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Denise Harmening - Modern Blood Banking & Transfusion Practices-F. A. Davis Company (2012) PDF
Denise Harmening - Modern Blood Banking & Transfusion Practices-F. A. Davis Company (2012) PDF
k KEL2 98.8 100 strong RBC only occ Enz. → AET+ ↓ ZZAP ↓++
Kpa KEL3 2 rare strong RBC only occ Enz. → AET+ ↓ ZZAP ↓++
Kell Kpb KEL4 99.9 100 strong RBC only occ Enz. → AET+ ↓ ZZAP ↓++
Jsa KEL6 .01 20 strong RBC only occ Enz. → AET+ ↓ ZZAP ↓++
Jsb KEL7 99.9 99 strong RBC only occ Enz. → AET+ ↓ ZZAP ↓++
†Kx — 99.9 99.9 weak RBC low occ Enz. → AET+ ↓ ZZAP ↓++
*This chart is to be used for general information only. Please refer to the appropriate chapter for more detailed information.
AET = 2-aminoethylisthiouronium bromide; ↑ = enhanced reactivity; → = no effect; ↓ = depressed reactivity; occ = occasionally; CGD = chronic granulomatious disease;
HDN = hemolytic disease of the newborn; HTR = hemolytic transfusion reaction; NRBC = non-red blood cell; RBC = red blood cell; WBC = white blood cell; ZZAP = dithiothreitol
plus papain.
• No human antibody to FY6 has been reported.
† It has been found that Kx is inherited independently of the Kell system; consequently it is no longer referred to as K15.
2682_IFC 22/05/12 12:11 PM Page 3
ANTIBODIES
Immunoglobin Clinical
Serology Comp. Class Optimum Significance
Stimulation Saline AHG Binding IgM IgG Temperature HTR HDN Comments
RBC occ yes no occ yes warm yes yes Very rarely IgA anti-D may be
produced; however, this is
invariably with IgG.
RBC occ yes no occ yes warm yes yes
RBC/NRBC occ yes no occ yes warm yes yes Anti-E may often occur without
obvious immune stimulation.
RBC occ yes no occ yes warm yes yes
RBC occ yes no occ yes warm yes yes Warm autoantibodies often appear
to have anti-e-like specificity.
RBC occ yes no occ yes warm yes yes
RBC occ yes no occ yes warm yes yes
RBC/NRBC occ yes no occ yes warm yes yes Anti-Cw may often occur without
obvious immune stimulation.
RBC occ yes no occ yes warm yes yes
RBC occ yes no occ yes warm yes yes Antibodies to V and VS present
problems only in the black population,
where the antigen frequencies are in
the order of 30 to 32.
RBC occ yes no occ yes warm yes yes
RBC occ yes some occ yes warm yes yes Some antibodies to Kell system have
been reported to react poorly in
low ionic media.
RBC no yes no rarely yes warm yes yes
RBC no yes no no yes warm yes yes Kell system antigens are destroyed
by AET and by ZZAP.
RBC rarely yes no rarely yes warm yes yes
RBC rarely yes no rarely yes warm yes yes Anti-K1 has been reported to occur
following bacterial infection.
RBC no yes no no yes warm yes yes
RBC no yes no occ yes warm yes yes The lack of Kx expression on RBCs
and WBCs has been associated with
the McLeod phenotype and CGD.
RBC rare yes some rare yes warm yes yes Fya and Fyb antigens are destroyed by
enzymes. Fy (a–b–) cells are resistant
to invasion by P. vivax merozoites, a
malaria-causing parasite.
RBC rare yes some rare yes warm yes yes
RBC no yes rarely no yes warm yes yes FY3 and 5 are not destroyed by
enzymes.
RBC no yes ? no yes warm FY5 may be formed by interaction of
Rh and Duffy gene products.
FY6 is a monoclonal antibody which
reacts with most human red cells
except Fy(a–b–) and is responsible
for susceptibility of cells to penetration
by P. vivax.
(Continued on inside back cover)
2682_FM_i-xvi 22/05/12 2:12 PM Page i
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Modern Blood
Banking &
Transfusion Practices
SIXTH EDITION
F. A. Davis Company
1915 Arch Street
Philadelphia, PA 19103
www.fadavis.com
Copyright © 2012 by F. A. Davis Company. All rights reserved. This product is protected by copyright. No part of it may be reproduced, stored in a retrieval
system, or transmitted in any form or by any means, electronic, mechanical, photocopying, recording, or otherwise, without written permission from the
publisher.
As new scientific information becomes available through basic and clinical research, recommended treatments and drug therapies undergo changes. The
author(s) and publisher have done everything possible to make this book accurate, up to date, and in accord with accepted standards at the time of publi-
cation. The author(s), editors, and publisher are not responsible for errors or omissions or for consequences from application of the book, and make no
warranty, expressed or implied, in regard to the contents of the book. Any practice described in this book should be applied by the reader in accordance
with professional standards of care used in regard to the unique circumstances that may apply in each situation. The reader is advised always to check
product information (package inserts) for changes and new information regarding dose and contraindications before administering any drug. Caution is
especially urged when using new or infrequently ordered drugs.
Modern blood banking & transfusion practices / [edited by] Denise Harmening.—6th ed.
p. ; cm.
Modern blood banking and transfusion practices
Rev. ed. of: Modern blood banking and transfusion practices / [edited by] Denise M. Harmening. c2005.
Includes bibliographical references and index.
ISBN 978-0-8036-2682-9–ISBN 0-8036-2682-7
I. Harmening, Denise. II. Modern blood banking and transfusion practices. III. Title: Modern blood banking and transfusion practices.
[DNLM: 1. Blood Banks—methods. 2. Blood Grouping and Crossmatching. 3. Blood Transfusion—methods. WH 460]
615'.39—dc23
2011047863
Authorization to photocopy items for internal or personal use, or the internal or personal use of specific clients, is granted by F. A. Davis Company for users
registered with the Copyright Clearance Center (CCC) Transactional Reporting Service, provided that the fee of $.25 per copy is paid directly to CCC, 222
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arranged. The fee code for users of the Transactional Reporting Service is: / + $.25.
2682_FM_i-xvi 22/05/12 2:12 PM Page v
Foreword
Blood groups were discovered more than 100 years ago, but book, I would guess that her teaching philosophies are close
most of them have been recognized only in the past 50 years. to my own. She has gathered a group of experienced scien-
Although transfusion therapy was used soon after the ABO tists and teachers who, along with herself, cover all of the
blood groups were discovered, it was not until after World important areas of blood transfusion science.
