLE 23-1. Transfusion Guidelines for RBCs in Infants Younger than 4 Months 23,26

1. Hematocrit <20% with low reticulocyte count and symptomatic anemia (tachycardia, tachypnea,
poor feeding).

2. Hematocrit <30% and any of the following:

a. On <35% oxygen hood.

b. On oxygen by nasal cannula.

c. On continuous positive airway pressure and/or intermittent mandatory ventilation on

mechanical ventilation with mean airway pressure <6 cm of water.

d. With significant tachycardia or tachypnea (heart rate >180 beats/minute for 24 hours,
respiratory rate >80 beats/minute for 24 hours).

e. With significant apnea or bradycardia (>6 episodes in 12 hours or 2 episodes in 24 hours

requir- ing bag and mask ventilation while receiving therapeutic doses of
methylxanthines).

f. With low weight gain (<10 g/day observed over 4 days while receiving  100 kcal/kg/day).
3. Hematocrit <35% and either of the following:

a. On >35% oxygen hood.

b. On continuous positive airway pressure/intermittent mandatory ventilation with mean
airway pressure  6-8 cm of water.

4. Hematocrit <45% and either of the following:

a. On extracorporeal membrane oxygenation.

b. With congenital cyanotic heart disease.

infants younger than 4 months because of
their immature immunologic status.
Multiple observational studies have
shown that alloimmunization to red cell
anti- gens is rare during the neonatal
period.9,29,30 For this reason, repeated typing
and screening, which are required for adults
and children old- er than 4 months, is
unnecessary in younger infants and
contributes to significant iatrogen- ic blood
loss. Also, the transfusion service should
avoid transfusing any components that may
passively transfer unexpected alloanti-
bodies or ABO-incompatible antibodies to
re-
cipients.31
Components for Neonatal Transfusion
Advances in neonatology now permit the sur-
vival of extremely premature infants with sur-
factant therapy, nitric oxide therapy, high-
frequency ventilators, and compliance with
transfusion practice guidelines. These
advanc- es have substantially decreased the
number of RBC transfusions administered in
this popula- tion. Most neonatal transfusions
are now given to VLBW infants.32
Aliquoting for Small-Volume
Transfusion
The purpose of creating small-volume aliquots
is to limit donor exposures, prevent circulatory
overload, and33-37
potentially decrease donor-
related risks. Several technical approaches
can be used to accomplish these goals and
minimize blood wastage.38
Small-volume RBC transfusion aliquots
are commonly made with a multiple-pack
576 ■ AABB T EC HNIC AL MANUAL
system.38,39 Quad packs, employed mostly by
blood centers, are produced from a single
unit of whole blood that is diverted into a
primary bag with three integrally attached
smaller bags. The plasma is then separated
and divert- ed into one bag during
component prepara- tion. The remaining red
cells are drawn into the smaller bags as
needed for transfusion. Each of the smaller
units has the same expira- tion date as the
original unit because the sys- tem’s original
seal has remained intact and a “closed
system” is maintained.
A hospital transfusion service can then re-
move (either by heat sealer or metal clips)
each aliquot as needed. For hospital
transfusion services without a sterile
connection device (Fig 23-1), this method
provides three aliquots
FIGURE 23-1. A sterile tubing welder
(reproduced with permission from Terumo
Medical Corp., Somerset, NJ).
from a single unit.38 However, blood compo-
nents may still be wasted when used in ali-
quots that are larger than the dose selected
for each patient based on body weight.
Hospital transfusion services that have a
sterile connection device have multiple addi-
tional options to produce aliquots, such as
transfer packs [eg, PEDI-PAK system
(Genesis BPS, Hackensack, NJ) Fig 23-2],
small-volume bags, or tubing with integrally
attached syring- es.38 Syringe sets (Fig 23-3)
offer the greatest accuracy for obtaining the
desired volume to be transfused based on
volume-per-weight calculations.38 Some
syringe sets have an at- tached 150-micron
in-line filter for use during the aliquoting
process so that, when issued by the blood
bank, the cells are ready to be placed on a
syringe pump without further manipula- tion
of the component at the bedside. This
process eliminates the need for the nurse to
transfer blood from the pack to a syringe at
the bedside for delivery by a syringe pump.
Re- moving this additional step reduces the
risk of contamination, mislabeling, or
damage to the unit that results in blood loss
or spillage.38
Reducing donor exposures is more
readily accomplished by this technique,
which en- ables a recipient to receive
multiple small-vol- ume transfusions from a
single unit until it reaches its expiration
date.40,41 Many hospital transfusion services
assign a single unit of RBCs to one or more
infants based on their weight.34-40
Once an aliquot is produced at either
the blood center or hospital blood bank, it
must be labeled with the expiration date and
the origin and disposition of each smaller
unit must be recorded. Aliquot expiration
dates vary from institution to institution, and
local standard operating procedures should
always be fol- lowed.
RBC Additive Solutions
Historically, transfused RBCs for children
con- tained CPDA-1 anticoagulant-
preservative so- lution.40 However, as ASs
(see Chapter 6) evolved to extend the shelf
life of RBCs, many experts began to
question their safety in neo- nates. One
concern is the large amount of ade-
 C H AP T E R 23 Neonatal and Pediatric Transfusion Practice ■ 577

FIGURE 23-2. Diagram of PEDI-PAK (reproduced with permission of Genesis BPS, Hackensack, NJ).

nine and mannitol in AS and its relation to
re- nal toxicity. Moreover, mannitol is a
potent diuretic with effects on fluid
dynamics that can result in fluctuations in
the cerebral blood flow of preterm infants.
Most evidence suggests that small-vol-
ume transfusions (5 to 15 mL/kg) containing
AS are safe for this patient population.
Specifi- cally, when AS-1 and AS-3 were
compared, no harmful effects were observed
in neonates re- ceiving small-volume,
simple transfusions.40-42 These transfusions
were as effective as CPDA-1 RBCs in
increasing hemoglobin levels in recip- ients.
Luban and colleagues used theoretical
calculations in a variety of clinical settings
to demonstrate that red cells preserved
in
extended-storage media present no major
risk when used for small-volume
transfusions.43
However, for infants with renal or
hepatic insufficiency, it is recommended
that AS solu- tion be removed from RBC
units, particularly if multiple transfusions
from the same unit are expected. The safety
of AS-preserved RBCs in trauma-related
massive transfusions, cardiac surgery, or
exchange transfusions is unknown in this
population. Therefore, AS-preserved RBC
units should be used with caution in these
settings.21,42-45
RBC Age
The age of RBC units and its impact on
patient outcomes has become a concern,
although

FIGURE 23-3. Syringe with filter (reproduced with permission from Charter Medical, Ltd, Winston-Salem, NC).
578 ■ AABB T EC HNIC AL MANUAL
clinical confirmation of the basis for this
con- cern is controversial. A randomized
controlled trial conducted in Canada, Age of
Red Blood Cells in Premature Infants
(ARIPI), randomly assigned low birthweight
infants to be trans- fused with RBCs that
were 7 days old or less (mean = 5.1 days, n
= 188) or with standard- issue RBCs divided
into aliquots and stored for 2 to 42 days
(mean = 14.6 days, n = 189).46 The primary
composite endpoints included necro- tizing
enterocolitis (NEC), intraventricular
hemorrhage (IVH), and bronchopulmonary
dysplasia. The ARIPI trial found no
differences in the primary endpoints between
infants in the two arms, suggesting that in
the study pop- ulation, the age of RBCs does
not affect these common morbidities of
prematurity.
ARIPI’s external validity has been ques-
tioned because of the study's liberal transfu-
sion strategy, use of SAG-M units, and
average duration of blood storage. These
practices do not reflect the transfusion
practices or storage solution and ages of
RBCs used in many cen- ters in the United
States.47 Thus, it has not been firmly
established that there is a causal relationship
between morbidities and transfu- sion of
older RBC units in premature infants.47
TABLE 23-2. Blood Components and Dosing of Small Volumes in Neonatal and Pediatric Patients 49
Component Dose
Red Blood Cells 10-15 mL/kg
Fresh Frozen Plasma 10-15 mL/kg
Platelets [whole-blood-derived 5-10 mL/kg or
(WBD) or apheresis] 1 WBD unit/10 kg recovery)†
(patients  10 kg)
Cryoprecipitated AHF 1-2 units/10 kg
*Dependent on anticoagulant-preservative solution: with 3 g/dL increment for CPD and CPDA-1 and 2 g/dL for AS-1, AS-3,
and AS-5.
†
Assumes  5.5 × 1010 platelets in 50 mL of plasma (whole-blood-derived) and  3.0 × 1011 platelets in 250-300 mL plasma
(apheresis).
CPD = citrate-phosphate-dextrose; CPDA-1 = citrate-phosphate-dextrose-adenine-1; AS = additive solution.
Specific Indications for RBCs
In neonates, symptomatic anemia is the
major indication for simple transfusion.
Specifically, a venous hemoglobin of <13
g/dL in the first 24 hours of life necessitates
clinical consideration of an RBC
transfusion.48 RBC transfusions are also
considered when approximately 10% of a
sick neonate’s blood volume has been re-
moved or lost. When 10 mL/kg of RBCs
with a hematocrit of >80% are transfused,
the expect- ed increase in hemoglobin
concentration in a neonate is approximately
3 g/dL. A similar vol- ume of RBCs with
AS usually has a hematocrit of 65%, and its
transfusion results in a project- ed
posttransfusion hemoglobin increase of
<3 g/dL (see Table 23-2 for blood
component dosing recommendations and
expected re- sults).49
Two randomized controlled trials, the
Premature Infants in Need of Transfusion
(PINT) and its follow up study [PINT
Follow- up Outcomes Study (PINTOS)] as
well as a University of Iowa study compared
the out- comes of restrictive (Hb = 7 g/dL)
vs liberal RBC transfusion triggers (Hb = 10
g/dL) in VLBW infants.50-52 The Iowa trial
revealed a lower rate of transfusion events
(3.3 vs 5.2; p=0.025) with the restrictive
strategy com-
Expected Increment
Hemoglobin increase 2-3 g/dL*
15%-20% rise in factor levels (assuming
100%
recovery)
50,000/μL rise in platelet count (assuming 100%
60-100 mg/dL rise in fibrinogen (assuming 100%
recovery)
 C H AP T E R 23 Neonatal and Pediatric Transfusion Practice
pared to the liberal strategy.52 However, rates of
periventricular leukomalacia and death were
higher in the restrictive arm. The PINT study
found no significant difference between the
two arms, which had the same thresholds as
the Iowa study, in the composite endpoint of
death or any of bronchopulmonary dysplasia,
reti- nopathy of prematurity (Stage >3), or
brain in- jury (periventricular leukomalacia,
intracranial hemorrhage Grade 4, or
ventriculomegaly).50,52 The PINTOS study
revealed that at 18 to 24 months after birth,
infants in the PINT study’s restrictive arm had
more neurodevelopmental impairments than
those in the liberal arm.50,51
In summary, these studies indicated that
maintenance of higher hemoglobin levels in
low birthweight infants may provide long-
term neurologic protection.50-52 Therefore,
whether a restrictive or liberal RBC
transfusion strategy should be adopted in
this population is not clear and requires
further prospective randomized controlled
trial data. The Transfu- sion of Premature
Infants Study is currently being conducted
in the United States.53
Exchange Transfusion
for Hyperbilirubinemia
Exchange transfusion in neonates involves re-
placement of one or two whole-blood vol-
umes. The primary purpose of this therapy is
to treat excessively high levels of
unconjugated bilirubin (hyperbilirubinemia).
In high con- centrations, bilirubin may cross
the blood- brain barrier; concentrate in the
basal ganglia and cerebellum of preterm and
full-term in- fants; and cause irreversible
damage, known as “kernicterus,” to the
central nervous sys- tem. Preterm and full-
term infants are suscep- tible to
hyperbilirubinemia because their im- mature
liver conjugates bilirubin poorly and their
incompletely developed blood-brain bar- rier
allows bilirubin transit. Phototherapy (use of
fluorescent ultraviolet lights) is the current
treatment of choice for hyperbilirubinemia;
exchange transfusion is reserved for patients
who fail phototherapy.
Two critical objectives of exchange trans-
fusions are the removal of unconjugated bili-
rubin and maximization of albumin binding of
■ 579
residual bilirubin. In addition, in antibody-
mediated hemolytic processes, exchange
therapy removes both free antibody and
anti- body-coated red cells, replacing them
with antigen-negative red cells.
Exchange transfusion needs to be per-
formed before the development of kernicterus.
In full-term infants, kernicterus rarely
develops at bilirubin levels less than 25
mg/dL. However, in ill VLBW infants,
kernicterus can occur at bil- irubin levels as
low as 8 to 12 mg/dL.54
A double-volume exchange transfusion
(two 85-mL/kg transfusions for full-term in-
fants and two 100-mL/kg transfusions for
VLBW infants) removes approximately 70%
to 90% of the circulating red cells and
approxi- mately 50% of the total bilirubin. 55
However, after the first exchange
transfusion, bilirubin levels may rise again
because of a re-equilibra- tion of the
extravascular tissue and plasma bil- irubin,
which may necessitate another ex- change
transfusion.56
Occasionally, exchange transfusion is
used to eliminate toxins, drugs, or chemicals
administered to the mother near the time of
delivery. Exchange transfusion is also used
when toxic doses have been administered to
the infant or accumulate at high levels in the
infant as a result of prematurity and/or an in-
born error of metabolism.57,58
Exchange Transfusion
CO MPON ENT CHOICE AN D PH YSIOLO GIC
EFFECTS. Typically, RBCs are resuspended
in ABO-compatible thawed Fresh Frozen
Plasma (FFP) for an exchange transfusion.
No single method of combining components
has been shown to be superior to another.
Most often, RBCs <5 to 7 days old and
stored in CPDA-1 are used to avoid high
levels of potassium and to maximize red cell
survival.59 When using AS- RBC units, some
blood banks elect to remove the additive-
containing plasma to reduce the volume
transfused.
Most transfusion services provide RBC
units that are hemoglobin S negative, cyto-
megalovirus (CMV) reduced-risk (leukocyte
reduced and/or CMV seronegative), and irra-
diated. Irradiation should be performed just
580 ■ AABB T EC HNIC AL MANUAL
before the exchange to prevent potentiation
of the potassium storage lesion. Some
experts recommend washing or removing
the super- natant of red cells that have been
irradiated to avoid the complications of
hyperkalemic car- diac arrhythmias.60
The glucose load administered during
ex- change transfusion can be high in some
cases, which stimulates the infant’s pancreas
to re- lease insulin and results in rebound
hypogly- cemia. Therefore, infant plasma
glucose levels should be monitored during
the first few hours following exchange
transfusion.
VO LU ME A N D HE MATO CR I T CONS I D ER -
AT I O N S . A double-volume exchange in
neo- nates rarely necessitates the infusion of
more than 1 RBC unit. The unit’s hematocrit
should be approximately 45% to 60%, and
the unit should have sufficient plasma
(based on esti- mated blood volume) to
provide clotting fac- tors.60 If the neonate’s
condition requires a higher postexchange
transfusion hematocrit, a small-volume RBC
transfusion may be given or a unit with a
higher hematocrit can be used for the initial
exchange transfusion. The re- constituted
blood should be well mixed to sus- tain the
intended hematocrit throughout the
exchange. The infant’s hematocrit and
biliru- bin can be measured by removing the
last ali- quot of the exchange unit.
VA S CUL A R ACCE SS . Umbilical venous
cath- eters are used for exchange
transfusions in preterm and full-term infants
just after birth. If umbilical venous catheters
are not available, small saphenous catheters
may be used.
T E C H N I Q U E S . Two exchange-transfusion
techniques are commonly employed:
isovolu- metric and manual push-pull. In
isovolumet- ric exchange transfusion, two
catheters of identical size provide vascular
access. The catheters allow simultaneous
withdrawal and infusion of blood and are
regulated by a single peristaltic pump. The
umbilical vein is typical- ly used for
infusion, and the umbilical artery is used for
withdrawal. The manual push-pull technique
uses a single vascular access portal with a
three-way stopcock attached to the unit of
blood, the patient, and a graduated discard
container. A standard filter and in-line blood
warmer are recommended.
With both techniques, the absolute
maxi- mum volume of each withdrawal and
infusion depend on the infant’s body weight
and hemo- dynamic status. Usually, no more
than 5 mL/ kg body weight or 5% of the
infant’s blood vol- ume is removed and
replaced during a 3- to 5- minute cycle.59
The exchange transfusion should not be
performed rapidly because sud- den
hemodynamic changes may affect cere- bral
blood flow and shift intracranial pressure,
contributing to IVH.61 A total double-volume
exchange transfusion typically takes 90 to
120 minutes.59
Platelet Transfusion Support
Mild-to-moderate thrombocytopenia (plate-
let count <150,000/µL) is the most common
hemostatic abnormality in ill preterm and
full- term infants, and it affects
approximately 20% of infants in neonatal
intensive care units.62 The causes of
thrombocytopenia include im- paired
platelet production, increased platelet
destruction, abnormal platelet distribution,
and/or platelet dilution secondary to massive
transfusion. The most common cause is in-
creased destruction of platelets that is
general- ly associated with a variety of self-
limited con- ditions.
Indications
Most platelet transfusions in preterm and
full- term infants are performed to treat
platelet counts less than 50,000/µL in the
presence of active bleeding.63
Prophylactic platelet transfusions in this
population are controversial (see Table 23-3
for transfusion indications and
thresholds).27,64 Unlike adult patients who
rarely have severe bleeding complications
until platelet counts decline to less than
10,000/µL, preterm infants with other
complicating illnesses may bleed at higher
platelet counts. This increased risk may be
attributable to 1) lower concentrations of
plasma coagulation factors, 2) circulation of
an anticoagulant that potentiates thrombin
inhibition, 3) intrinsic or extrinsic platelet
 C H AP T E R 23 Neonatal and Pediatric Transfusion Practice ■ 581

TABLE 23-3. Transfusion Guidelines for Platelets in Neonates and Older Children 23,26

With Thrombocytopenia
1. Platelet count 5,000 to 10,000/μL with failure of platelet production.
2. Platelet count <30,000/μL in neonate with failure of platelet production.
3. Platelet count <50,000/μL in stable premature infant:
a. With active bleeding, or
b. Before an invasive procedure, with failure of platelet production.
4. Platelet count <100,000/μL in sick premature infant:
a. With active bleeding, or
b. Before an invasive procedure in patient with DIC.
Without Thrombocytopenia
1. Active bleeding in association with qualitative platelet defect.
2. Unexplained excessive bleeding in a patient undergoing cardiopulmonary bypass.
3. Patient undergoing ECMO with:
a. A platelet count of <100,000/μL, or
b. Higher platelet counts and bleeding.
DIC = disseminated intravascular coagulation; ECMO = extracorporeal membrane oxygenation.

dysfunction/hyperreactivity, or 4) increased demonstrated to raise the platelet count of an

vascular fragility.63
A severe complication of prematurity is
IVH, which occurs in approximately 40% of
preterm neonates in the first 72 hours after
birth. Although prophylactic platelet
transfu- sions may increase platelet counts
and short- en bleeding times, this approach
has not been shown to reduce the incidence
of IVH, and the severity of
thrombocytopenia appears to be independent
of the risk if IVH >Grade 2.62,65 Hence, the
use of platelets in this situation and the
appropriate platelet dose remain contro-
versial.66 Posttransfusion platelet counts 15
to 60 minutes after transfusion can help
when platelet survival is evaluated; however,
these counts are not good predictors of
hemostatic efficacy.
Components and Dose
The use of whole-blood-derived platelets at
doses of 5 to 10 mL/kg body weight have been
average full-term newborn by 50,000 to
100,000/µL, depending on the concentration
of the platelet component used.14,62 A similar
dosing regimen is typically used with aphere-
sis platelets. For larger children (>10 kg),
trans- fusion of 1 platelet unit per 10 kg should
in- crease the platelet count by approximately
50,000 µL.14,67
When possible, the platelet component
should be ABO group specific/compatible
and should not contain clinically significant
and unexpected red cell antibodies.
Transfusion of ABO-incompatible plasma
should be avoided in children, and
especially in infants, because of their small
blood and plasma volumes.31 If it becomes
necessary to administer ABO-incom- patible
platelets in an infant, plasma may be
removed either by volume reduction or
wash- ing (see Method 6-14). The platelets
may then be resuspended in saline or
compatible plasma. However, routine
centrifugation to remove plasma from
platelets should be avoided
582 ■ AABB T EC HNIC AL MANUAL

because it is unnecessary and harmful to the
platelets.62,63
In addition, when platelets are stored in
a syringe, the pH has been shown to
decrease rapidly, a potential problem for an
already ill and acidotic recipient. 68-70
Therefore, when volume reduction in an
open system is used and the product is
placed in a syringe, the pro- cessing should
be done as close to the time of issuance as
possible and the product should be infused
within 4 hours of processing.
Plasma Transfusion Support
to Enhance Hemostasis
Infants must synthesize their own
coagulation factors because significant
amounts are not transplacentally transferred
from the mother. Furthermore, infants are
unable to produce normal levels of these
proteins in the early postnatal period.
Physiologically, low levels of vitamin K-
dependent factors (Factors II, VII, IX, and X)
and contact factors (Factor XI, Factor XII,
prekallikrein, and high-molecular-weight ki-
ninogen) contribute to altered coagulation test
results (see Table 23-4).71,72 Also, the naturally
occurring vitamin K-dependent anticoagu-
lants (proteins C and S) and the non-
vitamin- K-dependent antithrombin protein
are at low levels at birth. In spite of these
issues, the pro- coagulant and anticoagulant
systems are usu- ally in balance in healthy
newborns, so spon- taneous bleeding and
thrombosis are rare (see Table 23-4).72
However, the reserve capacity for both
systems is limited. Therefore, serious
bleeding may occur in sick premature
infants during the first week of life.
Cryoprecipitate and FFP can be transfused
to treat bleeding or clotting complications,
such as disseminated intravascular
coagulation (DIC).73,74
FFP
FFP is frequently used to replace coagulation
factors in preterm and full-term infants, par-
ticularly if multiple factor deficiencies are
present, such as hemorrhagic disease of the
newborn or vitamin K deficiency (Table 23-5).
The usual dose of FFP is 10 to 15 mL/kg,
which is expected to increase all factor activity
levels by 15% to 20% unless there is a marked
con- sumptive coagulopathy.64,68
To limit donor exposure for each
recipient while minimizing plasma wastage,
blood can be collected into a system with
multiple, inte-

TABLE 23-4. Screening Laboratory Tests for Hemostasis: Neonates vs Adults (reproduced with
permission)71

Preterm Neonates vs Neonates vs Older Approximate Age Adult

Full-Term Neonates Children/Adults Values are Reached
aPTT Longer Longer 16 years
Prothrombin time Longer Same or longer 16 years
INR Higher Same or higher 16 years
Thrombin time Longer Same or longer 5 years
Bleeding time Longer Shorter 1 months
PFA-100 Longer Shorter 1 months
ROTEM/TEG
Clotting time Same Shorter 3 months
Clot formation time Same Shorter 3 months
Maximal clot firmness Stronger Stronger 3 months
aPTT = activated partial thromboplastin time, INR = international normalized ratio.
 C H AP T E R 23 Neonatal and Pediatric Transfusion Practice ■ 583

TABLE 23-5. Transfusion Guidelines for Plasma Products in Neonates and Older Children 23,26

Fresh Frozen Plasma (FFP)

1. Support during treatment of disseminated intravascular dissemination.
2. Replacement therapy:
a. When specific factor concentrates are not available, including, but not limited to,
antithrombin; pro- tein C or S deficiency; and Factor II, Factor V, Factor X, and Factor XI
deficiencies.
b. During therapeutic plasma exchange when FFP is indicated (cryopoor plasma, plasma from which
the cryoprecipitate has been removed).
3. Reversal of warfarin in an emergency situation, such as before an invasive procedure with active
bleeding. Note: FFP is not indicated for volume expansion or enhancement of wound healing.
Cryoprecipitated AHF
1. Hypofibrinogenemia or dysfibrinogenemia with active bleeding.
2. Hypofibrinogenemia or dysfibrinogenemia while undergoing an invasive procedure.
3. Factor XIII deficiency with active bleeding or while undergoing an invasive procedure in the absence
of Factor XIII concentrate.
4. Limited directed-donor cryoprecipitate for bleeding episodes in small children with hemophilia A
(when recombinant and plasma-derived Factor VIII products are not available).
5. In the preparation of fibrin sealant.
6. von Willebrand disease with active bleeding, but only when both of the following are true:
a. Deamino-D-arginine vasopressin (DDAVP) is contraindicated, not available, or does not
elicit response.
b. Virus-inactivated plasma-derived Factor VIII concentrate (which contains von Willebrand factor)
is not available.

grally attached bags that create ready-to-
freeze aliquots.26 Once thawed, the aliquots
can be subdivided further for several
patients if they can be used within a 24-hour
period.
FFP for infants must be ABO
compatible and free of clinically significant
and unexpect- ed antibodies. Transfused
antibodies can reach high concentrations in
infants and chil- dren with very small
plasma volumes. A com- mon practice at
some institutions is to use group AB FFP
because a single unit can pro- vide multiple
small-volume doses for several neonates.
Cryoprecipitated Antihemophilic Factor
Cryoprecipitate transfusions are primarily
used to treat conditions resulting from de-
creased or dysfunctional fibrinogen (congeni-
tal or acquired) or Factor XIII deficiency.
Cryo- precipitate is usually given in
conjunction with platelets and FFP to treat
DIC in newborns. Typically, 1 unit is sufficient
to achieve hemo- static levels in an infant.
ABO-compatible cryoprecipitate is pre-
ferred because transfusion of a large volume of
ABO-incompatible cryoprecipitate may result
in a positive DAT result and, in very rare
cases, a mild hemolysis.75,76
Cryoprecipitate transfusion is not recom-
mended for patients with Factor VIII deficien-
cy because the standard therapy is to treat this
condition with recombinant Factor VIII prod-
ucts or virus-inactivated, monoclonal-anti-
body-purified, plasma-derived products.73,77
584 ■ AABB T EC HNIC AL MANUAL
Furthermore, cryoprecipitate should be used
only to treat von Willebrand disease if plasma-
derived, virus-inactivated concentrates con-
taining von Willebrand factor are not
available. See Table 23-5 for other guidelines
regarding cryoprecipitate use.64
Granulocyte Transfusion Support
Indications
The role of granulocyte transfusion for sepsis
in neonates is unclear, and this treatment is
rarely used. It is important to establish the fol-
lowing factors before the transfusion of granu-
locytes: 1) strong evidence of bacterial or fun-
gal septicemia; 2) absolute neutrophil count
less than 500/µL, chronic granulomatous dis-
ease, or leukocyte adhesion deficiency; and 3)
diminishing storage pool (such that 7% of nu-
cleated cells in the marrow are granulocytes
that are metamyelocytes or more ma-
ture).15,78,79 (See Table 23-6 for guidelines on
granulocyte transfusion support.)
Components and Dose
Granulocyte concentrates are produced by
standard apheresis techniques or by pooling
buffy coats from whole blood. A typical
dose for infants is 10 to 15 mL/kg body
weight, which is approximately 1 × 109 to 2
× 109 poly- morphonuclear cells/kg.15,78
Treatment should be administered daily until
an adequate neu- trophil count is achieved
and/or the patient shows clinical
improvement.
According to AABB Standards, these
com- ponents must be irradiated when the
patient is
TABLE 23-6. Transfusion Guidelines for
Granulocytes in Neonates and Older
Children23,26
1. Neonates or children with neutropenia
or granulocyte dysfunction with bacterial
sepsis and lack of responsiveness to
standard ther- apy.
2. Neutropenic neonates or children with
fungal disease not responsive to standard
therapy.
at risk of TA-GVHD, and the components
must be obtained from CMV-seronegative
donors to prevent virus transmission if the
recipient is CMV seronegative.28(p40)
Granulocytes must also be ABO compatible
with the red cells of the recipient infant
because of the significant red cell content in
these components.28(p37) Many institutions also
provide D-compatible components to
decrease Rh alloimmuniza- tion.
Intravenous immune globulin (IVIG) in
the treatment of early neonatal sepsis has
also been studied, but there is a lack of
agreement on its routine application to this
patient popu- lation.78,80-85
Transfusion Administration
Vascular Access
Vascular access is the most difficult aspect
of transfusion administration in patients
young- er than 4 months, particularly in
preterm in- fants who require long-term or
continuous in- travenous infusions. The
umbilical vein is most frequently cannulated
after birth to ad- minister fluids and
transfusions and to moni- tor central venous
pressure.86 Vascular cathe- ters (24-gauge)
and small needles (25-gauge) generally can
be safely used for RBC transfu- sions
without causing hemolysis if constant flow
rates are applied. The outcomes of trans-
fusions using smaller-gauge needles and
cath- eters have not been evaluated.
Pumps and Warming
When administered slowly, small-volume
transfusions typically do not require a blood
warmer; however, control of the rate and
vol- ume transfused is important.
Electromechani- cal syringe delivery pumps
administer the blood at a constant rate and
are able to provide adequate control. These
pumps cause mini- mal hemolysis and may
even be used with in- line leukocyte-
reduction filters.87,88 Although there are
several devices that can be used to transfuse
blood components, it is important to test and
validate the mechanical system cho-
sen for blood component administration.
 C H AP T E R 23 Neonatal and Pediatric Transfusion Practice
Filters and Transfusion Sets
All blood component transfusions require a
standard filter (150 to 260 microns), even if
the components have undergone leukocyte
reduc- tion before storage or at the
bedside.75 Micro- aggregate filters (20 to 40
microns) are occa- sionally used for simple
transfusions because of their small priming
volume. However, he- molysis may occur
when stored RBCs are ad- ministered
through these filters using negative
pressure.89
The plastic tubing in the administration
sets can add significant amounts of dead-
space volume to the transfusion and may
need to be accounted for when preparing a
transfu- sion dose. Pediatric infusion sets
created for platelets and other small-volume
components have less dead space than
standard sets.
Administration Rates
A lack of clinical studies and evidence-
based practices related to blood transfusions
in neo- nates has led to variability both in
rates of
transfusion and in devices used among insti-
tutions. The rate of RBC and other blood
com- ponent administration is dictated by
the clini- cal needs of the pediatric patient.
Despite concerns from neonatologists that
rapid blood infusion rates may adversely
affect intravascu- lar volume and electrolyte
levels, an increased
risk of IVH in these small and fragile
patients has not been clearly demonstrated.
Therefore, administering a simple
transfusion over 2 to 4 hours is adequate in
nonemergent situations. However, in states
of shock or severe bleeding, a rapid infusion
is often required.
Unique Therapies and Situations
in Neonates
Polycythemia
Neonatal polycythemia is defined as a venous
hematocrit >65% or a hemoblogin >22 g/dL at
any time during the first week after birth. Ap-
proximately 5% of all newborns develop poly-
cythemia, and this risk may be higher in small-
for-gestational-age neonates and infants of
diabetic mothers. Once the hematocrit rises
■ 585
above 65%, viscosity increases and oxygen
transport decreases. However, in neonates,
the exponential rise in viscosity can occur at
a he- matocrit as low as 40%.90 Congestive
heart fail- ure can result because infants
have limited ca- pability to increase their
cardiac output to compensate for
hyperviscosity. Central ner- vous system
abnormalities, pulmonary and re- nal failure,
and NEC can occur from the resul- tant
decreased blood flow.
A partial exchange is used to normalize
the hematocrit to between 55% and 60% and
improve tissue perfusion while maintaining
blood volume. This exchange is
accomplished by removing whole blood and
replacing it with normal saline or other
crystalloid solutions. Plasma is not used to
replace whole blood be- cause NEC has
been reported as a complica- tion of plasma
transfusion.91
The formula below can be used to ap-
proximate the volume of replacement fluid
re- quired and the volume of whole blood
that must be withdrawn for the partial
exchange:
(Blood (Observed Hct
Volume of – volume) × Desired Hct)
replacement =
Observed Hct
fluid
ECMO
ECMO is a prolonged treatment where
blood is removed from the patient’s venous
circulation, circulated through a machine to
remove CO2 and replenish O2, and then
returned to the pa- tient. ECMO has been
successfully used since the early 1980s to
provide gas exchange inde-
pendently of a patient’s lungs. ECMO allows
patients to recover without exposure to
aggres- sive ventilator support that can
cause baro- trauma and permanent lung
damage and to maintain the circulation of
oxygenated blood during cardiac surgery or
in patients with dis- ease when other forms
of treatment fail.92,93 In neonates and
children, ECMO has become a lifesaving
advance treatment of meconium as- piration
syndrome, persistent pulmonary hy-
pertension of the newborn, congenital dia-
phragmatic hernia, and respiratory failure
due
586 ■ AABB T EC HNIC AL MANUAL

to sepsis. It is also used for postoperative TRANSFUSION IN INFANTS

sup- port following cardiac surgery.
Because standardized guidelines for
transfusion practice in ECMO have not been
established, centers typically establish their
own criteria. Table 23-7 provides some
guide- lines for ECMO.94 Bleeding
complications are frequent during ECMO
treatment and may be caused by 1) systemic
heparinization, 2) plate- let dysfunction, 3)
thrombocytopenia, 4) other coagulation
defects, or 5) the nonendothelial ECMO
circuit. Hospital blood banks and trans-
fusion services must be in close communica-
tion with the ECMO staff and observe local
protocols to ensure safe, efficient, and
consis- tent care.
ECMO typically requires 1 to 2 units of
ABO- and Rh group-specific and crossmatch-
compatible RBCs for blood priming. In addi-
tion, 1 unit of group-specific FFP should be al-
located to the ECMO patient. RBC units are
usually negative for hemoglobin S, relatively
fresh (<5 to 7 days old), irradiated, and CMV
seronegative and/or leukocyte reduced. 43 Be-
cause the ECMO circuit consumes platelets,
higher platelet counts are often maintained.
OLDER THAN 4 MONTHS AND
CHILDREN
RBC Transfusion Support
RBC transfusions in infants older than 4
months and children are similar to transfu-
sions in adults. The most significant
differenc- es between this young group and
adults are 1) blood volume, 2) the ability to
tolerate blood loss, and 3) age-appropriate
hemoglobin and hematocrit levels. In these
infants and chil- dren, the most common
indication for RBC transfusion is to treat or
prevent tissue hypoxia caused by decreased
red cell mass, typically because of surgery,
anemia of chronic disease, or hematologic
malignancies. Chronic RBC transfusions are
the treatment of choice for children with
sickle cell disease (SCD) or thal- assemia.
These transfusions are administered to
combat tissue hypoxia and suppress endog-
enous hemoglobin production. Table 23-8
can help guide transfusion decisions in
patients older than 4 months.

TABLE 23-7. Blood Component Protocols for ECMO94

Clinical Scenario Urgency Components Blood Groups Storage

Cardiac arrest 5-10 min 2 units RBCs O-neg RBCs <14 days, AS

ECMO circuit disruption 5-10 min 2 units RBCs O-neg RBCs <14 days, AS

Progressive septic 30 min 2 units RBCs O-neg RBCs or <10 days,

shock (nonneonate) type specific any preservative

Neonate transferred for 1-2 hours 2 units RBCs O-neg RBCs <10 days,
ECMO 1 unit FFP AB plasma CPD or CPDA
1 unit platelets

Cardiac ICU 30-60 min 2 units RBCs Type specific <7 days, AS

Gradual respiratory or Hours to days 2 units RBCs Type specific <10 days, CPD
cardiac failure on
con- ventional
support
ECMO = extracorporeal membrane oxygenation; RBCs = Red Blood Cells; AS = additive solution; FFP =
Fresh Frozen Plasma; CPD = citrate-phosphate-dextrose; CPDA = citrate-phosphate-dextrose-adenine;
ICU = inten- sive care unit.
 C H AP T E R 23 Neonatal and Pediatric Transfusion Practice ■ 587

TABLE 23-8. Transfusion Guidelines for RBCs in Patients Older than 4 Months 23,26

1. Emergency surgical procedure in patient with significant postoperative anemia.

2. Preoperative anemia when other corrective therapy is not available.
3. Intraoperative blood loss >15% total blood volume.
4. Hematocrit <24% and:
a. In perioperative period, with signs and symptoms of anemia.
b. While on chemotherapy/radiotherapy.
c. Chronic congenital or acquired symptomatic anemia.
5. Acute blood loss with hypovolemia not responsive to other therapy.
6. Hematocrit <40% and:
a. With severe pulmonary disease.
b. On extracorporeal membrane oxygenation.
7. Sickle cell disease and:
a. Cerebrovascular accident.
b. Acute chest syndrome.
c. Splenic sequestration.
d. Aplastic crisis.
e. Recurrent priapism.
f. Preoperatively when general anesthesia is planned (target hemoglobin 10 mg/dL).
8. Chronic transfusion programs for disorders of red cell production (eg, -thalassemia major and
Diamond- Blackfan syndrome unresponsive to therapy).

Before receiving any RBC transfusion, Simple or partial-exchange transfusions

all pediatric patients older than 4 months
require ABO and Rh testing and screening
for the pres- ence of clinically significant
antibodies. Com- patibility testing should be
performed accord- ing to AABB
Standards.28(p39)
SCD
Chronic transfusion therapy has been shown
to reduce the risk of stroke in patients with
SCD by decreasing the proportion of RBCs
containing hemoglobin S, reducing sickling,
and preventing blood viscosity increases.95-98
Chronic transfusions can reduce the risk of re-
current stroke to <10% if hemoglobin levels
are maintained at between 8 and 9 g/dL with a
he- moglobin S level <30%.
can be administered every 3 to 4 weeks.
Eryth- rocytapheresis has also been used to
prevent iron overload in patients with SCD.
See Table 23-8 for a list of other
complications of SCD necessitating either
simple or chronic RBC transfusions.
Chronic transfusion therapy must be
pro- vided indefinitely because its cessation
can lead to a stroke.95,96 The Transcranial
Dopplers with Transfusions Changing to
Hydroxyurea trial is currently evaluating the
role of hydroxy- urea in stroke prevention
compared to chronic transfusions in patients
with SCD.99 Of note, RBCs for patients with
SCD should ideally be screened for
hemoglobin S and leukocyte re- duced to
prevent HLA alloimmunization and
588 ■ AABB T EC HNIC AL MANUAL

for donors of African ethnicity to decrease the

decrease platelet refractoriness in
preparation for possible stem cell
transplantation.

RBC ALLO IMM UNIZ AT I ON IN SCD. Patients

with SCD have the highest rates of
alloimmu- nization of any patient group.100-
102
These anti- bodies are produced against
common Rh, Kell, Duffy, and Kidd system
antigens. Many SCD treatment centers
perform thorough pheno- type analysis of a
patient’s red cells before be- ginning
transfusion therapy. This testing helps
reduce the rate of alloimmunization by
allow- ing preferential selection of
phenotypically similar units. 96,103,104
However, particularly for patients who are
not yet alloimmunized, this process remains
controversial because pheno- typically
compatible units may be difficult to
obtain.95,104
In academic institutions in the United
States and Canada, the most common
alloim- munization protocol for
nonalloimmunized patients with SCD is
pretransfusion phenotyp- ic matching for C,
E, and K antigens.105 Once patients have
developed a red cell antibody, extension of
matching to additional red cell antigens (Fy,
Jk, S) is often used to prevent further
alloimmunization.106 In a retrospective study,
children with SCD undergoing matched-
sibling-donor marrow transplanta- tion
demonstrated a decrease in RBC transfu-
sion requirements during transplantation
when they received phenotypically minor
RBC antigen-matched units.107 A recent
retrospec- tive review of patients with SCD
at Children’s Hospital of Philadelphia
showed that the chil- dren developed
alloimmunization to Rh anti- gens despite
being transfused with units from Rh-
matched minority donors, and 87% of allo-
immunized patients were identified with RH
genotyping as having RH variant alleles.108
This study’s results suggest that the role of
genotyp- ing of patients with SCD and
minority donors in alloimmunization rates
needs to be stud- ied.108 Method 2-23 can be
used to perform phenotyping on autologous
red cells of recent- ly transfused patients
with SCD.
Another strategy aimed at preventing
al-
loimmunization in patients with SCD is to
de- velop recruitment programs specifically
 character- ized by destruction of the patient’s
number of antigenically own red cells along with transfused cells. If
different RBC units from hyperhe- molytic syndrome is suspected,
donors of European case reports have shown that stopping
ethnicity that are transfusion and ad- ministering
transfused. Leukocyte corticosteroids in combination with IVIG is
reduction to prevent al- beneficial.112,113 These patients should also be
loimmunization to red monitored closely for the for- mation of
cell antigens remains autoantibodies.114
controversial.109, 110

However, HLA Thalassemia

alloimmuni- zation has
Thalassemia with severe anemia must be
recently been
treated with transfusion to improve tissue oxy-
demonstrated to oc- cur
genation and suppress extramedullary eryth-
more frequently in red
ropoiesis in the liver, spleen, and marrow. This
cell alloimmunized
approach also decreases many thalassemia
patients with SCD.111
complications. Maintaining target hemoglo-
OT HER COMPLICATIONS OF RB C TRANS- bin levels of 8 to 9 g/dL allows normal growth
FU SION S IN SCD. The and development in these patients. Super-
benefits of transfu- sion transfusion protocols aim for higher target he-
therapy in patients with moglobin levels (11-12 g/dL). Iron overload is
SCD should be weighed a potential nonpreventable complication of
against the
complications of
transfu- sion, such as
iron overload and minor
red cell antigen
alloimmunization, as
well as the risks of
increased donor
exposure during
erythrocy- tapheresis.
Some practitioners have
proposed that a
clinically successful
course of transfu- sions
that maintains the
hemoglobin S level at
<30% could, after
several years, be transi-
tioned to a strategy of
more limited transfu-
sions with a hemoglobin
S target of 40% to 50%
to reduce the risk of iron
overload.97 Patients with
SCD may also be at risk
of life-threaten- ing,
delayed hemolytic
transfusion reactions.
If a patient’s
hemoglobin level
decreases after
transfusion, the patient
might have de- veloped
“hyperhemolytic”
syndrome. This poorly
understood
phenomenon is
 C H AP T E R 23 Neonatal and Pediatric Transfusion Practice
this RBC transfusion protocol that must be
treated with chelation therapy beginning
early in childhood.115,116
Platelet and Plasma Support
The indications for platelet and plasma
trans- fusion support are similar for older
infants, children, and adults. Tables 23-3 and
23-5 pro- vide the indications for the
transfusion of platelets, FFP, and
cryoprecipitate. A recent study showed that
plasma transfusions were independently
associated with an increased risk of new or
progressive multiple organ dys- function,
nosocomial infections, and pro- longed
length of stay in 831 children admitted to
one institution’s pediatric critical care unit.117
Plasma transfusions should be consid- ered
with caution in this population.
In older infants and children, platelets
are
most often prophylactically administered dur-
ing chemotherapy. The transfusion threshold
for these patients is usually between 10,000
and 20,000/µL, although the platelet count
should not be the sole determinant for trans-
fusion. In the Prophylactic PLAtelet DOse
(PLADO) study, Slichter et al randomly as-
signed 1351 patients with hypoproliferative
thrombocytopenia to receive one of three pro-
phylactic platelet transfusion doses (low, me-
dium, or high) with the primary endpoint of
World Health Organization Grade 2 bleed-
ing.118 The dose of prophylactic platelets ad-
ministered had no effect on the incidence of
Grade 2 bleeding.118 A subgroup analysis of
the 198 children aged 0-18 years in the
PLADO study revealed that children were at a
higher risk of bleeding over a wider range of
platelet counts than adults.119 Therefore,
prophylactic platelet transfusions with a
threshold of 10,000/µL in concert with
different doses of platelets do not seem to
affect the bleeding rates of children or
adults.118,119 However, when children are
transfused, the use of ABO-com- patible
platelets is associated with better clini- cal
outcomes than use of non-ABO matched
platelets.31,120-122
■ 589
Granulocyte Support
For children older than 4 months, the indica-
tions for granulocyte transfusions—
persistent neutropenia or granulocyte
dysfunction in conjunction with a bacterial
and/or fungal in- fection(s)—are similar to
those previously mentioned for younger (<4
months) infants. The minimum granulocyte
dose for older chil- dren and adults is 1 ×
1010 cells/kg.23,78
Granulocyte components should be irra-
diated, ABO compatible, CMV seronegative
(if the recipient is CMV seronegative), cross-
match compatible with the recipient, and, ide-
ally, administered within 24 hours of collec-
tion. To obtain higher doses of granulocytes
for larger patients, donors can be mobilized
with steroids, growth factors [eg, granulocyte
colony-stimulating factor (G-CSF)], or a com-
bination. This approach can increase the
amount of granulocytes in the collection by
three or four times compared to products from
donors who receive steroid stimulation alone.
The National Heart, Lung, and Blood In-
stitute’s High Dose Granulocyte Transfusions
for the Treatment of Infection in Neutropenia:
Resolving Infection in Neutropenia with Gran-
ulocytes study is examining the effect of gran-
ulocyte transfusions plus standard microbial
therapy vs standard microbial therapy alone
for patients with neutropenia and severe in-
fections as well as the combined effect of G-
CSF and steroids on granulocyte donation
yield.
PREVENTION OF ADVERSE
EFFECTS OF TRANSFUSION IN
NEONATES, OLDER INFANTS,
AND CHILDREN
CMV Prevention
CMV may be transmitted to neonates trans-
placentally, during the birth process or breast-
milk feeding, as a result of personal contact
with the mother or nursery staff, or from trans-
fusion. With current technologies, the risk of
acquiring CMV from transfusion is between
1% and 3%.123
590 ■ AABB T EC HNIC AL MANUAL
The manifestation of CMV infection in
neonates is variable, ranging from asymptom-
atic seroconversion to death. Studies have re-
vealed that the rate of symptomatic transfu-
sion-transmitted CMV infection in infants is
low compared to the high rate of seropositivity
in adults. Moreover, symptomatic CMV infec-
tion is uncommon in neonates born to sero-
positive mothers.124
The risk of transfusion-transmitted
CMV infection is higher in multitransfused
low birthweight infants (<1200 g) born to
seroneg- ative mothers.13,123,125 For this
reason, these in- fants should receive only
CMV-reduced-risk blood. One approach is
to use blood from CMV-seronegative
donors.
Leukocyte Reduction
The benefits of transfusions of leukocyte-
reduced components for neonates include
re- ducing the risk of transfusion-transmitted
CMV.13,126,127 A recent study in Canada that
evaluated clinical outcomes in premature in-
fants (weighing <1250 g) before and after
na- tionwide implementation of universal
leuko- cyte reduction revealed no change in
mortality or bacteremia rates. However, rates
of retinop- athy of prematurity and
bronchopulmonary dysplasia decreased as
did length of hospital- ization.128 Other
known benefits of transfusing leukocyte-
reduced components include pre- vention of
febrile nonhemolytic transfusion reactions
and HLA alloimmunization.
TABLE 23-9. Irradiation Guidelines for Neonates and Older Children Who Require Cellular Blood
Components23,26
1. Premature infants weighing <1200 g at birth.
2. Any patient with:
a. Known or suspected cellular immune deficiency.
b. Significant immunosuppression related to chemotherapy or radiation treatment.
3. Any patient receiving:
a. Components from blood relatives.
b. HLA-matched or crossmatched platelet components.
Irradiation
Cellular blood components are irradiated to
prevent TA-GVHD in immunocompromised
recipients (see Table 23-9). Expert opinions
and practices differ on this topic. Therefore,
protocols should be based on the patient
pop- ulations served, equipment available,
and best practices. The processes of
irradiation, irradia- tor quality control, and
quality assurance are beyond the scope of
this chapter but are ad- dressed in Chapters
1 and 9.
Volume Reduction and Washing
The plasma volume of the component is
usu- ally reduced in transfusions in
premature in- fants who have renal ischemia
or compro- mised cardiac function. In 1993,
the AABB Committee on Pediatric
Hemotherapy stated that volume reduction
of platelet concentrates should be reserved
for infants who have total body fluid
restrictions.63 Methods for platelet volume
reduction have been published (see Method
6-13).129 However, optimal centrifuga- tion
rates and preparation methods remain
unclear. As with any platelet modification,
the total number of platelets typically
decreases and platelet activation may occur
with these procedures.130
Saline-washed RBCs and platelets are ad-
ministered in an effort to reduce the risk of ad-
verse reactions to certain components from
plasma, anticoagulant preservative solutions,
and high levels of potassium. Transfusion of
 C H AP T E R 23 Neonatal and Pediatric Transfusion Practice ■ 591

unwashed RBCs or platelet products
procured from the mother of an infant is
strongly dis- couraged.26 Maternal cells must
be washed for effective treatment of
hemolytic disease of the newborn and
neonatal alloimmune thrombo- cytopenia
when maternal RBCs and platelets are
transfused.
Massive Transfusion in the Pediatric
Setting
Trauma is the leading cause of death in in-
fants, children, and young adults aged 1 to
21 years. Although trauma rarely leads to
hemor- rhagic shock and massive
transfusion, resusci- tation after trauma can
be challenging.
The evidence to support pediatric MTP
is
limited, but several pediatric institutions use
MTPs to improve the outcomes of patients
who have experienced trauma. The
appropri- ate ratios of RBCs to plasma and
platelets have
not been well defined (ie, 1:1:1 or 2:1:1).
How- ever, studies indicate that an MTP in
the pedi- atric setting is feasible not only for
providing rapid and balanced blood product
support but also for decreasing the risk of
thromboembolic events.131-133 Furthermore,
when Hendrickson et al examined the impact
of coagulopathy in 102 pediatric trauma
patients, they found that abnormal
prothrombin time, activated partial
thromboplastin time, and platelet count at
emergency room admission were strongly
as- sociated with mortality (p = 0.005,
0.001, and
<0.0001, respectively).134 These investigators
did not examine the effect of MTP resuscita-
tion on coagulopathy and mortality, which
may provide insights into further optimiza-
tion of MTPs. Although adult studies have
demonstrated benefit from MTP, the role of
the MTP and the optimal ratio of blood com-
ponents still needs to be defined in the pediat-
ric setting.131-133

KEY POINTS

RBCs are the most frequently transfused blood product in children. Frequent blood loss, in- cluding iatrogenic losses from
Symptomatic anemia and/or a target hemoglobin level is a preferred strategy for neonatal
RBC transfusion rather than a milliliter-for-milliliter replacement of due to blood losses.
A full-term newborn has a blood volume of approximately 85 mL/kg whereas a preterm in- fant has a total blood volume o
In infants <4 months of age initial patient testing must include ABO and D typing of their
red cells and a screen for unexpected red cell antibodies, using either plasma or serum from the infant or mother. During an
Small-volume simple RBC (no matter what the storage solution) transfusions when admin-
istered slowly have been shown to have little effect on serum potassium concentrations in infants less than 4 months of age
During component preparation if aliquots are made with a sterile docking device, it is con-
sidered a “closed system” and the original units expiration date can be used for the new ali- quot product.
Dosing of RBC units with additive solutions such as AS-1, AS-3, and AS-5 should not exceed
10-15 mL/kg for neonates.
592 ■ AABB T EC HNIC AL MANUAL

8. Transfusion of ABO-incompatible plasma should be avoided in pediatric patients and espe-

cially in infants because of their small blood and plasma volumes. If ABO out-of-group
platelet transfusion becomes necessary, plasma may be removed either by volume reduc-
tion or washing.
9. Chronic RBC transfusion therapy reduces the risk of stroke in patients with sickle cell
dis- ease by decreasing the percentage of red cells containing hemoglobin S in order to
reduce sickling and prevent an increase in blood viscosity. Hemoglobin levels should be
main- tained around 8 to 9 g/dL with a hemoglobin S level of <30%.
10. Patients with sickle cell disease have the highest rates of alloimmunization to red cell
minor
antigens than any other patient group. The most commonly formed antibodies are to
Rh, Kell, Duffy, and Kidd system antigens. Many sickle cell treatment centers try to
prevent red cell alloimmunization by matching for phenotypically similar antigen
profiles on recipients. This strategy is not the same at all centers and is controversial
because obtaining enough phenotypically similar units may be difficult.
11. Currently it is recommended that low-birthweight infants born to CMV-seronegative moth-
ers receive CMV-reduced-risk blood for transfusion. These units could be from CMV-sero-
negative donors or CMV-positive donors whose donation has been leukocyte reduced.
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for treatment-induced hyoproliferative throm-
107. McPherson M, Anderson AR, Haight AE, et
bocytopenia. Blood 2011;120:748-60.
al. Transfusion management of HLA-
120. Larsson LG, Welsh VJ, Ladd DJ. Acute
matched sibling bone marrow transplant
intravas- cular hemolysis to out-of-group
(BMT) recipi- ents with sickle cell disease
platelet trans- fusion. Transfusion
(SCD). Transfusion 2009;49:1977-86.
2000;40:902-6.
108. Chou ST, Jackson,T, Vege S, et al. High
121. Heal JM, Blumberg N. The second century of
preva- lence of red blood cell
ABO: And now for something completely dif-
alloimmunization in sickle cell disease
ferent. Transfusion 1999;39:1155-9.
despite transfusion from Rh- matched
122. Blumberg N, Heal JM, Hicks GL, Risher WH.
minority donors. Blood 2013;122: 1062-
Association of ABO-mismatch platelet
71.
transfu- sions with morbidity and mortality in
109. Blumberg N, Heal JM, Gettings KF. Leukore-
cardiac surgery. Transfusion 2001;41:790-3.
duction of red cell transfusions is associated
123. Bowden RA, Slichter SJ, Sayers M, et al. A
with a decreased incidence of red cell alloim-
com- parison of filtered leukocyte-reduced and
munization. Transfusion 2003;43:945-52.
cy- tomegalovirus (CMV ) seronegative
110. Van de Watering L, Jermans J, Witvliet M, et
blood products for the prevention of
al. HLA and RBC immunization after filtered
transfusion-as- sociated CMV infection after
and buffy coat-dependent blood transfusion
marrow trans- plant. Blood 1995;86:3598-
in cardiac surgery: A randomized controlled
603.
trial. Transfusion 2003;43:765-71.
124. Mussi-Pinhata MM, Yamamoto AY, Rego
111. McPherson M, Anderson A, Jessup P, et al.
MAC, et al. Perinatal or early-postnatal
HLA Alloimmunization in pediatric sickle cell
cytomegalo- virus infection in preterm
dis- ease patients: Potential impact for
infants under 34 weeks gestation born to
transfusion
CMV-seropositive mothers within a high-
seroprevalence popula- tion. J Pediatr
2004;145:685-8.
 C H AP T E R 23 Neonatal and Pediatric Transfusion Practice
125. Bradley MT, Milam JD, Anderson DC. Use of
deglycerolized red blood cells to prevent post-
transfusion infection with cytomegalovirus in
neonates. J Infect Dis 1984;150:334-9.
126. Gilbert GL, Hayes K, Hudson H, et al.
Preven- tion of transfusion-associated
cytomegalovi- rus infection in infants by
blood filtration to remove leukocytes.
Lancet 1989;i:1228-31.
127. Strauss RG. Selection of white cell-reduced
blood components for transfusions during
early infancy. Transfusion 1993;33:352-7.
128. Fergusson D, Hebert PC, Lee SK, et al.
Clinical outcomes following institution of
universal leukoreduction of blood
transfusions in pre- mature infants. JAMA
2003;289:1950-6.
129. Moroff G, Friedman A, Robkin-Kline L, et al.
Reduction of the volume of stored platelet
concentrates for use in neonatal patients.
Transfusion 1984;24:144-6.
■ 597
130. Schoenfeld H, Muhn M, Doepfmer UR, et al.
The functional integrity of platelets in volume-
reduced platelet concentrates. Anesth Analg
2005;100:78-81.
131. Dehmer JJ, Adamson MD. Massive transfusion
and blood product use in the pediatric
trauma patient. Semin Pediatr Surg
2010;19:286-91.
132. Hendrickson JE, Shaz BH, Pereira G, et al. Im-
plementation of a pediatric trauma massive
transfusion protocol: One institution’s
experi- ence. Transfusion 2011;52:1228-36.
133. Chidester SJ, Williams N, Wang W, Groner JI. A
pediatric massive transfusion protocol. J Trau-
ma Acute Care Surg;73:1273-7.
134. Hendrickson JE, Shaz BH, Pereira G, et al. Co-
agulopathy is prevalent and associated with
adverse outcomes in transfused pediatric
trauma patients. J Pediatr 2012;160:204-9.
 C h a p t e r 2 4

Patient Blood Management

Mary Ghiglione, RN, MSN, MHA,

and Kathleen E. Puca, MD,
MT(ASCP)SBB

TR ANSFU S IO N OF BLOOD compo - DEFINITION AND SCOPE OF

nents plays a critical role in clinical
care. More than 13.5 million Red Blood
Cell (RBC) transfusions were administered
in US hospi- tals in 2011 at an estimated
cost of $10 bil- lion.1,2 Transfusion is the
single most com- monly billed procedure
for patients receiving hospital care.3
Blood transfusions can be life-saving,
but they can also be associated with
complica- tions. Transfusion-transmitted
infections, al- though rare, are of concern. In
addition, a growing body of literature
demonstrates an as- sociation between
transfusions and poorer pa- tient outcomes.4-
8 agement” may be fairly new, the concepts
The need to better understand the bene-
fits and risks associated with transfusion and
to control health-care costs has led to a grow-
ing interest by many organizations in initia-
tives to improve the quality, stewardship, and
utilization of blood components. Identifying
best practices—including provision of quality
health care without transfusion—is being ad-
dressed by professional societies, federal agen-
cies, hospital systems, and other stakeholders.
PATIENT BLOOD MANAGEMENT
Patient blood management (PBM) can be de-
fined as an evidence-based, multidisciplinary
approach to optimizing the care of patients
who might need transfusion. Evidence-based
guidelines for the use of blood components
and education for health-care providers on
best practices in blood transfusion are vital el-
ements of PBM. Employing a combination of
pharmacologic, surgical, and medical modali-
ties to conserve blood also plays an important
role in the overall care of patients.
Although the term “patient blood man-
have developed over time in parallel with
medical advances. A full discussion of this
history is be- yond the scope of this chapter.
However, two examples illustrate the shared
“drivers” of blood transfusion and PBM.
One example is the influence of
wartime injuries. The discoveries of blood
groups, crossmatching, and blood
preservation in the early years of the 20th
century enabled the use

Mary Ghiglione, RN, MSN, MHA, National Director, Patient Blood Management, Accumen, Inc, San Diego,
California, and Kathleen E. Puca, MD, MT(ASCP)SBB, Medical Director, BloodCenter of Wisconsin,
Milwau- kee, Wisconsin
The authors have disclosed no conflicts of interest.

599
600 ■ AABB T EC HNIC AL MANUAL
of banked blood in the treatment of
exsangui- nating wounds suffered in the
armed conflicts of the same period. Yet
transporting the blood was difficult, and
battlefield surgeons devel- oped techniques
to treat casualties when no transfusion was
possible.
A second example is the influence of Je-
hovah’s Witnesses, who cite Biblical passages
as the foundation for their refusal of blood
transfusion. By the middle of the 20th century,
blood transfusion had become universally ac-
cepted as a medical treatment for a wide range
of indications. Jehovah’s Witness patients had
to seek out those few physicians and hospitals
that would provide medical and surgical care
without blood transfusion. These surgeons and
hospitals became increasingly utilized by Je-
hovah’s Witnesses and other patients, and
blood conservation programs began to
flourish. With an emphasis on an
individualized, patient- centric approach, these
programs led the way toward the PBM
movement seen today.
Although the focus is frequently on sur-
gery, PBM encompasses the entire scope of a
patient’s hospital experience. It includes:
1. Efforts to identify and manage anemia and
bleeding risks before any treatment begins.
2. Use of blood-sparing surgical techniques
and intraoperative blood recovery meth-
ods.
3. Adjunctive strategies during intensive
care unit (ICU) and postoperative care
that decrease the need for transfusion.
4. Blood utilization review and feedback to
ordering physicians.
5. PBM education of all health-care
providers involved in patient care.
The scope of PBM activities is
discussed in detail in the section on Basic
Elements of a Patient Blood Management
Program.
THE RATIONALE FOR PATIENT
BLOOD MANAGEMENT
Blood transfusion became universally
accept- ed as a mainstream medical therapy
long be- fore all the risks and complications
associated with it were recognized. The
compelling rea-
sons for PBM include the risk of clerical
errors, errors in patient identification, the
threat of a new transfusion-transmitted
pathogen, and the mounting evidence that
transfusions are associated with adverse
patient outcomes. Other potential drivers of
PBM include in- creasing patient demand
for improved quality in health care, the
projected shrinking of the eligible donor
base, and increasing health-care costs.9,10
Risks of Transfusion
The infectious risks of transfusion that
became so widely known in the late 20th
century, which are now rare, as well as
unknown emerging pathogens continue to
be a concern. Improvements in donor
screening, develop- ment of effective tests,
and research into pathogen inactivation
technology have all played a role in
reducing the known infectious disease risks
of transfusion. Currently, the risk of either
hepatitis C virus transmission or hu- man
immunodeficiency virus transmission is less
than 1 in 1,000,000 donations, and the risk
of hepatitis B is less than one in 300,000.11
The noninfectious risks of transfusion,
including hemolytic transfusion reactions,
transfusion-related acute lung injury,
transfu- sion-associated circulatory
overload, and allergic reactions are more
common than in- fectious adverse events
and often go unrecog- nized. Chapter 27,
Noninfectious Complica- tions of Blood
Transfusion, discusses the diagnosis,
etiology, treatment, and prevention of these
risks in detail.
An emerging body of evidence
indicates that transfusion may not provide
the expected therapeutic outcomes long
assumed by both clinicians and patients.
This is especially the case in stable,
nonbleeding patients.12 Ran- domized
controlled trials assessing the effica- cy of
RBC transfusion have shown that restric-
tive transfusion strategies for nonbleeding
patients have no negative effect, and may
even improve outcomes for some
patients.8,13-16
Recent research is identifying a link be-
tween allogeneic transfusion and worse pa-
tient outcomes. Although many of these
stud- ies on blood transfusion are
observational,
 CH A P T E R 2 4
they have enrolled large numbers of patients
across many different specialties (eg, cardiolo-
gy, surgery, ICU, gastroenterology). Such
stud- ies have repeatedly concluded that a
strong as- sociation exists between RBC
transfusion and worse patient outcomes.5,8,9,17
Several studies have shown the risk of
nosocomial infection in ICU, surgical, and
trauma patients to be two to four times
higher in transfused patients than in
nontransfused patients.5 In addition, the risk
is dose-depen- dent (ie, the more blood the
patient receives, the greater the risk of
adverse events).18,19 RBC transfusion has
been associated, in a dose- related fashion,
with higher rates of cancer re- currence,
stroke, myocardial infarctions, renal
complications, acute lung injury and respira-
tory arrest, longer ICU and hospital stays,
and mortality both in the hospital and the
long term (5 years).5,17,20-24
These deleterious effects on clinical
out- comes are not solely related to
allogeneic RBC transfusions. One single-
center study of more than 800 critically ill
patients demonstrated that 1) transfusion
was independently associ- ated with the risk
of acute lung injury and 2) the risk was
higher with the transfusion of plasma-rich
blood components (platelets and plasma)
than with transfusion of RBC units.25 Plasma
transfusion in critically ill patients has
shown little benefit and has been associated
with worse patient outcomes.26,27
Thus, a growing body of literature
strong- ly suggests that for the nonbleeding
patient the benefit of transfusion is
outweighed by un- expected risks and worse
outcomes. The risks can be reduced only by
a shift from the more conventional
transfusion practice (where transfusion is
often a default decision) to a PBM approach
to transfusion practice (where the decision
to transfuse is an option consid- ered after
evaluation of the risks, benefits, and
alternatives specific to each patient).
The Patient’s Perspective
Despite advances in safety of the blood
supply, patients are concerned about needing
a blood transfusion during their
treatment.28,29 Wheth- er due to their
underlying illness, inadequate
Patient Blood Management ■ 601
communication, or their physician’s demean-
or, some patients trust that the provider who
orders the transfusion is acting in their best
in- terest. Too often, patients do not ask for,
or are not provided with, information on
alternative treatment options before
receiving a transfu- sion.28
Allogeneic transfusion is considered a
therapeutic intervention and, as with other
medical interventions, requires the patient to
provide informed consent.30,31 In order for
the patient to be adequately “informed,” the
con- sent discussion needs to include
information about the risks and benefits not
only of the transfusion option, but of the
alternatives to transfusion and the refusal of
treatment as well.32 The PBM approach,
which encompass- es all the options,
provides an excellent avenue for open
communication, joint consideration of
treatments, and the potential for improved
patient outcomes and satisfaction.
PBM strategies are a necessity for
patients for whom blood transfusion is not
an option due to medical status, cultural
influences, reli- gious beliefs, or
unavailability of compatible blood. From the
patient’s perspective, the ra- tionale for PBM
is the ability to make a more informed
choice, having access to a higher quality of
care, and potentially experiencing less risk.
The Physician’s Perspective
After “first do no harm,” a physician’s primary
responsibility is to ensure effective treatment
with minimal risk. Clinicians intend to do
what is best for their patients. But too often
they take blood transfusion for granted, as-
suming that benefits will be achieved and per-
ceiving the risks to be minimal.
Medical school curricula devote a bare
minimum of time for education on the risks
of, indications for, and alternatives to the use
of blood.33,34 Physicians often learn
transfusion practices based on the norms and
standards developed years ago. It is difficult
enough for them to keep up to date with
current research and best practices in their
own specialty, let alone to keep abreast of
developments in transfusion medicine.
602 ■ AABB T EC HNIC AL MANUAL
Studies show a wide variation in
transfu- sion practice. Whether or not a
patient re- ceives a blood transfusion is
often influenced by the hospital where the
patient is admitted or by the physician
providing the clinical care, rather than by
the patient’s clinical condi- tion.3,35-38
Transfusion based on physician
behavior can be improved with the
implementation of evidence-based clinical
practice guidelines and computer
“dashboards” identifying best practices
linked to blood utilization and pa- tient
outcomes. Providing individual physi- cians
with their usage data in comparison to that
of their colleagues can be a powerful tool
for process improvement.
Audits and evaluation of transfusion
practices, along with analysis of blood
utiliza- tion data, are tools hospitals can
apply to iden- tify best practices. From these
tools, guide- lines, algorithms, and protocols
can be developed and implemented across a
service line in an effort to assist individual
physicians in meeting quality standards and
improving patient care.
Thus, a PBM approach benefits not
only patients, but the physicians who treat
them. Physicians who use the patient-centric
ap- proach of PBM will gain a reputation for
better patient outcomes, shorter lengths of
stay, and reduced costs. In some facilities,
blood utiliza- tion data are included on a
physician “score- card,” acknowledging
those physicians whose transfusion practice
complies with the hospi- tal’s guidelines.
The Hospital’s Perspective
Economic Pressures
Due to the economic pressures facing hospi-
tals today, it is estimated that by 2020 one in
three hospitals will close or reorganize into
a different type of health-care service.39
Oppor- tunities to reduce costs are eagerly
sought by hospital administrators, and
reducing blood usage is no exception.
The acquisition cost of an RBC unit is
only a fraction of the total cost of that unit to
the hospital. Using activity cost-based
modeling, Shander et al2 reported that the
total cost for
blood- and transfusion-related activities for
surgical patients ranged from $522 to $1183
per unit transfused. They also found that
total annual blood expenditures correlated
not with the volume of surgeries performed,
but with the transfusion rate. The authors
concluded that reducing the proportion of
patients who receive transfusions, the
number of RBC units transfused per patient,
or both can be an im- portant cost-saving
strategy for hospitals.
In a model simulation, Zilberberg and
Shorr40 showed that universal adoption of a
re- strictive transfusion therapy (hemoglobin
less than 7 g/dL) in all ICU patients in US
hospitals could potentially result in cost
savings of near- ly $1 billion annually. Even
more important, the simulation indicated
that this strategy could improve quality of
care and patient out- comes by preventing
nearly 40,000 transfu- sion-attributable
complications.
A recent blood utilization project involv-
ing 464 US hospitals identified opportunities
for significant reductions in blood utilization
and costs.3 Several elements of a PBM
program were noted as important steps for
hospital leaders to consider.
Appropriate reimbursement for health-
care supplies and services, including blood
and transfusion, is a growing concern for
hos- pitals. Medicare reimbursements have
failed to keep pace with increased costs,
which have exacerbated the revenue
pressures. The Cen- ters for Medicare and
Medicaid Services (CMS) structure for
outpatient reimbursement iden- tified that in
2013, payments for the more fre- quently
transfused blood components would
decrease.41 An understanding of all the con-
tributing factors to the cost of transfusion
(in- cluding reimbursement) is vital for
hospitals to develop. The economic
pressures can be an additional incentive for
reducing unnecessary transfusions and
improving blood utilization.
Requirements for Accreditation
The Joint Commission promotes several
stan- dards related to hospital blood
administration42 and requires hospitals to
review the use of blood and blood
components in an effort to monitor and
improve practices. The Joint
 CH A P T E R 2 4
Commission also has developed PBM
Perfor- mance Measures, which include
seven areas for improvement.43 Although
they are not uni- versally practiced, the
measures can help hos- pitals to monitor
blood usage.
Both AABB and the College of
American Pathologists (CAP) promote
standards for transfusion service
laboratories. These re- quirements address
more specific, technical issues in quality
testing and patient safety re- lated to blood
administration.44,45 Often, it is the
responsibility of the transfusion service to
ensure that requirements related to transfu-
sion are met—even when the requirements
in- volve activities outside the purview of
the lab- oratory staff.
For instance, the CAP requirement
TRM.41025-Transfusionist Training Phase II
requires all personnel who administer blood
components to be trained in proper identifica-
tion of transfusion recipients. AABB
Standards for Blood Banks and Transfusion
Services re- quires a peer-review program for
monitoring and addressing transfusion
practices in all cat- egories of blood and blood
components.45(p91) Both require collaboration of
other hospital departments for compliance.
With the multi- disciplinary, team-oriented
approach of PBM, an additional benefit to
hospitals may be in- creased ease of
compliance, as well as more readily accepted
and implemented corrective actions and
process improvements.
The Community’s Perspective
Initiatives to limit the use of blood also
benefit the community by supporting a safe
and ade- quate blood supply for those who
need it. The increasing age of the population
and the di- minishing donor pool may have
significant impacts on blood availability. In
the next 10 to 20 years, it is expected that
the number of indi- viduals in the United
States who are more than 65 years of age
will increase at a faster rate than those aged
16 to 64, and make up 20% of the
population.46,47
A recent review of data from the Health
and Retirement Study reported that 31% of
Americans over the age of 65 received at least
Patient Blood Management ■ 603
one transfusion in a 10-year period.48
Although data are limited, it is estimated that
patients who are 65 or older receive 55% to
60% of RBC transfusions.47 Similar
population demograph- ics are expected in
Germany, where the pre- dicted shortfall in
the blood supply could be 47% by 2020.49
Blood availability may also be affected by
changes in donor eligibility and storage of
blood units. A better understanding of iron
de- pletion in frequent blood donors has
prompt- ed a discussion of increased donor
hemoglo- bin requirements and prolonged
donation intervals.50,51 If ongoing trials
confirm a rela- tionship between the age of
stored blood units and increased morbidity
and mortality follow- ing transfusion in
acutely ill adult patients, blood inventory
management will change.52-56
Any shortening of the shelf-life of the RBC
unit could put more pressure on diminishing
blood supplies and donor recruitment efforts
may not be able to make up for the loss.57
Im- plementing PBM strategies to reduce the
need for allogeneic blood and making sure
only ap- propriate transfusions of the correct
dose are given are two ways to support the
available blood supply for a community.
BASIC ELEMENTS OF A PBM
PROGRAM
As noted earlier in the section on Definition
and Scope of PBM, several PBM strategies
can be implemented to decrease the use of
alloge- neic blood transfusions. They
encompass all aspects of the patient’s
evaluation and clinical management. A
PBM approach is typically im- plemented in
elective surgical patients in the preoperative,
intraoperative, and postopera- tive phases of
care as outlined in Table 24-158 and in the
following sections. PBM strategies are also
relevant to medical patients. Although any
one of the strategies used individually will
be successful in decreasing the use of
alloge- neic blood transfusion, they are most
effective when combined and individualized
for the specific patient.59
604 ■ AABB T EC HNIC AL MANUAL

TABLE 24-1. Scope of Patient Blood Management

Nonsurgical or Preoperative ICU andBlood UtilizationMedical Postoperative

Intraoperative Review Education
■ Anemia screening and ■ Replacement ■ Replacement ■ Clinical prac- ■ In-service
management fluids fluids tice guidelines – grand rounds
– iron therapy – crystalloid – crystalloid – indications – conferences
– cautious use of – colloid – colloid – thresholds – reminders
EPO ■ Surgical techniques ■ Limit – restrictive (posters, data
■ Screen for – meticulous phlebot- strategies cards, etc)
bleeding risks and suturing omy ■ Blood Utiliza- – journal club
optimize – harmonic scalpel ■ Tolerate low tion or ■ Continuing
coagulation – suction control hemoglobin Transfu- sion edu- cation
– discontinue – rinsing swabs ■ Monitor Committee – certification
antico- agulants ■ Acute normovole- bleed- ing ■ Audits – CME/CE credit
and anti- platelet mic hemodilution ■ Wound – prospective – online tools
drugs ■ Intraoperative drain- age – concurrent (webinars,
– discontinue herbal blood recovery ■ Iron therapy – retrospective interactive
supplements, some ■ Hemostatics ■ Hyperbaric ■ Patient Blood modules)
vitamins – topical oxygen Management ■ Medical and
– address genetic thrombin ■ Point-of-care Coordinator nursing school
coagulation abnor- – fibrin sealant testing or curricula
malities – platelet gel Transfusion
■ Minimize ■ Point-of-care Safety Officer
crystalloids in testing
acute bleeding ■ Monitor acute
■ Limit phlebotomy bleeding and
■ Preoperative autolo- man- age
gous donation coagulation
■ Tolerate low
hemo- globin
■ Avoid hypothermia
■ Tolerate low
blood pressure
■ Positioning

Modified with permission from Becker J, Shaz B.58

ICU = intensive care unit; EPO = erythropoietin; CME = continuing medical education; CE = continuing education.