War II that blood transfusion science really started to become The chapters included in Part I, “Fundamental Concepts”
an important branch of medical science in its own right. In (including a section on molecular phenotyping), provide a
order to advance, transfusion science needs to be nurtured firm base on which the student can learn the practical and
with a steady flow of new knowledge generated from re- technical importance of the other chapters. The chapters in
search. This knowledge then must be applied at the bench. Part II, “Blood Groups and Serologic Testing,” and Part III,
To understand and best take advantage of the continual “Transfusion Practice” (including a new chapter on cellular
flow of new information generated by blood transfusion sci- therapy), provide enough information for medical technol-
entists and to apply it to everyday work in the blood bank, ogists without overwhelming them with esoteric and clinical
technologists and pathologists must have a solid understand- details. Part IV covers leukocyte antigens and relationship
ing of basic immunology, genetics, biochemistry (particularly (parentage) testing. The chapters in Part V, “Quality and
membrane chemistry), and the physiology and function of Compliance Issues” (including new chapters on utilization
blood cells. High standards are always expected and strived management and tissue banking), complete the scope of
for by technologists who work in blood banks and transfu- transfusion science. Part VI: Future Trends describes tissue
sion services. I strongly believe that technologists should un- banking as a new role for the transfusion service.
derstand the principles behind the tests they are performing, Although this book is designed primarily for medical
rather than performing tasks as a machine does. technologists, I believe it is admirably suited to pathology
Because of this, I do not think that “cookbook” technical residents, hematology fellows, and others who want to
manuals have much value in teaching technologists; they do review any aspect of modern blood banking and transfusion
have a place as reference books in the laboratory. During the practices.
years (too many to put in print) that I have been involved in
teaching medical technologists, it has been very difficult to GEORGE GARRATTY, PhD, FIBMS, FRCPath
select one book that covers all of the information that tech- Scientific Director
nologists in training need to know about blood transfusion American Red Cross Blood Services
science without confusing them. Southern California Region
Dr. Denise Harmening has produced that single volume. and
She has been involved in teaching medical technologists for Clinical Professor of Pathology and Laboratory Medicine
most of her career. After seeing how she has arranged this University of California, Los Angeles
vii
2682_FM_i-xvi 22/05/12 2:12 PM Page viii
2682_FM_i-xvi 22/05/12 2:12 PM Page ix
Preface
This book is designed to provide the medical technologist, basic concepts of genetics, blood group immunology, and
blood bank specialist, and resident with a concise and thor- molecular biology (including molecular phenotyping). Part II
ough guide to transfusion practices and immunohematology. focuses on blood groups and routine blood bank practices
This text, a perfect “crossmatch” of theory and practice, pro- and includes the chapters “Detection and Identification of
vides the reader with a working knowledge of routine blood Antibodies” and “Pretransfusion Testing.” It also covers cur-
banking. Forty-four contributors from across the country rent technologies and automation.
have shared their knowledge and expertise in 28 compre- Part III, “Transfusion Practice,” includes a new chapter
hensive chapters. More than 500 illustrations and tables called “Cellular Therapy” and covers the more traditional
facilitate the comprehension of difficult concepts not rou- topics of donor screening, component preparation, transfu-
tinely illustrated in other texts. In addition, color plates pro- sion therapy, transfusion reactions, and apheresis. Certain
vide a means for standardizing the reading of agglutination clinical situations that are particularly relevant to blood
reactions. banking are also discussed in detail in this section, including
Several features of this textbook offer great appeal to stu- hemolytic disease of the fetus and newborn, autoimmune
dents and educators, including chapter outlines and educa- hemolytic anemias, and transfusion-transmitted diseases.
tional objectives at the beginning of each chapter; case The human leukocyte antigens system and relationship
studies, review questions, and summary charts at the end of testing are discussed in Part IV of the book. In Part V, quality
each chapter; and an extensive and convenient glossary for and compliance issues are discussed, including a new chapter
easy access to definitions of blood bank terms. on utilization management. The chapters on quality man-
A blood group Antigen-Antibody Characteristic Chart is agement, transfusion safety and federal regulatory require-
provided on the inside cover of the book to aid in retention ments, laboratory information systems, and legal and ethical
of the vast amount of information and serve as a review of considerations complete the scope of practice for transfusion
the characteristics of the blood group systems. Original, services. Also included is the chapter “Tissue Banking: A
comprehensive step-by-step illustrations of ABO forward and New Role for the Transfusion Service,” which introduces an-
reverse grouping, not found in any other book, help the stu- other responsibility already in place in several institutions.
dent to master this important testing, which represents the This book is a culmination of the tremendous efforts of
foundation of blood banking. many dedicated professionals who participated in this project
The sixth edition has been reorganized and divided into by donating their time and expertise because they care about
the following sections: the blood bank profession. The book’s intention is to foster
improved patient care by providing the reader with a basic
• Part I: Fundamental Concepts
understanding of modern blood banking and transfusion
• Part II: Blood Groups and Serologic Testing
practices. The sixth edition is designed to generate an
• Part III: Transfusion Practice
unquenchable thirst for knowledge in all medical technolo-
• Part IV: Leukocyte Antigens and Relationship Testing
gists, blood bankers, and practitioners, whose education,
• Part V: Quality and Compliance Issues
knowledge, and skills provide the public with excellent
• Part VI: Future Trends
health care.
In Part I, the introduction to the historical aspects of red
blood cell and platelet preservation serves as a prelude to the DENISE M. HARMENING, PhD, MT(ASCP)
ix
2682_FM_i-xvi 22/05/12 2:12 PM Page x
Contributors
x
2682_FM_i-xvi 22/05/12 2:12 PM Page xi
Contributors xi
xii Contributors
Reviewers
Terese M. Abreu, MA, MLS(ASCP)CM Wyenona Hicks, MS, MT(ASCP)SBB Judith S. Levitt, MT(ASCP)SBB
Director, Clinical Laboratory Science Program Assistant Professor, Program in Clinical Laboratory Clinical Laboratory Manager
College of Arts and Sciences Sciences DeGowin Blood Center, Department of Pathology
Heritage University College of Allied Health Sciences University of Iowa Hospitals and Clinics
Toppenish, Washington, USA University of Tennessee Health Science Center Iowa City, Iowa, USA
Memphis, Tennessee, USA
Deborah Brock, MHS, MT(ASCP)SH Adjunct Faculty, Online Specialist in Blood Bank Beverly A. Marotto, MT(ASCP)SBB
Instructor, Medical Laboratory Technology Program (SBB) Certificate Program Blood Bank Manager, Blood Bank Department
Allied Health Department Rush University Lahey Clinic
Faculty Liaison for Professional Development Chicago, Illinois, USA Burlington, Massachusetts, USA
Academic Affairs Department
Tri-County Technical College
Shelly Hitchcox, RT (CSLT) Tina McDaniel, MA, MT(ASCP)
Medical Technologist Program Director, Medical Laboratory Technology
Pendleton, South Carolina, USA
Blood Bank Department School of Health, Wellness, & Public Safety
Lynne Brodeur, MA, BS (CLS) Fletcher Allen Healthcare Davidson County Community College
Lecturer Burlington, Vermont, USA Thomasville, North Carolina, USA
Department of Medical Laboratory Science
College of Arts & Sciences
Judith A. Honsinger, MT(ASCP) Dora E. Meraz, MEd, MT(ASCP)
Associate Professor Laboratory Coordinator, Clinical Laboratory Sciences
University of Massachusetts–Dartmouth
Health & Human Services Department Program
North Dartmouth, Massachusetts, USA
River Valley Community College College of Health Sciences
Cynthia Callahan, MEd, MLS(ASCP) Claremont, New Hampshire, USA The University of Texas at El Paso
Program Head, Medical Laboratory Technology El Paso, Texas, USA
School of Health & Public Services
Fang Yao Stephen Hou, MB(ASCP)QCYM, PhD
Stanly Community College
Assistant Professor, Clinical Laboratory Science Gretchen L. Miller, MS, MT(ASCP)
Department MLT Program Director, Assistant Professor
Locust, North Carolina, USA
College of Health Sciences Brevard Community College
Marquette University Heath Science Institute
Kay Doyle, PhD, MLS(ASCP)CM
Milwaukee, Wisconsin, USA Cocoa, Florida, USA
Professor and Program Director, Clinical Laboratory
Sciences/Medical
Laboratory Science Stephen M. Johnson, MS, MT(ASCP) Janis Nossaman, MT(ASCP)SBB
Department of Clinical Laboratory and Nutritional Program Director, School of Medical Technology Manager, Donor Collections and Transfusion
Sciences Saint Vincent Health Center Services
University of Massachusetts–Lowell Erie, Pennsylvania, USA Exempla St. Joseph Hospital
Lowell, Massachusetts, USA Denver, Colorado, USA
Vanessa Jones Johnson, MBA, MA, MT(ASCP)
Joyce C. Foreman, MS(CLS), MT(ASCP)SBB Program Director, Pathology & Laboratory Medicine Karen P. O’Connor, MT(ASCP)SBB
Blood Bank Team Leader Service Laboratory Instructor, Department of Medical
Clinical Laboratory Department Overton Brooks VA Medical Center Technology
Baptist Medical Center South Shreveport, Louisiana, USA College of Health Sciences
Montgomery, Alabama, USA University of Delaware
Douglas D Kikendall, MT(ASCP) Newark, Delaware, USA
Michelle Lancaster Gagan, MSHS, MT(ASCP) Blood Bank/Phlebotomy Supervisor
Instructor/Education Coordinator CLS Instructor, Blood Bank Department Janet Oja, CLS (NCA)
Medical Laboratory Technology Program Yakima Regional Hospital Immunohematology Instructor
Health and Human Services Department Yakima, Washington, USA Department of Medical Laboratory Sciences
York Technical College Weber State University
Rock Hill, South Carolina, USA Ogden, Utah, USA
xiii
2682_FM_i-xvi 22/05/12 2:12 PM Page xiv
xiv Reviewers
Susan H. Peacock, MSW, MT(ASCP)SBB, Barbara J. Tubby, MSEd, BS, MT(ASCP)SBB Meridee Van Draska, MLS(ASCP)
CQA(ASQ) Supervisor of Blood Bank Program Director, Medical Laboratory Science
Manager, Quality Assurance Department Guthrie Health Department of Health Sciences
Gulf Coast Regional Blood Center Sayre, Pennsylvania, USA Illinois State University
Houston, Texas, USA Normal, Illinois, USA
Amber G Tuten, MEd, MT(ASCP),
Emily A. Schmidt, MLS(ASCP)CM DLM(ASCP)CM
Clinical Instructor, School of Medical Technology Assistant Professor, Clinical Laboratory Science
Alverno Clinical Laboratory at St. Francis Hospital Program
and Health Centers Thomas University
Beech Grove, Indiana, USA Thomasville, Georgia, USA
2682_FM_i-xvi 22/05/12 2:12 PM Page xv
Contents
xv
2682_FM_i-xvi 22/05/12 2:12 PM Page xvi
xvi Contents
Chapter 6: The ABO Blood Group System • Procedure 6-1: Determination of the Secretor Property
Chapter 8: Blood Group Terminology and the • Procedure 8-1: Plasma Inhibition Studies
Other Blood Groups
Chapter 10: Pretransfusion Testing • Procedure 10-1: Preparation of Washed “Dry” Button of RBCs for Serologic Tests
• Procedure 10-2: Model One-Tube-Per-Donor-Unit Crossmatch Procedure
• Procedure 10-3: Saline Replacement Procedure
Chapter 20: Autoimmune Hemolytic Anemias • Procedure 20-1: Use of Thiol Reagents to Disperse Autoagglutination
• Procedure 20-2: Cold Autoadsorption
• Procedure 20-3: Prewarm Technique for Testing Serum Containing Cold Agglutinins
• Procedure 20-4: Adsorption of Cold Autoantibodies with Rabbit Erythrocyte Stroma
• Procedure 20-5: Dissociation of IgG by Chloroquine
• Procedure 20-6: Digitonin-Acid Elution
• Procedure 20-7: Autologous Adsorption of Warm Reactive Autoantibodies Application
• Heat and Enzyme Method
• ZZAP Method
• Procedure 20-8: Demonstration of Drug-Induced Immune Complex Formation
• Procedure 20-9: Detection of Antibodies to Penicillin or Cephalothin
• Procedure 20-10: EDTA/Glycine Acid (EGA) Method to Remove Antibodies from RBCs
• Procedure 20-11: Separation of Transfused from Autologous RBCs by Simple Centrifugation:
Reticulocyte Harvesting
Chapter 1
Red Blood Cell and Platelet Preservation:
Historical Perspectives and Current Trends
Denise M. Harmening, PhD, MT(ASCP) and Valerie Dietz Polansky, MEd,
MLS(ASCP)CM
OBJECTIVES
1. List the major developments in the history of transfusion medicine.
2. Describe several biological properties of red blood cells (RBC) that can affect post-transfusion survival.
3. Identify the metabolic pathways that are essential for normal RBC function and survival.
4. Define the hemoglobin-oxygen dissociation curve, including how it is related to the delivery of oxygen to tissues by transfused
RBCs.