Preoperative Strategies Anemia Assessment and Management

Patient evaluation and planning are essential
first steps of a PBM program. A detailed
medi- cal history and physical examination,
with special attention to family and personal
histo- ry of spontaneous or postoperative
bleeding and anemia, are vital to identifying
and sup- porting high-risk patients.
Medications that may affect coagulation
should also be as- sessed. Structured
questions can help with the evaluation,
which should be performed suffi- ciently
ahead of the planned surgery (eg, 30 days)
to allow for diagnostic testing and thera-
peutic interventions, as needed.60
Recognition and management of anemia in
the medical and preoperative patient is a
core principle of PBM. The World Health
Organiza- tion (WHO) defines anemia as a
hemoglobin level less than 13 g/dL in men
and less than 12 g/dL in premenopausal,
nonpregnant women.61 Clinical signs and
symptoms of ane- mia (tachycardia, chest
pain, shortness of breath, dizziness,
headache, depression, cold extremities, pale
skin, fatigue, and decreased cognitive
function) occur when the oxygen- carrying
capacities of the red cells are unable to meet
the oxygen demand of the tissues.
 CH A P T E R 2 4
Anemia is a common condition and is
es- timated to be as high as 75% depending
on the patient’s underlying conditions,
reason for surgery, and definition of anemia
used.62,63 Anemia has been independently
associated with increased perioperative
mortality and morbidity in patients
undergoing joint re- placement and cardiac
surgery.62,64 In a retro- spective study of
noncardiac surgery patients 65 years or
older, 30-day postoperative mortal- ity and
cardiac event rates increased by 1.6% for
every percentage-point decrease from the
normal hematocrit range.65 Even mild
anemia has been shown to be an independent
risk fac- tor for adverse outcomes in patients
undergo- ing colon surgery.66
If anemia is detected, the initial test re-
sults may suggest possible etiologies.67
Addi- tional laboratory testing such as
creatinine, reticulocyte count, iron and iron-
binding ca- pacity, ferritin, vitamin B12, and
folate may help in the diagnosis. Several
published guide- lines can assist the PBM
program in develop- ing algorithms for
identifying and treating anemia in medical
and preoperative pa- tients.67-70 Whenever
possible, an elective, high-blood-loss surgery
should be delayed un- til the anemia is
explained and appropriately treated.
Pharmacologic agents for anemia may in-
clude iron (both oral and intravenous prepara-
tions), folic acid, vitamin B12, or erythropoie-
sis-simulating agents (see Appendix 24-1).
The choice of therapeutic agent should be
guided by the underlying etiology of the
anemia, the patient’s other medical conditions,
and avail- able time to treat before any
surgery.
Intravenous (IV) iron replacement
using iron sucrose, ferric gluconate, or iron
dextran has been shown to quickly correct
iron defi- ciency anemia and may be the
preferred route over oral preparations in
patients scheduled for surgery or in the
postoperative period. The efficacy of IV
iron in treating anemia has been consistently
proven in reducing the need for allogeneic
blood transfusions in surgical pa- tients.
Erythropoietic stimulating agents (ESAs)
are also effective in increasing the hemoglo-
bin level if sufficient iron stores are present.
Patient Blood Management ■ 605
The use of ESAs to lower transfusion rates
in patients undergoing elective, orthopedic
sur- gery is well established.71 However, due
to the risk of thromboembolic events, tumor
growth, and death associated with ESAs, the
Food and Drug Administration (FDA) has
restricted its use, recommending more
conservative dosing and strict monitoring.72
Addressing Bleeding Risk
A second important aspect of PBM is
minimiz- ing the risk of bleeding. Assessing
a patient’s risk for bleeding in relation to the
type and complexity of surgery and the
presence of any pre-existing anemia and/or
coagulopathy can help formulate an
individualized plan. Estab- lishing protocols
for discontinuation or dose adjustment of
antiplatelet and anticoagulant drugs is an
important function of a PBM program and
an essential preoperative plan- ning tool.
Published guidelines such as those
devel- oped by the Society for Thoracic
Surgery and the American College of Chest
Physicians can provide a basis for
development of such proto- cols.73-75
Protocols help practitioners to opti- mize the
patient’s hemostasis before an elec- tive
surgery while minimizing risk for
thrombotic events.
Herbal supplements such as garlic,
gink- go, and ginseng, have also been known
to in- crease bleeding risk. Patients should
be asked about their use and ingestion
should be dis- continued before surgery.
Readers are direct- ed to more focused
reviews on the effect of herbal supplements
on coagulation.76,77
Preoperative Autologous
Blood Donation
Historically, preoperative autologous blood
donation (PAD) had been the primary means
to reduce the use of allogeneic blood. With
PAD the patient donates his/her own blood,
ideally 4 to 6 weeks, before surgery. The
goal of the preoperative donation is for
regeneration of the patient’s red cell mass
and return of the patient’s hemoglobin to the
precollection val- ue before surgery.
606 ■ AABB T EC HNIC AL MANUAL
With increasing safety of the blood
sup- ply, the number of autologous units
collected in the United States has
declined.1,78 Other fac- tors contributing to
the decline include high wastage of PAD
blood (45% or more is discard- ed),79,80
higher risk for preoperative anemia af- ter
donation,80,81 errors related to production and
handling of these units,83,84 and increasing
acquisition costs that may not be reimburs-
able.
In addition, a systematic review showed
that patients in a PAD program had a higher
likelihood of receiving transfusions
(allogeneic and/or autologous) compared to
those pa- tients who did not participate in
PAD because their hemoglobin did not
completely recover before surgery. 82
Therefore, patients in PAD programs incur a
higher overall risk to their health with a
greater likelihood of being trans- fused.
Despite these concerns, PAD may be a
reasonable option for patients with rare
blood types or multiple red cell
alloantibodies or for patients who refuse
allogeneic blood. In these situations,
advanced planning and evaluation of the
patient are crucial before PAD is at-
tempted. In order to mitigate the anemia in-
duced by PAD, the collection needs to occur
at least 4 to 6 weeks before surgery (earlier
if there are freezing capabilities for the
units) to allow sufficient time for recovery
of the pa- tient’s red cell mass.85
Intraoperative Strategies
Acute Normovolemic Hemodilution
Acute normovolemic hemodilution (ANH)
is the removal of whole blood from the
patient immediately before or shortly after
the begin- ning of the surgical procedure
into a standard blood bag containing
anticoagulant. Crystal- loid or colloid
solutions or both are reinfused to maintain
adequate circulatory volume. ANH lowers
the patient’s hematocrit, improves blood
fluidity, and increases cardiac output and
organ blood flow, offsetting the decline in
oxygen capacity of the diluted blood. 86 Any
blood lost from the patient during the
surgery is then diluted and has a reduced red
cell con- tent.
The ANH-collected whole blood is
typi- cally stored at room temperature (for
up to 8 hours) and is often reinfused near
the end of the procedure or whenever
transfusion is needed.87(p22) An added value
of ANH is that platelets and coagulation
factors remain viable when the product is
stored at room tempera- ture. Although
ANH is considered a relatively safe
procedure, reports on the clinical efficacy
and benefits are mixed.88 In some studies,
the use of ANH resulted in lower allogeneic
trans- fusion rates, fewer units transfused,
and de- creased complications,89,90 while
others failed to show any benefit.91,92
ANH is more likely to be of benefit in pa-
tients undergoing high-blood-loss procedures
(1500 mL or greater) who have preoperative
hemoglobin levels of 12 g/dL or higher. Poten-
tial contraindications for ANH include active
cardiac ischemia, restrictive or obstructive
pulmonary disease, renal failure, hemoglobin-
opathy associated with hemolysis, and known
coagulation abnormalities.93 Planning and co-
ordination by the surgical team are important
for ANH to be effective.
Intraoperative Blood Recovery
Intraoperative blood recovery is another
com- mon PBM strategy. Use of recovered
shed blood is efficacious in several
procedures, such as cardiothoracic,
orthopedic, neurolog- ic, vascular, and
trauma surgery. Reduction in allogeneic
RBC transfusions by 38%, with an average
savings of 0.68 unit of allogeneic RBCs per
patient has been reported.94
Intraoperative blood recovery involves
collecting shed blood from the surgical site,
centrifuging it, and washing it with normal
sa- line. During the centrifugation and
washing process, plasma, platelets, red cell
stroma, contaminants, and anticoagulant are
removed. The washed red cells are
transferred to a sepa- rate bag, and then
administered to the same patient. Ideally,
washed recovered blood should have a
hematocrit of at least 45% to 55%.
Concerns regarding the safety of blood
re- covery often surround its use in cancer and
obstetric surgeries. The concern is that un-
wanted material and cells (eg, tumor cells,
 CH A P T E R 2 4
bacteria, and amniotic fluid) from the
surgical field may end up in the washed
product and be re-infused to the patient.
However, the use of double suction setup
and leukocyte reduc- tion filters has been
shown to reduce these risks.95,96 In addition,
reviews do not support the theoretical
concern for increased risk of ei- ther
amniotic fluid emboli or cancer recur- rence,
although the quality of the studies has been
questioned.97,98 Prospective, appropriate- ly
powered studies are needed to define the
risks and benefits of recovered blood in
these controversial settings.
A recent novel approach for blood
recov- ery in surgeries involving
cardiopulmonary bypass (CPB) is the
Hemobag modified ultra- filtration system
(Global Blood Resources, LLC, Somers,
CT). After completion of CPB and
decannulation, residual whole blood from
the CPB circuit, which can be up to 2 liters,
is ultrafiltered and hemoconcentrated.
Excess water and inflammatory mediators
are re- moved, resulting in a whole blood
product with hematocrit of approximately
50%. In con- trast to washed, recovered
blood, blood pro- cessed with the
ultrafiltration system contains platelets,
plasma proteins, and coagulation factors that
have been preserved and concen- trated.99,100
The use of this device should theo- retically
reduce exposure to allogeneic blood more
than washed, recovered blood, especially
exposure to plasma and platelet
transfusions; however, the clinically relevant
impact of this newer technique remains
unknown.
Quality control and quality
management programs for autologous blood
products pre- pared by intraoperative blood
recovery devices are not yet as well
developed and accepted among hospitals as
other aspects of transfu- sion medicine.
Significant opportunity exists in hospitals
for collaboration between the transfusion
service, surgery, perfusion, anes- thesia, and
nursing departments in the devel- opment of
blood recovery programs. Guide- lines and
regulatory requirements for establishing,
enhancing, and maintaining quality and
safety for these particular transfu- sion
practices are described in the AABB Stan-
dards for Perioperative Autologous Blood
Col-
Patient Blood Management ■ 607
lection and Administration and other AABB
publications.58,87,101-103
Anesthesia and Surgical Techniques
Minimizing intraoperative bleeding is critical
for reducing the need for allogeneic transfu-
sion. Obvious measures include meticulous
surgical technique and rapid and rigorous
control of bleeding. Proper positioning of the
patient can reduce blood loss and is guided by
two basic principles—elevating the surgical
site above the level of the heart and avoiding
compression of venous drainage of the surgi-
cal field.104 The deliberate reduction of mean
arterial pressure, known as “controlled or de-
liberate hypotension,” in selected patients can
reduce blood flow to the surgical site and
thereby decrease blood loss. However, the
benefits must be balanced against potential
risk for organ ischemia.59,105
Maintaining normothermia is important
for optimal coagulation and hemostasis. Ef-
forts to keep the patient warm by using
warm IV fluids, air warming blankets and
by keeping the surgical suite warmer can
help to avoid hy- pothermia and thus
decrease surgical blood loss. Optimal fluid
management, including choice of infusion
fluids, volume, and timing of administration,
can also affect surgical blood loss and
transfusion requirements. Modern devices
for tissue dissection, such as harmonic
scalpels, water jet dissectors, and
electrocautery with argon beam coagulation
can minimize tissue damage and provide
bet- ter hemostasis at the incision site. Other
mea- sures influencing surgical blood loss
include use of tourniquets, direct control of
bleeding, infiltration of vasoconstrictors into
the surgi- cal wound, choice of ventilation
patterns, and choice of anesthesia.59,104-106
Point-of-Care Testing and
Transfusion Algorithms
Point-of-care testing (POCT) has several ad-
vantages over conventional laboratory testing
in that it can 1) be utilized whenever
necessary,
2) provide more rapid turnaround time, and
3) use minimal sample volumes for testing.
Use
608 ■ AABB T EC HNIC AL MANUAL
of POCT can provide information for timely
transfusion decisions and help to avoid
iatro- genic anemia. Several POCT devices
are avail- able that provide information on
coagulation, platelet function, and clot
stability; they are used in the surgical and
critical care set- tings.107,108
Transfusion based on clinical observa-
tion of bleeding and blood loss is highly em-
pirical and variable. Transfusion algorithms
based on POCT can enhance management of
bleeding by rapidly distinguishing between
bleeding due to coagulopathy and bleeding
of surgical origin and can provide goal-
directed transfusion therapy.
In cardiovascular surgical patients, the
use of thromboelastography-based transfu-
sion algorithms has generally shown a
reduc- tion in transfusion requirements,
particularly plasma and platelets, and a
decrease in blood loss compared to
clinician-directed and/or standard
laboratory-based transfusion algo- rithms. 109-
111
In a single center, small prospec- tive trial,
a therapy-guided algorithm based on
viscoelastic and aggregometric POCT was
su- perior to that based on conventional
laborato- ry testing for reducing allogeneic
RBC transfu- sions in coagulopathic,
complex cardiac surgery patients.112 The
authors attributed faster turnaround time and
the functional as- sessment of coagulation
by the POCT as an ad- vantage over the
conventional quantitative co- agulation tests.
It is important to note that institutions need
to establish their own algo- rithmic
decisions based on available coagula- tion
assays and therapeutic options.
Thromboelastography-guided algorithms
are also routinely used in liver transplant sur-
gery; however, the evidence to date is limited
in showing reduction in transfusion require-
ments.113 The use of thromboelastography in
trauma patients is emerging. Although recent
studies have shown its potential for early de-
tection of trauma-induced coagulopathy and
fibrinolysis for facilitating goal-directed thera-
py over conventional laboratory tests,114,115 on-
going research is needed to assess its
effective- ness in reducing transfusion
requirements.
Pharmacologic Agents
Hemostatic agents, such as antifibrinolytics,
desmopressin, and topical agents, can assist
coagulation during surgery in an effort to re-
duce the need for blood transfusion. Aminoca-
proic acid and tranexamic acid inhibit fibrino-
lytic activity, preventing premature breakdown
of the blood clot. Systematic reviews have
shown both agents to be safe and effective in
reducing surgical blood loss and the need for
RBC transfusion.116 Topical agents such as he-
mostats, sealants, and adhesives are gaining
importance as useful tools during surgery and
can reduce bleeding by causing blood to clot,
sealing vessels, or gluing tissues. More
focused reviews of these topical agents and
other phar- macologic agents are available. 117-
119
Brief de- scriptions of several
pharmacologic therapies that may help control
hemorrhage and/or pre- vent transfusion are
found in Appendix 24-1.
POSTOPERATIVE STRATEGIES
Postoperative Blood Recovery
Postoperative blood recovery involves the
col- lection and reinfusion of blood from
surgical drains and/or wounds. An adequate
amount of blood needs to be collected and
processed for this technique to be effective.
Thus, it is used mainly in cardiac and
orthopedic surgi- cal cases where the
volume of shed blood can be significant
(500 mL or more).
Postoperative recovered blood can be un-
washed or washed. For the unwashed
product, shed blood is collected and filtered
in a device until sufficient volume is
reached; then the blood is transferred to an
infusion bag for rein- fusion. For the washed
product, once suffi- cient shed blood is
collected, it is further pro- cessed by
washing and then transferred to a bag for
reinfusion.
In the past, reinfusion of unwashed shed
blood was popular as a blood conservation
technique in joint replacement surgery.
More recently, the increasing use of other
blood management strategies, particularly
antifibri- nolytics, has led to a decline in
surgical bleed-
 CH A P T E R 2 4
ing, making postoperative blood recovery
un- necessary.116 In addition, unwashed shed
blood is less desirable because it has a
hema- tocrit ranging from 20% to 30% and
contains activated clotting and complement
factors, in- flammatory mediators,
cytokines, and fat par- ticles that can
increase the risk for febrile reac- tions.120,121
For patients with substantial postopera-
tive blood loss, improved product quality and
safety—hematocrit ranging from 60% to 80%
with removal of contaminants—can be
achieved by using devices that wash and con-
centrate the postoperative wound drainage
blood. Maintaining competency of nursing
staff and higher cost for these devices may be
disadvantages for some hospitals. However,
when compared to allogeneic blood transfu-
sions, use of postoperative washed products in
joint replacement surgery is potentially com-
parable in cost.122
Limiting Phlebotomy-Related Blood
Loss
Minimizing the impact of phlebotomy on
the development of iatrogenic anemia is a
key strategy for reducing transfusion
requirement. Iatrogenic anemia related to
routine laborato- ry testing is common. In a
study of 145 West- ern European ICUs,
blood loss from phleboto- my averaged 41.1
mL per day per patient,123 which could result
in the loss of nearly a unit of blood for an
ICU stay of 1-2 weeks. A US retro- spective
database study of over 17,000 patients with
acute myocardial infarction who were not
anemic on admission showed that patients
who developed moderate to severe anemia
had experienced nearly 100 mL higher mean
blood losses from phlebotomy over the
course of their hospitalization than those
who did not develop anemia.124 In a single-
center retro- spective study of patients with
prolonged ICU stays, after day 21 the odds
of being transfused was independently
associated with phleboto- my volume.125
Reducing the quantity and frequency of
laboratory testing is a logical measure to re-
duce the amount of blood loss related to
phle-
Patient Blood Management ■ 609
botomy. Routine, standing orders are a com-
mon practice. Laboratory testing should be
ordered when clinically justified and the re-
sults are likely to change clinical
management. When ordered, collection for
laboratory test- ing should be consolidated
to minimize the number of tubes collected.
Another approach is the use of pediatric or
small volume tubes for sample collection.
This strategy can reduce blood loss from
laboratory testing by up to 70%.126,127
Smaller sample volumes, often less than 0.5
mL, can also be obtained with the use of
POCT devices.
Even the process of drawing the blood
can be a significant cause of blood loss. Pa-
tients with invasive lines (eg, central venous
catheters or arterial catheters) have a three-
fold increase in phlebotomy volume com-
pared to patients without such catheters. The
ease with which samples can be obtained
and the added requirement to discard the
first few mLs (up to 10 mL) each time the
line is ac- cessed can contribute to greater
blood loss.128
Such catheters should be discontinued
as soon as they are no longer required.
Orders for specimens drawn from these
catheters should be consolidated to
minimize the amount of blood otherwise
discarded. Use of a device for blood
sampling whereby the initial volume of
blood can be reinfused into the patient
instead of discarded has shown reduction in
mean phlebotomy volume by 50%, higher
hemoglo- bin levels, and fewer RBC
transfusions even when a restrictive
transfusion practice is im- plemented.127,128
Increased Tolerance of Anemia and
Transfusion Thresholds
A patient’s response to anemia is highly
indi- vidualized and dependent on his or her
ability to maintain adequate oxygen delivery
to the tissues. Tolerance is dependent on the
pa- tient’s volume status, his or her
physiologic re- serve (including cardiac,
pulmonary, and renal function), and the
dynamics of the anemia. The response
becomes one of the most impor- tant factors
in determining the need for trans-
fusion.129,130
610 ■ AABB T EC HNIC AL MANUAL
Patients with chronic anemia due to
chronic renal failure or slow gastrointestinal
bleeding often have physiologically adapted
to a lower hemoglobin level by increasing
their cardiac output, heart rate, or stroke
volume. Rapid blood loss from surgical
bleeding or trauma often results in patients
displaying he- modynamic instability, shock,
and other symptoms that require more rapid
volume re- placement.
Increasing a patient’s tolerance for
lower hemoglobin includes increasing
oxygen deliv- ery or decreasing oxygen
consumption, which can limit the need for
RBC transfusion. Strate- gies to optimize
the patient’s hemodynamic status and
oxygenation may include maintain- ing
normovolemia with intravenous fluids, use
of appropriate vasopressor agents, use of
sup- plemental oxygen or mechanical
ventilation, adequate pain control and/or
sedation, main- taining normothermia, and
avoidance and prompt treatment of any
infections.131
Incorporating evidence-based transfu-
sion protocols may further aid in reducing un-
necessary transfusions. Historically, hemoglo-
bin levels of 10 g/dL or less have been used as
the threshold for transfusion. Recent random-
ized control trials involving nonbleeding criti-
cally ill patients, post-cardiac-surgery patients,
elderly hip fracture patients with cardiac dis-
ease, and patients with upper gastrointestinal
bleeding have established evidence-based
transfusion thresholds. In all these trials, the
use of a more restrictive transfusion threshold
(eg, hemoglobin of 7 to 8 g/dL) has been
shown to decrease RBC transfusions without
increasing morbidity or mortality.8,132 In
addition, an arbitrary hemoglobin level or
“threshold” should not be the sole driver for
transfusion. Transfusion decisions should be
individualized and based not only on the he-
moglobin level but also on the patient’s clini-
cal signs and symptoms of anemia and their
ability to tolerate and compensate for the ane-
mia.133
Traditionally, physicians have ordered 2
units for RBC transfusion, a practice that
evolved without clear rationale. A unit of
blood can have varying effects on
hemoglobin and hematocrit, depending on
the patient’s total
blood volume and shifts in fluid between
fluid compartments. In the nonbleeding
patient, single-unit RBC transfusion
followed with clinical reassessment is
advocated.134,135 Often a single RBC unit
provides an adequate in- crease in
hemoglobin and relieves the patient’s
symptoms. The potential impact of imple-
menting a single-unit transfusion policy can
be significant. Based on a transfusion
thresh- old of 8 g/dL of hemoglobin and
adoption of a single-unit strategy, one center
predicted that half an RBC unit or more
could be saved per patient.136 When
combined with strict adher- ence to a
restrictive transfusion threshold, a single-
unit transfusion policy can have an even
greater impact.
BLOOD UTILIZATION REVIEW AND
CHANGING PHYSICIAN
BEHAVIOR
Changing the behavior of physicians whose
transfusion practices are embedded in tradi-
tion and habit can be challenging. The ele-
ments required to create a culture change in
physician transfusion practice include: 1) a
de- sire for change, 2) the provision of a
new be- havior practice, 3) a perception of
the new practice as safe and straightforward,
and 4) a presentation of change that is
nonthreatening to autonomy.119
More and more hospital administrators
are recognizing that the use of blood
products may worsen patient outcomes and
that the cost of allogeneic blood is
significant. Thus, a desire for change is
already under way. Strate- gies focused on
education of appropriate transfusion
practice, providing sound alterna- tives to
transfusion, and communication and
feedback by respected colleagues or
“champi- ons” are fundamental elements of
change in transfusion practices. Building a
team of dedi- cated stakeholders and
champions as the net- work for change
management is crucial for success of any
change and a key element for a successful
PBM program.
Studies have demonstrated the ability of
several interventions for changing
transfusion
 CH A P T E R 2 4
practice. Although a systematic review did
not find one intervention more effective than
an- other, the authors concluded that even
simple interventions appear to be
effective.137 Inter- ventions can be broadly
grouped into: 1) edu- cation, 2) adoption of
guidelines, 3) reminders, and 4) audits with
feedback.
Education can be in the form of sched-
uled conferences or one-on-one teaching.
One-on-one education with physicians, al-
though more time consuming, is likely to
have a more sustained effect. The
educational con- tent needs to be evidence-
based, provided by respected colleagues or
opinion leaders, and be ongoing. Providing
repeated sessions and easy access to
educational information, such as on the
hospital website, can help support
physicians as they consider changing their
practices.
Evidence-based transfusion guidelines
are the basis for improving appropriateness
in transfusion. Introduction of the guidelines
needs to be combined with education and re-
minders such as posters and pocket guide-
lines. Issuing a memo of the transfusion
guide- lines to physicians without follow-up
typically fails to reduce blood usage. 138
Transfusion guidelines can be reinforced
when they are used with computerized
provider order entry (CPOE) systems.
Incorporating decision sup- port tools into
CPOE transfusion order sets and requiring
physicians to document the in- dication
when outside of the guidelines can be
effective in improving practice.139,140
Interventions to change transfusion
prac- tice go hand in hand with auditing or
monitor- ing of transfusion practice. Audits
that occur at the time of the transfusion
order or during the 24 hours after the
transfusion and are accom- panied with
education provide a clear oppor- tunity for
changing behavior.141 Prospective audits
(approval before issuing the product)
performed manually require resources and
can be time consuming. The implementation
of CPOE can provide a more practical ap-
proach to monitoring each transfusion order.
Chapter 28 provides a detailed discussion of
blood utilization auditing.
Blood utilization reports that
incorporate benchmarking with the goal of
continuous
Patient Blood Management ■ 611
quality improvement are powerful tools to
change transfusion practice. Providing
physi- cians with outcome data in
relationship to their transfusion rates for a
surgical procedure or specific treatment as
well as comparison to their peers and best
practices are likely to mo- tivate changes.
Providing physicians with reg- ular reports
helps to maintain awareness and encourages
physicians’ interest in looking for additional
strategies to reduce avoidable
transfusions.142
A recent initiative to improve transfusion
practice has been the employment of
transfu- sion safety officers or patient blood
manage- ment coordinators. As change
agents they can provide regular education to
all staff involved in transfusion practice,
increase awareness of and access to
transfusion alternatives, develop a network
of caregivers and “champions” for the
promotion of optimal transfusions, and
provide utilization reports for continuous
im- provement. Working with the local
“champi- on,” these coordinators can be
instrumental in changing the culture and
reducing allogeneic transfusions.143
Medical Education
Education is an integral part of any PBM
initia- tive. Because behavioral change is
sometimes difficult to achieve, repetition and
reinforce- ment over time are typically
required for suc- cess. Because PBM is
multidisciplinary, differ- ent audiences may
respond to different approaches.
Educational delivery options may in-
clude journal club, grand rounds, webinars,
online courses, guest lecturers, conference
at- tendance, one-to-one instruction, self-
study, and blood usage review feedback.
Provision of continuing education credits for
educational activities can be an effective
motivator for par- ticipation.
Although ordering physicians and nurs-
ing staff need the greatest understanding of
PBM, other groups of hospital personnel
also benefit from educational opportunities.
For instance, information technology and
finan- cial personnel may not need to know
the most
612 ■ AABB T EC HNIC AL MANUAL

technical details, but they do need to under- TABLE 24-2. General Strategy for Implementing
stand the intent, benefits, and outcomes of a PBM Program
PBM efforts so they can effectively support
those efforts. 1. Education
a. A knowledgeable advocate
b. Executives
c. Core group (pharmacy, nursing, blood
PROGRAM DEVELOPMENT bank)
Successful implementation of a PBM d. Any department involved in PBM
program requires planning, education, and activi- ties (ordering clinicians, finance,
teamwork. It is important to first analyze the informa- tion technology)
current transfusion practices within the
2. Buy-in from executives
hospital. A needs assessment can help to
a. “C” suite (CEO, COO, CFO, CMO)
evaluate the current behaviors and
b. Medical executive committee
understand the hospital culture and
c. Blood utilization/transfusion committee
readiness for change.
d. Surgical services committee
Demonstrating the clinical benefits as
well as potential cost savings with a PBM 3. Business proposal
pro- gram is key to ensuring support and a. Background/current environment
buy-in at all levels. Care must be taken to b. Program description
ensure that clinical staff understand that the c. Financial aspects
primary ob- jective of a PBM program is to d. Risk/benefit analysis
improve patient outcomes, even though e. Summary/conclusion
hospital administra- tors may also focus on
the cost savings. 4. Teamwork
The specific activities that are carried a. Broad-based stakeholder group of
out as part of PBM program implementation those affected by PBM program
can vary by institution, and are defined by (multidisci- plinary emphasis,
the ex- ecutive management team of the identification of con- cerns, details to
facility. Guidance is available from a variety be addressed, enhanced buy-in)
of profes- sional organizations. The Society b. Smaller, more focused steering commit-
tee (baseline usage data, relationship to
for the Ad- vancement of Blood
strategic plan, implementation steps,
Management offers Ad- ministrative and
timeline, policy review, monitor progress
Clinical Standards for Patient Blood
checks)
Management Programs, which outlines 12
c. Effective meetings
standards related to the activities of a for- d. Milestone celebrations
mal, comprehensive, organization-wide
PBM program.144 A PBM program can be
designated as an activity level 1, 2, or 3
program as out- lined in AABB Standards
for a Patient Blood
Management Program.145(pp2-3) The responsibil-
ities for oversight and monitoring at each
5. Evaluation
ac- tivity level are described in Appendix
a. Possible improvements/lessons learned
24-2.
b. Study of metrics/outcome data
An effective PBM program involves a
c. Analysis/report of program success
mul- tidisciplinary effort with input and d. Possible program expansion
participa- tion from administration,
physician and
nursing leadership, laboratory, transfusion
medicine, ethics, registration, surgery, finance,
and technology personnel. Identifying cham-
pions who are willing to be spokespersons imple-
and drivers of the program is essential when
start- ing a program. A general strategy for
menting a PBM program
is summarized in Ta- ble
24-2. More detailed
descriptions of PBM
program implementation
are available.146,147
 CH A P T E R 2 4 Patient Blood Management ■ 613

KEY POINTS

Patient blood management (PBM) is an evidence-based, multidisciplinary approach to op- timizing the care of patients who m
Several factors have been drivers of PBM, including transfusion-associated risks, demand for improved quality of care, prom
Elements of PBM include: 1) managing anemia and bleeding risks before treatment begins,
2) intraoperative blood recovery and blood-sparing surgical techniques, 3) ICU and postop- erative strategies to reduce the ne
PBM can provide benefits to medical as well as surgical patients.
Preoperative anemia is common. Identifying and treating preoperative anemia is one of the fundamental elements of PBM.
PBM involves more than just transfusion avoidance. It involves use of pharmaceutical agents, blood recovery techniques, sur
A multidisciplinary team with “champions” is critical for success of the PBM program.
Ongoing blood utilization reviews with benchmarking and emphasis on patient outcomes are key elements of a successful pro

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147. Ozawa S, Thorpe E, Valenti J, Waters JH.
Devel- opment of a blood management
program. In: Waters JH, ed. Blood
management: Options for better patient care.
Bethesda, MD: AABB Press, 2008:33-82.
 APPENDIX 24-1

■
620

Pharmacologic Therapies for Supporting Patient Blood

Management*
Pharmacologic AgentSpecific DrugPrimary UseMechanism of ActionConsiderations
■

Intravenous (IV) Iron high-molecular- Used to treat iron Iron is an essential ■ Blood loss is a major cause of iron
Iron Therapy weight dextran; iron defi- ciency and ele- ment of defi- ciency.
low- molecular-weight iron-defi- ciency hemoglobin and the ■ Even under the best of circumstances,
dex- tran; iron anemia. actual site of oxygen oral iron is not well tolerated, and
gluconate; iron binding. It helps patients are often noncompliant due to
sucrose; iron trans- port oxygen to gastrointestinal (GI) symptoms.1
carboxy- methyl the tis- sues. ■ IV iron given concurrently with
There is substantial ESAs provides better response than
evi- dence that IV iron oral iron and allows lowering of ESA
is effective in treating dose in cer- tain patient
1
iron- deficiency ■ populations.
anemia in many High-molecular-weight dextran is
chronic condi- tions. asso- ciated with serious side

Erythropoietic Stimulat- Epoetin alfa: darbepoetin ■ Approved for treatment ■ ESAs stimulate ■ Response seen by day 5 to 7 in
ing Agents (ESAs) alfa. of anemia resulting from differ- entiation of iron- replete patients.
AABB T ECHNICAL M A NUAL

chronic kidney failure, stem cells into ■ One unit equivalent hematocrit increase
chemotherapy, certain immature red cells; by day 7; 3-5 units by day 28.
treatments for human increase rate of ■ Darbepoetin is an alternative used
immunodeficiency virus, mitosis and release of in renal and oncology settings.2,3
and also to reduce reticulo- cytes into
the number of blood the circula- tion; and
transfu- sions during induce hemoglobin
and after certain ■ forma- tion.
elective noncar- diac, A glycoprotein hor-
nonvascular sur- mone produced in
geries (hemoglobin the kidney, a growth
>10 g/dL but 13 factor for red cell
precursors in
 ■ Synthetic erythropoie-
Antifibrinolytics Epsilon-aminocaproic ■ Enhances hemostasis ■ Blocks fibrinolysis by
acid (EACA) associated with surgical
complications following
heart surgery; orthope-
Tranexamic acid (TA)6 dic surgery; some
(Similar to EACA, but hema- tologic
10 times as potent) disorders associated ■ TA provides
with throm-
bocytopenia; and mas-
sive traumatic bleeding
in the presence of fibri-
nolysis.
■ Society for Thoracic Surgery
tin produced by recom- 2011 guidelines report “It is
binant DNA technology. reasonable to use preoperative
erythropoietin plus iron, given
several days before cardiac surgery,
for preoperative anemia, in
candidates who refuse transfusion
(eg, Jehovah’s Witnesses), or in
patients who are at high risk for
4
■ postoperative anemia.”
Black box warning due to increased
risk for death, myocardial infarction,
stroke, venous thromboembolism,
thrombosis of vascular access, and
5
■ tumor progres- sion or recurrence.
CH A P T E R 2 4
inhibiting activation of Reduces blood loss and transfusion
plasminogen to plas- requirements in cardiac and joint
8
min, which is responsi- ■ replacement surgeries.
ble for limiting and Rapid intravenous administration
dissolving clots. should be avoided; may induce
hypo- tension, bradycardia, and/or
equivalent ■ arrhyth- mia.
antifibrinolytic effect Use with caution in hematuria of
as EACA but at only upper urinary tract origin, unless the
1/10 the concentration
Patient Blood Management
■ possible benefits outweigh risk.
and has a slower Contraindicated in patients with dis-
renal clearance (6-8 ■ seminated intravascular coagulation.
hours vs Skeletal muscle weakness with necrosis
of muscle fibers has been reported fol-
■
9
■ lowing prolonged use.
Must use renal dosing guidelines for
patients with impaired renal function.
(Continued)
621
 APPENDIX 24-1

■
622

Pharmacologic Therapies for Supporting Patient Blood Management*

Pharmacologic
(Continued)AgentSpecific DrugPrimary UseMechanism of ActionConsiderations
Antifibrinolytics ■ Dysfunctional uterine Adverse effects: nausea, vomiting,

■
■

(Continued) bleeding, menorrhagia diar- rhea, dizziness, disturbance of

associated with color vision, theoretic risk of
intrauter- ine device, thrombosis.9
conization of cervix,
post-/antepartum
hemorrhage, uterine/
■ vaginal surgery.
Oral bleeding, post-
dental extraction bleed-
ing in patients with
hemophilia, von Wille-
brand disease (vWD), or
Reversal Agents Vitamin K thrombocytopenia.
Reverses the Vitamin K is See clinical practice guidelines
anticoagu- lant effect necessary for the outlining the evidence-based
AABB T ECHNICAL M A NUAL

of warfarin. hepatic synthesis of management of vitamin K antagonist

coagulation factors initiation, monitor- ing, and treatment of
(Factors II, VII, IX, and complications.10,11
X) and anticoagulant
pro- teins (Protein C
Protamine Neutralizes unfraction- Adverse events associated with

■
■

and S).
ated heparin (UFH) admin- istration may include
Binds to heparin and
after cardiac surgery. hypotension, pul- monary edema
displaces antithrombin
Antidote when bleeding and anaphylaxis.

■
from heparin-antithrom-
complications are Excess protamine can lead to
■

asso- ciated with anticoag- ulation effect.

excessive heparin In cardiac surgery, dosing based
■

anticoagulation. on point-of-care testing allows

more appropriate dose and reduces
postop- erative bleeding.12
 ■ May be used for reversal of low-
molec- ular-weight heparin, but less
13
Coagulation Factor Prothrombin complex ■ 3-factor PCCs have only ■ PCCs are derived ■ effective. 3-factor PCCs do not
Concentrates concentrate (PCC) one approved from pooled human effectively lower the international
indication for the ■ plasma. 3-factor normalized ratio (INR); addition of
prevention and PCCs contain three small amount of Fresh Fro- zen
control of bleeding Vitamin-K-depen- dent Plasma (mean 2 units) increases
■ related to hemophilia coagulation fac- tors ■ likelihood of satisfactory INR
B. 4-factor PCCs are (Factors II, IX, and lowering.15 4-factor PCCs contain
approved for urgent X) and a small amount heparin and should not be used in
reversal of Vitamin K ■ of Factor VII. patients with heparin allergies or
antagonist (warfarin) 4-factor PCCs contain ■ heparin-induced
in life-threatening therapeutic levels of thrombocytopenia.14
bleeding.14 Factor VII (in addition Efficacy and safety of 3-factor PCCs
to Factors II, IX, and or 4-factor PCCs in the setting of
CH A P T E R 2 4

patients with severe or life-

threatening bleeding associated with
Recombinant Factor Approved by the Food Enhances thrombin ■ newer oral anticoagu- lants (eg,
VIIa (rFVIIa) and Drug gen- eration at the site dabigatran, rivaroxaban, apixaban)
Administration (FDA) of vas- cular injury. is unclear.16
for treatment of Black box warning issued in 2005
hemophilia A and B regarding the risk of thromboembolic
with inhibitors. complications. Efficacy of rFVIIa as a
Acceptable to use in general hemostatic drug remains
patients with con- unproven. Study results indicate
Patient Blood Management

genital Factor VII defi- increased risk of arterial thromboem-

ciency and patients bolic events. Use of rFVIIa outside its
with Glanzmann
■

thrombasthe- nia with

(Continued)
623
 APPENDIX 24-1
■
Pharmacologic Therapies for Supporting Patient Blood Management*
Pharmacologic
(Continued)AgentSpecific DrugPrimary UseMechanism of ActionConsiderations
Coagulation Factor Fibrinogen Approved by FDA for
Concentrates (Continued) concentrate treatment of bleeding
only in patients with
con- genital fibrinogen
defi- ciency.18
Other pharmaceuticals Desmopressin (DDAVP) ■ Useful in patients with
that can help with synthetic analog of mild hemophilia A or
hemo- stasis vasopressin19 Type I vWD (DDAVP
is contraindicated in
Type 2B and platelet-
type VWD).
■ May be useful in
patients with uremia or
cirrhosis; these patients
have pro- longed
bleeding times due to
complex disor- ders
of hemostasis.
624
■ Derived from pooled ■ Adverse reactions have included
■
human plasma. aller- gic and hypersensitivity
18
■ Precursor to fibrin; ■ reactions. Thromboembolic events
thrombin converts have been reported.18
fibrinogen to fibrin to ■ Use of fibrinogen concentrate in
form soluble fibrin obstet- ric hemorrhage and cardiac
clot, which is then surgery has been reported but is
stabilized by activated considered off- label and future
Factor XIII. studies are needed to prove
12
■ Raises circulating Fac- ■ efficacy.
tor VIII and vWF; Responsiveness to DDAVP in
unidentified effect on mild hemophilia A and type 1 VWD
platelets and endothe- is usu- ally confirmed by elective
lium.12 ■ challenge (trial).
■ Synthetic analog of Should be used with caution in
patients with fluid or electrolyte
AABB T ECHNICAL M A NUAL
the natural pituitary
hor- mone 8-arginine imbalance, as with cystic fibrosis;
vaso- pressin, an ■ patients may develop
antidiuretic hormone hyponatremia.
affecting renal water –Patients
Patients
whoundergoing from
may benefitprolonged
conservation. sur- gery with cardiopulmonary
bypass.20
– Patients experiencing excessive
postoperative bleeding or those
who are at high risk for
– bleeding.21-23 Patients taking
platelet-inhibiting drugs.24
– Patients with chronic renal failure
or liver dysfunction.
 Conjugated estrogen Dysfunctional uterine Precise mechanism of ■ Possible alternative or adjunct for

■
■
bleeding. action not known. cryo- precipitate or desmopressin
Uremia. May involve effect for the treatment of bleeding

■
■
on associated with renal failure.
mucopolysaccharide ■ Effectiveness of use in uremia
content of vessel patients is mixed.
wall; increase
synthesis of vWF by
Topical Hemostatic Mechanical hemostats Hemostats, sealants, endothelial cells.12 ■ Topical hemostatic agents can be
Agents (porcine gelatin, bovine and adhesives are Generally act by highly effective, but they must be
collagen, oxidized primarily used during com- pressing the used care- fully to avoid systemic
bleeding vessel, 1
regen- erated cellulose); surgery for a variety of ■ reactions.
biologi- cally active applications to help activating/aggre- gating Full descriptions and uses of the
hemostats (bovine with hemostasis when platelets, and/or vari- ous topical hemostatic agents
thrombin, human ligation, sutures, providing a scaffold for are avail- able.25
CH A P T E R 2 4

thrombin, recombinant compression, or cautery clot formation. Some

human thrombin); fibrin is not effective. The applications include
sealants, polyethylene types of bleeding where thrombin, which speeds
glycol polymer topical agents may be
sealants; synthetic used include: diffuse
raw sur- face
bleeding, oozing
venous-type bleeding,
bone bleeding, and
Pharmaceuticals used Oxytocin ■ Obstetric hemorrhage. Stimulates uterine con- Oxytocin is the standard treatment
Patient Blood Management

primarily in obstetrics ■ Used to treat uterine tractions. for postpartum hemorrhage.26-28

to control bleeding atony.
Obstetric hemorrhage.
Methergine ■ Increases the strength, Hypertension and toxemia are
Used to treat uterine
■ duration, and contrain- dications.26,28
■

atony.
frequency of uterine
contractions.
(Continued)
625
 APPENDIX 24-1

■
626

Pharmacologic Therapies for Supporting Patient Blood Management*

Pharmacologic
(Continued)AgentSpecific DrugPrimary UseMechanism of ActionConsiderations
Pharmaceuticals used Carboprost ■ Obstetric hemorrhage. Stimulates uterine con- Contraindicated in patients with pulmo-

■
■

primarily in obstetrics ■ FDA-approved to tractions. nary, cardiac, renal, and hepatic

to control bleeding treat uterine atony dis- ease.
(Continued) that has not Asthma is a relative

■
responded to con- contraindica- tion.26,28
Misoprostol ■ ventional treatment. Synthetic prostaglandin. Works most effectively when used
■ Obstetric hemorrhage. with other medications listed in this
Used to treat uterine table (synergistic action).26,27
atony. Proton pump inhibitors are more
Other drugs that Proton pump ■ Peptic ulcer. Reduces incidence
help with inhibitors ■ of upper GI effec- tive than H2-antagonists in
hemostasis Upper GI rebleeding after preventing persistent or recurrent
sclerotherapy by bleeding from peptic ulcer, although
increasing pH to this advantage seems to be more
above 4, which is evident in patients not having adjunct
necessary for clot sclerosis therapy.29
AABB T ECHNICAL M A NUAL

formation and
Octreotide ■ Variceal hemorrhage. ■ An octapeptide that Octreotide and somatostatin are at
■ Acute non-variceal upper mimics natural soma- least as effective as the conventional
GI bleeding. tostatin pharmacologi- vasoac- tive drugs and balloon
cally. tamponade in the treatment of
■ Long-acting analog variceal hemorrhage with the
of somatostatin (has advantage of fewer side effects.30
a much longer half-life
— approximately 90
min- utes, compared
to 2 to 3 minutes for
soma- tostatin).
 Reduces splanchnic

■
blood flow via
multifac- torial
mechanism.
Most effective when

■
combined with
sclero- therapy.
Eliminates vasospasm

■
complications seen
with vasopressin.
*Drugs approved for use in the United States as of April 2014. The table is intended to provide general information. Professionals seeking additional information and
individuals seeking
1. Goodnough LT, Shander A. Current status of pharmacologic therapies in patient blood management. Anesth Analg 2013;116:15-34.
2. Goodnough LT, Monk TG, Andriole GL. Erythropoietin therapy. N Engl J Med 1997;336:933-8.
3. Ross SD, Allen IE, Henry DH, et al. Clinical benefits and risk associated with epoetin and darbepoetin in patient with chemotherapy-induced anemia: A systematic review of the literature. Clin
CH A P T E R 2 4

Ther 2006;28:801-31.
4. Ferraris VA, Brown JR, Despotis GJ, et al. Society of Thoracic Surgeons Blood Conservation Guideline Task Force; Society of Cardiovascular Anesthesiologists Special Task Force on Blood Transfusion;
Inter- national Consortium for Evidence Based Perfusion. 2011 update to the Society of Thoracic Surgeons and the Society of Cardiovascular Anesthesiologists blood conservation clinical practice
guidelines. Ann Thorac Surg 2011;91:944-82.
5. Epogen (epoetin alfa) injection for intravenous or subcutaneous use package insert. Thousand Oaks, CA: Amgen, 2012. [Available at: http://pi.amgen.com/united_states/epogen/epogen_pi_hcp_english.pdf
(accessed on March 10, 2013).]
6. Cyklokapron tranexamic acid tablets and tranexamic acid injection package insert. New York, NY: Pfizer, 2014.
7. Use of desmopressin, antifibrinolytics, and conjugated estrogens in hemostasis. In: Goodnight SH, Hathaway WE. Disorders of hemostasis and thrombosis a clinical guide. 2nd ed. New York: McGraw-Hill,
2001:528-42.
8. Henry DA, Carless PA, Moxey AJ, et al. Anti-fibrinolytic use for minimising perioperative allogeneic blood transfusion. Cochrane Database Syst Rev 2011;(3):CD001886.
9. Ipema HJ, Tanzi M. Use of topical tranexamic acid or aminocaproic acid to prevent bleeding after major surgical procedures. Ann Pharmacother 2012;46:97-107.
10. Holbrook A, Schulman S, Witt DM, et al. Evidence-based management of anticoagulant therapy. Antithrombotic therapy and prevention of thrombosis. 9th ed. American College of Chest Physicians
Patient Blood Management

evidence- based clinical practice guidelines. Chest 2012;141(Suppl):e152S-e184S.

11. Patriquin C, Crowther M. Treatment of warfarin-associated coagulopathy with Vitamin K. Expert Rev Hematol 2011;4(6):657-67.
12. Bolan CD, Klein HG. Blood component and pharmacologic therapy for hemostatic disorders. In: Kitchens CS, Kessler CM, Konkle BA, eds. Consultative hemostasis and thrombosis. 3rd ed. Philadelphia:
Else- vier Saunders, 2013:496-525.
■

13. Garcia DA, Baglin TP, Weitz JI, et al. Parenteral anticoagulants. Antithrombotic therapy and prevention of thrombosis. 9th ed. American College of Chest Physicians
evidence-based
14. clinical practice guidelines. Chest 2012;141(Suppl):e24S-e43S.
Kcentra (Prothrombin Complex Concentrate, Human). Silver Spring, MD: Food and Drug Administration, 2013. [Available at: http://www.fda.gov/BiologicsBloodVaccines/BloodBloodProducts/ApprovedProd
ucts/LicensedProductsBLAs/FractionatedPlasmaProducts/ucm350130.htm (accessed April 1, 2014).]
(Continued)
627
 628

APPENDIX 24-1

■
Pharmacologic Therapies for Supporting Patient Blood Management*
(Continued)
■

15. Holland L, Warkentin TE, Refaai M, et al. Suboptimal effect of a three-factor prothrombin complex concentrate (Profilnine-SD) in correcting supratherapeutic international normalized ratio due to
warfarin overdose. Transfusion 2009;49:1171-7.
16. Siegal DM, Garcia DA, Crowther MA. How I treat target-specific oral anticoagulant-associated bleeding. Blood 2014;123:1153-8.
17. Simpson E, Lin Y, Stanworth S, et al. Recombinant factor VIIa for the prevention and treatment of bleeding in patients without haemophilia. Cochrane Database Syst Rev 2012;(3):CD005011.
18. RiaSTAP, Fibrinogen Concentrate (Human) for Intravenous Use, Lyophilized Powder for Reconstitution. Package insert. Kankakee, IL: CSL Behring, 2011. [Available at
http://labeling.cslbehring.com/PI/US/Ri- aSTAP/EN/RiaSTAP-Prescribing-Information.pdf (accessed on April 6, 2014).]
19. Desmopressin acetate injection package insert. Irvine, CA: Sicor Pharmaceuticals, 2014.
20. Salzman EW, Weinstein MJ, Weintraub RM, et al. Treatment with desmopressin acetate to reduce blood loss after cardiac surgery: A double-blind randomized trial. N Engl J Med
21. 1986;314:1402-6.
Cattaneo M, Harris AS, Stromberg U, et al. The effect of desmopressin on reducing blood loss in cardiac surgery: A meta-analysis of double-blind, placebo-controlled trials. Thromb Haemost
22. 1995;74:1064- 70.
23. Crescenzi G, Landoni G, Biondi-Zoccai G, et al. Desmopressin reduces transfusion needs after surgery: A meta-analysis of randomized clinical trials. Anesthesiology 2008;109:1063-
76.
24. Mongan PD, Hosking MP. The role of desmopressin acetate in patients undergoing coronary artery bypass surgery: A controlled clinical trial with thromboelastographic risk stratification.
Anesthesiology 1992;77:38-46.
25. Laupacis A, Fergusson D. Drugs to minimize perioperative blood loss in cardiac surgery: Meta-analyses using perioperative blood transfusion as the outcome. The International Study of Peri-operative
26. Trans- fusion (ISPOT) investigators. Anesth Analg 1997;85:1258-67.
AABB T ECHNICAL M A NUAL

27. Spotnitz WD. Hemostats, sealant, and adhesives: A practical guide for the surgeon. The American Surgeon 2012;78:1305-21.
28. Esler MD, Douglas J M. Planning for hemorrhage: Steps an anesthesiologist can take to limit and treat hemorrhage in the obstetric patient. Anesthesiol Clin North Am 2003;21:127- 44.
Soltani H, Hutchon DR, Poulose TA. Timing of prophylactic uterotonics for the third stage of labour after vaginal birth. Cochrane Database Syst Rev 2010;(8):CD006173.
29. Shields L. Uterotonic agents fact sheet. Obstetric hemorrhage toolkit. Stanford, CA: California Maternal Quality Care Collaborative, 2010. [Available at http://www.cmqcc.org/resources/934/download (ac-
30. cessed January 1, 2013).]
Gisbert JP, González L, Calvert X, et al. Proton pump inhibitors versus H2-antagonists: A meta-analysis of their efficacy in treating bleeding peptic ulcers. Aliment Pharmacol Ther 2001;15:917-
26.
 CH A P T E R 2 4 Patient Blood Management ■ 629

■ APPENDIX 24-2
Responsibilities for Activity Levels 1, 2, and 3 PBM Programs*

Activity Activity Activity

Item Responsibility Level 1 Level 2 Level 3
1 Evidence of institutional support for the patient blood management pro- X X X
gram at the executive level.
2 Patient outcomes related to transfusion. X X X
3 Budgeting to the level of care required by the implementation of X X X
these
PBM Standards.
4 Pretransfusion patient testing and evaluation. X X X
5 Assessment of potential need for blood usage. X X X
6 Ordering of blood, including completion of typing and antibody X X X
testing before procedure start time with a plan for antibody-
positive patients.
7 Identification and management of presurgical anemia before elective proce- X X X
dures for which type and screen or type and crossmatch is
recommended.
8 Preprocedure optimization of patient coagulation function including dis- X X X
continuation of medications and herbal supplements that impair coagu-
lation function.
9 Percentage of blood components wasted by component type (such as X X X
gen- eral red cells, rare unit red cells, general platelets, matched platelets,
plasma, AB plasma, cryoprecipitate, and granulocytes) and cause
(misordering, mis- handling, not released in a timely manner,
outdating in stock, etc).
10 Minimize blood loss due to laboratory testing. X X X
11 Process for identifying patients lacking identification. Standard 6.2.3 X X X
applies.
12 Processes to identify, prior to or upon admission, patients who X X X
may refuse transfusion under any circumstances.
13 Adverse events and incidents related to transfusion. X X X
14 Processes and/or equipment to facilitate rapid decision making with X X N/A
regard to anemia and coagulation management.
15 A plan by each service line to reduce perioperative blood loss. X X N/A
16 Strategies to reduce blood loss and manage anemia and coagulopathy in X X N/A
nonsurgical patients.
17 Treatment of massive blood loss (massive transfusion) including timely X X N/A
delivery of proper ratios of blood components.
18 Use of perioperative techniques consistent with current AABB X N/A N/A
Standards for PeriopERATIVE Autologous Blood Collection and
Administration.
19 An active program with evidence-based metrics and clinician feedback X N/A N/A
to ensure compliance with transfusion guidelines.
20 A formal program to care for patients who decline the use of X N/A N/A
blood or blood-derived products.
*Holcomb J, ed. Standards for a patient blood management program. Bethesda, MD: AABB, 2014:2-3.
 C h a p t e r 2 5

Transfusion Support for

Hematopoietic Stem Cell
Transplant Recipients

Christopher A. Tormey, MD, and Jeanne E. Hendrickson, MD

A L L O G E N E I C H E MA T O P O I E TIC
STEM
cell transplantation (HSCT) recipients
present a distinct set of challenges for blood
banks and transfusion services. When
consid- ering transfusion for an HSCT
recipient, one has to take into account not
only the complex- ities associated with the
patient’s underlying condition, but also
potential problems associ- ated with
recipient alloantibodies, donor pas- senger
lymphocytes, and different blood group
systems. Over the past two decades, HSCT
has become significantly more com- mon.
Moreover, many patients now receive
posttransplant care in community hospitals.
Thus, issues pertaining to the complexity of
transfusion support for HSCT recipients are
no longer relegated solely to academic
medical centers.
Given the increasing numbers of
patients undergoing HSCT from related and
unrelated donors and the wide variety of
clinical condi- tions that are currently
treated with this ap- proach, transfusion
medicine specialists must be prepared to
address the challenges associ- ated with
transfusion support for these pa- tients.1 This
chapter provides an up-to-date summary of
the most common and important issues
faced by blood bank physicians and other
medical practitioners at transfusion ser-
vices in their treatment of HSCT recipients.

Christopher A. Tormey, MD, Assistant Professor of Laboratory Medicine, Yale University School of
Medicine, New Haven, and Medical Director, Transfusion Service, VA Connecticut Healthcare System, West
Haven, Con- necticut and Jeanne E. Hendrickson, MD, Assistant Professor of Laboratory Medicine, Yale
University School of Medicine, and Associate Medical Director, Transfusion Medicine/Apheresis Service,
Yale-New Haven Hos- pital, New Haven, Connecticut
C. Tormey has disclosed no conflicts of interest. J. Hendrickson has disclosed a financial relationship
with Terumo BCT.
The views expressed in this article are those of the authors and do not necessarily reflect the position or
policy of the Department of Veterans Affairs or the United States government.

631
632 ■ AABB T EC HNIC AL MANUAL

IMPLICATIONS OF ABO- AND tion to blood component selection for

NON-ABO- ANTIGEN-
INCOMPATIBLE RED BLOOD
CELL TRANSPLANTATION FOR
TRANSFUSION
Incompatibilities in ABO blood group
antigens are not necessarily a barrier to
successful HSCT. In solid organ
transplantation, ABO compatibility may be
essential. However, plu- ripotent and early
committed hematopoietic progenitor cells
(HPCs) lack ABO antigens; thus, engraftment
of HPCs is uninhibited even in the presence of
circulating ABO antibodies. Nonetheless,
discrepancies in ABO and non- ABO antigens
between a donor and a recipient do play an
important role in transplantation and can
become a major complicating factor in the
transfusion support of HSCT recipients.
In allogeneic transplantation, the rela-
tionships between the ABO types of the
donor and recipient fall into four categories:
compat- ible, incompatible in the major
crossmatch, incompatible in the minor
crossmatch, and bi- directionally
incompatible.2-4 Table 25-1 lists potential
ABO combinations between donors and
recipients and indicates the associated
compatibility or incompatibility. ABO
incom- patibilities are present in 25% to
50% of donor/ recipient pairs, and,
therefore, careful atten-
transfu- sion is necessary.2,4-6 Although the
terms “major,” “minor,” and “bidirectional
incom- patibility” are most frequently used
in the con- text of the ABO system, they are
also used to describe the presence of other
red cell alloan- tibodies, such as anti-K or
anti-D, in the plas- ma of the donor and/or
recipient.
Major ABO Incompatibility
Major ABO incompatibility creates two chal-
lenges: 1) the potential for acute intravascular
hemolysis when ABO-incompatible donor red
cells within the graft are infused to a recipient
who has high ABO antibody titers and 2) the
ongoing production of ABO antibodies by host
immune cells directed against erythroid pro-
genitors and mature red cells produced by the
engrafting HPCs.
The first challenge is typically
addressed during the collection and/or
processing of HPCs. Techniques, such as
red cell reduction of a marrow graft product,
minimize the risk of hemolysis during
infusion. Some HPC prod- ucts, including
all umbilical cord blood cells, are
cryopreserved before administration, and
incompatible donor red cells may be lysed
during the freeze/thaw process. There is no
general consensus on the threshold level of

TABLE 25-1. Compatibility for HSCT by ABO Blood Group of the Donor and the Recipient

Donor ABO Group

Recipient
ABO Group O A B AB
O Identical Major* Major* Major*
A Minor Identical Bidirectional Major*
(major/minor)‡
B Minor† Bidirectional Identical Major*
(Major/minor)‡
AB Minor† Minor† Minor† Identical
*Due to a naturally occurring antibody (or antibodies) in the recipient (eg, the donor is group A and the recipient is
group O and has naturally occurring anti-A).
†
Due to a naturally occurring antibody (or antibodies) in the donor graft product (eg, the donor is group O and has
naturally occurring anti-A, and the recipient is group A).
‡
Due to naturally occurring antibodies in both the donor and recipient (eg, the donor is group A and the recipient is group B).
 CH APT E R 2 5 HSCT Recipient Transfusion Support
incompatible red cells that may be safely in-
fused, with currently acceptable volumes
ranging from 10 to 20 mL. As suggested by
some, the recipient’s antibody titer may also
be used in guiding facility policy.2 In the ab-
sence of red cell depletion or cryopreserva-
tion, plasmapheresis of the recipient
immedi- ately before graft infusion may be
warranted to reduce the circulating titer of
ABO antibodies.
The second challenge, the continuous
production of antibodies against the A and/or
B antigens of engrafted donor red cells and
erythroid precursors, can continue for up to 3
to 4 months after HPC infusion. As a result,
erythropoiesis is often delayed, with red cell
engraftment potentially occurring >40 days af-
ter transplantation. Time to red cell engraft-
ment may be even further prolonged with the
use of reduced-intensity or nonmyeloablative
conditioning regimens.5,7 In severe cases, pure
red cell aplasia (PRCA) can develop.5 There-
fore, HSCT involving major ABO
incompatibil- ity may render some recipients
transfusion dependent for several months
following trans- plantation. Fortunately, major
incompatibility tends not to affect engraftment
or the produc- tion of other myeloid-lineage
cells.
Minor ABO Incompatibility
Analogous to red cell depletion in major in-
compatibility, plasma depletion of the graft
product can significantly decrease donor
allo- antibodies in minor ABO
incompatibility. However, even if
isoagglutinins are not com- pletely removed,
the ensuing hemolysis is typ- ically mild and
self-limited.4 A more significant problem
with minor-incompatible HSCT re- sults
from the rapid generation of anti-A and/ or -
B by donor lymphocytes against residual
recipient red cells. This so-called “passenger
lymphocyte syndrome” occurs
approximately 5 to 16 days after infusion of
HPCs. The patient experiences acute,
immune-mediated hemo- lysis that can
result in morbidity and even mortality.
Fortunately, in most cases, the he- molysis is
not severe and eventually subsides with the
clearance of recipient red cells.4 If the
hemolysis is severe and potentially life
threat- ening, therapeutic
erythrocytapheresis can be
■ 633
used to exchange incompatible recipient red
cells with donor-compatible red cells. In pa-
tients believed to be at particularly high risk
of the passenger lymphocyte syndrome
(usually based on the type of posttransplant
immuno- suppression), prophylactic
erythrocytaphere- sis may be warranted
before transplantation.8
Bidirectional ABO Incompatibility
In bidirectional ABO incompatibility, compli-
cations arising from both major and minor
ABO-incompatible HSCT can occur in the re-
cipient. To prevent these complications, the
processing of HPC grafts might include the re-
duction of both red cell and plasma content.
The posttransplant period can be complicated
by the onset of hemolysis within days or
weeks (as occurs in minor incompatibility),
delayed engraftment of red cells, and/or, in
some cas- es, PRCA (as occurs in major
incompatibility).
Incompatibility Related to Non-
ABO Antigens
Red cell antigens of other blood group
systems can present challenges similar to the
ones de- scribed for ABO-incompatible
transplants. Overall, these incompatibilities
are generally less frequently encountered,
but the presence of red cell antibodies in the
patient (more common) and/or donor (less
common) re- quires attention and
approaches that are simi- lar to those
outlined above for ABO incompati- bility.
Special consideration of graft donor
selection may also be needed when reduced-
intensity or nonmyeloablative conditioning
regimens are used in alloimmunized recipi-
ents, and cognate graft donor antigen/recipi-
ent alloantibody pairs should be avoided if
at all possible.
BLOOD COMPONENT
CONSIDERATIONS
Selection of Blood Components
The selection of appropriate blood compo-
nents for recipients of allogeneic HSCT is
particularly problematic. When determining
634 ■ AABB T EC HNIC AL MANUAL
transfusion requirements for an allogeneic
HSCT recipient, it is vital that the blood
bank or transfusion service keep detailed
records of the patient’s pretransplant ABO
group and ABO antibody titers and the
donor’s ABO group. It is also imperative to
determine the stage of the transplant (ie,
preparative period, immediate posttransplant
period, or posten- graftment period with
absence of recipient red cells and/or
antibodies).9 Recommendations for optimal
component selection at each point in the
transplant process are shown in Table 25-2.
With regard to Rh(D), the recipient can
continue to receive the type of components
transfused before the transplantation as long
as the HSCT donor and recipient match.
How- ever, if the recipient is Rh negative
and the do- nor is Rh positive or vice versa,
only Rh-nega- tive Red Blood Cell (RBC)
units should be transfused to prevent
alloimmunization to the highly
immunogenic D-antigen. Given the small
risk of D alloimmunization from red cells
contained in Rh-positive platelet units, Rh-
negative platelets may be preferred if the
blood supply allows this. However, it has re-
cently been suggested that this practice be
re- evaluated.10
RBC Support
The majority of HSCT recipients require trans-
fusion support in the peritransplant period,
regardless of incompatibilities or blood group
antigen mismatches. Symptomatic anemia is
one of the most common indications for RBC
transfusion in HSCT recipients. In the absence
of symptomatic anemia, a patient’s status and
underlying comorbid conditions can be used
to determine whether RBC transfusion may be
warranted. Because specific RBC transfusion
thresholds in HSCT populations are lacking,
clinicians can rely on general RBC transfusion
guidelines. For instance, a threshold hemoglo-
bin level of 7 g/dL is likely appropriate for
most stable, nonpostoperative adult HSCT re-
cipients. However, a slightly higher threshold
of 8 g/dL is likely appropriate for adults with
preexisting heart disease, those at risk of end-
stage organ damage, or HSCT recipients in
the postoperative stage.11,12
The mechanisms underlying anemia and
RBC transfusion dependence in HSCT are
suf- ficiently exceptional to warrant brief
further discussion. After intensive
chemotherapy, se- rum erythropoietin (EPO)
levels increase rap- idly and peak in the first
week after treat- ment.13-15 Although
recipients of autologous HSCT maintain
adequate EPO levels through- out the
posttransplantation period, allogeneic
recipients do not.16-20 Allogeneic HSCT
recipi- ents experience a prolonged period of
inap- propriately low endogenous EPO
levels, which often necessitate prolonged
RBC transfusion support. Interestingly,
mixed results have been obtained by the
various researchers who have examined
recombinant EPO dosing after allo- geneic
HSCT.13-20 Larger studies are necessary to
determine whether EPO is capable of reduc-
ing the RBC transfusion burden and prevent-
ing the complications of HSCT, such as
PRCA. It is noteworthy that the US Food
and Drug Administration has recently
strengthened its warnings about EPO use
after some studies showed that EPO
treatment had adverse ef- fects (decreased
survival and/or tumor pro- gression) in some
patients with cancer.16 Al- though no studies
have specifically linked EPO use to poor
outcomes in HSCT patients, clini- cians
should be mindful of the potential risks of
EPO usage.
Platelet Support
Platelet recovery after allogeneic HSCT has
been studied extensively. Several factors are
strongly associated with the rate at which
pa- tients become platelet transfusion
indepen- dent, including: 1) the relationship
between the donor and the recipient (ie,
whether they are related vs unrelated), 2)
conditioning regi- men used, 3) presence of
graft-vs-host disease (GVHD) or
cytomegalovirus (CMV) infection, and 4)
HPC CD34+ cellular source/dose.17-23 To
summarize broadly, the findings of several
studies suggest that slower platelet engraft-
ment tends to be more common in patients
TABLE 25-2. Transfusion Support for Patients Undergoing HSCT by Type of ABO Incompatibility and Stage of
Transplant
ABO Blood Group Selection

Type of Incompatibility Transplant Stage RBCs Platelets* Plasma

Major incompatibility Preparative regimen Recipient Donor Donor
Transplantation Recipient Donor Donor

Recipient antibodies detected Recipient Donor Donor

Recipient antibodies no longer Donor Donor Donor

detected
CH APT E R 25

Minor incompatibility Preparative regimen Donor Recipient Recipient

Transplantation Donor Recipient Recipient
Recipient cells circulating
Donor Recipient Recipient
Recipient cells no longer circulating Donor Donor Donor

Bidirectional incompatibility Preparative regimen Group Group Group

Transplantation O Group AB AB Group
Recipient antibodies detected/recipient
O Group AB
cells circulating
HSCT Recipient Transfusion Support

AB
Recipient antibodies no longer detected/
Donor Donor Donor
recipient cells no longer circulating

*Due to the short shelf life and limited availability of group AB platelets, it may not always be possible to provide fully matched ABO platelet products as recommended in the
■

table. Therefore, blood banks and transfusion services may consider providing ABO-mismatched platelets that have been volume reduced to diminish their plasma
content.
635
636 ■ AABB T EC HNIC AL MANUAL
receiving grafts from unrelated donors, who
have higher-grade GVHD, or who have pre-
transfusion viral infections.17-20 There is also
increasing evidence to suggest that the
source of an allogeneic transplant—such as
cord blood HPCs [HPCs(C)] vs HPCs from
apheresis [HPC(A)]—is predictive of time to
platelet en- graftment. Several studies have
shown that the median time to platelet
engraftment for HPC(C) transplants is, on
average, significant- ly longer than for
recipients of HPC(A) or HPCs from
marrow.20-23 Thus, longer-term depen- dence
on platelet transfusion is more likely for
individuals in any of the above categories.
One major consideration for platelet
transfusion in HSCT recipients is
compatibili- ty. Plasma (including the
significant volume of plasma in platelets)
contains variable amounts of isoagglutinins.
Although transfusion of lim- ited quantities
of ABO-incompatible plasma and platelets
is common in routine transfu- sion, this
practice cannot be readily applied to the
recipients of allogeneic HSCT without
careful consideration.24 In ABO-
incompatible HSCT, the plasma-containing
components should be compatible with both
the donor and the recipient whenever
possible. Recommen- dations for component
selection are more ful- ly described in Table
25-2.
In addition to the risk of hemolysis, some
researchers have found other morbidities as-
sociated with ABO-incompatible platelet
transfusions. For instance, one pediatric study
showed that such transfusions, when com-
bined with the use of melphalan, correlated
with the development of hepatic venoocclu-
sive disease, possibly resulting from the bind-
ing of A- and/or B-antigens expressed on the
surface of hepatic endothelial cells.25 There-
fore, some centers have an institutional policy
requiring the transfusion of ABO-identical
RBCs and platelets only to HSCT recipients;
at least one group has noted that this policy
may be associated with improved patient
survival.26 There is an obvious need for
additional pro- spective studies to address this
issue.
In the context of platelet transfusion
support in HSCT recipients, the following
ad- ditional areas are discussed more
extensively below: 1) prophylactic vs
therapeutic transfu-
sions, 2) the transfusion “threshold,” 3) the ap-
propriate transfusion dose, and 4) HLA or
platelet antibodies.
Prophylactic vs Therapeutic
Platelet Transfusions
Platelet transfusions are frequently adminis-
tered for prophylaxis of bleeding, rather
than to treat acute hemorrhage, in HSCT
recipients. Although this has been a
traditional manage- ment approach for many
years, several recent studies have questioned
the utility and clinical benefit of this
practice. Two studies, both pub- lished by
Wandt and colleagues,27,28 examined the
assumption that prophylactic platelet
transfusion could be replaced by therapeutic
transfusion in recipients of autologous trans-
plants. The researchers found that although
patients assigned to a therapeutic (rather
than prophylactic) transfusion regimen had
some bleeding, there was no evidence of
life-threat- ening hemorrhage. Moreover,
therapeutic in- terventions resulted in
dramatic reductions in platelet usage.
However, Stanworth and colleagues29
concluded, based on a recently published
trial, that prophylactic platelet transfusions
were more effective in preventing
hemorrhage in patients with hematologic
ma- lignancies than in a nontransfused
control population. Therefore, questions still
remain regarding the nature of appropriate,
evidence- based prophylactic transfusion
strategies for patients with
thrombocytopenia. The results of the studies
conducted to date and future tri- als will
help shape prophylactic platelet trans-
fusion practice for HSCT recipients.30
Platelet Transfusion Threshold
The acceptable threshold for prophylactic
transfusion of platelets has been studied ex-
tensively for >20 years. One of the earliest es-
tablished thresholds to prevent spontaneous
hemorrhage was a platelet count of
<20,000/µL for patients undergoing
chemotherapy/HSCT.31 Over time and as
clinical trial data have ac- crued, studies have
consistently revealed that a platelet count
<10,000/µL in uncomplicated
thrombocytopenia (ie, in patients without co-
 CH APT E R 2 5 HSCT Recipient Transfusion Support
existing conditions such as fever, bleeding,
or bacteremia/sepsis) is a reasonable
threshold to prevent increased bleeding or
hemorrhage- related morbidity. 30,32-35
However, a study by Nevo and colleagues36
raises questions about this threshold. In this
study, HSCT recipients who received
transfusions at a platelet count
<10,000/µL had significantly increased non-
hemorrhagic mortality rates and reduced sur-
vival compared to those infused at counts
<20,000/µL. Although changes in the 10,000/
µL threshold should not be based on this
study alone, it is imperative that data
continue to be collected to examine the
appropriateness of this target platelet count
and, as noted previ- ously, whether
prophylactic platelet transfu- sions are
necessary at any platelet count.30
Platelet Dose
Another question that frequently arises with
platelet transfusion is what the optimal
plate- let dose is per transfusion episode. To
date, three large-scale clinical trials have
examined the question of platelet transfusion
dosing.37-39 A meta-analysis of aggregate data
from these three studies showed no
increased risk of sig- nificant bleeding
between low and standard platelet doses.30 It
is important to note that, as best illustrated in
the PLADO trial, selecting platelet doses for
patients based on body sur- face area may
be a critical factor in the provi- sion of
lower platelet doses. Drawbacks to a lower
dosing regimen may include a lower platelet
increment after infusion and the po- tential
for a greater number of platelet transfu-
sions over time.30,39 Clearly, as more data are
collected, lower platelet transfusion doses
may become accepted as routine clinical
practice for HSCT recipients.
HSCT Recipients with HLA or
HPA Antibodies
Some patients scheduled to undergo HSCT
possess antibodies against HLA and/or
human platelet antigen (HPA); both of these
types of antibodies may reduce the efficacy
of platelet transfusion, resulting in lower
corrected count increments.40 For more
thorough discussions
■ 637
of the evaluation and treatment of platelet re-
fractoriness driven by HLA and HPA antibod-
ies, see Chapters 18 and 19.
In addition to leading to potential prob-
lems with platelet transfusion, recipient allo-
antibodies against HPA or HLA could delay
platelet or white cell engraftment in some
transplant settings. This is analogous to what
occurs in the red cell lineage with major ABO-
mismatched transplants. The issue of HPA and
HLA matching in HSCT donor-recipient pairs
has also been raised by several groups because
of concerns that mismatches between donors/
recipients for HPA (which may serve as minor
histocompatibility antigens) may increase the
risk of GVHD, although two studies have
found otherwise.41,42
Plasma, Cryoprecipitated AHF,
and Factor Concentrate Support
There are no specific recommendations re-
garding the transfusion of plasma,
cryoprecip- itated antihemophilic factor, or
factor concen- trates in HSCT patients;
therefore, adherence to general guidelines
and/or expert recom- mendations for the
transfusion of these com- ponents is
advised.43 As discussed above and outlined
in Table 25-2, the most important is- sue for
plasma transfusion is the ABO group of the
recipient and engrafting cells.
For HSCT complicated by GVHD, there
has been interest in determining whether re-
combinant factor concentrates could be used
to treat bleeding complications. Dysregula-
tion of hemostasis is a well-known complica-
tion of GVHD. The utility of recombinant acti-
vated Factor VII (NovoSeven, Novo Nordisk,
Zurich, Switzerland), a potent procoagulant,
has been investigated. A multicenter, random-
ized controlled trial found no differences in
bleeding score for any of three doses (40, 80,
or 160 µg/kg) of Factor VIIa compared to
control treatment for hemorrhage associated
with GVHD in the setting of HSCT.44 The
results of this trial suggest that recombinant
Factor VIIa is likely to be ineffective as a first-
line therapy for GVHD-associated bleeding.44
However, Factor VIIa may still be useful as a
last-resort
638 ■ AABB T EC HNIC AL MANUAL
treatment for patients with intractable bleed-
ing.
Another possible hemostasis therapy
aris- es from the fact that GVHD often
results in the depletion of Factor XIII,
increasing the risk and severity of
gastrointestinal hemorrhage.45 The high
concentration of Factor XIII in cryopre-
cipitate makes cryoprecipitate a potentially
useful therapy for GVHD-related bleeding.
Formal studies are required to determine the
efficacy of this approach.
Transfusion Support for Autologous
Transplant Recipients
Because recipients of autologous transplants
are not exposed to foreign graft products
from donors, virtually all of the concerns
regarding major/minor incompatibility (and
their im- pact on transfusion support) are not
applica- ble to this patient group. Before,
during, and after HSCT, autologous
recipients should be supported by
transfusions of blood compo- nents as
needed and in compliance with stan- dard
protocols that are applicable to any trans-
fusion recipient. However, because of their
underlying immunosuppression, autologous
recipients may require specialized products
or components involving additional
processing steps. Such issues and concerns
(applicable to both autologous and
allogeneic transplant re- cipients) are
discussed in greater detail below.
PATIENTS WITH NEUTROPENIA
AND INFECTION THAT IS
UNRESPONSIVE TO
ANTIMICROBIAL THERAPY
Traditionally, infusions of fresh granulocytes
have been used to treat severe, antibiotic-
refractory bacterial or fungal infections in
pa- tients with absolute neutrophil counts
less than 500/µL.46,47 (For a more in-depth
discus- sion of granulocyte therapy, see
Chapter 18.) To date, one study has
demonstrated that neu- trophil transfusions
can be efficacious in treat- ing infection in
HSCT recipients with neutro- penia;
however, this Phase I/II study included only
11 participants.48 To more fully establish
the clinical efficacy of transfused granulocytes
in the setting of HSCT, a multicenter random-
ized controlled trial is currently under way. 49
The results of this study will be helpful in un-
derstanding the potential benefit of granulo-
cyte infusion for severely ill HSCT recipients.
SPECIAL PROCESSING OF
BLOOD COMPONENTS FOR
HSCT RECIPIENTS
The irradiation of cellular blood products
(eg, RBCs, platelets, and granulocytes) is
intended to inhibit the proliferation of donor
lympho- cytes, thereby preventing
transfusion-associ- ated GVHD, a reaction
that is almost uniformly fatal.9,50 Although it
is generally accepted that HSCT recipients
require irradiated compo- nents during, and
for at least 1 year after, transplantation, it is
unclear whether these pa- tients require
irradiated components after this time.
Despite the absence of evidence indicat- ing
that irradiation is essential after this time has
elapsed, many institutions provide irradi-
ated products indefinitely to HSCT
recipients. This is likely a prudent policy
given the poten- tial for lifelong
immunosuppression associat- ed with HSCT
and the possibility of relapse of the
malignancy for which the patient initially
underwent the HSCT.
Blood banks and transfusion services
must rigorously ensure irradiation of
compo- nents transfused to HSCT
recipients. Part of this vigilance is the
development of systems or policies to
identify patients who require irradi- ated
products. One approach to help reduce the
possibility of inadvertently releasing a
nonirradiated unit for transfusion to an
HSCT recipient is to require universal
irradiation of selected blood components. In
some institu- tions, all platelet products are
irradiated upon receipt from the regional
blood center. Al- though this strategy may
not be practical for other components, such
as RBCs, additional approaches may be
developed, such as irradi- ation of all
components issued to in- and out- patient
hematology/oncology wards.
 CH APT E R 2 5 HSCT Recipient Transfusion Support
The situation is equally complex with
re- gard to CMV, a pathogen linked to high
mor- bidity and mortality rates in HSCT
recipients. It has been proposed that
transfusions of com- ponents that were
leukocyte reduced before storage are as
efficacious in the prevention of CMV spread
as components collected from donors lacking
antibodies to CMV (CMV-sero- negative
donors).9 However, a meta-analysis of 829
recipients of CMV-seronegative compo-
nents and 878 recipients of leukocyte-
reduced components revealed that the risks
of CMV in- fection were 1.63% and 3.01%,
respectively, following transfusion.51
Because CMV is also acquired in the
community, conducting stud- ies addressing
this issue definitively remains logistically
difficult.
The major shortcomings of CMV-sero-
negative products include their limited
avail- ability and the fact that seronegative
donors may have recently acquired CMV
with viremia that is not detected by
serologic testing. In the absence of a large
supply of CMV-seronega- tive products or
antigen testing, leukocyte- reduced units can
reasonably be considered to be “CMV
reduced risk.” To better triage re- quests for
CMV-seronegative components, the CMV
status of recipients and donors should also
be considered. Some hospitals provide
CMV-seronegative units to HSCT recipients
who are CMV seronegative and received a
transplant from a CMV-seronegative donor,
al- though leukocyte-reduced components
may be equally efficacious.52
Special processing of transfusion units
may also be needed for HSCT recipients
who repeatedly experience common
transfusion reactions.50 For instance, HSCT
recipients may develop allergic symptoms,
such as pruritis, urticaria, or wheezing; the
severity of such symptoms may increase
over time. One ap- proach to prevent
repeated allergic reactions is to wash
components to remove supernatant plasma
using automated methods for RBCs or by
centrifugation and saline resuspension for
platelets. These modifications, in
conjunction with timely premedication
regimens, can help reduce the risk or
severity of recurrent allergic or febrile
transfusion reactions.
■ 639
SPECIAL CONSIDERATIONS
FOR TRANSFUSIONS IN
PEDIATRIC HSCT RECIPIENTS
In general, the considerations regarding
trans- fusion support of pediatric HSCT
recipients are similar to those for adults.53
However, indi- cations for transplant, stem
cell source, and donor selection may be
subtly different in these patient populations.
For example, chil- dren with inherited
diseases (such as sickle cell disease or
thalassemia major) may be more likely to be
treated with HSCT than adults. Fur-
thermore, high-dose chemotherapy with au-
tologous stem cell rescue is more often
utilized to treat some childhood
malignancies (includ- ing advanced stage
neuroblastoma) than ma- lignancies in
adults. In addition, cord blood may be
utilized for transfusions to treat some
diseases in pediatric recipients. For example,
HPC(C) transfusion often provides a
sufficient dose for a child, but not for an
adult, because of a child’s small size.
Special considerations are necessary for
transplantation in children (or adults) with
sickle cell disease during both the pre- and
peritransplant periods. Red cell phenotyping
of the stem cell donor is recommended to
pre- vent alloimmunizaton in the recipient
against the donated cells by guiding both
donor selec- tion and product processing. If
possible, stem cell donors who are negative
for cognate red cell antigens against which
recipients have red cell alloantibodies should
be considered.54 This is particularly
important for reduced- intensity or
nonmyeloablative conditioning regimens,
which can induce long-term mixed
chimerism.54 If antigen-positive donors are
utilized, then red cell reduction of the stem
cell product is recommended (even in the
absence of ABO incompatibility) to avoid a
transfusion reaction during stem cell
infusion.
Because of the association between
RBC components and HLA/HPA
alloimmunization, consideration should be
given to pretrans- plant HLA/HPA antibody
screening.55-57 The results of this screening
should be taken into consideration in
planning posttransplant platelet transfusions
to comply with the gener- al
recommendations to keep platelet counts
640 ■ AABB T EC HNIC AL MANUAL
above 50,000/µL (to prevent
cerebrovascular bleeding).58,59 Such results
may also be impor- tant in HPC donor
selection for non-HLA- matched,
nonmyeloablative transplants. An additional
consideration is to perform red cell
exchange transfusion before transplantation
with a goal of <30% to 45% hemoglobin S
to prevent vasoocclusive events resulting
from granulocyte colony-stimulating factor
admin- istration.60
Beta-thalassemia major and severe
aplas- tic anemia may be cured by matched
sibling HSCT in childhood, yet prior
transfusion ex- posure may adversely
influence engraftment and outcomes in
subsequent transfusions.61-63 Potential reasons
for this association include humoral or
cellular immunization to trans- fused
antigens (whether they are defined or other
minor histocompatibility antigens) or iron
overload. Early transplantation for eligible
patients may decrease the risk of graft rejec-
tion, and careful attention to iron status at
the time of transplantation is also
recommended. The intensity of conditioning
regimens is also likely a key factor in
outcomes of HSCT in pa- tients with beta-
thalassemia major or severe aplastic anemia,
and their long-term risk/ben- efit ratios
deserve careful consideration.
INFORMATION PORTABILITY
FOR HSCT RECIPIENTS
Many patients undergo HSCT far from their
usual medical institution. Before
transplanta- tion procedures, all of the
patient’s transfusion history (including red
cell, platelet, or leuko- cyte alloantibody
testing history) should be communicated to
the transplanting facility. Eventually, these
patients may return for fol- low-up care to
their primary care physicians,
KEY POINTS
Allogeneic HSCT recipients face distinct transfusion challenges because of their immuno- suppression, preexisting diseases, a
ABO compatibility is not critical in the selection of potential HSCT donors because pluripo- tent and early-committed HPCs l
and supporting such patients can be a
signifi- cant challenge for the transfusion
service.
The flow of information from the trans-
plant center to local providers is rarely
perfect; with increased complexity of care,
critical data can be overlooked. Some
institutions have cre- ated a process to deal
with such situations. For example, the
nationally networked Depart- ment of
Veterans Affairs medical center data- base
can be consulted to detect any ABO or Rh
discrepancies and determine whether a
partic- ular patient has ever had an
alloantibody de- tected at another network
hospital. If a trans- fusion service or blood
bank does have an interconnected hospital
laboratory informa- tion system, such
databases can be useful for gathering
information on transplant recipi- ents.
Regardless of the availability of an inter-
connected information system, the transfu-
sion service should consider developing a pol-
icy with its hematology-oncology colleagues
that clearly outlines the approach to patients
who receive their HSCT at an outside facility.
This policy should call for communicating the
following important information to a patient’s
usual health-care facility after that patient re-
turns from an off-site transplant facility: 1)
whether the transplant was autologous or allo-
geneic; and, 2) if the transplant was allogeneic,
the ABO type of the donor, and any auto- or
al- loantibodies developed by the patient
during care at that facility. In some cases, if
such in- formation is not completely
communicated, the transfusion service can
contact the trans- plant center and obtain the
required informa- tion at the time of the
patient’s first posttrans- plant visit. A portable
worksheet with these data should also be made
available to patients.
 CH APT E R 2 5 HSCT Recipient Transfusion Support ■ 641

3. ABO incompatibilities, which are present in 20% to 40% of donor/recipient pairs, fall into
three categories: 1) incompatible in the major crossmatch, 2) incompatible in the minor
crossmatch, and 3) bidirectionally incompatible. Such incompatibilities have the potential
to produce acute and, in the case of major incompatibility, ongoing intravascular hemolysis
and pure red cell aplasia. They can be partially mitigated by red cell and/or plasma deple-
tion of the graft before infusion.
4. Management approaches to transfusion are similar for adult and pediatric HSCT
recipients; however, indications for transplantation, stem cell source, and donor
selection may be sub- tly different.
5. When transfusion requirements for allogeneic HSCT recipients are determined, it is vital
that the blood bank or transfusion service keep detailed records of the recipient’s
pretrans- plant ABO group and the donor’s ABO group. It is also imperative to
determine the stage of the transplant.
6. Platelet concentrates are most frequently transfused to HSCT recipients to prevent an
acute hemorrhage. A platelet count threshold of 10,000/µL in uncomplicated
thrombocytopenia is widely accepted as safe for allogeneic recipients.
7. Cellular blood components are irradiated to inhibit the proliferation of donor lymphocytes,
thereby preventing transfusion-associated GVHD. Many services provide irradiated compo-
nents indefinitely to HSCT recipients.
8. RBC and platelet components that have been leukocyte reduced before storage are general-
ly considered to be equivalent to CMV-seronegative units in terms of CMV transmission
risk. Some studies, however, suggest that seronegative units may be marginally safer.

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 C h a p t e r 2 6
Therapeutic Apheresis
Robertson D. Davenport, MD
T H E R A P E U T I C A P H E R E SI S IS the
treatment of diseases through removal or
extracorporeal manipulation of blood com-
ponents or specific blood substances. It is dis-
tinct from blood component collection by
apheresis, which is covered in Chapter 7. Stan-
dards and guidelines for therapeutic aphere-
sis, clinical privileging, and documentation are
provided by AABB and The American Society
for Apheresis (ASFA).1-4
PRINCIPLES AND MODALITIES
The goal of therapeutic apheresis is to
remove a pathologic element from blood or
to modu- late cellular function by
manipulation such as through extracorporeal
photopheresis (Table 26-1), with or without
replacement of the re- moved element.
Apheresis can be performed manually, but
automated techniques are faster and more
efficient. In therapeutic plasma ex- change
(TPE), 1.0 or 1.5 plasma volumes are
typically processed. Larger volume
procedures can increase the risk of
coagulopathy, citrate toxicity, or electrolyte
imbalance, depending on the replacement
fluid. The effectiveness of apheresis in
removing pathologic substances depends on
the concentration of those sub-
Robertson D. Davenport, MD, Medical Director, Blood Bank and Transfusion Service, and Associate
Professor, University of Michigan Health System, Ann Arbor, Michigan
The author has disclosed no conflicts of interest.
stances in the blood, the volume of blood
pro- cessed, and the equilibrium between
blood and the substance’s extravascular
volume of distribution. The procedure is
most efficient at the beginning because the
amount of the com- ponent removed
decreases exponentially with time (Fig 26-
1). Continued production or mo- bilization
from the extravascular space will re- sult in
a less-than-expected reduction. A 1.0 plasma
volume exchange typically removes
approximately two-thirds of a substance if it
does not move significantly from the
extravas- cular to the intravascular space.
Continuous flow centrifuge apheresis
de- vices have a rotating channel designed
to in- troduce whole blood at one site, and
the blood elements subsequently separate by
density as blood flows through the channel.
The resulting layers of plasma, platelets,
leukocytes, or red cells can be removed
selectively. The compo- nent to be removed
is diverted into a collec- tion bag, while the
remaining blood compo- nents are mixed
with the replacement fluid and returned to
the patient. The device con- trols the flow
rates of blood withdrawal, anti- coagulant
solution and replacement fluid in- fusion, as
well as centrifuge speed to achieve optimal
separation.
645
646 ■ AABB TECHNICAL MANUAL

TABLE 26-1. Apheresis Modalities

Blood Component Replacement

Procedure Removed Typical Indication Fluid
Therapeutic plasma Plasma Reduction of an Albumin or plasma
exchange abnor- mal plasma
protein, eg,
autoantibody
Red cell exchange Red cells Sickle cell disease-related Red cells
complications
Leukapheresis Buffy coat Leukemia with As needed
leukostasis
Thrombocytapheresis Platelet-rich plasma Thrombocytosis As needed
Erythrocytapheresis Red cells Erythrocytosis None
Extracorporeal Buffy coat (reinfused) Chronic graft-vs-host None
photopheresis disease
Selective adsorption Specific plasma protein Hypercholesterolemia None
Rheopheresis High-molecular-weight Age-related macular None
plasma proteins degeneration

FIGURE 26-1. Theoretical removal of a substance by apheresis. For a substance that is strictly
intravascular, 36.8% of the initial concentration remains after a single blood volume exchange. However,
if the substance is also present in the extravascular space with a total volume of distribution equal to
twice the blood volume, 60.6% will remain after a single blood volume has been processed.
 CHA P TER 2 6
Intermittent centrifugation devices use
an alternative method in which a specified
vol- ume of whole blood is first withdrawn
into a centrifuge bowl. Blood withdrawal is
then stopped, and the extracorporeal blood
prod- uct is processed. The rest of the
procedure is similar to the continuous flow
process in that the blood is centrifuged, the
selected compo- nent is diverted into a
waste bag, and the re- mainder of the blood
is returned to the patient with appropriate
replacement fluid. The pro- cess can be
repeated for several cycles. Inter- mittent
centrifugation is most commonly used for
extracorporeal photopheresis (ECP), al-
though a continuous flow ECP device has
been introduced.5
Filtration devices also operate by contin-
uous flow. Anticoagulated whole blood is
passed through a microporous filter that al-
lows plasma to pass through but retains the
blood cells. The separated plasma can then be
diverted into a waste bag, or, as in the case of
selective adsorption, further processed and re-
turned to the patient. This type of device is not
suitable for cytapheresis. A variant of this
tech- nique is rheopheresis, or double-filtration
TPE, in which high-molecular-weight
molecules— primarily fibrinogen, low-density
lipoprotein (LDL), fibronectin, and von
Willebrand factor (vWF)—are removed,
reducing plasma viscosity.
In selective adsorption, blood or plasma
is passed over a column that has a high
affinity for a specific component, such as
immune globulin G (IgG) or LDL, and the
effluent is re- turned to the patient. This has
the advantage of highly specific removal of
the element of in- terest. However, it is
restricted in the United States to only a few
conditions for which affini- ty adsorbers are
available. Such techniques are more widely
employed outside of the United States.
INDICATIONS
Although there are many case reports of suc-
cessful treatment of a variety of diseases and
conditions by apheresis, there have been few
high-quality clinical trials. ASFA has
published
Therapeutic Apheresis ■ 647
clinical guidelines categorizing the indications
for apheresis in 78 disease states.2
1. Category I: Disorders for which apheresis
is accepted as first-line therapy, either as
a primary stand-alone treatment or in
con- junction with other modes of
treatment.
2. Category II: Disorders for which
apheresis is accepted as second-line
therapy, either as a stand-alone treatment
or in conjunction with other modes of
treatment.
3. Category III: Disorders in which the opti-
mal role of apheresis therapy is not estab-
lished. Decision making for patients should
be individualized.
4. Category IV: Disorders in which
published evidence demonstrates or
suggests that apheresis is ineffective or
harmful. Institu- tional review board
approval is desirable if apheresis
treatment is undertaken in these
circumstances.
TPE
The goal of TPE is to remove a pathogenic
mol- ecule, protein, or high-molecular-weight
com- plex from plasma. In addition, TPE may
be used to provide a deficient normal
substance, such as an enzyme or coagulation
factor. Indi- cations for TPE with the
recommended treat- ment course are listed in
Table 26-2. The fre- quency and duration of
treatment are guided by clinical judgment,
although laboratory test- ing may be helpful
for some indications.
Replacement fluids for TPE are
compared in Table 26-3. For most
indications, albumin is the preferred
replacement fluid, because it is isosmotic
with blood and has a smaller risk of adverse
reactions and infectious disease trans-
mission than plasma. Plasma is indicated for
thrombotic thrombocytopenic purpura
(TTP) or if coagulopathy is a concern. Red
Blood Cells (RBCs) may also be used to
prime the apheresis circuit when treating
small patients (ie, <10-20 kg) in whom the
extracorporeal vol- ume may exceed 10% to
15% of the patient’s blood volume.
Diseases in which a pathogenic
autoanti- body is targeted include acute and
chronic inflammatory demyelinating
polyradiculo-
648 ■ AABB T EC HNIC AL MANUAL

TABLE 26-2. Indications for Therapeutic Plasma Exchange

Typical Course (Number of

Categorytreatments)
Indication Modifying Conditions
Acute disseminated encephalomyelitis II QOD (3-6)
Acute inflammatory demyelinating I QOD (5-6)
polyneuropathy (Guillain-Barré After IVIG III
syndrome)
Acute liver failure III Daily (variable)
Amyloidosis, systemic IV
Amyotrophic lateral sclerosis IV
ANCA-associated rapidly progressive Dialysis dependence I Daily or QOD
glomerulonephritis (Granulomatosis DAH I (6-9)
with polyangiitis; Wegener granuloma-
tosis) Dialysis independence III

Antiglomerular basement membrane Dialysis dependent and no DAH III Daily or QOD
disease (Goodpasture syndrome) DAH (7-10)
I
Dialysis independence I
Aplastic anemia; pure red cell aplasia Aplastic anemia III Daily or QOD
(variable)
Pure red cell aplasia III
Autoimmunic hemolytic anemia: Severe WAIHA III Daily or QOD
WAIHA; cold agglutinin disease Severe cold agglutinin disease II (variable)

Burn shock resuscitation III Once

Cardiac transplantation Desensitization, positive cross- III Daily or QOD
match due to donor specific (variable)
HLA
antibody
Antibody-mediated rejection III
Catastrophic antiphospholipid II Daily (3-5)
syndrome
Chronic focal encephalitis (Rasmussen III QOD (3-6)
encephalitis)
Chronic inflammatory demyelinating I 2-3/week
polyradiculoneuropathy (variable)
Coagulation factor inhibitors Alloantibody IV
Autoantibody III Daily (variable)
Cryoglobulinemia Symptomatic/severe I QOD (3-8)
Dermatomyositis or polymyositis IV
Dilated cardiomyopathy, idiopathic NYHA II-IV III QOD (5)
 CHA P TER 2 6 Therapeutic Apheresis ■ 649

TABLE 26-2. Indications for Therapeutic Plasma Exchange (Continued)

Typical Course Number of

Categorytreatments)
Indication Modifying Conditions

Familial hypercholesterolemia Homozygotes with small II Weekly

blood (indefinite)
volume
Focal segmental glomerulosclerosis Recurrent in transplanted kidney I Daily or QOD
(variable)
HSCT, ABO incompatible Major HPC, Marrow II Daily (1-
3)
Major HPC, Apheresis II
Hemolytic uremic syndrome, atypical Complement gene mutations II Daily (variable)
Factor H antibodies I
MCP mutations IV
Hemolytic uremic
Shiga toxin associated IV Daily (variable)
syndrome, infection-
associated S. pneumonae associated III
Henoch-Schonlein purpura Crescentric III QOD (4-11)
Severe extrarenal disease III
Heparin-induced thrombocytopenia Precardiopulmonary bypass III Daily or QOD
Thrombosis III (variable)
Hypertriglyceridemic pancreatitis III Daily (1-3)
Hyperviscosity in monoclonal
Symptomatic I Daily (1-3)
gammopathies
Prophylaxis for rituximab I
Immune complex rapidly
III QOD (3-6)
progressive glomerulonephritis
Immune thrombocytopenia Refractory IV
Immunoglobin A nephropathy Crescentic III QOD (6-9)
Chronic progressive III
Inclusion body myositis IV
Lambert-Eaton myasthenic syndrome II Daily or
QOD
(variable)
Liver transplantation, ABO Desensitization, live donor I Daily or QOD
incompatible (variable)
Desensitization, deceased donor III
Antibody-mediated rejection III
Lung allograft rejection Antibody-mediated rejection III Daily or QOD
(variable)
(Continued)
650 ■ AABB T EC HNIC AL MANUAL

TABLE 26-2. Indications for Therapeutic Plasma Exchange (Continued)

Typical Course (Number of

Categorytreatments)
Indication Modifying Conditions

Multiple sclerosis Acute CNS inflammatory II QOD (5-7)

demye- linating disease
Chronic progressive III Weekly (variable)

Myasthenia gravis Moderate-severe I QOD (3-

7) Before thymectomy I
Myeloma cast nephropathy II Daily or QOD (5-7)
Nephrogenic systemic fibrosis III Daily or QOD (10-14)
Neuromyelitis optica (Devic syndrome) Acute II Daily or
QOD
(variable)
Maintenance III Weekly (variable)
Overdose, envenomation, and
Mushroom poisoning II Daily (variable)
poisoning
Envenomation III
Natalizumab and PML III
Paraneoplastic neurologic syndromes III QOD (5-6)
Paraproteinemic demyelinating
IgG/IgA I QOD (5-6)
polyneuropathies
IgM I
Multiple myeloma III
PANDAS; Sydenham chorea PANDAS exacerbation I QOD (5-6)
Sydenham chorea I
Pemphigus vulgaris Severe III Daily or
QOD
(variable)
Phytanic acid storage disease
II Daily or
(Refsum disease)
QOD
(variable)
POEMS syndrome IV
Posttransfusion purpura III Daily (variable)
Psoriasis IV
Red cell alloimmunization in pregnancy Before IUT availability III QOD (variable)
Renal transplantation, ABO compatible Antibody-mediated rejection I Daily or
QOD
Desensitization, living (5-6)
I
donor, positive crossmatch
due to donor-specific HLA
antibody
III
Desensitization, high PRA
deceased donor
 CHA P TER 2 6 Therapeutic Apheresis ■ 651

TABLE 26-2. Indications for Therapeutic Plasma Exchange (Continued)

Typical Course (Number of

Categorytreatments)
Indication Modifying Conditions

Renal transplantation, ABO Desensitization, live donor I Daily of QOD

incompatible (variable)
Antibody-mediated rejection II
Group A2/A2B into B, deceased IV
donor
Schizophrenia IV
Scleroderma (Progressive systemic
III 2/week (6)
sclerosis)
Sepsis with multiorgan failure III Daily (variable)
Stiff-person syndrome III QOD (4-5)
Sudden sensorineural hearing loss III QOD (3)
Systemic lupus erythematosus Severe II Daily or QOD
Nephritis IV (3-6)
Thrombotic microangiopathy, drug
Ticlopidine I Daily (variable)
associated
Clopidogrel III
Cyclosporine/tacrolimus III
Gemcitabine IV
Quinine IV
Thrombotic microangiopathy,
Refractory III Daily (variable)
HSCT associated
Thrombotic thrombocytopenic purpura I Daily (variable)
Thyroid storm III Daily (2-3)
Toxic epidermal necrolysis Refractory III Daily or
QOD
(variable)
Voltage-gated potassium
II QOD (5-7)
channel antibodies
Wilson disease Fulminant I Daily or QOD (3-5)
QOD = every other day; IVIG = intravenous immunoglobulin; DAH = diffuse alveolar hemorrhage; WAIHA = warm
autoim- mune hemolytic anemia; NYHA = New York Heart Association class; HSCT = hematopoietic stem cell
transplantation; MCP = membrane cofactor protein; CNS = central nervous system; PML = progressive multifocal
leukoencephalopathy; PANDAS = pediatric autoimmune neuropsychiatric disorders associated with streptococcal
infections; IUT = intrauterine transfusion.

neuropathy (AIDP and CIDP), clude renal transplantation with presensitiza-

antiglomerular basement membrane tion, and antibody-mediated organ transplant
antibody disease, and myasthenia gravis. rejection.
Examples of conditions in which the goal is
to remove an alloantibody in-
652 ■ AABB T EC HNIC AL MANUAL

TABLE 26-3. Comparison of Replacement Fluids

Replacement SolutionAdvantagesDisadvantages

Crystalloids Low cost 2-3 volumes

Nonallergenic required Hypo-
No viral risk oncotic
Lacks coagulation factors and
immunoglobulins
Albumin Iso-oncotic
Low risk of reactions Higher cost
Lacks coagulation factors and
immunoglobulins
Plasma Iso-oncotic
Normal levels of coagulation Viral transmission risk
factors, immunoglobulins and Citrate load
other plasma proteins ABO compatibility required
Risk of allergic reactions
Cryoprecipitate-reduced plasma Iso-oncotic
Reduced high-molecular-weight Same as plasma
von Willebrand factor and
fibrinogen
Normal levels of most
other plasma proteins

In myeloma with hyperviscosity, the goal
is to remove an excessive paraprotein (M pro-
tein). Measurement of plasma viscosity may
not be useful in guiding therapy in some pa-
tients because plasma viscosity may not corre-
late with symptoms. Normal plasma viscosity
ranges from 1.4 to 1.8 centipoise (cP).
Because most patients are not symptomatic
until the plasma viscosity is more than 4.0 or
5.0 cP, pa- tients with mild elevations may not
require treatment. In general, hyperviscosity
becomes a concern when M protein
concentrations reach 3 g/dL for IgM, 4 g/dL
for IgG, and 6 g/dL for IgA.6 Patients
receiving rituximab (anti- CD20) therapy for
IgM myeloma may experi- ence a transient
increase in M protein levels. Patients with
pretreatment IgM greater than 5 g/dL are at
particular risk of developing symptomatic
hyperviscosity.7
There are conflicting data regarding the
efficacy of TPE for treatment of acute renal
failure in myeloma. A randomized
controlled trial of TPE vs. conventional care
showed no difference in mortality or renal
function at 6 months.8 However, among
dialysis-dependent
patients, 43% in the TPE group and none in
the control group recovered renal function.
An- other randomized clinical trial showed
no benefit of TPE in a composite outcome
mea- sure of death, dialysis dependence, and
glo- merular filtration rate.9 However, biopsy
con- firmation of the renal diagnosis was not
required in this trial. Similarly, a
retrospective cohort study showed no
benefit of TPE in ei- ther reducing mortality
or preserving renal function.10 If TPE is to
be undertaken, biopsy confirmation of cast
nephropathy may be ad- visable.
Diseases in which immune complexes
may be pathogenic and can be removed by
apheresis include rapidly progressive
glomer- ulonephritis, cryoglobulinemia, and
vasculi- tis. Other indications include
conditions treat- ed by removal of protein-
bound drugs or toxins or high-concentration
lipoproteins.
In TTP, a deficiency of the vWF-cleaving
metalloprotease ADAMTS-13 results in accu-
mulation of high-molecular-weight vWF mul-
timers with subsequent intravascular platelet
activation and platelet-rich thrombi in the
 CHA P TER 2 6
microvasculature.11 In many cases, an inhibi-
tor of ADAMTS-13 can be demonstrated.
Plas- ma exchange is first-line treatment for
TTP, with the goal of removing both the
inhibitor and large vWF multimers while
simultaneous- ly replacing the deficient
enzyme. Secondary forms of
microangiopathic hemolytic anemia
associated with systemic lupus erythemato-
sus, hematopoietic progenitor cell transplan-
tation, chemotherapy, or immunosuppressive
medications may be clinically indistinguish-
able from idiopathic TTP. However, in many
cases, ADAMTS-13 activity has been shown
to be normal or only moderately reduced,
and the response to plasma exchange is
typically poor in these secondary forms of
microangio- pathic hemolytic anemias.
Transplant-associ- ated microangiopathic
hemolysis rarely re- sponds to apheresis and
probably represents a different disease
process.12
TPE to treat TTP is typically performed
daily until the platelet count and lactate
dehy- drogenase level are in the normal
range, but the intensity and duration of
treatment should be guided by the individual
patient’s course. After response has been
achieved, intermittent apheresis or plasma
infusion taper may be in- stituted, but the
efficacy of this approach in preventing
relapse has not been established.13 TPE
therapy has greatly improved the survival
rate in TTP; however, treatment failures do
oc- cur and cause major morbidity or
death.14
Hemolytic uremic syndrome (HUS) is a
similar condition that occurs more commonly
in children than adults. HUS may follow diar-
rheal infections with verotoxin-secreting
strains of Escherichia coli (strain 0157:H7) or
Shigella. Compared to patients who have clas-
sic TTP, those with HUS have more renal dys-
function and less prominent neurologic and
hematologic findings. Most patients with HUS
do not have antibody to ADAMTS-13 and
have normal activity of this protease.
Although diar- rhea-associated HUS rarely
responds to TPE, atypical HUS (aHUS)
caused by complement factor deficiencies or
autoantibodies to Factor H may respond;
however, patients with aHUS caused by
membrane cofactor protein muta- tions may
not respond to TPE. A terminal complement
inhibitor, eculizumab, may be ef-
Therapeutic Apheresis ■ 653
fective in the treatment of individuals with
aHUS.15
TPE is increasingly being used in the
treatment of central nervous system acute dis-
seminated encephalomyelitis. Experience in
treating chronic progressive multiple sclerosis
with TPE has been discouraging. However, a
randomized clinical trial in acute CNS inflam-
matory demyelinating diseases unresponsive
to steroids showed that TPE was beneficial.16
Early initiation of TPE is predictive of re-
sponse, and some clinical responses may not
manifest until later in follow-up.17 TPE may be
effective in neuromyelitis optica, even in the
absence of NMO antibodies.18
In focal segmental glomerulosclerosis
(FSGS), a circulating factor that increases glo-
merular permeability with resultant protein-
uria has been demonstrated.19 A recent study
has suggested that circulating urokinase re-
ceptor may be involved in the pathogenesis of
this disease.20 FSGS frequently recurs after re-
nal transplantation and can result in allograft
failure. TPE may effectively remove the
perme- ability factor and induce remission in
recur- rent FSGS following renal
transplantation. Re- sponse to TPE in the
primary form of the disease has not been well
studied.
TPE may be an adjunct to immunosup-
pression in the treatment or prevention of
an- tibody-mediated rejection (AMR) of
solid or- gan transplants. AMR presenting in
the early posttransplantation period may
respond bet- ter to TPE than later AMR.21
TPE before renal transplantation of an ABO-
incompatible kid- ney may be used to
prevent hyperacute rejec- tion, and
posttransplantation TPE is often used to
treat AMR that occurs in this set- ting. 22,23
TPE in conjunction with immuno-
modulatory therapies, such as intravenous
im- mune globulin (IVIG), before
transplantation can reduce the risk of
rejection in HLA-alloim- munized patients.24
Cytapheresis
The goal of cytapheresis is to remove
excessive or pathogenic leukocytes, platelets,
or red cells. In addition, in red cell exchange,
donor red cells are used to restore oxygen-
carrying
654 ■ AABB T EC HNIC AL MANUAL

flow velocity determined by

capacity. Indications for cytapheresis are listed
in Table 26-4.
In acute leukemia, high blast counts
(typ- ically >100,000/L) can result in
microvascu- lar stasis with headache, mental
status chang- es, visual disturbances, or
dyspnea. The leukocyte count at which a
patient may be- come symptomatic is
variable. Typically, pa- tients with acute or
chronic lymphocytic leu- kemia tolerate
higher cell counts than patients with
myelogenous leukemia, and cytapheresis
may not be required. Cytapheresis
commonly results in a less-than-predicted
reduction in leukocyte count, despite
excellent collection, because of mobilization
and re-equilibration of cells from
extravascular sites. Myelogenous leukemia
cells commonly have a higher densi- ty than
lymphocytic cells and can be difficult to
separate from red cells. Use of hydroxyethyl
starch enhances red cell sedimentation by
rouleaux formation and can improve the
effi- ciency of cytapheresis in acute
myelogenous leukemia. Massive
thrombocytosis, typically
>1,000,000/L, can occur in essential
throm- bocythemia, polycythemia vera, or
as a reac- tive phenomenon. Such patients
may be at risk of thrombosis or hemorrhage.
Reduction in platelet count is commonly
less than predicted because of mobilization
of platelets to the pe- ripheral blood,
primarily from the spleen.
Red cell exchange is most commonly
per- formed in the setting of sickle cell
disease (SCD). The goal is both to reduce
the burden of hemoglobin S and to provide
donor red cells containing hemoglobin A.
Acute chest syn- drome is a serious
complication of SCD, pre- senting as
dyspnea, chest pain, and cough, of- ten
accompanied by fever, leukocytosis,
decreasing hematocrit, and pulmonary infil-
trates. Respiratory failure can develop, and
death occurs in about 3% of cases. 25 Red cell
exchange is indicated for progressive infil-
trates and hypoxemia refractory to conven-
tional therapy and simple transfusion. 26,27 A
common goal is to reduce hemoglobin S to
less than 30% with a final hematocrit not to
ex- ceed approximately 30%. Red cell
exchange may also be indicated for
prevention of stroke in sickle cell anemia.
For patients with elevat- ed cerebral blood
 sickle cell disease.31
transcranial Doppler
imaging, transfusion re- ECP
duces the risk of ECP is a specialized procedure in which the
stroke.28,29 Chronic red buffy-coat layer is collected from peripheral
cell ex- change, typically blood, treated with 8-methoxypsoralen and
every 4 to 6 weeks, can ul- traviolet A light, and re-infused into the
be ef- fective in pa- tient. The treatment causes cross-linking
normalizing cerebral of leukocyte DNA, which prevents
blood flow while replication and induces apoptosis. The
minimizing iron overload procedure was de- veloped for the treatment
associated with simple of cutaneous T-cell lymphoma, although it is
transfusions. Red cell increasingly used for other indications
exchange may have a role (Table 26-5). ECP has com- plex
in other sickle cell immunomodulatory effects, including in-
syndromes, including duction of monocyte differentiation to den-
priapism, multiorgan dritic cells, alteration of T-cell subsets, and
failure, hepat- ic/splenic changes in cytokine production profiles.32
sequestration, ECP may be effective in acute and chronic
intrahepatic cho- lestasis. skin graft-vs-host disease (GVHD),
In addition, the technique although the role in non-skin GVHD is less
may be used for well defined.33
prevention of iron ECP for solid organ transplant rejection
overload, and pre- has been best studied in cardiac and lung
vention or management
of vaso-occlusive pain
crisis.
RBCs for
replacement must be
ABO com- patible and
negative for known
clinically sig- nificant
alloantibodies. For
sickle cell disease, the
RBCs should be
matched for C, E, and
K, if possible.30 It is
desirable to use
relatively fresh units to
maximize
posttransfusion red cell
survival. Units
containing either citrate-
phos- phate-dextrose-
adenine (CPDA)-1 or
additive solutions (AS)
may be used. It is
desirable that all units
used in a given
procedure contain the
same anticoagulant
solution so that they
have similar
hematocrits. Chronic
red cell ex- change may
carry a lower risk of
alloimmuni- zation than
simple transfusion in
 CHA P TER 2 6 Therapeutic Apheresis ■ 655

TABLE 26-4. Indications for Cytapheresis

Indication Modifying Conditions Procedure Category

Babesiosis Severe RCE I

High-risk population RCE II

Dermatomyositis or Leukocytapheresis IV
polymyositis

HSCT, ABO incompatible Minor HPC, Apheresis RCE III

Hereditary hemochroma- Erythrocytapheresis I

tosis

Hyperleukocytosis Leukostasis Leukocytapheresis I

Prophylaxis Leukocytapheresis III

Inclusion body myositis Leukocytapheresis IV

Inflammatory bowel Ulcerative colitis Adsorptive cytapheresis III/II

disease
Crohn’s disease Adsorptive cytapheresis III

Malaria Severe RCE II

Overdose Tacrolimus RCE III

Polycythemia vera and Polycythemia vera Erythrocytapheresis I

erythrocytosis
Secondary erythrocytosis Erythrocytapheresis III

Psoriasis Disseminated pustular Adsorptive cytapheresis III

Lymphocytapheresis III

Sickle cell disease, Acute stroke RCE I

acute
Acute chest syndrome, severe RCE II

Priapism RCE III

Multi-organ failure RCE III

Splenic sequestration; hepatic seques- RCE III

tration; intrahepatic cholestasis

Sickle cell disease, Stroke prophylaxis/ iron overload RCE II

Non-acute prevention

Vaso-occlusive pain crisis RCE III

Preoperative management RCE III

Thrombocytosis Symptomatic Thrombocytapheresis II

Prophylactic or secondary Thrombocytapheresis III

RCE = red cell exchange; HSCT = hematopoietic stem cell transplantation; HPC = hematopoietic progenitor cells.
656 ■ AABB T EC HNIC AL MANUAL

TABLE 26-5. Indications for Photopheresis

Indication Modifying Conditions Category

Cardiac transplantation Rejection prophylaxis II
Cellular or recurrent rejection II
Cutaneous T-cell lymphoma; Erythrodermic I
mycosis
fungoides; Sézary syndrome Nonerythrodermic III
Graft-vs-host disease Skin (chronic) II
Skin (acute) II
Non-skin (acute/chronic) III
Inflammatory bowel disease Crohn's disease III
Lung allograft rejection Bronchiolitis obliterans syndrome II
Nephrogenic sytemic fibrosis III
Pemphigus vulgaris Severe III
Psoriasis III
Scleroderma (Progressive systemic III
sclerosis)

transplantation. In a randomized clinical approved by the Food and Drug Administra-

trial, prophylactic photopheresis in
conjunction with previous generation
immunosuppres- sion (not including
calcinurin inhibitors or mycophenolate)
resulted in fewer rejection episodes,
decreased HLA antibodies, and re- duced
coronary artery intimal thickness, but no
difference in time to first episode, inci-
dence of hemodynamic compromise, or sur-
vival at 6 or 12 months.34,35 In recurrent
cardiac rejection, photopheresis may
decrease severi- ty of rejection and allow for
reduced dosage of immunosuppressives.36
ECP may have a role in the setting of
cardiac rejection with hemody- namic
compromise and for bronchiolitis oblit-
erans syndrome status after lung transplanta-
tion.37,38
Selective Adsorption
There are currently few established indica-
tions for selective adsorption of plasma pro-
teins (Table 26-6) and few devices have
been
tion (FDA).
In the two FDA-approved LDL apheresis
devices, selective removal of LDL can be
ac- complished by passing heparinized
plasma over a dextran sulfate column or
beads coated with anionic polyacrylate
ligands, or by pre- cipitation of heparin-LDL
complexes in acidi- fied plasma. Because
LDL production contin- ues, LDL apheresis
treatments must be repeated, typically at 2-
week intervals, indefi- nitely. There is
evidence that LDL apheresis re- duces the
incidence of major coronary events and
stroke.39 In addition, atherosclerotic plaque
regression can occur in some pa- tients. 40
Secondary effects of LDL apheresis that may
be beneficial include reduction in levels of
C-reactive protein, fibrinogen, tissue factor,
and soluble adhesion molecules.41,42 LDL
apheresis may also be used in the treat- ment
of primary or recurrent FSGS, although the
mechanism of action has not been well de-
fined.43
 CHA P TER 2 6 Therapeutic Apheresis ■ 657

TABLE 26-6. Indications for Selective Adsorption

Treatment
Indication Modifying Conditions Modality Category
Age-related macular degeneration, Rheopheresis I
dry
Chronic focal encephalitis (Rasmussen IA III
encephalitis)
Coagulation factor inhibitors Alloantibody IA III
Autoantibody IA III
Cryoglobulinemia Symptomatic/severe IA II
Dilated cardiomyopathy, idiopathic NYHA II-IV IA II
Familial hypercholesterolemia Homozygotes LDL apheresis I
Heterozygotes LDL apheresis II
Focal segmental glomerulosclerosis Primary, treatment resistant LDL apheresis *
Recurrent after renal trans- LDL apheresis
plantation
Immune thrombocytopenia Refractory IA III
Liprotein (a) hyperlipoproteinemia LDL apheresis II
Multiple sclerosis Acute CNS inflammatory IA III
demyelinating disease
Paraneoplastic neurologic syndromes IA III
Paraproteinemic demyelinating IgG/IgA/IgM IA III
polyneuropathies
Pemphigus vulgaris Severe IA III
Peripheral vascular diseases LDL apheresis III
Phytanic acid storage disease LDL apheresis II
(Refsum disease)
Sudden sensorineural hearing loss LDL apheresis III
Rheopheresis III
*FDA approved but ASFA category not yet assigned.
IA = immunoadsorption; LDL = low-density lipoprotein; NYHA = New York Heart Association class; CNS = central nervous
system.

IgG can be selectively removed by an alternative mechanism has been proposed

passing plasma over a column of
staphylococcal pro- tein A bound to silica. The
putative mecha- nism of action is the removal
of pathogenic au- toantibodies or immune
complexes, although
in ITP.44 Staphylococcal protein A adsorption
treatment can be performed manually or in
conjunction with automated TPE. Affinity col-
umns containing anti-IgG or ABO blood group
658 ■ AABB T EC HNIC AL MANUAL

substances have been tested in clinical trials, TABLE 26-7. Reported Frequency of Adverse
but are not currently approved for use in the Reactions to Apheresis*
United States.
ReactionFrequency (%)

ANTICOAGULATION Paresthesia 1.30

Acid-citrate-dextrose solution A (ACD-A), is Hypotension 0.91

the most commonly used anticoagulant, al- Urticaria 0.63
though heparin in combination with ACD-A is Nausea 0.39
also used, particularly in the setting of large-
volume leukapheresis for hematopoietic pro- Shivering 0.29
genitor cell collection. Current automated Flushing 0.16
apheresis devices control the administration
rate of citrate to achieve anticoagulation Dyspnea 0.15
while minimizing the risk for hypocalcemia. Vertigo 0.17
Hepa- rin anticoagulation is necessary for
Arrhythmia 0.11
LDL
apheresis and may be desirable for selected
patients undergoing TPE who are Abdominal pain 0.12
particularly susceptible to hypocalcemia, Anaphylaxis 0.02
such as small children, or in the setting of
severe metabolic alkalosis or renal failure. Total 4.25
With citrate anticoag- ulation, coagulation *Adapted from Matsuzaki. 40

monitoring is not gener- ally necessary,

although monitoring of ionized calcium may
be helpful for selected patients. The infused
citrate in the returned blood is rapidly
metabolized and rarely causes system- ic
anticoagulation.
ADVERSE EFFECTS
Although therapeutic apheresis is very safe,
complications do occur. An adverse event
oc- curs in about 4% of procedures (Table
26-7), but the majority are mild.45,46
Symptomatic hy- pocalcemia from infusion
of citrate with the returned blood is the most
common adverse effect of apheresis.
Perioral and digital pares- thesias are the
most common symptoms. Nau- sea may also
occur. Tetany is rare. Cardiac ar- rhythmia is
very rare, but patients with preexisting
hypocalcemia or significant pro- longation
of the QT interval should be moni- tored
carefully. Calcium supplementation may
alleviate the symptoms of citrate toxicity. A
typical supplementary dose is 10 mL of 10%
calcium gluconate per liter of albumin in-
fused. Citrate also chelates ionized magne-
sium, so it is possible that hypomagnesemia
contributes to the symptoms of citrate
toxicity.
However, one randomized clinical trial
showed no benefit to adding magnesium
dur- ing leukapheresis with continuous IV
calcium supplementation.47 Metabolism of
citrate leads to a mild metabolic alkalosis,
which can exacerbate hypocalcemia, and
may cause hy- pokalemia.48
Allergic reactions are most common
with plasma replacement, although they may
oc- cur with albumin as well. Most reactions
are mild, characterized by urticaria or
cutaneous flushing. More severe reactions
can involve the airways with dyspnea,
wheezing, and (rarely) stridor. Most allergic
reactions respond quick- ly to intravenous
diphenhydramine. Anaphy- laxis is very rare
but can occur. Patients with TTP who
receive large volumes of plasma are most at
risk of allergic reactions. Premedica- tion
with an antihistamine, or possibly ste- roids,
is not necessary for routine apheresis but
may be indicated for patients with repeat- ed
or previous severe reactions.
Respiratory difficulty during or immedi-
ately following apheresis can have many
 CHA P TER 2 6
causes, such as pulmonary edema, pulmo-
nary embolism, air embolism, obstruction of
the pulmonary microvasculature, anaphylac-
tic reactions, and transfusion-related acute
lung injury (TRALI).49 Hemothorax or
hemo- pericardium resulting from vascular
erosion due to a central venous catheter is
rare, but when it occurs it is typically
unsuspected, and may be fatal.50 Pulmonary
edema that results from volume overload or
cardiac failure is usu- ally associated with
dyspnea, an increase in di- astolic blood
pressure, and characteristic chest radiograph
findings. Acute pulmonary edema may also
arise from damage to alveolar capil- lary
membranes secondary to an immune re-
action or to vasoactive substances in plasma
or colloid solutions prepared from human
plasma. Predominantly ocular reactions
(peri- orbital edema, conjunctival swelling,
and tear- ing) have occurred in donors
sensitized to the ethylene oxide gas used to
sterilize disposable plastic apheresis kits.
51,52
Hypotension during apheresis can be a
sign of citrate toxicity; hypovolemia; or a
vaso- vagal, allergic, drug, or transfusion
reaction. Hypovolemia can occur early in a
treatment of a small patient when the return
fluid consists of the saline used to prime the
apheresis cir- cuit. Vasovagal reactions are
characterized by bradycardia and
hypotension. Such reactions usually respond
well to a fluid bolus and plac- ing the patient
in the Trendelenburg position.
When hypotension occurs during
plasma or red cell exchange, a transfusion
reaction such as TRALI, acute hemolysis,
bacterial con- tamination, or anti-IgA-
related anaphylaxis should be considered.
Hypotension is more frequent in children,
the elderly, neurology pa- tients, anemic
patients, and those treated with intermittent-
flow devices that have large ex- tracorporeal
volumes. Continuous-flow devic- es typically
do not have large extracorporeal volumes
but can produce hypovolemia if re- turn
flow is inadvertently diverted to a waste
collection bag, either through operator over-
sight or device malfunction. Hypovolemia
may also be secondary to inadequate volume
or protein replacement. During all
procedures, it is essential to maintain careful
and continuous records of the volumes
removed and returned.
Therapeutic Apheresis ■ 659
When plasma is exposed to foreign sur-
faces of plastic tubing or filtration devices,
the kinin system can be activated, resulting
in pro- duction of bradykinin. Infusion of
plasma con- taining bradykinin can cause
abrupt hypoten- sion. Patients taking
angiotensin-converting enzyme (ACE)
inhibitors are more susceptible to the
hypotensive reactions because the drugs
block enzymatic degradation of bradyki-
nin.53 Hypotensive reactions are more likely
during selective adsorption procedures be-
cause the devices expose plasma to a very
large surface area. Because some ACE
inhibi- tors have a long duration of action,
stopping the drug only the day before the
procedure may not be sufficient to prevent a
reaction.
Intensive TPE without plasma replace-
ment causes depletion of coagulation
factors. A 1.0 plasma volume exchange will
typically reduce coagulation factor levels by
25% to 50%, though Factor VIII levels are
less affect- ed.54 Levels of fibrinogen, a
large molecule without extravascular
distribution, are re- duced by about 66%. If
the patient has normal hepatic synthetic
function, coagulation factor levels typically
return to near normal within 2 days. Thus,
many patients can tolerate TPE ev- ery other
day for 1 or 2 weeks without develop- ing
significant coagulopathies that require
plasma replacement.
Bleeding as a consequence of coagula-
tion factor depletion is rare but has been re-
ported. For patients at risk, plasma may be
used for replacement at the end of the proce-
dure. Apheresis can also cause thrombocyto-
penia, particularly with HPC collection.
Inten- sive TPE can cause
hypogammaglobulinemia. Serum levels of
IgG and IgM recover to about 40% to 50%
of the preapheresis level at 48 hours. 54 The
absolute immunoglobulin level at which a
patient becomes at risk of infections has not
been established.
Albumin-bound drugs are removed by
TPE. This can result in subtherapeutic levels
of medications unless dosage adjustments are
made. High-molecular-weight biologicals
such as IVIG, antithymocyte globulin, and
monoclonal antibodies have a long intravas-
cular half-life and are readily removed by
apheresis. TPE shortly after administration of
660 ■ AABB T EC HNIC AL MANUAL
such drugs should be avoided because it
may significantly impair their effectiveness.
In ad- dition, intensive apheresis reduces the
con- centration of potentially diagnostic
plasma constituents, so blood for testing
should be drawn before initiating a course of
treatment.
Collapsed or kinked tubing,
malfunction- ing pinch valves, or improper
threading of tub- ing may damage red cells
in the extracorporeal circuit. Instrument-
related hemolysis has been reported in
0.06% of therapeutic aphere- sis
procedures.45 Hemolysis can also occur with
the use of incompatible replacement flu- ids
such as 5% dextrose solution (eg, D5W used
to dilute 25% albumin) or ABO-incom-
patible plasma. The operator should
carefully observe plasma collection lines for
pink dis- coloration suggestive of hemolysis.
Other types of equipment failure such as
problems with the rotating seal, leaks in the
plastic, and roller pump failure are rare.
Fatalities during apheresis are rare.
Death has been reported in 0.006% to 0.09%
of thera- peutic procedures.45,55 Most
fatalities are at- tributable to underlying
medical conditions.
VASCULAR ACCESS
Therapeutic apheresis requires good vascular
access to achieve adequate flow rates.
Periph- eral access generally requires at
least a 17- gauge needle for blood
withdrawal and at least an 18-gauge catheter
for return. Patients with- out adequate
peripheral veins or who require multiple
frequent procedures may require central
venous catheters. Central venous cath- eters
for apheresis must have rigid walls to ac-
commodate the negative pressure generated
in the withdrawal line. Peripherally inserted
central catheters (PICC lines) typically do
not support the high flow rates used in
apheresis and should not be used. At least
two lumens are required for continuous-flow
procedures, although single-lumen catheters
can be used for intermittent-flow
procedures. An implant- ed subcutaneous
port may be an option for some patients
requiring long-term treatment, such as
chronic red cell exchange.
The choice of placement site for a
central catheter is influenced by the
expected dura- tion of treatment. Subclavian
or internal jugu- lar access is generally
preferable for treat- ments lasting up to
several weeks. Femoral access should be
used only temporarily be- cause of the
higher risk of infection. Patients requiring
long-term treatment usually have a tunneled
catheter. With proper care, tunneled
catheters can be used for prolonged periods.
Good catheter care is very important to
maintain patency and prevent complications.
Catheters need to be flushed regularly. Hepa-
rin or 4% trisodium citrate is usually placed in
each lumen after each use to prevent occlu-
sion by clots. If a port becomes clotted, instil-
lation of a fibrinolytic agent such as urokinase
or recombinant tissue plasminogen activator
may restore patency. Routine dressing care is
essential to prevent insertion site infections.
An arteriovenous (AV) fistula may also be
used for apheresis, but apheresis personnel
should be suitably trained before attempting to
access an AV fistula.
Venous access devices may cause further
vascular damage, sometimes resulting in
thrombosis. Infrequently, they may result in
severe complications such as pneumothorax
or perforation of the heart or great vessels.
Other complications include arterial
puncture, deep hematomas, and
arteriovenous fistula formation. Bacterial
colonization often com- plicates long-term
placement and may lead to catheter-
associated sepsis, especially in pa- tients
who are receiving immunosuppressants.
Inadvertent disconnection of catheters may
produce hemorrhage or air embolism.
PATIENT EVALUATION
All patients should be evaluated by a
physician familiar with apheresis before
treatment be- gins. The indication, type of
procedure, fre- quency and number of
treatments, and the goal or endpoint should
be documented in the patient’s medical
record. The nature of the procedure, the
expected benefits, the possible risks, and the
available alternatives must be explained to
the patient, and the patient’s con-
 CHA P TER 2 6 Therapeutic Apheresis ■ 661

sent must be documented. The procedure cluding hypocalcemia, hypokalemia, and

should be performed only in a setting where
there is ready access to care for untoward
reac- tions, including equipment,
medications, and personnel trained in
managing serious ad- verse events such as
anaphylaxis.
The assessment should include evalua-
tion of the indication, medical conditions
that may affect the patient’s ability to
tolerate apheresis, vascular access, and
medications. Some points to consider
include the following:
■ Transfusion history: History of transfusion
reactions and special blood component re-
quirements.
■ Neurologic status: Mental status and the
ability to consent and cooperate.
■ Cardiorespiratory status: Adequate ventila-
tion and oxygenation, hyper- or hypovole-
mia, and cardiac arrhythmia.
■ Renal and metabolic status: Fluid balance,
alkalosis, and electrolyte abnormalities,
in-
hypomagnesemia.
■ Hematologic status: Significant anemia or
thrombocytopenia, coagulopathy, bleed-
ing, or thrombosis.
■ Medications: Drugs with high albumin
binding, immunoglobulins, and biologics.
Appropriate laboratory monitoring is dic-
tated by the indication, type and frequency of
procedures, and concomitant medical condi-
tions. In general, it is wise to obtain a
complete blood cell count, type and screen,
and electro- lytes assessment before starting
treatment. If at all possible, diagnostic studies
such as tests for infectious disease, pregnancy,
ADAMTS-13 activity, glomerular basement
membrane anti- body, or acetylcholine
receptor antibody should be completed before
the first treat- ment, particularly if plasma is
used for re- placement. Coagulation
monitoring may be appropriate when albumin
is used for replace- ment during frequently
repeated procedures.

KEY POINTS

Therapeutic apheresis treats disease by removal or extracorporeal manipulation of patho- logic plasma substances, white ce
The American Society for Apheresis (ASFA) has published guidelines and recommendations for the use of therapeutic aph
Anticoagulation is usually accomplished with citrate, although heparin may be used, partic- ularly for selective adsorption,
Albumin is the most commonly used replacement fluid for therapeutic plasma exchange, but plasma may be indicated for p
Adverse effects of apheresis are usually mild but may include symptomatic hypocalcemia, hypotension, urticaria, and naus
Vascular access for apheresis may be accomplished through peripheral veins, but a central venous catheter or an AV fistula
Medical evaluation of the apheresis patient should focus on the indication, type of proce- dure, frequency and number of tr
662 ■ AABB T EC HNIC AL MANUAL

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 C h a p t e r 2 7

Noninfectious Complications of
Blood Transfusion

Catherine A. Mazzei, MD; Mark A. Popovsky, MD;

and Patricia M. Kopko, MD

S T A T I S T I C A LL Y, T H E G R E A T E S
T sion practices. One of the main purposes of
risk of morbidity and mortality from
transfusion is from a transfusion reaction or
the noninfectious complications of blood
transfusion. In fact, transfusion-related acute
lung injury (TRALI), hemolytic transfusion re-
actions (HTRs), and transfusion-associated
circulatory overload (TACO) are the three
most commonly reported causes of
transfusion- related mortality.1 It is these
noninfectious complications that are
addressed in this chap- ter.
HEMOVIGILANCE
Hemovigilance may be defined as the
collec- tion of information on the
complications of transfusion, analysis of
these data, and subse- quent data-driven
improvements in transfu-
developing a hemovigilance program is to
im- prove reporting of transfusion-related
adverse events. It is widely believed that
the major noninfectious complications of
transfusion are both underrecognized and
underreported. The US Biovigilance
Network is a nation-
al collaboration between government and
nongovernment organizations. The network
develops and enhances surveillance systems
designed to track adverse reactions and inci-
dents associated with blood collection;
blood transfusion; and the transplantation of
cells, tissues, and organs. Transfusion safety
was the first issue that the network
addressed.
Definitions and classification schemes
are outlined in detail in the appendices of the
National Healthcare Safety Network Biovigi-
lance Component protocol with the goal of

Catherine A. Mazzei, MD, Medical Director, American Red Cross, Northern California Blood Services
Region, Oakland, California; Mark A. Popovsky, MD, Associate Clinical Professor, Harvard Medical School,
Boston, Massachusetts, and Vice President and Chief Medical Officer, Haemonetics Corporation, Braintree,
Massa- chusetts; and Patricia M. Kopko, MD, Clinical Professor of Pathology, University of California, San
Diego, Cali- fornia
C. Mazzei and P. Kopko have disclosed no conflicts of interest. M. Popovsky has disclosed a financial
relation- ship with Haemonetics Corporation.

665
666 ■ AABB T EC HNIC AL MANUAL
improving the quality of national
surveillance data.2 The Centers for Disease
Control and Prevention intends to publish
the results of system participation to allow
benchmarks and best practices to be
established.
RECOGNITION AND
EVALUATION OF A SUSPECTED
TRANSFUSION REACTION
Identification of a Transfusion Reaction
As with many necessary medical therapies,
ad- verse effects cannot always be
accurately pre- dicted or avoided. The
transfusing physician should be aware of
such risks when discussing the need for
transfusion with a patient. In- formed
consent for transfusion may include a
discussion of the risks of infectious disease
and serious noninfectious complications,
such as TRALI, and HTRs.3 Furthermore,
medical staff administering blood
components should be well aware of the
signs and symptoms of possible reactions.
These staff should be pre- pared to mitigate
the current episode and pre- vent future
similar reactions when possible.
Many common clinical signs and symp-
toms are associated with more than one type
of adverse reaction (see Table 27-1). Early
rec- ognition, prompt cessation of the
transfusion, and further evaluation are key
to a successful outcome. The signs and
symptoms that may be indicators of a
transfusion reaction include the following:
■ Fever, generally defined as a 1 C rise in
temperature above 37 C [the most common
sign of an acute HTR (AHTR)].4(p907)
■ Chills with or without rigors.
■ Respiratory distress, including wheezing,
coughing, and dyspnea.
■ Hyper- or hypotension.
■ Abdominal, chest, flank, or back pain.
■ Pain at the infusion site.
■ Skin manifestations, including urticaria,
rash, flushing, pruritus, and localized
edema.
■ Jaundice or hemoglobinuria.
■ Nausea/vomiting.
■ Abnormal bleeding.
■ Oliguria/anuria.
Clinical Evaluation and Management
of a Transfusion Reaction
The evaluation of a suspected transfusion re-
action involves a two-pronged investigation
combining clinical evaluation of the patient
with laboratory verification and testing. The
nurse or transfusionist must treat the patient
by administering supportive care at the
direc- tion of the physician, discontinue the
transfu- sion of the implicated component,
and con- tact the blood bank for directions
on the investigation. When an AHTR is
suspected, several steps must be taken
immediately.
Patient-focused steps are as follows:
1. Stop the transfusion immediately but
keep the line open with saline.
2. Document the clerical recheck between the
patient and the component. The labels on
the component, patient records, and
patient identification should be examined
to detect any identification errors. Trans-
fusing facilities may require repeat ABO
and Rh typing of the patient using a new
sample.5(p88) (See “Standard Laboratory
Investigation of a Transfusion Reaction”
section below.)
3. Contact the treating physician immediately
for instructions on patient care.
Component-focused steps are as
follows:
1. Contact the transfusion service for direc-
tions on investigating the cause of the
AHTR. Most transfusion services use a
stan- dardized form to document all of the
infor- mation available on both the patient
and the component.
2. Obtain instructions concerning the return
of any remaining component, associated
intravenous fluid bags, and tubing.
3. Identify appropriate samples (blood and
urine) to send to the laboratory.
The transfusion service determines
whether the blood donor center should be
no- tified of the AHTR.
 CH A P T E R 2 7 Noninfectious Complications of Blood Transfusion ■ 667

(Continued)
(acetaminophen, no aspirin)
Leukocyte-reduced blood
Antipyretic premedication
Rule out hemolysis (DAT,
inspect for hemoglobinemia,

WBC antibody screen†

repeat patient ABO)
Rule out bacterial
contamination
Therapeuti
Type Incidence Etiology Presentation Diagnostic Testing Approach

Hemolytic ABO Rh mismatch:Red cell Chills, fever, hemoglobinuria,Clerical

hypotension,
check renal
DAT failure with
Keep
oliguria,
urine D
1 in 40,000incompatibility Visual inspection (free Hb) Repeat patien
with fluids a
head- ache, vomiting

AHTR: 1 in 76,000 Further tests as indicated to detect hemo

Analgesics
Fever, chills/rigors,

Fatal HTR: Pressors fo

1 in 1.8 million
TABLE 27-1. Categories of Adverse Transfusion Reactions and Their

Antibody to donor
versal leukoreductionkines in platelet unit
0.1 to 1% with uni-Accumulated cyto-
Acute (<24 hours) Transfusion Reactions—Immunologic

WBCs

Urticarial 1:100-1:33 Antibody to donor plasma

Urticaria,
proteins
pruritis, flushing Rule out hemolysis (DAT, inspect
Antihistamin
for hem
(1%-3%) premedicati

antihistamin
nonhemo- lytic
Management*

Febrile,
 Type

TRALI
TABLE 27-1. Categories of Adverse Transfusion Reactions and Their Management*
668

(Continued)
■

Anaphylactic 1:20,000-1:50,000 Antibody to Hypotension, urticaria, bron-Rule out hemolysis (DAT, Trendelenburg (feet-up) posi-
donor plasma chospasm (respiratory dis-inspect for hemoglobinemia, tion
proteins tress, wheezing), local edema, repeat patient ABO) Fluids
(includes IgA, anxiety Anti-IgA Epinephrine (adult dose:

Incidence
haptoglobin, C4) IgA, quantitative 0.2-
Cytokines 0.5 mL of 1:1000 solution SC
or IM; in severe cases,

1:1,200-1:190,000
1:10,000 IV, initial rate
1mcg/minute) Antihistamines,
corticosteroids, beta-2
agonists

Etiology
AABB T ECHNICAL M A NUAL

WBC antibodies in donor

Hypoxemia,
Presentation

(occasionally
respiratory
in recipient),
fail- ure,
Rule
other
hypotension,
hemolysis
Diagnostic Testing

fever,
outWBC-activating
(DAT,

WBC crossmatch Chest X-ray

agents
bilateral
Rule out cardiogenic pulmonary
inspect
Deferral
Approach

edema
Supportive
Therapeuti

of i
compo

WBC antibody screen in donor and recip

inpulmona
for hem
Acute (<24 hours) Transfusion Reactions—Nonimmunologic

Air embolus
Circulatory over- load
Transfusion- associated

<1%

Rare
Varies
sepsis
Hypotension asso- Dependent on Inhibited metabolism Flushing, hypotension Rule out hemolysis (DAT, Withdraw ACE inhibition
ciated with ACE clinical setting of bradykinin with inspect for hemoglobinemia, Avoid albumin volume
inhibition infusion of brady- repeat patient ABO) replace- ment for
kinin (negatively plasmapheresis Avoid
charged filters) or bedside leukocyte filtra- tion
activators of

by component Bacterial
prekallikrein

(see Infectious

Volume overload

Air infusion via line

contamina- tion
Dis- ease
Fever,

Nonimmune hemo- Rare Physical or Hemoglobinuria, hemoglobi- Rule out patient hemolysis Identify and eliminate
lysis chemical nemia (DAT, inspect for hemoglobi- cause
destruction of nemia, repeat patient ABO)
Screening,

blood (heating, Test unit for hemolysis

CH A P T E R 2 7 Noninfectious Complications of Blood Transfusion

freezing, hemolytic
drug or solution
chills, hypotension
Chapter 8)

added to blood)
■

Sudden shortness of breath, X-ray

acute for
Gram’s stain

(Continued)
669

cyanosis,
intravascular air
pain, cough,
Culture of component Patient(until

Place
ments)
culture

IV diuretic (
sensit
Broad spec

Dyspnea, orthopnea, cough,Chest X-ray tachycardia, hypertension,R

Upright pos

hypotensio
legs elevate
patien
Rule out hemolysis (DAT, inspect for hem
 Type
TABLE 27-1. Categories of Adverse Transfusion Reactions and Their Management*
670

(Continued)

Hypothermia
■

Hypocalcemia (ion- Dependent on clinicalRapid citrate infusion Paresthesia, tetany, Ionized calcium PO calcium supplement for mild
ized calcium; citrate setting (massive transfusion arrhythmia Prolonged Q-T interval symptoms during therapeutic
toxicity) of citrated blood, on electrocardiogram apheresis procedures
delayed metabolism Slow calcium infusion while

Incidence
monitoring ionized calcium lev-

settingblood
of citrate, apheresis
procedures) els in severe cases

Delayed (>24 hours) Transfusion Reactions—Immunologic

Etiology
Alloimmuni- 1:100 Immune response to Positive blood group Antibody screen Avoid unnecessary
zation, red cell (1%) foreign antigens on antibody screening test DAT transfusions Leukocyte-
antigens RBCs reduced blood
Alloimmuni- WBCs and
1:10 Platelet refractoriness, Platelet antibody Avoid unnecessary
AABB T ECHNICAL M A NUAL

zation, HLA anti- (10%) platelets (HLA)

delayed hemolytic reaction, screen HLA antibody transfusions Leukocyte-
gens
hemolytic disease of the screen reduced blood
newborn
Fever, decreasing
Hemolytic 1:2500-11,000 Anamnestic Antibody screen Identify antibody
hemoglobin, new
Presentation

immune response DAT Transfuse compatible RBCs

to red cell positive antibody screening Tests for hemolysis (visual as needed
Dependent on clinicalRapid infusion of cold Cardiac arrhythmia
antigens test, mild jaundice inspection for hemoglobine-
mia, LDH, bilirubin, urinary
hemosiderin as clinically
indicated)
Diagnostic Testing

Central body temperature

Approach
Therapeuti

Employ bloo
Graft-vs-host Rare Donor lymphocytes Erythroderma, maculopapu- Skin biopsy Corticosteroids, cytotoxic
disease engraft in lar rash, anorexia, nausea, HLA typing agents

Iron overload
recipient and vomiting, diarrhea, hepatitis, Molecular analysis for Irradiation of blood components
mount attack on pancytopenia, fever chimerism for patients at risk (including
host tissues components from related
donors and HLA-selected com-
ponents)
IVIG
Posttransfusion Rare Recipient platelet Thrombocytopenic Platelet antibody screen
purpura antibodies (apparent purpura, bleeding 8-10 and identification HPA-1a-negative platelets
alloantibody, usually days after transfusion Plasmapheresis
anti-HPA-1a)
destroy autologous

Typically after >100 RBC

platelets
Delayed (>24 hours) Transfusion Reactions—Nonimmunologic

Multiple
*For platelet refractoriness, see chapter on platelet and granulocyte antigens and antibodies; for septic transfusion reactions, see chapter on transfusion-transmitted diseases. For a

unitstransfusions with
recent summary of transfusion reactions, see Popovsky.4
†
Blood group antibody screening test.

Diabetes,
AHTR = acute hemolytic transfusion reaction; HTR = hemolytic transfusion reaction; DIC = disseminated intravascular coagulation; DAT = direct antiglobulin test; IV =

obligate
intravenous; Hb = hemoglobin; LDH = lactate dehydrogenase; CRYO = cryoprecipitated antihemophilic factor; FFP = fresh frozen plasma; WBC = white blood cell; PO = by
mouth; SC = subcutaneous; IM = intramuscular; IgA = immunoglobulin A; ACE = angiotensin-converting enzyme; TRALI = transfusion-related acute lung injury; RBC = Red Blood Cell;
CH A P T E R 2 7 Noninfectious Complications of Blood Transfusion

cirrhosis,
IVIG = intravenous immunoglobulin; HPA = human platelet antigen.

iron load
cardiomy-
■

Serum
in transfusion-
opathyferritin
671

dependent
Endocrine function tests
Liver patient
enzymesIron chelato
672 ■ AABB T EC HNIC AL MANUAL
Standard Laboratory Investigation of a
Transfusion Reaction
When the laboratory receives notice of a
possi- ble transfusion reaction, several steps
are per- formed by technologists:
1. Clerical check of the component bag,
label, paper work, and patient sample.
2. Repeat ABO testing on the
posttransfusion sample.
3. Visual check of pre- and posttransfusion
samples to look for evidence of
hemolysis (which may not be visible if
<50 mg/dL of hemoglobin is present).
4. Direct antiglobulin test (DAT) on a post-
transfusion sample.
5. Report of findings to the blood bank
super- visor or medical director, who
may request additional studies, tests, or
quarantine of cocomponents generated
from the same donor collection.
The transfusion service must retain any
patient records that are related to transfusion
reactions, clinically significant antibodies,
or special transfusion requirements.5(pp74-77)
Addi- tional information regarding special
products, such as irradiated or washed
components re- quired for a particular
patient, may be re- tained by the transfusion
service as well.
In an increasingly mobile society,
patients often present for treatment at a
facility that has no ready access to the
patient’s medical re- cords. When the patient
is able to give a thor- ough history,
transfusion services are able to share
medical information in a timely manner.
When this is not possible, medical warning
bracelets or wallet identification cards may
benefit patients with multiple alloantibodies
whose strength may decrease over time.
Specialized Laboratory Investigations
for Selected Reactions
Additional laboratory evaluation may be re-
quired for investigations of some nonhemoly-
tic transfusion reactions, such as anaphylaxis,
sepsis, or TRALI, as described in their respec-
tive sections.
Acute or Immediate Transfusion
Reactions
Acute or immediate transfusion reactions
oc- cur within 24 hours of the administration
of a component and often during the
transfusion. Acute transfusion reactions
include hemolysis, both immune and
nonimmune mediated; transfusion-related
sepsis; TRALI; severe aller- gic reactions,
including anaphylaxis; TACO;
complications of massive transfusion; air
em- bolism; febrile nonhemolytic
transfusion reac- tions (FNHTRs); and mild
allergic reactions, such as urticaria or rash.
The clinical signifi- cance of an acute
transfusion reaction often cannot be
determined by the patient’s clinical history
or signs and symptoms alone but re- quires
laboratory evaluation.
AHTRs
Presentation
Rapid hemolysis of as little as 10 mL of
incom- patible blood can produce symptoms
of AHTR. The most common presenting
symp- tom is fever with or without
accompanying chills or rigors. A patient
with a mild reaction may have abdominal,
chest, flank, or back pain. If a patient has a
severe AHTR, hypoten- sion, dyspnea, and
flank pain may be present and, in some
cases, progress to shock with or without
accompanying disseminated intravas- cular
coagulation (DIC). Red or dark urine may
be the first sign of intravascular hemolysis,
particularly in the anesthetized or uncon-
scious patient, who may also present with
oli- guria or, in rare cases, DIC. The severity
of the symptoms of this reaction is related to
the amount of incompatible blood
transfused. Prompt recognition of the
reaction and imme- diate cessation of the
transfusion can prevent grave consequences.
Differential Diagnosis
Many of the signs and symptoms of an im-
mune-mediated AHTR also occur in other
acute transfusion reactions. Fever with or
without chills and accompanied by hypoten-
sion may also develop in transfusion-related
 CH A P T E R 2 7 Noninfectious Complications of Blood Transfusion
sepsis and TRALI. Hemolysis may not be
de- tected immediately and does not occur
in sep- sis and TRALI. Respiratory difficulty
is not typ- ically a symptom of an AHTR.
Immediate treatment requirements for acute
hemolysis are identical to those for sepsis—
stop the transfusion and maintain
hemodynamic sta- bility—and can be
instituted while the diagno- sis is being
determined.
The patient’s underlying disease process
can make the diagnosis of any AHTR
extremely difficult. Patients with glucose-6-
phosphate dehydrogenase (G6PD)
deficiency, autoim- mune hemolytic anemia,
or sickle cell disease present particularly
complicated situations when symptoms such
as fever and hypoten- sion occur. In these
patients, autoantibodies and multiple
alloantibodies delay the serolog- ic
diagnosis of an AHTR and make identifica-
tion of the responsible antibody a challenge.
Acute hemolysis may also result from
nonim- mune mechanisms, as described in
the “Non- immune-Mediated Hemolysis”
section below.
Pathophysiology
The interaction of preformed antibodies with
red cell antigens is the immunologic basis for
AHTRs. The most severe reactions are associ-
ated with transfusions of red cells that are
ABO incompatible with the recipient, resulting
in acute intravascular destruction of the trans-
fused cells. Transfusion of ABO-incompatible
plasma, as can occur with apheresis platelets,
may also cause hemolysis of the patient’s red
cells. Although this form of acute hemolysis is
not usually clinically significant or character-
ized by typical hemolytic symptoms, it can be
severe if donors have high titers of ABO anti-
bodies. Although rare, the most common cir-
cumstance is when group O platelets from do-
nors with high titers of anti-A are transfused to
group A recipients.6,7
When preformed immunoglobulin M
(IgM) or IgG antibodies recognize
correspond- ing red cell antigens,
complement activation may occur, resulting
in intravascular hemoly- sis,
hemoglobinemia, and, eventually, hemo-
globinuria. IgM antibodies are strong activa-
■ 673
tors of complement, and IgG antibodies,
when present at sufficient concentrations
and of the relevant subclass, may activate
complement as well.
Complement activation involves C3
cleavage with the ensuing production of
C3a, an anaphylatoxin, which is released
into the plasma, and C3b, which coats the
red cells. If complement activation proceeds
to comple- tion, which is characteristic of
ABO antibodies, a membrane attack
complex is assembled on the red cell
surface, and intravascular lysis oc- curs.
C5a, an anaphylatoxin that is 100 times
more potent than C3a, is produced as part of
this hemolysis. C3a and C5a promote the re-
lease of histamine and serotonin from mast
cells, leading to vasodilation and smooth-
muscle contraction, particularly of bronchial
and intestinal muscles. C3a and C5a are
recog- nized by many other cell types as
well, includ- ing monocytes, macrophages,
endothelial cells, platelets, and smooth
muscle, and are in- volved in the production
and release of cyto- kines, leukotrienes, free
radicals, and nitric ox- ide. The end result
may include wheezing, flushing, chest pain
or tightness, and gastroin- testinal
symptoms. These symptoms may also be
caused by release of bradykinin and norepi-
nephrine caused by antigen-antibody com-
plex stimulation.
Phagocytosis of IgG-coated red cells leads
to cytokine release, which plays a role in pro-
ducing the effects of acute hemolysis. 8 Inter-
leukin-8 (IL-8), which activates neutrophils,
and tumor necrosis factor alpha (TNF),
which activates the coagulation cascade, have
also been found after in-vitro incubation of
incompatible group O whole blood and group
A or B red cells.9 Other cytokines involved in
the pathogenesis of AHTRs include IL-1, IL-
6, and monocyte chemoattractant protein 1
(MCP-1). In a mouse model of HTR,
transfusion of incompatible red cells resulted
in very high plasma levels of MCP-1 and IL-6
and lower levels of TNF.10
If complement activation does not pro-
ceed to completion, which typically happens
with non-ABO antibodies, the red cells can
un- dergo extravascular hemolysis where cells
coated with C3b and/or IgG are rapidly
674 ■ AABB T EC HNIC AL MANUAL

mated the risk of clinical or laboratory evi-

removed from the circulation by phagocytes.
In extravascular hemolysis, the
consequences of complement activation,
including release of anaphylatoxins and
opsonization of red cells, may still have
adverse effects.
Coagulation abnormalities associated
with AHTRs may be caused by various
mecha- nisms. The “intrinsic” pathway of
the clotting cascade may be activated by
antigen-antibody interaction, resulting in
activation of Factor XII, also known as
“Hageman factor.” Activa- tion of the
Hageman factor can result in hypo- tension
through its effect on the kinin system. The
kinin system produces bradykinin, which in
turn increases vascular permeability and
causes vasodilation. Activated complement,
TNF, and IL-1 may increase the
expression of tissue factor. Tissue factor,
expressed by leuko- cytes and endothelial
cells, can activate the “extrinsic” pathway
and is associated with the development of
DIC. DIC is an often life- threatening
consumptive coagulopathy. Its
characteristics include microvascular
thrombi formation with ischemic organ and
tissue damage; consumption of platelets,
fibrinogen, and coagulation factors; and
activation of fi- brinolysis with resultant
production of fibrin- degradation products.
The end result of these activations can vary
from generalized oozing to uncontrolled
bleeding.
Shock may be a component of DIC.
Hypo- tension, caused by the release of
vasoactive amines, kinins, and other
mediators, produc- es a compensatory
vasoconstrictive response that further
aggravates organ and tissue dam- age. Renal
failure may occur as well. Free he-
moglobin impairs renal function, but
compro- mised renal cortical supply is
thought to be the major contributing factor
in renal failure. In addition, antigen-
antibody complex deposi- tion,
vasoconstriction, and thrombi formation
contribute to the development of renal
vascu- lar compromise.

Frequency
The frequency of AHTRs is not easy to deter-
mine. The authors of a review article based on
data from several surveillance systems esti-
 evidence at this time.
dence of ABO HTR to be The addition of the diuretic furosemide
1:80,000 and the risk of a (40-80 mg intravenously in adults, 1-2
fatal ABO HTR to be 1 mg/kg in children) promotes increased urine
in 1.8 million.11 Of the output and further enhances renal cortical
transfusion-related blood flow.13 If urine output remains
fatalities reported to the diminished af- ter a liter of saline has been
Food and Drug infused, acute tu- bular necrosis may have
Administration (FDA) occurred, and the pa- tient may be at risk of
from 2007 to 2011, 23% developing pulmonary edema. At this point,
(in 50 patients) were a nephrologist should be consulted for
caused by HTRs.1 further management of the pa- tient.
Oliguric renal failure may be complicat- ed
Treatment by hyperkalemia and subsequent cardiac
arrest; therefore, these patients should be
Prompt recognition of monitored with telemetry. Metabolic
an AHTR and immedi- acidosis and uremia often necessitate the
ate cessation of the institution of dialysis.
transfusion are crucial. DIC is an equally serious component of
The unit of blood an AHTR. DIC is extremely difficult to treat
should be returned to and may be the first indication that
the blood bank for a hemolysis
transfusion reaction
investi- gation. The
infusion of saline
should be con- tinued to
maintain venous access,
treat hypo- tension, and
maintain renal blood
flow, with a goal of a
urine flow rate >1
mL/kg/hour. Saline
infusion alone may not
be adequate therapy, and
the urine output must be
carefully moni- tored so
as not to cause volume
overload in the patient.
Studies have concluded
that low-dose dopamine
may improve renal
function initial- ly, but
no solid evidence exists
that it can pre- vent
renal failure.12 However,
these studies, which did
not include patients
with AHTRs, did show
a 25% increase in urine
output in the acute
setting. Thus, low-dose
dopamine (1-5
µg/kg/minute) may still
have a role in treating
the complications of an
AHTR, but no recom-
mendations regarding
its use may be made
based on published
 CH A P T E R 2 7 Noninfectious Complications of Blood Transfusion
has occurred in an anuric or anesthetized pa-
tient. Traditional therapy for DIC includes
treating or removing the underlying cause
and providing supportive care via the
administra- tion of platelets, frozen plasma,
and cryopre- cipitated antihemophilic factor
(AHF).
The administration of heparin to treat
DIC associated with HTRs is controversial.
First, the underlying condition for which the
patient required transfusion may be a contra-
indication to heparin administration. For ex-
ample, in patients with recent surgery or
active bleeding, heparin can exacerbate
bleeding. Second, there is some thought that
because the precipitating event is limited to
hemolysis of the volume of blood
transfused, the risks of administering
heparin do not justify its use. Those in favor
of heparin point out, however, that the most
unstable patients (those with the most severe
reactions) are those who received larger
volumes of incompatible blood and are thus
the most likely to develop DIC.14 In the
worst cases, DIC can become a self-
sustaining vicious cycle of inflammation
and consump- tive coagulopathy.
Recent studies of optimal treatment for
DIC have evaluated the targeting of various
components of the inflammatory and
coagula- tion cascades. Activated protein C
has shown some benefit in patients with
sepsis and DIC; however, no new
therapeutic agents have been added to the
arsenal to treat DIC associated with AHTRs
in recent years.15,16
Unconscious or anesthetized patients
may receive multiple units of incompatible
blood before acute hemolysis is recognized.
Because the severity of an AHTR is related
to the amount of incompatible red cells
trans- fused, red cell exchange transfusion
may be considered. Some severe reactions
to a single unit of strongly incompatible
blood may re- quire exchange transfusion as
well. Antigen- negative blood must be used
for the red cell exchange. In the case of
acute hemolysis caused by a non-ABO
antibody, the blood bank must be given
adequate time to identify the appropriate
units for further simple red cell transfusion
or planned red cell exchange. Likewise,
plasma and platelets that will not contribute
to hemolysis should be chosen.
■ 675
Prompt initiation of therapy to aggres-
sively manage hypotension, renal blood
flow, and DIC provides the greatest chance
of a suc- cessful outcome. Furthermore,
consultation with appropriate medical
specialists early in the course of treatment
will ensure that the pa- tient receives
hemodialysis, cardiac monitor- ing, and
mechanical ventilation when needed.
Prevention
The most common events leading to the trans-
fusion of a component in error are the very
things that the laboratory evaluates when an
AHTR is suspected. Clerical and human errors
involving patient, sample, and blood unit
identification are the most common causes of
mistransfusion and, therefore, AHTRs. The
risk of a near miss is 1:1,000, wrong blood
given is 1:15,000, and ABO-incompatible
transfusion is 1:40,000.11 Institutional policies
and proce- dures should be in place to
minimize the likeli- hood of such errors, and
corrective and preventive action programs
should target con- tinual reduction of the
numbers of such errors. However, no one
method for reducing the number of errors is
foolproof. Products avail- able to increase
patient safety include technol- ogy-based
solutions, such as radiofrequency
identification chips, handheld bar-code scan-
ners, and “smart” refrigerators similar to sys-
tems used for pharmacologic agents.
The prevention of potential hemolysis
from the administration of minor ABO-
incom- patible platelets remains a challenge
in pa- tients with HLA antibodies. A number
of op- tions, including anti-A or anti-B
titration of the product, limitations in total
amount of incom- patible plasma transfused
from platelets, and volume reduction, may
offer some benefit. The use of platelet
additive solutions is also promising because
these solutions replace plasma, which
contains ABO antibodies and plasma
proteins.17
Nonimmune-Mediated Hemolysis
Transfusion-associated hemolysis can also
be due to several nonimmune-mediated
causes.
676 ■ AABB T EC HNIC AL MANUAL
Before issue, improper shipping or storage
temperatures as well as incomplete
deglycero- lization of frozen red cells can
lead to hemoly- sis. At the time of
transfusion, using a needle with an
inappropriately small bore size or em-
ploying a rapid pressure infuser can cause
me- chanical hemolysis, which may result
from the use of roller pumps as well.
Improper use of blood warmers or the use of
microwave ovens or hot waterbaths can
cause temperature- related hemolysis. Few
fluids are approved by FDA for transfusion
with Red Blood Cells (RBCs). 5(p45) Infusion
of RBCs simultaneously through the same
tubing with hypotonic solu- tions or some
pharmacologic agents may cause osmotic
hemolysis; for safe administra- tion, RBCs
and these solutions or agents should be
given via alternate venous access lo-
cations. In rare cases, hemolysis may be
caused by bacterial contamination of the
RBC unit. Not infrequently, patients may
also expe- rience hemolysis as part of their
underlying disease process. Of note is that
although a neg- ative DAT result usually
indicates no evidence of an immune-
mediated cause of hemolysis, complete
destruction of incompatible trans- fused red
cells may be associated with a nega- tive
DAT result.
When both immune and nonimmune
causes of hemolysis have been excluded, the
possibility of an intrinsic red cell membrane
defect in the recipient or even in the trans-
fused cells should be considered. Cells with
these defects, such as G6PD deficiency,
have increased fragility when challenged
with par- ticular stressors and may undergo
coinciden- tal hemolysis.
Treatment
Hemolysis of nonimmune etiology may
cause symptoms whose severity depends on
the de- gree of hemolysis and amount of
component transfused. In all cases, the
transfusion should be discontinued and
appropriate care should be administered.
(See the earlier section on the treatment of
AHTRs for details on manag- ing
hypotension and declining renal function.)
Prevention
As is true for the mitigation of any type of
transfusion reaction, written procedures for
all aspects of the manufacture and
transfusion of blood and components should
always be fol- lowed. Prompt recognition of
nonimmune he- molysis and robust root
cause analysis may prevent additional
occurrences.18
Transfusion-Related Sepsis
Presentation
Fever (particularly a temperature of 38.5 C or
101 F) and shaking chills and hypotension
during or shortly after transfusion are the
most frequent presenting symptoms of
transfusion- related sepsis. In severe cases,
the patient may develop shock with
accompanying renal fail- ure and DIC.
Differential Diagnosis
The abruptness of onset and severity of the
signs and symptoms associated with transfu-
sion-related sepsis may be very similar to
those of AHTRs. Mild cases may be
confused with FNHTRs. The key to
diagnosing transfu- sion-related sepsis is
culturing the same organism from both the
patient and the re- mainder of the
component. The returned component should
be visually examined in suspected cases of
posttransfusion sepsis. Par- ticular attention
should be paid to any color changes,
especially brown or purple discolor- ation in
an RBC component and bubbles/ frothiness
in a platelet component. A Gram’s stain
should be performed on the returned
component.
Treatment
If transfusion-related sepsis is suspected, the
transfusion should be stopped immediately
and supportive care of the patient should be
initiated. Broad-spectrum antibiotics may be
indicated. (See Chapter 8 for a more detailed
discussion of bacterial contamination of
transfused blood, including frequency data
and prevention strategies.)
 CH A P T E R 2 7 Noninfectious Complications of Blood Transfusion
FNHTRs
Presentation
An FNHTR is usually defined as the occur-
rence of 1 C rise in temperature above 37
C that is associated with transfusion and for
which no other cause is identifiable.
Accompa- nying symptoms may include
shaking, chills, an increased respiratory rate,
a change in blood pressure, and anxiety. In
some instanc- es, the patient may be afebrile
but have the re- maining constellation of
symptoms. Symp- toms usually occur during
transfusion but may occur up to 4 hours
after. Most FNHTRs are be- nign, although
they may cause significant dis- comfort and
even hemodynamic or respirato- ry effects.
Differential Diagnosis
The symptoms associated with an FNHTR
may be present in several other types of
transfusion reactions, the most serious of
which are HTRs, sepsis, and TRALI. Each of
these other reac- tions has signs, symptoms,
and associated lab- oratory results that help
distinguish them from an FNHTR once an
investigation is begun; rec- ognition of
FNHTR requires diagnosis by ex- clusion.
Fever may commonly occur as a compo-
nent of a patient’s underlying illness. In a pa-
tient who has been experiencing spiking fevers
during the course of admission, it may be
diffi- cult to rule out an FNHTR. Hemolysis
along with any other signs or symptoms of a
serious reaction must be ruled out in a patient
who ex- periences fever associated with
transfusion.
Pathophysiology
Recipient leukocyte antibodies may cause fe-
brile transfusion reactions. As few as 0.25 ×
109 residual leukocytes in a blood component
can produce a temperature elevation in the
recipi- ent. FNHTRs may also be the result of
accu- mulated cytokines in a cellular blood
compo- nent.19 This mechanism may be
particularly relevant in reactions that occur
after the trans- fusion of platelets. Some
FNHTRs are attribut- able to recipient
antibodies, particularly HLA
■ 677
antibodies that react with antigens on trans-
fused lymphocytes, granulocytes, or
platelets. Cytokine release in the recipient in
response to these antigen-antibody reactions
may contrib- ute to the severity of the
reaction. Whatever the initiating cause,
cytokine release is the common event
leading to symptoms of FNHTR.
Treatment
When an FNHTR is suspected, the
transfusion should be discontinued and a
transfusion re- action work-up initiated.
Antipyretics (ie, acet- aminophen) should be
administered, and the patient may be safely
transfused once the symptoms subside. For
more severe reactions that include rigors,
meperidine may be neces- sary where it is
not contraindicated.
In general, the remainder of the
implicat- ed component and donor-related
co-compo- nents should not be transfused.
Many times, an FNHTR does not develop
until after the transfusion has been
completed. If a portion of the component
remains, the laboratory work- up to exclude
hemolysis must be completed before the
transfusion is resumed. This may be difficult
to accomplish within an acceptable amount
of time. Among the few valid situa- tions in
which transfusion of the remainder of the
component should be considered is when the
component is a medically indicated rare unit
and a significant volume remains un-
transfused. In that circumstance, the blood
bank’s medical director should be consulted
before proceeding with caution because bac-
terial contamination of the component might
have been the underlying cause of the reac-
tion.20,21
Prevention
Prestorage leukocyte reduction, especially if
performed at the time of collection, signifi-
cantly decreases the frequency of FNHTRs.22
Premedication with acetaminophen may be
beneficial in further reducing the residual
rate of FNHTRs and has not been shown to
impair the ability to detect serious
complications of transfusion.21
678 ■ AABB T EC HNIC AL MANUAL

hypotension,
Allergic Reactions
Presentation
Most allergic transfusion reactions (ATRs)
are mild, but their spectrum can range from
a sim- ple allergic reaction (urticaria) to life-
threaten- ing anaphylaxis. Symptoms
generally occur within seconds or minutes
of the start of the transfusion. In rare cases,
the symptoms may take several hours to
develop. If symptoms do not appear until
more than 4 hours later, they may represent
an allergic reaction that is unre- lated to the
blood transfusion. An ATR is diag- nosed in
the same way as any other allergic re-
action.
The mildest form of ATR is urticaria, or
hives. An outbreak of swollen, raised, red
ar- eas (wheals) on the skin appears
suddenly as a result of the body’s adverse
reaction to an al- lergen. Hives usually cause
itching (pruritus) but may also burn or sting.
Hives can appear anywhere on the body and
vary greatly in size. They can last from
hours to several days before fading but often
respond quickly to treatment with
antihistamines. More extensive cases may
be accompanied by angioedema, where the
swelling is caused by fluid accumulation be-
neath the skin instead of on the surface. It is
a deep swelling, often around the eyes and
lips, and generally lasts longer than
urticaria. An- gioedema can, rarely, involve
the throat, tongue, or lungs, causing
respiratory distress.
More serious are anaphylactic
transfusion reactions, which include the
symptoms of urti- caria and angioedema in
the majority of cas- es. 23 Severe
hypotension, shock, and loss of
consciousness may also occur. In addition,
the respiratory system is often involved,
with pa- tients experiencing dyspnea,
wheezing, and stridor. Gastrointestinal
disturbances such as nausea, vomiting,
diarrhea, and cramping, af- fect
approximately 30% of these patients. Car-
diovascular manifestations in addition to
hypotension may include tachycardia, ar-
rhythmia, or cardiac arrest.

Differential Diagnosis
It is important to distinguish anaphylaxis
from other reactions characterized by
 Mast cell activation (Type I
dyspnea, and/or loss of hypersensitivity), resulting in degranu-
consciousness. The lation, with the release of allergic mediators
most common reaction occurs. Secondary mediators—including cyto-
that may be mistaken kines and lipid mediators—are also
for anaphylaxis is a generated and released. Recent studies
vasovagal reaction, suggest that the mechanisms underlying
which is characterized most ATRs have not been fully elucidated.
by hypotension, A combination of recipi- ent, donor, and
diaphoresis, component factors is likely in- volved,
nausea/vomiting, which is supported by the incidence of these
weakness, bradycardia, reactions compared to the prevalence of IgA,
and sometimes loss of haptoglobin, or C4 deficiency. Recipients with
consciousness. an atopic predisposition appear to have a
Urticaria, angioedema, higher rate of ATRs, and certain donors’
pruritus, and respiratory plate- lets are associated with an increased
symp- toms, such as risk. In- creased understanding of the
wheezing or stridor, are underlying mechanisms of ATRs is crucial
symp- toms of to improving prevention.24,25
anaphylaxis but do not ATRs that progress beyond urticaria may
occur in vaso- vagal occur in IgA-deficient patients. These
reactions. The anaphy- lactic reactions are caused by anti-
respiratory symptoms of IgA in the
anaphylaxis may be
suggestive of an acute
asthma attack or
TRALI. However, the
classic symptoms of
allergy, including
urticaria, an- gioedema,
and pruritus, do not
occur in asth- ma
attacks or TRALI.
Fever, a prominent
symptom of HTR and
bacterial contamina-
tion, is not a feature of
anaphylaxis.
Patients who take angiotensin-convert-
ing enzyme (ACE)
inhibitors and undergo
plasma exchange
sometimes develop hypo-
tensive reactions that
mimic anaphylaxis when
albumin is used as a
replacement fluid.