5. Explain how transfusion of stored blood can cause a shift to the left of the hemoglobin-oxygen dissociation curve.
6. State two FDA criteria that are used to evaluate new preservation solutions and storage containers.
7. State the temperature for storage of RBCs in the liquid state.
Continued
1
2682_Ch01_001-025 28/05/12 12:22 PM Page 2
OBJECTIVES—cont’d
8. Define storage lesion and list the associated biochemical changes.
9. Explain the importance of 2,3-DPG levels in transfused blood, including what happens to levels post-transfusion and which
factors are involved.
10. Name the approved anticoagulant preservative solutions, explain the function of each ingredient, and state the maximum
storage time for RBCs collected in each.
11. Name the additive solutions licensed in the United States, list the common ingredients, and describe the function of each
ingredient.
12. Explain how additive solutions are used and list their advantages.
13. Explain rejuvenation of RBCs.
14. List the name and composition of the FDA-approved rejuvenation solution and state the storage time following rejuvenation.
15. Define the platelet storage lesion.
16. Describe the indications for platelet transfusion and the importance of the corrected count increment (CCI).
17. Explain the storage requirements for platelets, including rationale.
18. Explain the swirling phenomenon and its significance.
19. List the two major reasons why platelet storage is limited to 5 days in the United States.
20. List the various ways that blood banks in the United States meet AABB Standard 5.1.5.1: “The blood bank or transfusion service
shall have methods to limit and to detect or inactivate bacteria in all platelet components.”
21. Explain the use and advantages of platelet additive solutions (PASs), and name one that is approved for use in the
United States.
Chapter 1 Red Blood Cell and Platelet Preservation: Historical Perspectives and Current Trends 3
program Dr. Drew established became the model for the paid and provide nearly all of the blood used for transfusion
national volunteer blood donor program of the American in the United States.
Red Cross.1 Traditionally, the amount of whole blood in a unit has been
In 1943, Loutit and Mollison of England introduced the 450 mL +/–10% of blood (1 pint). More recently, 500 mL
formula for the preservative acid-citrate-dextrose (ACD). +/–10% of blood are being collected. This has provided a small
Efforts in several countries resulted in the landmark publi- increase in the various components. Modified plastic collection
cation of the July 1947 issue of the Journal of Clinical Inves- systems are used when collecting 500 mL of blood, with the
tigation, which devoted nearly a dozen papers to blood volume of anticoagulant-preservative solution being increased
preservation. Hospitals responded immediately, and in 1947, from 63 mL to 70 mL. For a 110-pound donor, a maximum
blood banks were established in many major cities of the volume of 525 mL can be collected, including samples drawn
United States; subsequently, transfusion became commonplace. for processing.5 The total blood volume of most adults is 10 to
The daily occurrence of transfusions led to the discov- 12 pints, and donors can replenish the fluid lost from the
ery of numerous blood group systems. Antibody identifi- 1-pint donation in 24 hours. The donor’s red cells are replaced
cation surged to the forefront as sophisticated techniques within 1 to 2 months after donation. A volunteer donor can
were developed. The interested student can review historic donate whole blood every 8 weeks.
events during World War II in Kendrick’s Blood Program Units of the whole blood collected can be separated into
in World War II, Historical Note.2 In 1957, Gibson intro- three components: packed RBCs, platelets, and plasma. In
duced an improved preservative solution called citrate- recent years, less whole blood has been used to prepare
phosphate-dextrose (CPD), which was less acidic and platelets with the increased utilization of apheresis platelets.
eventually replaced ACD as the standard preservative used Hence, many units are converted only into RBCs and plasma.
for blood storage. The plasma can be converted by cryoprecipitation to a clot-
Frequent transfusions and the massive use of blood soon ting factor concentrate that is rich in antihemophilic factor
resulted in new problems, such as circulatory overload. (AHF, factor VIII; refer to Chapter 13). A unit of whole
Component therapy has solved these problems. Before, a sin- blood–prepared RBCs may be stored for 21 to 42 days, de-
gle unit of whole blood could serve only one patient. With pending on the anticoagulant-preservative solution used
component therapy, however, one unit may be used for mul- when the whole blood unit was collected, and whether a pre-
tiple transfusions. Today, physicians can select the specific serving solution is added to the separated RBCs. Although
component for their patient’s particular needs without risk- most people assume that donated blood is free because most
ing the inherent hazards of whole blood transfusions. Physi- blood-collecting organizations are nonprofit, a fee is still
cians can transfuse only the required fraction in the charged for each unit to cover the costs associated with col-
concentrated form, without overloading the circulation. lecting, storing, testing, and transfusing blood.
Appropriate blood component therapy now provides more The donation process consists of three steps or processes
effective treatment and more complete use of blood products (Box 1–1):
(see Chapter 13, “Donor Screening and Component Prepa-
1. Educational reading materials
ration”). Extensive use of blood during this period, coupled
2. The donor health history questionnaire
with component separation, led to increased comprehension
3. The abbreviated physical examination
of erythrocyte metabolism and a new awareness of the prob-
lems associated with RBC storage.
The donation process, especially steps 1 and 2, has been RBC Membrane
carefully modified over time to allow for the rejection of
donors who may transmit transfusion-associated disease to Basic Concepts
recipients. For a more detailed description of donor screen-
ing and processing, refer to Chapter 13. The RBC membrane represents a semipermeable lipid bi-
The nation’s blood supply is safer than it has ever been layer supported by a meshlike protein cytoskeleton struc-
because of the donation process and extensive laboratory ture (Fig. 1–1).8 Phospholipids, the main lipid components
screening (testing) of blood. Currently, 10 screening tests for of the membrane, are arranged in a bilayer structure com-
infectious disease are performed on each unit of donated blood prising the framework in which globular proteins traverse
(Table 1–1). The current risk of transfusion-transmitted and move. Proteins that extend from the outer surface and
hepatitis C virus (HCV) is 1 in 1,390,000, and for hepatitis B span the entire membrane to the inner cytoplasmic side of
virus (HBV), it is between 1 in 200,000 and 1 in 500,000, the RBC are termed integral membrane proteins. Beneath
respectively.6,7 the lipid bilayer, a second class of membrane proteins, called
The use of nucleic acid amplification testing (NAT), peripheral proteins, is located and limited to the cytoplasmic
licensed by the Food and Drug Administration (FDA) since surface of the membrane forming the RBC cytoskeleton.8
2002, is one reason for the increased safety of the blood supply.
Refer to Chapter 18, “Transfusion-Transmitted Diseases” for a
detailed discussion of transfusion-transmitted viruses.
Advanced Concepts
RBC Biology and Preservation Both proteins and lipids are organized asymmetrically within
the RBC membrane. Lipids are not equally distributed in the
Three areas of RBC biology are crucial for normal erythrocyte
two layers of the membrane. The external layer is rich in gly-
survival and function:
colipids and choline phospholipids.9 The internal cytoplas-
1. Normal chemical composition and structure of the RBC mic layer of the membrane is rich in amino phospholipids.9
membrane The biochemical composition of the RBC membrane is ap-
2. Hemoglobin structure and function proximately 52% protein, 40% lipid, and 8% carbohydrate.10
3. RBC metabolism As mentioned previously, the normal chemical compo-
sition and the structural arrangement and molecular inter-
Defects in any or all of these areas will result in RBCs sur-
actions of the erythrocyte membrane are crucial to the
viving less than the normal 120 days in circulation.
normal length of RBC survival of 120 days in circulation.
In addition, they maintain a critical role in two important
RBC characteristics: deformability and permeability.
Table 1–1 Current Donor Screening Tests Deformability
for Infectious Diseases To remain viable, normal RBCs must also remain flexible,
TEST DATE TEST REQUIRED deformable, and permeable. The loss of adenosine triphos-
phate (ATP) (energy) levels leads to a decrease in the phos-
Syphilis 1950s phorylation of spectrin and, in turn, a loss of membrane
Hepatitis B surface antigen (HBsAg) 1971 deformability.9 An accumulation or increase in deposition
of membrane calcium also results, causing an increase in
Hepatitis B core antibody (anti-HBc) 1986 membrane rigidity and loss of pliability. These cells are at a
Hepatitis C virus antibody (anti-HCV) 1990 marked disadvantage when they pass through the small
(3 to 5 µm in diameter) sinusoidal orifices of the spleen, an
Human immunodeficiency virus 19921 organ that functions in extravascular sequestration and
antibodies (anti-HIV-1/2)
removal of aged, damaged, or less deformable RBCs or frag-
Human T-cell lymphotropic virus 19972 ments of their membrane. The loss of RBC membrane is
antibody (anti-HTLV-I/II) exemplified by the formation of “spherocytes” (cells with a
Human immunodeficiency virus 1999 reduced surface-to-volume ratio; Fig. 1–2) and “bite cells,”
(HIV-1)(NAT)*,** in which the removal of a portion of membrane has left a
permanent indentation in the remaining cell membrane
Hepatitis C virus (HCV) (NAT) ** 1999 (Fig. 1–3). The survival of these forms is also shortened.
West Nile virus (NAT) 2004 Permeability
Trypanosoma cruzi antibody 2007 The permeability properties of the RBC membrane and the
(anti-T. cruzi) active RBC cation transport prevent colloid hemolysis and
control the volume of the RBC. Any abnormality that in-
*NAT-nucleic acid amplification testing
**Initially under IND starting in 1999 creases permeability or alters cationic transport may decrease
1 Anti-HIV-1 testing implemented in 1985 RBC survival. The RBC membrane is freely permeable to
2 Anti-HTLV testing implemented in 1988
2682_Ch01_001-025 28/05/12 12:22 PM Page 5
Chapter 1 Red Blood Cell and Platelet Preservation: Historical Perspectives and Current Trends 5
I = integral proteins
Spectrin P = peripheral proteins
ankyrin-band 3
Phospholipids interaction
Fatty acid
chains GP-C Membrane
F-actin surface
I Lipid
I I I bilayer
GP-B 3 3 GP-A
7
P 2.1
4.2 6 P
P Membrane
P
cytoskeleton
Adducin Protein 4.1
Spectrin dimer-dimer
Alpha chain Spectrin- Ankyrin interaction
Spectrin Beta chain actin-4.1-adducin
interaction
Figure 1–1. Schematic illustration of red blood cell membrane depicting the composition and arrangement of RBC membrane proteins. GP-A = glycophorin A; GP-B =
glycophorin B; GP-C = glycophorin C; G = globin. Numbers refer to pattern of migration of SDS (sodium dodecyl sulfate) polyacrylamide gel pattern stained with Coomassie
brilliant blue. Relations of protein to each other and to lipids are purely hypothetical; however, the positions of the proteins relative to the inside or outside of the lipid bilayer
are accurate. (Note: Proteins are not drawn to scale and many minor proteins are omitted.) (Reprinted with permission from Harmening, DH: Clinical Hematology and
Fundamentals of Hemostasis, 5th ed., FA Davis, Philadelphia, 2009.)
water and anions. Chloride (Cl–) and bicarbonate (HCO3–) shape and makes it more rigid. When RBCs are ATP-
can traverse the membrane in less than a second. It is spec- depleted, Ca2+ and Na+ are allowed to accumulate intracel-
ulated that this massive exchange of ions occurs through a lularly, and K+ and water are lost, resulting in a dehydrated
large number of exchange channels located in the RBC mem- rigid cell subsequently sequestered by the spleen, resulting
brane. The RBC membrane is relatively impermeable to in a decrease in RBC survival.
cations such as sodium (Na+) and potassium (K+).