Pathophysiology
ATRs are attributed to
hypersensitivity reac-
tions to allergens in the
component caused by
preformed IgE antibody
in the recipient. In most
cases, the causative
plasma protein or
antigen is not identified.
 CH A P T E R 2 7 Noninfectious Complications of Blood Transfusion
recipient. Although IgA deficiency is present
in approximately 1:700 people of European
an- cestry, only a small percentage of these
people ever make antibodies against IgA.
Those who do are divided into two groups
based on IgA levels and the type of antibody
formed. People with absolute IgA deficiency
(<0.05 mg/dL) may form class-specific
antibodies that are of- ten associated with
anaphylactic reactions. Those with decreased
but detectable amounts of IgA, or relative IgA
deficiency, can form sub- class-specific
antibodies (eg, anti-IgA1 or anti- IgA2) that
typically result in less severe reac- tions.26
Although precautions should be taken
when transfusing an IgA-deficient patient, it
must be kept in mind that the majority of
ATRs are caused by allergens to substances
other than IgA.26 Other well-known triggers
include patient antibodies against
haptoglobin,27 peni- cillin, and the C4
determinant of comple- ment.28 Patients
taking ACE inhibitors can ex- perience
transfusion reactions that are thought to
result from dual actions on bradykinin: inhi-
bition 1) of its catabolism by the ACE
inhibitor and 2) of bradykinin by low levels
of prekalli- krein activity in plasma protein
fraction.
Frequency
ATRs are quite common, with an overall fre-
quency of approximately 1% to 3% of
transfu- sions. ATRs also represent 12% to
33% of all transfusion reactions.29,30
Urticaria is relatively common, whereas
anaphylactic reactions oc- cur much less
often. As with any ATR, anaphy- laxis
occurs most commonly during the trans-
fusion of plasma or platelets; it has an
incidence of 1:20,000 to 1:50,000 transfu-
sions.29,31 Of the transfusion-associated
fatali- ties reported to the FDA from 2007 to
2011, 6% (in 12 patients) were caused by
anaphylaxis.1
Treatment
Urticaria is the only transfusion reaction in
which the administration of the component
may be routinely resumed after prompt treat-
ment. When a patient develops symptoms,
the transfusion should be paused so that an
anti- histamine, typically 25 to 50 mg
diphenhydr-
■ 679
amine, may be administered. Once the
symp- toms have dissipated, the transfusion
may be resumed and a laboratory work-up
need not be initiated.30
If the symptoms do not subside—or, in
the case of severe urticaria or urticaria
accom- panied by hypotension if dyspnea,
significant edema, or gastrointestinal
symptoms do not subside—the transfusion
must be stopped and the reaction promptly
treated. Severe urticari- al reactions may
require treatment with meth- ylprednisolone
(125 mg intravenously) or prednisone (50
mg orally). Once a severe reac- tion or
developing anaphylaxis is identified, action
should be promptly initiated to main- tain
oxygenation and stabilize hypotension.
Epinephrine (1:1000) may be administered
in- tramuscularly or subcutaneously at an
adult dose ranging from 0.2 to 0.5 mL or a
pediatric dose of 0.01 mL/kg. If symptoms
persist, the dose may be repeated every 5 to
15 minutes up to three times, unless
palpitations, extreme anxiousness, or
tremors occur. If the patient is unconscious
or in shock, epinephrine may be given
intravenously at a dilution of 1:10,000 (100
µg/mL) and an initial rate of 1 µg/minute.
Such patients ideally receive cardiac
monitor- ing because of the epinephrine’s
arrhythmic potential.
Supplemental oxygen should be adminis-
tered, and the airway should be maintained.
Hypotensive patients should be placed in the
Trendelenburg position and supported with
crystalloids. If bronchospasm is present,
respi- ratory symptoms may not respond to
the epi- nephrine, and addition of a beta-2
agonist or aminophylline may be required.
Patients who do not respond, possibly
because of the use of a beta-adrenergic
blocker or an ACE inhibitor, may respond to
the addition of glucagon as a 1-mg bolus
intravenously or by continuous in-
fusion.23,32
Prevention
Premedication with an antihistamine (25 to
50 mg diphenhydramine) 30 minutes before
transfusion may be helpful in patients with
a history of multiple or severe urticarial
trans- fusion reactions. Routine prophylaxis
may also
680 ■ AABB T EC HNIC AL MANUAL
be beneficial for patients undergoing
multiple transfusions, as in plasma
exchange; however, routine antihistamine
premedication of all pa- tients receiving a
transfusion has not been shown to decrease
the risk of ATR.33 If antihis- tamines are not
sufficient, prednisone (20-50 mg orally) or
parenteral steroids may be of benefit. For
patients whose reactions are se- vere,
unrelenting, and unresponsive to pre-
medication, washing red cells or platelets
may be considered. Plasma reduction or
replace- ment in platelet products may also
be benefi- cial. Alternatively, the
administration of de- glycerolized RBCs has
met with some success when reactions have
occurred even after RBCs were washed with
2 L of saline.
The plasma used for transfusions for
pa- tients with diagnosed IgA deficiency
who pro- duce anti-IgA should be from IgA-
deficient do- nors (<0.05 mg/dL). If the
local blood center cannot provide these
components, they may be available through
the American Rare Donor Program (see
Method 3-13). Other cellular components
(RBCs and platelets) can be de- pleted of
plasma proteins through washing. IgA
deficiency without the presence of anti-IgA or
without a history of an anaphylactoid/
anaphylactic reaction does not warrant the
use of IgA-deficient or plasma-depleted
com- ponents. Intravenous immune globulin
(IVIG) products from various manufacturers
have different IgA amounts. The
manufacturer should be contacted to obtain
detailed infor- mation about a product and
lot number.34 Au- tologous donation
programs may also be con- sidered for
patients with IgA deficiency once they have
recovered.
TRALI
Presentation
Clinical signs and symptoms of TRALI
typical- ly include fever, chills, dyspnea,
cyanosis, hy- potension, and new-onset
bilateral pulmonary edema.35 An increase in
blood pressure fol- lowed by hypotension is
not uncommon. TRALI can be life
threatening or fatal. Symp- toms arise within
6 hours of transfusion, with most cases
becoming evident within 1 to 2 hours after
the end of transfusion. In addition
to the signs and symptoms traditionally
asso- ciated with TRALI, there is a growing
apprecia- tion that the disorder can be
associated with a dramatic transient
neutropenia or leukope- nia.36
All plasma-containing components, in-
cluding whole blood, RBCs, platelets, cryopre-
cipitated AHF, and fresh frozen plasma
(FFP), have been implicated in TRALI.
Transfusion volumes as small as 15 mL
have led to TRALI.
TRALI is a form of acute lung injury
(ALI). The American-European Consensus
Conference37 defined ALI as acute hypoxemia
with a PaO2/
FiO2 ratio of 300 mm Hg and bilateral pulmo-
nary edema on frontal chest radiograph. The
Canadian Consensus Conference38 relied on
this definition of ALI when creating its diag-
nostic criteria for TRALI: 1) ALI with hypox-
emia and PaO2/FiO2 300 or SpO2 <90% on
room air, 2) no preexisting ALI before transfu-
sion, 3) onset of symptoms within 6 hours of
transfusion, and 4) no temporal relationship
with an alternative risk factor for ALI. The
pan- el also defined “possible TRALI” using
the same criteria as for TRALI, with the
exception that possible TRALI occurs in the
setting of an alternative risk factor for ALI.
Although the lung injury in ALI is usually
irreversible, the lung injury in TRALI is most
often transient. Approximately 80% of pa-
tients with TRALI improve within 48 to 96
hours. The remaining 20% of patients who do
not improve rapidly have either a protracted
clinical course or a fatal outcome. In one of
the largest studies of TRALI, 100% of the
patients required oxygen support and 72%
required mechanical ventilation.39
Differential Diagnosis
The three main conditions that need to be
dis- tinguished from TRALI are 1)
anaphylactic transfusion reactions, 2)
TACO, and 3) transfu- sion-related sepsis. In
anaphylactic transfu- sion reactions,
bronchospasm, laryngeal ede- ma, severe
hypotension, erythema (often confluent),
and urticaria are prominent symp- toms.
Fever and pulmonary edema are not as-
sociated with anaphylactic reactions. The
clin- ical presentation of TACO is very
similar to that
 CH A P T E R 2 7 Noninfectious Complications of Blood Transfusion
of TRALI, with respiratory distress,
tachypnea, and cyanosis as the most
prominent features. The key distinction
between TACO and TRALI is that the
pulmonary edema in TACO is car- diogenic,
whereas it is noncardiogenic in TRALI.
High fever with hypotension and vas- cular
collapse are prominent features of trans-
fusion-related sepsis. Respiratory distress is
infrequently associated with these reactions.
With rapid onset of respiratory distress, in
ad- dition to TACO and TRALI, coincident
myocar- dial infarction and pulmonary
embolus as well as other possible causes of
ALI should be con- sidered.
Pathophysiology
The precise mechanism of lung injury in
TRALI has not been determined. TRALI
has been associated with the infusion of
antibod- ies to leukocyte antigens and of
biologic re- sponse modifiers (BRMs).39
Infusions of these antibodies or BRMs are
thought to initiate a sequence of events that
results in cellular acti- vation and damage of
the basement mem- brane. Pulmonary
edema occurs secondary to leakage of
protein-rich fluid into the alveolar space.
BRMs accumulate in some cellular com-
ponents during storage. BRMs enhance the
polymorphonuclear cell oxidative burst, are
soluble in chloroform, and consist of a mixture
of lysophosphatidylcholines.40
Antibodies to HLA Class I antigens,
HLA Class II antigens, and human
neutrophil anti- gens (HNAs) have been
associated with TRALI. These antibodies can
be formed after exposure to foreign antigens
via pregnancy, transfusion, or
transplantation. In the majority of TRALI
cases with antibodies, the source of the anti-
body is the donor, not the recipient.
A two-event model of the mechanism
of TRALI has been hypothesized.40 In the
first event, generation of biologically active
com- pounds activates pulmonary vascular
endo- thelial cells and primes neutrophils,
resulting in sequestration of neutrophils in
the pulmo- nary microvasculature. This first
event can result from a variety of
physiologic stressors, including sepsis,
surgery, and massive transfu-
■ 681
sion, and it predisposes the recipient to
devel- op TRALI if a second event is
experienced. The infusion of BRMs or
antibodies is the second event. These
stimuli, which ordinarily do not activate
neutrophils, activate the primed neu-
trophils in the pulmonary microvasculature,
which results in pulmonary endothelial dam-
age, capillary leakage, and pulmonary
edema.
Frequency
Although the true incidence of TRALI is un-
known, TRALI is the leading cause of
transfu- sion-related mortality reported to
the FDA, with more than 34 cases reported
in 2007.41 Re- cent efforts to decrease the
amount of trans- fusable plasma collected
from female donors appear to be decreasing
the number of TRALI fatalities in the
United States.1
In 2006, the year before many blood
cen- ters implemented measures to reduce
the risk of TRALI from plasma transfusions,
35 fatal cases of TRALI, 22 of which were
associated with transfusion of FFP, were
reported to the FDA. Since 2008, the year
after many blood centers implemented such
measures, the numbers of TRALI fatalities
and of TRALI fatal- ities associated with FFP
transfusions reported to the FDA have
decreased each year.
Treatment
Treatment of TRALI consists of respiratory
and circulatory support. Treatment should be
as intensive as dictated by the clinical
picture. Oxygen supplementation with or
without me- chanical ventilation is required
in almost all cases. Pressor agents may be
needed to sup- port blood pressure.
Diuretics are not indicat- ed because TRALI
is not related to volume overload.
Administration of corticosteroids has not
been shown to improve the clinical outcome
in TRALI or acute respiratory distress
syndrome.42
Prevention
Strategies to reduce the risk of TRALI are
com- plicated by a number of factors.
Although approximately 10% of blood
donations con- tain HLA and/or HNA
antibodies, TRALI
682 ■ AABB T EC HNIC AL MANUAL
occurs in fewer than 1:1000 transfusions.39,40
There is no mechanism to identify which pa-
tients are at risk for developing TRALI. In
2013, AABB announced that a new standard
in the 29th edition of Standards for Blood
Banks and Transfusion Services would
require that plas- ma products and whole
blood collected for transfusion be from male
donors, never-preg- nant female donors, or
females who have been tested since their
last pregnancy and found to be negative for
HLA antibodies.5
In 2014 the AABB issued Association
Bul- letin 14-02, providing guidance on how
to im- plement the new TRALI risk
reduction require- ment. In this bulletin,
AABB outlined a number of considerations
for implementation of TRALI risk reduction
requirements in plas- ma components and
whole blood intended for transfusion.43
Although the measures required by
AABB and described in Association Bulletin
14-02 are expected to reduce the risk of
TRALI, it is important to recognize they do
not eliminate TRALI because they do not
decrease the risk of TRALI from RBCs,
platelet concentrates, or cryoprecipitate.
HLA antibody screening ad- dresses only
the risk of TRALI from HLA anti- bodies. A
practical test to screen the blood supply for
HNA antibodies is not available. In addition,
none of these risk-reduction mea- sures
addresses the risk of TRALI from BRMs.
TACO
Presentation
It is well known that transfusion can precipi-
tate acute pulmonary edema caused by vol-
ume overload, but only recently has this
prob- lem been recognized as an important
complication. Patients older than 70 years
and infants are at greatest risk, although all
trans- fusion recipients are susceptible to
some de- gree.44 Whereas large volumes of
components and nonblood fluids are most
frequently im- plicated, modest volumes can
also precipitate TACO. High flow rates are
frequently cofactors. TACO has no diagnostic
signs or symp- toms. Within 1 to 2 hours of
transfusion, pa- tients may develop any or all
of the following: gallop; jugular venous
distension; elevated
central venous pressure; dyspnea;
orthopnea; new ST-segment and T-wave
changes on elec- trocardiogram; elevated
serum troponin; and likely elevated brain
natriuretic peptide (BNP), a cardiac
marker.45 Increased blood pressure
characterized by a widening of the pulse
pres- sure is characteristic of TACO.
Radiographs show a widened cardiothoracic
ratio.
Differential Diagnosis
TACO is frequently confused with TRALI
be- cause both types of reactions produce
pulmo- nary edema. It is possible for TACO
and TRALI to occur concurrently in the
same patient. The timelines and the clinical
presentation are similar, but hypertension is
a constant feature of TACO, whereas it is
only an infrequent and transient
manifestation of TRALI. Further- more,
rapid improvement with diuresis or ino-
tropic agents is consistent with TACO.
In congestive heart failure, BNP levels
are elevated. Several studies have shown
that a posttransfusion-to-pretransfusion BNP
ratio of 1.5 with a posttransfusion level at
least 100 pg/mL as a cutoff yielded a
sensitivity and specificity greater than 80% in
TACO.46 Howev- er, a recent study in the
intensive care setting found that BNP was
only of moderate value for distinguishing
TACO from TRALI.47 With rapid onset of
respiratory distress, possible causes of ALI,
such as coincident myocardial infarction,
pulmonary embolus, and others, should be
considered in addition to TACO and TRALI.
Frequency
The incidence of TACO is unknown, but
sever- al studies suggest that TACO is much
more common than previously known. In the
gener- al population, one group found TACO
in 1:707 recipients of RBCs, and 20% of the
affected pa- tients received a single unit of
RBCs. In studies of older orthopedic patients
undergoing hip or knee replacement, TACO
was found in 1% and 8%, respectively.48,49 In
one report from the Quebec hemovigilance
network, the incidence was 1:5000
components, and 1.3% of the cases resulted in
death.50 In the critical care setting, 1:356 units
transfused resulted in TACO.51 From 2007 to
2011, 15% of the transfusion-
 CH A P T E R 2 7 Noninfectious Complications of Blood Transfusion
associated fatalities reported to the FDA (in
32 patients) were a consequence of TACO.1
Although RBCs are most commonly
asso- ciated with TACO, a recent
prospective surveil- lance study found that
FFP was a frequent cause of this
complication.52 In this study, 4.8% of
patients developed TACO with a prevalence
rate of 1.5%. Of the 24 reactions, 14 (58%)
oc- curred in the intensive care unit.
Treatment
As soon as symptoms suggest TACO, the
trans- fusion should be stopped. The
symptoms should be treated by placing the
patient in a seated position, providing
supplementary oxy- gen, and reducing the
intravascular volume with diuretics. If
symptoms persist in con- firmed TACO,
administration of additional di- uretics or
therapeutic phlebotomy in 250-mL
increments is appropriate.
Prevention
In the absence of ongoing and rapid blood
loss, components should be administered
slowly, particularly in patients at risk of
TACO (ie, pediatric patients, patients with
severe anemia, and patients with congestive
heart failure). Rates of 2 to 4 mL/minute and
1 mL/ kg of body weight per hour are the
most fre- quently cited, despite a paucity of
data on ap- propriate infusion rates. Total
fluid input and output must be monitored.
Complications of Massive
Transfusion
The potential complications of massive
trans- fusion, usually defined as the receipt
of more than 10 RBC units within 24 hours,
include metabolic and hemostatic
abnormalities, im- mune hemolysis, and air
embolism. Metabolic abnormalities can
depress ventricular func- tion. Hypothermia
from refrigerated blood, ci- trate toxicity,
and lactic acidosis from under- perfusion
and tissue ischemia, which are often
complicated by hyperkalemia, can
contribute to this effect. Although metabolic
alkalosis caused by citrate metabolism may
occur, it is not likely to be clinically
significant. Patients
■ 683
who lose blood rapidly may have
preexisting or coexisting hemostatic
abnormalities or de- velop them during
resuscitation. Hemostatic abnormalities may
include dilutional coagu- lopathy, DIC, and
liver and platelet dysfunc- tion.
Citrate Toxicity
PATHOPHYSI OLOGY AND MA NIFESTA-
TION S. Plasma, whole blood, and platelets
contain citrate as an anticoagulant. When
large volumes of these components are
trans- fused rapidly, particularly in the
presence of liver disease, plasma citrate
levels may rise, binding calcium and ionized
calcium and re- sulting in hypocalcemia. In
patients with a normally functioning liver,
citrate is rapidly metabolized; thus, these
symptoms are tran- sient.53 Hypocalcemia is
more likely to cause manifestations in
patients who are hypother- mic or in shock.
A decrease in ionized calcium levels in-
creases neuronal excitability, which in the
con- scious patient leads to symptoms of
perioral and peripheral tingling, shivering,
and light- headedness, followed by a diffuse
sense of vi- bration, muscle cramps,
fasciculations, spasm, and nausea. In the
central nervous system, hy- pocalcemia is
thought to increase the respira- tory center’s
sensitivity to carbon dioxide, causing
hyperventilation. Because myocardial
contraction is dependent on the intracellular
movement of ionized calcium, hypocalcemia
depresses cardiac function.54
TREATM EN T AN D PREVENT I ON . Unless the
patient has a predisposing condition that
hin- ders citrate metabolism, hypocalcemia
caused by citrate overload during massive
transfusion can usually be treated by
slowing the infusion. Calcium replacement
should be considered when the calcium
concentration falls to below 50% of its
normal value or the symptoms of
hypocalcemia are evident.55
Hyperkalemia and Hypokalemia
PATHOPHYSIOLOGY. When RBCs are
stored at 1 to 6 C, the intracellular
potassium gradual- ly leaks into the
supernatant plasma or addi-
684 ■ AABB T EC HNIC AL MANUAL
tive solution. Although the concentration in
the supernatant may be high (see Chapter 6)
because of the small volume, the total extra-
cellular potassium load is less than 0.5 mEq
for fresh RBC units and only 5 to 7 mEq for
units at expiration. These potassium
concentrations rarely cause problems in the
recipient because rapid dilution,
redistribution into cells, and ex- cretion
blunt the effect. However, hyperkale- mia
can be a problem in patients with renal
failure, premature infants, and newborns re-
ceiving large transfusions, such as in cardiac
surgery or exchange transfusion; otherwise,
hyperkalemia is typically a transient effect
during very rapid transfusions.
Hypokalemia occurs more frequently
than hyperkalemia after transfusion because
potassium-depleted donor red cells reaccu-
mulate this ion intracellularly, and citrate
me- tabolism causes further movement of
potassi- um into the cells in response to the
consumption of protons. Catecholamine re-
lease and aldosterone-induced urinary loss
can also trigger hypokalemia in the setting
of massive transfusion.56
TREATMEN T A N D PRE VEN TION . No treat-
ment or preventive strategy for hypokalemia
and hyperkalemia is usually necessary,
provid- ed that the patient is adequately
resuscitated from the underlying condition
that required massive transfusion. 57 For
infants receiving small-volume transfusions
infused slowly, units may be used safely
until the expiration date. 58 Although
washing of RBC units results in very low
levels of potassium, there is no evi- dence
that this is indicated for routine RBC
transfusions, even in patients with impaired
renal function.59
Hemostatic Abnormalities in Massive
Transfusion
PATHOPHYSIOLOGY. Coagulopathy can oc-
cur in massive transfusion, particularly
when the lost blood is initially replaced with
RBCs and asanguineous fluids.
Coagulopathy in massive transfusion is
frequently ascribed to the dilution of
platelets and clotting factors as patients lose
hemostatically active blood, and
enzymatic activity is reduced as the core
body temperature lowers if a blood warmer
is not used. Mortality rates associated with
hemo- static abnormalities range from 20%
to 50%.60 The high rate of mortality results
from hypo- thermia, metabolic acidosis, and
coagulopa- thy.61
Studies of military and civilian trauma
pa- tients demonstrated a progressive
increase in the incidence of microvascular
bleeding (MVB) characteristic of a
coagulopathy with increasing transfusion
volumes that typically occurs after
replacement of two to three blood volumes
(20 to 30 units).62,63 Although platelet counts,
coagulation parameters, and levels of
selected clotting parameters correlate with
the volume transfused, contrary to
expectations from a simple dilutional model,
the relation- ship is marked by tremendous
variability. Moreover, there is frequently
discordance be- tween the laboratory
assessment and the clini- cal evidence of
bleeding. It has been suggested that the
platelet deficits play a more important role
in causing the bleeding than the coagula-
tion deficiencies.
MVB typically occurs when the platelet
count falls below 50,000 to 60,000/µL.
Howev- er, no simple relationship can be
determined between a patient’s coagulation
test results and the onset of bleeding. The
etiology of bleeding (elective surgery vs
massive trauma) may play a role as well.64
Subsequent studies have refined these
observations. Significant platelet
dysfunction has been demonstrated in
massively trans- fused patients.65,66 Low
fibrinogen and platelet counts are better
predictors of hemostatic fail- ure than
elevations of prothrombin time (PT) and
partial thromboplastin time (PTT), sug-
gesting that consumptive coagulopathy is an
important factor in MVB in addition to dilu-
tion.63 Similarly, Harke and Rahman67
showed that the degree of platelet and
clotting abnor- malities correlates with the
length of time that the patient is
hypotensive, suggesting that shock is the
most important cause of DIC. In aggregate,
Collins68 concluded that “coagulop- athy in
heavily transfused patients was due to
hypoperfusion, not transfusion.” More
recent- ly, more powerful hemostatic assays
have been
 CH A P T E R 2 7 Noninfectious Complications of Blood Transfusion
introduced that may be more predictive of
blood component requirements.69
These data may not be generalizable to
patients undergoing massive transfusion in
the controlled setting of the operating room,
where hypotension caused by volume loss is
prevented. In this context, coagulation factor
levels may have priority over platelet prob-
lems. Murray and colleagues 64 documented
that excessive bleeding in elective surgery pa-
tients transfused with more than one blood
volume (RBCs and crystalloid) corresponded
to a prolongation in PT and PPT compared to
patients with normal hemostasis.
TREATMENT AND PREVENTION. The dilu-
tional model of coagulopathy in massive
transfusion suggests that prophylactic re-
placement of hemostatic components based
on the volume of RBCs or whole blood
trans- fused prevents the development of a
bleeding diathesis. No specific regimen has
yet been shown to be superior to any other
in prospec- tive studies, and this is a
controversial topic.70 According to AABB
guidelines, there are insuf- ficient data to
recommend for or against a 1:1 RBC to
plasma transfusion ratio in massive
transfusion.71
Previously, the predominant thinking
was
that the replacement of platelets and
coagula- tion factors in the massively
transfused surgi- cal and trauma patient
should be based on the identification of a
specific abnormality using platelet counts,
international normalized ra- tio, activated
PTT, and fibrinogen levels. Fre- quent
monitoring of these laboratory values serves
to avoid overuse of platelets and plasma
products (FFP and Cryoprecipitated AHF)
by anticipating the specific components
needed while avoiding dilutional
coagulopathy. It is imperative that the
laboratory provide results of these tests
rapidly. Intraoperative and post- operative
laboratory testing, such as thrombo-
elastography, may be useful.
US E OF AD JUN CT THER APIES IN M A SS
IVE
TR ANS F US I O NS . Recombinant Factor
VIIa (rFVIIa), a 50-kDa analog of Factor
VIIa, is li- censed in the United States for
the treatment with inhibitors of bleeding in
patients with he-
■ 685
mophilia A or B. However, the off-label use of
rFVIIa is growing in several areas, including
to treat hemorrhagic bleeding in trauma and
sur- gery.72,73 The recombinant product works
by targeting the site of tissue damage, where it
binds to tissue factor. This complex activates
Factor X to Factor Xa and, ultimately, Factor
IXa. In patients with hemophilia, rFVIIa by-
passes the need for Factor VIII or Factor IX
through the activation of the small amounts of
Factor X on activated platelet surfaces at the
site of injury. Studies of the efficacy of rFVIIa
have produced mixed results.74 In the absence
of definitive data, transfusion services should
establish guidelines on the reasonable use of
rFVIIa. In contrast, antifibrinlytics may have a
role in controlling massive bleeding from trau-
ma. The 2010 CRASH-2 study concluded that
tranexamic acid should be given as early as
possible in the trauma patient.75
Air Embolism
Air embolism can occur if blood in an open
system is infused under pressure or if air en-
ters a central catheter while containers or
blood administration sets are being changed.
Air embolism has been reported in
association with intraoperative and
perioperative blood recovery systems that
allow air into the blood infusion bag. The
minimum volume of air em- bolism that is
potentially fatal for an adult is
approximately 100 mL.76 Symptoms include
cough, dyspnea, chest pain, and shock.
If air embolism is suspected, the patient
should be placed on the left side with the
head down to displace the air bubble from
the pul- monic valve. Aspiration of the air is
sometimes attempted. However, proper use
and inspec- tion of infusion pumps,
equipment for blood recovery or apheresis,
and tubing couplers are still essential to
prevent this complication.
Hypothermia
Blood warmers may be used to prevent
hypo- thermia. Proper procedures for the use
of blood warmers should be followed
because
686 ■ AABB T EC HNIC AL MANUAL
overheating may damage or destroy red
cells, causing hemolysis and serious
transfusion re- actions, including fatalities.
Blood warmers must have a temperature
monitor and warning system to decrease the
risk of over- heating.5(p6)
DELAYED TRANSFUSION
REACTIONS
Delayed transfusion reactions occur days to
months or even years after transfusion. As
with acute transfusion reactions, the
consequences may be severe but are often
treatable.
DHTRs
Presentation
Fever and anemia occurring days to weeks
af- ter transfusion of an RBC component are
char- acteristic of a DHTR. The hemolysis
associated with a DHTR is usually not brisk,
but some pa- tients may develop jaundice
and leukocytosis. In a DHTR, the hemolysis
is primarily extra- vascular, so although
hemoglobinuria may oc- cur in rare cases,
acute renal failure and DIC are not generally
present. In some cases, the hemolysis occurs
without causing clinical symptoms. These
patients present with unex- plained anemia
or do not experience an in- crease in
hemoglobin concentrations follow- ing
transfusion.
Differential Diagnosis
Fever with hemolysis may also occur well
after transfusion when the component has
been contaminated with an intracellular red
cell parasite, such as malaria or babesiosis.
Fever without hemolysis may be an
indication of graft-vs-host disease (GVHD)
[described in the transfusion-associated (TA)-
GVHD section be- low] or transfusion-
transmitted viral disease (see Chapter 8).
Hemolysis resulting from anti- body
production by donor passenger lympho-
cytes may occur after transplantation of a
minor ABO-incompatible organ (eg, trans-
plantation of a group O liver in a group A
pa- tient).
Pathophysiology
After transfusion, transplantation, or
pregnan- cy, a patient may make an antibody
to a red cell antigen that he or she lacks. Red
cell anti- bodies may cause a delayed
transfusion reac- tion if the patient
subsequently receives a unit of blood
expressing the corresponding red cell
antigen. Primary alloimmunization occurs
anywhere from days to months after a
transfu- sion of antigen-positive RBCs,
depending on the immunogenicity and dose
of the antigen.
Approximately 1% to 1.6% of RBC
transfu- sions are associated with antibody
formation, excluding antibodies to antigens
in the Rh sys- tem. D-negative blood is
usually transfused to D-negative patients, so
the frequency attribut- able to anti-D is
relatively low. Newly formed alloantibodies
are routinely detected during pretransfusion
screening (see Chapters 15 and 16).
Recently transfused or pregnant patients
must have samples drawn for compatibility
testing within 3 days of the scheduled
transfu- sion to ensure identification of any
potential new alloantibodies.5(p36) A 5-year
retrospective study of alloimmunization
showed that 11 of 2932 patients (0.4%) had
developed new anti- bodies, including anti-
E, anti-K, and anti-Jka, within 3 days of their
transfusion.77
DHTRs and delayed serologic transfusion
reactions (DSTRs; rapid development of
allo- antibody in the absence of laboratory
evi- dence of hemolysis) rarely, if ever,
occur as a result of primary immunization
and are gener- ally associated with
subsequent transfusions. Antibody titers
may slowly decrease after the initial
immune response, with as many as 30% to
40% of alloantibodies becoming undetect-
able over months to years. Antibodies
against antigens of some blood group
systems, such as the Kidd system, frequently
exhibit this behav- ior. Subsequent
transfusion of an antigen-pos- itive unit
triggers an anamnestic response, with
production of antibody occurring over the
next several days to weeks after the trans-
fusion. The rapidity of antibody production
and hemolytic potential of the antibody
com- bine to influence the clinical
presentation. Blood group antibodies
associated with DHTRs/DSTRs include
those of the Kidd,
 CH A P T E R 2 7 Noninfectious Complications of Blood Transfusion
Duffy, Kell, and MNS systems, in order of
de- creasing frequency.78(pp358-89)
In a DHTR/DSTR, antibodies may be
found in the serum, on transfused red cells, or
both. Routine antibody screening and anti-
body identification should be possible. If
transfused red cells are still present, the DAT
result may be positive. When the DAT result is
positive, an eluate should be performed and
the antibody identified. If a segment from the
unit is available, antigen typing may confirm
the diagnosis.
Frequency
As with AHTRs, the estimated rate of
DHTRs varies widely from study to study.
Some of this variation is the result of the
practice of consid- ering DSTRs and DHTRs
as one category. Also, improvements in
laboratory techniques have contributed to
the increased number of DSTRs detected. It
is known that these reactions oc- cur much
more frequently than AHTRs, with
estimates of approximately 1:2500 for either
type of delayed reaction and a frequency
that is twice as high for DSTRs.79 These
reactions are likely to be greatly
underrecognized be- cause most patients do
not undergo red cell antibody screening
following transfusion.80
Treatment
The treatment of DHTRs consists of
monitor- ing the patient and providing
appropriate sup- portive care. The most
frequent therapy is correction of the anemia
by transfusing anti- gen-negative RBCs as
needed. When a DSTR is identified by the
laboratory, the patient’s phy- sician and the
transfusion service director should be
notified so that unrecognized he- molysis
may be appropriately identified and treated.
Prevention
DHTRs/DSTRs caused by known antibody
specificities can be prevented by the transfu-
sion of antigen-negative RBCs. It is
essential to obtain prior transfusion records
for the recipi- ent because alloantibodies
may have been identified that are no longer
detectable but re-
■ 687
quire transfusion of antigen-negative RBCs.
Many institutions have programs to provide
at least partially phenotypically matched
blood for patients with sickle cell disease
and some other patients who have developed
multiple alloantibodies. Patients with sickle
cell disease may develop a complication
known as “sickle cell HTR,” where
autologous and allogeneic cells are
destroyed (see Chapter 23).
Refractoriness to Platelet Transfusions
See Chapter 18.
TA-GVHD
Presentation
The clinical manifestations of TA-GVHD typi-
cally begin 8 to 10 days after transfusion, al-
though symptoms can occur as early as 3
days and as late as 30 days. Signs and
symptoms in- clude a maculopapular rash,
fever, enterocoli- tis with watery diarrhea,
elevated liver func- tion test results, and
pancytopenia. The rash begins on the trunk
and progresses to the extremities. In severe
cases, bullae may develop.81
Unlike GVHD after allogeneic marrow
transplantation, TA-GVHD leads to profound
marrow aplasia, with a mortality rate higher
than 90%. The time course of the reaction is
very rapid; death typically occurs within 1 to 3
weeks of the first symptoms.
Differential Diagnosis
Because the clinical manifestations of TA-
GVHD appear several days after a
transfusion, it may be difficult to associate
the patient’s symptoms with the transfusion.
The symp- toms can easily be attributed to
other condi- tions, including drug reactions
and viral ill- ness. In cases of TA-GVHD, a
skin biopsy reveals a superficial
perivascular lymphocytic infiltrate, necrotic
keratinocytes, compact or- thokeratosis, and
bullae formation. Molecular techniques,
including HLA typing, cytogenet- ics, and
chimerism assessment, can be used to make
the diagnosis.82
688 ■ AABB T EC HNIC AL MANUAL
Pathophysiology
Billingham83 has proposed three
requirements for GVHD to develop in a
patient. First, there must be differences in
the HLA antigens expressed between the
donor and the recipi- ent. Second,
immunocompetent cells must be present in
the graft. Finally, the host must be incapable
of rejecting the immunocompetent cells.
In TA-GVHD, viable transfused lympho-
cytes mount an immunologic attack against
the transfusion recipient. Consistent with Bill-
ingham’s work, the three primary factors that
determine the risk of developing TA-GVHD
are the degree of recipient immunodeficiency,
number of viable T lymphocytes in the trans-
fusion, and degree of a population’s genetic di-
versity. Risk factors for developing TA-GVHD
include leukemia, lymphoma, use of immuno-
suppressive drugs administered for transplant
or myeloablative chemotherapy, congenital
immunodeficiency disorders, and the neona-
tal state.84,85
The number of viable lymphocytes in a
transfusion can be affected by the age,
leuko- cyte-reduction status, and irradiation
status of the component.86 Unfortunately, the
mini- mum number of viable lymphocytes
required to cause TA-GVHD is unknown.
Although cur- rent leukocyte-reduction
technologies signifi- cantly reduce the
number of lymphocytes in a component,
leukocyte reduction does not eliminate the
risk of TA-GVHD. There are well-
documented cases in which transfusion of
leu- kocyte-reduced blood components have
caused TA-GVHD.87
Although patients who are immunocom-
promised are at risk of developing TA-GVHD,
the disorder has also been reported in transfu-
sion recipients with an intact immune sys-
tem.82 TA-GVHD can occur after a transfusion
from a donor who is homozygous for an HLA
haplotype to a heterozygous recipient (one-
way haplotype match). In this circumstance,
the recipient’s immune system is unable to
recognize the HLA-homozygous transfused
lymphocytes as foreign. In contrast, the trans-
fused lymphocytes are able to recognize the
host cells as foreign and are able to mount
an immunologic attack on the host.
The degree of genetic diversity in a popu-
lation also affects the risk of developing TA-
GVHD. For example, the estimated risk of de-
veloping TA-GVHD ranges from 1:874 in
Japan to 1:16,835 in France.82 The difference
is the re- sult of the lower diversity in HLA
antigen ex- pression in the Japanese
population than in the French population.
Treatment
Treatment of TA-GVHD has been attempted
with a variety of immunosuppressive agents.
Unfortunately, the disorder is almost
uniform- ly fatal; only rare cases of
successful treatment, many involving some
form of stem cell trans- plant, have been
reported. Therefore, empha- sis is placed on
prevention of the disorder.
Prevention
The only reliable way to prevent TA-GVHD is
by irradiation of cellular blood components.
AABB Standards requires a minimum dose of
25 Gy (2500 cGy) delivered to the central por-
tion of the container and a minimum of 15 Gy
(1500 cGy) elsewhere.5(p25) AABB Standards
also requires irradiation of cellular blood
compo- nents when 1) the patient is identified
as being at risk of TA-GVHD, 2) the donor is a
blood rel- ative of the recipient, and 3) the
donor is se- lected for HLA compatibility by
typing or crossmatching.5(p40) These standards
are mini- mum requirements for irradiation of
cellular blood components, and institutions
may choose to administer irradiated
components to other categories of patients
(see Table 27-2).
Posttransfusion Purpura
Presentation
Posttransfusion purpura (PTP) is a relatively
uncommon complication of transfusion, and
its true incidence is therefore difficult to
esti- mate. Nonetheless, more than 200 cases
have been reported in the literature, and data
from the Serious Hazards of Transfusion
(SHOT) program in the United Kingdom
suggest that
 CH A P T E R 2 7 Noninfectious Complications of Blood Transfusion ■ 689

TABLE 27-2. Clinical Indications for Irradiated Components

Well-documented indications
Intrauterine transfusions
Prematurity, low birthweight, or erythroblastosis fetalis in newborns
Congenital immunodeficiencies
Hematologic malignancies or solid tumors (neuroblastoma, sarcoma, Hodgkin disease)
Peripheral blood stem cell/marrow transplantation
Components that are crossmatched, HLA matched, or directed donations (from family members or other
related donors)
Fludarabine therapy
Granulocyte components
Potential indications
Other malignancies, including those treated with cytotoxic agents
Donor-recipient pairs from genetically homogeneous populations
Usually not indicated
Patients with human immunodeficiency
virus Full-term infants
Nonimmunosuppressed patients

the disorder may be more common than previ-
ously believed.87 Hospitals that participated in
SHOT reported 44 cases of PTP in 8 years.
Patients typically present with wet
purpura and thrombocytopenia 9 days (range
= 1-24), on average, after a transfusion.88
The thrombo- cytopenia is often profound,
with platelet counts of <10,000/µL.
Bleeding from mucous membranes and the
gastrointestinal and uri- nary tracts is
common. Mortality rates in large case series
range from 0 to 12.8%, primarily due to
intracranial hemorrhage.67,68
PTP has most commonly been
associated with transfusions of RBCs or
whole blood; however, the disorder has also
been associated with transfusions of
platelets or plasma.
Differential Diagnosis
The differential diagnosis of PTP includes
con- sideration of other causes of
thrombocytope- nia, such as autoimmune
thrombocytopenic purpura, thrombotic
thrombocytopenic pur- pura, heparin-
induced thrombocytopenia, DIC, and drug-
induced thrombocytopenia. Al- though the
diagnosis of PTP can be obvious in
patients with previously normal platelet
counts and no other significant medical ab-
normalities, it can be a challenge in patients
with multiple medical problems. Platelet se-
rology studies may aid in the diagnosis.
Pathophysiology
The pathogenesis of PTP is related to the pres-
ence of platelet-specific alloantibodies in a pa-
tient who has previously been exposed to
platelet antigens via pregnancy or transfusion.
The female-to-male ratio of affected patients
is 5 to 1. Antibodies against human platelet an-
tigen 1a (HPA-1a), located on glycoprotein
IIIa, are identified in about 70% of PTP cases.
Antibodies to HPA-1b, other platelet antigens,
and HLA antigens have also been implicated
in PTP.88
The reason for the concomitant destruc-
tion of autologous platelets in this disorder
is unknown; three theories have been
advanced. According to the first theory,
immune com- plexes bind to platelets
through the Fc recep- tor, causing
destruction of platelets.89 The sec- ond
theory posits that the patient’s platelets
690 ■ AABB T EC HNIC AL MANUAL
absorb a soluble platelet antigen from donor
plasma, making them susceptible to immune
destruction. According to the third theory,
the platelet alloantibody has autoreactivity
that develops when a patient is re-exposed
to a foreign platelet-specific antigen. 88 The
third theory currently has the most support.
The use of leukocyte reduction filters may
decrease the incidence of PTP. In the three
years prior to the implementation of
universal leukocyte reduction, in the United
Kingdom, there were 10.3 cases of PTP per
year, com- pared to 2.3 cases per year after
universal leu- kocyte reduction (p<0.001).87
Treatment
Because the duration of thrombocytopenia
in untreated patients is about 2 weeks, it can
be difficult to assess the effectiveness of
therapies for PTP. Steroids, whole-blood
exchange, and plasma exchange have all
been used to treat PTP. The current
treatment of choice for PTP is IVIG.88
Patients respond within 4 days, on av- erage,
and some respond within hours.
Prevention
PTP typically does not recur with
subsequent transfusions. However, there are
four case re- ports of PTP recurrence.
Therefore, for pa- tients with previously
documented PTP, efforts should be made to
obtain components from antigen-matched
donors. Autologous dona- tions and directed
donations from antigen- matched donors and
family members may also be appropriate.
Because PTP has also oc- curred after
transfusions of deglycerolized re- juvenated
or washed RBCs, such manipula- tions are
not indicated for the prevention of
recurrence.88
Iron Overload
A unit of RBCs contains approximately 250
mg of iron. The average rate of excretion of
iron is approximately 1 mg per day. As red
cells are destroyed, the majority of the released
iron cannot be excreted and is stored in the
body as
hemosiderin and ferritin. Transferrin
becomes saturated after the administration
of 10 to 15 RBC units to a nonbleeding
patient.90 As iron accumulates in the
reticuloendothelial system, liver, heart,
spleen, and endocrine organs, tis- sue
damage leading to heart failure, liver fail-
ure, diabetes, and hypothyroidism may
occur. Patients who are chronically
transfused for diseases such as thalassemia,
sickle cell dis- ease, and other chronic
anemias are at greatest risk for iron
overload. Prevention of the accu- mulation
of these toxic levels of iron by reduc- ing
the body’s iron stores through the use of iron
chelators is therefore extremely impor- tant.
A cumulative dose of 50 to 100 RBC units
can cause significantly greater morbidity
and mortality than the underlying anemia.91
Iron chelators bind to iron in the body
and tissues and help remove it through the
urine and/or feces. The development of iron-
chelating drugs, such as parenteral deferox-
amine and the oral agent deferiprone,
greatly reduced the complications of iron
overload in chronically transfused patients,
leading to im- proved quality of life.
Deferiprone is more ef- fective than
deferoxamine in reducing myo- cardial
siderosis.92
The newest agent, deferasirox, has a
much longer half-life than deferoxamine and
may be administered in one daily oral dose.
Al- though in-vivo studies are in the early
stages, deferasirox has similar rapid access
to intracel- lular iron stores in cultured
myocytes as defer- iprone.93 Unlike
deferiprone, which has occa- sionally been
associated with agranulocytosis, the most
frequent side effects of deferasirox are
transient gastrointestinal distress and mildly
increased creatinine levels that are rare- ly
of clinical significance. Deferasirox treat-
ment for a median of 2.7 years showed
efficacy in children and adults with sickle
cell disease, resulting in a significant dose-
dependent de- cline in their serum ferritin
levels without pro- ducing any new adverse
events.94 Lower doses of deferasirox have
also been used to reduce iron overload in
non-transfusion-dependent patients with
minimal side effects.94
 CH A P T E R 2 7 Noninfectious Complications of Blood Transfusion ■ 691

TABLE 27-3. How to Contact the FDA

MethodContact Details

E-mail fatalities2@fda.hhs.gov
Telephone/voicemail 240-402-9160
Fax 301-827-6748, Attn: CBER Fatality Program Manger
Express mail US Food and Drug Administration
CBER Office of Compliance and Biologics
Quality Document Control Center
10903 New Hampshire Avenue
WO71, G112
Silver Spring, MD 20993-0002
FDA = Food and Drug Administration; CBER = Center for Biologics Evaluation and Research.

FATALITY REPORTING formation for the FDA. The report should

REQUIREMENTS
When the death of a patient results from a
re- action to or complication of a
transfusion, cur- rent good manufacturing
practice regulations require that the fatality
be reported to the FDA by the facility that
performed the compatibili- ty testing.95 The
director of the Office of Com- pliance and
Biologics Quality at the FDA Cen- ter for
Biologics Evaluation and Research must be
notified as soon as possible by telephone,
express mail, or facsimile or electronic mail,
followed by the submission of a written
report within 7 days. Table 27-3 lists the
contact in-
con- tain the patient’s medical records,
including laboratory reports, and the autopsy
results when available. The patient’s
underlying ill- ness may make determination
of the cause of death difficult. If there is any
clinical suspicion that the transfusion may
have contributed to the patient’s death, that
possibility should be investigated. Most
transfusion-associated fa- talities are caused
by acute hemolysis, TRALI, or TACO.
Investigations of these cases must at- tempt
to rule out laboratory, transfusion ser- vice,
or blood administration errors.96

KEY POINTS

The blood supply is safer today than at any time in history. Hemovigilance and blood man- agement programs decrease the
Many transfusion reactions have signs or symptoms that may be present in more than one type of reaction. Early recognitio
Acute intravascular hemolytic reactions are often caused by sample or patient misidentifi- cation and are thus, for the most
Allergic reactions range from urticaria (hives) to anaphylaxis. Patients experiencing severe reactions should be checked for
Recognition of TRALI requires diagnosis by exclusion. Most patients recover from TRALI with supportive care.
TACO can be confused with TRALI because both feature pulmonary edema. TACO should be suspected in nonbleeding pa
692 ■ AABB T EC HNIC AL MANUAL

7. The most common complications of massive transfusion are hemostatic abnormalities.

Each institution should develop its own massive transfusion protocol that takes into ac-
count the availability of appropriate laboratory testing.
8. TA-GVHD has a much more acute and severe course than GVHD after marrow or stem
cell transplantation. TA-GVHD is fatal in >90% of cases and can be prevented by
irradiation of blood components.
9. PTP is a serious but rare complication in which antibodies to human platelet antigens
result in destruction of autologous and allogeneic platelets.
10. Iron overload is perhaps the longest-lasting long-term complication of transfusion. Oral
iron chelators greatly increase compliance with therapy.
11. TRALI is the leading cause of transfusion-related mortality reported to the FDA.
12. Recipient fatalities must be reported to the FDA by the compatibility testing facility as
soon as possible after a fatal complication of transfusion has been confirmed.

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45. Pomper GJ. Febrile, allergic, and non-immune
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61. Engstrom M, Schott U, Rommer B, Reinstrup
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Coagu- lation defects associated with massive
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51. Rana R, Fernandez-Perez E, Khan SA, et al.
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52. Narick C, Triulzi DJ, and Yazer MH. Transfu-
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in massive transfusion. Bibl Haematol
54. Olinger GN, Hottenrott C, Mulder DG, et al.
1980;46: 179-88.
Acute clinical hypocalcemic myocardial de-
68. Collins JA. Recent developments in the area of
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massive transfusion. World J Surg
1987;11:75- 81.
69. Plotkin AJ, Wade CE, Jenkins DH, et al. A re-
duction in clot
formation rate and
strength as- sessed by
thromboelastography is
indicative of
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transfusion requirements in patients with
penetrating injuries. J Trauma 2008;64:S64-8.
70. Reed RI, Ciaverella D, Heimbach DM, et al.
Prophylactic platelet administration during
massive transfusion: A prospective, random-
ized double-blinded clinical study. Ann Surg
1986;203:40-8.
71. Roback JD, Caldwell S, Carson J, et al. Evi-
dence-base practice guidelines for plasma
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72. Hedner U, Erhardson E. Potential role for
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73. Meyer E, Uhl L, Complications of massive
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74. Clark AD, Gordon WC, Walker ID, et al.
“Last- ditch” use of recombinant Factor VIIa
in pa- tients with massive hemorrhage is
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75. The CRASH-2 collaborators. The importance
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em- bolism. Arch Intern Med 1982;142:2173-
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77. Schonewille H, van de Watering LMG,
Loomans DSE, Brand A. Red blood cell
alloan- tibodies after transfusion: Factors
influencing incidence and specificity.
Transfusion 2006;46: 250-6.
78. Mollison PL, Engelfriet CP, Contreras M.
Blood transfusion in clinical medicine. 10th
ed. Ox- ford, UK: Blackwell Science, 1997.
79. Vamvakas EC, Pineda AA, Reisner R, et al.
The differentiation of delayed hemolytic and
de- layed serologic transfusion reactions:
Inci- dence and predictors of hemolysis.
Transfu- sion 1995;35:26-32.
80. Schonewille H, van de Watering LMG, Brand
A.Additional red blood cell alloantibodies
af- ter blood transfusions in
nonhematologic al- loimmunized patient
cohort: Is it time to take precautionary
measures? Transfusion 2006;46: 630-4.
81. Rühl H, Bein G, Sachs UJH. Transfusion-
asso- ciated-graft-versus-host disease.
Transfus Med Rev 2009;23:62-71.
82. Jacobson CA, Anderson KC, Alyea EP.
Transfu- sion-associated graft-versus-host
disease. In: Popovsky MA, ed. Transfusion
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83. Billingham R. The biology of graft-versus-
host reactions. In: The Harvey lecture series,
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84. Anderson KC, Weinstein HJ. Transfusion-
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85. Leitman SF, Tisdale JF, Bolan CD, et al. Transfu-
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86. Klein HG. Transfusion-associated graft-ver-
sus-host disease: Less fresh blood and more
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gray (Gy) for an aging population. Transfusion
2006;46:878-80.
87. Williamson LM, Stainsby D, Jones H, et al. The
impact of universal leukodepletion of the
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posttransfusion purpura and transfusion-as-
sociated graft-versus-host disease. Transfu-
sion 2007;47:1455-67.
88. McFarland JG. Posttransfusion purpura. In:
Popovsky MA, ed. Transfusion reactions. 4th
ed. Bethesda, MD: AABB Press, 2012:263-87.
89. Shulman NR, Aster RH, Leitner A, et al. Immu-
noreactions involving platelets. V. Post-trans-
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antibody against a genetically controlled
platelet antigen. A proposed mechanism for
thrombocytopenia and its relevance in “auto-
immunity.” J Clin Invest 1961;40:1597-620.
90. Ley TJ, Griffith P, Nienhuis AW. Transfusion
haemosiderosis and chelation therapy. Clin
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91. Sharon BI. Management of congenital hemo-
lytic anemias. In: Simon TL, Snyder EL, Sol-
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fusion medicine. 4th ed. Bethesda, MD:
AABB Press, 2009:448-69.
92. Cario H, Janka-Schaub G, Jarisch A, et al. Re-
cent developments in iron chelation therapy.
Klin Padiatr 2007;219:158-65.
a 93. Neufeld EJ. Oral chelators deferasirox and de-
feriprone for transfusional iron overload in
thalassemia major: New data, new
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94. Taher AT, Porter J, Viprakasit V, et al. Defera-
sirox reduces iron overload significantly
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696 ■ AABB T EC HNIC AL MANUAL

95. Code of federal regulations. Title 21, CFR Part

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transfusion.
 C h a p t e r 2 8

Approaches to Blood
Utilization Auditing

Alan Tinmouth, MD, FRCPC, MSc, and

Simon Stanworth, FRCP, FRCPath, DPhil

A U D I T I N G TH E USE of blood trans-

fusions is a required function for all
hos-
pital transfusion services.1 Both The Joint
Commission and AABB require health-care
in- stitutions to monitor the use of blood
compo- nents. The Joint Commission also
requires hospitals to collect data to monitor
the perfor- mance of processes that involve
risks or may result in sentinel events,
including the use of blood and blood
components (Standard PI.3.1.1).2 The
AABB requires all facilities to have a peer-
review program “that monitors and
addresses transfusion practices for all cat-
egories of blood and components” including
“usage and discard” and “appropriateness of
use.”3(p91) In addition, federal regulations per-
taining to Medicare and Medicaid require
that hospitals make recommendations to the
med-
ical staff regarding improvements in transfu-
sion procedures.1
Other countries also require hospitals to
establish processes to audit and monitor
transfusion practices. For example, in
Canada, both the Canadian Standard
Association and the Canadian Society for
Transfusion Medicine require hospitals to
monitor blood transfusion use, and
compliance with the Canadian Stan- dard
Association’s requirements will be man-
dated by federal law. In England, the
Depart- ment of Health’s Better Blood
Transfusion initiatives from 1997 to 2012
and the subse- quent Patient Blood
Management program have promoted and
continued to encourage hospital
participation in national audits. This
includes a specific program of National
Com- parative Audits for hospitals in
transfusion.

Alan Tinmouth, MD, FRCPC, MSc, Head, General Medicine and Transfusion Medicine, The Ottawa
Hospital, and Scientist, University of Ottawa Centre for Transfusion Research, Clinical Epidemiology
Program, Ottawa Hospital Research Institute, Ottawa, Ontario, Canada, and Simon Stanworth, FRCP,
FRCPath, DPhil, Consul- tant Haematologist, Oxford University Hospitals NHS Trust, and Honorary Senior
Clinical Lecturer, University of Oxford, Oxford, United Kingdom
A.Tinmouth is supported by a Research Award from the Department of Medicine, The Ottawa Hospital;
and has disclosed a financial relationship with Amgen. S. Stanworth has disclosed no conflicts of
interest.

697
698 ■ AABB T EC HNIC AL MANUAL
Factors that help ensure high levels of
partici- pation in national audits in England
are:
1. The audits contribute to “Quality Account”
reports required of National Health Service
(NHS) hospitals (Health Act 2009).
2. Data are used by the Care Quality
Commis- sion, an independent regulator
of all health and social care services in
England.
3. NHS hospitals participating in a national
audit may receive a discount from the
NHS Litigation Authority, which
manages negli- gence and other claims
against the NHS in England.
4. The National Blood Transfusion
Committee oversees, promotes, and
supports all national audits.
In a similar vein, The Joint Commission
announced a certification program starting
in 2014 to further recognize accredited
hospitals that implement system-wide
measures to im- prove blood utilization.
A number of different methods can be
employed to meet the requirements to moni-
tor or audit blood transfusion use. However,
the general objective of all blood utilization
programs is to ensure the appropriate use of
blood components. Alternative or comple-
mentary goals may include reducing inappro-
priate use and, as a result, reducing costs.
Historically, audits of blood use have concen-
trated on controlling or reducing the total
number of blood components transfused and/
or individual “overtransfusions.” This focus
was based on the assumption that inappropri-
ate use consisted primarily of overtransfusion.
Unnecessary transfusions result in unneces-
sary costs and adverse events. However, with
increased attention on limiting patients’ expo-
sure to blood components, undertransfusion
may be a concern. In addition, the dose of
blood transfusion requests, which may result
in overuse or underuse, might also need to be
assessed in audits. However, neither of these
issues has historically been a major focus of
the monitoring of blood transfusion utiliza-
tion.4
The methods used to audit blood use de-
pend on the desired objectives. Monitoring
trends in the total number of blood units
transfused, perhaps to control ordering or in-
ventory management, requires a different
pro- cess from that required to limit
inappropriate transfusion requests. In all
instances, the transfusion service
(technologists and/or phy- sicians) and the
institutional transfusion med- icine or blood
utilization committee must work together to
perform the appropriate au- dit(s) and
follow-up.
RATIONALE FOR MONITORING
BLOOD UTILIZATION
The primary motivation for monitoring
blood utilization is to identify instances
when blood product utilization is less than
optimal. Inter- ventions can then be
implemented to change transfusion practice.
Therefore, audits or mon- itoring must be
combined with a recognized process to
effectively provide feedback on the findings
to the appropriate health-care profes-
sionals.
Improving or ensuring optimal use of
blood transfusions is important for several
reasons. Blood is a biologic agent associated
with many possible adverse events,
including both infectious and noninfectious
complica- tions.5,6 Many of these
complications are well known, others are not
usually recognized by physicians or the
general population, and some potential
complications are still not fully understood.
Unnecessary complications from
inappropriate blood transfusions need to be
avoided. In addition, blood components are
a scarce and expensive resource. Transfusing
a single unit of Red Blood Cells (RBCs) has
been estimated to cost from $400 to $760
when all the associated costs are included,
and many regions have experienced
shortages.6-8
Through the careful monitoring of
blood utilization, instances of inappropriate
blood component use can be identified and
correc- tive actions can be taken. If a “real-
time” or prospective audit system is used,
then a mem- ber of the transfusion medicine
service can in- tervene, which may result in
a change in the transfusion request before
the issue of a blood unit. A retrospective
review does not alter the current transfusion
episode but it does identi-
 C H AP T E R 28 Approaches to Blood Utilization Auditing
fy issues that can be addressed through
inter- ventions designed to change future
transfu- sion practice.
A variety of interventions have been
used to change transfusion practice (Table
28-1). One of the most common
interventions for change is audit and
feedback, which is defined by the Cochrane
Effective Practice and Organi- zation of
Care Group as “any summary of clini- cal
performance of health care over a specified
period of time.” However, there is less
under- standing of the effective elements of
audit and feedback or how the feedback
should be struc- tured to produce changes in
behavior in differ- ent settings. Following
the delivery of an intervention, ongoing
monitoring allows for assessment of the
sustained effectiveness of the intervention
and need for ongoing or addi- tional
intervention.
In summary, audits serve the dual func-
tion of 1) identifying areas of concern in the
use of blood products and 2) monitoring
inter- ventions or changes in the identified
areas.
TYPES OF TRANSFUSION
AUDITS
Audits of transfusion practice can examine in-
dividual blood transfusion requests and/or ag-
gregate transfusion data. Reviews of
individual requests can be performed either
prospective- ly in real time or retrospectively.
Reviews of in- dividual transfusions may also
be performed (retrospectively) very shortly
after the event
TABLE 28-1. Use of Interventions to Change Transfusion Practice with Different Types of Audits
Intervention
Individual education/feedback
Group education/feedback/teaching
Guideline dissemination
Incorporation of reminders/guidelines into
transfusion request form or computerized
order entry system
++ = very complementary to audit type; + = can be complementary to audit type; – = less complementary or not
complemen- tary to audit type.
■ 699
(usually within 24 hours); this has been
termed a “concurrent review” because it still
affords the ability to provide timely
feedback on indi- vidual transfusion
episodes.9 More remote ret- rospective audits
allow for the review of aggre- gate
transfusion data, which can then be analyzed
in several ways. Reviews of individual and
aggregate transfusion data are not mutu- ally
exclusive. Because they may serve differ-
ent functions or purposes, they can, in fact,
be complementary.
Prospective (“Real-Time”) Audits
In a prospective audit, individual transfusion
requests are reviewed in “real time” (ie,
before the issue of the blood component).
An elec- tronic review using a computer-
based algo- rithm and/or a manual review by
technolo- gists is undertaken whereby the
request is compared to local transfusion
audit criteria. Clinical data (eg, hematocrit
and indication for RBC transfusion) are
required to perform the review. Ideally, this
information is obtained from the clinical
staff as part of the transfusion request
process10,11 or pretransfusion blood values are
automatically retrieved from the laboratory
information system.12,13 With com- puter
provider/physician order entry (CPOE), the
institutional guidelines can be incorporat- ed
into the request process.11 Laboratory staff
can also obtain these data from the
laboratory information system,14 although
this is more labor intensive and difficult to
implement
Prospective Audit Concurrent Audit Retrospective Audit
++ ++ +
– – ++
+ + +
++ ++ +
700 ■ AABB T EC HNIC AL MANUAL
universally.10 Transfusions that do not meet
the audit criteria are flagged and the request-
ing physician is contacted by the transfusion
medicine service before the blood
component is issued to discuss the need for
the transfu- sion.
A prospective audit system must be
orga- nized such that undue delays in filling
transfu- sion requests do not occur. For
example, when CPOE is used to screen the
appropriateness of a transfusion request that
does not comply with the institution’s
guidelines, that request may still be
completed after the ordering clini- cian
enters the rationale into the information
system.11 Delays in obtaining laboratory re-
sults and the uncertainties of clinical cases
also need to be identified and reflected in
the audit process.
Given these issues and constraints, the
criteria for undertaking a review as part of a
prospective audit may need to be less rigid
than the guidelines for the optimal
utilization of blood components because
prospective au- dits might delay some
transfusions that fail to meet audit criteria
for appropriateness.13 Therefore, a prac- tice.14,15,17-20,22,23 Although these reports
mechanism must be in place to ensure that
emergency and urgent transfu- sions are not
unduly delayed. Specific clinical areas, such
as emergency departments and operating
suites, where delays in filling trans- fusion
requests might result in adverse clinical
events might be excluded from prospective
transfusion audits.
Prospective audits also have the
potential to generate animosity in the
ordering physi- cian when a transfusion
request is questioned. Engaging requesting
physician groups and broadly consulting
them during the develop- ment of the audit
guidelines and process can help reduce
friction and ensure the long-term success of
a prospective audit system. In addi- tion,
interactions with the requesting physi- cian
often require the involvement of a trans-
fusion medicine physician or, at least, a
senior technologist.14,15 To reduce the
potentially onerous time requirements for
technologists and physicians in a practice. However, follow-up with the re-
prospective audit system, selective audits of
either specific clinical areas (eg, obstetrics
or orthopedics) and/or specific time periods
may be used.
The potential benefits of a prospective
audit include the ability to intervene directly
and to change a transfusion request before
the component is issued. As a result, an
unneces- sary transfusion may be stopped 12
or a more appropriate component or dose
may be or- dered.16 Ideally, the immediate
intervention stemming from a prospective
audit also results in long-term changes in the
transfusion prac- tice of the ordering
physician. However, the benefits of this
immediate intervention must be weighed
against the additional work re- quired by the
transfusion service, including the
transfusion medicine physician.
A number of reports from single institu-
tions have demonstrated the effectiveness of
prospective audits in reducing the total num-
ber of units transfused,14,15,17 the number of
units transfused per patient,18-20 the propor-
tion of patients transfused,18,19,21,22 and the
number of inappropriate transfusions19,22,23
(Table 28-2). Many of these studies used
pro- spective audits in conjunction with
other in- terventions to change transfusion
attest to the potential utility of prospective
audits to change transfusion practice, the
sustainability of the changes is not known.
In one study, the initial reduction in
inappropriate transfusions following the
implementation of a prospective audit
system did not persist when inappropri- ate
transfusion rates were reexamined 3 years
later.27
Concurrent Audits
A concurrent audit is a review of an
individual transfusion request that occurs in
the 12 to 24 hours following the transfusion
episode.9,13 As such, it involves processes
similar to those of the prospective audit.
However, because a concurrent audit is a
posttransfusion review, it cannot lead to any
alterations in the individual transfusion
event. Therefore, the concurrent audit is
designed only to alter future transfu- sion
questing physician occurs while the transfu-
sion event remains fresh in his or her
memory. It is hoped that this immediate
contact im-
 Study
TABLE 28-2. Studies of the Effectiveness of Prospective and Retrospective Audits in Changing Transfusion
Practice

Cohn 201313 Concurrent audit RBCs 5%-14% per - - -

Arnold 201124 Education, form, month - - -
FFP
audit/feedback 14%
Tavares 201115 80.8%
FFP
Education, prospective audit
Sarode 201014 RBCs 12.6%
- - -
Prospective audit, guidelines, FFP

Interventions
C HA P TER 28

- - - 59.9%
education
Platelets - - - 25.4%
RBCs - - - 15%
Yeh 200617 Prospective audit, audit/
FFP
feedback - - - 74%
Platelets - - - 14%
RBCs - -
Rubin 200125 Audit/feedback, education 2% -
Capraro 200126 Audit/feedback, education RBCs - - -
FFP 26%
7%
Platelets 6%
RBCs
Tobin 200127 Prospective audit, +4% (increased use) - -
FFP
guidelines +13% (increased
Blood Components

Platelets use)
28
FFP +14% (increased
Approaches to Blood Utilization Auditing

Hameedullah 2000 Audit/feedback, guidelines, use) 21% - -

edu- cation
Reduction
Reduction

Prospective audit, form

Rehm 199818 RBCs - - 26% -
inin

Joshi 199729 Retrospective audit, guidelines RBCs 20.7% 50% - -

■

(Continued)
Proportion
Inappropriateof
701

Reduction
Patients
Transfusions
in Number
Transfused
of Units
 Study
TABLE 28-2. Studies of the Effectiveness of Prospective and Retrospective Audits in Changing Transfusion Practice
702

(Continued)
■

Tuckfield 199723 Prospective audit, form RBCs 13% - - -

FFP 10% - - -
Platelets 10% - - -
FFP 7% - 35% 31%
Cheng Prospective audit, form
199619 Platelets 10% - 22% 17%

Interventions
RBCs - - 12% 62%
Morrison Retrospective audit,
199330 education, guidelines, form
Prospective audit, guidelines
Littenberg 199522 RBCs - 4.1% - 1%
Brandis 199431 Retrospective audit, education - - 19%
RBCs 29%
Hawkins 199421 Prospective audit - - 55%
FFP -
Rosen 199332 Retrospective audit, form, RBCs - - 27%
21%
guidelines FFP
AABB T ECHNICAL M A NUAL

- - 18% 9%
Platelets - - 23% 15%
FFP - - - 46%
Ayoub 198933 Retrospective audit, education,
guidelines
Blood Components

Retrospective audit,
Giovanetti 198834 RBCs 67% 10.9% - -
guidelines
Solomon 198820 FFP - - - 52%
Prospective audit, retrospective
Reduction

audit, guidelines, education,

Reduction

Shanberge form FFP - - - 77%

inin

198735
Concurrent audit, education,
Handler 198336 guidelines RBCs - - - -
RBCs = Red Blood Cells; FFP = Fresh Frozen
Proportion

Plasma.
Inappropriateof
Reduction
Patients
Transfusions
in Number
Transfused
of Units
 C H AP T E R 28 Approaches to Blood Utilization Auditing ■ 703

proves the chance of changing the implementation of massive transfusion

physician’s future transfusion behavior. proto- cols) or communication with the
As with the prospective audit, the transfusion service might not be readily
concur- rent review can be conducted by a recalled in retro- spective audits.
computer algorithm or manually using
transfusion audit criteria. The review may Retrospective Audits
occur at the time of the transfusion or
Retrospective audits of blood utilization are
shortly after the request is filled. Requests
commonly performed by hospital teams or
that do not meet the audit cri- teria are
blood transfusion services. The results
flagged for subsequent review by a se- nior
should be reviewed by the hospital’s
technologist or transfusion medicine phy-
transfusion med- icine committee. The
sician. When performing the review, the
frequency of such audits varies from one
transfusion medicine physician may have
institution to another. Com- puterized
ac- cess to additional laboratory results that
systems may facilitate ongoing utili- zation
help to determine the appropriateness of
review across many specialties.11 Unlike
each re- quest. However, the reviewer must
prospective and concurrent reviews, retro-
also con- sider the fact that these additional
spective audits can be used to examine
results were not available to the ordering
aggre- gate transfusion data and trends in
physician at the time he or she made the
transfusion utilization. The results may be
request. Because con- current audits do not
informative for understanding wider
delay the filling of trans- fusion requests,
differences in transfu- sion practice across
these audits can assess urgent transfusions
different specialty groups. Individual
and use stricter criteria for appro- priateness
transfusion requests can also be reviewed
than prospective audits.
Following the identification of an inap- for appropriateness as part of a retro-
propriate transfusion request, the ordering spective audit, but doing so may be cumber-
physician may be contacted by telephone or some unless the data are collected prospec-
e- mail to discuss the case. The less tively or the laboratory information system
immediate nature and ability to use written can link data on transfusion events and labo-
communica- tion may reduce any animosity ratory results.
Retrospective reviews can analyze data in
from the order- ing physician and result in a
a variety of ways. The simplest analysis is
more meaningful dialogue. Shanberge and
an examination of the total numbers of
colleagues35 report- ed a 77% reduction in
blood components transfused and patients
the number of Fresh Frozen Plasma units
trans- fused. However, these data might
transfused after the intro- duction of a
show con- siderable temporal variability that
concurrent audit system com- bined with a
limits meaningful analysis. Further analysis
new guideline and an education program.
is re- quired to make observations regarding
Conducting concurrent audits is time
differences in blood utilization. The mean or
consuming, and a transfusion medicine
median number of units transfused per
physi- cian usually needs to review
hospi- talized patient and/or procedure
inappropriate transfusion requests and
provides a more meaningful summary of
follow up with the re- questing physicians.13
blood compo- nent use. Simply dividing the
For these reasons, per- forming a concurrent
total number of units transfused by the total
audit of all transfusion requests may not be
number of pa- tients who received a
feasible. Using selective audits, as discussed
transfusion is not an ap- propriate statistical
for prospective audits, is an option to reduce
analysis; any observed changes in total
the workload involved. Concurrent audits
blood component use, partic- ularly during a
may be particularly relevant for certain
short period, could be skewed by a small
patient groups, such as patients treated for
number of patients who required a large
trauma or others in the emergency
number of units. The proportion of pa- tients
department, where the urgent need for blood
transfused should also be determined and
precludes a prospective audit, and accurate
can be further examined by procedure or
time lines (key to determining the
appropriate
704 ■ AABB T EC HNIC AL MANUAL
clinical specialty. All of these analyses com-
prise the minimal set required to monitor
dif- ferences in blood utilization. Additional
analy- ses could include assessments of
appropriate and inappropriate transfusion
rates, although these analyses require
collecting additional clinical data and
reviewing individual transfu- sion requests,
which may be labor-intensive exercises.
Utilization trends by individual product,
individual physician, or clinical service can
be analyzed. For health-care institutions
with multiple sites, similar clinical services
at different sites can also be compared.
These additional layers of analysis may
provide im- portant information to identify
variations in practice and detect
inappropriate transfusion practices. These
areas could then be targeted by interventions
to improve transfusion prac- tice.
In general, a retrospective review is less
labor intensive than a prospective or concur-
rent review, particularly because it involves
less immediate attention from a technologist
or transfusion medicine physician. The
amount of time required to perform the
review depends on the amount of detail
desired.
The use of retrospective audits and the
provision of dedicated feedback to clinicians
have been effective in reducing the total
num- ber of units transfused,20,24,29-33 number
of units transfused per patient,30-32,37,38
proportion of patients transfused,26,28,36 and
number of inap- propriate transfusions24,29,34
(Table 28-2). The long-term durability of
these changes in trans- fusion practice
following the introduction of a retrospective
audit has not been evaluated.
INTERVENTIONS TO CHANGE
TRANSFUSION PRACTICE
As previously described, the results of
transfu- sion audits should ideally be used in
conjunc- tion with interventions to effect
change and improve transfusion practice.
Thus, audits should be considered a critical
component of a larger process to optimize
the utilization of blood components. This
process can be more widely conceived as
dissemination, knowledge transfer, or
implementation work, where the
goal is to increase the uptake of research re-
sults and/or best practices into daily prac-
tice.39
The process of closing the gap between
knowledge and action involves first
distilling knowledge, usually in the form of
guidelines. National or international
guidelines can sim- ply be adopted;
however, local development of guidelines
with the involvement of local stake- holders
may increase adherence to guide- lines.40
Audits then serve the purpose of identi-
fying gaps between the guidelines (best
practices) and current practice (action).
When important gaps are identified through
audits, interventions can be developed to
target prac- tice changes. To improve the
chance of achiev- ing meaningful and
lasting changes in prac- tice, interventions
should be carefully chosen and designed.
Unfortunately, the process of
identifying the best interventions has not
been well de- fined. Ideally, the facilitators
of and barriers to change are first identified
so that any selected intervention targets
these identified fac- tors.41,42 Concurrent,13
retrospective,37 and prospective21 audits have
been effective indi- vidual interventions in
changing transfusion practice. However,
other interventions, includ- ing
dissemination of guidelines,11,20,28,30,32-35 ed-
ucation,15,20,24,30,33,36 introduction of new trans-
fusion request forms, and computer order
entry,24,30,32 have been commonly used in
con- junction with audits.
EFFECTIVENESS OF
MONITORING AND
INTERVENTIONS TO CHANGE
TRANSFUSION PRACTICE
Most published studies on auditing efforts
have shown that prospective and
retrospective audits reduce either the total
amount of blood transfused or the
proportion of inappropriate transfusions
(Table 28-2). These reductions occurred in
studies that used a concurrent au- dit alone,13
a prospective audit alone,21 or a ret-
rospective audit and feedback alone.37 The
prospective audit study showed a reduction
in the utilization of RBCs and frozen plasma
but
 C H AP T E R 28 Approaches to Blood Utilization Auditing
not platelets.21 The retrospective audit study
showed a reduction in the utilization of
RBCs but not frozen plasma or platelets. 37
The re- sults from these two studies might
suggest that prospective and retrospective
audits used in conjunction with other
interventions are more effective in
improving blood utilization. How- ever, the
poor designs of the studies (almost all of
which were uncontrolled, single-center, be-
fore-and-after studies) that examined inter-
ventions to change transfusion practice and
the possibility of publication bias (eg,
because the results of studies in which an
intervention did not result in an
improvement in transfu- sion practice were
not published) do not allow the true or
relative effectiveness of individual, or
combinations of, interventions to be deter-
mined.43,44
SELECTING AN AUDIT PROCESS TO
MONITOR TRANSFUSIONS
How transfusions should be monitored de-
pends on a number of factors that are
specific to each institution.45 The first step in
deter- mining how to monitor transfusions is
to de- cide which type of audits to use. The
prospec- tive and concurrent audits serve
identical purposes and cannot be used
jointly to assess an individual transfusion
episode. Prospective audits require the
greatest amount of time from laboratory
staff and transfusion medi- cine physicians,
including 24-hour coverage and support
from the laboratory information system.
These requirements may present diffi-
culties for smaller hospitals and busy
transfu- sion medicine services, respectively.
In gener- al, concurrent reviews are more
practical in smaller hospitals or hospitals
with limited re- sources for laboratory staff
or transfusion medicine physicians because
transfusions can be reviewed in the 12 to 24
hours following the transfusion episode. If a
universal prospective audit of all blood
components is not feasible, then the
prospective audit could be limited to
particular components (eg, intravenous im-
mune globulin or recombinant Factor VIIa)
or specific clinical areas. Retrospective
reviews supplement prospective
concurrent audits
■ 705
of individual transfusions by providing data
on trends in the use of transfusions.
Lists of the steps for implementing pro-
spective, concurrent, and retrospective re-
views are provided in Tables 28-3, 28-4, and
28-5, respectively. A transfusion request
form or order entry system incorporating
guidelines and/or clinical information (see
sample in Ap- pendix 28-1) can aid in
identifying inappropri- ate transfusion
requests through either pro- spective or
concurrent audits.46 Similarly laboratory
information systems and comput- er
algorithms can be used to screen transfu-
sions for appropriateness in all types of au-
dits.13,47 Reviews may be more frequent in
larger transfusion services or less frequent in
smaller hospitals. The retrospective data can
allow smaller hospitals to compare their
trans- fusion practices (eg, number of units
trans- fused per patient by diagnosis-related
group) with those of other similar
institutions.
CONCLUSIONS
Auditing transfusion practice is a required
function for all transfusion services.
Transfu- sion audits provide information on
levels of compliance with standards and
frequency of unnecessary transfusions.
Audits combined with guidelines are clearly
essential to evaluate transfusion practices at
individual institutions, and they permit the
identification of subopti- mal transfusion
practices.
In general, the findings of most audits
continue to show unnecessary use of blood
outside established guidelines. The
translation of research findings into hospital
practice is often slow and haphazard, and
this applies as much to transfusion as to
other branches of health care. Many
interventions are undertak- en by hospitals
to change their transfusion practices, but
there are real uncertainties about their
effectiveness and durability.
There is a need for research that defines
the determinants of appropriate transfusion
behavior to better guide the design and
selec- tion of interventions that can produce
opti- mal changes in transfusion practice.
Ideally, the selection of audits and
or
interventions should be based on an
assessment of local
706 ■ AABB T EC HNIC AL MANUAL

TABLE 28-3. Steps for Implementing a Prospective Audit System to Monitor Blood Component
Utilization
1. The extent and frequency of the audit process is determined.
■ The audit may be performed on all blood components or only on specific blood products.
■ Areas with urgent needs for transfusion (eg, emergency or operating rooms) may be excluded from
the audit to avoid delays in transfusion for their patients.
■ To limit resource demands, a selective audit may be used. For example, transfusion requests may be
audited only from a single ward, selected on a rotating basis, because of its high transfusion volume
or a perceived problem in its transfusion practice.
■ Similarly, audits may be performed only during specific hours to limit off-hour work by laboratory
staff and transfusion medicine physicians.
2. The requirements for clinical information at the time the transfusion requests are met.
■ This may be part of the request form (Appendix 28-1) or computer order entry.
3. Criteria for appropriate or inappropriate indications are developed for transfusions.
■ Audit criteria should be less stringent than optimal transfusion guidelines to reduce audit workload
and conflicts with requesting physicians.
■ Audit criteria should be developed in conjunction with a transfusion medicine committee and
various medical specialists who use significant amounts of blood products to increase acceptance of
transfusion audits.

4. Transfusion requests are screened by laboratory staff, transfusion nurses, or a computer algorithm to
iden- tify inappropriate requests.
5. For all inappropriate transfusion requests, the requesting physician is contacted by the transfusion
medi- cine service.
■ The requesting physician is usually contacted by a transfusion medicine physician or senior technologist.
■ A predefined mechanism is developed to override/bypass a review if blood is required urgently and
to avoid unacceptable delays in filling a transfusion request.
6. Any decisions to change the transfusion request are made in conjunction with the ordering physician.

TABLE 28-4. Steps for Implementing a Concurrent Audit System to Monitor Blood Component
Utilization
1-4. The same as for the Prospective Audit.
5. All inappropriate transfusion requests are subsequently reviewed by senior laboratory staff or a
transfu- sion medicine physician who discusses the transfusion request with the requesting
physician.
■ Contact with the requesting physician is made within 24 hours of the transfusion so that the
physician remembers the clinical details. This time frame is optimal for providing feedback and
potentially chang- ing future transfusion behavior.
■ Contact can be made by telephone or electronically.
 C H AP T E R 28 Approaches to Blood Utilization Auditing ■ 707

TABLE 28-5. Steps for Implementing a Retrospective Audit System to Monitor Blood Component
Utilization
1. Determine the extent and frequency of the audit reviews.
■ The frequency of the data reviews needs to be based on the volume of transfusions and resources
avail- able. Records should not be reviewed more than 6 months after transfusions were
administered.
■ The components to be reviewed need to be determined and should include all conventional
components and frequently transfused blood derivatives.
2. Determine the outcomes.
■ Totals for the numbers of transfusions and patients transfused are the simplest data to collect
but pro- vide a limited understanding of, and ability to monitor, changes in transfusion utilization.
Providing data on utilization by patient (ie, mean or median number of units per patient) and
proportion of patients transfused provides a greater understanding of utilization.
■ The appropriateness of transfusions can also be reported if clinical and/or laboratory data are
available. These can be aggregate data from prospective or concurrent reviews, or the laboratory
information sys- tem can link laboratory data to data on transfusion events.
3. Review the audit data.
■ Audit data should be reviewed by the transfusion medicine service and the transfusion medicine
commit- tee.
■ Data may be analyzed on individual physicians, departments, or diagnoses.
■ Data may be compared within the institution or with data from similar institutions to evaluate overall
utili- zation rates for blood components.
■ Data should be shared with relevant departments and physicians.
4. Determine whether any additional interventions to optimize transfusion practice are warranted.

barriers to and facilitators of change. Further fective in certain circumstances or to

audits then allow the monitoring of any
changes in transfusion practice.
Future studies are required to determine
what interventions are likely to be the most
ef-
compare different interventions, including
their cost-ef- fectiveness. Nonetheless,
audits remain a criti- cal part of the process
to evaluate blood utili- zation and improve
transfusion practice.

KEY POINTS

Auditing the use of blood components is a necessary function for all transfusion medicine services and is required by AABB
Different forms of audits (prospective, concurrent, or retrospective) may be used depending on the objectives of the audit a
Prospective audits require the review of transfusion requests before issue of components, which permits the opportunity to
708 ■ AABB T EC HNIC AL MANUAL

4. Concurrent audits review individual transfusion requests in the 12 to 24 hours

following a transfusion episode and involve similar processes as the prospective audit.
The individual transfusion events cannot be altered, as they have already occurred, but
feedback to the physician within a short period after the transfusion offers the
opportunity to change future transfusion practice.
5. Retrospective audits of transfusion offer the opportunity to review aggregate transfusion
data. Reviewing individual transfusions for appropriateness may be more difficult,
given the remoteness of the transfusion event. Feedback to individual clinicians or
clinical servic- es should be part of a retrospective audit process to help ensure the
optimal use of compo- nents.
6. All forms of audits can be used alone to effect changes in transfusion practice. Audits
may also be combined with other interventions such as dissemination of guidelines,
education, and the introduction of new transfusion request forms. Ideally the selection
of interventions includes local stakeholders and an assessment of local factors that may
affect the effective- ness of interventions.
7. Published studies evaluating the effect of prospective and retrospective audits on transfu-
sion practice have generally demonstrated a reduction in either the total amount of
blood transfused or the proportion of inappropriate transfusions. Because of the poor
quality of published studies, the true relative effectiveness of individual interventions or
combina- tions of interventions cannot be inferred.

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42. Legare F, O’Connor AM, Graham ID, et al.
Pri- mary health care professionals’ views
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43. Wilson K, MacDougall L, Fergusson D, et al.
The effectiveness of interventions to reduce
physician’s levels of inappropriate transfusion:
What can be learned from a systematic review
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44. Tinmouth A, MacDougall L, Fergusson D, et
al. Reducing the amount of blood transfused:
A systematic review of behavioral
interventions to change physicians’
transfusion practices. Arch Intern Med
2005;165:845-52.
45. Qu L, Kiss JE. Blood utilization review. In:
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 C H AP T E R 28 Approaches to Blood Utilization Auditing ■ 711

■ APPENDIX 28-1
Transfusion Order Form in Use at St. Vincent Indianapolis Hospital Since 2001

Used with permission from Hannon T, Gross I. Transfusion guidelines: Development and impact on blood management. In: Saxena S, ed. Th
 C h a p t e r 2 9

The Collection and Processing of

Hematopoietic Stem Cells

Scott A. Koepsell, MD, PhD; Eapen K. Jacob, MD; and

David H. McKenna Jr, MD

HEMATOPOIETIC STEM CELLS (HSCs) poietic precursors to generate a niche that

are primitive pluripotent cells capable
of self-renewal and differentiation into any
cells of hematopoietic lineage (lymphocytes,
mono- cytes, granulocytes, erythrocytes, and
plate- lets), including committed and
lineage- restricted progenitor cells, unless
otherwise specified, regardless of tissue
source [eg, mar- row, mobilized peripheral
blood, or umbilical cord blood (UCB)].1
Clinically, HSCs are able to fully reconstitute
the functions of marrow when transplanted
into susceptible recipients. For this reason,
HSC transplantation has been increasingly
utilized to treat a diverse array of
hematologic and nonhematologic diseases
and conditions.
In vivo, HSCs are concentrated in the
marrow, where mesenchymal elements—such
as osteogenic progenitor cells, osteoblasts, ad-
ipocytes, mesenchymal stem/stromal cells,
and endothelial cells—interact with hemato-
supports and regulates hematopoiesis.2
Although the cell-surface antigen CD34
is not specific to HSCs, CD34 is used to
identify and quantitate HSCs by flow
cytometry in cel- lular products intended for
use in transplanta- tion. Historically, CD34
quantitation by flow cytometry suffered
from significant interin- strument and
interprotocol variability, a limi- tation that
has been addressed by more rigor- ous
quality control (QC) procedures and assay
standardization.3,4
CLINICAL UTILITY
Myriad indications exist for HSC transplanta-
tion that range from nonneoplastic immune
disorders to malignancies. An abbreviated list
is presented in Table 29-1. In general, indica-
tions vary with patient age because immuno-
deficiencies and inborn errors of metabolism

Scott A. Koepsell, MD, PhD, Assistant Professor, Department of Pathology and Microbiology, University of
Nebraska Medical Center, Omaha, Nebraska; Eapen K. Jacob, MD, Assistant Professor, Department of
Labora- tory Medicine and Pathology, Mayo Clinic, Rochester, Minnesota; and David H. McKenna Jr, MD,
Associate Professor, Department of Laboratory Medicine and Pathology, University of Minnesota Medical
School, Min- neapolis, Minnesota
S. Koepsell and E. Jacob have disclosed no conflicts of interest. D. McKenna has disclosed a financial
relation- ship with Novartis, Inc.

713
714 ■ AABB T EC HNIC AL MANUAL

TABLE 29-1. Diseases Treated with are more common in a pediatric population,
Autologous or Allogeneic Transplantation whereas the number of patients with a clonal
disorder in their marrow or a hematologic ma-
Hematologic Cancers lignancy is greater in adults. Ultimately, the
Leukemia decision to perform HSC transplantation re-
quires a complex integration of many vari-
Lymphoma ables. These variables include patient goals,
Myeloma prognosis, disease progression, previous ther-
apy, age, availability of a suitable HSC source
Marrow Failure States/Clonal Disorders of (ie, marrow, mobilized peripheral blood, or
Marrow UCB), and type of transplant (ie, autologous
Aplastic anemia vs allogeneic, and myeloablative vs
nonmyeloab-
lative).
Fanconi anemia
Autologous Transplantation
Pure red cell
In general, autologous HSC transplantation is
aplasia

Amegakaryocytosis
used for hematopoietic rescue after high-dose
Paroxysmal nocturnal hemoglobinuria antineoplastic therapy. The antitumor effect
Myelofibrosis of the transplant comes solely from the
chemo-
therapy and radiotherapy used during the con-
Myelodysplasia ditioning phase of transplantation. For older
Inborn Errors of Metabolism/Congenital Immuno-
patients who are not traditional candidates
deficiency
for autologous transplantation or patients
with
other significant morbidities, reduced-intensi-
Mucopolysaccharidoses ty induction chemotherapy can broaden the
Leukodystrophies
clinical utility of HSC transplantation.
Donor requirements for autologous
Osteopetrosis transplants are based on the donor’s disease
state. The patient must be healthy enough to
Severe combined immunodeficiency
undergo mobilization (as described later in
syndrome Wiskott-Aldrich syndrome this chapter) and procurement of HSCs
either by peripheral blood apheresis
Pediatric Cancers
collection or marrow aspiration. Significant
Wilms tumor levels of prior chemotherapy, radiation, or
ongoing marrow
disease involvement may make the mobiliza-
Neuroblastoma tion and collection of HSCs unfeasible due to
Ewing sarcoma the reduced quality or number of HSCs.
Eligibility requirements are not
Medulloblastoma mandated by the Food and Drug
Rhabdomyosarcoma Administration (FDA) for autologous HSC
transplantation [Title 21,
Code of Federal Regulations (CFR) Part
Hemoglobinopathies 1271.90], so the use of screening question-
Thalassemia naires to identify relevant communicable dis-
ease is not required. However, a general health
Sickle cell disease assessment is needed per AABB Standards for
Autoimmune Diseases Cellular Therapy Product Services (CT Stan-
dards).1 AABB CT Standards also requires labo-
Solid Tumors
r hepatitis B virus; hepatitis C
a
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y

t
e
s
t
i
n
g

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o
r

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u
m
a
n

i
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u
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o
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e
f
i
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i
e
n
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y

v
i
r
u
s

(
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I
V
)

1
/
2
;
 CH A P T E R 2 9 Hematopoietic Stem Cells
virus; syphilis; human T-cell lymphotropic vi-
rus, Types I and Type II (HTLV-I/II); and cyto-
megalovirus (CMV) because the autologous
products are cryopreserved and stored with
other products, so the presence of these virus-
es would pose a contamination risk.
Allogeneic Transplantation
Indications for allogeneic HSC transplantation
vary. However, in general, allogeneic trans-
plantation is used to treat malignant condi-
tions only when both the induction antineo-
plastic therapy and the transplanted cells, due
to the graft-vs-neoplasm (GVN) effect, are
therapeutic. For patients with an inborn error
of metabolism, congenital immunodeficiency,
or other diseases and conditions in which
germline mutations are present in the patient’s
cells, allogeneic HSC transplants offer thera-
peutic benefit by helping to replace the defi-
cient cellular machinery.
In the allogeneic setting, screening and
infectious disease testing are mandated in
the United States to determine whether the
trans- plantation of mobilized peripheral
blood or UCB poses a risk of transmission
of a relevant communicable disease to the
recipient [Title 21 CFR 1271.3(r)]. This
screening and testing includes the
administration of a screening questionnaire,
a physical examination, a re- view of the
relevant medical records, and ap- plicable
testing [Title 21, CFR Parts 1271.3(s) and 21
CFR 1271 Subpart C]. Although marrow
products are administered under Sections
375 and 379 of the Public Health Services
Act, mar- row screening and testing are very
similar to mobilized peripheral blood and
UCB screen- ing and testing because the
administration of all of these products is
governed by the stan- dards of various
accrediting bodies, such as the AABB, the
Foundation for the Accredita- tion of
Cellular Therapy (FACT),4 and the Na-
tional Marrow Donor Program (NMDP).5
For UCB transplantation, the screening
and testing process is performed on the
moth- er and her samples (see Chapter 30).
Relevant communicable diseases that can be
transmit- ted by transplanted HSCs to the
recipient or to the people who handle the
components and
■ 715
for which there is an FDA licensed screening
test [Title 21, CFR Part 1271.3(r)] include
HIV, hepatitis B and C, human transmissible
spon- giform encephalopathy, Treponema
pallidum, HTLV-I/II, and CMV. Donor
questionnaires have been developed to assist
with screening6 and medical record review
for these diseases using FDA guidance
documents.7
In the United States, infectious disease
testing for HSC donors must be performed
in a Clinical Laboratory Improvement Act
certified laboratory as mandated by the FDA.
If screen- ing or testing detects a risk of a
relevant com- municable disease, the
potential HSC donor is considered
ineligible. All parties (the donor, the
recipient, and their physicians) are in-
formed of the donor’s eligibility status, and a
risk-benefit analysis is performed to deter-
mine whether the donor’s HSCs should be
used. If a decision is made to proceed with a
transplant of the ineligible donor’s HSCs,
the urgent medical need, as defined by the
FDA [Title 21, CFR Part 1271.3(u)], is
documented. Depending on the institution’s
accrediting body and the circumstances,
such as when the HSCs are from a related
first- or second-degree ineligible donor, the
requirement for docu- mentation of an
urgent medical need may vary. Finally, in
addition to screening and test- ing for
infectious diseases, the donor’s medical
evaluation is used for determining whether
the donor’s health is adequate to allow that
do- nor to undergo the HSC mobilization
and pro- curement process (described
below).
Histocompatibility
In addition to infectious diseases, donor char-
acteristics that may affect transplant out-
comes include histocompatibility with the
recipient, gender, age, parity, and ABO com-
patibility. Of these characteristics, histocom-
patibility is the most important. In general, if a
healthy HLA-matched related donor is avail-
able, that donor is selected instead of an HLA-
matched unrelated donor. However, recent
data have shown that in patients with acute
myeloid leukemia or myelodysplastic syn-
drome, an HSC transplant from an HLA-
716 ■ AABB T EC HNIC AL MANUAL
matched, unrelated donor may be as
effective as a transplant from a sibling.8,9
Major histocompatibility antigens were
first studied in depth in various animal
models for skin transplantation. 10(pp97-111) In
humans, these are HLA antigens and are
categorized into Class I (HLA-A, -B, and -
C) and Class II (HLA-DR, -DQ, and -DP).
These highly poly- morphic molecules are
essential in determin- ing graft survival as
well as the likelihood that the recipient will
develop graft-vs-host dis- ease (GVHD). As
molecular techniques have supplanted
serologic methods, the resolution has
improved and the number of antigens that
can be compared has increased. Matching at
the antigen level has been replaced by allele-
level matching for the final selection of
periph- eral blood and marrow HSC
components for a given recipient.
HLA matching in HSC transplantation
has been reviewed in detail elsewhere and is
summarized here only briefly.11 HLA matching
has an important impact on outcomes, espe-
cially in low-risk patients. Allogeneic trans-
plantation using either mobilized peripheral
blood or marrow HSCs with high-resolution
(allele-level) mismatching at HLA-A, -B, -
C, and -DR1 is associated with a 5% to
10% de- crease in survival with each
mismatch.12 The results are similar, although
the evidence is a bit less clear, for HSC
grafts with mismatches in Class II HLA-DQ
and -DP. For mobilized peripheral blood
HSC grafts, allele-level mis- matching is
probably as detrimental to surviv- al as
antigen-level mismatching, although the
data come from smaller studies. Many
centers now match at high resolution for 8 to
10 loci (HLA-A, -B, -C, -DR, and -DQ).
High-resolu- tion 8/8 and 10/10 matched
transplants of HSCs from unrelated donors
are at least in part responsible for the
improvement in outcomes of matched
unrelated transplants compared to matched
related donors.12
Not surprisingly, HSC transplants in re-
cipients who have antibodies against donor
HLA [known as donor-specific antibodies
(DSAs)] have adverse outcomes.13 The level at
which DSAs have a significant impact is less
clear, as is the appropriate course of action
when DSAs are detected. Despite the difficulty
of predicting graft failure based on the pres-
ence of DSAs, screening HSC transplant
candi- dates for DSAs may become more
routine as more data emerge on this topic.14
UCB has several unique characteristics
with regard to HLA matching. The level of
HLA matching required for UCB is less
stringent than that required for marrow and
mobilized peripheral blood HSCs. Data on
UCB indicate that matching at 4 of 6 loci is
sufficient for HLA-A and -B at the antigen
level and for HLA- DRß1 at the allele level,
provided that a suffi- cient cell dose is
achieved.15 As the number of mismatched
alleles increases (from one to two), a higher
total nucleated cell (TNC) dose is needed to
overcome their deleterious effect, including
the use of combined double-cord
transplants.16 In addition, early evidence
indi- cates that noninherited, maternal HLA
may be permissive when considering donor-
recipient mismatches.17 Unit-to-unit
matching of 4 of 6 loci in the setting of
double-cord blood trans- plantation is also
performed at various institu- tions; however,
firm evidence to support this practice is not
available at this time.
Other Donor Characteristics
For marrow and mobilized peripheral blood
HSC donors, other factors that may have a
positive effect on transplant outcomes include
male gender, younger age, nulliparity, ABO
and CMV status matching, and greater size of
the donor relative to the recipient. Other than
HLA, only donor age appears to be associated
with survival.11 ABO incompatibility has been
reviewed extensively. ABH antigens found on
red cells, platelets, and neutrophils would sug-
gest that ABO incompatibility would affect
outcomes. However, inconsistent results on
outcomes—such as survival, nonrelapse mor-
tality, GVHD, and graft failure—of both major
and minor incompatible transplants have
been observed.18 The risk of delayed red cell
engraftment, pure red cell aplasia, increased
transfusion requirements, and both immedi-
ate and delayed hemolysis is increased in ma-
jor ABO-incompatible allogeneic HSC trans-
plantation.
 CH A P T E R 2 9 Hematopoietic Stem Cells
Other characteristics that are specific to
UCB HSC donation and may affect
outcomes include issues related to maternal
history, unit collection, processing, and unit
storage.19 Characteristics of the UCB unit
have been uti- lized in a scoring system, the
“Cord Blood Ap- gar,” to determine the
utility of the unit after consideration of the
TNC count, CD34 positiv- ity, colony-
forming-unit count, mononuclear cell
content, and volume.20
DETERMINATION OF GRAFT
SOURCE
The choice of the source of HSCs for
allogeneic transplantation is determined by
several vari- ables, including the availability
of an ade- quately matched donor. As
described above, marrow and mobilized
peripheral blood prod- ucts in general need a
higher level of HLA matching than UCB units.
For this reason, a UCB transplant may be the
best alternative when only 1 and 2 allele-
mismatched donor units are available. In
addition, for some cen- ters, the relatively
rapid availability of UCB units compared to
marrow and peripheral blood products plays a
substantial role in graft choice in patients with
an immediate need for transplantation. A
typical unrelated marrow or peripheral blood
donor search may take months, whereas a
search for UCB takes sever- al days to a few
weeks.21
GVHD and GVN Effect
GVHD and GVN effect, although linked in
terms of their cause, have opposite
outcomes. In GVHD, donor lymphocytes
attack host-re- cipient tissues in the skin,
lung, liver, gastroin- testinal tract, and other
organs. GVHD may be categorized as acute
or chronic, and it occurs in more than 40%
of individuals who receive an allogeneic
HSC transplant. GVHD is associ- ated with
significant morbidity and mortality. Not
surprisingly, the risk of GVHD is related to
the graft source. Consequently, GVHD risk
in- creases as the lymphocyte content of the
HSC product increases. Various techniques
have been used to decrease the risk of
GVHD, in-
■ 717
cluding T-cell depletion, use of
antithymocyte globulin, and pharmacologic
measures.22
The GVN effect is also thought to be
driv- en by donor lymphocytes. In recent
years, mo- bilized peripheral blood has
greatly outpaced marrow as an HSC source
due to its easier col- lection and the belief
that GVN effect may be improved. The first
randomized controlled tri- al comparing
marrow and mobilized peripher- al blood
HSC grafts in unrelated donor mye-
loablative transplantations found that
chronic GVHD, but not acute GVHD, risk
was higher in patients who received a
peripheral blood HSC transplant.23
Kinetics
The kinetics of engraftment are affected by
many variables, such as degree of HLA match-
ing, dose of HSCs, and use of granulocyte
colony-stimulating factor (G-CSF). In
addition, the characteristics of the stem cell
source may play a role. For instance, stem
cells in UCB ap- pear to have a greater
regenerative capacity than those in marrow
and peripheral blood.24 In general, the rate of
engraftment is predicted by the number of
progenitor cells in each graft. This is greatest
in mobilized peripheral blood, followed by
marrow, UCB, and other HSC sources.
In a meta-analysis of studies that com-
pared related transplantation with donor-mo-
bilized peripheral blood and marrow, the
me- dian time to neutrophil engraftment was
14 vs 21 days and to platelet engraftment
was 14 vs
22 days, respectively.25 These findings have
been corroborated by a more recent meta-
analysis of studies of allogeneic related trans-
plantation in which mobilized peripheral
blood vs marrow HSC engraftment was 15 vs
21 days for neutrophils and 13 vs 21 days for
platelets, respectively.26 The more rapid en-
graftment of peripheral blood compared to
marrow HSCs was also seen in a study of
unre- lated allogeneic transplantation in which
me- dian neutrophil engraftment occurred at
15 and 19 days, respectively, and median
platelet engraftment occurred at 20 and 27
days, re- spectively.27 These findings were
confirmed in the randomized controlled trial
of Anasetti
718 ■ AABB T EC HNIC AL MANUAL
and colleagues23 in which the relative differ-
ences in time to engraftment was 5 days
short- er for neutrophils and 7 days shorter
for plate- lets. A comparison of adult
unrelated marrow to UCB HSC grafts
showed that engraftment occurred earlier
with both neutrophils (medi- an of 18 days
for matched marrow, 20 for mis- matched
marrow, and 27 for mismatched UCB) and
platelets (median of 29 days for matched
marrow and mismatched marrow, and 60
days for mismatched UCB).28 In the set- ting
of reduced-intensity UCB transplantation,
median neutrophil engraftment times ap-
proaching those of mobilized peripheral
blood and marrow HSC transplants have
been ob- served.24
Survival
The most important outcome measure for any
transplant procedure is patient survival. For
recipients of transplants from matched related
donors, mobilized peripheral blood may offer
an advantage in overall and disease-free
survival over marrow HSC grafts, at least in
re- cipients with late-stage hematologic malig-
nancies.25 In recipients of myeloablative trans-
plants from unrelated donors for hematologic
malignancies, a recent randomized controlled
trial indicated that marrow and mobilized pe-
ripheral blood sources have equivalent effects
on survival, with marrow grafts associated
with less chronic GVHD but more graft fail-
ure.23
Based on these findings, the rapid in-
crease in the use of mobilized peripheral
blood relative to marrow in recent years may
change. This may not be true, however, in
nonmye- loablative patients or transplant
recipients at high risk of infection or graft
failure. For pedi- atric transplant recipients,
marrow may actu- ally be preferred over
mobilized peripheral blood, although reports
differ.29,30 In addition, when mismatched
marrow and mismatched UCB were
compared, there was no difference in the rate
of overall mortality in adults with leukemia;
however, recipients of matched marrow
HSC grafts did have a lower overall
mortality rate.28
Transplant physicians face a complex
choice when determining which donor and
stem cell source is best for their patients based
on numerous factors, including the patient’s
disease, disease stage, age, and comorbidities.
As more data are collected, the choice of HSC
graft may evolve.
COLLECTION/SOURCES OF
HSCS
Regardless of the source of HSCs, standards
of both FACT and AABB require all
institutions that collect HSCs to have a
procedure in place to obtain informed
consent from the donor or the donor’s
representative, as dictated by local laws.1(pp16-
17,20-21),4
The informed consent pro- cess should
include providing information to the donor
regarding the risks/benefits of the procedure,
the tests performed on the donor that are
designed to protect the recipient, al-
ternative collection methods, and protection
of their health information. In addition, the
donor should be given the opportunity to ask
questions as well as to refuse donation. The
risks that are specific to each collection
proce- dure are discussed below.
Another requirement for all facilities
that collect HSCs is to provide donor access
to medical care based on the risks and
clinical situation associated with each type
of dona- tion. Specifically, procedures
should be in place to provide medical or
emergency care to donors who experience
adverse effects. Clear- ance from the donor’s
physician for HSC col- lection should be
documented.
Marrow HSC Collection
In addition to undergoing relevant donor
screening, infectious disease testing, and
HLA compatibility testing, marrow donors
must also be physically suitable for
donation. Mar- row harvest is an invasive
procedure per- formed under sterile
conditions in the operat- ing room under
anesthesia. Therefore, the donor must be
able to tolerate the type of an- esthesia
required to successfully perform the harvest.
Another consideration is the donor’s medical
history. Autologous donors and some
 CH A P T E R 2 9 Hematopoietic Stem Cells ■ 719