RBC volume and water homeostasis are maintained by
controlling the intracellular concentrations of sodium and Metabolic Pathways
potassium. The erythrocyte intracellular-to-extracellular
ratios for Na+ and K+ are 1:12 and 25:1, respectively. The
300 cationic pumps, which actively transport Na+ out of Basic Concepts
the cell and K+ into the cell, require energy in the form of The RBC’s metabolic pathways that produce ATP are mainly
ATP. Calcium (Ca2+) is also actively pumped from the inte- anaerobic, because the function of the RBC is to deliver
rior of the RBC through energy-dependent calcium-ATPase oxygen, not to consume it. Because the mature erythrocyte
pumps. Calmodulin, a cytoplasmic calcium-binding pro- has no nucleus and there is no mitochondrial apparatus for
tein, is speculated to control these pumps and to prevent oxidative metabolism, energy must be generated almost
excessive intracellular Ca2+ buildup, which changes the exclusively through the breakdown of glucose.
2682_Ch01_001-025 28/05/12 12:22 PM Page 6
PHOSPHOGLUCONATE
PATHWAY
(oxidative)
H2O2
GP
EMBDEN-MEYERHOF
PATHWAY GSH GSSG
(non-oxidative)
Glucose GR
ATP
HK NADP NADPH
ADP
Glucose 6-P 6-P-Gluconate
G-6-PD
GPI 6-PGD CO2
Fructose 6-P Pentose-P
ATP PFK
ADP
Fructose 1,6-diP
METHEMOGLOBIN A
REDUCTASE
PATHWAY Glyceraldehyde DHAP
LUEBERING-RAPAPORT
Hemoglobin NAD PATHWAY
R GAPD
Methemoglobin NADH
HK Hexokinase 1,3-diP-Glycerate DPGM
GPI Glucose-6-phosphate isomerase ADP 2,3-diP-Glycerate
PFK Phosphofructokinase PGK DPGP
ATP
A Aldolase 3-P-Glycerate
TPI Triose phosphate isomerase
GAPD Glyceraldehyde-3-phosphate dehydrogenase PGM
PGM Phosphoglycerate mutase
E Enolase 2-P-Glycerate
PK Pyruvate kinase
LDH Lactic dehydrogenase E
DPGM Diphosphoglyceromutase P-Enolpyruvate
DPGP Diphosphoglycerate phosphatase
ADP
G-6-PD Glucose-6-phosphate dehydrogenase PK
6-PGD 6-Phosphogluconate dehydrogenase ATP
GR Glutathione reductase Pyruvate
GP Glutathione peroxidase NADH
LDH
DHAP Dihydroxyacetone-P NAD
PGK Phosphoglycerate kinase Lactate
R NADH-methemoglobin reductase
Figure 1–4. Red cell metabolism. (Reprinted with permission from Hillman, RF, and Finch, CA: Red Cell Manual, 7th ed., FA Davis, Philadelphia, 1996.)
2682_Ch01_001-025 28/05/12 12:22 PM Page 7
Chapter 1 Red Blood Cell and Platelet Preservation: Historical Perspectives and Current Trends 7
20 pH Decreased
10 2,3-DPG Decreased
Normal P50 = 28 mm Hg
0 Oxygen dissociation curve Shift to the left (increase in
hemoglobin and oxygen affinity;
0 10 20 30 40 50 60 70 80 90 100
less oxygen delivered to tissues)
PO2 (mm Hg)
Plasma K+ Increased
Figure 1–5. Hemoglobin-oxygen dissociation curve. (Reprinted with permission
from Harmening, DH: Clinical Hematology and Fundamentals of Hemostasis, Plasma hemoglobin Increased
5th ed., FA Davis, Philadelphia, 2009.)
2682_Ch01_001-025 28/05/12 12:22 PM Page 8
are stored, 2,3-DPG levels decrease, with a shift to the left the transfusion of RBCs with low 2,3-DPG levels and in-
of the hemoglobin-oxygen dissociation curve, and less oxy- creased affinity for oxygen include: an increase in cardiac
gen is delivered to the tissues. It is well accepted, however, output, a decrease in mixed venous (pO2) tension, or a
that 2,3-DPG is re-formed in stored RBCs, after in vivo cir- combination of these.11 The physiological importance of
culation, resulting in restored oxygen delivery. The rate of these effects is not easily demonstrated. This is a complex
restoration of 2,3-DPG is influenced by the acid-base status mechanism with numerous variables involved that are
of the recipient, the phosphorus metabolism, the degree of beyond the scope of this text.
anemia, and the overall severity of the disorder.11 It has Stored RBCs do regain the ability to synthesize 2,3-DPG
been reported that within the first hour after transfusion, after transfusion, but levels necessary for optimal hemoglo-
most RBC clearance occurs.14 Approximately 220 to bin oxygen delivery are not reached immediately. Approxi-
250 mg of iron are contained in one RBC unit.17 Therefore, mately 24 hours are required to restore normal levels of
rapid RBC clearance of even 25% of a single unit of blood 2,3-DPG after transfusion.19 The 2,3-DPG concentrations
delivers a massive load of hemoglobin iron to the monocyte after transfusion have been reported to reach normal levels
and macrophage system, producing harmful effects.13 as early as 6 hours post-transfusion.19 Most of these studies
have been performed on normal, healthy individuals. How-
ever, evidence suggests that, in the transfused subject whose
Anticoagulant Preservative Solutions capacity is limited by an underlying physiological distur-
bance, even a brief period of altered oxygen hemoglobin
Basic Concepts affinity is of great significance.14 It is quite clear now that
Table 1–3 lists the approved anticoagulant preservative so- 2,3-DPG levels in transfused blood are important in certain
lutions for whole blood and RBC storage at 1°C to 6°C. The clinical conditions. Studies demonstrate that myocardial
addition of various chemicals, along with the approved function improves following transfusion of blood with high
anticoagulant-preservative CPD, was incorporated in an 2,3-DPG levels during cardiovascular surgery.11 Several in-
attempt to stimulate glycolysis so that ATP levels were vestigators suggest that the patient in shock who is given
better maintained.18 One of the chemicals, adenine, incor- 2,3-DPG–depleted erythrocytes in transfusion may have
porated into the CPD solution (CPDA-1) increases ADP already strained the compensatory mechanisms to their
levels, thereby driving glycolysis toward the synthesis of limits.11,20–22 Perhaps for this type of patient, the poor
ATP. CPDA-1 contains 0.25 mM of adenine plus 25% more oxygen delivery capacity of 2,3-DPG–depleted cells makes
glucose than CPD. Adenine-supplemented blood can be a significant difference in recovery and survival.