allogeneic donors may have had previous have significant decreases in their
radi- ation therapy to the pelvis, which may hemoglobin concentrations after the
limit the amount of marrow available for procedure. As a result, almost all marrow
harvest in the posterior iliac crest. Similarly, donors donate autologous Red Blood Cells
previous chemotherapy may limit the (RBCs) before the procedure, and 76% of
number of nucle- ated cells that can be donors receive at least 1 autolo- gous RBC
aspirated from the mar- row space. For unit during or shortly after marrow harvest.31
autologous donors, a signifi- cant tumor If the donor requires an allogeneic RBC or
burden in the marrow space is a platelet transfusion before or during the
contraindication for collection of HSCs by procedure, the units should be irradiated to
marrow harvest because the graft would be prevent viable leukocytes from these blood
contaminated by tumor cells. products from contaminating the graft. Mar-
Physically, the donor must be able to row donors should be made aware that they
tol- erate the volume loss associated with might need to undergo a transfusion as part
marrow harvest, which means that young or of the informed consent process.
small do- nors may not be suitable. The Be
The Match Registry limits the volume Peripheral Blood HSC Collection
collected from a marrow donor to 20
Pharmacologic methods for mobilizing
mL/kg.5 Typically, the vol- ume of the
HSCs from the marrow into the peripheral
harvest requested is dictated by the
circula- tion combined with apheresis
recipient’s weight, with a minimum of 2.0 to
technology have made peripheral HSC
3.0 × 108 nucleated cells/kg needed to
collection the most common procedure for
facilitate efficient engraftment. Thus, during
HSC donation.32 Be- cause peripheral
harvest, checking the TNC count midway
collection of HSCs requires only vascular
through the procedure can help with
access, most apheresis proce- dures used to
estimating the total volume needed.
collect HSCs are performed on an outpatient
Alternatively, CD34 quantita- tion midway
basis, with minimal side effects. However,
through the procedure or on the final
donors with poor vascular access or who
product for QC may also be performed,
may need a number of apheresis proce-
depending on the institution’s policies.
dures for the collection of a sufficient
The marrow harvest technique varies
number of HSCs may require the placement
considerably depending on institutional prac-
of a cen- tral line, which imposes an
tice. In general, an 11- to 14-gauge needle on a
additional risk. AABB CT Standards
syringe flushed with anticoagulant is inserted
requires that the place- ment of any central
into the posterior iliac crest, and approximate-
line be confirmed before HSC collection is
ly 5 mL of marrow is aspirated. The needle
initiated.1(p48)
and syringe are then rotated to a different
HSCs can be mobilized into the peripher-
vector, and the aspiration is repeated.
al circulation using a variety of chemothera-
Vigorous aspi- ration is avoided to prevent
peutic agents, hematopoietic growth factors,
significant periph- eral blood contamination of
or receptor antagonists. For most healthy
the product. The aspirated marrow is collected
allo- geneic HSC donors, sufficient numbers
into a large col- lection bag containing
of HSCs can be mobilized with the
anticoagulant and me- dia and/or an infusible-
administration of hematopoietic growth
grade electrolyte solu- tion. The process is
factor, often G-CSF, alone. G-CSF is
repeated utilizing different bone sites until the
administered once per day at a dose of 5 to
collection volume target, based on TNC count
20 µg/kg, and doses are often rounded to the
or donor volume limit, is reached.
nearest vial size.33 Total white cell count and
Serious complications of marrow
CD34 percentage can be moni- tored to
harvest are rare. However, minor
determine the optimal collection time, which
complications, such as pain at the site of
is usually 3 to 4 days after initiation of G-
harvest, fatigue, insomnia, nausea,
CSF treatment. The side effects of G-CSF,
dizziness, and anorexia, occur fre- quently
which are common and mild, include bone
but resolve in most donors by 1 month after
pain, myalgia, headache, insomnia, flu-like
the procedure.31 Marrow donors often
720 ■ AABB T EC HNIC AL MANUAL
symptoms, sweating, anorexia, fever, chills,
and nausea.33 Potentially serious complica-
tions, such as splenic rupture, are rare. Other
growth factor preparations are available, in-
cluding a pegylated form of G-CSF that
offers the advantage of a one-time dose in a
majority of donors.
For some autologous donors and rare al-
logeneic donors, mobilization of HSCs can
be challenging, potentially requiring
additional pharmacotherapy to mobilize an
adequate number of cells and facilitate
efficient engraft- ment. The minimum
number of cells needed for transplantation is
commonly cited as 2× 106 CD34+ cells/kg,
although 5 × 106 CD34+ cells/kg is more
desirable.34 In autologous do- nors, a
chemotherapy drug, such as cyclo-
phosphamide, can be added to the G-CSF
regi- men. Although the number of HSCs
collected can be increased by using a
combination of chemotherapy and G-CSF,
complications such as cytopenias and
additional apheresis may outweigh the
potential benefits of this strate- gy.35 If the
benefits of adding chemotherapy to G-CSF
to stimulate peripheral HSC mobiliza- tion
outweigh the risks, this mobilization strat-
egy may be utilized successfully in patients
with significant tumor burden.
Patients who are poor mobilizers may
benefit from the addition of plerixafor, a
che- mokine (C-X-C motif ) receptor 4
antagonist, in combination with G-CSF.
Various clinical studies have been published
demonstrating that plerixafor in combination
with G-CSF can increase HSC collection
yields. A number of clinical scenarios where
plerixafor may be uti- lized have been
published, and patients with multiple
myeloma or lymphoma who have dif-
ficulty mobilizing HSCs may benefit from
plerixafor therapy to collect a sufficient
quan- tity of HSCs during donation.34
Peripheral collection of the HSCs is
per- formed using an apheresis device
according to the manufacturer’s instructions.
For most allo- geneic donors, sufficient
numbers of HSCs can be obtained with one
to two collection proce- dures. Up to 20% of
donors experience minor
apheresis/collection-related adverse events,
such as citrate toxicity, nausea, fatigue,
chills, hypertension, hypotension, allergic
reactions,
or syncope.32 Autologous donors experience
similar collection-related side effects, which
can be problematic in donors who require
multiple apheresis procedures due to poor
mobilization. Depending on the donor
scenar- io, large-volume apheresis
techniques may be used to limit the number
of total procedures.36 AABB CT Standards
requires a complete blood count to be
performed within 24 hours before the
procedure begins for all mobilized donors,
and this is especially important for
autologous donors because the apheresis
procedure can deplete platelets.1(p47)
UCB HSC Collection
UCB collection is discussed in detail in Chap-
ter 30 and is not addressed here.
PROCESSING OF HSCS
Processing methods for HSCs can be
divided into routine methods, which are
usually cen- trifuge based, and specialized
methods that involve a variety of
technologies. Routine methods include
volume (plasma) reduction, red cell
reduction, buffy coat preparation,
thawing/washing, and filtration. Volume re-
duction is performed in the settings of minor
ABO-mismatched allograft (marrow or periph-
eral blood) transplantation to reduce the
amount of incompatible plasma and prevent
fluid balance/overload issues in small
patients and/or patients with renal disease or
cardiac failure. Volume reduction may also
be per- formed before cryopreservation (eg,
for UCB banking where storage space is
limited or dur- ing cell concentration
optimization).
Classically, red cell reduction employs
sedimenting agents (eg, hydroxyethyl
starch) to reduce red cell content. These
agents are used to prevent hemolytic
transfusion reac- tions when major ABO
incompatible marrow HSC allografts and
allografts with other clini- cally relevant red
cell antigens (eg, Kell, Kidd) are
transplanted. Red cell reduction before
freezing also limits the amount of lysed red
cell fragments and free hemoglobin on
infusion and may be particularly important
for patients with renal failure. Red cell
reduction may also
 CH A P T E R 2 9 Hematopoietic Stem Cells
be useful when storage space is limited. Be-
cause apheresis instruments collect mononu-
clear cells efficiently, with very little red cell
content, peripheral blood-derived HSCs gen-
erally do not require red cell depletion.
Buffy coat concentration of marrow in-
volves centrifugation and harvesting of the
white cell fraction and can be performed
with an apheresis or cell-washing device.
Manual centrifugation may be used when
product vol- ume is too low for apheresis or
cell washing devices. Buffy coat preparation
is usually used to reduce the unit volume for
cryopreservation or as a method of red cell
reduction before fur- ther manipulation (eg,
immunomagnetic se- lection).
The thawing procedure for all HSCs, re-
gardless of source, is similar. Although this
procedure is straightforward, it should be
done carefully because frozen plastic
contain- ers are prone to break for a variety
of reasons.37 The product should be handled
with care while it is verified to determine
the product’s identity and ensure the
integrity of the bag. The product is then
placed into a clean or ster- ile plastic bag
and submerged in a 37 C water- bath. If the
freezing bag breaks, the product may be
recovered using this approach, but a risk-
benefit discussion with the patient’s phy-
sician should take place to determine the
course of the patient’s care.
Gentle kneading allows the thaw proce-
dure to proceed relatively quickly while pre-
venting recrystallization and consequent cell
damage/death. A hemostat should be used to
prevent loss of the product if the bag breaks,
and the contents should be aseptically divert-
ed into a transfer bag. A sample should also be
sent for culture.
Washing the HSCs removes lysed red
cells, hemoglobin, and cryoprotectant [di-
methyl sulfoxide (DMSO)]. Although UCB
is typically red-cell depleted before
cryopreser- vation, it remains the primary
HSC product that is routinely washed. This
practice is changing, however, and Chapter
30 discusses alternative approaches to UCB
preparation for infusion. Historically, most
institutions based their UCB processing
methodology, including the
thawing/washing procedure, on the proce-
■ 721
dure originally described by Pablo Rubinstein
of the New York Placental Blood Program.38
Briefly, the thawing process involves slow, se-
quential addition of a wash solution (eg, 10%
dextran followed by 5% albumin), transfer into
an appropriately sized bag for centrifugation,
and resuspension of cell pellet(s) before deliv-
ery to the patient care unit for infusion. Many
laboratories perform two centrifugation steps,
removing the supernatant from the first spin
and centrifuging that portion a second time
before combining the two cell pellets. This ap-
proach optimizes cell recovery.39
Marrow harvest typically involves se-
quential filtration in the operating room or
the laboratory to remove bone spicules,
aggre- gates, and debris. Opinions regarding
the use of standard blood filters upon
infusion of HSCs vary, however. Whether to
use a standard blood filter (>170 microns) is
up to the individ- ual cell processing
laboratory and/or trans- plant center. If an
institution opts to use a standard blood filter,
the laboratory should validate its filtration
process.
SPECIALIZED CELL-
PROCESSING METHODS
Specialized cell-processing methods are
used to optimize product purity and potency
be- yond levels obtained through routine
meth- ods. Several of these methods, which
require unique reagents and instrumentation,
are dis- cussed in other chapters. For this
reason, the descriptions of these methods in
this chapter are brief and focus on their
application to HSCs.
Elutriation
Counter-flow centrifugal elutriation is a spe-
cialized method that separates cell popula-
tions based on two physical characteristics—
size and density (sedimentation coefficient). A
centrifuge is used to separate the cell popula-
tions of a cell product based on density alone.
However, if fluid/media is passed through the
chamber housing the cells in the direction that
is opposite (counterflow) to the centrifugal
force, adjustment of flow rate and/or centrifu-
722 ■ AABB T EC HNIC AL MANUAL
gation speed allows the separation of cell pop-
ulations based on size as well. Through this
process, cells with “signature” size/density
profiles can be separated from the rest of the
cells. Historically, this method was used for T-
cell depletion of HSC grafts. In more recent
years, the method has been used to enrich
monocytes for preparation of dendritic-cell
vaccines.
Cell-Selection Systems
Immunomagnetic cell selection systems
incorporating monoclonal-antibody-based
technologies to target cell-surface antigens
(eg, CliniMACS system, Miltenyi Biotec
Ber- gisch, Gladbach, Germany) have
become a widely used method of cell
depletion/enrich- ment at many institutions.
These methods in- volve the isolation of the
cell type of interest by either positive
selection (target cells retained) or negative
selection (target cells depleted). Monoclonal
antibodies (eg, anti-CD34 for HSC isolation)
are coupled to 50-nm ferromagnetic
particles. Magnetically labeled target cells
are retained in the process as the cell
suspension passes through a column in
which a magnetic field is generated.
Unlabeled cells pass through the column and
are collected in a neg- ative-fraction bag.
Target cells are then re- leased from the
column by removal of the magnetic field
from the column, which allows passage of
the cells into a separate collection bag.
Cell Expansion
Because the dose of nucleated, CD34+, and
colony-forming cells has a positive
correlation with patient outcomes, much
effort has been focused on ex-vivo
expansion of HSCs and progenitors. It is
thought that successful ex- pansion
enhances hematopoietic engraftment while
reducing transfusion dependence, risk of
infection, and duration of hospitalization. In
recent years, UCB has become the focus of
expansion trials due both to the higher
prolif- erative and self-renewal capacity of
HSCs from UCB and the limit on cell
quantity in a UCB collection. Most
expansion cultures contain a cytokine
cocktail that includes stem cell factor,
FLT-3 ligand, and thrombopoietin along
with novel and/or proprietary ingredients.
The me- dia, culture vessels, and culture
duration used vary from protocol to
protocol.
CRYOPRESERVATION
Methods for cryopreservation must be used
because HSCs may need to be stored for
weeks to years prior to being transplanted. 40
Most cell-processing laboratories use the
cryopro- tectant DMSO, usually at 10% final
concentra- tion, and a source of plasma protein
for cryo- preservation of HSCs. DMSO is a
colligative cryoprotectant; it diffuses rapidly
into the cell, reducing the osmotic stress on the
cell mem- brane. DMSO prevents dehydration
injury by moderating the nonpenetrating
extracellular solutes that form during ice
formation. It also slows extracellular ice
crystal formation. Some laboratories add
hydroxyethyl starch (HES), which allows the
use of a decreased concentra- tion of DMSO
(eg, 5% DMSO and 6% HES). HES is a
nonpenetrating (extracellular),
macromolecular cryoprotectant. This high-
molecular-weight polymer likely protects the
cell by forming a glassy shell, or membrane,
around the cell, retarding the movement of
water out of the cell and into the extracellular
ice crystals.
The HSCs may be frozen at a controlled
rate or a noncontrolled rate, in which the
HSC product is simply transferred into a
freezer bag and placed in a –80 C
mechanical freezer. Con- trolled-rate
freezing is favored in the clinical laboratory
setting, and it utilizes computer
programming to incrementally decrease
HSC product temperature in a closely
monitored fashion. The controlled rate
freezing protocols used vary from institution
to institution.
In general, the HSC product is placed in
the chamber and initially cooled at a rate of
1 C/minute. When the temperature decreases
to approximately –14 C to –24 C, the HSC
product begins to transition from a liquid to
a solid. At this time, the freezer undergoes a
pe- riod of supercooling to counteract the
latent heat of fusion that is released by the
phase change. Following solidification of
the HSC product, cooling proceeds at the
rate of 1 C/
 CH A P T E R 2 9 Hematopoietic Stem Cells
minute until the product has reached –60 C.
At this point, the product is cooled at a
controlled rate determined by the institution
until it reaches –100 C. Following both
controlled-rate and noncontrolled-rate
freezing, the HSC product is transferred to a
storage freezer. An increasing number of
laboratories store HSCs
in the vapor phase of liquid nitrogen (LN 2) at
temperatures below –150 C; however, some
laboratories do store cells in the liquid phase
of LN2.
QC
QC testing in the clinical cell therapy
laborato- ry serves two purposes: determine
the suit- ability and safety of the cellular
product for the patient and monitor overall
laboratory practic- es. QC testing is aimed at
characterizing the safety, purity, identity,
potency, and stability of the cellular product.
The extent of QC testing is primarily
dependent on the complexity of
manufacturing the product and the nature of
the clinical experience (ie, standard practice
vs a clinical trial).
Common QC tests for HSCs include
cell count and differential, viability, CD34+
cell enumeration, sterility testing, and
colony- forming unit assays. Cell count and
differential are performed on a hematology
analyzer. Cell viability may be determined
using a variety of methods, including trypan
blue, acridine or- ange, and 7-
aminoactinomycin D (flow cy- tometry).
Microscope-based methods utilizing vital
dyes or fluorescent stains may be particu-
larly useful for a quick assessment of overall
nucleated cell viability. Flow cytometry-
based analysis can be useful when cell
population- specific viability needs to be
determined. Most CD34+ cell-enumeration
strategies are based on guidelines of the
International Society for Cellular Therapy.41
Sterility testing at most in- stitutions is
performed using an automated microbial
detection system.
The clonogenic assay (most commonly
used to count colony-forming units) is the
only truly functional assessment of HSCs rou-
tinely performed in clinical laboratories. The
results of this assay correlate with the speed
and likelihood of engraftment of HSCs from
■ 723
marrow, peripheral blood, or UCB.42-45
Howev- er, the similar correlation between
results and engraftment speed and likelihood
as well as the more rapid availability of
results have made CD34+ cell enumeration
the accepted, albeit surrogate, QC test for
graft potency. The clonogenic assay is still
useful, despite difficul- ties in
standardization, especially for HSCs that are
stored for a long time (eg, UCB bank- ing).46
SHIPPING AND TRANSPORT OF
HSC CELLULAR PRODUCTS
Shipping and transport of cellular therapy
products allow the geographic separation of
donors and recipients. The two terms are
given specific definitions by accreditation
organiza- tions.1(pp35-36),4(pp13-14) With shipping,
the product leaves the control of trained
personnel in the facilities involved in the
distribution and re- ceipt of the product.4(pp13-
14)
Conversely, with transport the transfer of a
product between or within facilities occurs
under the control of trained personnel.
Three issues are particularly important to
ensure the safe delivery of HSC products:
product integrity, safety of the personnel in-
volved in the transport, and compliance with
applicable regulations and standards. The
necessary conditions for shipping and trans-
port vary depending on the type of product, its
state (fresh or cryopreserved), and the dis-
tance involved. These issues are reviewed in
depth elsewhere.47
During shipment, the product must be
placed in a secondary container that can pre-
vent leakage and is validated for the tempera-
ture range required for the product and the
anticipated duration of shipping. This tem-
perature range may be defined in standards
(eg, <–150 C for cryopreserved cord blood) or
by an institutional protocol.4 For fresh prod-
ucts, several studies have shown that ship-
ment at 2 C to 8 C can maintain CD34+ cell
via- bility more effectively than shipment at
room temperature particularly for shipping
times of between 24 and 72 hours. 48-50 This
effect ap- pears to be more pronounced for
peripheral
724 ■ AABB T EC HNIC AL MANUAL

also noted that the addition of 1%

blood products than marrow products and
for products with higher concentrations of
HSCs.
Cryopreserved products are shipped in
dry shippers charged with LN2. These contain-
ers maintain temperatures below –150 C for
up
to 2 weeks and their temperatures are
continu- ously monitored.1(p35) If the
products are shipped to a noncontiguous
facility or on pub- lic roads, an appropriately
labeled outer con- tainer must be used to
further protect the product during transport
and shipping.4 De- pending on the mode of
transport (eg, air or ground), additional
federal government re- quirements must be
met. If products are shipped abroad,
international requirements must be met. If
high-dose conditioning thera- py has been
given to the recipient, shipment using a
qualified courier is required.4 The product
should not be x-rayed; instead, it should be
manually inspected, if necessary.
Appropriate records must accompany the
product.
The receiving institution must have pro-
tocols in place for receipt and inspection of
the product for acceptability for
transplant.1(pp36-37),4

PATIENT CARE
Once an HSC product is ready for infusion, it
should be delivered to the patient care unit
without delay. After the physician approves
the product for infusion and proper
identification procedures are carried out, the
product is in- fused by intravenous (IV) drip
directly into a central line, typically without a
needle or pump. Some institutions use a
standard blood filter at the bedside. To
maximize cell dose, the product bag and IV
tubing may be flushed with sterile saline after
the bag empties. Sterile saline also may be
added directly to the bag if the flow rate
becomes too slow.
HSC products are usually infused as
quickly as the patient can tolerate,
particularly for thawed cells that have not
been washed or diluted, to lessen the DMSO
toxicity to the cells. Although Rowley and
Anderson51 con- cluded that DMSO is not
toxic to HSCs at clini- cally relevant
concentrations (ie, 5% or 10%) at either 4 C
or 37 C for up to 1 hour of incuba- tion, they
 wash- ing, or dilution). Reactions often
DMSO to culture dishes attributed to DMSO (eg, nausea, vomiting,
suppressed colony- cough, and headache) are less common with
forming units. However, infusions of smaller volumes and/or
these studies were washed/diluted prod- ucts.52,53 HSC products
performed on fresh are usually well tolerat- ed, however.
cells, and studies of the Because the possibility of a severe reaction
ef- fects of DMSO on does exist, aggressive IV hydration (eg, 2 to
HSCs that have been 6 hours before and 6 hours after infu- sion,
previ- ously with the use of diuretics as needed) and use
cryopreserved are of prophylactic antiemetics, antipyretics,
limited. The possible and antihistamines may be warranted.
functional defect due to The transplant physician and the
DMSO coupled with the medical director of the cell therapy
not-infrequent need to laboratory should be notified immediately of
hold clinical prod- ucts an unexpected or moderate-to-severe
(because of patient- reaction. An investigation should begin and
care-related issues) raise include laboratory testing (eg, direct
concern about the antiglobulin test, antibody titer, Gram’s stain,
possibility of cell inju- or culture) that targets the signs/ symptoms
ry from the infusion of of the patient.
thawed, unwashed HSC Data on clinical outcomes (eg, engraft-
products. ment) and adverse events should be
The patient’s vital reviewed regularly and discussed with the
signs should be checked institutional
before infusion,
immediately after in-
fusion, and 1 hour after
infusion, at a mini-
mum. All of the
monitoring information
should be captured on
the accompanying in-
fusion form. When
completed, this form
should be returned to
the laboratory. If an ad-
verse reaction occurs,
more frequent monitor-
ing is required.
Reactions
associated with HSC
infusion may be very
similar to those that
occasionally occur with
blood transfusion (ie,
allergic, he- molytic,
and febrile reactions
and those due to
microbial
contamination).
However, some re-
actions may be less
likely depending on the
cell-processing
technique used (eg, red
cell re- duction, plasma
reduction, postthaw
 CH A P T E R 2 9 Hematopoietic Stem Cells ■ 725

quality management group. Quarterly product and requires licensing or an exemp-

reviews are reasonable for engraftment
analysis. The medical director’s review
should include as- sessment of the HSC
product’s quality indica- tors (eg, dose,
viability, and colony-forming units),
associated deviations, and presence of
infusion reactions, with a focus on potential
laboratory-related component affecting any
less-than-optimal outcome.
tion from licensing from the FDA as part of
an investigational new drug (IND)
application. HSCs from unrelated donors
facilitated through the Be The Match
Registry may be ad- ministered under their
IND (BB-IND 6821) or an institutionally
held IND. Similarly, HSCs can now be
obtained from FDA-licensed UCB sources
or administered under an institutional IND.

OTHER REGULATORY
CONSIDERATIONS
The relevant regulations with regard to HSC
collection are discussed earlier in this
chapter. In general, HSCs that are minimally
manipu- lated and collected for
transplantation in an autologous fashion or
transplanted to a first- or second-degree
relative are regulated solely under Section
361 of the Public Health Service Act and are
subject to the jurisdiction of the Center for
Biologics Evaluation and Research of the
FDA. If HSCs are manufactured in a way
that alters their relevant biological
characteris- tics (eg, if they are genetically
modified, ex- panded ex vivo, or combined
with a drug) or the cells are intended for
transplantation into a non-first- or second-
degree relative, then the HSC product is
subject to regulation under Ti- tle 21, CFR
Part 1271, as a drug and/or biologic
CONCLUSION
The indispensable, lifesaving role of HSCs
in medicine has been established, especially
for patients with hematologic disorders. As
the understanding of HSC biology increases
and the ability to engineer HSC grafts
expands, the clinical applications of HSCs
will likely contin- ue to grow. Along with the
fast-paced growth in the use of HSCs,
emerging novel technolo- gies and
approaches to manipulate HSCs are adding
to the challenge of ensuring that HSCs
continue to serve as a safe and effective
cellu- lar therapy product for patient use.
Addressing this challenge will require
regulatory agencies and accrediting bodies
to continue to update and modify their
applicable rules, regulations, and standards.

KEY POINTS

Hematopoietic stem cells (HSCs) derived from the patient being treated (autologous) or a donor (allogeneic) can be used to
Autologous HSCs in general are used to rescue the marrow function of a patient undergoing
high-dose chemotherapy and/or radiotherapy.
In addition to rescuing the marrow function of a patient undergoing high-dose chemother- apy and/or radiotherapy, allogene
Regardless of the HSC donor source, AABB CT Standards requires laboratory testing for hu- man immunodeficiency virus
Screening and testing for infectious diseases in allogeneic HSC donors is mandated by the
Food and Drug Administration (FDA), and when a risk for a communicable disease is dis- covered, the donor is ineligible (
Allogeneic HSC donors are chosen with regard to their histocompatibility with the recipient.
ABO-Rh compatibility between the donor and recipient is not necessary.
726 ■ AABB T EC HNIC AL MANUAL

7. HSCs can be obtained by aspiration of marrow, collection of umbilical cord blood, or by pe-
ripheral blood mobilization followed by collection with an apheresis machine.
8. HSCs usually require minimal manipulation, and can be stored following
cryopreservation with the cryoprotectant, dimethyl sulfoxide.
9. Specialized HSC manipulation techniques can be used to reduce the HSC product
volume, lysed cells, red cells, and cryoprotectant depending on the recipient’s clinical
needs.
10. Quality control (QC) is essential for providing a safe and efficacious HSC product.
Common QC testing includes cells counts (CD34+ cell enumeration, total nuclear cell
count), micro- bial contamination testing, and viability testing.
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 C h a p t e r 3 0

Umbilical Cord Blood Banking

Aleksandar M. Babic, MD, PhD, and

Donna M. Regan, MT(ASCP)SBB

OV ER TH E P A S T 25 years, umbilical spite their less stringent HLA-matching re-

cord blood (UCB) has evolved into an
important source of hematopoietic stem
cells (HSCs) for transplantation. Classified
by the Food and Drug Administration
(FDA) as a bio- logic drug, stem cells
derived from UCB are also used in a
growing number of large, multi- institutional
trials in immunotherapy and re- generative
medicine. This chapter summarizes the
practical aspects of creating an inventory
that provides UCB cells for clinical use,
recent research, economic considerations in
balanc- ing inventory size with the potential
for unit selection, and the regulatory
structure that
keeps pace with product innovation.
BACKGROUND
UCB has become a widely accepted source
of HSCs for transplantation in pediatric and
adult patients lacking an HLA-matched do-
nor.1-6 The advantages of UCB-derived HSCs
over other HSCs include their higher
prolifera- tive and self-renewal capacity and
their associ- ation with a lower incidence of
graft vs host disease (GVHD), particularly
acute GVHD, de-
quirements.4,5,7-11 In-vitro and in-vivo animal
research suggests that these attributes of
UCB transplantation are due to the primitive
nature and naïve immune system of UCB.12-
14
Trans- plant candidates, especially those
with rare HLA types, are often successful in
finding an acceptable UCB unit.
Furthermore, the search time, in general, to
find a donor is markedly re- duced for UCB
transplant recipients than for recipients of
HSCs from other sources because the
cellular characteristics, donor screening/
eligibility, and HLA typing of UCB units are
de- termined at the time of banking.15
The first UCB bank, established by Dr. Hal
E. Broxmeyer, provided the donor graft for
the historic 1988 UCB transplant as well as
four additional HLA-matched sibling trans-
plants.16,17 As UCB gained initial acceptance
as an HSC source in the related transplant
set- ting, the possibilities for the use of
unrelated HSC units became apparent and
supported the rationale for the establishment
of unrelat- ed cord blood banks (CBBs). The
first unrelat- ed CBB was established by Dr.
Pablo Rubin- stein at the New York Blood
Center in 1992.18

Aleksandar M. Babic, MD, PhD, Medical Director, and Donna M. Regan, MT(ASCP)SBB, Executive
Director, St. Louis Cord Blood Bank and Cellular Therapy Laboratory, SSM Cardinal Glennon Children’s
Hospital, St. Louis, Missouri
The authors have disclosed no conflicts of interest.

729
730 ■ AABB T EC HNIC AL MANUAL
CBBs in Dusseldorf and Milan opened
shortly thereafter. Recent reports indicate
that more than 600,000 unrelated UCB units
are banked worldwide for potential clinical
use, and near- ly 30,000 unrelated UCB
transplants have been performed to date.19 In
2011 alone, the World Marrow Donor
Association reported that 4093 UCB
products were shipped to hospitals for
transplantation to unrelated patients in 47
countries; in comparison, 3,743 marrow
grafts were performed in that year.19 In
addition, a number of private CBBs have
been established worldwide for families to
bank the UCB of their infants for future use
by family members. Two of the largest
private CBBs have recently reported that
they have stored UCB stem cells from
700,000 newborns, and each of these CBBs
has distributed approximately 250 UCB
products for traditional transplantation and
regenerative applications.
The sections of this chapter describe the
current generally accepted practices for
UCB banking (primarily from a public
CBB’s per- spective), including donor-
related issues, col- lection and processing
methods, and storage and shipment as well
as recommended trans- plant center-related
activities for product preparation and
infusion. The chapter contin- ues with a
brief discussion of UCB banking economics
and how inventories can be creat- ed to meet
the needs of a diverse patient popu- lation
and address cell dose limitations while
continuing to serve as cost-effective means
of providing access to therapy in an
“affordable health care” environment. The
chapter finish- es with an overview of
standards and regula- tions.20
DONOR-RELATED ISSUES
Recruitment
Most women agree to donate the UCB of their
infants when they become aware that the UCB
that is normally discarded as medical waste
can be recovered and used to save the life of
another individual. However, a woman’s ability
to donate UCB may be limited by the
availabil- ity of a CBB servicing the area in
which she gives birth.
Engaging pregnant women and making
arrangements before they give birth to
donate their infant’s UCB is critical for
achieving the desired results. Early
education of pregnant women allows for
more complete medical screening,
comprehensive donor protection, availability
of adequate supplies to collect UCB, and
acquisition of truly informed con- sent from
the woman. Recruitment by a public CBB
typically begins with education of the
physician/midwife by the CBB and the
distri- bution of informational materials to
obstet- rics offices or prenatal class staff.
This ap- proach enlists support for UCB
donation while providing information to
obstetric staff. This information empowers
staff to introduce the idea of UCB donation
to pregnant women and respond to their
initial inquiries while refer- ring more
detailed questions to CBB person- nel.
The informational materials that CBBs
distribute to obstetrics office and prenatal
class staff about UCB donation address
basic information, such as the name of the
CBB, whether the CBB is public or private,
the cost (or lack thereof ) of donation, names
of partici- pating hospitals, medical uses of
UCB, and how UCB is collected. The
materials also dis- cuss the risks of UCB
donation to the mother and infant and
whether the donated unit will be available
for the donating family’s use. Within an
informational packet, a CBB may in- clude
the health history questionnaire, which is
used to solicit information about the preg-
nancy, risk factors of the parents, and
medical history of first-degree family
members.
The CBB explains to potential donors
the need for their written permission
(informed consent) to collect and store the
UCB for later use in transplantation or
research. Participat- ing mothers must
understand that their blood will be drawn
and tested for certain infections, such as
hepatitis and human immunodefi- ciency
virus, to reduce the risk of transmission of
disease through the transplantation of their
infant’s UCB. The CBB emphasizes that all
in- formation and test results obtained
during the process are held strictly
confidential to pro- tect the identity and
privacy of donors and
 C H A P TER 30 Umbilical Cord Blood Banking
that the family will not be approached to do-
nate more cells after the infant is delivered.
The role of the pregnant woman’s
physi- cian cannot be understated. During
pregnan- cy, a woman trusts her physician to
provide re- liable care and advice.
Therefore, a woman’s decision to donate or
store her UCB can be as simple as
acceptance of a recommendation of- fered
by her physician. In addition, a woman’s
decision to donate may be influenced by re-
cruitment materials designed to appeal to all
ethnic groups.
In spite of broad awareness campaigns
and recruitment efforts, some women may
not be aware of UCB donation or may not
have registered to participate when they
arrive at a hospital to give birth. For this
reason, informa- tional material on UCB
donation should also be available in the
labor and delivery area. The extent to which
women not previously in- formed about
UCB donation can be recruited at this stage
(ie, delivery) varies. However, in most cases,
pregnant women have been previ- ously
informed of the option of UCB donation but
simply have not registered or completed the
necessary documentation.
Pregnant women may also be solicited
for UCB donation by private CBBs. For a
fee, these banks will arrange for UCB to be
collected and held in long-term storage for
use only by that family. These CBBs provide
written or video information to pregnant
women that is specif- ic to their program.
Consent
Consent must be obtained from the pregnant
woman for UCB collection, processing, test-
ing, storage, and medical use.21-26 Although
the UCB actually belongs to the newborn
infant, consent is obtained from the mother
because her infant cannot provide it and
testing of her blood for transmissible
diseases is required. Consent from the father
is not necessary and does not increase the
safety of the UCB for transplantation.27
Although consent to collect UCB must
be obtained before delivery of the infant,
CBBs use different approaches to obtain
consent for further manufacturing, testing,
and use of
■ 731
UCB. A CBB may use a single consent form
covering all activities. Presented in the
prena- tal period, a single consent process
affords the pregnant woman adequate time
to seek infor- mation and consider her
options under low- stress circumstances.
Other CBBs may use a phased consent
process that permits collec- tion and, if the
resulting harvest meets criteria for banking,
allows the bank to approach the mother later
for permission to screen, process, test, and
store the product. This latter process
facilitates collection from mothers who have
not previously been introduced to UCB
banking.
Criteria have been suggested that
protect the woman’s ability to make an
informed deci- sion during childbirth. The
Advisory Council on Blood Stem Cell
Transplantation (ACBSCT) has
recommended that each CBB develop a
policy on informed consent that considers
the stage of labor and stress of the woman,
the amount of counseling on UCB donation
that she has received, and the amount of
time available for an adequate discussion of
UCB donation.28,29 Final judgment regarding
the woman’s ability to give intralabor consent
should rest with the obstetric staff.
In October 2011, the FDA classified UCB
banking as a manufacturing rather than an in-
vestigational activity when a CBB distributes
products for specified indications. If not li-
censed, manufactured UCB products may still
be distributed for nonlicensed (clinical re-
search) applications as an investigational new
drug (IND).
Health History and Medical Evaluation
It is incumbent on the CBB to provide a safe
product by minimizing the transmission of ge-
netic or infectious diseases. A donor-eligibility
determination, based on donor screening and
testing for relevant communicable disease
agents and diseases, is required for all donors
of cells or tissue, including UCB cells. A
robust medical health history designed to
solicit information on exposures to infectious
diseas- es and history of symptoms indicating
genetic disorders is obtained by interviewing
the mother and reviewing her medical record.
The
732 ■ AABB T EC HNIC AL MANUAL
father’s medical history may be solicited to
identify any issues that might affect the
quality of the UCB, but this is not required.
If not se- cured before delivery of the infant,
the history must be obtained no later than 7
days after de- livery.24(p73) The history
questionnaire may be self-administered with
follow-up by CBB staff, or it can be
completed through direct inter- view by
CBB staff or hospital staff who are ade-
quately trained in, and capable of, answering
the woman’s questions.
The approach to screening and testing
the mother is admittedly more conservative
than the approach for the infant donor. It is
the mother’s circulation that nourishes the
fetus during pregnancy and because of this
shared physiology, her infectious exposures
are rele- vant to determining the eligibility
of her in- fant’s UCB for transplantation.
Also associated with risk are certain
maternal conditions dur- ing labor and
delivery, such as fever, prolonged time after
membrane rupture, or administra- tion of
antibiotics—all of which suggest possi- ble
bacterial contamination that could be
transmitted through the UCB product.
Medical screening includes an
extensive family genetic history because the
infant has not had an opportunity to
manifest many in- herited diseases. If first-
degree relatives have a history of
malignancy or parents have been treated
with chemotherapy and/or radiation, the
infant’s UCB cannot be donated. In addi-
tion, if UCB is used to compensate for defi-
ciencies associated with specific genetic
disor- ders, the product is tested for the
presence of the targeted enzyme when such
testing is available.
Infants are not required to be retested or
reexamined at 6 to 12 months of age to
evalu- ate their eligibility to donate UCB
because of the difficulty of locating some
families, con- cerns about parental and
infant privacy, the difficulty of maintaining
the records required to trace families, and
cost. In spite of the diffi- culties of
reconnecting with a donor family, however,
some CBBs follow up with families at
various intervals after birth to ensure that
the infant has not developed any
transmissible diseases and instill confidence
in the suitabili- ty of the infant’s UCB for
transplantation.
Donor Testing
The second step in determining donor
eligibil- ity is accomplished through
laboratory testing. The strategy for UCB
donation is similar to that used for whole
blood donation except that the tests are
performed on maternal blood and not the
blood of the infant donor. This ap- proach is
used because it is assumed that in- fectious
diseases present in the UCB originat- ed
from the mother and, thus, infectivity is
likely to be detected in a maternal blood
sam- ple.
Because testing is conducted on mater-
nal blood, it is necessary to obtain the moth-
er’s consent for testing as well as for collection
of the UCB. It is also important to note that the
testing reagents used must be approved by
FDA for use in donor screening rather than di-
agnostic testing. In addition, the sample types
collected must be cleared for use in this type
of testing, and the laboratory must be autho-
rized to perform the test. In the United States,
no donor tests have been approved for UCB
samples; therefore, infectious disease testing is
performed on a maternal sample. The samples
for infectious disease testing must be collected
on the day of UCB collection or within 7 days
before or after the delivery.24(p40)
The CBB may perform testing or issue
a contract to a laboratory to conduct testing
to rule out collections from donors with
hemo- globinopathies. Certain CBBs store
units from donors who have sickle cell trait
or alpha thal- assemia trait because these
units may have unique HLA types needed
for transplantation in patients from minority
populations. Units with homozygous
abnormal hemoglobin or compound
heterozygous abnormal hemoglo- bin are
unsuitable for transplant. UCB units selected
for transplant in a patient with an in- herited
disease should be tested for that dis- ease
before being used.30 Therefore, it is im-
portant to archive appropriate UCB and
maternal ancillary samples for additional
and/ or confirmatory testing.
Regardless of who obtains the medical
history or tests maternal or UCB samples,
the final donor eligibility must be
determined by
 C H A P TER 30
the CBB medical director and approved by
its quality control unit.22,24(pp1-2,71-72)
UCB COLLECTION
The methods to collect UCB consist of
cleans- ing of the cannulation site and
aseptic collec- tion. Typically, a sterile
collection bag contain- ing citrate-
phosphate-dextrose (CPD) solution
anticoagulant is used, although acid-citrate-
dextrose and lyophilized heparin are also ac-
ceptable anticoagulants for UCB collection.
After delivery, the umbilical cord is
clamped, cut, and separated from the infant.
The clamping must be timed so as not to
inter- fere with routine delivery practice.
UCB can be collected before (in utero) or
after (ex utero) the placenta has been
delivered. There are ad- vantages and
disadvantages to each method, but neither
appears to be better overall.31 A brief
discussion of the two methods is provid- ed
below.
Regardless of whether the UCB is
collect- ed in vivo vs ex vivo, the process of
UCB collec- tions is essentially identical. As
in whole-blood collection, the venipuncture
needle is at- tached to the collection bag.
After an appropri- ate umbilical vein is
identified, the site is cleansed. Typically, this
involves wiping the cord with isopropanol
and then scrubbing it with a broad-spectrum
topical microbicide (eg, povidone iodine)
for at least 30 seconds. Alternatively, a few
CBBs have chosen to use a broad-spectrum
antimicrobial formulation of 2%
chlorhexidine gluconate/70% isopropyl al-
cohol in place of traditional iodophors.
Subse- quently, a hemostat is placed on the
tubing a few inches from the needle. The
hemostat is opened once the vein has been
accessed, al- lowing blood to flow into the
bag. Removal of the hemostat before
puncturing the vein may allow air to
contaminate the tubing. While the UCB is
being collected (a 3- to 5-minute pro- cess),
the UCB should be gently mixed with the
anticoagulant to prevent clotting. The
umbili- cal cord appears collapsed when the
collection is complete. Placement of the bag
on a labora- tory scale during collection
allows the collec- tor to monitor the volume
and provides an
Umbilical Cord Blood Banking ■ 733
additional means to assess completion of
col- lection.
Once the collection is complete, the
tub- ing is “stripped,” forcing the blood
inside the tubing into the collection bag and
allowing it to mix with the anticoagulant.
This can be ac- complished most easily with
a specialized blood-banking tool (ie, a tube
stripper), al- though newer bags include a
filtered vent to eliminate the need for
additional equipment. The UCB unit can
then be packed, stored, and transported to
the cell-processing laboratory in a
temperature-controlled environment.
In-Utero Collection
In-utero collection is generally performed by
the obstetrician or nurse/midwife after the
newborn has been delivered and assessed
and the umbilical cord has been clamped
and cut. If the well-being of the newborn
and/or moth- er is in question, the collection
is not attempt- ed. If a decision to collect the
UCB is made, care must be taken to
maintain sanitary condi- tions. Unless a
sterile bag or extension set is used, the
traditional supplies are not sterile and
inadvertent misplacement could contam-
inate the surgical field.
For logistical reasons, it is advisable to
have preassembled collection kits available
for in-utero collection. An instructional
packet may be included in these kits. This
packet might contain, for example, a donor
informa- tion form; description of the
collection proce- dure; list of collection kit
contents; maternal medical history form;
consent form(s); and packaging, storage,
and shipping instructions. Collection kits
also include items such as one or two
collection bags, antimicrobial supplies,
tubing closures, appropriate sample tubes for
infectious disease testing, sample tube
labels, product labels, biohazard and other
appropri- ate stickers (eg, for indicating
temporary stor- age temperatures), and a
secondary specimen and product bag. In
some cases, the hospital’s maternal label
may be used to identify mater- nal tubes and
UCB products because these la- bels include
two forms of identification and precautions
are taken to protect donor confi- dentiality
on these labels.
734 ■ AABB T EC HNIC AL MANUAL
Validated containers and/or processes
are used to ship the UCB. Acceptable
tempera- tures during shipping can range
from cold to ambient, depending on the
shipping distance and conditions, and are
defined based on sta- bility studies.
Temperature during transport may be
monitored depending on the risk to the UCB
involved in the shipping process.
The primary advantage of in-utero com-
pared to ex-utero collection is the substantial-
ly lower cost because dedicated CBB
collection personnel are not required. As a
result, the only costs are for collection and
shipping sup- plies. In addition, several studies
have suggest- ed that in-utero collection
methods may result in the collection of greater
volumes of cells and higher counts of
nucleated and CD34+ cells and colony-
forming units (CFUs).32,33
In addition, the initial costs and efforts
associated with education and training of the
physician/midwife collectors need to be con-
sidered. However, once obstetrics practice
group members are trained, their hands-on
experience should improve the quality of their
collections. In-service and educational ses-
sions can provide refresher courses and dem-
onstrate appreciation of clinicians’ support for
UCB collection. Subsequently, a program of
continued competency assessments, collec-
tion-site visits, and regular communication
can be utilized to maintain high-quality UCB
collections.
Ex-Utero Collection
Ex-utero collections are often performed by
dedicated UCB collection staff. The advantag-
es of this approach over in-utero collection are
its use of standardized collection methods,
limited personnel involvement, and greater
control.
After delivery, the cord is clamped at a
time that does not interfere with the physi-
cian’s routine practice. The placenta is
imme- diately taken by CBB staff to a
suitable loca- tion, where it is suspended in
a device to allow collection of blood by
gravity. The collection proceeds as described
above. The placenta and cord are more
accessible in the ex-utero setting and,
therefore, the use of manipula-
tion, such as application of pressure to the
cord (“milking”), can increase the collection
volume. Caution must be used to prevent
ma- ternal contamination and lysis of cells.
Because blood clotting readily occurs
during UCB collection, which adversely
affects the volume and number of cells
collected, it is important to minimize delays
during the col- lection process. Wong and
colleagues hypoth- esized that a higher
incidence of macroscopic clots in ex-utero
collections resulted in the col- lection of
lower nucleated cell and CFU counts.34
Because it occurs outside the delivery
room with all of its activity, ex-utero
collection may inherently provide better
process control (ie, a lower risk of microbial
contamination and labeling errors), but it
raises the costs as- sociated with collection.
In addition, a lack of availability of
collectors on all shifts may limit the
opportunity for a facility to participate in
UCB collection. Some investigators have
ques- tioned these points.35 Because data
demon- strate that in-utero and ex-utero
collection methods are equivalent, the
choice of UCB collection method should be
based on the needs and capabilities of each
UCB collection program.31
UCB PROCESSING
Methods
In the early 1990s, UCB units were often
stored in an unmanipulated state without
volume re- duction (ie, red cells and/or
excess plasma were not removed before
storage) in an effort to conserve the number
of stem cells in the product. 18,36 Concern
about infusion-related complications
associated with red cell incom- patibility,
free hemoglobin, dimethyl sulfoxide (DMSO),
and logistical issues surrounding limited
storage capacity motivated an evalua- tion
of processing methods to minimize the red
cell content and size of the final UCB unit
while limiting stem cell loss.
At present, a variety of processing tech-
niques for volume reduction and removal of
red cells are employed, and the majority of
these methods involve sedimentation,
centrif-
 C H A P TER 30 Umbilical Cord Blood Banking
ugation, and/or filtration. The most common
means of reducing red cell content is the use
of sedimenting agents, such as hydroxyethyl
starch (HES), gelatin, polygeline, and dex-
tran.37,39 One of the earliest methods for pro-
cessing UCB utilizing HES was developed by
Pablo Rubinstein.39 Modifications of his meth-
od have been commonly used by other pro-
grams.40 An alternative preparation method
involves density separation by layering UCB
cells over Percoll or Ficoll-Hypaque. This
method ultimately results in a mononuclear
cell-enriched product essentially devoid of red
cells.41 Also, utilization of commercially avail-
able leukocyte reduction filters and semiauto-
mated methods results in similar in-vitro cell
recovery to the standard HES method.37,42
Initially, UCB was processed with other
HSC products in laboratories that were part
of a research facility, hospital transfusion
ser- vice, or blood center. However, the
methodol- ogy for processing UCB products
has evolved to involve dedicated processing
laboratories designed to comply with current
good manu- facturing practice (cGMP) by
using more stan- dardized processing
techniques for volume reduction, removal of
red cells, and cryo- preservation.
An important driving force behind most
significant recent changes in UCB
processing was a set of recommendations
made by FDA as part of its process for the
submission of bio- logics license
applications (BLAs). These rec-
ommendations provide guidance on how to
provide assurances of the safety, purity,
poten- cy, and effectiveness of UCB
products. These recommendations have
compelled the indus- try to standardize its
manufacturing processes by using reagents
that have been approved for human use and
systems and supplies that have been cleared
by the FDA. To date, the fol- lowing
systems have been approved by the FDA for
the processing of UCB:
■ Automation technology incorporated into
the SEPAX Cord Blood Processing System
manufactured by Biosafe SA (Lake
Geneva, Switzerland). This technology
offers a closed and sterile processing
system that harvests nucleated cells and
enriches stem
■ 735
cells from UCB. The technology is adapt-
able to a large-scale processing environ-
ment. The SEPAX machine, which is
essen- tially composed of a centrifuge
with piston position sensors surrounding
the chamber and pneumatic pump
system, is operated using computer-
controlled protocols to achieve
component separation. Consum- able kits
consist of a large syringe-type bar- rel
separation chamber with bags and tub-
ing connected via a stopcock assembly.
Biosafe’s SEPAX system received FDA
clear- ance in January 2007 and a
European CE mark of approval in 2001.
■ The AXP (AutoXpress Cord Blood
Process- ing System), manufactured by
ThermoGen- esis (Rancho Cordova, CA), is
an automat- ed, functionally closed system
that harvests stem-cell-rich buffy coat from
UCB. The system reduces a unit of UCB to
a precise volume chosen by the operator
and selec- tively isolates mononuclear cells.
The sys- tem includes the AXP device, a
docking sta- tion, a processing set, and
XpressTRAK software. The AXP system
received 510(k) clearance from the FDA in
October 2007.
■ PrepaCyte-CB, a UCB-processing system
manufactured by CytoMedical Design
Group (St. Paul, MN), received FDA
510(k) clearance in January 2009.
PrepaCyte-CB is a functionally closed
sterile bag system composed of three
integrally attached pro- cessing and storage
bags containing the PrepaCyte-CB
separation solution. While isolating
nucleated cells, PrepaCyte-CB re- moves
most red cells and nucleated red cells from
the final processed UCB unit. The system
requires plasma extractors and a standard
laboratory centrifuge to sedi- ment and
concentrate desired cells.
Each of these methods has advantages
and disadvantages with respect to cell recov-
ery, red cell removal, processing time, and
cost. Each laboratory must therefore validate
and determine which processing method is
most appropriate for its situation. Cellular
function, product integrity, and safety must
be considered when developing a validation
plan. Nucleated and CD34+ cell recovery,
viability,
736 ■ AABB T EC HNIC AL MANUAL

potency (eg, ability to form CFUs), and Cryopreservation and Long-Term

sterility testing (before and after
cryopreservation) are typical validation
performance measures. Fac- tors to consider
when choosing a processing method include
workload, processing time, cost, maximization
of storage time, and pro- duction efficiency
(number of UCB units pro- cessed per staff
member per day).
Quality Control Testing and
Characterization
Quality control (QC) testing to assess the ade-
quacy of the product and process is essential.
QC testing is usually performed on receipt
(prior to manipulation) of a UCB unit and be-
fore cryopreservation. Typical QC assays in-
clude counts of nucleated cells (white cells
plus nucleated red cells), mononuclear cells
(monocytes plus lymphocytes), and CD34+
cells; hematocrit; CFU assay; and viability and
sterility (aerobic, anaerobic, and fungal) test-
ing. All testing methods must be validated for
their intended use.
UCB products used for allogeneic trans-
plant must be tested for HLA Class I and Class
II antigens, including HLA-A, -B, and -DRB1
loci; HLA-C and HLA-DQB typing is recom-
mended.22 In this setting, ABO/Rh typing is
performed primarily to assist clinicians with
red cell and plasma product support after in-
fusion. Table 30-1 lists these basic tests.
Storage
Successful outcomes of UCB transplantation
hinge on the CBB’s ability to preserve the
in- tegrity of UCB over substantial lengths
of time. Freezing and storage methods must
be robust enough to ensure that the quality
of the UCB unit is maintained for many
years.
The most common method of UCB
cryo- preservation consists of freezing the
cells in a cryogenic bag in the presence of
10% DMSO as a cryoprotectant.43-45 One of
the main advan- tages of using cryogenic
bags over freezing vi- als is the ability to
perform post-thaw confir- matory HLA
typing and QC testing using an integrally
attached tubing segment. Both AABB and
the Foundation for the Accredita- tion of
Cellular Therapy (FACT) mandate the use
of cryogenic bags with attached segments
for the freezing of UCB.22,24(p53)
It has been well established that slow
cooling in a programmable controlled-rate
freezing device at a rate of 1 C/minute
results in adequate recovery of CD34+
cells/human progenitor cells.44,45 Such
instruments com- pensate for the heat of
fusion phase, mitigate process variability,
and limit cell damage. As with all critical
processes involved in collect- ing and
storing UCB, the freezing process must be
validated and procedures must be in place
that govern the use and operation of the con-
trolled-rate freezer (or equivalent), expected

TABLE 30-1. Summary of QC Testing for UCB

TestMethod(s)

Cell count Hematology analyzer, manual

differential CD34+ cell enumeration Flow cytometry (single or dual
platform)
Viability assay Dye exclusion (light and/or fluorescence microscopy), flow cytometry
Clonogenic assay CFU (most commonly used in clinical laboratories), LTC-ICs
Sterility testing Aerobic, anaerobic, and fungal culture
HLA typing Molecular
ABO/Rh typing Serology
Hematocrit Standard/routine
QC = quality control, UCB = umbilical cord blood, CFU = colony-forming unit, LTC-ICs = Long-term culture-initiating cells.
 C H A P TER 30 Umbilical Cord Blood Banking
cooling rates and freezing curve parameters,
endpoint freezing temperature, addition of
cryoprotectant, final cell and cryoprotectant
concentrations, and storage temperature. Be-
cause equipment failures can occasionally
oc- cur, simplified passive freeze methods
can also be validated to provide satisfactory
cryo- preservation of human stem cell
products.46,47
Both AABB and FACT require cryopre-
served UCB to be stored at <–150 C.22,24(p54)
Once frozen, UCB units are typically trans-
ferred into a monitored liquid-nitrogen (LN2)
storage container and either overwrapped
and
immersed in liquid (at –196 C) or in vapor
phase to minimize the potential for cross-
contamination during long-term storage.
To ensure the safety and stability of the
stored UCB units, control measures should
be established for product security and
segrega- tion, storage container monitoring,
inventory control, and duration of storage.
Storage con- tainers must be located in a
secure area to pre- vent unauthorized access.
For units in which transmissible disease
screening and testing re- sults are positive or
the testing is incomplete, there must be a
designated quarantine storage area or
process to prevent accidental release. To
ensure that temperature and LN2 levels are
continuously maintained, a monitoring sys-
tem with local and remote alarm capabilities
must be in place. Alarm limits should be set
to allow adequate time for staff to respond
to no- tifications and react before inventory
is com- promised. All UCB products and
reference sample storage locations should be
cataloged using an inventory-management
system that allows for rapid retrieval. The
duration of stor- age and product expiration
dates should be defined in operating
procedures, even when expiry dates have yet
to be determined.
Studies of the long-term effects of ultra-
low-temperature storage of UCB units have
shown that retention of viability and/or
prolif- erative function of UCB cells frozen
in the liq- uid phase of LN2 remains
acceptable for at
least 23 years.48,49 However, each CBB must es-
tablish and maintain a written testing
program designed to assess the integrity,
potency, safe- ty, and stability of drug
products manufac- tured under its facility-
specific manufacturing
■ 737
and storage conditions. The CBB’s UCB
pro- gram must address individual products
before distribution (lot release testing) and
demon- strate the stability of the drug’s
active ingredi- ents, used in determining
expiration dates.
Post-Thaw Stability and Other Testing
Accreditation organizations require UCB
products to have integrally attached
segments that are representative of the UCB
product and used to verify the results of
HLA typing.24(p78) One of these segments can
be used for confir- matory testing—a
process in which product identity is
confirmed and potency is evaluated before
the UCB unit is released to a transplant
facility. Extended HLA typing, viability,
poten- cy, or stability testing can be
performed using other replicate aliquots of
the UCB unit (reten- tion samples).
Progenitor assays, CD34+ cell enumeration,
viability testing, or others tests performed
on segment material before distri- bution
may be useful in determining the prod- uct’s
potency. Traditionally performed as an
internal QC process, the results of these tests
are not released due to their lack of demon-
strated correlation with engraftment.
Howev- er, transplant centers, knowing that
these re- sults are available, are requesting
them from CBBs when determining which
UCB units to select. Accordingly, studies are
under way to assist with interpretation of the
variable re- sults obtained when testing is
performed on the limited material in these
samples.
SHIPMENT
With a number of commercial carriers avail-
able, UCB can be routinely transported to
pro- cessing laboratories or transplant
facilities within hours to a few days. It is
important to ensure that UCB units and
products are prop- erly packaged and
shipped to prevent damage or deterioration
during shipment. This pro- cess is the
responsibility of the CBB. Validated
packaging and shipping procedures should
demonstrate that acceptable temperatures
and unit integrity are maintained. 24(p35) Each
CBB needs to define the shipping conditions
(temperature, type of shipping container,
and
738 ■ AABB T EC HNIC AL MANUAL
packaging material) for the transport of its
fresh and frozen UCB products.
Shipping methods must also be designed
to protect the safety of the personnel in-
volved.22,50 The FDA, International Air Trans-
port Association (IATA), US Department of
Transportation (DOT), AABB, and FACT
have established packaging and labeling
require- ments for the shipping of
biologics.22,24,51-53 IATA requires that shipping
containers be able to withstand extreme
external temperature variability and that
primary outer containers be leakproof,
constructed to resist breakage, and durable
enough to withstand pressure changes and
falls. In addition, CBBs must package UCB in
a secondary inner container, such as a plastic
resealable bag, with enough absorbent material
to contain the contents of the product in the
event of a leak or break.22,52
Shipping Fresh UCB
The requirements for the transport of freshly
collected UCB are not well defined, and
each facility must establish transport
temperature criteria and acceptable limits
that are com- mensurate with the risks
involved. Available options include transport
at room tempera- ture or use of insulated,
precooled stabilizing packs. Wada and
colleagues observed a 1% drop in viability
for every 4-hour increase in transport time
for recently collected UCB units that were
shipped at ambient temperature.54 However, a
series of studies has shown that UCB can be
preserved in CPD for up to 48 hours
before processing and retains satis- factory
cell recovery rates and progenitor con-
tent.55-58
Shipping Cryopreserved Units
An advantage of selecting a frozen UCB prod-
uct over a fresh marrow or apheresis product is
that the frozen UCB product can be shipped
and secured at the transplant facility before
the recipient is conditioned for transplant.
Cryopreserved products are shipped to trans-
plant facilities in portable LN2 “dry” shipping
containers to maintain the products in a fro-
zen state. Insulated containers are used that
allow LN2 to be absorbed into the vessel wall,
creating an ultracold environment. FACT re-
quires that LN2 dry shippers be validated to
maintain temperature of <–150 C for at least
48 hours beyond the time of delivery to the
trans-
plant facility.22
For a dry shipper to be fully effective, it
must be charged or filled with LN2 24 hours
be- fore the estimated time of release from the
processing laboratory. This allows for com-
plete absorption of the LN 2 into the wall of the
shipping container. Improperly prepared dry
shippers present a risk of LN2 leakage, and
CBBs may be subject to civil/criminal penal-
ties from US DOT in the event of a spill.
Dry shippers are vulnerable to incident
handling and can be compromised if they
are mistreated or positioned incorrectly.
This will result in warm conditions and
thawing of the product, rendering it
unusable for the clinical procedure. To
minimize the risk of product loss, shipping
instructions must be clear and the conditions
of transport, particularly tem- perature, must
be continuously monitored.
Transport Labeling and Record
Requirements
AABB and FACT require continuous
monitor- ing of the temperature during
shipment, which is accomplished through
the use of a data logger.22,24(p35) According to
AABB and FACT, the shipping container
must include the name, address, and
telephone number of the shipping and
receiving institutions; the phras- es “medical
specimen,” “do not irradiate” (if applicable),
and “do not x-ray”; and biohazard labels (as
appropriate).22,24(p68) Biological prod- ucts
must be packaged and shipped in compli-
ance with all applicable governmental
regula- tions. Transportation records that
identify the shipping facility, date and time
the unit was shipped and received, courier,
and contents of each shipping container
should be main- tained.22
RECEIPT OF UCB FOR
TRANSPLANTATION
UCB transplantation is a coordinated effort
potentially involving several entities—the reg-
 C H A P TER 30
istry, such as the National Marrow Donor
Pro- gram (NMDP), CBB, and cell-processing
labo- ratory/clinical team at the transplant
center. The process is initiated by the
placement of a reservation of or order for a
UCB unit, usually by the clinical program’s
transplant coordina- tor, based on the
institutional algorithm or guidelines. The
coordinator may rely on the laboratory or
medical director to answer ques- tions
related to a prospective UCB unit, in-
cluding those associated with technical
issues and donor medical history. It is
advisable to initiate involvement of the cell-
processing lab- oratory early in the unit-
selection process.59
Once the unit’s quality is confirmed and it
is approved for shipment, the coordinator
should notify the cell-processing laboratory of
the impending arrival of a UCB unit, including
any special handling requirements (eg, size
and dimensions of product canister or indica-
tions of risk factors requiring quarantine).
When the unit is received at the laboratory, it
should be carefully unpacked and examined to
verify its labeling and integrity before it is
placed into the LN2 storage tank. The dry ship-
per may be weighed to check for excessive
LN2
loss (in general, greater than 10 pounds).
Because both AABB and FACT
standards require continuous temperature
monitoring of cryopreserved products during
shipment, temperature-monitoring devices
must be in- cluded in the shipment. Upon the
unit’s arriv- al, the technologist should
confirm that the UCB remained within the
acceptable tempera- ture range during
shipment. If the tempera- ture-monitoring
device displays a digital tem- perature, the
temperature when the unit is unpacked
should be recorded. If the device does not
show a temperature or the data can- not be
downloaded, the CBB should forward a
copy of the data when they become
available.
If the device for continuous temperature
monitoring cannot be located, a question and an integrally attached segment
temperature- measuring device from the
transplant center should be inserted for
several hours to ensure that the shipper can
maintain an acceptable temperature. The
CBB should be notified that no temperature-
monitoring device was pres- ent in the
shipment, and documentation of
Umbilical Cord Blood Banking ■ 739
the CBB’s shipper validation should be re-
quested.
The identity of the UCB unit should be
verified at the time of inspection, before the
unit is placed in storage. The product label
should indicate the appropriate minimum
partial label requirements—unique product
identifier (ie, unit number) and proper prod-
uct name. All other product information
should be attached to the product on a tie tag
and/or in the accompanying paper work.
Whether affixed or attached, the CBB paper
work that accompanies the UCB and the
docu- ments generated by the transplant
program/ coordinator should be compared.
Any incon- sistencies should be immediately
and appro- priately investigated.
The unit information, including donor
medical history and infectious disease
testing results, should again be reviewed for
com- pleteness. If there are any comments
not pre- viously communicated to the
receiving labora- tory, particularly any
related to the donor’s medical history, they
should be referred to the receiving
laboratory’s medical director. The medical
director should determine whether the
information is significant and requires any
further action and/or notification of the
trans- plant physician. If any infectious
disease test- ing is found to be incomplete or
the results are positive, the unit should be
placed into quar- antine storage, and the
appropriate personnel (eg, laboratory
supervisor, medical/laboratory director,
coordinator, and/or transplant physi- cian)
should be notified. The product label/tie tag
must be updated accordingly (eg, to in-
clude a biohazard label) and a special
medical release form may need to be
completed as well.
Any further testing deemed necessary by
the transplant center (eg, additional HLA or
viability testing or CFU count) should be
performed as soon as possible on an
integrally attached segment, if one
accompanies the unit. If a unit’s identity is in
is not available, a rapid (Class I serologic)
HLA test may be per- formed along with the
standard product test- ing on the thawed
product (ie, on the day of the transplant
procedure).60
740 ■ AABB T EC HNIC AL MANUAL
Instructions for handling and preparing
frozen UCB products are provided by the
UCB manufacturer in the shipment.
However, the receiving laboratory may use
alternative thaw- ing methods based on its
own validated proce- dures, clinical
protocol, or physician prefer- ence.
THAWING AND WASHING OF
UCB
Coordination of the transplant event requires
consistent communication among all mem-
bers of the clinical team. In the days before
the planned transplantation, the coordinator
should verify the infusion date with the
clini- cal team, and both the coordinator and
labora- tory should again review all relevant
records. Subsequently, the patient care unit
should be contacted at least 1 day before the
day of transplant to schedule an infusion
time. Given the wide variety of types of
products received from an increasing
number of CBBs, the most appropriate
thawing procedure must be deter- mined
based on the transplant center’s vali- dated
method or CBB’s recommendations. There
may be considerations if the product was
manufactured to reduce red cell and plas-
ma content (RBC reduced) or to reduce
plas- ma content only (red cell replete)
before cryo- preservation. The choice of the
thawing procedure may be further affected
by the re- quirements of the clinical protocol
in which the patient is enrolled.
Currently, three different practices for
preparing UCB products for infusion are
avail- able: the traditional thaw-and-wash
method, the thaw-and-dilution technique,
and the bed- side thaw method.61,62
Despite the potential advantage of the
bedside product preparation method in mini-
mizing potential cell loss from postthaw ma-
nipulation, this approach is not
recommended because of the difficulty of
rescuing the prod- uct at the bedside if the
bag’s integrity is com- promised and the
adverse effect of prolonged exposure of
thawed cells to DMSO if the infu- sion is
delayed. Furthermore, this method lacks the
capacity for process control and product
assessment. Finally, this method
should not be used with red cell-replete
prod- ucts according to recent FACT
requirements.
The traditional washing method
original- ly described in 1995 is
recommended if the product’s exposure to
DMSO, free hemoglobin, and volume
approach critical limits for recipi- ents,
particularly pediatric patients or those with
underlying cardiac, pulmonary, or sensi-
tizing conditions.39 More recently, a dilution
or simple reconstitution approach has gained
support, primarily due to concerns about cell
loss during the wash step.63,64 Both methods
are initiated in similar fashion:
■ The UCB product is carefully removed
from the storage tank and a thorough
inspection is performed to evaluate the
container’s in- tegrity. Verification of the
product’s identity on its label is
conducted by two technolo- gists.
■ The unit is sealed in a clean or sterile trans-
parent bag (ie, a plastic zipper bag) and
submerged in a 37 C waterbath of clean or
sterile water or saline. Gentle kneading of
the UCB bag during thawing helps acceler-
ate the thawing process while preventing
recrystallization and consequential cell
damage or death. If a leak is discovered af-
ter initiation of the thaw procedure, the site
of the container break is determined and a
hemostat is employed or the product is po-
sitioned to prevent further escape of the
cellular material. The contents can then be
aseptically transferred into a transfer bag
under a biological safety cabinet.40
■ The thaw solution is used for product
dilu- tion and should be prepared in
advance. It typically contains 10%
dextran and a pro- tein source, usually
human serum albumin in a final
concentration of approximately 2.5% to
4.2%. The supplementation of the thaw
solution with protein (albumin) has been
proven to restore osmolarity and ex- tend
cell viability.39 Subsequently, a volume of
solution at least equal to the volume of
the UCB product is gradually added to
the bag while it is gently mixed. The
product and solutions are drained into a
labeled transfer bag and left to equilibrate
for 5 minutes. At this stage, if the product
is to be
 C H A P TER 30 Umbilical Cord Blood Banking
reconstituted or diluted only, samples are
removed for product testing.
■ If the product is to be washed, the labeled
transfer bag is centrifuged at 400-600 × g
for 15 minutes at 10 C. The supernatant
is ex- pressed, leaving behind a pellet of
washed UCB cells. If it is the policy of
the transplant laboratory to centrifuge the
supernatant, the second labeled transfer
bag is spun at 400 × g for 15 minutes at
10 C, the superna- tant is again
expressed, and the two cell pellets are
combined into one labeled bag.
■ The UCB cells are resuspended in a
volume of thaw solution that is
appropriate for the weight of the patient
while additional con- sideration is given
to the potential for fluid overload.
■ Some laboratories filter the resuspended
product with a standard blood filter (at
170- 260 microns).
■ Samples are removed for QC tests, such as
total nucleated cell (TNC), CD34+, and
CD3+ cell counts; microbial culture (bacte-
ria, fungus); confirmatory ABO/Rh typing;
CFU assay; and viability testing. Final vol-
ume, cell dose, and cell recovery are deter-
mined. After completion of paper work and
labeling, the unit can be released for infu-
sion).58
It is anticipated that thawing procedures
based on the type of product processed will
be standardized through the collaboration of
UCB bankers and transplanters.
INFUSION OF UCB
To facilitate the product infusion procedure,
it is necessary to maintain good
communication between the patient and the
physician over- seeing the infusion. Hence,
it is a good practice to again describe the
general process, includ- ing graft selection,
cell processing, infusion, potential side
effects/adverse reactions, and plans for
premedication. This conversation should
take place on, or shortly before, the day of
the transplant procedure. A procedure for
infusion is outlined below.50 The general ap-
proach and potential adverse reactions are
■ 741
similar to those for infusions of other HSC
products.65
Once the UCB product has been thawed
and washed, it should be delivered to the pa-
tient care unit without delay. Subsequently, a
form acknowledging the receipt of the UCB
should be signed by a nurse, who then
notifies the patient’s physician of the UCB
unit’s arriv- al. After the physician approves
the infusion and proper identification
procedures are fol- lowed, the unit is infused
by intravenous (IV) drip or syringe push
directly into a central line without a needle
or a pump. Some institutions use a standard
blood filter at the bedside. If a filter is used
in the laboratory after the thaw/ wash, a
second standard blood filter at the bedside is
not needed.
The thawed UCB must be transfused as
soon as clinically possible. Timely infusion is
most likely to be challenging with UCB that
has not been washed or diluted. Although
Rowley and Anderson66 concluded that DMSO
is not toxic to HSCs at clinically relevant con-
centrations (ie, 5% or 10%) at either 4 C or 37
C for up to 1 hour of incubation, they also
noted that the addition of 1% DMSO to culture
dish- es suppresses CFU assay results. It is
impor- tant to note that these studies were
performed on fresh cells.67 Studies of the
effects of DMSO on HSCs that have been
previously cryopre- served are limited.
However, some investiga- tors have noted
similar suppression of colony formation when
thawed UCB samples are not washed and are
immediately placed into CFU culture. 67 Thus,
the possible functional defect due to DMSO
coupled with the not infrequent need to hold
clinical products raises the con- cern that cell
injury could occur with thawed UCB,
particularly when cells are not washed or
diluted.
The unit bag and IV tubing should be
flushed with sterile saline after the unit bag
empties to maximize cell dose. Sterile saline
may be added directly to the unit bag if the
flow rate becomes unusually slow. Because
the final volume of a UCB unit is relatively
low (roughly 60 to 100 mL with the
thaw/wash methods described above), the
infusion rate is slow (5-10 mL/minute) and
the infusion pro- cess is typically
completed within 15 to 30
742 ■ AABB T EC HNIC AL MANUAL

re-
minutes of the unit’s receipt in the patient
care unit.
It is recommended that the patient’s
vital signs be checked before infusion,
immediate- ly after infusion, and 1 hour
after infusion at a minimum. Should an
adverse reaction occur, more frequent
monitoring is recommended. The transplant
physician and the medical di- rector of the
cell therapy laboratory should be notified
immediately of an unexpected or moderate
to severe reaction. A prompt investi- gation
should include confirmation of product
identity, a product inspection, and the initia-
tion of any appropriate laboratory testing
(eg, direct antiglobulin test, antibody titers,
Gram’s stain, or culture). All of the
monitoring results should be captured on the
accompanying in- fusion form, which
should be returned to the laboratory. Serious
adverse reactions must then be
communicated as appropriate to the NMDP,
distributing CBB, and/or the IND ap-
plication or license holder for reporting to
the FDA.
Reactions associated with UCB infusion
may be very similar to those that
occasionally occur with blood transfusion
(ie, allergic, he- molytic, or febrile reactions
and those due to microbial contamination).
However, with combinations of the various
processing meth- ods for UCB (eg, red cell
depletion, plasma re- duction, and postthaw
wash step), some types of reactions to UCB
(ie, those attributed to red cell antigens and
plasma proteins) may be less likely to occur
with UCB than other blood transfusions.
Likewise, if the UCB has under- gone red
cell depletion and/or wash steps, re- nal
failure due to infusion of red cells and free
hemoglobin should occur less often than
with infusions of HSCs from other sources.
Reac- tions (ie, nausea, vomiting, coughing,
and headache) often attributed to DMSO
should be less common with UCB infusions
as well.68,69 Bacterial contamination, which
oc- curs in up to 5% of collections, is
unusual in released UCB units because
CBBs exclude these units from their
inventories.70-72
UCB infusions are usually very well
toler-
ated; if reactions occur, they are typically mild
and readily managed by the clinical team.65
However, because the possibility of a severe
 Improving the efficiency of the collection
action exists, aggressive process in estab- lished centers could increase
IV hydration is recom- the number of products eligible for further
mended (eg, for 2 to 6 manufacture and their likelihood of selection
hours before and 6 while offsetting the associated costs related to
hours after infusion education, staff- ing, and transportation.
with diuretics as Collaborating to iden- tify alternative uses for
needed). In addition, clinical-grade products could also help CBBs
administration of achieve self-sufficiency.
prophylactic an- The transition to a full cGMP setting has
tiemetics, antipyretics, been accompanied by spending money to
and antihistamines is ex- pand UCB inventories. The facility used
suggested. to manufacture human cells, tissues, and
cellu- lar and tissue-based products
(HCT/Ps) must be maintained in a clean,
ECONOMIC ISSUES sanitary, and orderly manner to prevent the
The establishment of a introduction, transmis- sion, or spread of
diverse and sustainable communicable diseases, as described in Title
inventory from which 21, CFR Part 1271.190(b). To minimize the
approximately 1% of risk of product contamination, manufacturers
products are distributed (CBBs) may upgrade their facil-
for clinical use is an
expensive undertaking
for a CBB. One of the
main reasons for this
selectivity in product uti-
lization is the strong
association of successful
outcomes of UCB
transplantation with the
grade of the HLA match
and cell dose.73 As a
result, UCB selection has
recently been direct- ed
toward utilization of
products with high TNC
content. This finding
further complicates the
strategy of banking
products from donors
who are not well
represented in registries
due to the reduced
volume and cellular
content in the collections
from these populations.
To limit the negative economic impact of
these trends, CBBs must
expand their collec- tion
efforts to increase the
proportion of units with
higher TNC counts.74 In
addition, ex- panding the
number of collection sites
to al- low broader
participation of donors
could support
diversification efforts.
 C H A P TER 30 Umbilical Cord Blood Banking
ities to offer International Organization for
Standardization Class 5 processing areas,
de- fine robust cleaning and disinfection
plans, enhance environmental monitoring to
ensure continued control of the
manufacturing areas, and develop
comprehensive stability plans to establish
expiry dates of products manufac- tured
under these conditions. Facility retrofit-
ting, process redesign, and hiring of
additional staff to accommodate upgraded
activities in- crease the fixed cost of UCB
manufacturing.
Whereas pharmaceutical drug manufac-
turers can recover the cost of research and
de- velopment along with a substantial profit
mar- gin from the consumer, it is
improbable that CBBs will be able to
incorporate increasing costs of UCB product
manufacture into the amounts invoiced to
clinical programs for product acquisition.
Doing so may encourage transplant centers
to pursue alternative means of treatment for
their patients, a strategy that would be
detrimental to the survival of UCB
manufacturers. Therefore, public CBBs must
be innovative and resourceful in all aspects
of their business strategy.
The Stem Cell Therapeutic and
Research Act
The Stem Cell Therapeutic and Research
Act of 2005 was passed by Congress and
signed by President George W. Bush in
December 2005 as Public Law 109-129. In
October 2010, Presi- dent Obama signed the
Stem Cell Therapeutic and Research
Reauthorization Act of 2010 (Public Law
111-264). The implementation of both acts
is managed by the Health Resources and
Services Administration (HRSA) of the US
Department of Health and Human Services
(DHHS). Provisions in these acts that are
spe- cific to UCB include:
■ The CW Bill Young Cell Transplantation
Program, intended to increase the
numbers of UCB units and establish an
outcomes da- tabase to collect data to
characterize and refine all aspects of
UCB transplantation. Through the Stem
Cell Therapeutic Out- comes Database,
the Center for Interna- tional Blood and
Marrow Transplant Re-
■ 743
search received a contract to collect data
on all allogeneic (related and unrelated)
HSC transplantations performed in the
United States and those that were
completed with products procured
through the program but performed
outside the United States.
■ Solicitation and subsequent contractual
agreements with CBBs to collect and store
at least 150,000 new UCB units that meet
specific criteria comprise the National Cord
Blood Inventory (NCBI). CBBs with a con-
tract from the NCBI also provide UCB
units that do not meet these criteria for
research studies.
■ Requirement for the US Government Ac-
countability Office to report on efforts to
increase UCB unit collection for the
NCBI.
■ Establishment of the ACBSCT to make rec-
ommendations to the Secretary of DHHS.
This legislation not only validated UCB
as a credible therapeutic option but also
provid- ed financial support to CBBs for the
creation of larger inventories. However,
federal funding does not cover the costs
associated with man- ufacturing and is
limited in terms of appropri- ations. In the
2010 reauthorization, Congress challenged
CBBs to expand their collection while
lowering costs and improving the effi-
ciency of UCB unit collection without
decreas- ing the quality of the UCB units
collected. In addition, CBBs are required to
demonstrate ongoing measurable progress
toward achiev- ing self-sufficiency in their
collection and banking operations.
REGULATIONS AND
STANDARDS
FDA
In 1997, the FDA announced a novel, risk-
based, tiered approach to the regulation of
so- matic cells and tissues.75 The approach
out- lined in the FDA’s proposed approach
to regu- lation of cellular and tissue-based
products (February 27, 1997) was followed
by the good tissue practice (GTP)
regulations of 2004 “requiring cells and
tissues to be handled ac- cording to
procedures designed to prevent
744 ■ AABB T EC HNIC AL MANUAL

contamination and preserve [cell and] tissue
function and integrity.”51 This historic docu-
ment has set the framework for cell therapy
laboratories by identifying the agency’s
expec- tations regarding the prevention of
disease transmission and laboratory process
controls. The GTP requirements are
intended to 1) pre- vent unknowing use of
contaminated tissue at risk of transmitting
infectious disease, 2) pre- vent improper
processing of tissue in ways that could cause
damage or risk of contamina- tion, and 3)
ensure the safety and efficacy of products
that are more than minimally manip-
ulated.75,76 Based on these principles, all hu-
man cellular and tissue-based products in-
tended for use in unrelated recipients,
regardless of degree of manipulation or
poten-
tial risk, are required to comply with the re-
quirements for donor testing and screening,76
establishment registration,77 and the current
GTP regulations.51
Since 2004, CBBs have been required to
register with the FDA as manufacturers of
UCB51 and demonstrate compliance with
GTPs. For CBBs that manufacture and/or
store more than minimally manipulated
products (eg, UCB that is activated,
expanded, or genet- ically modified) or
combine UCB with non- tissue components,
the FDA requires submis- sion of an IND
application and adherence to licensure
application requirements. Table 30-2
summarizes the FDA’s regulations
governing HCT/Ps, including UCB.

TABLE 30-2. Summary of FDA Regulations Regarding Human Cells, Tissues, and Cellular
and Tissue-Based Products (HCT/Ps) and Hematopoietic Progenitor Cells-Cord (HPC-
Cs)75
Products RegulatedSpecific Product

HCT/Ps that are or will be Administration.

regu- lated as biological
products

HPC-Cs that are currently

subject to biologics license
application (BLA)
requirements

HPC-C that is currently subject

to investigational new drug
require- ments

FDA = Food and Drug

■ All allogeneic, unrelated ogic reconstitution in patients with disorders affecting the
HCT/Ps derived from cord and hematopoietic sys- tem that are inherited, acquired, or result from
peripheral blood myeloablative treatment.
■ HCT/Ps ■ FDA intends to grant licensure to products manufactured according to
that are recommended establishment and process controls and that comply
more with all applicable regulatory requirements.
than
minimall ■ When no satisfactory alternative treatment is available, HPC-Cs that
y are not FDA licensed and are:
manipul 1. Allogeneic, unrelated, and minimally manipulated.
ated 2. Intended for use in unrelated donor HPC transplantation
(eg, procedures in conjunction with an appropriate preparative
expande regimen for hemato- poietic and immunologic reconstitution in
d, acti- patients with disorders affecting the hematopoietic system that
vated, are inherited, acquired, or result from myeloablative treatment.
or 3. Manufactured in a US cord blood bank and intended to be
genetic used in the United States.
ally ■ Manufactured in a US cord blood bank before BLA approval and do
modifie not meet licensing requirements.
d). ■ Prospectively manufactured in the United States and for which there
■ HCT/Ps that are combined is no satisfactory alternative.
with a drug, device, or
biological product.
■ HCT/Ps
that are
intende
d for
nonhom
ologous
use (eg,
for
cardiac
repair).
■ Allogene
ic,
unrelate
d, and
minimall
y
manipul
ated
cells
intended
for use
in
unrelate
d donor
HPC
transpla
ntation
procedur
es in
conjuncti
on with
an
appropri
ate
preparati
ve
regimen
for
hematop
oietic
and
immunol
 C H A P TER 30 Umbilical Cord Blood Banking
Until 2011, minimally manipulated
UCB intended for use in unrelated recipients
was not licensed. However, the FDA has
long con- sidered licensure as a way of
improving the safety and quality of UCB by
instituting re- quirements that would be
legally enforce- able.51 The FDA believed that
compelling clini- cal safety and efficacy data
existed to support the development of
product standards as an approach toward
licensure. In 1998, the FDA issued a request
for the public to submit com- ments
regarding establishment controls, pro- cess
controls, and product standards for UCB
manufacturers.78 After reviewing the submit-
ted information, the FDA determined that
suf- ficient data did exist to support the
develop- ment of processing and product
standards for granting licensure.
In October 2009, the FDA published its
fi- nal guidance, which described ways to
assist UCB manufacturers in submitting a
BLA.79 Originally published with
indications restrict- ed to “hematopoietic
reconstitution in pa- tients with hematologic
malignancies, certain lysosomal storage and
peroxisomal enzyme deficiency disorders,
primary immunodefi- ciency diseases, bone
marrow failure, and beta thalassemias,” the
guidance was modified by the FDA in 2011
to broaden the indications to “use in
unrelated donor hematopoietic pro- genitor
cell transplantation procedures in con-
junction with an appropriate preparative
regi- men for hematopoietic and
immunologic reconstitution in patients with
disorders af- fecting the hematopoietic
system that are in- herited, acquired, or
result from myeloablative treatment.” This
guidance applies to hemato- poietic
progenitor cells (HPCs), UCB [HPCs- cord
(HPC-Cs)] manufactured by US UCB
establishments and non-US UCB establish-
ments that distribute UCB in the United
States. For establishments that manufacture
UCB products for autologous use or use by
a first- or second-degree blood relative, the
FDA encourages the same manufacturing
recom- mendations and applicable
regulations be fol- lowed.20
The FDA recognizes that there will be
cir-
cumstances in which the best available UCB
unit for a patient is not a licensed unit. There
■ 745
may be units from UCB establishments in
which a BLA is pending; units that do not
meet licensing requirements, or units that come
from non-US CBBs that have chosen not to
apply for licensure. Under such circumstances,
the FDA requires submission of an IND
application. FDA published the guidance for
such applica- tions as companion documents
to the BLA.80
In March 2014, FDA updated the 2009
BLA and 2011 IND guidance documents.
The BLA guidance document was updated
to ex- pand the indications for use, clarify
the type of clinical data required in the
application, and replaced the term “HPC-C”
with “HPC, Cord Blood.” Similar revisions
were made in the IND guidance document,
in addition to clari- fying that a table of
contents is required for all IND applications.
In summary, the FDA regulates HPCs,
HPC-C blood for unrelated allogeneic use as
a drug under the Food Drug and Cosmetic
Act and as biological products under the
Public Health Services Act. Therefore,
regulations ap- plicable to HPC-Cs include:
1. Title 21, CFR Part 201 and 610 Subpart G
– Labeling.
2. Title 21, CFR Part 202: Prescription drug
advertising.
3. Title 21, CFR Parts 210 and 211: cGMP.
4. Title 21, CFR Part 600: Biological
products, general.
5. Title 21, CFR Part 601: Licensing.
6. Title 21, CFR Part 610: General biological
products standards.
7. Title 21, CFR Part 1270 (Section 351 of the
Public Health Services Act): HCT/Ps with
systemic effect intended for use in unre-
lated persons.
8. Title 21, CFR 1271 (Section 361 of the
Public Health Services Act): HCT/P
registration and listing, donor eligibility,
and GTP.
In the event that one regulation is in
con- flict with another, the one that is more
specifi- cally applicable to UCB supersedes
the one that is more general.
In combination, standards regulating
bio- logic drugs are akin to those for
pharmaceuti- cal manufacturing, and their
application to a
746 ■ AABB T EC HNIC AL MANUAL

cellular therapy product, such as UCB, is ment, materials, records, storage,

chal- lenging. In contrast to pharmaceutical
drugs, which are chemicals, are
manufactured in tab- let lots, and can be
terminally sterilized, UCB products are
composed of viable cells, are manufactured
in single-unit lots, and must be processed
aseptically. Recognizing the value and
unique nature of each UCB product, FDA
has been very cautious in regulating UCB to
avoid preventing patients from having
access to existing or prospectively
manufactured products. At the time of this
writing, the FDA has approved the BLAs of
five CBBs in the United States (see Table
30-3).
AABB and FACT
The AABB and FACT are the two
professional organizations that have
established standards for HPCs, including
UCB, in the United States. 22,24 AABB has
incorporated the require- ments for UCB
processing facilities in its Stan- dards for
Cellular Therapy Product Services.24 FACT
and NetCord combined their efforts to
establish a unique set of standards for UCB-
re- lated activities.22 Both AABB and FACT
have created standards that align with the
FDA’s GMP and GTP requirements and
center on quality systems essentials. These
standards cover donor suitability, collection,
processing controls, document controls,
facility manage-
distribution, quality audits, quality plans,
errors/deviations, labeling, personnel,
equipment, validation, outcome reviews,
and adverse event reporting. In 2009, the
ACBSCT recommended that both AABB
and FACT be recognized as accreditation
organizations for the NCBI. Both organiza-
tions incorporated specific standards to en-
sure that CBBs comply with the contracts
held with HRSA.
In a post-licensure era, it is expected that
UCB banking will continue to evolve to bal-
ance the creation of large inventories of
safely manufactured products that not only
meet de- mands for cell dose and matching
from clini- cians, but also target new
populations and ap- plications for use of this
abundantly available resource. Currently,
there are several ongoing clinical trials
involving HSCs and HPC expan- sion
approaches to overcome limited per-unit cell
doses and the lengthy time required to
achieve an absolute neutrophil count of
500/µL; UCB-derived natural killer, T-
regulatory, and dendritic cells for
immunotherapeutic treat- ments; and UCB-
derived mesenchymal stem or stromal cells
to enhance engraftment and regenerative
applications. However, optimal methods for
production and clinical applica- bility of
these new products have yet to be es-
tablished.

TABLE 30-3. Approved Cord Blood Biologic License Applications*

Product NameManufacturer

Hemacord New York Blood Center (New York, NY)

HPC-Cord Blood University of Colorado Cord Blood Bank (Denver, CO)
Ducord Carolinas Cord Blood Bank (Durham, NC)
Allocord St. Louis Cord Blood Bank (St. Louis, MO)
HPC-Cord Blood LifeSouth Community Blood Centers (Gainesville, FL)
*As of March 2014.
 C H A P TER 30 Umbilical Cord Blood Banking ■ 747

KEY POINTS

1. UCB stem cells have a higher proliferative capacity and immune tolerance than stem cells
from adult sources, as demonstrated by the decreased incidence of GVHD in UCB
recipients despite the less stringent HLA-matching requirements for UCB transfusion.
These features allow the use of more flexible matches with UCB, which provide the ability
to support pa- tients with rare HLA types and permit matches across ethnic lines.
2. UCB has evolved from being considered biologic waste to acceptance as a viable
source of HSCs. More than 30,000 unrelated donor UCB transplant procedures have
been performed, and there are an estimated 600,000 UCB units banked worldwide for
use as an HPC source. In 2008, UCB surpassed marrow as the most frequently utilized
source of donor cells for un- related transplantation.
3. Engaging pregnant women and arranging donation of their infant’s UCB in advance is
criti- cal for achieving the best results. Early education allows for more complete
medical screen- ing, comprehensive donor protection, availability of adequate supplies,
and acquisition of thorough informed consent.
4. UCB can be collected before (in utero) or after (ex utero) the placenta has been
delivered. There are procedural and economic advantages and disadvantages to each
method. When performed properly, both are acceptable for providing high-quality
collections.
5. Current methods of manufacturing UCB products involve reduction of red cell and plasma
fractions before cryopreservation. Methods generally involve sedimentation and/or centrif-
ugation and may be performed manually or with automated devices that have been cleared
for use by the FDA.
6. Transplant centers must determine the most appropriate thawing procedure for the
prod- ucts received based on the manufacturer’s instructions, red cell volume, patient-
specific variables, and their own validated method. Current practices for preparing
UCB for infusion consist of bedside thawing, the traditional thaw-and-wash method,
and a thaw-and-recon- stitution technique.
7. Following the announcement of a tiered approach to regulating UCB in 1997, the FDA pub-
lished a guidance document in 2009 defining a process by which CBBs can submit a BLA
to the FDA for UCB. Through this process, five public CBBs have been authorized to
manufac- ture HPC products from UCB for allogeneic use.
8. The clinical utility of several cell types within UCB (eg, mesenchymal stem and
immune cells) is being investigated for nonhematopoeitic applications,
immunomodulation, and re- generative medicine.
9. To ensure their economic sustainability, CBBs must adapt to recent trends in product selec-
tion, improve collection efficiency, collaborate to identify alternative uses for clinical-grade
products, and successfully transition to a full cGMP and licensure environment.

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al. Wash-out of DMSO does not improve HLA match on transplantation outcome in
the speed of engraftment of cord blood
transplan- tation: Follow-up of 46 adult
patients with
 C H A P TER 30
1061 cord blood recipients with hematologic
malignancies. Blood 2010;115:1843-9.
74. Bart T, Boo M, Balabanova S, et al. Impact of
selection of cord blood units from the United
States and Swiss registries on the cost of bank-
ing operations. Transfus Med Hemother 2013;
40:14-20.
75. Food and Drug Administration. Proposed
approach to regulation of cellular and tissue-
based products. CBER Office of Communica-
tion, Outreach, and Development, 1997.
[Available at http://www.fda.gov/downloads/
BiologicsBloodVaccines/GuidanceComplian
ceRegulatoryInformation/Guidances/Tissue/
UCM062601.pdf (accessed December 6,
2013).]
76. Food and Drug Administration. Eligibility de-
termination for donors of human cells, tissues,
and cellular and tissue-based products; Final
Rule. Fed Regist 2004;69:29786-834.
77. Food and Drug Administration. Human
cells, tissues, and cellular and tissue-based
prod- ucts; establishment registration and
listing; fi- nal rule. Fed. Regist
2001;66:5447-69.
78. Food and Drug Administration. Request for
proposed standards for unrelated allogeneic
peripheral and placental/umbilical cord
blood
Umbilical Cord Blood Banking ■ 751
hematopoietic stem/progenitor cell products;
request for comments. Fed Regist 1998;63:
2985.
79. Guidance for industry: Minimally manipulat-
ed, unrelated allogeneic placental/umbilical
cord blood intended for hematopoietic recon-
stitution for specified indications. (October
2014). Silver Spring, MD: CBER Office of
Com- munication, Outreach, and
Development, 2009. [Available at
http://www.fda.gov/down
loads/BiologicsBloodVaccines/Guidance
ComplianceRegulatoryInformation/Guidanc
es/Blood/UCM187144.pdf (accessed Decem-
ber 6, 2013).]
80. Food and Drug Administration. Guidance for
industry and FDA staff: IND applications for
minimally manipulated, unrelated allogeneic
placental/umbilical cord blood intended for
hematopoietic and immunologic reconstitu-
tion in patients with disorders affecting the
hematopoietic system. (March 2014) Silver
Spring, MD: CBER Office of Communication,
Outreach, and Development, 2014. [Available
at: http://www.fda.gov/BiologicsBloodVac
cines/GuidanceComplianceRegulatoryInfor
mation/Guidances/CellularandGeneTherapy/
ucm388218.htm (accessed April 8, 2014).]
 C h a p t e r 3 1

Tissue-Derived Non-Hematopoietic
Stem Cell Sources for Use in
Cell-Based Therapies

Yameena Jawed, MD; Brian Johnstone, PhD;

Sreedhar Thirumala, PhD; and Keith March, MD,
PhD

RE G E NER A T I V E ME DI CI NE HOLD S
great promise for the treatment of some
of the most intractable diseases afflicting hu-
mankind. Each year brings new findings that
advance the science of regenerative medicine
toward becoming clinical reality. In fact,
achieving this goal seems closer now than ever
with the recent success in growing functional
human livers in the laboratory. 1 However, the
reality is that, although these results are prom-
ising, their clinical translation still will require
many years, if not decades, of further research
and refinement before patients receive organs
grown in the laboratory.
Nevertheless, the use of stem cells has al-
ready shown great promise for treating a wide
variety of diseases in clinical trials. Each
month, new successes are reported in treat-
ments of even the most serious life-threaten-
ing diseases, such as amyotrophic lateral
scle- rosis (Lou Gehrig disease).2
Interestingly, there is scant evidence that
these salutary effects oc- cur through the
regeneration of degenerated tissues by the
implanted stem cells. In fact, there is very
little evidence in animal disease models or,
where possible, human patients that
transplanted cells are capable of long- term
engraftment in sufficient numbers to account
for the observed effects. Therefore, al-
ternative mechanisms have been invoked to
explain the measurable benefits of
mesenchy- mal stem cell (MSC) therapies.
The prevalent

Yameena Jawed, MD, Postdoctoral (Research) Fellow, Indiana Center for Vascular Biology and Medicine,
Indi- ana University School of Medicine; Brian Johnstone, PhD, Preclinical Director, Center for Regenerative
Medi- cine, Director, Cardiovascular Ischemia and Vasculogenesis Core, and Associate Research Professor,
Indiana University School of Medicine; Sreedhar Thirumala, PhD, Senior Scientist, Cook General
BioTechnology, LLC; and Keith March, MD, PhD, Director, Indiana Center for Vascular Biology and
Medicine, Director, Vascular and Cardiac Center for Adult Stem Cell Therapy, and Professor of Medicine,
Physiology, and Biomedical Engi- neering, Indiana University School of Medicine, Indiana University,
Indianapolis, Indiana
The authors have disclosed no conflicts of interest.

753
754 ■ AABB T EC HNIC AL MANUAL
hypotheses of “paracrine support” are dis-
cussed in detail below. This new understand-
ing has led to a widespread adoption of the
more general phrase “cell-based therapy” to
encompass the panoply of beneficial effects of
treatment with stem cells from a variety of
sources—effects that are independent of sta-
ble tissue engraftment and tissue regenera-
tion. In practice, though, the term “regenera-
tive medicine” is still widely used, especially
by the lay public, to refer to any stem cell-
based treatment.
Strictly speaking, regenerative medicine
is simply defined as the “process of
replacing or regenerating human cells,
tissues or organs to restore or establish
normal function.”3 The origins of cellular
therapy date back to 1968 with the first bone
marrow transplant of he- matopoietic stem
cells to restore the hemato- poietic system in
dysfunctional or ablated marrow.4 From
these origins, there has been great progress
in identifying many different sources of
potentially therapeutic stem and progenitor
cells in the adult system. These findings
have also been translated to early- stage
clinical trials to test the effectiveness of
these approaches in a number of diseases
that are not currently addressed by
traditional pharmacologic therapies or
surgery.
Treatment with both autologous and allo-
geneic stem and progenitor cells has been
evaluated. Autologous cell therapy has the ad-
vantage of involving fewer potential
regulatory hurdles and, in some cases, such as
recon- structive surgery, is arguably within the
tradi- tional scope of the practice of medicine
and, therefore, outside regulatory oversight.
Autol- ogous cell-based therapies, although
attractive and perhaps more tractable than
allogeneic cell-based therapies, are practical
only when the source of cells is abundant and
their ex- traction is without significant
morbidity or en- hanced mortality. For these
reasons, autologous therapy has been limited
to cells from marrow and adipose tissues;
however, the former is mostly composed of
hematopoietic cells, whereas the latter is made
up of approximately 15% to 30% MSCs on
isolation.4,5 Allogeneic therapies, however,
usually require the mass production of stem
cells originally obtained
from genetically nonidentical donor tissue in
carefully controlled environments with
estab- lished controls to ensure quality and
safety.
Much research has focused on the
ability of these regenerative therapies to
replace or- gan transplants. In 2006, an
article was pub- lished on what was labeled
the “first artificial- ly grown organ.”5 In this
study, scientists were able to produce
sections of a bladder and pro- vide some
degree of urinary continence to pa- tients
with spina bifida. Currently, most re- search
relates to tissue and organ progenitor cells
that either increase or support the func-
tionality of preexisting tissues or induce new
organ structures to form in situ, a process
technically termed “organ reconstruction.”
The reconstruction of bioengineered organs
or even for a regenerative therapy treatment
po- tentially requires a massive number of
stem cells. For many stem-cell-mediated
therapies, the number of cells needed to
effectively treat a patient greatly surpasses
the number of cells available from donors.
MSC SOURCES
Tissue sources of stem cells used for cell-
based therapies are varied and cover nearly
every or- gan system in the body. Based on
the develop- mental stage of the donor, these
cells can be classified as embryonic, fetal, or
adult. Embry- onic stem cells are primordial
cells isolated from the eight-cell blastula
that has the capac- ity to form any cell type
found in the human body. Stem cells isolated
from postnatal or re- productive tissues are
known as “adult stem cells.” Most of these
cells were discovered and defined based on
such attributes as the ability to proliferate
for many generations in culture as well as
their intrinsic property of differenti- ation in
response to environmental or chemi- cal
stimuli. The ability to differentiate along
multiple lineages (eg, mesodermal, ectoder-
mal, and endodermal) is important. The hall-
marks of pluripotentiality and
multipotentiali- ty distinguish stem cells
from progenitor cells; the latter have
undergone partial differentia- tion in situ
and, thus, are committed to differ- entiate
terminally into a single cell type (eg,
endothelial progenitor cells or
preadipocytes,
 CH A P T E R 3 1
which form endothelial cells and adipocytes,
respectively).
The most studied adult multipotent cells
are mesoderm-derived MSCs, alternatively
termed “multipotent stromal cells,” which
originate from the stromal compartment of
skeletal muscle, reproductive organs
(umbili- cal cord and uterus), amnion,
marrow, and ad- ipose tissues. The recent
advances in tech- niques to reprogram
terminally differentiated cells to induce
pluripotency (termed “induced pluripotent
stem cells”) have opened up a vast source of
stem cells from nonembryonic tis- sues.
Induced pluripotent cells present excit- ing
new opportunities for cell-based therapies
and are covered in detail in Chapter 33.
Mar- row-derived MSCs (BM-MSCs), which
are the nonhematopoietic cells isolated from
marrow stroma, are also discussed in detail
in Chapter 33 and are only briefly discussed
here, mostly to compare them with MSCs
derived from oth- er tissues and to discuss
their current thera- peutic uses.
BM-MSCs were the first
nonhematopoiet-
ic stem cells discovered and, due to having
been studied in great detail, are considered the
archetypal MSCs. Because the numbers and
quality of MSCs decline with the aging of do-
nors and due to the invasiveness of the proce-
dures required to procure marrow, other
sources of MSCs were sought. Perhaps the
most primitive adult MSCs are those obtained
from umbilical cord tissue, umbilical cord
blood, amnion, and amniotic fluid.6-10 The
dental pulp of the third molar is another
source of MSCs.11 It has been recently recog-
nized that adipose tissue is a rich and abun-
dant source of cells with stem cell properties.12
Additional sources of MSCs are placenta and
uterine endometrial cells shed during men-
ses.13,14 Each of these tissue-specific MSCs is
discussed in more detail below.
MSCs from different tissue sources
differ with respect to prevalence,
proliferative capac- ity, immunophenotype,
and differentiation potential (Table 31-1),
which may be impor- tant variables
governing the clinical utility of each MSC
type. In the cases of low abundance in
source tissues or difficulty of obtaining tis-
sue specimens, MSCs must be first isolated
Tissue-Derived Stem Cells ■ 755
from the nondesirable cell types (such as
leu- kocytes and endothelial cells) and then
ex- panded under conditions that are
permissive for maintaining stem-cell-like
properties. Be- cause of the cost and time
required to generate sufficient numbers of
qualified cells under current good
manufacturing practice (cGMP) conditions,
the effort is only economically practical
when culture expansion yields suffi- cient
numbers of MSCs at lower passages to treat
many hundreds, if not thousands, of pa-
tients.
Most adult tissues contain low percentag-
es of MSCs that are not available in sufficient
abundance, with the exception of adipose tis-
sues. BM-MSCs constitute only a small frac-
tion (approximately 1 in 3.4 × 104 cells) of
cells in adult marrow.28 Although the
frequency of MSCs in fetal tissues may be
higher, these tis- sues are comparatively quite
small and limited in availability, and their use
is associated with ethical concerns when their
collection in- volves fetal destruction.29
Examples of MSCs found in low numbers in
source tissues but that are highly expandable
are muscle-derived MSCs (M-MSCs), cord
blood MSCs (CB- MSCs), umbilical cord
MSCs (UC-MSCs), pla- cental MSCs (Pl-
MSCs), endometrial lining MSCs (EL-MSCs),
and dental pulp MSCs (DP- MSCs).
Adipose-derived stem cells (ASCs),
which are phenotypically similar to BM-
MSCs, are more than 500-fold more prevalent
per gram of fat than per gram of marrow.30
Most patients have excess fat that can be easily
and safely ex- tracted for processing to isolate
ASCs. This property makes adipose tissue an
ideal source of MSCs for either autologous or
allogeneic applications. ASCs are perhaps the
only type of MSC available in numbers that
are sufficient for immediate or “point-of care”
use in thera- peutic applications without the
need for cul- ture expansion.31-33 Extraction of
adipose tissue is accomplished with minimally
invasive tech- niques, such as lipoaspiration,
with little asso- ciated morbidity even on
removal of tissue volumes as large as 1-3
liters. Thus, the procurement and use of ASCs
is a uniquely practical approach to point-of-
care autolo- gous applications.
TABLE 31-1. Comparison of MSCs Derived from Different Tissue
756

Sources
■

Study (Reference
9 CB + +++ + CB-MSCs have less adipogenic CD105 + CD90 + CD106 +
BM ++ + ++ differentia- tion potential. Osteogenesis CD105 + CD90 ++ + CD106

Number)
AT +++ ++ +++ and chondro- genesis are similar. + CD105 + CD90 + ++

MSC SourceUnits)
AT BM-MSCs have superior osteogenic ++ + CD106 +
15
BM capacity. AT-MSCs are least CD34 +++
16
CB osteogenic.
CD34 (null)
AM

Frequency (Colony- Forming

17 AM-MSCs and AF-MSCs have neurogenic, MSCs from AM, AF, BM– CD105+,
AF
osteogenic, chondrogenic, adipogenic, CD90+, CD73+
BM
hepatogenic and endothelial MSC from AM, AF-CD44+, CD49e+,
differentia- tion. AM-MSCs also CD54+, CD166+
differentiate into car- diomyocyte-like
cells in vivo.
18 UC +++ +++ + CD106 HLA-ABC +++CD146 +++
AABB T ECHNICAL M A NUAL

Neurogenesis is higher in UC-MSCs. +

19 BM + + +++ CD106 +++HLA-ABC ++++ CD146 +
UC- MSCs have higher osteogenesis
and neu- rogenesis than BM-MSCs.
20 Pl + +++ + CD49d ++
Osteogenesis and chondrogenesis are
BM + + +++ CD49d +
comparable but Pl-MSCs do not maintain
adipogenesis as readily as BM-MSCs.
21 SV +++ +++ + VEGFR-2 ++ CD10 +
Adipogenesis is highest in SV-MSCs and
PM +++ +++ + VEGFR-2 ++ CD10 ++
AT-MSCs. Chondrogenesis is superior in
Cell Proliferation Potential in

SkM +++ +++ +++ + VEGFR-2 + CD10

SV-MSCs. BM-MSCs, SV-MSCs, and
AT +++ ++ ++ ++ VEGFR-2 + CD10
PM-
BM + +++ + + + VEGFR- + CD10
MSCs have the highest osteogenic 2 ++ +
VitroSenescenceMultilineage Differentiation Potential

Immunophe
22 DP +++ ++ + All dental cells express higher levels CD106 ++
23 PD ++ ++ + of neuronal markers than BM-MSCs. CD106 +
BM + + +++ PD- MSCs express higher levels of CD106 ++
ED +++ +++ + tendon- specific markers, whereas DP- + CD106
MSCs retain weaker and chondrogenic +
and adipogenic potential compared
with BM-MSCs.
24 AF + +++ + CD44 + CD105 +
Osteogenesis and adipogenesis are
BM +++ + +++ CD44 ++ CD105 ++
similar.
DP
25 All MSCs express CD44, CD105, and CD90.
UC All MSCs undergo osteogenesis,
All are negative for CD34 and CD45.
AT chondro- genesis, and adipogenesis. BM-
BM MSCs and AT-MSCs show a loss of
CH A P T E R 3 1

osteogenic differ- entiation potential with

increasing num- bers of passages, and
DP-MSCs have an increasing
26 CB +++ +
AT + ++
BM ++ +++
UC ++
27 AT-MSCs have more efficient No statistically significant difference
AT
+ adipogene- sis and neurogenic
Tissue-Derived Stem Cells

differentiation.
+ = expressed; ++ = strongly expressed; +++ = very strongly expressed.
MSC = mesenchymal stem cell; CB = cord blood; BM = bone marrow; AT = adipose tissue; AM = amniotic membrane; AF = amniotic fluid; UC = umbilical cord; Pl = placenta;
SV = synovium; PM = periosteum; SkM = skeletal muscle; DP = dental pulp; PD = periodontal ligament; ED = exfoliated deciduous teeth.
■
757
758 ■ AABB T EC HNIC AL MANUAL
Periadventitial cells (called “pericytes”),
which support the integrity of microvessels
(capillaries and arterioles), have been recently
discovered to possess multilineage potential.34
Traktuev et al highlighted the perivascular lo-
cation and presumptive prepericyte or peri-
cyte function of MSCs within adipose tissue. 35
The extension of this finding to recognize the
perivascular location of MSCs throughout the
body led to the current hypothesis that peri-
cytes are the source of all MSCs; this can ex-
plain both the high phenotypic similarity of
MSCs from different tissue sources as well as
the broad distribution of these cells, in associ-
ation with blood vessels, throughout the
body.35-37 It has also been proposed that the bi-
ological function of perivascular MSCs is inti-
mately related to systemic circulation in that
once activated, they either secrete trophic fac-
tors that promote endogenous repair into the
blood or may even mobilize to home to in-
jured tissues, where they provide structural
units for repair through differentiation.38
PROPERTIES OF CLINICAL
RELEVANCE
MSCs display several key complementary
properties that are of potential clinical use:
immunomodulation, paracrine effects, and
differentiation. Immunomodulatory func-
tions include immunosuppression or
immune stimulation, depending on the
signals in the microenvironment. 39 In the
laboratory envi- ronment, MSCs can be
induced to differenti- ate into endothelial
cells, neural cells, astro- cytes,
cardiomyocytes, skeletal myocytes,
chondrocytes, adipocytes, osteocytes, and
other cells that are developmentally derived
from the endoderm and exoderm.40-43 The
low immunogenicity of MSCs also makes
them a relatively safe allogeneic cell source
in com- parison with certain other cell types,
including embryonic stem cells.44 There is
evidence from preclinical and, in some
cases, clinical studies to suggest that each of
these properties con- tributes to the
therapeutic efficacy of MSCs.
The paracrine effects of MSCs are
increas- ingly recognized as an important, if
not the primary, mechanism of action to
promote tis-
sue rescue and repair as well as to modulate
the immune system.45 The demonstrated
abili- ty of MSCs to effect change in disease
models and patients in the absence of long-
term en- graftment and differentiation in
numbers re- quired to account for damaged
tissue replace- ment can be most easily
explained by paracrine mechanisms. Thus,
exogenously ad- ministered MSCs home to
the site of injury or disease through signals
that are primarily in- flammatory and reside
temporarily at the site where they secrete
paracrine factors with pro- survival,
antiinflammatory, and repair-induc- ing
properties before clearance from the sys-
tem.46
Although the evidence supports the pre-
dominant function of secreted factors in the
diminution of injury or disease, there is also
evidence that cell-mediated contact may be
an important immunomodulator during the
peri- od of residency of MSCs at the target
site.47 The concept of paracrine support by
MSCs was first directly demonstrated in
diseases of pe- ripheral tissues and the
central nervous sys- tem by treatment of
disease models with the cocktail of factors
present in media condi- tioned by the growth
of BM-MSCs and ASCs.48 In another early
demonstration, ablating pro- duction of a
single factor (hepatocyte growth factor) by
stable small interfering ribonucleic acid
knockdown abolished the salutary effects of
ASCs in a mouse hind-limb ischemia model
of peripheral vascular disease.46
ISOLATION AND EXPANSION
Obtaining sufficient numbers of MSCs com-
monly requires their extensive expansion in
cell culture. The isolation and expansion of
MSCs from marrow aspirate is covered in de-
tail in Chapter 33. Liposuction surgery is a
well-tolerated and safe procedure for produc-
ing a large amount of lipoaspirate that can be
processed to yield ASCs.
The method of ASC isolation in
different laboratories varies subtly, but it
usually in- volves enzymatic dissociation
from adipocytes and extracellular matrix,
filtration to remove particulates, and low-
speed centrifugation to separate adipocyte
and nonadipocyte cells.
 CH A P T E R 3 1 Tissue-Derived Stem Cells ■ 759
connective tissue known as

Following surgical removal of subcutaneous

fat, lipoaspirate samples are washed exten-
sively in phosphate-buffered saline to
deplete erythrocytes and then treated by
continuous agitation with collagenase Type I
solution for 30 minutes to 1 hour at 37 C. It
is important that the collagenase also
contain neutral pro- teases, presumably to
liberate cells from the extracellular matrix
fragments; these cells are removed by
subsequent filtration. The solu- tion is
neutralized with an equal volume of serum-
containing medium and centrifuged at low
speed (approximately 300 × g for 10 min-
utes). The buoyant adipocytes migrate in op-
position to centrifugal force. The
sedimenting cells are a heterogenous
mixture termed the “stromal-vascular
fraction” (SVF), which con- tains variable
proportions of endothelial cells, pericytes,
ASCs, and resident leukocytes (pri- marily
monocytes and macrophages).47-50 The cell
pellet can then be resuspended in an 160
mM NH4Cl solution (for 10 minutes at room
temperature) to lyse erythrocytes, and the
sus-
pension is passed through a 100-micron cell
strainer to remove debris. After an
additional low-speed centrifugation, the
cells are resus- pended in growth media,
such as DMEM/F12 with 10% fetal bovine
serum, for counting and are then placed in
tissue culture flasks and
grown under typical conditions of 5% CO2 at
37 C.
Enrichment of ASCs occurs through
plat- ing on uncoated tissue-culture plastic
in the presence of a rich medium that is not
permis- sive for leukocyte and endothelial
cell attach- ment; thus, the latter cells are
removed with a media change after
overnight culture. After an initial lag phase,
ASCs proliferate with dou- bling times of 36
to 48 hours, depending on the growth
medium used. The cell surface marker
profile of ASCs is altered with their
expansion. For example, the CD34 marker
expression of cells in culture decreases
rapidly and becomes unmeasurable after two
or three passages.34,36 Conversely, the CD105
and CD166 markers, which are expressed at
low levels in fresh iso- lates, increase during
culture.51
The umbilical cord consists of two
arter- ies and a vein supported by a
surrounding ma- trix of gelatinous
 either dispase II (2.4 U/mL for 1 hour) or
trypsin- EDTA (various concentrations and
“Wharton’s jelly.” Stromal cells incubation times have been used) to release the
associated with the matrix epithelial cells. The Pl-MSCs are then released
include specialized fibroblast- through subsequent digestion with collagenase
like cells, mast cells, and (0.75 mg/mL) and deoxyribonuclease (0.075
MSCs.52,53 Methods for mg/ mL).57 Human chorionic Pl-MSCs are
isolation of UC-MSCs are liberated similarly after removal of the
varied and differ be- tween surrounding lay- ers and treatment with 2.4
laboratories; however, in all U/mL dispase II at 37 C, followed by 1 to 3
cases, the vein and arteries hours of treatment with collagenase II (270
are first removed, followed by U/mL) or collagenase A (0.83 mg/mL). For
processing of the remaining both chorionic and amni- onic Pl-MSCs, the
cord tissue into smaller liberated cells are centri- fuged at 200 × g for
fragments (reviewed by Can 10 minutes to obtain the cell pellet, which is
and colleagues54). In some then resuspended, count- ed, and cultured.13
protocols, the interior of the An alternative to digesting
cord is scraped to remove the
Wharton’s jelly before
mechanical disruption. The
pieces are placed in an
enzymatic solution of collage-
nase (typically Type I) and,
depending on the protocol,
other proteolytic enzymes to
digest the matrix and release
the stromal cells. Vari- ous
digestion times, from 30
minutes to 16 hours, have
been employed, depending on
the enzymes used and their
concentration.53
Alternative protocols
involve the conduct of
explant culture with the cord
fragments.56 For protocols
involving enzymatic
digestion, the homogenate is
filtered through a 100-mi-
cron filter to remove debris.
UC-MSCs from the total cell
population can be selected by
ei- ther fluorescence-activated
cell sorting or cul- ture at low
density to identify colony-
forming cells. Typical
mesenchymal cell surface
mark- ers, such as CD90,
CD105, and CD73, are ex-
pressed by UC-MSCs;
however, the level of ex-
pression varies depending on
the isolation method used.56
Isolation of amnion-
derived Pl-MSCs is
accomplished by first removing
the chorion, followed by
digesting them at 37 C with
760 ■ AABB T EC HNIC AL MANUAL
placental tissue is to isolate Pl-MSCs present
in the amniotic fluid. This method relies on re-
moval of amniocytes by an initial culture peri-
od and then transferal of nonadhering Pl-
MSCs to a new culture dish, where they are
allowed to adhere and proliferate. 8 Each
method for Pl- MSC isolation includes
culturing to expand the cells.58
DP-MSCs are isolated from the interior
pulp chamber of extracted molars. The pulp
tissue is removed from the crown and root re-
gions and then digested with a combination
collagenase Type I (3 mg/mL) and dispase
(4 mg/mL) for 1 hour at 37 C.59 Single-cell
sus- pensions are separated from debris by
passage through a 70-micron filter. Cells
proliferate well in -MEM supplemented with
20% fetal calf serum (FCS), L-ascorbic acid 2-
phosphate (100 µM), and L-glutamine (2
mM).60,61
Because extraction and digestion of tis-
sues are not needed to isolate cells, EL-MSCs
are a convenient therapeutic cell source. Isola-
tion of EL-MSCs is easily accomplished by
culturing menstrual-blood-containing, density-
gradient-purified mononuclear cells on un-
coated plastic vessels and selecting adherent
cells after a brief overnight incubation in com-
plete media, such as DMEM supplemented
with 20% FCS.14 Adherent EL-MNCs are ex-
panded in the same medium for multiple gen-
erations until sufficient numbers of cells are
obtained.
M-MSCs are pluripotent stem cells that
are distinct from committed progenitors
(such as satellite cells in skeletal muscle)
and can be isolated from skeletal, smooth,
and cardiac muscle tissue.62,63 The most
common tech- nique to isolate M-MSCs is
based on their ad- hesion characteristics and
uses a process termed the “modified preplate
technique.” In this method, the muscle mass
is isolated by a biopsy and dissected into
pieces with razor blades or scalpels. The
tissue is dissociated us- ing enzymes, such
as collagenase II (1%) and dispase II (2.4
U/mL).64,65 The dissociated cells are then
resuspended in culture medium, which is
then placed in collagen-coated flasks.
Within minutes to hours of seeding, the first
cells, including fibroblastic-like and
myoblast cells, attach. The supernatant
containing the
nonadherent fraction is then transferred to a
new collagen-coated plate. The slowly
adher- ing cells, which contain M-MSCs,
are allowed to adhere and grow over several
days to weeks, after which the proliferating
cells are expand- ed by passaging.
STANDARDIZATION OF METHODS
FOR ISOLATION AND EXPANSION OF
CLINICAL PRODUCT
Although many countries have implemented
regulations governing the manufacture and
use of stem cells for cell therapy, there is
still significant variation in the procedures
used for isolation, expansion, preservation,
and ship- ping, which are critical
components of clinical translation.66,67 It is
imperative that cGMP re- quirements and
quality-control systems be used to ensure, to
the greatest extent possible, consistency in
stem cell identity and potency.
These practices are established at most
companies that are pursuing federal
regulato- ry approval for off-the-shelf
allogeneic stem cell products. This
regulatory approval re- quires careful
documentation of manufactur- ing processes
and product characterization (termed
“chemistry, manufacturing, and con- trols”).
These companies include Mesoblast Ltd.
(Melbourne, Australia), Athersys Inc.
(Cleveland, OH), Medistem Inc. (San
Diego, CA), and Pluristem Therapeutics Inc.
(Haifa, Israel).
Autologous MSC therapies are also
typi- cally required to demonstrate
consistency of a product for regulatory
approval when the cells are derived from
multiple donors. Two com- plementary
models for autologous cell prepa- ration are
currently employed: the centralized
manufacturing facility and point-of-care de-
vices. The former is typified by Aastrom
Biosci- ences Inc. (Ann Arbor, MI) and RNL
Bio (Seoul, South Korea). Devices for
isolation in the oper- ating theater are in
development (and, in lim- ited cases,
approved for marketing) by such companies
as Cytori Therapeutics (San Diego, CA),
Tissue Genesis Inc. (Honolulu, HI), and
Ingeneron (Houston, TX).
 CH A P T E R 3 1
CELL PRODUCT BANKING AND
MANAGEMENT OF SUPPLY
CHAIN AND END USE
Manufacturing, storage/banking, and distri-
bution are all critical for ensuring a steady
supply of cellular products to clinics around
the world. Fortunately, these processes can
be modeled on those already established by
the biological drug industry, which has more
than two decades of experience with supply-
chain optimization. The biologics model
must be adapted to cellular therapeutics to
address key logistical considerations, such
as maintaining the viability of cellular
therapeutics during shipping as well as after
receipt at a clinical fa- cility. Many biologics
are stored as a lyophi- lized powder or in
suspension or solution at normal freezer or
refrigeration temperatures (ie, –20 C).
Extended storage of cells requires
temperatures below –80 C, and these cells
are most commonly stored in liquid-nitrogen
tank facilities.
Defining optimal and reproducible con-
ditions for long-term storage, transport, and
revival of MSCs is critical for maintaining cell
viability and, thus, potency. Defining these
conditions is particularly important for alloge-
neic MSCs produced in a centralized facility
under cGMP controls but also needs to be ad-
dressed for autologous cells that will be used
for repetitive treatments and the avoidance of
repeated extractions and isolations of cells.
Good results with long-term storage at
–196 C (temperature of liquid nitrogen) or
<–150 C (temperature of vapor-phase nitro-
gen) have been obtained with typical cell
cryo- preservation protocols using media
contain- ing serum and dimethyl sulfoxide
(DMSO), provided that cooling and thawing
rates are carefully controlled.68 Although
both DMSO and animal serum present
concerns, there are currently few acceptable
alternatives to their use; thus, most protocols
require that these media be depleted before
the cells are used for the treatment of
patients.69-73
Clinical banking requires strict adher-
ence to cGMP requirements for cell product
processing, storage, and shipment to the end
user to ensure preservation of viability and
Tissue-Derived Stem Cells ■ 761
therapeutic potency. Consequently, closed-
system containers are required to meet phar-
maceutical quality standards for packaging,
storage, and distribution of cellular products
at low temperatures. An ideal
cryopreservation container is stable despite
exposure to a wide range of temperatures for
extended periods and allows selective access
to the sample when desired for retrieval. As
the demand for cell therapy increases,
commercial-scale cell ther- apy product
manufacturing and banking will require the
containers to be compatible and integrated
into the automated fill lines tradi- tionally
used in pharmaceutical settings to en- able
lot sizes of several hundred to several
thousand doses.
Although freezers that maintain cryo-
preservation temperatures are likely to be
available in large hospitals, especially those
af- filiated with universities, these freezers
are not available within most clinical
practices. Smaller- scale solutions, such as
liquid-nitrogen Dewar flasks or dry ice in
insulated coolers, can be employed for
relatively short-term storage; however, use
of these flasks or coolers may be associated
with logistical and safety issues. Al-
ternatively, a just-in-time approach may be
taken where therapeutic cells are shipped di-
rectly from centralized facilities in insulated
containers that have sufficient dry ice to
main- tain a steady temperature for a few
days to a week. The cells are thawed
immediately before use, washed to remove
cryoprotective agents, and resuspended in
diluent for administra- tion.
Another option is to use practices estab-
lished for the shipment and short-term stor-
age of tissue products. In general, viable tis-
sues are placed in biocompatible containers
in contact with nutrient-rich media and
shipped at ambient temperatures within
insulated con- tainers via overnight courier
service. The shelf life of certain tissues
packaged and shipped in this manner is
approximately 1 week. An ex- ample is
Apligraf (manufactured and distribut- ed by
Organogenesis, Canton, MA), a living cell
skin graft approved by the US Food and
Drug Administration for the treatment of
chronic venous leg and diabetic foot ulcers.
This prod- uct has a shelf life of 10 days
after it is shipped.
762 ■ AABB T EC HNIC AL MANUAL

Regardless of the method used to

transfer cellular therapy products to the end
user, there is a critical need to manage the
supply chain’s integrity and ensure that the
cell transport and clinical site storage run
efficiently while ad- verse incidents are
minimized. Particularly with autologous or
type-matched therapeu- tics, and with
downward cost pressures due to increased
competition and falling reimburse- ment
rates, it is critical to avoid errors and in- FIGURE 31-1. Number of registered clinical trials
terruptions in the delivery of these therapies of therapeutic uses of mesenchymal stem cells
to hospitals and private clinics. (as of March 2014).
NR = not reported.

THERAPEUTIC APPLICATIONS OF
MSCS
Cell therapy using BM-MSCs or ASCs is cur-
rently being investigated in clinical trials for a
wide variety of diseases that are not adequate-
ly addressed by current pharmacological or
surgical approaches. These trials include some
that evaluate culture-expanded autologous
and allogeneic MSCs and ASCs and others
that involve autologous ASCs that can be
freshly isolated in the context of SVF using
any of sev- eral point-of-care systems.
The number of clinical studies
evaluating these therapies has steadily
increased over the last decade, and the
number of publications relating to stem cell
preclinical as well as clini- cal studies over
time demonstrates this trend.
The majority of these studies are early-phase
trials (Fig 31-1). As can be seen in Fig 31-2,
the types of diseases under investigation are
wide ranging and affect essentially all organs
and systems in the body. The target
populations in- clude both pediatric (65
studies) and adult (286 studies) patients.
Immunomodulation and Skeletal
Repair
The most intensively investigated class of dis-
orders treated with MSCs is immune system
diseases (58 trials), which include graft-vs-
host disease (GVHD; 23 trials) and autoim-
mune disorders, such as multiple sclerosis

70
60
50
Number of Trials

40
30
20
10
0

Disease Category

FIGURE 31-2. Diseases for which mesenchymal stem cell therapy is under investigation (as of March
2014). CNS = central nervous system.
 CH A P T E R 3 1
(13 trials), Type 1 diabetes (10 trials), and lu-
pus (4 trials).
The potential of MSC treatment for
modi- fying these diseases is supported by a
substan- tial volume of literature
demonstrating the immunomodulatory
effects of these cells in preclinical studies of
animal diseases. Admin- istration of MSCs
significantly reduced the incidence and
severity of GVHD after trans- plantation of
major-histocompatibility-com- plex-
mismatched blood and tissues in animal
models.74-77 These preclinical results have
been translated into seminal early trials and
then successful Phase II and III clinical trials
with BM-MSCs for prevention and
treatment of acute and chronic GVHD after
allogeneic he- matopoietic stem cell
transplantation.78-80 The success of the Phase
II and III trials led to regu- latory approval
of the first licensed stem cell therapy,
Prochymal (Osiris Therapeutics), in North
America for treatment of refractory GVHD.
This therapy is also available in Canada.
The discovery in 1998 that MSCs
differen-
tiate into cartilage under the influence of
transforming growth factor-beta ushered in a
new era of investigations of MSC
performance in a multitude of chondrogenic
conditions.81 At the time of publication, 28
clinical studies were evaluating the use of
MSCs to treat osteo- arthritis and
rheumatoid arthritis. Similarly, clinical trials
have demonstrated that MSCs can be used
successfully to repair various bone defects,
including critical size defects in the long
bone, hard palate reconstruction, and
calvarial defects.82-84
Cardiovascular and Cerebral Diseases
MSCs are being investigated for stand-alone
treatment of ischemic heart diseases as well
as in combination with coronary artery
bypass surgery and left ventricular device
implanta- tion. Early open-label,
uncontrolled trials of MSCs and ASCs have
had safety-related pri- mary endpoints. The
APOLLO trial demon- strated that the
intracoronary delivery of au- tologous SVF
to patients with acute ST segment elevation
myocardial infarction was safe.85 The
complementary PRECISE trial was a
Tissue-Derived Stem Cells ■ 763
clinical trial of SVF administration in the
treat- ment of symptomatic,
nonrevascularizable ischemic myocardium.
The POSEIDON trial evaluated both
allogeneic and autologous BM- MSCs as a
therapy for ischemic cardiomyopa- thy and
found that both cell types were safe when
delivered directly into the myocardi- um.86
Two Phase II randomized, double-blind,
placebo-controlled trials of
intramyocardially injected autologous BM-
MSCs for heart failure have been recently
initiated in Europe and the United States to
confirm the positive effects of these cell
treatments observed in early open- label
Phase I/II trials.87,88
Diseases of peripheral limb vascular in-
sufficiency are also under clinical investiga-
tion as targets for MSC therapy. In six
patients with Buerger disease, 24 weeks of
treatment with multiple intramuscular ASC
injections had positive clinical outcomes,
including de- creased pain.89 Intramuscular
injection of ASCs also reduced symptoms of
diabetic foot and atherosclerotic obliterans.90
A random- ized, placebo-controlled trial of
BM-MSC in- jected directly into the
affected limb of patients with critical limb
ischemia demon- strated safety as well as
indices of efficacy.91
Central nervous system trials involving
MSC treatment have been dominated by in-
vestigations of the treatment of ischemic
stroke (nine trials).92 Additional diseases for
which MSCs are being evaluated include Al-
zheimer disease, Parkinson disease, spinal
cord injury, amyotrophic lateral sclerosis, and
multiple sclerosis.93-96 The safety and efficacy
of intravenous autologous BM-MSC infusion
in patients with severe middle cerebral artery
ischemic stroke was evaluated in a 5-year fol-
low-up study in which the mortality rate of the
treated group was lower than that of the
control group, and no major side effects were
observed during the follow-up period.97
Inflammation Modulation and Barrier
Repair
The antiinflammatory and proendothelial
sur- vival activities of MSCs suggest that
they might have therapeutic utility for
diseases involving systemic inflammation
and associated endo-
764 ■ AABB T EC HNIC AL MANUAL
thelial barrier dysfunction, such as sepsis,
acute lung injury, and pancreatitis. Related po-
tentially therapeutic properties in this context
include immunomodulation; differentiation
into lung epithelial cells; secretion of antimi-
crobial peptides; as well as other cytoprotec-
tive factors affecting endothelium, such as ke-
ratinocyte growth factor and angiopoietin.98-101
Preclinical studies in murine models have
shown that MSCs can be beneficial in acute
lung injury induced by lipopolysaccharide,
pneumonia, or systemic sepsis.102 In one study,
ASC therapy decreased oxidative stress and
minimized ischemia/reperfusion-induced dam-
age to the rat lung.103 Phase I trials of both
BM- MSCs and ASCs to treat acute respiratory
dis- tress syndrome are under way. Intravenous
UC-MSC therapy has also been shown to alle-
viate fibrosis, improve histologic scores, and
decrease inflammatory cytokine levels in rats
with chronic pancreatitis.104
Gastrointestinal Diseases
Intestinal diseases are an additional category
of disorders for which MSC treatment is cur-
rently undergoing investigation. The largest
number of trials (11) address Crohn’s disease.
In a Phase I trial of 10 patients with fistulizing
Crohn’s disease, local injections of BM-MSCs
resulted in complete fistula closure in seven
patients and partial closure in three. 105 In 2012,
a Phase III trial investigating the safety and ef-
ficacy of allogeneic ASCs for the treatment of
complex perianal fistulas in patients with
Crohn’s disease was initiated.106
Treatment of end-stage, chronic liver
dis- eases, primarily cirrhosis, using MSCs
is un- der active investigation. A Phase I/II
clinical study, in which eight patients with
end-stage liver disease were treated with
predifferentiat- ed autologous BM-MSCs
injected into either the portal vein or
peripheral vein, showed sig- nificant
improvement in liver function and clinical
parameters.107 A placebo-controlled trial of
30 patients with decompensated liver
cirrhosis demonstrated that systemic infu-
sions of UC-MSCs were safe and improved
liv- er function at 1 year.108 An open-label
trial of peripheral venous delivery of UC-
MSCs to pa-
tients with biliary cirrhosis also showed that
the treatment was safe and improved liver
function.109 Autologous BM-MSCs were ad-
ministered to 53 patients with liver failure due
to complications of hepatitis B infection, and
patient responses were evaluated at early and
late intervals.110 Within 3 weeks of the infu-
sions, there was a significant improvement in
liver function in treated patients compared to
the 105 matched patients admitted in the
same time frame. Although indices of safety at
the early and late time points (approximately
3.5 years) were no different in the treatment
and control groups, the benefit of the treat-
ment did not persist at the late time point.
CURRENT RESEARCH AND
DEVELOPMENT: FOCUS ON
CELL CULTURE AND HANDLING
In the context of the positive results and lack
of apparent safety concerns from numerous
clin- ical studies conducted on MSCs to
date, there are many issues that must be
routinely addressed before regulatory
approval and widespread adoption of MSCs
in the clinical setting. For many culture
protocols, animal- derived supplements,
such as bovine serum (which is also
commonly included in preserva- tion
media), have historically been used as a
source of growth factors and to facilitate
post- thaw cell culture. However, the use of
bovine serum and the xenogeneic proteins it
contains could stimulate immune reactions
in the host. In addition, bovine serum must
be obtained from sources that minimize the
risk of trans- mitting prions and other as-
yet-unidentified zoonotic diseases.111,112
Furthermore, DMSO’s toxicity at
concentrations currently used to
cryopreserve MSCs along with the
undesirable osmotic shock to the cells during
its addition and removal may render DMSO
undesirable to preserve MSCs intended for
clinical use.
Considerable effort has been invested in
identifying substitutes for bovine serum and
DMSO; both of these agents must be
depleted by washing the cells upon thawing
and before injection.69-73 For example, there
has been con- siderable effort directed at
developing serum-
 CH A P T E R 3 1
free, or at least animal-serum-free,
cryopreser- vation media containing cell
protectants oth- er than DMSO.113,114
Strategies for eliminating or reducing the
exposure of MSCs to DMSO and
technologies to remove DMSO from thawed
cells have been proposed in the litera- ture
but rarely implemented in clinical set- tings.
An important consideration that has re-
strained efforts to alter the growth
conditions of MSCs is that such
modifications might alter the phenotype,
genetic stability, or subsequent biological
properties of MSCs.115-118 Alterations that could
influence these properties are not limited to
serum components because micro- nutrient
levels can also influence genomic sta- bility
and biological properties.119 A particular
concern regarding the modification of media
constituents is that much of the preclinical
and clinical data that underlie our
knowledge regarding the potential benefits
and safety of MSCs may not be directly
relevant to cells grown in medium
containing bovine serum substitutes. 120 The
absence of continuity be- tween the
extensive amount of previous pre- clinical
data on MSC safety and efficacy may
increase the regulatory burden for obtaining
licensing to market MSCs as drugs.
In addition to addressing issues of
manu-
facturing and storage, the adequate demon-
stration that exogenously administered
MSCs possess an exceedingly low potential
to form tumors or promote endogenous
tumor forma- tion has been required to
obtain regulatory ap- proval for MSCs.
Published reports indicate a lack of
consensus on the tumor-formation po-
tential of multipotent MSCs. Long-term cul-
tures of MSCs have been reported to be
associ- ated with genomic instability,
resulting in the accumulation of genetic
alterations that have the potential to become
manifest as malignant transformation.121-123
Conversely, other studies have found no
indication of chromosomal ab- errations
with extended cultures of MSCs and
ASCs.124-127 These opposing findings may be
due to subtle differences in the MSC
cultures examined, culture conditions, or
methods for detecting genetic abnormalities.
Regardless, tumor formation resulting
from MSC or ASC treatment is indeed very rare
Tissue-Derived Stem Cells ■ 765
and has been calculated to occur at a
frequen- cy of 10-9.128 There has been at least
one report of MSC transformation in vivo to
form tu- mors.19 These studies emphasize
the need to thoroughly test highly expanded
MSCs to be used in clinical trials for genetic
alterations. These results also may suggest
that, whenever possible, lower-passage
MSCs, or ASCs and umbilical MSCs,
should be used because these cells require
less expansion due to their much greater
availability after their initial recovery from
tissue.
Tumorigenesis may also be promoted
by the recruitment of MSCs to the tumor
stroma. There are conflicting reports
regarding wheth- er human MSCs support
tumor formation. A number of
investigations have shown that MSCs
enhance tumor growth, whereas others have
reported that MSCs are associated with
tumor suppression.130-141 These discrepant re-
sults may be due to the different
experimental systems used to assess tumor
promotion. In addition, the conditions for
each MSC prepa- ration might have differed
in substantial ways that influenced the
outcomes.
Given this still-evolving understanding,
it is important to assess each MSC
preparation for tumorigenic potential and
carefully con- sider the exclusion of patients
with active or recent cancer diagnoses in the
context of clini- cal applications.
CONCLUSIONS AND FUTURE
DIRECTIONS
It is evident that the field of cell-based
thera- pies holds great promise and is on the
cusp of widespread commercialization,
which will have an extensive impact on the
health-care field in the coming years. Today,
the potential market value of stem cell
therapy has been es- timated at $5.1 billion
with adult stem cells holding a majority
share (more than 80%) of the market.142
An important concern revolves around
the potential expense of both autologous and
allogeneic cell-based therapies. Much
emphasis over the last several years has
focused on off-the- shelf allogeneic cell
therapy products, which are considered to be
more suitable for clinical
766 ■ AABB T EC HNIC AL MANUAL

adoption and successful commercialization.
However, allogeneic therapies may be chal-
lenging to produce cost-effectively due to the
complexities involved in their manufacturing
and storage. For allogeneic use, cells from a
sin- gle qualified source must be massively
expanded and stored for a long time to treat
large num- bers of patients. Because
successful commer- cialization is heavily
influenced by costs, the manufacturing and
banking of these massive number of cells in a
safe, robust, and cost- effective manner while
preserving key thera- peutic properties will be
critical. These param- eters are significantly
affected by the source of material, isolation
and manufacturing meth- ods, safety, banking,
shipping, and tracking.
Finally, the successful use and ultimate
potential for widespread adoption of these
therapies will be affected by regulatory re-
quirements for the production and marketing
of any cell-based therapies, including issues
ranging from product characterization to
safe- ty testing and clinical trial design. As
the field continues to mature, the regulatory
pathway and its challenges are becoming
progressively refined. It is hoped that this
evolution will con- tinue to lead to the
establishment of a better- defined path for
permitting the field to test the promise of
cell therapy more efficiently and meet the
expected future demands for these
treatments.

KEY POINTS

Multipotent mesenchymal stem cells (MSCs) can be harvested from a diverse array of tis- sues, including marrow, adipose tiss
The hallmarks of pluripotentiality and multipotentiality distinguish stem cells from progen- itor cells.
The beneficial properties of these therapeutic cells appears to be more related to transitory effects produced by secreted substa
The key complementary properties that are of potential clinical use are immunomodula- tion, paracrine effects, and differentia
The method of MSC isolation usually involves enzymatic dissociation, filtration, and low- speed centrifugation.
Early clinical experience suggests that cellular therapies are safe and effective; however, lon- ger-term studies are required to f
Key to future commercial success will be developing well-characterized and consistent therapeutic cell products through estab

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 C h a p t e r 3 2
Human Allografts and the Hospital
Transfusion Service
Lance D. Trainor, MD, and Rita A. Reik, MD
TH E SU R G I CA L U SE of human tis-
sue allografts continues to expand. Every
year, member organizations of the American
Association of Tissue Banks (AATB) recover
tis- sue from more than 30,000 donors and
provide more than 2 million tissue grafts for
transplan- tation.1 Many surgical specialties,
including orthopedics, plastic surgery,
urology, neuro- surgery, sports medicine,
trauma, and recon- structive surgery, use
human tissue. Increas- ingly sophisticated
grafts are being developed by tissue
suppliers to meet diverse clinical needs.
There is no regulatory requirement for
the activities of a tissue-dispensing service
to be managed by any particular individual
or de- partment within a hospital. However,
because ordering, receiving, storing,
dispensing (issu- ing), tracking, tracing,
investigating adverse events, and managing
recalls are functions performed by both
transfusion and tissue-dis- pensing services,
the AABB recommends a centralized tissue-
dispensing model located within the
transfusion service.2
Lance D. Trainor, MD, Divisional Medical Director, LabCorp, Dublin, Ohio, and Rita A. Reik, MD, Chief
Medi- cal Officer, OneBlood, Lauderhill, Florida
The authors have disclosed no conflicts of interest.
TISSUE TRANSPLANTATION
Allografts are selected for transplantation
based on intrinsic qualities that meet the sur-
geon’s functional requirements for the
patient. Bone, tendons, and corneas are the
most fre- quently implanted human tissues;
others in- clude cartilage, skin, veins, dura
mater, fascia, and heart valves. Most tissues
are procured from deceased donors within
24 hours of death after appropriate consent
(also known as “authorization”) is obtained
from a desig- nated legal authority (eg, the
donor’s next of kin) or by first-person
authorization if the do- nor registered his or
her wishes before death. Strict adherence to
aseptic surgical recovery techniques
minimizes contamination of tis- sues with
microorganisms from the donor’s skin and
intestinal flora as well as the sur- rounding
environment.
Responsible persons at tissue banks de-
termine donor eligibility based on an evalua-
tion of 1) answers provided by the next of kin
or other knowledgeable person to questions
773
774 ■ AABB T EC HNIC AL MANUAL
about the donor’s travel and medical history
and any high-risk behaviors, 2) available
medi- cal records, 3) the autopsy report (if an
autopsy was performed), 4) a thorough
physical assess- ment of the donor to identify
evidence of high- risk behaviors or active
communicable disease,
5) the suitability of any blood sample
collected for infectious disease testing, and
6) the cir- cumstances of death. A history of
high-risk ac- tivity that increases the
possibility of transmis- sion of disease leads
to the rejection of the donor. Allografts,
similar to blood products, are released for
transplantation only after the donor has been
tested for relevant communi- cable diseases
and the results are determined to be
acceptable (see Table 32-1).
Background and Definitions
Human tissue banking in the United States
started more than 60 years ago at the US Navy
Tissue Bank. The use of tissue allografts, now
common, has evolved into a highly innovative
and continuously changing field. Collabora-
tion between tissue bank scientists and end-
user surgeons has produced a wide variety of
life-saving and life-enhancing tissue grafts.
Allografts are tissues transferred
between individuals of the same species.
Allografts may be processed to remove cells
or carefully pre- served to maintain cellular
viability. They can be derived from a single
tissue or multiple tis- sues acting as a
functional unit. During pro-
TABLE 32-1. Required Infectious Disease Testing of Human Allografts 3
Infectious AgentTest Performed
Hepatitis B
Hepatitis C (HCV)
Human immunodeficiency virus (HIV)
Human T-cell lymphotropic virus (HTLV)*
Syphilis
*Required for tissues that are rich in viable leukocytes only.
cessing, human tissue allografts may be
com- bined with other biocompatible agents
to achieve desired handling and functional
char- acteristics. Bone allografts, due to
their hard and rigid nature, can be precisely
machined for compatibility with surgical
instrumenta- tion.
Autografts are tissues implanted into the
individual from whom they were removed.
For example, bone that has been surgically
re- moved from a patient’s ilium can be
shaped to desired dimensions and implanted
into the vertebral disk space of the same
patient. The autograft bone promotes fusion
of adjacent vertebrae, providing stability and
relief from the pain of degenerative disc
disease or trau- ma. Advantages of
autografts include the elim- ination of
communicable disease transmis- sion risk
and the ready availability of graft material.
Disadvantages include morbidity as-
sociated with the additional surgical proce-
dure for the patient, including pain and
poten- tial surgical-site infection. In
addition, the quality (eg, strength) and
quantity of autolo- gous tissue may not be
adequate for the in- tended use, and removal
of the patient’s tissue may adversely affect
function at the site from which it was
removed.
Isografts, which are tissues transferred be-
tween genetically identical individuals, such
as identical twins, are uncommonly used.
Hepatitis B surface antigen
Hepatitis B core antibody (IgM and IgG)
Hepatitis C antibody
HCV nucleic acid
testing
HIV-1 and HIV-2 antibodies
HIV-1 nucleic acid testing
HTLV-I and HTLV-II antibodies
Nontreponema- or treponema-specific assay
 CH A P T E R 3 2
Xenografts are tissues transplanted from
one species to another; a growing number of
medical products are being developed from
highly processed nonhuman animal tissues.
Tissue Processing
After tissue has been procured from a de-
ceased donor, it is processed using aseptic
technique in a controlled environment in a
fa- cility designed to prevent contamination
or cross-contamination. Similar to good
manu- facturing practice, good tissue
practice (GTP) requires the facility to
establish and maintain controls over
temperature, humidity, ventila- tion, and air
filtration. Thorough cleaning and
disinfection of processing rooms and equip-
ment are required to ensure a proper
environ- ment.
Tissues are debrided of extraneous soft
tissue and cut to specification. In some
cases, more than 100 grafts can be produced
from a single donor. Precision bone grafts,
including a growing array of spinal
implants, are crafted using sophisticated,
computer-aided cutting devices to meet the
exacting specifications re- quired by surgical
instrumentation; precisely milled allografts
allow surgeons to operate more quickly and
efficiently.
During processing, various solutions,
in- cluding antibiotics, alcohols, and
surfactants, may be used to reduce or
eliminate bacterial contamination and
remove extraneous lipids and other biologic
material. Depending on the type of graft,
graft sterilization may or may not be
possible; grafts containing viable cells or a
fragile matrix cannot be subjected to
steriliza- tion methods without destruction
of cellular or matrix integrity. Ionizing
radiation is the most frequently used
sterilization technique. Ethylene oxide and
proprietary methods of tis- sue sterilization
may also be used by tissue processors.
Several methods of tissue preservation
are available for long-term storage of human
allografts, including freezing and lyophiliza-
tion (freeze drying). Both processes destroy
cell viability. Tissue integrity can be
enhanced through cryopreservation, a
process in which tissues are frozen in a
protective medium at a
Tissue Services in the Hospital ■ 775
steady, controlled rate. The use of cryoprotec-
tants, such as glycerol or dimethyl sulfoxide,
minimizes cell damage caused by cell shrink-
age and intracellular ice formation during
freezing.
Refrigerated storage can preserve
cellular viability in osteochondral allografts
for which preservation of living
chondrocytes is essen- tial. These grafts,
which consist of an intact ar- ticular surface
with associated soft tissue and bone, are
used for treating traumatic and de-
generative joint conditions. Refrigeration is
not suitable for long-term storage of
allografts.
Clinical Uses of Allografts
Allografts are used in a variety of surgical pro-
cedures. Cadaveric human bone can be used
to replace bone lost to degenerative disease,
trauma, or malignancy. Allogeneic human
bone has unique healing characteristics, in-
cluding osteoconductive and osteoinductive
properties. In vivo, it acts as a scaffold that al-
lows recipient capillary growth into the graft
(osteoconductivity) and provides stimulation
for the production of new bone (osteoinduc-
tivity) by exposing the patient’s osteogenic
progenitor cells to bone morphogenetic pro-
teins (BMPs), growth factors in bone that in-
duce new bone formation. The result is creep-
ing substitution, in which bone remodeling
occurs through osteoclastic resorption of the
implanted tissue and osteoblastic generation
of new bone.
A variety of human tissues are used for
transplantation.4 Selected clinical uses are
list- ed in Table 32-2. Bone-tendon (Achilles
ten- don) or bone-ligament-bone (patellar-
tibia ligament) grafts are routinely used for
anterior cruciate ligament repair. The
implanted ten- don or ligament spans the
joint space, and the bone provides an anchor
into the femur and/ or tibia, restoring joint
stability. Crushed bone subjected to acid
demineralization can be used alone or
suspended in a biologically compatible
carrier and applied to exposed bone
surfaces. BMPs in demineralized bone
stimulate osteogenesis, fusion of adjacent
bones, and healing. Cadaveric skin can be
used as a temporary wound dressing for
severe
776 ■ AABB T EC HNIC AL MANUAL

TABLE 32-2. Clinical Uses of Selected Human Allografts

Allograft TissueSurgical Use

Amnion Wound covering

Conjunctiva surface repair
Corneal ulcer repair
Bone (cortical, corticocancellous, and cancellous) Skeletal reconstruction
Spinal fusion
Dental implant placement
Bone-tendon (Achilles tendon)
Anterior cruciate ligament repair
Bone-tendon-bone (patellar ligament)
Posterior cruciate ligament repair
Tendon (semitendinosus, gracilis, and peroneus
Rotator cuff restoration
longus)
Biceps tendon rupture repair
Cardiac valves (aortic and pulmonary) Valvular insufficiency correction
Congenital cardiac defect
repair
Cartilage (costal) Facial reconstruction
Cornea Keratoconus repair
Traumatic scarring reversal
Corneal ulcer excision
Decellularized skin Hernia repair
Soft tissue reconstruction
Gingival restoration

Demineralized bone Dental implant

placement Spinal
fusion
Dura mater Dural defect/cerebrospinal leak repair
Fascia lata Soft tissue reconstruction
Pelvic floor support
Meniscus Meniscus replacement
Osteoarticular/osteochondral graft (bone and
Joint restoration
joint cartilage)
Pericardium Dura patch
Eyelid reconstruction
Soft tissue reconstruction
Sclera Eye enucleation
Scleral ulcer repair
Eyelid repair
Skin Protection of underlying tissues from severe burns
Veins/arteries Coronary artery bypass
grafting Tissue
revascularization Aneurysm
repair
Dialysis access shunts
 CH A P T E R 3 2
burns, protecting underlying tissues from
de- hydration and environmental pathogens.
Hu- man skin can be processed to remove
cellular elements, producing an acellular
collagen ma- trix that provides a scaffold for
revasculariza- tion and cellular
incorporation in soft tissue reconstructive
surgery. In addition, donor tis- sues can be
used to treat a variety of ocular conditions.
Thinning, scarring, or clouding of the cornea
may be treated by corneal trans- plantation.
Sclera and amnion-derived al- lografts can
be used to treat glaucoma, scleral ulcers, and
traumatic injuries.
Although bone and soft tissue allografts
may provoke a recipient immune response,
such responses are not usually clinically
signif- icant, presumably due to a lack of
residual cel- lular material in processed grafts. 5
Therefore, it is not necessary to match most
allografts to the recipient’s HLA or ABO type.
Possible excep- tions include cryopreserved
veins and arteries, for which some clinicians
request ABO com- patibility, and frozen,
unprocessed bone al- lografts containing
marrow or red cells. Devel- opment of Rh(D),
Fy(a), and Jk(b) antibodies in recipients
following transplantation of un- processed
bone allografts has been document- ed.6 If
unprocessed bone is to be used for an Rh(D)-
negative female of childbearing poten- tial, her
future offspring may be at risk of he- molytic
disease of the fetus and newborn. Rh Immune
Globulin prophylaxis should be con- sidered if
the Rh type of the red cells or marrow
contained in the allograft is positive or un-
known.
Disease Transmission through Tissue
Transplantation
Rare, sporadic transmissions of infectious
dis- eases, including human
immunodeficiency vi- rus (HIV), hepatitis C
virus (HCV), hepatitis B virus (HBV), and
Creutzfeldt-Jakob disease (CJD), have been
documented following tissue implantation.7
In addition, bacterial and fun- gal infections
from allografts have resulted in morbidity
and death. Potential sources of
contaminating agents include donor flora,
premortem infection, and environmental
contaminants in recovery and processing fa-
Tissue Services in the Hospital ■ 777
cilities. Advances in screening, testing, and
processing methods continue to improve the
safety profiles of human tissue products.
REGULATIONS AND
STANDARDS
The Food and Drug Administration (FDA)
reg- ulates the activities of tissue banks and
tissue distribution intermediaries, which are
estab- lishments or persons engaged in the
recovery, screening, testing, labeling,
processing, stor- age, and/or distribution of
human tissues for clinical use, under Title
21, Parts 1270 and 1271 of the Code of
Federal Regulations (CFR).3 These entities
manufacture what the FDA classifies as
human cells, tissues, and cel- lular and
tissue-based products (HCT/Ps). Ex- amples
of common tissue products are listed in
Table 32-3.
The three rules of 21 CFR Part 1271
con- cern 1) registration of tissue bank
establish- ments, 2) donor eligibility, and 3)
GTP related to handling HCT/Ps.
Compliance with current GTP regulations is
required of tissue banks and tissue
distribution intermediaries to control
contamination and cross-contamination that
may lead to transmission of disease. Tissue-
dispensing institutions, such as hospitals,
den- tal offices, and surgical centers that
provide and use tissue within their own
facility are not subject to this oversight
except under certain circumstances (ie,
redistribution of allografts or autografts to
affiliated institutions located at a different
address or attempts to sterilize autografts).
For a hospital–based tissue-dispensing
service, regulatory oversight is governed by
voluntary accrediting organizations. Title
21, CFR Parts 1270 and 1271 do not apply
to facili- ties that only receive, store, and
dispense tis- sue, and engage in no further
manufacturing or redistribution of the tissue
product. Howev- er, The Joint Commission,
AABB, College of American Pathologists
(CAP), Association of periOperative
Registered Nurses (AORN), AATB, and Eye
Bank Association of America (EBAA) all
publish standards that apply to the practices
of tissue-dispensing services.8-13 The
778 ■ AABB T EC HNIC AL MANUAL

TABLE 32-3. Examples of Common Cell are safe, available, and of high quality.
and Tissue Products8,14 AATB’s standards pertain to institutional
and quality program requirements; donor
Amniotic membrane
authorization/ informed consent; donor
Bone and demineralized bone matrix screening and test- ing; and tissue recovery,
Bone marrow
processing, release, and distribution.12
EBAA’s scope encompass- 13
es all aspects of eye banking. Accreditation
Bone paste, powder, or by these organizations is based on verified
putty Cancellous chips compliance with established standards and
periodic inspections. Both organizations
Cardiac (heart) valves
serve as scientific and educational resources
Cartilage for the donation and transplantation com-
Cornea munities. A tissue-dispensing service may
find AATB and EBAA accreditation of a sup-
Dermal matrix plier to be valuable in assessing that suppli-
Dura mater er’s qualifications.
Embryos
HOSPITAL TISSUE SERVICES
Fascia
Hematopoietic stem cells There is no requirement for a tissue-dispens-
ing service to be managed in any particular de-
Ligaments partment or by any specific individual. The
Meniscus AABB supports a tissue-dispensing service
within the transfusion service, which has ex-
Ocular tissues
pertise in providing human-derived products
Oocytes that are perishable, potentially infectious,
Pericardium and sometimes in short supply and that
require
temperature-controlled storage. The tasks of
Peripheral blood stem cells ordering, receiving, storing, distributing
Sclera (issu- ing), tracking, and tracing products as
well as investigating adverse events,
Semen and sperm
including com- plaints, recalls, and look-
Skin back investigations, are activities that are
Tendons common to the transfu- sion service and
tissue-dispensing service.
Umbilical cord blood stem cells
Vascular grafts (veins and arteries) Responsibility for Hospital-Based
Tissue Services
The Joint Commission requires that
location of a tissue-dispensing service in a organiza- tions assign oversight
hospital or medical facility and the scope of responsibility for their tissue program, use
its responsibilities dictate which standards standardized procedures in tissue handling,
are applicable. All of these standards are maintain traceability of all tissues, and have
updated regularly; therefore, a periodic a process for investigating and reporting
review of the most recent versions is adverse events. Either a central- ized or
required to guarantee ongoing compliance. decentralized process is permitted to manage
The AATB and EBAA are voluntary these activities. In either model, des- ignated
accred- iting organizations dedicated to oversight is required to coordinate tis- sue-
ensuring that human tissues intended for related activities and ensure standardiza-
transplantation tion of practices throughout the
organization. The Joint Commission
standards apply to
 CH A P T E R 3 2
human and nonhuman cellular-based trans-
plantable and implantable products,
including both tissue allografts and certain
medical de- vices, as classified by the FDA.
Written Standard Operating
Procedures
Hospital tissue services must have written pro-
cedures (printed or electronic) for all functions
pertaining to the acquisition, receipt, storage,
issuance, and tracing of tissue grafts as well as
procedures for investigating adverse events
and handling recalls. Manufacturers’ instruc-
tions for handling tissues should be incorpo-
rated into standard operating procedures
(SOPs). When the blood bank or transfusion
service is responsible for tissue, the AABB re-
quires the medical director to approve all
medical and technical policies and procedures
pertaining to tissue.9(p2)
Tissue Supplier Qualifications and
Certification
In contrast to blood banks, tissue processors
and distributors often specialize in providing
particular types of products (grafts). As a re-
sult, a hospital tissue-dispensing service
may need to acquire human tissue products
from more vendors than the number of
vendors from which a transfusion service
commonly obtains blood products.
Tissue suppliers should be selected
based
on their ability to reliably provide high-quality
tissues that meet expectations for availability,
safety, and effectiveness. The tissue service
should establish minimal criteria for the quali-
fication of prospective suppliers. According to
The Joint Commission, tissue supplier require-
ments must include evidence of current FDA
registration and state licensure if such licen-
sure is required. A written process for review
and approval of suppliers is expected and
should contain the elements listed in Table 32-
4. A list of approved suppliers that includes
documentation of each supplier’s qualifica-
tions, certifications, and appropriate licenses
or permits should be developed and main-
tained. Tissue services should establish proce-
Tissue Services in the Hospital ■ 779
dures to receive or monitor evidence of
com- pliance or noncompliance, such as
reviewing FDA warning letters and tissue
allograft recalls or market withdrawals.
Accreditation by AATB and/or EBAA may
be desirable.
Qualification information for each sup-
plier should be reviewed and approved
annu- ally by the hospital tissue service.
During these reviews, the performance of
suppliers in meet- ing the transplant
facility’s needs should be evaluated. Each
year, the tissue service should determine
whether the supplier remains regis- tered
with the FDA; whether AATB and/or EBAA
accreditation is current can also be con-
firmed. FDA web postings should be
reviewed for information related to closures,
recalls, or MedWatch reports. FDA
inspection reports on tissue processors can
be requested from the FDA through the
Freedom of Information Act. Complaints
from transplanting surgeons con- cerning
the supplier’s tissue should be re- viewed
along with any reports of infections that
might have been caused by transplanted
allografts. Further use of a particular tissue
supplier should depend on approval by the
tis- sue service’s director. Hospital
management may consider establishing a
committee of in- ternal stakeholders,
including transplant phy- sicians, to provide
oversight in the approval of tissue suppliers.
Inspection of Incoming Tissue
Allografts
Before being placed in storage, tissue
allografts must be inspected upon receipt
from a tissue supplier to ensure that the
packaging remains intact and the label is
complete, appears to be accurate, and is
adequately affixed and legible before
acceptance into inventory. The incom- ing
inspection results should be recorded along
with the date, time, and name of the staff
person who conducted the inspection.
The Joint Commission requires that
hos- pitals verify package integrity and
ensure that the transport temperature was
controlled and acceptable. Inspection of the
shipping con- tainer for evidence of residual
coolant (eg, wet ice for refrigerated grafts or
dry ice for frozen grafts) may be useful to
determine that the re-
780 ■ AABB T EC HNIC AL MANUAL
TABLE 32-4. Vendor Qualification Criteria for Human Allografts
CriterionDocumentation/Performance
FDA registration
FDA inspection findings
Voluntary accreditation, if available
State license and registration, if required by state law
annually)
Reliable supply of needed tissues
Transparency of the organization
Medical consultation
Quality assurance resources
New or trial tissue product support
Professionalism of sales representatives
FDA = Food and Drug Administration, AATB = American Association of Tissue Banks, EBAA = Eye Bank Association of
America; eHCTERS = Human cell and tissue establishment registration.
quired tissue-specific storage environment
was maintained during transportation.
Many distributors use “validated” ship-
ping containers that are tested to maintain
re- quired temperatures for a specified
period. If such a container was used, the
receiver of the tissue simply needs to verify
that there is no damage to the container and
that it has been received and opened within
the specified time frame.
Tissues requiring “ambient
temperature” (defined as the temperature of
the immediate environment) for storage and
shipping do not need to have the
temperature verified upon re- ceipt.
However, the temperature of tissues re-
quiring “room-temperature” storage should
be verified and documented if the
manufacturer
Current Form FDA 3356 /eHCTERS Query
Form 483 findings, if any
Warning letters and responses, if any
Current AATB accreditation for tissues
Current EBAA accreditation for ocular
tissues
Copy of state license and registration (to be reviewed
Adequate notification of tissue
shortages Ability to meet special
requests
Suitable expiration dates for tissue products
Willingness to provide information regarding donor-
selection and tissue-processing processes
Accessibility of the tissue supplier’s medical director
Accessibility of the tissue supplier’s quality
assurance staff
Willingness to provide information on newly released
tissue products
Approval sought by representatives through desig-
nated channels before promoting or providing tissue
within the hospital
has specified a temperature storage range in
the package insert.
Tissue Storage
As with blood components, tissue grafts are
stored under various conditions (see Table
32- 5). The appropriate storage conditions
depend on the nature of the tissue, method
of preser- vation, and type of packaging.
Hospital tissue services should store tis-
sue allografts according to the processor’s
instructions in the package insert. Storage
devices can include “ambient” and/or room-
temperature cabinets, refrigerators,
mechani- cal freezers, and liquid-nitrogen
storage units. Continuous temperature
monitoring of refrig-
 CH A P T E R 3 2 Tissue Services in the Hospital ■ 781

TABLE 32-5. Storage Conditions for Commonly Transplanted Human Tissue 12

Human Tissue Storage Conditions Temperature*

Cardiac and vascular Frozen, cryopresERVed –100 C or colder
Musculoskeletal Refrigerated Above freezing (0 C) to 10 C
Frozen, cryopresERVed and non- –20 C to –40 C
cryopreserved (temporary storage
for 6 months or less)
Frozen, cryopresERVed and non- –40 C or colder
cryopreserved (long-term storage)
Lyophilized Ambient†
Reproductive Frozen, cryopresERVed Liquid nitrogen (liquid or vapor
phase)
Skin Refrigerated Above freezing (0 C) to 10 C
Frozen, cryopresERVed –40 C or colder
Lyophilized Ambient†
*Warmest target temperature unless a range is listed.
†
Ambient temperature monitoring not required for lyophilized tissue.

erators and freezers is required. Room-tem-
perature storage equipment need only be
monitored if this is required by the
allograft’s package insert. Storage
equipment should have functional alarms
and emergency back- up capability.
Lyophilized tissues whose pack- age insert
instructions specify ambient tem- perature
or colder storage can tolerate a very broad
range of temperatures and their temper-
atures do not require monitoring.
Storage SOPs should address steps to
be taken in case of excursions from
allowable temperature limits or in the event
of equip- ment or power failure. Emergency
backup al- ternatives, including
arrangements for tempo- rary storage,
should be described in written procedures.
Tissue Allograft Traceability and
Record Keeping
Proper management of human allografts re-
quires that the hospital tissue service docu-
ment all of the steps taken in tissue handling
as they occur and maintain comprehensive
records of these steps. The records should be
accurate, legible, and indelible. They must
identify the staff who have handled the
tissue and the dates when the tissue was
handled as well as the time when the tissue
was accepted, prepared, or processed. The
records should be detailed and provide a
clear history of all ac- tions performed.
Documentation of the tissue supplier, unique
numeric or alphanumeric identifier(s) of the
allograft, its expiration date, and the
recipient’s name must be maintained for all
tissue grafts used.
Tissue service records need to permit bi-
directional traceability of all tissues from the
donor and tissue supplier to the recipient(s)
or other final disposition, including the
discard of tissue because of damage to
package integ- rity, opening of the tissue
package in the oper- ating room without use,
or expiration. Records should be retained for
10 years, or longer if re- quired by state or
federal law, after distribu- tion,
transplantation, discard, or expiration
(whichever occurs last).
782 ■ AABB T EC HNIC AL MANUAL
Tissue usage information cards or other
systems supplied with the allograft by the
tis- sue bank must be completed and
returned to the tissue source facility. This
information helps maintain the traceability
of the allograft and expedites market
withdrawals or recalls when necessary. The
tissue supplier may also use this information
to better understand al- lograft utilization,
obtain positive or negative feedback, and
better meet the needs and ex- pectations of
customers.
Recognizing and Reporting Adverse
Events Possibly Caused by Allografts
Use of human-derived medical products,
such as tissue allografts, carries risks that
must be balanced with clinical benefits.
Human al- lografts have been associated
with bacterial, viral, fungal, and prion
transmissions. In addi- tion, allografts may
have structural defects that sometimes lead
to unsuccessful outcomes as a result of
donor selection or processing fac- tors.
Hospital tissue services are required to
have procedures to investigate in a timely
way any infections suspected to be caused
by a tis- sue allograft. The Joint
Commission requires that allograft-
transmitted infections and other severe
adverse events be immediately report- ed by
the hospital to the tissue supplier.
Transplant surgeons play a critical role in
the identification of allograft-associated ad-
verse outcomes and need to immediately noti-
fy the hospital tissue service when they sus-
pect such events. Prompt notification of
adverse events enables the hospital tissue ser-
vice to investigate the cause, report the issue
to the tissue supplier, and institute corrective
action, including sequestration of any other
suspect allografts. The investigation of infec-
tions and other adverse events requires coop-
eration between the tissue-dispensing service,
clinicians, and tissue supplier. Consultation
with the hospital infection-control depart-
ment or an infectious disease specialist may
be beneficial. Early notification can prevent an
untoward outcome for other recipients of al-
lografts from an implicated donor.
State health departments have lists of
communicable diseases that must be reported
when they are newly diagnosed. A new
diagno- sis of HIV or viral hepatitis in a tissue
allograft recipient where the allograft is
suspected as a possible source must be
reported to the state department of health by
the patient’s physi- cian or the director or
medical director of the hospital tissue service.
An epidemiologic in- vestigation may be
needed to establish wheth- er the tissue
allograft was the source of the re- cipient’s
infection. Early notification of the state
department of health can result in timely
assistance with the investigation.
Recalls and Look-Back Investigations
A tissue product recall or market withdrawal
occurs when a tissue allograft is determined
by the tissue supplier to be compromised or
po- tentially infectious. The supplier may
recall all of the tissues from a specific donor
or process- ing lot, sequester tissues in
inventory, and no- tify hospitals to which
the allografts were shipped. The hospital
may be instructed by the supplier to
quarantine the allografts in in- ventory,
identify recipients, and/or notify the
transplanting surgeon(s) of the recall. Sur-
geons should evaluate the circumstances of
the recall and notify the recipient as
appropri- ate that a tissue graft was recalled.
Look-back investigation can be triggered
when a tissue donor is subsequently found to
have been infected with HIV, human T-cell
lymphotropic virus Types I or II, HBV, HCV,
or other infectious disease known to be
transmit- ted by tissue grafts. Because most
tissues are procured from deceased donors,
infectious disease testing of subsequent donor
blood samples is not possible. Look-back
investiga- tions involving tissue grafts are
uncommon.
Tissue Autograft Collection, Storage,
and Use
Surgical reconstruction using the patient’s
own tissues often has advantages over the
use of cadaveric tissue. These advantages
include ready availability, faster
incorporation, appro-
 CH A P T E R 3 2
priate size or shape, and relative safety from
viral disease transmission.
A bone flap removed during
decompres- sive craniectomy is a commonly
used example of tissue autograft use. In this
procedure, a sec- tion of skull is excised by
the neurosurgeon to reduce intracerebral
pressure caused by brain swelling due to
trauma, stroke, or surgery. After removal, the
skull fragment is rinsed, packaged, frozen,
and stored for future reim- plantation during
a procedure known as “cra- nioplasty.”
Written procedures should address the
collection, microbial testing, packaging,
stor- age, and issuance of tissue autografts
for reim- plantation. Bacterial testing by
obtaining ap- propriate cultures should be
performed after surgical removal and before
packaging. Auto- grafts should not be
collected from patients with systemic
infections or if the tissue is in close
proximity to an infected area. Autografts
may be stored at the medical facility where
they are collected or at an off-site, FDA-
regis- tered tissue bank. Procedural
recommenda- tions have been published by
the AATB and AORN.
TRANSFUSION SERVICE
SUPPORT FOR ORGAN
TRANSPLANTATION
Organ transplantation relies on the
coordinat- ed activities of organ-
procurement organiza- tions (OPOs) and the
hospital transplant team. A successful
transplant program requires an
interdisciplinary team that has well-defined
policies, procedures, and communication
pathways. The transfusion service provides
appropriate compatibility testing and
transfu-
KEY POINTS
Surgical use of human allografts has grown dramatically in recent years, with more than 2 million tissue grafts used annual
Not all tissue allografts are sterile. Depending on the type of allograft, sterilization may not be possible because it could jeo
Tissue Services in the Hospital ■ 783
sion support before, during, and after trans-
plantation.
OPOs evaluate potential donors, obtain
authorization (consent), and prepare organs
for transportation. The United Network for
Or- gan Sharing (UNOS) coordinates the US
organ transplant system. The evolving
UNOS guide- lines for organ allocations are
designed to achieve equitable distribution of
life-saving organs. Factors such as severity
of recipient ill- ness, geographic proximity,
and donor-recipi- ent compatibility are
considered. UNOS pro- vides detailed
policies and other relevant information on
its website.15
Depending on the organ, ABO and/or
HLA compatibility may be important factors
for transplantation success. ABO antigens ex-
pressed on the vascular endothelium of organs
constitute strong histocompatibility barriers.
Major incompatibility between donor ABO an-
tigens and recipient plasma can result in acute
humoral rejection of the transplanted organ
and threaten a successful outcome. As a result,
UNOS requires two separate determinations
of ABO type for both 1) transplant candidates
prior to listing their needs on the Organ Pro-
curement and Transplantation Network wait-
ing list and 2) donors prior to making an organ
available for transplant. ABO-incompatible or-
gan transplants are sometimes performed fol-
lowing a conditioning regimen that may in-
clude plasma exchange.
Services that may be required by a trans-
plant program include provision of cytomega-
lovirus-reduced-risk components, blood irra-
diation, massive transfusion support, ABO
subgroup typing, and immunohematology ref-
erence testing. Transfusion services should
understand and address such expectations of
the transplant team.
784 ■ AABB T EC HNIC AL MANUAL

of the graft and adversely affect in-vivo performance. Methods of sterilization include
the use of ionizing radiation or ethylene oxide and some proprietary processes and
techniques.
3. Rare examples of HIV, HCV, HBV, CJD, and bacterial and fungal disease transmission
from allografts have been documented. Therefore, like blood components, allografts are
released for use only after infectious disease testing has been completed and the results
deemed ac- ceptable.
4. In general, bone and soft tissue allografts do not need to be matched for HLA or ABO type.
5. Hospital-based tissue services are not subject to FDA regulatory oversight if their activities
are limited to receiving, storing, and dispensing tissue to a requesting surgeon. However,
The Joint Commission, AABB, CAP, AORN, AATB, and EBAA publish standards that
apply to such services.
6. Tissue banks are engaged in the recovery, screening, testing, labeling, processing,
storage, and distribution of human tissues. They are regulated by the FDA under the
Title 21, CFR Parts 1270 and 1271 as manufacturers of HCT/Ps.
7. No regulatory or standard-setting organization requires a hospital tissue-dispensing
service to be managed by a particular department or individual. However the AABB
favors a cen- tralized model for managing this activity within the transfusion service.
REFERENCES

1. American Association of Tissue Banks. About

AATB. McLean, VA: AATB, 2013. [Available at
http://www.aatb.org/About-AATB (accessed
9.
December 8, 2013).]
2. Eastlund DT, Eisenbrey AB for the Tissue
Com- mittee. Guidelines for managing tissue
al- lografts in hospitals. Bethesda, MD: 10.
AABB, 2006.
3. Code of federal regulations. Title 21, CFR
Parts 1270 and 1271. Washington, DC: US 11.
Govern- ment Printing Office, 2014 (revised
annually).
4. Woll JE, Smith DM. Bone and connective tis- 12.
sue. Clin Lab Med 2005;25:499-518.
5. Malinin TI. Preparation and banking of bone
and tendon allografts. In: Sherman OH,
Minkoff J, eds. Arthroscopic surgery. Balti- 13.
more, MD: Williams and Wilkins, 1990:65-
86. 14.
6. Cheek RF, Harmon JV, Stowell CP. Red cell
allo- immunization after a bone allograft.
Transfu- sion 1995;35:507-9.
7. Eastland T, Warwick, RM. Diseases transmit-
ted by transplantation of tissue and cell al-
lografts. In: Warwick E, Brubaker SA, eds.
Tis- sue and cell clinical use: An essential
guide. West Sussex, United Kingdom:
Blackwell, 2012:72-113.
15.
8. The Joint Commission. Transplant safety. In:
Comprehensive accreditation manual for hos-
pitals: The official handbook. Oakbrook Ter-
race, IL: The Joint Commission, 2014:TS1.
Levitt J, ed. Standards for blood banks and
transfusion services. 29th ed. Bethesda, MD:
AABB, 2014.
College of American Pathologists. Standards
for laboratory accreditation. Northfield, IL:
CAP, 2012.
Association of Perioperative Registered Nurs-
es. Perioperative standards and recommended
practices. Denver, CO: AORN, 2013.
Dock N, Osborne J, Brubaker S, et al. Stan-
dards for tissue banking. 13th ed. McLean,
VA: American Association of Tissue Banks,
2012.
Eye Bank Association of America. Medical
standards. Washington, DC: EBAA, 2011.
Food and Drug Administration. FDA regula-
tion of human cells, tissues, and cellular and
tissue based products (HCT/Ps) product list.
Rockville, MD: Food and Drug
Administration, 2013. [Available at
www.fda.gov/Biologics
BloodVaccines/TissueTissueProducts/Regula
tionofTissues/ucm150485.htm (accessed No-
vember 13, 2013).]
United Network for Organ Sharing. Richmond,
VA: UNOS, 2013. [Available at
www.unos.org (accessed December 8, 2013).]
 C h a p t e r 3 3

Blood and Marrow-Derived

Nonhematopoietic Stem Cell
Sources and Immune Cells
for Clinical Applications

Mickey B. C. Koh, MD, PhD; Edward R. Samuel, PhD, MSc; and

Garnet Suck, PhD, MSc

HE M A T O P O I E T I C STEM CELL
transplantation (HSCT) is an established
modality of treatment for a wide range of he-
matologic diseases, including leukemias and
lymphomas. One of the most powerful mecha-
nisms for cure is the presence of the graft-vs-
tumor (GVT) or graft-vs-leukemia (GVL)
effect, which is mediated largely by mature
immune effector cells, such as T and natural
killer (NK) cells.1 The immune response that is
generated is derived from a complex system of
interac- tions involving antigen recognition
and pre- sentation mediated by dendritic cells
(DCs). DCs are also known as “professional
antigen-
presenting cells” and are key to
orchestrating the adaptive immune response
in an antigen- specific manner.
The success of HSCT has led to
enormous interest in the isolation,
characterization, ex- pansion, and
modification of these immune cells to
augment the powerful GVT response. Most
of these cells form part of the hemato-
poietic stem cell (HSC) graft infused into
pa- tients and can be derived from blood.
In fact, the discovery of the GVT effect
came from the observation that depleting T
cells from the graft resulted in a greater risk
of leukemia relapse. Conversely, the
occurrence

Mickey B. C. Koh, MD, PhD, Director, Stem Cell Transplantation, St. George’s Hospital and Medical
School, London, United Kingdom, Medical Director, Cell Therapy Facility, Health Sciences Authority,
Singapore; Edward R. Samuel, PhD, MSc, Clinical Scientist and Senior Research Associate, Department of
Haematology, University College London Medical School, Royal Free Campus, London, United Kingdom;
and Garnet Suck, PhD, MSc, Division Director, Productions, Institute for Transfusion Medicine, German Red
Cross Blood Dona- tion Service North-East nonprofit GmbH, Berlin, Germany
The authors have disclosed no conflicts of interest.