stored at 1°C to 6°C for 35 days; the other anticoagulants It is apparent that many factors may limit the viability
are approved for 21 days. Table 1–4 lists the various chem- of transfused RBCs. One of these factors is the plastic ma-
icals used in anticoagulant solutions and their functions terial used for the storage container. The plastic must be
during the storage of red cells. sufficiently permeable to CO2 in order to maintain higher
pH levels during storage. Glass storage containers are a
matter of history in the United States. Currently, the ma-
jority of blood is stored in polyvinyl chloride (PVC) plastic
Advanced Concepts bags. One issue associated with PVC bags relates to the
plasticizer di(ethylhexyl)-phthalate (DEHP), which is used
It is interesting to note that blood stored in all CPD preser-
in the manufacture of the bags. It has been found to leach
vatives also becomes depleted of 2,3-DPG by the second
from the plastic into the lipids of the plasma medium and
week of storage. The reported pathophysiological effects of
RBC membranes of the blood during storage. However, its
use or that of alternative plasticizers that leach are impor-
tant because they have been shown to stabilize the RBC
Table 1–3 Approved Anticoagulant membrane and therefore reduce the extent of hemolysis
Preservative Solutions during storage. Another issue with PVC is its tendency to
STORAGE break at low temperatures; therefore, components frozen
NAME ABBREVIATION TIME (DAYS) in PVC bags must be handled with care. In addition to
PVC, polyolefin containers, which do not contain DEHP,
Acid citrate-dextrose ACD-A 21
(formula A) *
are available for some components, and latex-free plastic
containers are available for recipients with latex allergies.5
Citrate-phosphate CPD 21
dextrose
Citrate-phosphate- CP2D 21
Additive Solutions
double-dextrose
Citrate-phosphate- CPDA-1 35
Basic Concepts
dextrose-adenine Additive solutions (AS) are preserving solutions that are
added to the RBCs after removal of the plasma with or
* ACD-A is used for apheresis components.
2682_Ch01_001-025 28/05/12 12:22 PM Page 9
Chapter 1 Red Blood Cell and Platelet Preservation: Historical Perspectives and Current Trends 9
ACD-A = Acid citrate-dextrose (formula A); CPD = Citrate-phosphate dextrose; CP2D = Citrate phosphate double dextrose; CPDA-1 = Citrate phosphate dextrose adenine;
2,3-DPG = 2,3-diphosphoglycerate; ATP = adenosine triphosphate
without platelets. Additive solutions are now widely used. glucose. AS-1 and AS-5 also contain mannitol, which pro-
One of the reasons for their development is that removal tects against storage-related hemolysis,23 while AS-3 con-
of the plasma component during the preparation of RBC tains citrate and phosphate for the same purpose. All of the
concentrates removed much of the nutrients needed to additive solutions are approved for 42 days of storage for
maintain RBCs during storage. This was dramatically packed RBCs. Table 1–5 lists the currently approved addi-
observed when high-hematocrit RBCs were prepared. The tive solutions.
influence of removing substantial amounts of adenine and
glucose present originally in, for example, the CPDA-1
anticoagulant-preservative solution led to a decrease in
viability, particularly in the last 2 weeks of storage.14 Advanced Concepts
RBC concentrates prepared from whole blood units col- Table 1–6 shows the biochemical characteristics of RBCs
lected in primary anticoagulant-preservative solutions can be stored in the three additive solutions after 42 days of stor-
relatively void of plasma with high hematocrits, which causes age.4,24,25 Additive system RBCs are used in the same way
the units to be more viscous and difficult to infuse, especially
in emergency situations. Additive solutions (100 mL to the
RBC concentrate prepared from a 450-mL blood collection) Table 1–5 Additive Solutions in Use
also overcome this problem. Additive solutions reduce hema-
in North America
tocrits from around 70% to 85% to around 50% to 60%. The
ability to pack RBCs to fairly high hematocrits before adding STORAGE
additive solution, also provides a means to harvest greater NAME ABBREVIATION TIME (DAYS)
amounts of plasma with or without platelets. Box 1–2 sum- Adsol (Baxter Healthcare) AS-1 42
marizes the benefits of RBC additive solutions.
Currently, three additive solutions are licensed in the Nutricel (Pall Corporation) AS-3 42
United States: Optisol (Terumo
1. Adsol (AS-1; Baxter Healthcare) Corporation) AS-5 42
2. Nutricel (AS-3; Pall Corporation)
3. Optisol (AS-5; Terumo Corporation)
Table 1–6 Red Cell Additives: Biochemical
The additive solution is contained in a satellite bag and
Characteristics
is added to the RBCs after most of the plasma has been
expressed. All three additives contain saline, adenine, and AS-1 AS-3 AS-5
Chapter 1 Red Blood Cell and Platelet Preservation: Historical Perspectives and Current Trends 11
storage without rejuvenation. The need to transfuse Current Trends in RBC Preservation
RBCs within 24 hours of thawing has limited the use of Research
frozen RBCs.
Recently, an instrument (ACP 215, Haemonetics) has Advanced Concepts
been developed that allows the glycerolization and deglyc-
Research and development in RBC preparation and preser-
erolization processes to be performed under closed system
vation is being pursued in five directions:
conditions.28 This instrument utilizes a sterile connecting
device for connections, in-line 0.22 micron filters to deliver 1. Development of improved additive solutions
solutions, and a disposable polycarbonate bowl with an 2. Development of procedures to reduce and inactivate the
external seal to deglycerolize the RBCs. RBCs prepared level of pathogens that may be in RBC units
from 450-mL collections and frozen within 6 days of blood 3. Development of procedures to convert A-, B-, and AB-
collection with CPDA-1 can be stored after thawing at type RBCs to O-type RBCs
1°C to 6°C for up to 15 days when the processing is con- 4. Development of methods to produce RBCs through bio-
ducted with the ACP 215 instrument. The deglycerolized engineering (blood pharming)
cells, prepared using salt solutions as in the traditional pro- 5. Development of RBC substitutes
cedures, are suspended in the AS-3 additive solution as a
final step, which is thought to stabilize the thawed RBCs.