785
786 ■ AABB T EC HNIC AL MANUAL
of graft-vs-host disease (GVHD), which is
also mediated by T-cells, was protective and
re- duced the chance of relapse.1
In addition to HSCs and their
differentiat- ed progeny, the marrow
contains nonhemato- poietic cells, often
referred to as “stromal cells,” which include
mesenchymal stem cells (MSCs). In addition
to providing stromal sup- port for
hematopoietic cells, MSCs give rise to
structural components, such as bone, carti-
lage, tendon, and fat. Their wide-ranging
and potent differentiation properties have
generat- ed considerable clinical interest in
their use for cell therapy. MSCs also appear
to exert immu- nosuppressive effects and are
therefore of in- terest as immunomodulators.
Self-renewing pluripotent stem cells are
rare and difficult to isolate. The
demonstration that pluripotent stem cells
can be derived from differentiated cells,
including skin fibro- blasts and blood, has
transformed the field of stem cell therapy.
The use of these cells has the potential to
have myriad applications in all ar- eas of
medicine.
IMMUNE CELLS FOR CLINICAL
THERAPY
Cellular immunotherapy is the exploitation
of immune cells to target and treat diseases.
This approach is most established in cancer
pa- tients, where these novel treatment
modalities are used to overcome the
limitations and tox- icities of chemotherapy
and radiotherapy.
Considerable progress in cellular
process- ing technology has allowed for
advanced and complex immune-cell
manipulations. It is now possible to
characterize immune cells with high
precision, isolate specific cell types with
high purity, and engineer and amplify them
ex vivo in sophisticated culture systems that
comply with good manufacturing practice
(GMP) requirements. The adoption of auto-
mation has also allowed for more
reproducible immune cell expansion.
Blood banks have been instrumental in
these advances in cellular immunotherapy
due to their preexisting infrastructure, which
includes the GMP-compliant processing of
blood units for transfusion and meticulous
quality-assurance systems embedded into
the processes. In addition, the stringent
donor- selection processes, widespread use
of auto- mation, use of mandatory release
criteria for blood units, and requirements of
traceability and hemovigilance of blood
banks have been incorporated into cellular
immunotherapy.
Donor Lymphocyte Infusion: A
Post- HSCT Treatment
Although allogeneic HSCT can achieve long-
lasting cures in many patients, relapse still oc-
curs in some patients and is often associated
with a poor prognosis.2 A second HSCT is
pos- sible but is often associated with high
toxicity and morbidity risk, especially if the
relapse oc- curs within a year of the first
transplant.
Clinical Efficacy of Donor
Lymphocyte Infusions
The discovery that T cells are one of the
main drivers of the GVT or GVL response
led to the development of donor leukocyte
(lymphocyte) infusions (DLIs) after HSCT.
One of the key ob- servations in the
development of DLIs was that patients with
chronic myeloid leukemia (CML) that
relapsed after HSCT could achieve long-
lasting remissions with the administration of
DLIs.3
Lymphocytes for DLI are collected from a
donor via leukapheresis using a process simi-
lar to peripheral blood stem cell collections,
except that prestimulation with a colony-stim-
ulating factor or mobilizing agent is not usual-
ly required. The leukapheresis product is usu-
ally then aliquoted into doses starting from as
low as 1  106 CD3-positive cells/kg recipient
weight, followed by half- or 1-log increments.
The initial dose is often infused fresh, and ali-
quots for subsequent doses are cryopreserved.
The efficacy of DLIs is dependent on the
type and aggressiveness of the underlying dis-
ease and the disease burden at the time of re-
lapse. It has been demonstrated that patients
with relapsed CML benefit most from DLIs
with an 80% complete response rate for those
in cytogenetic relapse; patients in hematologic
relapse respond less well.3 In comparison, only
 CH A P T E R 3 3 Blood/Marrow-Derived Non-HPC and Immune Cells
15% to 40% of patients with relapsed acute
myeloid leukemia (AML) respond to DLIs,
and patients with acute lymphocytic leukemia
(ALL) have virtually no response to DLI. The
lack of efficacy of DLI in acute leukemias
com- pared to CML is thought to be related to
a lack of antigen expression on tumor cells and
the more rapid proliferative kinetics associated
with acute leukemias.4
The starting dose for DLIs is often in
the order of 1 × 106 CD3-positive cells/kg.5
Lower starting doses have been
administered, espe- cially in the unrelated
transplant setting. Re- sponse is usually not
immediate and can take as long as 3 months.
For this reason, subse- quent doses of DLIs
are often administered as far apart as 3
months and in incremental doses half to 1
log apart so that responses can be ad-
equately gauged.3
There is no exact correlation between
dose and response. Patient response can be
difficult to predict because it depends on the
disease, stage of relapse, patient factors,
HLA matching, and donor characteristics.6
Complications of DLIs
The major complication of DLI is GVHD,
which is caused by alloreactive donor T cells
attacking healthy host cells. In early studies,
up to 50% to 90% of patients developed
GVHD after DLI. However, more recently, the
rate of GVHD following DLI has declined due
to an improved understanding of the biology
of DLIs and predictive risk factors for GVHD
in this setting. The GVHD that occurs after
DLI can be very severe and require systemic
immune sup- pression, which can lead to
significant mor- bidity and mortality as a result
of opportunis- tic infections.
Another major complication of DLI is
the development of marrow aplasia, which
is thought to be due to the immune-mediated
destruction of host hematopoeisis.6
The variable results in the initial studies
of DLIs in patients with frank disease relapse
together with the treatment’s potential toxici-
ties have resulted in crucial modifications to
this T-cell therapy to increase its GVT activity
while minimizing its side effects. These modi-
■ 787
fications include the use of CD8-depleted
and alloantigen-depleted DLIs and the
introduc- tion of a “suicide” gene, such as
the Herpes simplex virus-tyrosine kinase
gene, into the T cells to selectively eliminate
them if GVHD de- velops.4
A group of researchers at University
Col- lege London Hospital has also
pioneered a strategy of using a dose-
escalating DLI regimen to treat residual or
progressive dis- ease or mixed donor
chimerism after reduced- intensity, T-cell-
depleted transplantation.5 This strategy has
resulted in an impressive 70% response rate
in patients with low-grade lym- phomas or
Hodgkin disease.
New Directions in DLI
Applications: Targeted and Antigen-
Specific T-Cell Therapy
Adoptive immunotherapeutic approaches em-
ploying more targeted or antigen-specific T-
cell populations have been used successfully
in the treatment of hematologic malignancies
and solid tumors as well as viral infections af-
ter HSCT. To develop immunotherapies using
cytotoxic T lymphocytes (CTLs; T cells ex-
pressing CD8), potential target antigens on tu-
mor cells or viruses must first be identified.
These antigens must be capable of providing
epitopes for specific immune responses and
be present in sufficient quantity and duration
to engage responder T cells.4
A step forward in efficiently isolating
T cells was achieved with the development of
the major histocompatibility complex (MHC)
multimer method. In this method, high-affini-
ty binding of MHC molecules to the T-cell re-
ceptor is achieved through oligomerization of
MHC molecules (multimers) as ligands. 7 This
method allows specific detection and isola-
tion of antigen-specific T cells through
fluores- cence-activated cell sorting in a flow
cytome- ter or the use of closed magnetic
selection systems, such as the automated
CliniMACS in- strument (Miltenyi Biotec,
Bergisch Gladbach, Germany). The multimer
method has recently been improved with the
invention of the novel streptamer technology,
which has entered clinical testing.8,9
788 ■ AABB T EC HNIC AL MANUAL
Adoptive transfer of donor-derived, virus-
specific CTLs constitutes an important novel
treatment of life-threatening viral infections
after HSCT and an alternative to traditional
antiviral drug therapy, such as ganciclovir. Cy-
tomegalovirus (CMV) reactivation is a com-
mon complication of HSCT. Infusions of CTLs
generated against the CMV pp65 antigen have
achieved good clearance of the virus with few
side effects and limited GVHD. Similarly, do-
nor-derived Epstein-Barr virus (EBV)-specific
CTLs have been successfully used for both
prophylaxis and treatment of EBV-associated
lymphoproliferative disease (LPD). In one
study, more than 60 patients were prophylacti-
cally treated with these CTLs and none devel-
oped LPD, compared to 11.5% of patients who
developed LPD in a historical, nontreated con-
trol group.4 Significantly, gene-marked donor
CTLs have been demonstrated to persist in
DLI recipients for as long as 7 years.4
A recent major breakthrough has been
achieved with the development of
engineered T cells bearing chimeric antigen
receptors (CARs) composed of antibody-
binding do- mains connected to domains
that activate T cells. This strategy was
developed to overcome T-cell tolerance,
which has been a limitation of CTL
populations that have been isolated and
expanded based on recognition of tumor-as-
sociated antigens. One such CAR construct
utilizes an antibody-binding domain with
specificity for the B-cell antigen CD19 cou-
pled with CD137 (a costimulatory receptor
in T cells) and the CD3-zeta signaling
domain.10 The construct’s efficacy was
recently demon- strated in a clinical pilot
trial in which autolo- gous CAR-T cells
directed against CD19-bear- ing advanced
chronic lymphocytic leukemia (CLL) with
more than 1000-fold expansion in vivo and
demonstration of trafficking to the marrow.
Significantly, memory CAR-T cells were
generated, and two out of three patients with
advanced CLL achieved complete remis-
sion with persistence of T cells for more
than 6 months.10 This proof of concept has
just been extended into the treatment of B-
cell ALL with impressive results and the
attainment of com- plete remissions in some
treated patients.11
NK Cells
Particularly promising candidates for cellular
immunotherapy are NK cells. These large
granular lymphocytes of innate immunity are
characterized by a CD3–CD56+ phenotype in
humans and are natural defenders against ma-
lignancies and infectious diseases. Their cyto-
toxicity is immediate and they do not require
antigen priming. Autologous NK cell attack is
prevented via recognition of HLA Class I
mole- cules by cognate NK-cell inhibitory
killer im- munoglobulin-like receptors (KIRs).
In-Vivo Expansion
Early cellular immunotherapy protocols in-
volving NK cells employed short-term
stimula- tion in vitro with high-dose
interleukin (IL)-2 for 1 to 5 days, generating
a heterogeneous lymphokine-activated killer
(LAK) cell product with a relatively low
representation of cytotox- ic NK cells. A
better understanding of NK cell biology and
recent advances in cell selection,
monitoring, and expansion technologies
have enabled the development of more
promising new NK-cell-therapy
approaches.12
An important key finding was the obser-
vation that alloreactive NK cells in the graft
ex- erted a potent antileukemic effect in the
con- text of T-cell-depleted, HLA-
haploidentical HSCT.13 Significantly, there
were no relapses in a group of patients with
high-risk AML when NK cells exerted their
effects due to donor- patient KIR mismatching,
compared to a 75% relapse rate when no NK
alloreactivity was present.14
NK cells have also been administered in a
non-HSCT setting. Patients with poor-progno-
sis AML, Hodgkin disease, metastatic melano-
ma, or renal carcinoma were given haploiden-
tical NK cells.15 In-vivo expansion of these
cells occurred with assistance from the
lymphope- nia induced by a lympho-depleting
prepara- tive regimen, such as
cyclophosphamide and fludarabine. This
regimen induced a marked increase in
endogenous IL-15 levels, which is essential for
donor NK cell expansion and sur- vival in the
patient. Significantly, these infused and in-
vivo-expanded NK cells did not induce GVHD
but did produce complete remissions
 CH A P T E R 3 3 Blood/Marrow-Derived Non-HPC and Immune Cells
in 5 of 19 patients with AML. These results
of- fer another proof of the ability of NK
cells to separate GVL from GVHD with the
aim of ex- ploiting the former while
minimizing the lat- ter. As in the
haploidentical transplant setting, the effect
was more marked when KIR-ligand
mismatched (indicating alloreactive) donors
were used.
In clinical protocols involving NK cells,
ef- fectors are enriched by magnetic CD3
deple- tion followed by CD56 selection or
through combined CD3 (T-cell) and CD19 (B-
cell) de- pletion using the CliniMACS
(Miltenyi Bio- tech) to reduce the risk of T-
cell-mediated GVHD and EBV infections.16
Ex-Vivo Expansion
An alternative method to in-vivo expansion of
NK cells is expansion of NK cells in long-
term, GMP-compatible culture systems that
gener- ate large numbers of highly cytotoxic
effectors. This method has been established
for clinical “off-the-shelf” production of the
highly cyto- toxic NK cell line NK-92
[Conkwest (Del Mar, CA) proprietary line].17,18
In contrast, GMP- compatible expansion of
primary NK cells has remained challenging,
although promising protocols are under
development.
New cell-culture media, including
serum- free media, are now available.
Furthermore, novel growth factors, such as
IL-15, are being used instead of the
traditional IL-2 to further optimize culture
conditions with efficient bi- asing of
proliferation of the NK-cell popula- tion
within LAK cultures after long-term ex-
pansion.19
Highly purified NK cells are more chal-
lenging to expand. However, purified NK cells
can be grown on feeder cell sources of either
autologous or allogeneic origin. An elegant
way to obtain autologous feeder cells is to re-
tain the nonselected, NK-cell-negative frac-
tion after a magnetic isolation procedure. Fol-
lowing irradiation of this by-product to inhibit
unwanted cell proliferation during culture,
these cells can be seeded as a feeder layer to
support NK-cell stimulation and proliferation.
Other protocols have been established that in-
volve the use of allogeneic cell sources. Expo-
■ 789
nential NK-cell expansion, for example, was
achieved with the EBV-transformed lympho-
blastoid cell line or the leukemic cell line
K562, engineered to present both IL-15 and
the co- stimulatory molecule CD137 (4-1BB)
on the surface (K562-CD137/IL-15). These
protocols are undergoing clinical testing.16,19
Automated cell processing within biore-
actors, such as Wave™ (GE Healthcare Life
Sci- ences, Freiburg, Germany) or G-Rex
(Wilson Wolf Manufacturing, New Brighton,
MN), pro- vides a developing platform for the
mass pro- duction of NK cells in less time with
better re- producibility.16 These methods are
also used for other immune effector therapies.
Further- more, shipment of fresh NK cells
under IL-2 protection is feasible without the
need for a controlled incubator environment
(37 C and
5% CO2) and facilitates international
distribu- tion of such products.20
NK-cell therapy products are generally
safe and well tolerated, especially when in-
vivo injections of IL-2 are not required.
How- ever, NK-cell products are not entirely
risk free. Two cases of significant hemolytic
anemia af- ter treatment with minor ABO-
mismatched NK-cell-enriched products have
been report- ed that were presumably due to
the presence of isoagglutinin-producing
passenger B lym- phocytes in the NK-cell
product.21
Cytokine-Induced Killer Cells:
Polyclonal T Cells with NK-Cell
Features
The traditional LAK cell culture protocol
has been further developed to generate a
popula- tion of rapidly expanding polyclonal
T cells known as “cytokine-induced killer”
(CIK) cells.22 Culture conditions for CIK
expansion are designed to obtain an
increased propor- tion of superior cytotoxic
CD56+CD3+, double- positive T cells or NK-
like T cells. The genera- tion of such CD56+
T cells is promoted in this procedure
through the initial addition of gam- ma
interferon (IFN; 1000 U/mL). The T-cell-
activating, anti-CD3 monoclonal antibody
OKT3 (50 ng/mL) and IL-2 (300 U/mL) are
added 24 hours later, and the cultures are
maintained under permanent IL-2-stimula-
tion for 21 to 28 days.23 The heterogeneous
CIK
790 ■ AABB T EC HNIC AL MANUAL
population at the end of culture comprises a
very small proportion (about 2%) of NK cells.
Most of the cells (>90%) in the culture are T
cells, of which about 35% express the double-
positive phenotype CD3+CD56+.
Characteristics of CIK Cells
CIK cells combine features of NK-cell
(CD56+CD3–) and T-cell (CD56–CD3+)
effectors. The different pathways involved in
CIK cyto- toxicity are under active
investigation. Similar to NK cells, CIK cells
have cytolytic potential that is immediate and
independent of antigen priming.24 However,
studies have shown that target cell recognition
requires direct MHC Class I engagement, a
mechanism that is not yet fully understood. As
with NK cells, the criti- cal role of the
activating receptor NKG2D in tu- mor lysis
has been demonstrated for targets, such as
myeloma cells, that express its ligands MIC
A/B or ULBPs.
Clinical Trials
Results from clinical trials using autologous
or allogeneic CIK cells in a variety of
hematologic malignancies and solid tumors
have been re- ported. Overall, CIK cell
therapy was well toler- ated and associated
with a low risk of GVHD. 25 Results for
cancer clearance and increased survival
varied. Patients with a lower tumor burden,
such as those who had undergone HSCT, are
probably the most likely to benefit from
CIK effectors. In this sense, CIK cells may
be used as an alternative to DLI. Strategies
to increase the cytolytic potential of CIK
cells, in- cluding co-administration of IL-2
and engi- neered CARs, are currently being
investigated.
DCs in Immunotherapy
DCs play a critical role in the regulation of
the adaptive immune response. DCs have
been called “sentinels of the immune
system,” and they possess the ability to
elicit a primary im- mune response in
resting naïve T lympho- cytes. Naïve CD8+
T cells, for example, differ- entiate into
CTLs through interaction with cognate
antigens presented by mature DCs on their
MHC Class I receptors.26 DCs are capable
of eliciting full T-cell activation upon engage-
ment of costimulatory molecules (eg,
B7:CD28 engagement) and mediate cytotoxic
responses against a broad range of
malignancies. Partic- ularly attractive is the
intrinsic potential of DCs to gain immunologic
memory for com- bating subsequent tumor
invasions.27
Defining DCs and Their Subsets
Unlike T cells, no single cell surface marker
is exclusively expressed on DCs that can be
used to define these cells. Instead, a
combination of morphology and various
surface markers is used, including the
expression of MHC Class II antigens and
absence of markers that define other
lineages, such as CD3 and CD19. DCs also
express a variety of adhesion molecules,
including CD11a (lymphocyte function-
asso- ciated antigen 1); the intercellular
adhesion molecule 1 family; and
costimulatory mole- cules, such as CD80
and CD86. Two additional markers of
mature DCs in humans are CD83 and
CMRF-44.28
In the blood, DCs are present in
different subsets that are distinguished by
CD303 (BDCA2, CLEC4C; plasmacytoid
DC), CD1C (or BDCA1; myeloid DC), and
CD141 (BDCA3 or thrombomodulin;
myeloid DC) markers. Different functions
could be attributed to these subsets, with
CD141-positive myeloid DCs playing a role
in eliciting CD8 T-cell re- sponses.29
The rarity of DCs has made it difficult
to study them, although they were first
described more than 30 years ago.26 The
ability to gener- ate large numbers of DCs
from CD34+ marrow precursors or CD14+
monocytes in vitro has expanded the field of
DC-based cellular im- munotherapy.
Expansion Protocols
Initial tissue-culture protocols for the genera-
tion of DCs (the first-generation DC vaccines)
used a combination of granulocyte macro-
phage colony-stimulating factor (GM-CSF)
with IL-4 for the stimulation of monocytes in
peripheral blood mononuclear cells (PBMCs)
to differentiate into DCs. However, the result-
ing DC vaccines usually contained a mixture
of
 CH A P T E R 3 3 Blood/Marrow-Derived Non-HPC and Immune Cells
immature or only partially mature DCs and
of- ten other additional cell types. Since
then, re- finements of protocols (known as
“second- generation” DC expansion
protocols) have been described using several
new cytokine/ GM-CSF combinations that
include IFN, tu- mor necrosis factor
(TNF), or IL-15.29 One such cytokine cocktail
consists of TNF, IL-1, IL-6, and
prostaglandin 2 for additional DC stimu-
lation. More recently, “third-generation” DC
vaccines have been developed that are polar-
ized toward a cytotoxic immune response
(in- nate and Type 1 immunity) and are
currently being tested in clinical trials.29,30
In-vitro manipulation of DCs includes
loading (pulsing) with tumor recognition
mol- ecules, such as tumor-antigen-derived
pep- tides, recombinant tumor antigens, or
tumor- derived ribonucleic acid or
deoxyribonucleic acid. Furthermore, DC
tumor-hybrid vaccines have been created
with the potential to acti- vate a combined
CD4+ and CD8+ T-cell re- sponse through
simultaneous presentation of several tumor
antigens, including a direct fu- sion of DCs
to leukemic blasts, which are easi- ly
obtainable from patients with leukemia.31,32
However, release of suppressive factors,
such as TGF, by the tumor cell fused in the
hybrid may limit the vaccine’s efficacy.31
Cancer Vaccine Approved by Food
and Drug Administration
One of the successes of cellular therapy has
been in the treatment of prostate cancer. A
first-generation DC-based vaccine, sipuleucel-
T (Provenge; APC 8015; Dendreon
Corpora- tion, Seattle, WA), was approved
by the US Food and Drug Administration
(FDA) based on a 4-month improvement in
survival compared to placebo in the pivotal
randomized clinical trial of men with
castration-resistant metastat- ic prostate
cancer.33 This is an autologous cel- lular
immunotherapy designed to stimulate a
patient’s own immune system to respond
against the prostate cancer. Sipuleucel-T
con- sists of autologous PBMCs that are
enriched for antigen-presenting DCs that
have been ac- tivated ex vivo with a
recombinant fusion pro- tein, prostatic acid
phosphatase (PAP), fused
■ 791
to GM-CSF. PAP is an immunogenic
prostate antigen whereas GM-CSF is an
immune-cell activator. Each dose is
manufactured by ob- taining a patient’s T
cells via leukapheresis and shipping them to
the company to manufacture the vaccine.
After this process, the patient’s own cells are
reinfused into the patient to treat the prostate
cancer. Provenge is administered
intravenously in a three-dose schedule given
at about 2-week intervals.
The effectiveness of Provenge was
studied in 512 patients with metastatic
prostate cancer that was refractory to
hormone treatment in a randomized,
double-blind, placebo- controlled,
multicenter trial.33 The median survival
duration for patients receiving Provenge
treatments was 25.8 months com- pared to
21.7 months for those who did not re- ceive
the treatment.
MSCs
MSCs are a rare subset of cells of nonhemato-
poietic origin that were first discovered in
1968 by Friedenstein as an adherent fibroblast-
like population after isolation from the
marrow, where they represent 0.001% to
0.01% of total cells.34 It was subsequently
shown that MSCs could be isolated from
various tissues, includ- ing amniotic fluid,
adipose tissue, umbilical cord blood, dental
pulp, muscle connective tissue, and placenta,
and could be rapidly ex- panded in vitro,
offering the potential for use in clinical
cellular therapy.35 The rarity of MSCs has
meant that the majority of research to date has
relied on isolating them from the marrow or
adipose tissue before expansion in culture, a
prerequisite for clinical-scale therapies. MSCs
are often referred to as “multipotent” due to
their capacity for differentiation into cells of
mesodermal lineage, including osteo- blasts,
chondrocytes, adipocytes, and, under specific
conditions, several nonmesodermal cell types,
including neurons.36 It is this capac- ity for
multipotency that has generated a great deal of
interest in using MSCs for tissue engi- neering
and regenerative medicine.
Expanded MSC cultures do not all possess
the same level of multipotency. Although a
low frequency of self-renewing
progenitors has
792 ■ AABB T EC HNIC AL MANUAL
been identified among marrow-derived
MSCs, it remains unclear whether MSCs
from other tissues share these properties.
This has result- ed in the use of the term
“multipotent MSCs” as an alternative to
“MSCs” because “multipo- tent MSCs” does
not imply that the cells have “stem-cell-
like” properties or self-renewal ability
without differentiation (Table 33-1).37
The defining characteristics of MSCs
have often differed among investigators,
which prompted the publication of the
minimal cri- teria for the definition of the
phenotypic and functional properties of MSCs
by the Interna- tional Society of Cellular
Therapy (ISCT).38 The ISCT guidelines for
defining MSCs are listed in Table 33-2.
MSCs have been identified as
modulators of several effector functions and
immune re- sponses through their
interaction with both the innate and adaptive
immune systems. They play a key role in the
support of hemato- poiesis, contributing to
the maintenance of the highly specialized
microenvironment of the marrow through
the regulation of HSC numbers and control
of their maturation and differentiation. 39
MSCs have been shown to be capable of
exerting a profound immunosup- pressive
effect on both polyclonal and anti- gen-
specific T-cell responses through induc- tion
by cellular or humoral stimuli. They largely
have no immunologic restriction, meaning
that they can be used without HLA
matching.40 The immunoregulatory functions
of MSCs, including the suppression and
inhi- bition of T-, B-, and NK-cell functions
and DC activity, therefore offer a promising
option for the treatment of immune-
mediated disorders,
TABLE 33-1. The Multipotentiality of MSCs
Self-RenewalDifferentiationTransdifferentiation
Marrow cavity Mesodermal lineage
Connective stromal cells
Cartilage cells
Fat cells
Bone cells
MSCs = mesenchymal stem cells.
including GVHD, autoimmunity, and solid-
or- gan-transplant rejection. The suppressive
properties displayed by culture-expanded
MSCs are promoted through the expression
of indolamine 2,3-dioxygenase and other
effector molecules, many of which are
augmented by IFN- stimulation.41,42
Isolation and Expansion of MSCs
The isolation and initial expansion of MSCs is
performed in three distinct phases over a peri-
od of 3 to 4 weeks:
1. Isolation and seeding.
2. Cell passaging.
3. Final harvest.
The clinical production of MSCs for ther-
apeutic use is carried out in large tissue-cul-
ture flasks, although MSC production can be
industrialized in 25,000-cm2 culture chambers
or CellSTACKsTM (Sigma-Aldrich Corning, New
York, NY). Marrow aspirates/harvests remain
the most common starting material for the iso-
lation of MSCs. Following harvest, marrow is
collected into preservative-free heparin be-
fore transportation to the laboratory, where
the first step is the isolation of bone-marrow
mononuclear-cell (BMMNC) fraction by light-
density centrifugation. At this stage, MSCs are
isolated either by their ability to adhere to tis-
sue culture plastic, as described below, or are
directly purified using monoclonal antibodies
to CD105, CD146, or CD271 by immunomag-
netic or flow-cytometric sorting.43
Ectodermal lineage Endodermal lineage
Epithelial cells Muscle cells
Neurons Gut epithelial cells
Lung cells
 CH A P T E R 3 3 Blood/Marrow-Derived Non-HPC and Immune Cells
TABLE 33-2. Criteria for Identification of
MSCs
■ Adherence to tissue culture plastic
■ Phenotype (levels of expression)
Positive (95%
Negative (2%
+)
+)
■ CD73
■ CD45
■ CD90
■ CD34
■ CD105
■ CD14
■ CD11b
■ CD79
■ CD19
■ HLA-DR
■ Demonstration of in-vitro differentiation
into osteoblasts, adipocytes,
chondrocytes
MSCs = mesenchymal stem cells.
For isolation by plastic adherence,
BMMNCs are seeded onto tissue-culture
flasks at a density ranging from 0.5 × 105 to
5 × 105 BMMNCs per cm.2 The BMMNCs
are cultured in alpha-modified minimum
essential medi- um supplemented with 10%
fetal bovine se- rum (FBS). Alternative
MSC expansion proto- cols have also been
described using human platelet lysate
autologous serum as an alterna- tive source
of serum and growth-factor-sup- plemented,
serum-free media.35
Despite the prescreening of FBS for use
in clinical studies, its application in clinical-
grade cellular therapies still raises concerns
because, theoretically, it is a putative source of
prions and virus transmission. These concerns
make the future use of serum-free media in all
clinical products an attractive option.
The propagation of adherent cells is
maintained by the removal of nonadherent
cells on the third day and their subsequent
feeding every 3 to 5 days with fresh culture
me- dium. Cultures are reviewed daily for:
■ Adherence of cells to flasks.
■ Shape of adherent cells (round vs
fibrocytic appearance).
■ Confluence of cells.
■ Amount of debris in medium as an
indica- tion of medium change.
■ 793
Adherent cells consistently achieve 80%
confluence after 9 to 14 days, at which point
the cells are passaged following enzymatic
dis- sociation from tissue culture flasks with
tryp-
sin. Passaging of cell cultures once they
have achieved confluence is often a
prerequisite when an attempt is made to
propagate cells in sufficient quantity for
therapeutic use. Howev- er, continual
passage of MSCs can lead to rep- licative
exhaustion and alterations in pheno- type,
which may play a role in modifying their
regenerative and immune-suppressive prop-
erties or potentially limiting their survival
and function in vivo.44 The malignant
transforma- tion potential of MSCs can be
eliminated by reducing cell passaging,
although it has also been reported that
MSCs can be safely ex- panded in vitro until
the 25th passage.45 MSCs are harvested and
can be cryopreserved at the recommended
therapeutic dose or adminis- tered “fresh.”
Immunophenotyping by flow cytometry
is an essential part of the quality assurance/
quality control process of manufacturing us-
ing the cell-surface markers CD73, CD90,
and CD105. Differentiation of MSCs into a
specific lineage often requires the addition of
a com- plex array of growth factors that are
selected based on the desired mature cell
phenotype.
Standardization of MSC isolation and
ex- pansion is now gathering pace among
the cell therapy community as MSCs are
increasingly used in clinical trials and
manufactured under GMP conditions. The
technology for culturing large numbers of
expanded MSCs for clinical use has also
undergone significant develop- ment.
Devices, such as The Quantum Cell Ex-
pansion System (Terumo BCT, Lakewood,
CO), a closed automated hollow fiber
bioreactor culture system with disposable
sets, are now commercially available and
designed to repro- ducibly grow adherent
cells in GMP environ- ments while
minimizing both space and oper- ator
variability.
Clinical Applications of MSCs
The main indications for use of MSCs are in
the treatment of GVHD, Crohn disease,
cardio- vascular disorders, multiple
sclerosis, spinal
794 ■ AABB T EC HNIC AL MANUAL
cord injury, liver disease, diabetes mellitus,
and various bone and cartilage defects.
MSCs can be used without HLA matching
because they do not induce GVHD and are
not rejected by recipient T cells. These
characteristics favor banking of
cryopreserved MSCs from third- party
donors. Such banked MSCs can be re-
leased and shipped as required for multiple
clinical applications as “off-the-shelf” prod-
ucts.
One of the earliest studies that illustrated
the regenerative properties of MSCs involved
their use in treating three children with osteo-
genesis imperfecta. 46 This disease is caused by
a deficiency in the production of Type I colla-
gen and results in defective connective tissue
formation, leading to multiple fractures, bony
deformities, and shortened stature. Following
the infusion of unmanipulated allogeneic
marrow, MSCs from within the graft migrated
to the bone and gave rise to osteoblasts, where
their presence correlated with an improve-
ment in bone structure and function. The
same group of researchers has since modified
their treatment protocol and demonstrated its
safety and efficacy after administering two
doses of marrow-derived, ex-vivo-expanded
MSCs following standard marrow transplanta-
tion in patients with this disease.47
The therapeutic role of MSCs in HSCT
has been largely targeted toward alleviation of
GVHD following transplantation. However,
the codelivery of MSCs at the time of
transplanta- tion has received attention
because of their potential role in graft
tolerance and engraft- ment. Marrow-derived
MSCs have been used successfully for the
treatment of steroid-re- fractory, severe, acute
GVHD, for which there is currently no
established definitive therapy. The clinical
benefits of infusing MSCs to treat GVHD
were first demonstrated when haploi- dentical
MSCs were administered in two trans- fusions
to a 9-year-old boy with treatment-re- sistant,
Grade IV acute GVHD of the gut.48 This has
triggered great interest over the past de- cade,
and subsequent Phase I/II trials have
demonstrated both the safety and efficacy of
the use of MSCs in HSCT in the setting of ste-
roid-refractory GVHD.49
Optimal therapeutic doses have not
been defined, although clinical responses
have been documented with infusions of
MSCs as low as
0.8 × 105/kg. However, a Phase III, industry-led
clinical trial examining the transfusion of high
doses of MSCs (2 × 106/kg) for the treatment
of steroid-refractory GVHD showed no
increase in overall complete response rate.44
The clinical use of MSCs for tissue regen-
eration has received the most interest for
cardiovascular repair.50 Many trials have had
encouraging results in the improvement of
cardiac function after injection of autologous-
marrow-derived MSCs. Adipose-tissue-
derived MSCs are also currently being
investigated as part of two industry-led clinical
trials, APOLLO and PRECISE, exploring their
safety, feasibility, and efficacy in patients with
either acute or chronic myocardial infarction.51
Although the therapeutic effects of
MSCs in cardiac repair have been
acknowledged in the scientific community,
several issues re- main unresolved,
including the optimal dose and the
mechanism of improvement in cardi- ac
function. The differentiation of MSCs into
cardiomyocytes remains controversial, and
the literature in this field is still unclear and
contradictory. There have been a number of
recent publications providing evidence that
in vitro, MSCs may be induced to exhibit
cardio- myocyte-like features, but the exact
culture conditions are not clear. A number
of com- pounds are involved in their
generation through modulation of signaling
pathways. There has been no direct evidence
of cardio- myocyte differentiation in vivo
following ad- ministration of MSCs for
cardiac repair, but functional improvement
has been observed. These observations in
preclinical and human trials have led to the
hypothesis that the im- provements are
related to the paracrine ef- fects of
transplanted cells, which induce myo-
cardial repair by releasing signals, including
cytokines and chemokines, into the
surround- ing tissue rather than generation
of de-novo cardiomyocytes.50-52
The multipotency of MSCs has also be-
come attractive to industry, and two
commer- cially driven clinical trials involve
third-party MSCs manufactured by Osiris
Therapeutics
 CH A P T E R 3 3 Blood/Marrow-Derived Non-HPC and Immune Cells
Inc. (Columbia, MD) for use in patients with
GVHD or Crohn’s disease and mesenchymal
precursor cells manufactured by Mesoblast
Ltd. (New York, NY) for the treatment of car-
diovascular, orthopedic, and neurologic dis-
ease. Although the clinical efficacy of such
MSC products has yet to be fully evaluated in
many of the proposed disease indications,
their application as cellular therapies contin-
ues to evolve rapidly.
MSCs and Tissue Engineering
The use of MSCs in tissue engineering has
de- veloped rapidly over the last few years
with the advent of scaffolds to improve the
outcomes of stem cell applications by
offering more precise targeting of MSCs
combined with the possibil- ity of making
them biodegradable, depending on the
therapy. Scaffolds can be used in a number
of ways: 1) seeding of MSCs onto scaf-
folds in vitro and implantation after a short
in- cubation period, 2) MSC seeding and
subse- quent differentiation into lineage-
specific cell types in short-term culture (1-2
weeks) before implantation, and 3)
induction of differentia- tion in culture
before seeding onto scaffolds and
implantation shortly afterwards.
In 2008, the first fully tissue-engineered
bronchial transplant was reported. 53 In this
case, a nonimmunogenic, decellularized
piece of human donor trachea was reseeded
with autologous marrow-derived MSCs. The
MSCs were differentiated into chondrocytes
in vitro before surgical implantation to
replace a bron- chus that had been
previously damaged by tu- berculosis
infection. The paucity of donor or- gans has
limited the use of this approach. As a result,
the use of biosynthetic nanocomposite
material seeded with MSCs has emerged as
a therapeutic option for tracheal
replacement.54
INDUCED PLURIPOTENT STEM
CELLS
The archetypical pluripotent cell is the embry-
onic stem cell (ESC), which is derived from
embryos and is capable of long-term self-
renewal and differentiation into all cells and
tissues in the human body. The uniqueness of
■ 795
these capabilities was called into question
when Gurdon55 demonstrated that the nuclei
of differentiated cells actually retain all of
the genetic information in pluripotent stem
cells. The cloning of Dolly the sheep, for
example, showed that the genome of even
fully special- ized cells remains genetically
totipotent; that is, the genome can support
the development of an entire organism.
The next key finding was that
manipula- tion of transcription factor
expression can change a cell’s fate.56
Experiments revealed that lineage-associated
transcription factors can change cell fate
when these factors are ec- topically
expressed in certain heterologous cells.
Lineage-associated transcription factors help
establish and maintain cellular identity
during development by driving the
expression of cell-type-specific genes while
suppressing lineage-inappropriate genes.
The third contributory result was ob-
tained from the Nobel Prize-winning work
of Yamanaka,57 who screened a pool of 24
pluri- potency-associated candidate genes
for fac- tors. He determined that a core set of
four transcription factors comprising Klf4,
Sox2, c-Myc, and Oct4 was sufficient to
produce in- duced pluripotent stem cells
(iPSCs). This re- search was initially
conducted in murine mod- els, but the
approach was also successful with adult
human skin fibroblasts. Many other in-
vestigators since then have demonstrated
that this cocktail of genes can be used to
derive iPSCs from other somatic cell
populations, in- cluding keratinocytes,
neural cells, and stom- ach and liver cells.
Now that iPSCs can be generated, the
pri- mary question is whether iPSCs and
ESCs are molecularly and functionally
equivalent. Comparisons of their genome-
wide-expres- sion patterns and global
histone modifications have demonstrated a
high degree of similarity between ESCs and
iPSCs. However, unlike ESCs, iPSCs are
derived from somatic tissues and therefore
do not raise the ethical issues as- sociated
with ESCs.58 The ability to obtain starting
material from skin, blood, and umbili- cal
cord immediately opens up the horizon for
translational clinical applicability.59
796 ■ AABB T EC HNIC AL MANUAL
Tumorigenicity
The use of integrating viruses in the genera-
tion of iPSCs raises concern about the
poten- tial for oncogenesis and teratoma
formation. iPSC-derived cells do indeed
have a propensi- ty to form tumors after
transplantation, and human iPSC-derived
blood progenitor cells also appear to
undergo premature senes- cence.60 Their
translation to human applica- tion will need
to overcome this safety concern. Recent
approaches to generate iPSCs free of
potentially harmful mutagenic effects have
used either vectors that do not integrate into
the host cell genome or site-specific
integrat- ing vectors to direct the integration
away from known oncogenic sites. Finally,
these tech- niques have been refined so that
iPSC genera- tion no longer requires such
integrating vec- tors.60
Therapeutic Applications
The scope for iPSC-based clinical cell therapy
in the future is bewilderingly vast. The last
few years have seen continual refinements in
tech- niques to allow for large-scale GMP
manufac- turing. At the time of this writing, a
new trial has just received conditional
approval in Japan to use iPSCs to treat age-
related macular de- generation.
In-Vitro Generation of
Hematopoietic Cells
One of the most exciting prospects for using
iPSCs in cellular therapy is that of in-vitro
gen- eration of hematopoietic cells,
including erythrocytes. Current blood
transfusion prac- tice is very safe, but its
past has been fraught with infectious disease
transmission. Blood banks continue to
contend with issues of red cell compatibility
and the need to have suffi- cient donor pools
and adequate inventory sys- tems. These
issues can potentially be resolved with
large-scale manufacturing processes us- ing
iPSCs.61
Disease Modeling
One important application of iPSCs is the
modeling of human diseases by generating
specific tissue types of interest followed by the
induction of necessary pathologic changes.
This process essentially replicates the disease
in a Petri dish. However, this approach may be
limited to single-gene disorders, such as sickle
cell disease. For now, it remains unclear
whether multigenic diseases, such as diabetes
or Alzheimer’s disease, are equally amenable
to in-vitro modeling using iPSCs.62
Drug Testing
If disease modeling using iPSCs is
successful and reproducible, these ex-vivo-
derived pathologic tissues can be subjected
to phar- maceutical testing to identify
potential new drugs that are more potent and
targeted than existing drugs.62
In conclusion, iPSCs hold great promise
for regenerative medicine and pharmaceuti-
cal evaluation. They represent a potentially
vi- able source of non-HSCs to treat a wide
variety of diseases. However, the path to
final fruition of their promise is laden with
several challeng- ing hurdles, and work is
still necessary to over- come these
difficulties.
REGULATORY AND OVERSIGHT
ACTIVITIES
The cell therapy field has developed along the
same lines as blood banking. Both fields share
the strengths of multidisciplinary teams (phy-
sicians, nurses, laboratory officers, quality
managers, infectious disease experts, and im-
munologists). Both fields also share an em-
phasis on large-scale manufacture, automa-
tion, product release criteria, computer
systems for tracking and inventory, cold
chains for transportation, product traceability,
and monitoring of side effects.
Thus far, the majority of cell therapy
products have been manufactured from
sourc- es from a mixture of blood banks,
academic
 CH A P T E R 3 3 Blood/Marrow-Derived Non-HPC and Immune Cells
GMP facilities, and commercial companies.
Harmonization in the terminology of cell-
ther- apy products is being undertaken by
the Cellu- lar Therapy Coding and Labeling
Group, a technical subgroup of ICCBBA
that promotes the use of ISBT 128 (the
information technolo- gy standard for
transfusion medicine and transplantation)
labeling. This harmonization will, of course,
facilitate the transportation of such products
across national borders. In ad- dition, the
Alliance for Harmonization of Cell Therapy
Accreditation consists of representa- tives
from a variety of organizations involved in
HSCT and cell therapy. Its primary aim is to
create a single set of quality, safety, and
profes- sional requirements for cellular
therapy.
Cell therapy products need to be gov-
erned under suitable regulatory frameworks,
including national ones that cover cells, tis-
sues, and organs. Such national regulatory
frameworks for cell-therapy products are not
in existence worldwide but will be important
as the field develops and the need grows for
cross-border transportation of cells.
Cellular products can be classified as
ei- ther substantially or minimally
manipulated. Substantially manipulated cell-
therapy prod- ucts are treated as biologics.
In the United States, these are known as
“351” human cells, tissues, and cellular and
tissue-based products and are regulated by
the Center for Biologics Evaluation and
Research of the FDA. In Eu- rope,
substantially manipulated products are
known as advanced therapeutic medicinal
products (ATMPs). They are manufactured
in accordance with national legislation and
over- sight by the European Medicines
Agency.
KEY POINTS
Hematopoietic stem cell transplantation (HSCT) is well established for the treatment of he- matologic diseases. A graft-vs-
Recent technological advances allow good manufacturing practice (GMP)-compliant isola- tion, characterization, expansio
Donor lymphocyte infusion (DLI) has demonstrated considerable benefit as post-HSCT treatment in relapsed patients, part
■ 797
The question that arises next is how the
issuance and inventories of these biologics
will be managed in the local hospital
environment. The hospital pharmacy is the
natural domain for medicinal products
oversight. However, the unique qualities of
cell therapy products ren- der them more
suitable for the sphere of activ- ity and
concern of the blood bank and GMP
processing facility.
Most importantly, the maturation of the
cell-therapy field and its clinical applicability
in many fields of medicine will mean that
more patients will be treated with this novel
therapeutic option. Because living cells are of-
ten administered to patients, short-term as
well as longer-term side effects and potential
toxicities should be systematically monitored.
Such monitoring systems have been in place
for pharmaceuticals (pharmacovigilance pro-
grams) and blood (hemovigilance programs),
and these programs could be extended to cel-
lular therapy products (cellulo-vigilance pro-
grams).
CONCLUSION
The history of cell therapy has seen great ad-
vances of late with the entrance into the mar-
ket of the first FDA-approved therapeutic can-
cer vaccine, Provenge, as well as the dramatic
successes of antigen-specific T-cell therapy in
hematologic diseases. These successes will
certainly spur continuing interest in the devel-
opment of clinical trials. The attraction of an
“off-the shelf,” third-party product is that it
will increase the number of patients who can
be treated and heighten interest in the inter-
national transportation of such products.
798 ■ AABB T EC HNIC AL MANUAL

obtained from leukapheresis and constitute mainly T cells. A major side effect is graft-vs-
host disease (GVHD) as a consequence of a healthy host cell attack by alloreactive T cells.
4. NK cells are innate immune cells, do not require antigen priming, and do not induce
GVHD. Current promising developments to improve clinical outcome include treatment
with allo- reactive NK cells, mismatched for their inhibitory receptors, as well as in-
vivo or ex-vivo ac- tivation and expansion of NK cells.
5. Cytokine-induced killer cells (CIK) cells or NK-like T cells are expanded in long-term cul-
tures. Trials with CIK cell therapy have thus far demonstrated good tolerability with a low
risk of GVHD and some efficacy. They may be applied as an alternative to DLI.
6. Dendritic cells (DCs) play a key role in regulating the adaptive immune response to an
anti- gen-specific manner. A DC-based vaccine, sipuleucel-T (Provenge) is approved
by the US Food and Drug Administration as an autologous immunotherapy against
prostate cancer.
7. Mesenchymal stem cells (MSCs) possess potent differentiation properties
(multipotency) and have sparked considerable clinical interest. They are generally
isolated from marrow or adipose tissue and expanded in vitro. They can give rise to
structural components, such as bone, cartilage, tendon, and fat, and appear to exert
immunosuppressive functions. Indica- tions for applications of MSCs include GVHD,
Crohn disease, cardiovascular disorders, cord injury, liver disease, diabetes mellitus and
bone and cartilage defects.
8. Promising therapeutic effects of autologous MSCs in regenerative cardiac repair have been
reported although the potential for differentiation of MSCs into cardiomyocytes remains
controversial and more results are needed.
9. Third-party MSCs can be applied “off-the-shelf” without HLA matching and are not reject-
ed by recipient T cells.
10. The field of stem cell therapy has been transformed with the discovery that self-induced
pluripotent stem cells (iPSCs) can be generated from differentiated cells, including
kerati- nocytes, neural cells, and stomach and liver cells. The use of iPSCs involves
considerably fewer ethical issues compared to that of embryonic stem cells.
11. iPSCs can potentially be applied in a wide range of medical conditions and for in-vitro gen-
eration of hematopoietic cells. Techniques for large-scale GMP-compliant manufacturing
of iPSCs are being developed.
12. Cellular products are classified as either minimally or substantially manipulated, with a
more stringent regulatory framework for production of the latter in the US and Europe.

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 Index
Page numbers in italics
refer to figures or tables
A in transfusion reaction evaluation, 672
A antigen, 292-293, 295, 302, 457
A(B) phenotype, 296, 298
ABO compatibility
of apheresis platelets, 169
of Cryoprecipitated AHF, 375, 554, 583
of granulocytes, 173, 375, 525, 554,
584 in hemolytic transfusion reactions,
673 of HSC transplants, 632-633, 635,
716
of organ transplants, 491, 492, 783
of plasma, 375, 379, 554, 583, 635
of platelets, 375, 457, 513-514, 554
after HSCT transplantation, 635, 636
in hemolytic transfusion reactions,
675 in pediatric patients, 581, 589
of RBCs, 375, 378-379, 504, 554, 635
of tissue transplants, 777
of Whole Blood, 375
ABO discrepancies, 296, 298-301
ABO hemolytic disease of the fetus and
newborn, 566-567
ABO system, 291-301
acquired B phenotype, 296-297, 298
antibodies of
anti-A and anti-B, 293, 297
anti-A1, 297, 301
anti-A,B, 297
antigens of, 291, 292-293, 457
B(A) and A(B) phenotypes, 296, 298
biochemistry of, 292-293, 294, 302
in development and aging, 293
genetics of, 259, 266, 273, 293-295
O alleles, 295
phenotypes of, 292
subgroups of, 292-293, 295-296, 298, 300
ABO testing
of blood components, 225-226, 297-298, 378
with cold autoagglutinins, 300, 301,
438 comparison with previous records,
378 discrepancies in, 296, 298-301
hemolysis in, 297
interpretation of, 292, 296
for organ transplantation, 783
in pediatric patients, 385, 574, 587
in prenatal studies, 563
reagents for, 298
of recipients, 297-298, 375
of umbilical cord blood, 736
with warm autoagglutinins, 432
ABTI antigen, 361
Accidents, 45
Accreditation, 90, 91, 603-604, 698
ACE inhibitors. See Angiotensin-converting
enzyme inhibitors
Acid-elution stain (Kleihauer-Betke), 565-566
Acid elutions, 429
Acquired B phenotype, 296-297, 298
Activated partial thromboplastin time, 519
Activated protein C, 532
Acute disseminated encephalomyelitis, 653
Acute lung injury, 680. See also Transfusion-
related acute lung injury
Acute normovolemic hemodilution, 606
ADAMTS-13, 652-653
Additive solutions, 136-137, 138, 576-577
Additives, in serologic testing, 376-377
Adipose-derived stem cells, 755, 758-759, 762,
794
Adsorption, 407-408
allogeneic, 433-434
autologous cold, 438-439
autologous warm, 432-433
and elution, 409
Adverse reactions
to apheresis, 169, 170, 658-660, 720
in blood donors, 20, 22, 142-146
to donor lymphocyte infusions, 787
to G-CSF, 719-720
to HSC infusions, 724-725, 742
to IVIG, 529, 532
management of, 20-23
related to medical devices, 97
related to tissue allografts, 782
to transfusion (See Transfusion reactions)
transfusion-transmitted diseases (See
Transfusion-transmitted diseases)
AET. See 2-aminoethylisothiouronium
bromide
Age
of blood samples, 370-371, 410-411, 417
803
804 ■ AABB T EC HNIC AL MANUAL

of donors, 119, 120

to platelet antigens, 515-516, 567-568, 637,
effect on antigens and antibodies, 293,
689-690
300, 410
prevention of, 329-330, 515
of RBC units, 574, 577-
red cell, 391, 670
578 Agglutination
in sickle cell disease, 283, 329-330, 379, 588,
in antiglobulin testing, 372
639
assays utilizing, 241-242, 279-
1-antitrypsin, 532
280
1-proteinase inhibitor, 532
interpreting and grading reactions, 372
mixed-field, 278, 298 American Rare Donor Program, 421
principles of, 371 Aminocaproic acid, 608, 621-622
spontaneous 2-aminoethylisothiouronium bromide (AET),
in ABO testing, 298, 300, 301 301, 405, 407
dispersing with sulfhydryl reagents, 432, Amniotic fluid-derived MSCs, 759-760
438 Amotosalen-UV treated plasma, 153, 205
in Rh testing, 332 Anaphylactic reactions, 658, 668, 678-679, 680
Agreements, 6-7, 9-10, 108 Anemia
AHG serum, 372 acute, 500-502
AIDS, 124, 125, 182 and bleeding prevention, 508-509
Air embolism, 669, 685 chronic, 502, 610
Alarm systems, 36, 214 defined, 604
Albumin, bovine (reagent), 376, 417 in delayed transfusion reactions, 686
Albumin solutions (colloid), 526, 647, 652 fetal (See Hemolytic disease of the fetus and
Aliquoting components, 224, 575-576, 583 newborn)
Alleles, 262-264 hemolytic (See Hemolytic anemia)
defined, 256 in HSC transplantation, 634
frequencies of, 274, 275 iatrogenic, 609
polymorphic, 264 in infants, 571-572, 578-579
terminology for, 279, 280 iron deficiency, 605, 620
Allergic reactions management of, 605
to apheresis, 658 preoperative, 604-605
in HSCT patients, 639 RBC transfusions in, 500-502
to latex, 45 screening donors for, 121
to transfusions, 547-548, 667-668, 678-680 signs and symptoms of, 604
Alloantibodies, 391-392. See also Antibodies tolerance, 609-610
Allogeneic adsorption, 433-434 Anesthesia, 607
Allogeneic HSC transplantation, 715-718. Angioedema, 678
See also Hematopoietic stem cell Angiotensin-converting enzyme inhibitors,
transplantation 659, 669, 678, 679
Allografts, 774. See also Tissue Ankylosing spondylitis, 493-494
Alloimmunization. See also Antibodies Antibiotics, in donors, 122, 129
in delayed hemolytic transfusion Antibodies
reactions, 686 autoreactive (See Autoantibodies)
in hemolytic disease of the fetus and detection of (See Antibody detection)
newborn, 562 drug-induced, 392, 440-444, 465-466, 516
HLA to Factor VIII, 527
in HSC transplant recipients, 637, 639- granulocyte, 466-469
640, 716 HLA (See HLA antibodies)
management of, 670 identification of (See Antibody
in organ transplant recipients, 488-489 identification)
and plasma transfusions, 151 platelet (See Platelet antibodies)
in platelet refractoriness, 458, 459- to reagent components, 298, 300, 417
461, red cell (See Red cell antibodies)
488-489, 515-516 structure of, 246-248
in TRALI, 489, 681, 682 Antibody-dependent cellular cytotoxicity, 419
in transfusion reactions, 489, 490, 677

Antibody detection, 375-377
with autoantibodies, 432-435, 438-439
by ELISA, 243-244
frequency of testing, 419
of granulocyte antibodies, 469
of HLA antibodies, 460, 487
interpreting results of, 381, 382
methods for, 376-378
in pediatric patients, 385, 574-575, 587
of platelet antibodies, 243, 456, 463-464,
465-466
in prenatal evaluations, 563
reagents for, 376-377, 393, 396
with rouleaux present, 416
solid-phase assays for, 243
specimen requirements for, 370-371
Antibody identification
anomalous reactions in, 416-417
of antibodies to high-prevalence antigens,
394-395, 415-416
of antibodies to low-prevalence antigens,
416
with antibodies to reagent components,
417
with autoantibodies, 432-435
autologous control in, 400, 403-404, 417-
419
complex problems, 402, 403-404
exclusion (“rule-out”) in, 398
factors affecting, 410-411
frequency of testing, 419
immunohematology reference labs for,
419 interpreting results of, 397
with multiple antibodies, 408, 410, 412,
414 with no discernible specificity, 411-412
positive and negative reactions in, 397-398
with positive DAT, 401, 404, 415-416,
417-
419
preanalytical considerations for, 392-
393 in prenatal evaluations, 563
probability values in, 400
reagents for, 393, 396
and selection of blood, 274, 379, 419-421
specimen requirements for, 393
test methods for, 396
adsorption, 407-408
alteration in pH, 406
combined adsorption-elution, 409
dispersing autoagglutination, 438
elution, 408-409
enzymes, 402, 405
flowcharts for, 403-404
identification panels, 393, 396-400
inactivation of antigens, 405, 406-
407 increased incubation time, 405
INDEX ■ 805
increased serum-to-cell ratio, 405
inhibition techniques, 406
LISS and PEG, 402
phenotyping autologous red cells, 401-
402
selected cells, 397, 398
temperature reduction, 405
titration studies, 409-410, 563
variations in antigen expression in, 410,
411-412
Antibody screen. See Antibody detection
Antibody specificity prediction method,
460 Anticoagulant medications
in blood donors, 129
reversal of, 517-518, 519, 622-623
Anticoagulant-preservative solutions, 136, 137
Anticoagulation, in apheresis, 658
Antifibrinolytic agents, 522, 608, 621-622
Antigen capture assays, 464-465
Antigen-matching, 281, 329-330, 379, 588, 654
Antigens. See also specific blood groups
acquired, 296-297, 298
antithetical, 263-264
of blood group collections, 361-
362 of blood group systems, 278-
279 chromosomal location of,
259-261 granulocyte, 466-468
high-prevalence, 361-362, 415-416, 421
HLA, 475-476, 480-483
inactivation of, 405, 406-407
low-prevalence, 362, 416
methods for detecting, 241-243, 244-246,
279-285
platelet, 243, 453-458
prevalence of, 274
terminology for, 278-279, 280
variations in expression of, 410
Antiglobulin test, 371-372
in crossmatching, 380
direct, 372, 425-430
indirect, 372
reagents for, 372, 396
sources of error in, 373-374
use of IgG-coated cells, 376
Antihistamines, 548, 679-680
Antiplatelet agents, 122, 129, 169, 509
Antipyretics, 547
Antithrombin, 531
Apheresis
complications of, 169, 658-660, 720
component collection by, 167-176
adverse donor reactions in, 169, 170
donation requirements for, 121, 122,
123, 168-169, 171
granulocytes, 168, 172-173, 175-176
806 ■ AABB T EC HNIC AL MANUAL
instrumentation for, 168, 173-176
multicomponent donations, 171-172, 174
plasma, 168, 170, 173-174
platelets, 168-170, 174-175
quality control of, 171
RBCs, 168, 171-172, 175
HSCs collected by (See Peripheral blood
HSCs)
records of, 170, 172
therapeutic (See Therapeutic apheresis)
Aplastic anemia, 640
Arterial puncture, in donors, 145
Arthritis, 763
Aspirin, 122, 169,
509 Assessments
of blood utilization, 24-25, 611, 697-707
competency, 7-8
external, 25, 86-87
internal, 24
management of, 13, 23-26
proficiency testing, 25-26, 91
quality indicators, 24
risk, 98-99, 102
Audits, transfusion, 25
concurrent, 699, 700, 703, 706
prospective, 611, 699-700, 701-702, 706
retrospective, 699, 701-702, 703-704, 707
selecting a process for, 705
Auto control. See Autologous control
Autoadsorption. See Adsorption
Autoagglutination
in ABO typing, 298, 299, 300, 301
dispersing with sulfhydryl reagents, 432,
438
in Rh testing, 332
Autoantibodies
cold
in ABO testing, 300, 301, 438
adsorption of, 438-439
antibody identification with, 418
in cold agglutinin disease, 308-309,
437-
439
in mixed AIHA, 439
in paroxysmal cold hemoglobinuria,
440 in Rh testing, 332, 438
use of sulfhydryl reagents with,
438 in warm autoimmune
hemolytic
anemia, 431
defined, 391
disease associations with, 392
distinguishing from alloantibodies, 283
drug-induced, 443
low-affinity, 437
in phenotyping problems, 401
platelet, 461, 465, 568
warm
ABO testing with, 432
adsorption of, 432-435
with alloantibodies, 418-419, 432-435,
438-439
antibody identification with, 418-419
disease associations with, 392
mimicking alloantibodies, 434-435
in mixed-type AIHA, 439
in phenotyping problems,
401 Rh testing with, 332,
432
specificity of, 435
transfusion with, 436-437, 506
in warm AIHA, 431-435
Autoclaving biohazardous waste, 55
Autografts, 774, 782-783. See also
Tissue Autoimmune hemolytic anemia
classification of, 430
cold agglutinin disease, 437-439
DAT-negative, 437
mixed-type, 439-440
paroxysmal cold hemoglobinuria,
440 serologic findings in, 431
transfusion in, 436-437, 439-440, 506
warm, 430-437
Autoimmune neutropenia, 468
Autoimmune thrombocytopenic purpura, 463,
465, 517, 568
Autologous adsorption, 432-433, 438-439
Autologous blood
collection of
by acute normovolemic
hemodilution, 606
by intraoperative blood recovery,
606- 607
low-volume units, 141
by preoperative blood donation, 605-
606 donor selection for, 120, 131
infectious disease testing of, 190-
191 for rare phenotypes, 421
separation from transfused cells, 401
Autologous control
in antibody identification, 400, 403-404
positive, 404, 417-419
in pretransfusion testing, 377, 382
Autologous HSC transplantation, 638, 714-
715, 718-719. See also Hematopoietic stem
cell transplantation
Automated testing systems, 378
Autosomal inheritance, 265-267
B
B antigen, 292-293, 302
acquired, 296-297, 298
on platelets, 457

B(A) phenotype, 296, 298
Babesiosis, 126, 201
Bacterial contamination, 198-199
detecting, 198-199
inspecting components for, 149, 224
during phlebotomy, 141, 198
of platelets, 150, 198-199, 221
of RBCs, 198
reactive testing results for, 188
of tissue allografts, 777, 782
in transfusion-associated sepsis, 669,
676
Band 3 glycoprotein, 352-353
Bernard Soulier syndrome, 456
Bg (Bennett-Goodspeed) antigens, 362, 480,
490
Bilirubin, 562, 564, 579
Bioassays, for radiation monitoring,
61 Biocontamination control system,
41 Biohazardous waste, 53-55, 108
Biological product deviations, 22, 89, 90
Biological products, regulations for, 84, 85
Biological response modifiers, 681
Biological safety cabinets, 49, 50-51
Biologics license applications, 735, 744, 745,
746
Biosafety, 47-55
biological safety cabinets for, 49, 50-51
biosafety levels, 48, 73
Blood-borne Pathogens Standard, 47
decontamination for, 49, 52
in donor room, 53
emergency response plan for, 53
engineering controls for, 49-52
hazard identification and communication
for, 48-49
in laboratory, 53
personal protective equipment for, 52
safe work practices for, 52-53
standard precautions, 48
storage of biohazards, 52, 54-55
training for, 48
waste management, 53-55
Biovigilance. See Hemovigilance
Bleeding
acute, transfusion for, 501-502
and anemia, 508-509
assessing risk of, 518-521, 605
microvascular, 684
Bleeding disorders. See Coagulopathy
Blood administration
baseline assessment of recipient, 546
blood warmers for, 548, 572, 584, 685-
686 delays in starting transfusion, 550
delivering blood to patient area, 549-550
INDEX ■ 807
emergency equipment for, 549
emergency release, 158, 227-228, 385-
386,
506, 555-556
errors in, 551, 675
filters for, 551-552, 554, 585
identification procedures in, 550-551
infusion pumps for, 548-549, 584
infusion rates for, 553, 554, 585
infusion sets for, 551-552, 585
IV solutions for, 552
medical history in, 546
medical order for, 546-547, 551, 699-700,
703-704
monitoring patient in, 553, 555
in neonates, 584-585
out-of-hospital, 556
patient consent in, 545-546,
601 patient education in, 546
post-administration events, 555
premedications for, 547-548, 677, 679-680
pressure devices for, 549
starting the transfusion, 552-
553 for surgery and trauma,
555-556 syringe infusion
pumps for, 549
transfusion reactions in, 553, 555, 666
venous access for, 547, 580, 584
Blood-borne Pathogens Standard, 47, 53
Blood clots, in RBCs, 149
Blood collection
additive solutions for, 136-137, 138, 576-
577
adverse donor reactions in, 20, 22, 142-
146
anticoagulant-preservative solutions for,
136, 137
autologous, 605-606
blood containers for, 136-139, 140
of blood samples, 141, 368, 370, 384
of components by apheresis, 167-176
in disaster planning, 100-102
donation intervals for, 120, 122
donor and component identification in,
139-140
donor care after, 142
equipment quality control, 38
mobile sites for, 41
phlebotomy in, 140-141
process of, 141
safety of, 41, 53
of umbilical cord blood, 733-734
volume collected, 120, 141-142, 143
Blood component selection
ABO/Rh compatibility in, 375, 378-379, 504,
513-515, 554, 635
after non-group-specific transfusions, 396
808 ■ AABB T EC HNIC AL MANUAL

with clinically significant antibodies, 274,

phenotyping, 281, 329-330, 379, 420,
379, 419-421
588, 654
for exchange transfusions, 574, 579-
traceability of, 225
580 for HSC transplantation, 633-634
transporting, 100-101, 146-147, 213-214,
for intrauterine transfusions, 564
215-220, 225
for massive transfusions, 386
volume reduction of, 157, 223, 513-514,
of platelets, 375, 459-461, 513-514, 581-
581-582, 590
582
washed, 223, 590-591, 639
rare types, 421
Blood containers
of red cell components, 375, 378-379,
additive solutions in, 136-137, 138
504 for red cell exchange, 654
in urgent situations, 228, 385-386 for aliquots, 576
anticoagulant-preservative solutions in,
in warm autoimmune hemolytic anemia,
136, 137
435-436
configurations of, 136-137, 139
Blood components. See also specific
diversion pouch on, 139, 141
components
modification by sterile connecting
administration of (See Blood
device,
administration)
139, 140
aliquoting, 224, 575-576, 583
bacterial contamination of, 198-199, 224 properties of, 136
CMV-reduced-risk, 190, 525, 564, 639 Blood donation. See Blood collection; Donors
costs of, 602 Blood exposure, 44-45, 47-48, 49, 53, 124
cryopreservation of, 155 Blood group genomics, 278-287
density of, 143 for antigen-negative blood donors, 285
expiration of, 213, 215-220, 551 to confirm D type of donors, 285
discrepancies between phenotype and
identification of, 139-140, 226, 384-385,
genotype, 240, 285-287, 402
549, 550, 551
inspection of, 148, 149, 224, 226, 385, 549, to distinguish alloantibody from
autoantibody, 283
550-551
to predict phenotype of recently transfused
irradiated, 155-156, 222, 590, 688, 689
patients, 240, 281
issuing, 226, 384-385, 549-550
labeling, 139, 158-159, 226, 384-385 to predict phenotype when red cells are
leukocyte reduction of, 154-155, 222-223, coated with IgG, 283, 401-402, 436
in prenatal practice, 283-285
504-505, 590
Blood group systems. See also specific blood
monitoring use of, 697-707
groups
ordering, 226, 367-368, 546, 699-700, 703-
clinical significance of antibodies in, 338-
704
340, 413-414
pooling, 156-157, 223-224
defined, 279
preparation and processing of, 146-148,
genetics of, 255-279
221-224
chimerism, 278
quality control of, 159-160
gene mapping, 259-261, 277-278
quarantine of, 157-158, 189, 224
inheritance patterns, 265-273
rare, 421
population genetics, 274-275
receiving into inventory, 225
principles of, 256-258, 261-264
records of, 384-385
relationship testing, 276-277
regulations regarding, 83
terminology for, 278-279, 280
retrieval of prior donations, 187-188,
Blood loss
189-
phlebotomy-related, 609
190
signs and symptoms of, 501
return and reissue of, 226-227, 550
transfusion for, 501-502
selection of (See Blood component
Blood management. See Patient
selection)
blood management
shortages of, 101-
Blood order schedules,
102
storage of, 213-214, 215-220, 221, 503-504 227 Blood pressure
of donors, 120
testing
hypotension
ABO and Rh, 225-226, 297-298, 378
infectious disease, 182-191, 192-204
 INDEX ■ 809

during apheresis, 659 donor lymphocyte infusions for, 786-

associated with ACE inhibitors, 659, 787, 788
669, natural killer cells in, 788
678 platelet transfusions in, 507
deliberate, 607 prostate, 791
in recipients, 674, 679 Carboprost, 626
Blood pressure cuffs, 38 Cardiac patients
Blood recovery, 606-607, 608-609 platelet transfusions in, 509
Blood samples red cell transfusions in, 500-
age of, 370-371, 410-411, 417 501 treatment with MSCs,
from arterial or central lines, 609 763, 794
collection of, 141, 368, 370, 384 Carriers, of traits,
confirming linkage with blood requests, 261 Cash reserves,
370 104 Catheters
for hemoglobin/hematocrit screening, 121 for apheresis, 660
hemolyzed, 370 drawing specimens from, 609
incompletely clotted, 370 for neonates, 580, 584
labeling, 370, 547 for transfusions, 547
lipemic, 370 Cause-and-effect diagrams, 27, 28
for PCR, 235, 236 CCI. See Corrected platelet count increment
for pretransfusion testing, 368, 370-371, CD11a/CD11b, 468
393, 547 CD34 cells, 723, 736
retention and storage of, 371 CD36, 458
transportation and shipment of, 63 CD59, 361
Blood spills, 53, 54 CD109, 456-457
Blood transfusions. See Transfusions CD177, 466-467, 469
Blood utilization review, 25, 697-707 Cefotetan, 442
interventions to change transfusion Ceftriaxone, 442
practice, 610-611, 699, 704-705 Cell counters, 38
rationale for, 698-699 Cell counts, of HSCs, 723, 736
selecting audit process, 705 Cell division, 258, 262, 263
types of audits Cell washers, 36
concurrent, 699, 700, 703, 706 Cellular immunotherapy
prospective, 611, 699-700, 701-702, 706 with cytokine-induced killer cells, 789-790
retrospective, 699, 701-702, 703-704, with dendritic cells, 790-791
707 donor lymphocyte infusions, 786-788
Blood volume, of pediatric patients, 572 with induced pluripotent stem cells, 795-
Blood warmers, 37, 548, 572, 584, 685- 796 with natural killer cells, 788-789
686 regulation and oversight of, 796-797
Bombay phenotype, 292, 293, 303 Centrifugation, in component preparation,
Bone grafts, 123, 775 147-148
Bone marrow. See Marrow Centrifuges, 36, 38
Bovine serum, 764-765, 793 Centromere, 257
Brain natriuretic peptide, 682 Cephalosporins, 442, 443
Bruising, in donors, 145 Ceppellini effect, 321, 323
Buerger disease, 763 Cerebral diseases, 763
Buffy-coat concentration of marrow, 721 cGMP. See Good manufacturing
Buffy-coat method of platelet preparation, practice cGTP. See Good tissue
146, 147, 150, 156-157 practice
Chagas’ disease, 126, 200-201
C Change control, 33
C1-esterase inhibitor deficiency, 522 Charts
Calcium supplementation, 658, 670, 683 Pareto, 27, 29
Calibration, equipment, 10, 11, pedigree, 265, 266
33 Cancer process flow, 27
in blood donors, 126, run and control, 24, 33
128 leukemia Check cells, 376
cytapheresis in, 654
810 ■ AABB T EC HNIC AL MANUAL

Chemical hygiene plan, 55

in blood donors, 126, 128-129
Chemical/organic solvent elutions, 429
in hemolytic transfusion reactions, 674
Chemical safety, 55-60
in massive transfusion, 591, 684-685
chemical categories, 57, 76-77 in neonates, 582-583
chemicals found in blood banks, 74- plasma transfusions for, 517-
75 emergency response plan for, 59, 522 Codominant inheritance, 265
78-82 engineering controls for, 58 Cold agglutinin disease, 437-439
hazard identification and communication, antibody detection in, 438-439
56-58 serologic findings in, 431, 437-438
personal protective equipment for, 58 specificity of autoantibodies in, 308-309,
safe work practices for, 58-59 392, 439
training for, 56 Cold autoadsorption, 438-439
waste disposal, 60
Cold autoantibodies
Chemiluminescence assays, 419 ABO typing with, 300, 301, 438
Chemotherapy, 720 adsorption of, 438-439
Chido/Rodgers system, 260, 338, 356-357, antibody identification with, 418
406
in cold agglutinin syndrome, 308-309, 437-
Chido substance, 406
439
Chikungunya virus, 202
in mixed AIHA, 439
Children. See Neonates; Pediatric patients
in paroxysmal cold hemoglobinuria,
Chimeric antigen receptors, 788
440 phenotyping with, 401
Chimerism, 278, 298, 489-490
in Rh testing, 332, 438
Chlamydia trachomatis, 191
use of sulfhydryl reagents with, 438
Chloroquine diphosphate, 407
in warm autoimmune hemolytic anemia,
Choline transporter-like protein 2, 467-468
431
Chromatids, 257, 258
Cold-reactive alloantibodies, 300, 301
Chromosomes, 256-258, 259-261, 277-278.
Cold stress. See Hypothermia
See also Genes
Collections, blood group, 361-362
Chronic granulomatous disease, 348-349,
Colloid solutions, 526
524,
Colony-forming cell assays, 723, 736
525
Colton system, 260, 339, 355
Circular of Information for the Use of Human
Column agglutination technology, 377, 396
Blood and Blood Components, 158-159
Committee, for transfusion oversight, 24-
Circulatory overload, transfusion-associated,
25 Communications, emergency plans for,
669, 682-683
105-
Cirrhosis, 518-519, 764
107
Cis position, 272, 329
Compatibility testing. See Pretransfusion
Citrate toxicity, 658, 670, 683
testing
CJD. See Creutzfeldt-Jakob disease
Competency assessments, 7-8
Clean rooms, 40-41
Complement
Cleaning and decontamination, 49, 52
in acute transfusion reactions, 673-674
Clinical Laboratory Improvement
in autoimmune hemolytic anemia, 431,
Amendments (CLIA), 89-90
437
Clocks, 37
role in immunity, 248-250
Clonogenic assays, 723, 736
Complications. See Adverse reactions
Clotting factors. See Coagulation factors
Complotype, 478
CMV. See Cytomegalovirus
Computer crossmatch, 380-381
Coagulation factors
Computer systems
concentrates, 124, 126, 526-527, 637-638
alternative systems for, 20
in Cryoprecipitated AHF, 153-154, 522-
backup of, 18, 19-20
523
management of, 19-20
half-lives of, 521
security of, 19
and INR, 520
validation of, 15-16
in neonates, 582
Computerized provider order entry, 611, 699,
replacement of, 524
700
in Thawed Plasma, 221
Concurrent audits, 699, 700, 703, 706
Coagulopathy
in apheresis, 659
 INDEX ■ 811

Confidentiality of information, 19 Cordocentesis, 563-564, 567, 568

Consanguineous mating, 266 Corrected platelet count increment, 458, 512,
Consent 516
for apheresis, 170 Corrective action, 26, 27
for blood donation, 119 Corticosteroids, 172, 548
for cord blood donation, Cost collection, 361
731 for donation of HSCs, Costs, health-care, 602
718 for transfusion, 545- Counter-flow centrifugal elutriation, 721-722
546, 601 CPOE. See Computerized provider order entry
Contact lists, in disaster planning, 105 Creutzfeldt-Jakob disease, 202-203
Containers and plasma derivatives, 526
for blood collection, 136-139, 140 screening donors for, 125, 126
for MSC storage, 761 transmission through transplantation, 777
for shipping variant, 125, 202-203
dry shippers, 724, 738, 739 Crohn’s disease, 764
HSCs, 724-725, 738 Cromer system, 260, 339, 357-358
quality control intervals for, 38 Cross-reactive groups, 482
temperature of, 724-725, 734, 738, 739, Crossing-over, gene, 270-271, 479
779-780 Crossmatch-to-transfusion ratios, 227, 381
validation of, 147, Crossmatching, 379-381
225 Contamination with alloantibodies, 420-421
bacterial, 198-199 antiglobulin test in, 380
detecting, 198-199 computer, 380-381
inspecting components for, 149, 224 HLA (lymphocyte), 487, 491-492
during phlebotomy, 141, 198 immediate-spin, 380
of platelets, 150, 198-199, 221 “in-vivo,” 419, 506-507
of RBCs, 198 interpretation of results, 381, 382
reactive testing results for, 188 in pediatric recipients, 385, 574-575
of tissue allografts, 777, 782 platelet components, 460-461, 489, 516
in transfusion-associated sepsis, 669, Cryoprecipitate Reduced Plasma, 152, 219-
676 220, 652
fungal, 777, 782 Cryoprecipitated AHF, 153-154
in PCR, 236 ABO compatibility of, 375, 554, 583
Continuity of Operations Plan, 102-109 coagulation factors in, 153-154, 522-523
alternate facilities in, 103-104 dose of, 523
cash reserves in, expiration of, 157, 218, 222
104 decision trees in HSC transplantation, 637
in, 103 elements of, indications for, 522-523
103 infusion of, 554
emergency communications plan in, for pediatric patients, 578, 583-584
105- 107 pooled, 157, 218, 222, 224, 523
essential functions in, 103 preparation of, 153
insurance in, 104 storage of, 153, 218, 222
logistics in, 107-108 thawing, 222
security and safety in, 104 topical application of, 523
staffing in, 108-109 transportation and shipping of, 218
utilities in, 108 Cryopreservation
vital records in, 104 agents for, 155, 722
Contracts, 9-10, 108 of HSCs, 722-723
Control charts, 24, 33 of platelets, 155
Control process, 3 of RBCs, 155
Controls of tissue allografts, 775
autologous, 377, 382, 400, 403-404, 417- Crystalloids, 501-502, 526, 652
419 CTL2, 467-468
IgG-coated cells, 376 Cytapheresis, 653-654, 655
for Rh typing reagents, 331-332
Copper sulfate, 38, 64, 121
Cord blood. See Umbilical cord blood
Cord blood banks, 729-730, 742-746
812 ■ AABB T EC HNIC AL MANUAL

Cytokine-induced killer cells, 789-790

Dengue virus, 202
Cytomegalovirus, 190
Density, of blood cells and components, 143
and CTL infusions, 788
Derivatives. See Plasma derivatives
in neonates, 589-590
Design output, 3, 33
preventing with leukocyte reduction, 190, Designated donations, 130
590, 639 Desmopressin, 624
screening donors for, 191
Dexamethasone, 525
Cytomegalovirus-reduced-risk products, 190,
DIC. See Disseminated
525, 564, 590, 639
intravascular coagulation
Cytotoxic T lymphocytes, 787-788
Diego system, 259, 338, 352-354
D Dimethyl sulfoxide (DMSO)
for cryopreservation of products, 155, 722,
D antigen, 323-328 761
antibody to (anti-D) functional effect on HSCs, 741
clinical significance of, 338 toxicity to, 724, 764-765
in HDFN, 338, 562 2,3-Diphosphoglycerate (2,3-DPG), 574
and partial D, 328 Diploid, defined, 258
passively acquired, 566 Direct antiglobulin test, 425-430
and platelet transfusions, false positive/negative results in, 373-374
515 in tissue transplantation, method for, 426-427
777 titrations of, 563 positive test
and weak D, 328 after HSC transplantation, 428
clinical considerations for, in antibody identification, 401, 404, 415-
328 D epitopes on RHCE, 416, 417-419
325, 326 causes of, 426
D-negative, 321, 327
in cold agglutinin syndrome, 431
D-positive, 323-325, 327
drug-induced, 428, 440-444, 448-451
elevated D, 325, 327
elution with, 428-430
in fetus, 283-284
evaluation of, 427-430
nonfunctional RHD, 325
medical history in, 427-428
partial D, 324, 326, 327, 328, 333
in mixed-type AIHA, 431,
in recipients, 327-328,
439
375 testing for (See Rh
in paroxysmal cold hemoglobinuria,
testing) weak D, 323-324
431, 440
in donors, 285, 327, 328
predicting phenotype with, 283
in fetus, 565
in warm autoimmune hemolytic
in prenatal patients, 565
anemia, 431
in recipients, 327-328,
in pretransfusion testing, 372, 377
375
principles of, 426-427
DARC glycoprotein, 349-351
reagents for, 372, 426-427
DAT. See Direct antiglobulin
specimens for, 427
test
in transfusion reaction evaluation, 426,
DAT-negative autoimmune hemolytic
672 Direct thrombin inhibitors, 518
anemia, 437
Directed donations, 131
DDAVP. See Desmopressin
Disaster management
Decontamination procedures, 49, 52,
for blood and transfusion services, 100-
55
102 business operations planning in, 102-
Deep vein thrombosis, in donors, 145
109 cycle of, 98
Deferasirox, 690
emergency management agencies in, 99-100
Deferiprone, 690
lessons learned from recent disasters, 112
Deferoxamine, 690
overview of, 97
Deferrals. See Donor deferrals
planning team in, 102
Deglycerolization, 155, 222
regulatory issues in, 109-111
Delayed transfusion reactions
risk assessment in, 98-99
hemolytic, 250, 588, 670, 686-687
testing disaster plans, 111-112
management of, 670-671
Disease modeling, 796
serologic, 686, 687
Dendritic cells, 785, 790-791

Disinfectants
to clean work surfaces, 49, 52
for venipuncture, 140-141, 198
Disposal, waste, 54-55, 60, 63, 108
Disseminated intravascular coagulation, 674-
675
Dithiothreitol (DTT)
to disperse autoagglutination, 301, 432,
438 for inactivating blood group antigens,
405,
407, 413-414
Diversion pouches, 139, 141, 198
DMSO. See Dimethyl sulfoxide
DNA, 232-233, 237
DNA-based assays. See Nucleic acid analysis
Documents, 13, 16-18. See also Records
Dolichos biflorus lectin, 295, 301
Dombrock system, 260, 339, 354-355
Donath-Landsteiner test, 312, 440
Donation identification number, 135, 139-
140,
159, 551
Donor deferrals
for allogeneic donors, 122-126
for bleeding conditions or blood diseases,
126, 128-129
blood donation intervals for, 122, 123, 169,
170 for blood exposure, 124
for blood transfusions, 123, 125
for bone, skin or dura mater grafts, 123, 126
for cancer, 126, 128
for clotting factor concentrates, 126
developing criteria for, 127-130
during disasters, 101, 110
for drugs taken by donor, 122, 125, 129-
130,
169
for heart and lung conditions, 126, 129
for hemoglobin/hematocrit, 121
for incarceration, 125
for infectious diseases, 124-126
for needlesticks, 124
for piercings, 124
for pregnancy, 122
for reactive infectious screening tests, 187-
188, 189
records of, 118, 119
regarding sexual contacts, 124, 125
for tattoos, 124
for transplants, 123
for travel outside the US or Canada,
125, 126
for vaccinations or shots, 123
Donor History Questionnaire, 121-127,
130 Donor lymphocyte infusion, 786-788
Donor room, biosafety in, 53
Donor selection
for autologous donations, 118, 131
INDEX ■ 813
blood-center-defined criteria for, 127-
130 consent of donor in, 119-120
for directed donations, 131
Donor History Questionnaire in, 121-
127 education of donor in, 119-120
for exceptional medical need,
130 for frequent donors, 130
hemoglobin or hematocrit in, 120,
121 for HSC transplantation, 714-
717 identification of donor, 118-119
overview of, 117-118
physical examination in, 120
registration process in, 118-
119
Donor-specific antibodies, 716
Donor testing
ABO, 297-298
for blood group antigens, 285, 379,
420 for cord blood donation, 732-733
for HSC transplantation, 714-716
for infectious diseases, 182-191, 192-204
Rh, 285, 327, 328
silenced or nonexpressed genes in, 286
weak D, 327
Donors
adverse reactions in, 20, 22, 142-146
age of, 119, 120
of apheresis RBCs, 123, 171
autologous, 131, 190-191, 605-606
with bleeding conditions or blood diseases,
126, 128-129
with cancer, 126, 128
consent of, 119, 170, 718, 731
deferral of (See Donor
deferrals) designated or
directed, 130-131
donation intervals for, 120, 122, 123, 169,
170
educational materials for, 119-120, 122
effect of disasters on, 101,
110 family members as, 421
frequent or repeat, 130
general health of, 122
hearing- or vision-impaired, 120
with heart and lung conditions, 126, 129
hemoglobin/hematocrit in, 120, 121
for HLA-matched platelets, 488-489
for HSC transplantation, 714-717
identification of, 118-119, 139
legally incompetent, 119
leukapheresis, 122
medications taken by, 122, 129-130, 169
non-English-speaking, 120
notification of abnormal test results, 189
phlebotomy of, 140-146
physical examination of, 120
plasmapheresis, 122, 170
814 ■ AABB T EC HNIC AL MANUAL
plateletpheresis, 122, 168-169
postphlebotomy care of, 142
pregnancy in, 122
qualification requirements for, 121-127
with reactive screening tests, 186-190
records of, 119
registration of, 118-119
sexual contacts of, 124, 125, 126
temperature of, 120
of tissue allografts, 773-774
and TRALI, 682
of umbilical cord blood, 730-733
Dosage effect, 264, 393, 410
Dosimeters, 61, 156
2,3-DPG, 574
Drug-induced immune hemolytic anemia,
440-444
antibodies in, 441-443
classification of, 441-443
drugs associated with, 448-451
laboratory investigation of, 443-444
mechanisms of, 441
Drug-induced thrombocytopenia, 462, 465-
466
Drug testing, with induced pluripotent stem
cells, 796
Drugs
administered for leukapheresis, 172
antiplatelet agents, 122, 129, 169,
509
causing positive DAT, 428, 440-444, 448-
451
hemostatic agents, 608, 621-622
intravenous use of, 125
platelet antibodies induced by, 465-466,
516
pretransfusion medications, 547-548,
677,
679-680
removal of, in apheresis, 659-660
taken by donors, 122, 129-130,
169
Dry ice, 225
Dry shippers, 724, 738, 739
DTT. See Dithiothreitol
Duffy system, 349-351
antibodies of, 338, 350
antigens, 259, 349-350
Duffy glycoprotein, 350-351
and malaria, 351
phenotypes and genotypes, 349
silenced alleles in, 287
Dura mater transplants, 126
Dysfibrinogenemias, 523
E performance intervals for, 36-
ECMO. See Extracorporeal membrane
oxygenation
Education
for donors, 119-120, 122
for patients, 546
for physicians, 611-612
for umbilical cord blood donation, 730-731
Electrical safety, 46-47
ELISA. See Enzyme-linked immunosorbent assay
Elutions, 408-409, 428-430
Elutriation, 721-722
Embolism, air, 669, 685
Emergency Communications Plan, 105-107
Emergency equipment, 549
Emergency management. See Disaster
management
Emergency Management Agencies, 99, 100,
106-107, 111-112
Emergency release of blood, 158, 227-228, 385-
386, 506, 555-556
Emergency response plans, 44
for biohazards, 53
for chemical spills, 59, 78-82
for electrical emergency, 47
for fires, 46
for radiation safety, 62
Emergency showers, 58
Employees. See Personnel
End-product test and inspection, 33
Endothelial barrier dysfunction, 763-
764 Engineering controls
for biosafety, 49-52
for chemical safety,
58 for electrical
safety, 47 for fire
prevention, 46
general guidelines for, 44, 72
for radiation safety, 62
Engraftment, in HSC transplantation, 717-
718 Enhancement media, 376-377, 396, 417
Enzyme-linked immunosorbent assay, 243-
245, 464, 465-466
Enzymes, 377, 402, 405, 413-414
Epinephrine, 679
EPO. See Erythropoietic stimulating agents
Epsilon-aminocaproic acid (EACA), 608, 621-
622
Equipment
apheresis, 168, 173-176, 645-646
critical, 10-11
decontamination of, 49, 52
emergency, 549
management of, 10-11, 13
manufacturers directions for, 11
personal protective, 44, 52-53, 70-71
quality control of, 159
alarm systems, 36
38 thermometers, 37

regulations for, 83-84, 87
for transfusions, 548-549
validation of, 15
Er antigens, 361
Ergonomics, 41-42
Errors
in ABO and Rh typing, 300,
332 fatalities due to, 551
identification, 551, 675
mislabeled specimens, 370
quarantine release, 192, 195-196
sources of, in antiglobulin test, 373-
374 wrong blood in tube, 378, 384
Erythroblastosis fetalis, 561
Erythrocytapheresis, 646, 655
Erythroid phenotypes, 345, 362-363
Erythropoiesis, in neonates, 572
Erythropoietic stimulating agents, 572, 605,
620-621, 634
Estrogen, conjugated, 625
Ethnic groups, differences in
in antibody identification, 392-393, 394-
395, 415
in Duffy system, 349
in Kell system, 347
in Kidd system, 351-352
in MNS system, 341
in Rh system, 318-319, 320, 321, 323,
324,
325, 327
Exchange transfusions
blood warmer for, 572
component choice for, 574, 579-580
for hemolytic disease of the fetus and
newborn, 564
for hyperbilirubinemia, 579
techniques for, 580
vascular access for,
580 volume of, 580
Executive management, 5
Expiration, of components, 215-220, 551
Exposure control plan, 47, 48
Extracorporeal membrane oxygenation,
585- 586
Extracorporeal photopheresis, 646, 654,
656
Eyewashes, 72
F FFP. See Fresh Frozen Plasma
Face shields,
71 Facilities
alternate, during disasters, 103-
104 clean rooms in, 40-41
design and workflow of,
40 ergonomic design of,
41-42 housekeeping in, 40
licensure and registration of, 84, 86
INDEX ■ 815
mobile sites, 41
quality management of, 5-6, 7, 12
regulatory agencies for, 39-40, 68-69
restricted areas in, 41
safety program of, 7, 12, 42-64
Factor VIIa, recombinant, 528, 623, 637-638,
685
Factor VIII
antibodies to, 527
concentrates, 524, 526-527
in Cryoprecipitated AHF, 153, 154, 522
Factor IX complex concentrate, 518, 527-528,
623
Factor IX concentrates, 524, 527
Factor Xa inhibitors, 518
Factor XIII, 638
Failure modes and effects analysis, 28
False positive/negative test results, 332, 373-
374
Fatalities
during apheresis, 660
of blood donors, 20, 145-146
due to identification errors, 551
due to TRALI, 681
due to transfusions, 20, 691
of employees, 45
related to medical devices, 87
reporting, 20, 45, 87, 145, 691
Fatigue, in donors, 145
Fc receptors, 248, 249, 250, 466
FDA. See Food and Drug Administration
Febrile nonhemolytic transfusion reactions,
489, 548, 667, 677
Fenwal apheresis systems, 168, 173, 174,
175 Fetal and neonatal alloimmune
thrombocytopenia, 461, 567-568
Fetal bovine serum, 764-765, 793
Fetal hemoglobin, hereditary persistence
of, 362
Fetomaternal hemorrhage, 565-
566 Fetus
genotyping, 283-284, 330, 563, 567
hemolytic disease in, 561-564, 566-567
pretransfusion testing of, 563
thrombocytopenia in, 461, 567-568
transfusions in, 563-564
weak D in, 565
Fever, 489, 667, 676, 677, 686
Fibrin clots, 300
Fibrinogen
abnormalities of, 522, 523
in Cryoprecipitated AHF, 153-154, 522
Fibrinogen concentrate, 529, 624
Ficin, 402
816 ■ AABB T EC HNIC AL MANUAL

Filters
preparation of, 147, 151
Leukocyte reduction, 154, 223, 552, 554
quarantine, 152
microaggregate, 551-552, 585
as replacement fluid in apheresis, 522, 647,
standard in-line, 551, 554, 585
652
Fire prevention, 45-46
storage of, 151, 218-219
First aid, 44-45
thawing, 151, 221
Fish-bone diagrams, 27, 28
transfusion of, 522, 554
Flow cytometry, 246
transportation and shipping of, 218-219
in HLA antibody testing, 487, 491
Fume hoods, 58
to measure fetomaternal hemorrhage, 565
Fungal contamination, 777, 782
to measure residual leukocytes, 154-155
in platelet antibody detection, 460, 464, G
465 in red cell survival studies, 419
Fludarabine, 443 G-CSF. See Granulocyte colony-stimulating
Fluids, replacement, in apheresis, 522, 647, factor
Gastrointestinal diseases, 764
652
Fluorescent methods for nucleic acid Gel-column agglutination technology, 377,
396
analysis, 238-240
Gene locus, 256
Focal segmental glomerulosclerosis, 653
Genes, 256-258
Food and Drug Administration
of blood group systems, 259-261, 277-278
inspections by, 86-87
frequencies of, 275-276
regulations and guidance
of major histocompatibility complex, 476-
for biological products, 83, 84, 85
480
cGMP, 7, 8, 20, 213, 742, 760, 761
mapping, 259-261, 277-278
cGTP, 8, 20, 87, 743-744, 775, 777
mutation of, 264
for HPCs, 87-89, 743-746
nucleic acid structure in, 232-
for infectious disease testing, 182, 184,
233 position effect, 272-273
187-188
silent or amorphic, 264, 267, 286-
for licensure and registration, 84, 86
287 suppressor or modifier, 273
for medical devices, 83-84, 87
syntenic, 271
for recalls and withdrawals, 89
Genetic principles
for tissues, 777
alleles, 262, 264, 275, 276
reporting adverse events to, 20, 22, 87
blood group gene mapping, 259-261, 277-
reporting fatalities to, 20, 87, 145,
278
691 variances to requirements, 11
cell division, 258, 262, 263
Forensic testing, 493
chimerism, 278
Forms, 17, 18. See also Documents; Records
genes and chromosomes, 256-
Forssman system, 261, 340, 360
258 genotype and phenotype,
Freeze-thaw elutions, 429
261-262 inheritance patterns,
Freezers, 36, 214
265-273
Freezing
autosomal, 265-267
cryoprotective agents for, 155, 722, 761
gene interaction and position effect,
Fresh Frozen Plasma, 151
272-273
hematopoietic progenitor cells, 722-723,
independent segregation and
736-737
independent assortment, 270
platelets, 155
linkage and crossing-over, 270-271, 272,
RBCs, 155
479
Frequency, allele (gene), 274, 275-276
linkage disequilibrium, 271, 479-480
Fresh Frozen Plasma, 151
pedigrees, 265, 266
ABO compatibility of, 375, 554, 583, 635
sex-linked, 267-269
aliquoting, 583
of major histocompatibility complex, 476-
expiration of, 151, 218-219, 221
480
leukocyte content of, 505
polymorphism, 264
in massive transfusion, 502, 685
population genetics, 274-276
for pediatric patients, 578, 582-583, 589
relationship testing, 276-277
X chromosome inactivation, 258, 261

Genotype
defined, 261-262
DNA-based assays for, 282
for antigen-negative blood donors, 282,
285
to distinguish alloantibody from
autoantibody, 282, 283
of fetus, 283-284, 330, 563, 567
platelet genotyping, 465
in prenatal practice, 283-285, 330
in recently transfused patients, 240,
281,
282, 330
of Rh system, 285, 330
in sickle cell disease patients, 330
when red cells are coated with IgG,
282,
283, 401-402, 436
frequencies of, 275-276
nomenclature for, 280
and phenotypes, 240, 261-262, 285-287,
402
Gerbich system, 260, 339, 357
Gill system, 261, 340, 359-360
Glanzmann thrombasthenia, 455
Globoside collection, 260, 309, 310, 311, 340
Gloves, 70-71
and latex allergy, 45
use in donor room, 53, 140
Glycerolization of red cells, 155
Glycine-HCl/EDTA, 407
Glycoproteins, platelet, 453, 454-455, 455-
457,
458
GMP. See Good manufacturing
practice Goggles, safety, 71
Gonorrhea, in donors, 124
Good manufacturing practice
for component manufacturing, 213
for HCT/Ps, 742
for MSCs, 760, 761
for nonconforming events, 20
for training personnel, 8
for work environment, 7
Good tissue practice
for infectious disease testing,
87 for nonconforming events,
20 for tissue processing, 775,
777 for training personnel, 8
for umbilical cord blood, 743-744
Grading test results, 372
Graft-vs-host disease
and donor lymphocyte infusions, 787
in HSC transplantation, 638-639, 717
transfusion-associated, 671, 687-688
HLA system in, 489-490, 688
in neonates, 573
treatment with MSCs, 762, 763, 794
Graft-vs-tumor effect, 715, 717, 785
INDEX ■ 817
Granulocyte agglutination test, 469
Granulocyte colony-stimulating factor
(G-
CSF)
for HSC mobilization, 719-720
in leukapheresis, 172-173, 524, 525
side effects of, 719-720
Granulocyte immunofluorescence test, 469
Granulocytes
ABO compatibility of, 173, 375, 525, 554,
584
antigens and antibodies, 466-469, 681, 682
apheresis collection of, 168, 172-173
CMV-reduced-risk, 525
dose of, 584
expiration of, 217
HLA-matched, 525
in HSC transplantation patients, 638
increasing yields of, 172-172, 525
indications for, 525, 584, 589
infusion of, 173, 523-526, 554
irradiation of, 173, 217,
525 laboratory testing of,
173 in pediatric patients,
584
storage of, 173, 217, 221, 525
transportation and shipping of, 217
Growth factors, recombinant, 172-173, 719-
720
Growth hormone, in donors, 129
GTP. See Good tissue practice
GVHD. See Graft-vs-host disease
H
H system, 301-304
alloanti-H, 303, 339
autoanti-H, 303
autoanti-HI, 303, 339
biochemistry and genetics of, 260, 292-293,
301-303
Bombay phenotype, 292, 293, 303
H antigen, 292, 293, 301
Para-Bombay phenotype, 303
transfusion practice for, 304
Hand washing, 72
Haploid, defined, 258
Haplotype
ancestral, 480, 493
defined, 271
of HLA system, 478, 479, 480
of Rh system, 319, 320, 321-322
Hardy-Weinberg equilibrium, 275-276
Hazardous areas of facilities, 41
Hazardous materials
biohazards, 47-55
chemicals, 55-60
classification of, 56
818 ■ AABB T EC HNIC AL MANUAL

identification and communication of, 43-

histocompatibility in, 491, 715-716
44 for biosafety, 48-49
history of, in blood donors, 123
for chemical safety, 56-
incompatible
58 for electrical safety,
bidirectional ABO, 633, 635
47 for fire safety, 46
major ABO, 632-633, 635, 716
radioactive, 60-63
minor ABO, 633, 635, 716
safety plan for, 42
related to non-ABO antigens,
shipping, 63
633 patient care in, 724-725
waste management of, 53-55, 63-64
patient survival after, 718
Hazards, risk assessment of, 98-99,
positive DAT after, 428
102 HBsAg. See Hepatitis B surface
products for (See Hematopoietic stem cells)
antigen HBV. See Hepatitis B virus
records of, 640
HCT/Ps (human cells, tissue, and cellular
regulation of, 87, 88, 89, 725, 743-746
and tissue-based products). See also Stem
standards for, 746
cells; transfusion support for
Tissue autologous recipients, 638
biological product deviations of, 22 component processing, 638-639
infectious disease testing on donors of, Cryoprecipitated AHF, 637
191- factor concentrates, 637-638
192, 714-715, 774 incompatible transplants, 632-633
regulation of, 87-89, 743-746, 777-778, information portability, 640
796-
in patients with HLA/HPA
797
antibodies, 637, 639-640
HCV. See Hepatitis C virus
in patients with neutropenia and
HDFN. See Hemolytic disease of the fetus and
infection, 638
newborn
in pediatric patients, 639-640
Health history questionnaires. See Donor
plasma, 637
history questionnaire
platelets, 634, 635, 636-637
Heart disease, in blood donors, 126, RBCs, 634
129 Heart transplants, 493-494
selection of components, 633-634, 635
Heat elutions, 429
transfusion reactions after, 639
Heating blocks, 37
Hematopoietic stem cells
Hemagglutination, 241-242, 279-280, 396
collection of, 718-720, 733-734
Hematocrit
cryopreservation of, 722-723, 736-737
in blood donors, 121
donors of, 714-717
of blood in exchange transfusions,
infusion of, 724-725
580 in neonates, 585
irradiation of, 638
of red cell components, 149
processing, 720-722
as transfusion threshold, 692
quality control of, 723, 736
of Whole Blood, 148
regulation of, 87, 88, 89, 725
Hematologic disorders, in blood donors, 128-
shipping and transport of, 723-724, 737-738
129
sources of, 717-720
Hematoma, in donors, 145
storage of, 736-737
Hematopoietic growth factors, 172-173, 719-
thawing, 721
720 Hematopoietic progenitor cells. See
washing, 721, 740-741
Hematopoietic stem cells
Hemizygous, defined, 263, 267
Hematopoietic stem cell transplantation
Hemoglobin
ABO typing discrepancies in, 298, 300
in blood donors, 120, 121
adverse reactions to, 724-725, 742
decreased, tolerance for, 609-
allogeneic, 632-638, 715
610 in infants, 571-572, 578-
autologous, 638, 714-715
579, 585 in Red Blood Cells,
and CMV, 639
149
diseases treated with, 713-714
in red cell apheresis donors, 171
donor lymphocyte infusion after, 786-
testing methods for, 121
788 donor requirements for, 714-717
as transfusion threshold, 499-501, 505-506,
engraftment kinetics in, 717-718
578-579, 610
graft-vs-host disease after, 638-639, 717,
794 graft-vs-neoplasm effect in, 717

Hemoglobin F, 362
Hemoglobin S, 564, 587, 588
Hemoglobin substitutes, 507
Hemoglobinometers, 38
Hemoglobinopathies, 587-589. See also
Sickle
cell disease
Hemolysis
in ABO testing, 297
in antiglobulin tests, 372
extravascular, 250, 430, 673-674
in hemolytic disease of the fetus and
newborn, 562
intravascular, 251, 430, 672-673
nonimmune, 660, 669, 675-676
passenger lymphocyte syndrome, 633
in patient samples, 370, 672
Hemolytic anemia
autoimmune
classification of, 430
cold agglutinin disease, 437-439
DAT-negative, 437
mixed-type, 439-440
paroxysmal cold hemoglobinuria,
440 serologic findings in, 431
transfusion in, 436-437, 439-440, 506
warm, 430-437
drug-induced immune, 440-444
neonatal, 561-564, 566-567
positive DAT in, 426, 428
Hemolytic disease of the fetus and newborn,
561-564
ABO, 566-567
antibodies associated with, 338-340, 347-
348, 413-414, 562
blood selection and administration in,
564 diagnosis of, 562-563
maternal alloimmunization in, 562
neonatal management in, 564
pathophysiology of, 562
pregnancy monitoring in, 563
preventing, 565-566
testing in, 563
antibody titers, 409, 563
elutions, 429
Rh testing, 283-285, 332, 563, 565
treatment of, 563-564
Hemolytic transfusion reactions
acute, 667, 672-675
antibodies associated with, 338-
340, 413-414
clinical evaluation and management of,
666, 667, 672
due to misidentification errors, 551
elutions in, 429
HLA antibodies in, 490
INDEX ■ 819
delayed, 250, 588, 670, 686-687
intravascular hemolysis in,
251 nonimmune, 669, 675-
676
Hemolytic uremic syndrome, 653
Hemophilia, 524, 526-527, 528
Hemorrhage
and anemia, 508-509
assessing risk of, 518-521, 605
fetomaternal, 565-566
intraventricular, 581
transfusion for, 501-502
Hemostasis
during massive transfusion, 591, 684-685
in neonates, 582-583
tests for, 518-521, 582
Hemostatic agents, 608, 621-627
Hemovigilance, 22, 33, 665-666
Heparin, 462, 465-466, 658, 675
Heparin-induced thrombocytopenia, 462,
465-466, 517, 528
Hepatitis, non-A,non-B, 179, 182, 196
Hepatitis B immune globulin, 45
Hepatitis B surface antigen, 179, 196
Hepatitis B virus, 196
employee exposure to, 44-45
nucleic acid testing for, 186,
196
prophylaxis for, 44
reactive testing results for, 187, 188, 189
residual transfusion risk of, 192, 194, 196
screening donors for, 124, 126, 179, 184,
191, 196
transmission through transplantation, 774,
777, 782
Hepatitis C virus, 196-197
employee exposure to, 44-45
nucleic acid testing for, 185-186, 197
reactive testing results for, 187, 188, 189
residual transfusion risk of, 192-193, 194,
197
screening donors for, 124, 126, 179, 184,
191, 197
transmission through transplantation, 774,
777, 782
Hepatitis E virus, 203-204
Herbal supplements, 605
Hereditary hemochromatosis, 476
Hereditary persistence of fetal hemoglobin
syndrome, 363
Heredity, genetics of. See Genetic principles
HES. See Hydroxyethyl starch
Heterozygous, defined, 263-264
High-prevalence antigens
901 series, 361-362
antibodies to, 415-416,
564 blood selection for,
421
820 ■ AABB T EC HNIC AL MANUAL
High-titer, low-avidity antibodies, 409-410
Histocompatibility. See HLA system
HIV. See Human immunodeficiency virus
Hives, 667, 678, 679-680
HLA alleles, 483-484
HLA antibodies
detection of, 460, 487
donor specific, 716
in HSC transplant recipients, 637, 639-
640,
716
management of, 670
and plasma transfusions, 151
in platelet refractoriness, 458, 459-461,
488-
489, 515-516
in TRALI, 489, 681, 682
in transfusion reactions, 489, 490, 677
HLA antigens
Bg, 362, 480, 490
Class I and Class II, 475, 480-482, 484
configuration of, 481
cross-reactive groups, 482
identification of, 484-487
nomenclature for, 481-482
on platelets, 458
“public,” 482-483
“splits,” 482
HLA-matched platelets, 459-460
HLA Matchmaker program, 460, 488-489
HLA system
biochemistry, tissue distribution and
structure of, 480-484
biologic function of, 484
and chimerism, 489-490
disease associations with, 493-494
genetics of, 476-480
absence of antigens, 478-479
crossovers, 479
finding HLA-identical siblings, 478
linkage disequilibrium, 479-480
organization of, 476-478
patterns of inheritance in, 478-
480 in graft-vs-host disease, 489-
490, 688
importance of, 475-476
HLA typing
cellular assays for, 486-487
crossmatching, 487, 491-492
DNA-based assays for, 485-486, 493
in forensic testing, 493
of Granulocytes, 525
lymphotoxicity assays for, 486
of platelets, 459-460, 488, 516
in relationship testing, 493
in transplantation, 491-493, 715-
716 HNA. See Human neutrophil
antigens Homolog, defined, 267
Homozygous, defined, 263-264
Hook effect, 245
Hospitals, 91, 602-603
Housekeeping, 40
HPA. See Human platelet
alloantigens HSC. See
Hematopoietic stem cells HSCT. See
Hematopoietic stem cell
transplantation
HTLV. See Human T-cell lymphotropic virus
Human immunodeficiency virus, 194-196
educating donors about, 119
employee exposure to, 44-45
nucleic acid testing for, 185-186,
195
reactive testing results for, 187, 188,
189 reporting new cases of, 782
residual transfusion risks for, 192-193, 194,
195
screening donors for, 124, 125, 126, 182,
184, 191, 194-196
transmission through transplantation, 774,
777, 782
Human neutrophil antigens, 466-468
Human platelet alloantigens, 453-
457 Human resources, 7-9, 12, 109
Human T-cell lymphotropic virus,
197
reactive testing results for, 187, 188, 189
screening donors for, 184, 191, 197
transmission through transplantation, 774,
782
Human urea transporter 11, 352
Hydatid cyst fluid, 312, 406
Hydrops fetalis, 561
Hydroxyethyl starch, 172, 722
Hyperbilirubinemia, 579
Hyperhemolytic syndrome, 588
Hyperkalemia, 555, 683-684
Hyperviscosity, 652
Hypocalcemia, 555, 658, 670, 683
Hypofibrinogenemia, 522-523
Hypogammaglobulinemia, 300, 529, 659
Hypoglycemia, 580
Hypokalemia, 683-684
Hypotension
in apheresis, 659
associated with ACE inhibitors, 659, 669,
678
deliberate, 607
in transfusion recipients, 674, 679
Hypothermia, 548, 572-573, 607, 670, 685-686
Hypovolemia, 526, 659
I
I and i antigens, 306-309
anti-I, 308, 340
anti-i, 308

in cold agglutinin disease, 308-309, 392,
439
disease associations with, 307, 392
genetics of, 260, 307-308
phenotypes, 306-307
transfusion practice with, 309
Iatrogenic anemia, 609
ICAM-4, 355-356
Identification
of blood components
before administration, 226, 551
before issue, 226, 549-550
donation identification number, 135,
139-140, 159
labeling, 158-159, 384-385
of donors, 118-119, 139
of equipment, 11
errors in, 551, 675
of personnel, 19
of persons issuing blood,
385 of phlebotomists, 370,
547
of problems and solutions, 26-29
of recipients, 226, 368-370, 384-385, 547,
549, 550, 551
of umbilical cord blood, 739
Idiopathic thrombocytopenia, 463, 465, 517,
568
IgA, 247, 248, 678-679, 680
IgE, 247, 248
IgG, 247, 248, 249, 573
IgG-coated cells (check cells), 376
IgM, 246, 248
cold-reactive autoagglutinins, 437-439
in complement activation, 249
dispersing autoagglutination caused by,
301, 438
in infants, 573
in intravascular hemolysis, 251
structure of, 247
Immediate-spin crossmatch, 380
Immune complexes, 652
Immune thrombocytopenic purpura, 463,
465,
517, 568
Immunity
antibodies in, 246-248
complement in, 248-250
extravascular hemolysis in,
250 Fc receptors in, 248, 249,
250
in infants, 573
intravascular hemolysis in, 251
Immunoglobulin products, 526, 529-531, 532
Immunoglobulins, 246-248. See also specific
immunoglobulins
Immunohematology reference laboratories,
419
Immunomagnetic cell separation, 722
INDEX ■ 821
Immunomodulation, 504, 758, 762-763, 764
In-vivo testing, 419, 506-507
Incarceration, donor history of, 125
Incidence, defined, 274
Incinerators, hospital/medical/infectious
waste, 55
Incubation times, 405
Incubators, platelet, 36, 214
Independent assortment, 270
Independent segregation, 270
Indian system, 260, 339, 358-359
Indirect antiglobulin test, 372, 373-374
Induced pluripotent stem cells, 755, 795-
796 Infants. See Neonates; Pediatric
patients Infection, in transplant patients,
638, 782 Infectious disease screening
approaches to, 183
of autologous donations, 190-191
for Babesia, 201
for bacterial contamination, 198-199
for chikungunya virus, 202
for CMV, 190
for dengue virus, 202
for HBV, 196
for HCV, 196-197
for HEV, 203-204
historical overview of, 179-182
for HIV, 194-196
in HSC transplantation donors, 191-
192, 714-715
for HTLV, 197
international variations in, 192
logistics of, 183
for malaria, 199-200, 201-202
nucleic acid testing, 185-186
for parvovirus B19, 203
for prions, 202-203
reactive test results in, 186-190
residual infectious risks of transfusion, 192-
194
serologic testing, 185
for syphilis, 198
in tissue transplantation, 774
for Trypanosoma cruzi, 200-
201 for umbilical cord blood,
732
in the United States, 184
for West Nile virus, 200
Infectious diseases
emerging agents, 203, 204
safety precautions for, 47-
55
screening for (See Infectious
disease screening)
transmitted by plasma derivatives, 203
transmitted by tissue transplantation, 777,
782
822 ■ AABB T EC HNIC AL MANUAL
Infectious waste, 53-55
Inflammation modulation, 763-764
Information management, 13, 19-20
Infusion pumps, 548-549, 584
Infusion rates, 553, 554, 555, 585
Infusion sets, 551-552, 585
Inheritance patterns
autosomal, 265-267
crossing-over, 270-271, 479
gene interaction and position effect, 272-
273
independent segregation and independent
assortment, 270
linkage, 270-271, 272
linkage disequilibrium, 271, 479-480
of major histocompatibility complex, 478-
480
pedigrees, 265, 266
sex-linked, 267-269
Inhibition tests, 406
Injuries, 45, 49, 87
INR. See International normalized
ratio Inspections
of components
before administration, 550-551
before release, 224, 226, 385, 549
after preparation, 148, 149
documentation of, 224
of incoming supplies, 10
of tissue grafts, 779-780
Insulin, bovine, 129
Insurance, 104
Integrins, 455-456
Internal event reports, 20, 21-22
International normalized ratio (INR), 518,
519,
520
Intraoperative blood recovery, 606-
607 Intrauterine transfusions, 563-564
Intravenous immune globulin, 529-
531
ABO discrepancies with, 300
adverse effects of, 529, 532
in antibody identification problems, 392
applications of, 530-531
for fetal and neonatal immune
thrombocytopenia, 568
in HDFN, 564
in platelet refractoriness, 516
positive DAT with, 428
Intravenous solutions, 552
Intraventricular hemorrhage, 581
Inventory, blood
during disasters, 101-102, 110
management of, 227-228
and patient blood management, 603
receiving components into, 225
Investigational new drug application, 731, 744,
745
Iron, supplemental, 605, 620
Iron chelation therapy, 690
Iron deficiency anemia, 605, 620
Iron overload, 588, 671, 690
Irradiated products, 155-156, 222
expiration of, 215, 217, 222
granulocytes, 173, 217, 525
in HSC transplantation, 638
indications for, 688, 689
for intrauterine transfusions, 564
for pediatric patients, 590
platelets, 156, 217, 459-460
potassium leak in, 573
quality control of, 156
RBCs, 156, 215
storage of, 215, 217
transportation of, 215, 217
Whole Blood, 215
Irradiators, blood, 37, 62, 155-156
Ishikawa diagrams, 27, 28
Isografts, 774
Isohemagglutinins, 292
Issuing components, 226
delivering blood to patient area, 549-550
identification of recipient and component
before, 384-385, 549-550
inspections prior to, 226, 385, 549
reissue, 226-227, 550
in urgent situations, 158, 227-228, 385-386,
506, 555-556
ITP. See Immune thrombocytopenic purpura
IVIG. See Intravenous immune globulin
J-K
John Milton Hagen system, 260, 339, 359
JR system, 261, 340, 360
Juran’s Quality Trilogy, 2-3
Karyotype, 256
Kell system, 345-349
allele frequencies in, 275-
276 antibodies of, 338, 347-
348
antigens of, 259, 346-347
biochemistry and genes of, 259,
346 functional aspects of, 348
in HDFN, 284, 347-348, 562, 563
Kmod, 348
Ko (null) phenotype, 348
phenotypes of, 346
position effect in, 272-273
Kernicterus, 562, 564, 579
Kidd system, 351-352
antibodies of, 338, 352

antigens of, 259, 351-352
genetics of, 259
Kidd glycoprotein, 351, 352
phenotypes of, 351
in transfusion reactions, 352
Kidney transplantation, 491-492
Kleihauer-Betke acid-elution test, 565-566
Knops system, 260, 339, 358
Kx system, 348-349
antibodies of, 339
genetics of, 258, 260, 261, 269
McLeod phenotype, 258, 261, 269, 348
L phenotypes of, 304-305
Labels
for biohazardous materials, 49
for blood components, 139, 158-159, 226,
384-385
for blood samples, 370, 547
control of, 17, 18
for hazardous chemicals, 57-58
ISBT 128 system for, 159
for shipments, 738
for umbilical cord blood,
739 Laboratories
biosafety precautions for, 53
regulations for, 89-91
Laboratory coats, 70
Lan system, 261, 340, 360-361
Landsteiner-Wiener system, 260, 339, 355-356
Latex allergies, 45, 136
LDL apheresis, 656
Leadership, organizational, 5-6, 12, 105
LEAN, process improvement, 28-29
Lectins, 295, 301
Leukapheresis
collection of granulocytes by, 175-
176 donation intervals for, 120, 122
indications for, 646, 654, 655
instrumentation for, 168, 175-
176 Leukemia
in blood donors, 126, 128
cytapheresis in, 654
donor lymphocyte infusions for, 786-787,
788 natural killer cells in, 788
platelet transfusions in, 507
Leukocyte-reduced components, 504-505
expiration, transportation and storage of,
216, 217
leukocyte content in, 154-155, 505
for pediatric patients, 590
platelets, 154, 156, 169-170, 217, 505
poststorage filtration, 222-223
prestorage filtration, 154-155, 223, 552
to prevent CMV infection, 190, 590, 639
INDEX ■ 823
RBCs, 149, 154, 216, 504-505
to reduce alloimmunization, 515
to reduce incidence of PTP, 690
Leukocyte reduction filters, 154, 223, 552, 554
Leukocytes, in components, 154-155, 505
Lewis substance, 406
Lewis system, 304-306
antibodies of, 305-306, 338
antigens of, 259, 302, 304
biochemistry and synthesis of, 302,
304 disease associations with, 306
expression in children, 305
genetics of, 259, 304-305
saliva testing for, 406
transfusion practice with, 306
Licensure, of facilities, 84, 86
Likelihood ratio, 277
Linkage, 270-271, 272
Linkage disequilibrium, 271, 479-480
Lipemic samples, 370
Liquid nitrogen
for shipping, 724, 738
for storage, 723, 737, 761
Liquid Plasma, 152, 220
LISS, 376, 402, 417
Liver, transplantation of, 493-494
Liver disease, 764
LKE antigen, 309, 310, 313
Look-back investigations, 187-188, 189-190, 782
Low-prevalence antigens, 362, 416
Low-volume units, 141, 142,
149 Lui freeze thaw elution, 429
Luke antigen, 309, 310, 313
Lung conditions, in blood donors, 126,
129 Lung transplants, 493-494
Lutheran system, 344-345
antibodies of, 338, 345
antigens of, 259, 344-345
genetics of, 259, 267, 272, 273
Lymphocyte crossmatching, 487
Lymphocytes, T cytotoxic, 787-788
Lymphocytotoxicity assays, 485, 486, 487
Lymphoproliferative disease, 788
Lyonization, 258, 261
M
MACE (modified antigen capture ELISA), 464
MAIGA (monoclonal antibody immobilization
of granulocyte antigens), 469
MAIPA (monoclonal antibody immobilization
of platelet antigens assay), 464
Major histocompatibility complex, 475. See
also HLA system
Class I and Class II antigens in, 480-482
824 ■ AABB T EC HNIC AL MANUAL
genetics of, 476-480
public antigens, 482-483
split and cross-reactive groups in, 482
Major histocompatibility complex multimer
method, 787
Malaria, 201-202
and Duffy glycoprotein, 351
screening donors for, 125, 126, 202
Markers, defined, 255
Market withdrawals, 782
Marrow
autologous, 714-715
collection of, 718-719
cryopreservation of, 722-723
donor requirements for, 714-716
engraftment kinetics of, 717-718
histocompatibility of, 715-716, 717
infectious disease testing of, 191, 715
infusion of, 724-725
MSCs derived from, 755, 762, 763
processing, 720-722
quality control of, 723
red cell reduction in, 720-721
regulations for, 87, 88, 89, 725
shipping and transport of, 723-724
transplantation outcomes using, 718
Marrow aplasia, 787
Masks, 71
Massive transfusions
complications of, 591, 683-685
component replacement in, 501-502, 685
defined, 228, 386
Factor VIIa, recombinant in,
685 in pediatric patients, 591
pretransfusion testing in, 228, 330, 386
Material safety data sheets, 57, 58
Materials management, 9-10, 12
Maximum surgical blood order schedules, 227
McLeod phenotype, 258, 261, 269, 348-349
2-ME. See 2-mercaptoethanol
Mechanical hemolysis, 660, 676
Media, working with, 107
Medical devices, regulations for, 83-84, 87
Medical history
in antibody identification, 392
in blood donor selection, 121-
127 for cord blood donation,
731-732
in evaluation of positive DAT, 427-428
of recipients, 546
Medical waste, 53-55
Medication Deferral List, 129-130
Medications. See Drugs
Meiosis, 258, 263
Membrane attack complex, 249-250
2-mercaptoethanol (2-ME), 407, 432, 438
Mesenchymal stem cells
autologous vs allogeneic use,
754 future directions for, 765-
766 identification criteria for,
793
isolation and expansion of, 758-760, 792-
793
multipotentiality of, 754-755, 791-792
properties of, 756-757, 758, 786, 791-792
research and development on, 764-765
shipping, 761-762
sources of, 754-758
standardization of methods for, 760
storage and banking, 761
therapeutic applications of, 762-764, 793-
795 and tissue engineering, 795
tumor-forming potential of, 765
Message mapping, 107
Messenger RNA, 233
Metabolic abnormalities, in infants, 573-
574 Methergine, 625
Methyldopa, 443
Methylene blue-treated plasma, 153, 205
Microaggregate filters, 551-552, 585
Microarray assays, 245
Microlymphocytotoxicity tests, 485, 486, 487
Microsatellites, 277
Microvascular bleeding, 684
Minisatellites, 277
Misoprostol, 626
Mitosis, 258, 262
Mixed-field agglutination, 278, 298, 362
Mixed leukocyte culture, 486-487
Mixed-type autoimmune hemolytic anemia,
431, 439-440
MNS system, 337, 341-344
antibodies of, 338, 343-344, 406
antigens of, 259, 341,
344 effect of enzymes
on, 314 genetics of, 259,
341, 343
glycoproteins of, 341, 342,
343 linkage disequilibrium in,
271 phenotypes of, 341
S–s–U– phenotype, 343
Mobilization regimens
for granulocytes, 172-173
for HSCs, 719-720
Molecular immunohematology. See
Blood group genomics
Monoclonal antibody immobilization of
granulocyte antigens, 469
Monoclonal antibody-specific immobilization
of platelet antigen, 464
Monocyte monolayer assay, 419
Mortality. See Fatalities
MSC. See Mesenchymal stem cells

Multiple sclerosis, 653
Multiplex nucleic acid amplification, 238
Multipotent stromal cells. See
Mesenchymal
stem cells
Mutations, genetic, 264, 362-363
Myeloma, IgM, 652
N in HSC transplant patients, 638
Nageotte hemocytometry, 154-155
Narcolepsy, 494
NAT (nucleic acid testing), 185-186
in HBV testing, 196
in HCV testing, 197
in HIV testing, 195
Natural killer cells, 788-790
Near-miss events, 26, 33
Needlesticks, 49, 124, 141
Neisseria gonorrhea, 191
Neonatal alloimmune neutropenia, 468
Neonatal alloimmune thrombocytopenia,
461,
567-568
Neonates (younger than 4 months)
ABO and Rh typing in, 300, 385,
574
ABO antigens and antibodies in, 293,
300 anemia in, 571-572
antibody screening in, 385
antigenic variations in, 305, 410,
411 blood volume in, 572
compatibility testing in, 385, 574-575
ECMO in, 585-586
erythropoietic response in, 572
hemoglobin in, 571-572, 578-579
hemolytic disease in, 561-564, 566-567
hemostasis in, 582-583
hypothermia in, 572-573
immunologic status of, 573
Lewis antigens in, 305
metabolic problems in, 573-574
neutropenia in, 468, 525
polycythemia in, 585
thrombocytopenia in, 461, 567-568, 580-
581 transfusion-associated GVHD in,
573 transfusions in
administration of, 584-585
age of units for, 577-578
aliquots for, 224, 575-576, 583
of Cryoprecipitated AHF, 578, 583-584
dosing for, 578
exchange, 564, 574, 579-580
of FFP, 578, 582-583
of granulocytes, 584
indications for, 574, 575, 578-579
of platelets, 578, 580-582
of RBCs, 574-580
safety of additive solutions in, 576-577
INDEX ■ 825
transfusion thresholds in, 578-579
vascular access for, 547, 580, 584
Nerve injury, in donors, 145
Neutralization techniques, 406
Neutropenia
autoimmune, 468
granulocyte transfusions for, 523-525, 584,
589
of infancy, 468
neonatal alloimmune, 468
NMDP registry, 725
Nomenclature
for blood group systems, 259-261, 278-279,
280
for granulocyte antigens, 467
for HLA system, 481-482, 483-484
for human platelet alloantigens, 453-
455 of Rh system, 318-319, 319, 320
Nonconformances
biological product deviations, 22, 89, 90
classification of, 22-23
internal event reports, 20, 21-22
management of, 13, 20-23
Nonimmune-mediated hemolysis, 660, 669,
675-676
NOR phenotype, 311
Normovolemic hemodilution, 606
Notifications, of reactive screening tests, 189-
190
Nucleic acid analysis
detection of amplification products in, 238-
240
for genotyping, 282
for antigen-negative blood donors, 282,
285
to distinguish alloantibody from
autoantibody, 282, 283
of fetus, 283-284, 330, 563, 567
platelets, 465
in prenatal practice, 283-285, 330
in recently transfused patients, 240, 281,
282, 330
of Rh system, 284-285, 287, 330
in sickle cell disease patients, 330
when red cells are coated with IgG, 282,
283, 401-402, 436
for granulocyte antigens, 469
in HLA typing, 485-486, 493
hybridization-based methods of, 233
for infectious diseases, 185-186, 195, 196,
197 isolation of nucleic acids in, 233
nucleic acid sequence-based amplification
in, 237-238
overview of, 231-232
826 ■ AABB T EC HNIC AL MANUAL
for platelet antigens, 465
polymerase chain reaction in, 233-237,
280-
281
in pretransfusion testing, 378
in relationship testing, 277
reverse-transcriptase PCR in, 236-237
of single nucleotide polymorphisms,
240 transcription-mediated
amplification in,
237-238
in West Nile virus testing, 200
Nucleic acid sequence-based amplification,
237-238
Nucleic acids, structure of, 232-233
O Partial D, 324, 326, 327, 328, 333
Obstetrics, controlling bleeding in, 625-626
Octreotide, 626-627
Oh (Bombay) phenotype, 292, 293, 303
Ok system, 260, 339, 359
Oligonucleotide probes, 485
Orders, physician
auditing, 697-707
computerized provider order entry, 611,
699, 700
pretransfusion, 381, 383, 384, 546-547
sample form for, 711
surgical blood orders, 227
verifying prior to transfusion,
551
Organ transplantation
ABO compatibility in, 491, 492,
783 history of, in blood donors,
123 HLA testing in, 491-492, 493
kidney, 491-492
paired donations, 492
positive DAT after, 428
regenerative therapy in, 754
rejection of, 653, 654, 656
transfusion support for, 783
Organizations
for emergency management, 99-100, 109-
110
for quality system regulation, 1-2
for safety, 39-40, 68-69
structure of, 5-6, 12
Orientation programs, 7-8
Osteoarthritis, 763
Osteogenesis imperfecta, 794
Outpatients, 368, 556
Oxytocin, 625
P transfusion algorithms in, 608
P blood groups, 309-313
antibodies of, 312, 338
antigens of, 259, 260, 309-312
biochemistry of, 310-311
disease associations with, 312-313, 392, 440
genetics of, 259, 260
molecular biology of, 311-312
phenotypes of, 309-310
transfusion practice with, 312
P1 substance, 312, 406
P1PK system, 259, 309, 338
Panel-reactive antibody, 487, 492
Panels, red cell, 393, 396-400
Papain, 402
Para-Bombay phenotype, 303
Pareto analysis, 27, 29
Paroxysmal cold hemoglobinuria, 312, 392,
431, 440
Partial thromboplastin time, 519
Parvovirus B19, 187, 203, 313
Passenger lymphocyte syndrome, 633
Paternal samples, testing
DNA-based testing, 284-285, 563
in relationship testing, 276-277
Pathogen reduction technology, 204,
205 and emerging infectious agents,
194 for plasma, 153
for plasma derivatives, 203, 526
to reduce risk of septic reactions,
199 Patient blood management
activity levels of, 612, 629
acute normovolemic hemodilution in, 606
anemia assessment in, 604-605
anesthesia in, 607
auditing in, 24-25, 697-707
bleeding risk assessment in, 605
blood recovery in, 606-607, 608-
609
blood utilization review in, 604, 611
changing physician behavior in, 610-
611 coordinators for, 611
definition of, 599
increased tolerance of anemia in, 609-610
limiting phlebotomy in, 609
medical education in, 604, 611-612
pharmacologic agents in, 608, 620-627
point-of-care testing in, 607-608
preoperative autologous blood donation in,
605-606
program development, 612
rationale for, 600-603
responsibilities for, 629
scope of, 600, 604
surgical blood orders in, 227
surgical techniques in, 607
transfusion thresholds in, 610
 INDEX ■ 827

PBSC (peripheral blood stem cells). See Personal protective equipment, 44, 70-71
Peripheral blood HSCs for biosafety, 52-53
PCC. See Prothrombin complex concentrates for chemical safety,
PCR. See Polymerase chain reaction 58 gloves, 45, 53, 70-
PEDI-PAK system, 576, 577 71
Pediatric patients (older than 4 months). See Personnel
also Neonates accidents and injuries in, 45, 49
ABO antigens and antibodies in, 293, 300 blood exposure in, 44-45, 47-48,
CMV prevention in, 589-590 49 competency assessment of, 7-
Lewis antigens in, 305 8 contact list of, 105
pretransfusion testing in, 300, disaster plans for, 108-109
587 essential, 105, 109
thrombocytopenia in, 581 hepatitis prophylaxis for, 44
transfusions in identification of, 19
aliquoting for small volumes, 224, 575- latex allergies in, 45
576 orientation program for, 7-8
of CMV-reduced-risk components, 590 protective equipment for, 44, 52-53, 70-71
of Cryoprecipitated AHF, 578, 583 records, 19
of FFP, 578, 583, 589 safety monitoring programs for, 44
of granulocytes, 584, 589 selection of, 7
in HSC transplantation patients, 639- staffing plan, 9, 108-109, 110-111
640 training (See Training)
of irradiated components, 590 PF4 ELISA, 465-466
of leukocyte-reduced components, 590 pH, 406, 411
massive, 591 pH meters, 37
of platelets, 578, 581, 582, 589, 590 Phenotype. See also specific blood groups
of RBCs, 578, 586-589 calculations for, 274, 421
with sickle cell disease, 587-588, 639 defined, 261-262
syringe infusion pumps for, 549 and genetic mutations, 264
with thalassemia, 588-589, 640 and genotypes, 240, 261-262, 285-287, 402
vascular access for, 547 nomenclature for, 280
of volume-reduced components, 590 prevalence of, 274
of washed components, 590-591 rare, 394-395, 421
Pedigrees, 265, 266 Phenotyping
Peer review, 24-25, 697-707 antigen-matching, 281, 329-330, 379, 588,
PEG. See Polyethylene glycol 654
Penicillin, 442 autologous red cells, 401-402
Performance improvement standards, 26 with DNA-based assays, 280-281, 282
Periadventitial cells, 758 for antigen-negative donors, 282,
Pericytes, 758 285 to confirm D type of donors,
Peripheral blood HSCs 285, 330 to distinguish alloantibody
allogeneic, 715-717 from
autologous, 714-715 autoantibody, 282, 283
collection of, 718, 719-720 in prenatal practice, 283-285, 330
cryopreservation of, 722-723 in recently transfused patients, 240, 281,
donor eligibility of, 714-717 282, 330
engraftment kinetics of, 717-718 when red cells are coated with IgG, 282,
infectious disease testing on donors of, 283, 401-402, 436
191, 714-715 donor units, 420
infusion of, 724-725 solid-phase assays for, 242-243
mobilization of, 719-720 Phlebotomy. See also Blood
patient survival with, 718 collection
processing, 720-722 adverse reactions to, 142-
quality control of, 723 146 blood loss due to, 609
regulations for, 87, 88, 89, 725 for collection of blood samples, 368,
shipping and transport of, 723-724 370 disinfection methods for, 140-141
of donors, 140-146
vein selection for, 140
828 ■ AABB T EC HNIC AL MANUAL
Photopheresis, 646, 654, 656
Physical assessment
of apheresis patients, 660-661
of donors, 120
of recipients, 546
Physicians
changing behavior of, 610-611, 704-705
educating, 611-612
perspective on patient blood management,
601-602
Physiologic anemia of infancy, 571-572
Piercings, 124
Piperacillin, 442, 443
Pipettes, recalibration of, 37
PlA1 (HPA-1) antigen, 455,
567 Plasma
ABO compatibility of, 375, 554, 583, 635
aliquoting, 583
coagulation factors in, 221
collection by apheresis, 168, 170
cryoprecipitate reduced, 152, 219-220, 652
donors of, 682
expiration of, 151, 218-220, 221
fresh frozen (See Fresh Frozen Plasma)
frozen within 24 hours after Phlebotomy,
152
liquid, 152, 220
pathogen-reduced, 153, 205
preparation of, 147-148, 151
for pretransfusion testing, 392, 393
recovered (for manufacture), 152-153, 220,
526
as replacement fluid in apheresis, 522,
647,
652
solvent/detergent-treated, 153, 205
source, 170, 526
storage of, 151, 218-220
thawed, 151, 152, 219, 221, 522
thawing, 221
transfusion of (See Plasma transfusions)
transportation and shipping of, 218-220,
225
Plasma derivatives
1-antitrypsin, 532
activated protein C, 532
albumin, 526
antithrombin, 531
clotting factor concentrates, 526-527
fibrinogen concentrate, 529, 624
infectious disease screening for, 203
intravenous immune globulin, 529-531,
532
pathogen reduction for, 203, 526
prothrombin complex concentrates, 527-
528, 623
recombinant Factor VIIa, 528, 623
Plasma exchange. See Therapeutic
plasma exchange
Plasma transfusions
to correct PT/INR, 518, 519-520
dose and timing of, 521
and HLA antibodies, 151
indications for, 517-518, 522
in cirrhosis patients, 518-519
in correction of anticoagulation, 518, 519
in HSC transplantation, 635, 637
in massive transfusion, 502, 685
in pediatric patients, 578, 582-583, 589
infusion of, 554
types of plasma for, 522
Plasmapheresis, 170
consent for, 170
donation intervals for, 120, 122, 170
instrumentation for, 168, 173-
174 plasma transfusion in, 522
red cell losses in, 170
therapeutic plasma exchange, 647-653
adverse effects of, 658-660
in hemolytic disease of the fetus and
newborn, 564
indications for, 647, 648-651, 651-653
replacement fluids for, 646, 647, 652
Platelet antagonists, 122, 129, 169,
509 Platelet antibodies
anti-HPA, 455-457
autoantibodies, 461, 465, 568
in autoimmune thrombocytopenic
purpura, 463, 568
detecting, 243, 456, 463-464, 465-466, 639-
640
drug-induced, 462, 465-466
in fetal and neonatal alloimmune
thrombocytopenia, 455, 456, 461,
567
in HSC transplant patients, 637
in platelet refractoriness, 515-
516
in posttransfusion purpura, 455, 456, 461-
462, 689-690
Platelet antigens
ABO antigens,
457
GPIV/CD36, 457, 458
GPVI, 457, 458
HLA antigens, 458, 488
HPA, 453-457
Platelet counts
corrected platelet count increment, 458,
512, 516
in infants, 568
in plateletpheresis donors, 169
posttransfusion platelet recovery, 458,
512
as transfusion threshold, 507-509, 581, 589,
636-637

Platelet disorders
drug-induced thrombocytopenia, 462,
465-
466, 516
fetal and neonatal alloimmune
thrombocytopenia, 461, 567-568
immune thrombocytopenia purpura, 463,
465, 517, 568
in massive transfusion, 684
platelet transfusion refractoriness, 458-
461 posttransfusion purpura, 461-462,
671, 688-
690
Platelet factor 4 ELISA, 465-466
Platelet gel, 222, 517
Platelet incubators, 36, 214
Platelet-rich plasma, 146, 148, 150, 517
Platelet transfusions
ABO compatibility of, 375, 457, 513-514,
554
after HSCT transplantation, 635, 636
in hemolytic transfusion reactions, 675
in pediatric patients, 581, 589
contraindications to, 517
to correct thrombocytopathy, 509-510
dosage of, 510-511, 581, 637
in fetus, 567-568
in HSC transplantation, 634, 635, 636-637
indications for, 507, 509-510, 580-581
infusion of, 554
in massive transfusion, 502, 685
in pediatric patients, 578, 580-582, 589
prophylactic vs therapeutic, 507, 510-511,
636
refractoriness to, 458-461
ABO compatibility in, 457
causes of, 459
HLA antibodies in, 458, 459-461, 488-
489, 515-516, 670
HLA-matched platelets for, 459-460,
488-489
HPA antibodies in, 453-457
managing, 514, 516-517
platelet crossmatching for, 460-461, 489,
516
preventing alloimmunization in, 515-
516
selection of platelets for, 459-461, 488-
489
response to, 512
Rh matching, 515
thresholds for, 507-509, 581, 589, 636-637
unit type and age, 511, 512, 513
Plateletpheresis, 168-170
adverse reactions to, 169
donor selection and monitoring in, 120,
122, 168-169
instrumentation for, 168, 174-175
INDEX ■ 829
records of, 170
therapeutic, 654
volume collected in, 169
yields from, 510
Platelets, Apheresis
additive solution in, 217
agitation of, 214, 221
bacterial contamination of, 198-199,
221
biochemical changes in storage of, 221, 511,
513
collection of, 168-170
compared to Whole-Blood-derived
Platelets, 511, 512
crossmatching, 460-461, 489, 516
donors of, 168-169
expiration of, 217
HLA matched, 459-460, 488-489, 516
irradiated, 156, 217, 459-460
laboratory testing of, 169-170
leukocytes reduced, 169-170, 217, 505
pathogen reduction for, 205
storage of, 214, 217
transfusion of (See Platelet
transfusions) transportation and
shipping of, 217 volume-reduced, 157,
223, 513-514
washed, 223
Platelets (Whole-Blood-derived), 150-151
agitation of, 150, 214, 221
bacterial contamination of, 150, 198-199,
221
biochemical changes in storage of, 221, 511,
513
clumping in, 148
compared to Apheresis Platelets, 511, 512
cryopreservation of, 155
expiration of, 150, 156, 216-217, 224
irradiated, 156, 217
leukocytes-reduced, 154, 156, 218, 505
pathogen reduction for, 205
pooled, 156-157, 217, 223-224, 512
preparation of, 146, 147-148, 150
storage of, 150-151, 214, 216-217, 221
transfusion of (See Platelet transfusions)
transportation and shipping, 150-151, 216-
217
visual inspection of, 148, 199
volume-reduced, 157, 223, 581-582, 590
washed, 223, 590-591
Plerixafor, 720
Point-of-care testing, 607-608
Policies, 11, 17, 18
Polyagglutination, 300, 332
Polycythemia, 585
Polyethylene glycol (PEG), 376-377, 402
830 ■ AABB T EC HNIC AL MANUAL
Polymerase chain reaction, 233-
237 in genotyping, 280-281
in HLA typing, 485
oligonucleotide probes, 485
problems with, 235-236
real-time, 238-240
reverse transcriptase, 236-237
sequence-based typing, 486
sequence-specific primers, 485-486
Polymorphism, 264, 276-277
Pooled components, 156-157, 223-224
Cryoprecipitated AHF, 157, 218, 224, 523
platelets, 156-157, 217, 223-224, 512
Reconstituted Whole Blood, 224
storage, transportation, and expiration of,
217, 218, 223-224
Population genetics, 274-276
Position effect, 272-273
Postoperative blood recovery, 608-609
Posttransfusion platelet recovery, 458,
512
Posttransfusion purpura, 461-462, 671, 688-
690
Postzone effect, 242
Potassium, 573-574, 683-684
PPR. See Posttransfusion platelet
recovery Preadmission testing, 368
Pregnancy
in blood donors, 122
in patient history, 370-371, 392
testing during (See Prenatal studies)
umbilical cord blood recruitment in, 730-
731
Premedication, 547-548, 677, 679-680
Prenatal studies
ABO/Rh testing, 563
antibody detection, 563
antibody identification, 416
antibody titration, 409, 563
DNA-based testing, 283-285
on fetus, 283-284, 330, 563
of paternal samples, 284-285, 563
in pregnant women, 284, 330
in fetal and neonatal immune
thrombocytopenia, 567
Preoperative autologous donation, 131, 605-
606
Preservatives, antibodies to, 417
Pressure devices, 549
Pretransfusion testing
ABO and Rh typing, 375
after non-group-specific transfusions, 386
antibody detection and identification,
375-
378, 381
antiglobulin test, 371-372, 373-374
with autoantibodies, 432, 438-439
autologous control, 377
and blood availability, 384
blood samples for, 368, 370-371, 384,
547 in cold agglutinin disease, 438-439
comparison with previous records, 378
component selection, 375, 378-379
crossmatching, 379-381
donor unit testing, 378
identification of recipients, 368-
370 in massive transfusions, 228,
386
orders for, 381, 383, 384, 546-547
in pediatric recipients, 385, 574-575,
587 reading and interpreting reactions,
372,
381, 382
requests for transfusion, 367-368, 383, 546,
699-700, 703-704
serologic testing, principles of, 371-372
tubeless methods, 377-378
turnaround times, 547
in urgent situations, 227-228, 385-
386 Prevalence, of phenotypes, 274
Preventive action, 26, 27
Primed lymphocyte typing, 486, 487
Primers, PCR, 235-236
Prions, 202-203
Probability values, in antibody
identification, 398, 400
Proband, 265
Problem identification and resolution, 26-29
Procedures, 11, 17, 18, 23
Process
capability, 33
control, 3, 33
flow charts, 27
improvement, 13, 26-29
management, 3-4, 11, 13, 14-16
validation, 14
Processes, 11, 17, 18
Production, principles of, 4
Proficiency testing, 25-26, 91
Propositus, 265
Prospective audits, 699-700, 701-702, 706
Prostate cancer, 791
Protamine, 622
Protein analysis, 240-247
agglutination-based methods for, 241-242
ELISA for, 243-245
flow cytometry for, 246
protein microarrays for, 245
SPRCA for, 242-243
Western blotting for, 245-246
Protein C, 532
Protein microarrays, 245
Prothrombin complex concentrates, 518, 527-
528, 623
 INDEX ■ 831

Prothrombin time, 519-520, Quality oversight, 5-6

521 Proton pump inhibitors,
626 Provenge, 791
Prozone effect, 241-242
PRP. See Platelet-rich plasma
Pseudogenes, 476, 478
Psoralen-treated plasma, 153, 205
PT. See Prothrombin time
PTT. See Partial thromboplastin time
Public antigens, 482-483
Pulmonary disease, in blood donors, 126, 129
Pulmonary edema, 659, 680, 681, 682
Pulse, of donor, 120
Pumps, infusion, 548-549, 584
Q for ABO testing, 298
Quad packs, 576
Qualification
defined, 33
of equipment, 15
of personnel, 7
of suppliers, 9, 779, 780
Quality assurance, 1-2, 34
Quality control, 16
of blood components, 159-160,
171 of copper sulfate solution, 38
defined, 2, 34
of equipment, 36, 37
of HSCs, 723, 736, 739, 741
performance intervals for, 36-38
records of, 16
unacceptable results for, 16
Quality improvement, 3
Quality indicators, 24, 34
Quality management systems
Code of Federal Regulations references, 35
components of, 4-5, 12-13
customer focus, 6-7, 12
documents and records, 13, 16-19
equipment management, 10-11, 13
facilities, work environment and safety, 7,
12
general concepts of, 1-5
human resources, 7-9, 12
information management, 13, 19-20
management of nonconforming events,
13,
20-23
monitoring and assessment, 13, 23-26
organization and leadership, 5-6, 12
process improvement, 13, 26-29
process management, 11, 13, 14-16
quality control performance intervals, 36-
38 suppliers and materials management,
9-10,
12
terminology of, 33-34
Quality planning, 2-3
Quality System Essentials, 2
Quarantine
of collected blood, 157-158
of nonconforming products, 224, 225
of repeatedly reactive units, 187-188, 189
Quarantine FFP, 152
Quarantine release errors, 192, 195-196
R
Radiation safety, 60-63
Raph system, 260, 339, 359
RBCs. See Red Blood Cells
Reagents
antibodies to components of, 298, 300, 417
antiglobulin, 372, 396, 426-427
bovine albumin, 376
chloroquine diphosphate, 407
contamination of, 332
DTT, 405
for elutions, 429
enhancement media, 396, 417
enzymes, 377, 402, 405
glycine-HCl/EDTA, 407
LISS, 376, 402
manufacturers directions for, 11
PEG, 376-377, 402
for phenotyping, 420
quality control intervals for, 38
red cells, 376, 393, 396
for Rh testing, 326, 327, 331-332
sulfhydryl, 407
ZZAP, 407
Real-time PCR, 238-240
Recalls, 89, 90, 782
Recipients
ABO and Rh testing, 297-298, 327-328,
375 antibody detection in, 357-377
baseline assessment of, 546
consent of, 545-546, 601
crossmatching in, 379-381
education of, 546
identification of, 226, 368-370, 384-385,
549, 550, 551
immunocompromised, 190
medical history of, 392, 546
monitoring during and after transfusions,
553, 555
pediatric patients (See Neonates; Pediatric
patients)
phenotyping, 329-330, 401-402
records of, 378, 672
tracing (look-back), 187-188, 189-190, 782
832 ■ AABB T EC HNIC AL MANUAL

of unknown identity, 368, 370

Red Blood Cells, Deglycerolized
weak D in, 327-328, 375
expiration, storage, and transportation of,
Recombination, 271
215, 222
Reconstituted Whole Blood, 224
leukocyte content of, 505
Records
preparation of, 155, 222
altering or correcting, 18-19
quality control of, 222
apheresis, 170, 172
rejuvenated, 216
blood component, 384-385
Red Blood Cells, Frozen
checking before blood issue, 226, 384-385,
expiration, storage, and transportation of,
550
215
comparing testing results to, 378
preparation of, 155
confidentiality of, 19
rejuvenated, 216
donor, 119
thawing and deglycerolizing, 155, 222
electronic, 18, 19
Red cells, in-vitro generation of, 796
HSC transplantation, 640
Red Blood Cells, Leukocytes Reduced, 504-505
management of, 13, 16, 18-19
expiration, storage, and transportation of,
personnel, 19
216
protection of, during disasters, 104, 111 leukocyte content of, 149, 505
quality control, 16 prestorage filtration, 154
storage of, 19 Red Blood Cells (RBCs)
of tissue allografts, 781-782 additive solutions for, 136-137, 138, 576-
transfusion, 226, 555, 640, 672 577
Recovered Plasma, 152-153, 220
age of, 574, 577-578
Red Blood Cell transfusion, 499-507 aliquoting, 224, 575-576
ABO/Rh compatibility of, 375, 378-379, 504, anticoagulant-preservative solutions for,
554, 635 136, 137
in autoimmune hemolytic anemias, 436- bacterial contamination of, 198
437, 439-440, 506 biochemical changes of storage in,
in chronic anemia, 502 221,
dose of, 505-506, 610 503-504
in emergency release, 227-228, 385-386, collected by apheresis, 171-172
506, 555-556 cryopreservation of, 155
exchange transfusion, 579-580 deglycerolized, 155, 215, 222, 505
hemoglobin targets in, 499-501, 505- expiration of, 149, 215-216
506 in hemorrhagic shock, 501-502 frozen, 155, 215, 222
in HSC transplantation, 634 hemoglobin/hematocrit of, 149
of incompatible units, 506-507 irradiated, 156, 215
indications for, 499-502, 574, 575, 578- leukocyte content of, 505
579 leukocytes reduced, 216, 504-505
infusion of, 554, 585 low-volume units, 141, 142,
intrauterine, 563-564 149 pathogen reduction of,
leukocyte reduced, 504-505 205 phenotyping, 420, 588
in massive transfusion, 228, 386, 501- preparation of, 147-148
502, rare, 421
683-685 in red cell exchange, 654
in pediatric patients, 574-580, 586-589 rejuvenated, 216
restrictive vs liberal strategies for, 499-501 storage of, 214, 215-216, 221, 410-411
Red Blood Cells, Apheresis, 171-172 substitutes for, 507
collection of, 168, 175 survival studies of, 419, 506-507
donation requirements for, 120, 122, 123, transfusion of (See Red Blood Cell
171 transfusion)
expiration, storage, and transportation of, transportation and shipping of, 215-216,
216 225
leukocytes reduced, 216 visual inspection of, 149
quality control of, 171 washed, 216, 223, 505, 590-591
records of, 172

Red cell antibodies. See also specific blood
groups
with autoantibodies, 432-435
and with HDFN, 338-340, 347-348, 413-
414,
562
and with hemolytic transfusion reactions,
338-340, 413-414, 686-687
clinical significance of, 338-340, 347-348,
376, 392, 413-414, 419
defined, 391
detection of (See Antibody
detection) disease associations with,
392 distinguishing alloantibodies
from
autoantibodies, 283
dosage effect of, 264, 393, 410
effect of DTT on, 413-414
effect of enzymes on, 413-414
to high-prevalence antigens, 392-393,
394-
395, 415-416, 564
high-titer, low-avidity, 409-410
identification of (See Antibody
identification)
low-affinity, 437
to low-prevalence antigens, 416
multiple, 412, 414
naturally occurring, 391, 392
nonhemolytic, 251-252
in selection of units, 379, 419-421
serologic reactivity of, 413-414
in sickle cell disease, 329-330, 379,
588 in tissue transplant patients, 777
Red cell exchange, 646, 653-654, 655,
675 Red cell losses, in apheresis, 170,
171 Red cell reduction, 720-721
Red cell substitutes, 507
Reference laboratories, 419
Refrigerators, 36, 214
Regenerative medicine, 753-754. See also
Stem cells
Registration
of donors, 118-119
of facilities, 84, 86
Regulatory issues, 83-91
for biological products, 84, 85
for blood-related devices, 83-84, 87
for cellular therapy products, 760, 796-
797
for emergencies, 109-111
FDA inspections, 86-87
hospital regulations and accreditation, 91,
603-604
for HSCs, 87-89, 725, 743-746
licensure and registration, 84, 86
medical laboratory laws and regulations,
89-91
INDEX ■ 833
for quality systems, 1-2
for radioactive materials, 60-61
recalls and withdrawals, 89, 90
safety, 39-40, 60-61, 68-69
for tissue, 777-778
Reissuing blood products, 227-228, 550
Rejuvenated RBCs, 216
Relationship testing, 276-277, 493
Relative risk, 494
Remedial action, 26, 27
Renal failure, 652, 674
Reports
of adverse events related to tissue
grafts, 782
facility quality, 27
of fatalities, 20, 45, 87, 145, 691
of injuries, 45, 87
internal event, 20, 21-22
Requests for transfusion, 367-368, 383, 546,
699-704
Requirement, defined, 34
Respiratory distress, 658-659, 680-681
Restriction fragment length polymorphism
analysis, 238
Retrospective audits, 699, 701-702, 703-704,
707
Reverse transcriptase PCR, 236-237
RFLP. See Restriction fragment length
polymorphism analysis
Rh Immune Globulin, 565-566
antepartum administration of, 564, 565
in antibody identification problems, 392
dosage for, 565-566
positive DAT after, 428
postpartum administration of, 565-566
serology and mechanism of, 566
use in platelet transfusions, 515
Rh system, 317-333
antibodies of, 331, 338
antigens of, 259, 318-319, 321-330
C/ c and E/ e, 328-330
D, 323-328
G, 328
characterization of, 317, 319
clinical considerations for, 328
ethnic differences in, 318-319, 320, 321,
325, 326, 327
genes and proteins of, 259, 272, 273, 319,
320, 321, 325
genotypes, 321, 322-323
haplotypes in, 319, 320, 321-322
phenotypes, 321-323
RhAG, 261, 331, 340, 360
Rhnull, 273, 331
terminology for, 318-319, 319, 320
834 ■ AABB T EC HNIC AL MANUAL
Rh testing
with autoagglutinins, 332, 432, 438
of blood components, 225-226, 285, 327,
328, 378
for C, c, E, e antigens, 322-323
comparison with previous records, 378
in component selection, 379, 515
for D antigen, 327-328, 330
discrepancies in, 328, 333
DNA-based assays, 284-285, 287,
330 false-positive/negative results in,
332 of fetus, 283-284, 330, 563
in hemolytic disease of the fetus and
newborn, 332, 563, 565
in HSC transplantation, 634
in multitransfused patients, 330
in pediatric patients, 332, 385, 574, 587
phenotyping, 322-323
in prenatal evaluation, 284, 330, 563
reagents for, 326, 327, 331-332
of recipients, 327-328, 375
for sickle cell disease patients, 283, 329-
330 for weak D, 327, 328, 375, 565
RhAG system, 261, 319, 331, 340, 360
Rheopheresis, 646, 647
Rheumatoid arthritis, 763
Riboflavin-treated plasma, 153, 205
Risks
assessment of, 98-99, 102
of bleeding, 605
relative, 494
of transfusion, 192-194, 600-601
RNA, 233
Rodgers blood group. See
Chido/Rodgers Rodgers substance, 406
Root cause analysis, 27, 28, 29
Rosette test, 565
Rotational thromboelastometry, 520
Rouleaux
in ABO testing, 298, 300
in antibody detection/identification, 416
in Rh testing, 332
saline replacement technique for, 301,
417
Run charts, 24
S Sequence-based typing, 486
Safe work practices
for biosafety, 52-
53
for chemical safety, 58-
59 for electrical safety,
47 for fire prevention,
47
general guidelines for, 44, 72
for radiation safety, 62
Safety goggles, 71
Safety program
accidents and injuries, 45, 49
biosafety, 47-55, 73
chemical safety, 55-60, 74-82
in disaster management, 104
electrical safety, 46-47
emergency response plan, 44
employee health services, 44-
45 engineering controls, 44, 72
fire prevention, 45-46
first aid and follow-up, 44-45
hazard identification and communication,
43-44
hepatitis prophylaxis, 44
latex allergies, 45
management controls, 42-43, 44
personal protective equipment, 44, 70-71
quality management of, 7, 12
radiation safety, 60-63
regulations and recommendations for, 39-
40, 68-69
safe work practices, 44, 72
safety officers, 42, 56, 61
safety plan, 42
shipping hazardous materials, 63
training, 43
waste management, 63-64
Saline replacement technique, 301, 417
Samples, blood. See Blood samples
Sandwich ELISA, 244
Scianna system, 259, 339, 354
Scoring reactions, 372
SD (solvent/detergent-treated) plasma, 153,
205
Sda antigen, 361-362, 372, 406
Sda substance, 406
Secretors
genetics of, 301, 302, 303, 304-305
linkage with Lutheran group, 271, 272
Security, 104
Sedimenting agents, 172
Segments, of RBCs, 149
Selective absorption, 646
Sepsis, transfusion-associated, 198, 199, 669,
676
Sequence-specific oligonucleotide probes, 485
Sequence-specific primers, 485-486
Serotonin release assay, 466
Serum proteins, in typing discrepancies, 298,
301, 332
Serum-to-cell ratio, 405
Services, critical, 4, 9-10
Sex-linked inheritance, 267-269
 INDEX ■ 835

Sexual contacts, of blood donors, 124, 125, Source Plasma, 170

126 Specific gravity, of blood cells
Sharps injuries, 49, 141 and components, 143
Shipping Specification, defined, 34
blood components, 146-147, 150-151, Spills
213- blood, 53, 54
214, 215-220, 225 chemical, 59, 78-82
containers for, 38, 225, 724-725, radioactive, 62
738 in disaster planning, 107-108 “Splits,” 482
frozen products, 225, 724, 738 SPRCA. See Solid-phase red cell adherence
hazardous materials, 63 testing
HSCs, 723-724, 734, 737-738 SSOP. See Sequence-specific oligonucleotide
labeling, 738 probes
monitoring temperature during, 213-214, SSP. See Sequence-specific primers
724-725, 738, 739, 779-780 Staffing, 9, 108-109, 110-111
MSCs, 761-762 Standard Operating Procedures. See
plasma derivatives, 220 Procedures
samples, 63 Standard precautions, 48
tissue, 220, 779-780 Standards
Shock, 501-502, 674 for biosafety, 47
Short tandem repeat analysis, 277 for performance improvement, 26
Showers, emergency, 58 for quality management, 1- 2
Siblings, 266, 478 for tissue transplantation, 778-779
Sickle cell disease Staphylococcal protein A absorption, 657
alloimmunization in, 329-330, 379, 588, Stem Cell Therapeutic and Research Act, 743
639 Stem cells
blood selection in, 379, 587-588, 654 adipose-derived, 755, 758-759, 762, 794
delayed transfusion reactions in, 588, 687 embryonic, 795
genotyping for, 283, 330 hematopoietic
in HSCT patients, 639 collection of, 718-720, 733-734
in pediatric patients, 587-588, 639 cryopreservation of, 722-723, 736-737
red cell exchange in, 587, 654 donors of, 714-717
separation of transfused from autologous infusion of, 724-725
cells in, 401 irradiation of, 638
transfusion in, 379, 587-588 processing, 720-722
Side effects. See Adverse reactions quality control of, 723, 736
Signs, safety, 46, 48-49, 57, 58 regulation of, 87, 88, 89, 725
Silent mutations, 264, 267, 286-287 shipping and transport of, 723-724, 737-
Single nucleotide polymorphisms, 240, 264 738
Sipuleucel-T, 791 sources of, 717-720
Six Sigma, 28-29 storage of, 736-737
Skeletal repair, with MSCs, 763 thawing, 721
Skin appearance, in donors, 120 washing, 721, 740-741
Skin grafts, 123, 775, 777 induced pluripotent, 755, 795-796
Solid-phase red cell adherence testing mesenchymal
for detection of HLA antibodies, autologous vs allogeneic use, 754
487 future directions for, 765-766
for detection of platelet antibodies, 463- identification criteria for, 793
464 for phenotyping red cells, 242-243 isolation and expansion of, 758-
for platelet crossmatching, 460 760,
for pretransfusion testing, 377, 396 792-793
Soluble substances, 406 multipotentiality of, 754-755, 791-792
Solutions properties of, 756-757, 758, 786, 791-792
additive, 136-137, 138, 576-577 research and development on, 764-765
anticoagulant-preservative, 136, 137 shipping, 761-762
intravenous, 552 sources of, 754-758
Solvent/detergent-treated plasma, 153, 203,
204, 205
836 ■ AABB T EC HNIC AL MANUAL

standardization of methods for, 760

techniques in, 607
storage and banking of, 761
temperature regulation in,
therapeutic applications of, 762-764,
607 transfusion algorithms
793-795 in, 608
and tissue engineering, 795 Survival studies of red cells, 419, 506-507
tumor-forming potential of, 765 Syncope, 144-145
Sterile connection devices, 37, 139, 140, 576 Syntenic genes, 271
Sterility testing, of HSCs, 723, 736 Syphilis, 198
Storage reactive testing results for, 187, 188, 189
of biohazardous material, 52, 54-55 screening blood donors for, 124, 184,
of blood components, 213-214, 215-220, 198 screening tissue donors for, 774
221 Syringe aliquoting devices, 576
biochemical changes in, 221, 503-504, Syringe infusion pumps, 549, 584
511, 513
Cryoprecipitated AHF, 153, 218, 222 T
granulocytes, 173, 217, 221, 525
T-activation, 300
plasma, 151, 218-220
TA-GVHD. See Transfusion-associated
platelets, 150-151, 216-217, 511, 513
graft- vs-host disease
RBCs, 214, 215-216, 221, 503-504
TACO. See Transfusion-associated circulatory
red cell antigen deterioration with,
overload
410- 411
Tattoos, 124
Whole Blood, 147, 215
Temperature
of blood samples, 371
of antibody reactivity, 405, 413-414
in disaster plan, 108
of collected whole blood, 147
equipment for, 214
for component storage, 213-214, 215-220,
of hazardous chemicals, 58-59
221
of HSCs, 736-737
of donors, 120
liquid nitrogen, 723, 737, 761
monitoring systems for, 214
of MSCs, 761-762
of recipients, 546, 676, 677
of plasma derivatives, 220
regulation of, during surgery, 607
of records, 19
for shipping containers, 213-214, 225, 734,
temperature for, 213-214, 215-220, 221
738, 739, 779-780
of tissue grafts, 220, 775, 780-781, 783
Teratogens, 122, 129
Storage lesion, 221, 503-504, 511, 513
TerumoBCT apheresis systems, 168, 174, 175
STR. See Short tandem repeat analysis
Testing. See also specific testing methods;
Stroke, 763
Pretransfusion testing
Stromal-vascular fraction, 759, 762, 763
grading results of, 372
Sulfhydryl reagents, 407, 438
of incoming supplies,
Suppliers, 9-10, 12, 779, 780
10 method validation,
Supplies, critical, 9-10, 11, 107, 108
14
Surgery
point-of-care, 607-608
acute normovolemic hemodilution in, 606
regulations for, 90
anemia assessment before, 604-605
Thalassemia, 588-589, 640
anesthesia in, 607
Thawed Plasma, 151, 152, 219, 221,
assessing bleeding risk in, 605
522 Thawing
blood administration in, 555-556
Cryoprecipitated AHF, 222
blood ordering practices for, 227
devices for, 37
blood recovery in, 606-607, 608-
frozen RBCs, 222
609 deliberate hypotension in, 607
HSCs, 721
fluid management in, 607
plasma, 151, 221
maintaining normothermia in, 607
Therapeutic apheresis
pharmacologic agents in, 608, 620-
adverse effects of, 658-660
627 point-of-care testing in, 607-608
anticoagulation in, 658
positioning in, 607
cytapheresis, 653-654, 655
preoperative autologous donation for,
extracorporeal photopheresis, 646, 654, 656
605- 606 indications for, 646, 647, 648-651, 655, 656,
657

modalities of, 645, 646, 647
patient evaluation in, 660-661
principles of, 645, 646, 647
replacement fluids in, 522, 646, 652
selective absorption, 656-658
therapeutic plasma exchange, 646, 647-
653 vascular access in, 660
Therapeutic plasma exchange, 647-
653 adverse effects of, 658-660
in hemolytic disease of the fetus and
newborn, 564
indications for, 646, 647, 648-651, 651-653
replacement fluids for, 646, 647, 652
Thermal amplitude studies, 419, 438
Thermometers, 37
Thrombocytapheresis, 646, 654, 655
Thrombocytopathy, 509-510
Thrombocytopenia
drug-induced, 462, 465-466, 516
fetal and neonatal alloimmune, 461, 567-
568
heparin-induced, 462, 465-466, 517, 528
immune, 463, 465, 517, 568
management of, 514
in pediatric patients, 580-581
in plasmapheresis, 659
platelet transfusions in, 507-517
posttransfusion purpura, 461-462, 671,
688-
690
thrombotic thrombocytopenic purpura,
517, 522, 652-653, 713
Thrombocytosis, 654
Thromboelastography, 520-521, 608
Thrombosis, in blood donors, 145
Thrombotic thrombocytopenic purpura, 517,
522, 652-653, 713
Time, incubation, 405
Timers, 37
Tissue
autografts, 782-783
collecting, 773, 782-783
expiration of, 220
hospital-based services for, 778-783
infectious disease testing on donors of,
191-
192, 774
oversight responsibility for, 778-779
processing, 775
recall of, 782
receipt and inspection of, 779-780
regulations and standards for, 87, 88, 89,
777-778
standard operating procedures for, 779
storage of, 220, 775, 780-781, 783
suppliers of, 779, 780
traceability and records of, 781-782
transplantation of, 773-777
INDEX ■ 837
ABO compatibility in, 777
adverse events in, 782
antibody development after, 777
background of, 774-775
clinical uses for, 775-777
disease transmission through, 777
donor eligibility for, 773-774
history of, in blood donors, 123,
126 look-back investigations in,
782 and MSCs, 795
organ transplantation, 783
transfusion support for, 783
types of grafts for, 774
transporting, 220, 779-780
Tissue banks and distributors, 777
Tissue engineering, 795
Titration of antibodies, 409-410, 563
Topical hemostatic agents, 608, 625
TPE. See Therapeutic plasma exchange
Traceability, 225, 781-782
Training
biosafety, 48
cGMP and cGTP,
8 chemical safety,
56
disaster, 109, 111-112
electrical safety, 47
fire safety, 46
general safety, 43
new employees, 7-8
radiation safety, 61-62
Traits, 256, 265
TRALI. See Transfusion-related acute lung
injury
Tranexamic acid, 608, 621-622
Trans position, 272
Transcription-mediated amplification, 237-
238
Transfusion-associated circulatory overload,
669, 682-683
Transfusion-associated graft-vs-host disease,
671, 687-688
HLA system in, 489-490, 688
in neonates, 573
Transfusion-associated sepsis, 198, 199, 669,
676
Transfusion committee, 24-25
Transfusion reactions
acute hemolytic, 667, 672-675
air embolus, 669, 685
allergic, 547-548, 667, 678-680
alloimmunization, 670 (See also
Alloimmunization)
anaphylactic, 658, 668, 678-679, 680
biovigilance programs in monitoring, 665-
666
838 ■ AABB T EC HNIC AL MANUAL
clinical evaluation and management of,
666, 667-671
coagulopathy, 591, 684-685
delayed hemolytic, 250, 588, 670, 686-687
febrile nonhemolytic, 489, 548, 667, 677
graft-vs-host disease, 671, 687-688
in HSCT patients, 639
hyperkalemia and hypokalemia, 555, 683-
684
hypocalcemia, 555, 658, 670, 683
hypotension, 659, 669, 678
hypothermia, 548, 572-573, 670, 685-686
identification of, 553, 555, 666
iron overload, 588, 671, 690
laboratory investigation of, 672
nonimmune hemolysis, 669, 675-676
platelet refractoriness, 458-461
posttransfusion purpura, 461-462, 671,
688-
690
records of, 672
reporting, 22
signs and symptoms of, 666, 667-671
TACO, 669, 682-683
TRALI, 668, 680-682
transfusion-related sepsis, 198, 199, 669,
676 Transfusion-related acute lung injury,
468,
489, 668, 680-682
Transfusion-related immunomodulation, 504
Transfusion safety officers, 611
Transfusion thresholds
in pediatric patients, 578-579
for platelet transfusions, 507-509, 581, 589,
636-637
for red cell transfusions, 499-501, 578-
579,
610, 634
Transfusion-transmitted diseases
Babesiosis, 201
chikungunya virus, 202
dengue virus, 202
hepatitis B virus, 192, 194, 196
hepatitis C virus, 192-193, 194, 196-197
human immunodeficiency virus, 194-196
human T-cell lymphotropic virus, 197
malaria, 201-202
parvovirus B19, 203
prions, 202-203
safety precautions for, 47-55
syphilis, 198
Trypanosoma cruzi, 200-201
West Nile virus, 200
Transfusions
administration procedures for (See Blood
administration)
algorithms for, 608
auditing, 25, 697-707
in blood donors, 123, 125
chimerism after, 489-490
consent for, 545-546, 601
costs of, 602
of Cryoprecipitated AHF, 522-523, 583-584
during disasters, 101
documentation of, 226, 555
exchange, 564, 574, 579-580
fatalities due to, 20, 551, 681, 691
of granulocytes, 173, 523-526, 584
guidelines for, 611, 704
and HLA system, 488-490
in HSC transplantation, 633-
640 intrauterine, 563-564
massive, 228, 386, 501-502, 591, 683-685
in medical history, 123, 370-371, 392, 417-
418, 428, 546
monitoring appropriateness of, 697-707
non-group-specific, 396
in operating room and trauma, 555-556
in organ transplantation, 783
out-of-hospital, 556
in pediatric patients, 574-591
of plasma, 517-522, 582-583
of plasma derivatives, 526-532
of platelets, 507-517, 580-581
of RBCs, 499-507, 574-580
requests for, 367-368, 383, 546, 603-604,
699-700
risks of, 192-194, 600-601
selection of components for, 375, 378-379
in urgent situations, 227-228, 385-386, 506,
555-556
of Whole Blood, 502-503
Transmissible spongiform encephalopathy,
202-203
Transplantation. See specific types
of transplantation
Transportation
of blood components, 146-147, 150-151,
213-214, 215-220, 225
containers for, 38, 225, 724-
725 in disaster plan, 107-108
of frozen products, 225, 724,
738 of hazardous materials, 63
of HSCs, 723-724, 734, 737-738
labeling requirements, 738
monitoring temperatures during, 213-214,
738, 739, 779-780
of MSCs, 761-762
of plasma derivatives, 220
of samples, 63
of tissue, 220, 779-780
Trauma, blood administration in, 555-556
Travel, by blood donors, 125, 126, 202

Treponema pallidum, 191, 198
TRIM. See Transfusion-related
immunomodulation
Trypanosoma cruzi, 184, 187, 190, 192, 200-
201 TTP. See Thrombotic thrombocytopenic
purpura
Tumorigenicity, of stem cells, 765, 796
Two-unit red cell collection, 120, 122, 123,
168,
171-172
Type and crossmatch, 383, 384
Type and hold, 381, 383
Type and screen, 382, 383, 384
U Vascular access
UCB. See Umbilical cord blood
Ulex europaeus lectin, 295
Umbilical cord blood
antigen expression on, 410, 411
DAT testing on, 427
for transplantation
advantages of, 729
cell expansion, 722
collection, 733-734
consent for collection, 731
cryopreservation, 736-737
donor recruitment for, 730-731
donor testing, 715, 716, 717, 732-
733 economic issues regarding, 742-
743 engraftment kinetics, 717-718
health history and medical evaluation,
731-732
HLA matching, 716, 736
infusion, 741-742
labeling, 738, 739
legislation regarding, 743
patient survival, 718
post-thaw testing, 737
processing, 734-736
quality control testing, 736, 739, 741
receipt of, 738-740
regulations and standards, 87, 88, 89,
743-746
shipping, 734, 737-738
storing, 736-737
thawing and washing, 721, 740-741
Umbilical cord blood banks, 729-730, 742-743
Uniforms, 70
United Network for Organ Sharing, 783
Urgent release of blood, 227-228, 385-386,
506,
555-556
Urine neutralization, 362, 406
Urticaria (hives), 667, 678, 679-680
Utilities, in disaster plan, 108
Utilization of blood. See Blood utilization
review
INDEX ■ 839
V
Vaccines, 44, 123, 791
Validation
of computer systems, 15-16
definition of, 34
of equipment, 15
of processes, 14
of shipping containers, 147,
225 of test methods, 14
validation plans, 14-15
Vapors, hazardous, 59
Variable number of tandem repeats (VNTR),
277
for apheresis, 660
in pediatric patients, 547, 580, 584
for transfusions, 547
Vasovagal reactions, 143-145, 169, 659, 678
Vector-borne diseases, 199-202
Vel system, 261, 340, 361
Venipuncture, 140-141
Verification, defined, 34
Viability assays for HSCs, 723, 736
View boxes, 37
Viruses
donor screening for, 194-197, 200
transmitted by tissue transplants, 774, 782
Viscoelastic coagulation testing, 520-521, 608
Viscosity, plasma, 652
Vital signs, 546, 553, 555, 724, 742
Vitamin K, 518, 622
Volume of blood
in exchange transfusions, 580
in intrauterine transfusions, 564
in neonatal transfusions, 578
in pediatric patients, 572
in whole blood collections, 120, 141-142, 143
Volume overload. See Transfusion-associated
circulatory overload
Volume-reduction
of HSCs, 720
of platelets, 157, 223, 513-514, 581-582, 590
von Willebrand disease, 129, 523, 524, 584
von Willebrand factor, 154, 523
W
Warfarin, 518, 519, 622
Warm autoantibodies
ABO testing with, 432
adsorption of, 432-435
with alloantibodies, 418-419, 432-435, 438-
439
antibody identification with, 418-
419 disease associations with, 392
840 ■ AABB T EC HNIC AL MANUAL

mimicking alloantibodies, 434-435

adverse donor reactions in, 142-
in mixed-type AIHA, 439
146 containers for, 136-139, 140
in phenotyping problems,
donation intervals for, 120, 122
401 Rh testing with, 332,
donor and blood identification in, 139-
432
140
specificity of, 435
donor care after, 142
transfusion with, 436-437, 506
handling after, 146-147
in warm AIHA, 431-435 phlebotomy, 140-141
Warm autoimmune hemolytic anemia, 430-
process of, 141
437
volume collected, 120, 141-142, 143
adsorption testing in, 432-435
expiration of, 148-149, 215
autoantibody specificity in, 435
hematocrit of, 148
blood selection in, 435-436
indications for, 502-503
serologic characteristics of, 431-432
irradiated, 215
serologic problems of, 432
leukocyte content of, 505
transfusions in, 436-437
processing, 146, 147-148
Warmers, blood, 37, 548, 572, 584, 685-686
reconstituted, 149, 224
Washed components, 223
storage of, 215
HSCs, 721, 740-741
temperature for, 147
for pediatric patients, 573-574, 590-591
transportation of, 146-147, 215, 225
platelets, 223, 590-591, 639
Wipe tests, 61
RBCs, 216, 223, 505, 590-591, 639
Withdrawals, 89, 90
Waste management, 63-64
WNV. See West Nile virus
biohazardous, 53-55
Work environment, 7, 12, 39-42
chemical, 60
Work instructions, 17
disposal, 54-55
Wristbands, patient, 368
radioactive, 63
Wrong blood in tube, 378, 384
treating, 55
Waterbaths, 37 X-Z
WB. See Whole Blood
Weak D, 323-324 X-borne genes, 267-269
in donors, 285, 327, 328 X chromosome inactivation, 258, 261
in fetus, 565 Xenografts, 775
in prenatal patients, 565 Xg system, 354
in recipients, 327-328, antibodies of, 339, 354
375 genes and antigens of, 259, 354
testing for, 327, 328, 375, 565 inheritance pattern of, 258, 268
West Nile virus, 200 XK gene, 258, 260, 261, 269, 348
reactive testing results for, 187, 188, 190 Yt system, 259, 338, 354
screening donors for, 184, 186, 192, 200 Zygosity, 264, 393, 410
Western blotting, 245-246 ZZAP, 407, 438
Whole Blood, 148-149
ABO compatibility of, 375
collection of, 135-146