These storage conditions are based on the parameters used Improved Additive Solutions
in a study by Valeri and others that showed that RBC prop-
Research is being conducted to develop improved additive
erties were satisfactorily maintained during a 15-day pe-
solutions for RBC preservation. One reason for this is be-
riod.28 Further studies will broaden the conditions that can
cause longer storage periods could improve the logistics of
be used to prepare RBCs for subsequent freezing with
providing RBCs for clinical use, including increased benefits
closed system processing.
associated with the use of autologous blood/RBCs.
Chapter 1 Red Blood Cell and Platelet Preservation: Historical Perspectives and Current Trends 13
curve. Another problem was the product’s short half-life, due A 2008 meta-analysis of 16 clinical trials involving 3,711
to dissociation of the hemoglobin molecule into α and β patients and five different HBOCs found a significantly in-
dimers that were filtered by the kidneys and excreted in the creased risk of death and myocardial infarction associated with
urine.32 Scientists began to look for ways to chemically mod- the use of HBOCs.38 These findings make their widespread
ify the hemoglobin molecule to overcome these problems. clinical use unlikely in the near future; however, some experts
Cross-linking, polymerization, and pegylation produced believe that HBOCs hold more promise than PFCs.33,39
larger, more stable molecules. This reduced some, but not Table 1–11 lists the advantages and disadvantages of HBOCs.
all, of the adverse effects.
To date, four generations of HBOCs have been developed.33 Perfluorocarbons
HBOCs have been produced from human, bovine, and recom- Perfluorocarbons (PFCs) are synthetic hydrocarbon struc-
binant hemoglobin. Bovine hemoglobin has several advantages tures in which all the hydrogen atoms have been replaced
over human hemoglobin. It has a lower oxygen affinity and bet- with fluorine. They are chemically inert, are excellent gas
ter oxygen uploading in ischemic tissues, and its availability is solvents, and carry O2 and CO2 by dissolving them. Be-
not dependent upon an adequate supply of outdated human cause of their small size (about 0.2 microns in diameter),
RBCs. However, concerns about potential immunogenicity and they are able to pass through areas of vasoconstriction and
transmission of prions have been raised.37 Although several deliver oxygen to tissues that are inaccessible to RBCs.
HBOCs have progressed to phase II and III clinical trials, cur- PFCs have been under investigation as possible RBC sub-
rently none have been approved for clinical use in humans in stitutes since the 1970s. Fluosol (Green Cross Corp.) was
the United States or Europe. Development of several products approved by the FDA in 1989 but was removed from the
was terminated following clinical trials in which serious ad- market in 1994 due to clinical shortcomings and poor
verse side effects were discovered. Two HBOCs are still in clin- sales. Four other PFCs have proceeded to clinical trials.
ical trials in the United States and Europe: Hemopure (OPK One, Perftoran (Perftoran), is in clinical use in Russia and
Biotech) and Hemospan (Sangart). Hemopure was approved Mexico. Two others are no longer under development, and
for clinical use in South Africa in 2001 and a related product, one (Oxycyte, Oxygen Biotherapeutics) is currently being
Oxyglobin, has been used to treat canine anemia in the United investigated as an oxygen therapeutic for treatment of
States and Europe since 1998. An interesting side note is that wounds, decompression sickness, and traumatic brain in-
a Spanish cyclist admitted to using Oxyglobin in the 2003 Tour jury.40 Refer to Table 1–12 for further details, and review
de France. He crashed after experiencing nausea. Table 1–10 of PFCs. Table 1–13 for the advantages and disadvantages
summarizes the history and status of several HBOCs. of Perfluorochemicals.
HemAssist (DCLHb) Baxter Diaspirin cross-linked Hgb from First HBOC to advance to phase III clini-
outdated human RBCs cal trials in United States. Removed
from production because of increased
mortality rates.
PolyHeme (SFH-P) Northfield Laboratories Polymerized and pyridoxalated Underwent phase II/III clinical trials in
human Hgb United States. Did not obtain FDA ap-
proval. No longer produced.
Hemopure (HBOC-201) Originally Biopure; currently Polymerized bovine Hgb Still in phase II/III clinical trials in
OPK Biotech United States and Europe. Approved
for use in S. Africa (2001).
Oxyglobin Originally Biopure; now Polymerized bovine Hgb Approved for veterinary use in United
OPK Biotech States and Europe.
Hemospan (MP4) Sangart Polyethylene glycol (PEG) at- In phase II trials in United States; phase
tached to the surface of Hgb III in Europe.
from human RBCs
HemoLink Hemosol Purified human Hgb from out- Abandoned due to cardiac toxicity.
dated RBCs, cross-linked and
polymerized
HemoTech HemoBioTech Derived from bovine Hgb Limited clinical trial outside of
United States.
Table 1–11 Advantages and Disadvantages Table 1–13 Advantages and Disadvantages
of Hemoglobin-Based Oxygen of Perfluorochemicals
Carriers ADVANTAGES DISADVANTAGES
ADVANTAGES DISADVANTAGES
Biological inertness Adverse clinical effects
Long shelf-life Short intravascular half-life
Lack of immunogenicity High O2 affinity
Very stable Possible toxicity
Easily synthesized Retention in tissues
No antigenicity (unless bovine) Increased O2 affinity
Requirement for O2 administration when
No requirement for blood-typing Increased oncotic effect infused
procedures
Deep-freeze storage temperatures
Chapter 1 Red Blood Cell and Platelet Preservation: Historical Perspectives and Current Trends 15
Dense granules or bodies are smaller and fewer in num- platelets then become irreversibly swollen, aggregate to-
ber (2 to 10 per platelet) and appear as dense opaque gether, or lyse, and when infused, will not circulate or func-
granules in transmission electron microscopy (TEM) tion. During storage of PCs, the pH will remain stable as
preparations.8 Dense granules contain storage ADP, ATP, long as the production of lactic acid does not exceed the
ionic calcium, serotonin, and phosphates. Platelet ADP and buffering capacity of the plasma or other storage solution.
ATP are present in two cellular pools—a metabolic pool
and a storage pool. The metabolic pool meets the platelet’s
ongoing metabolic needs, and the storage pool, which is
located in the dense granules, is released when the platelet Advanced Concepts
is stimulated.19 Lysosomes contain microbicidal enzymes, The platelet storage lesion results in a loss of platelet qual-
neutral proteases, and acid hydrolases. Glycogen granules ity and viability. When platelets deteriorate during storage,
are also found within the organelle zone and function in their membranes lose their ability to maintain normal lipid
platelet metabolism. The estimated 10 to 60 mitochondria asymmetry and phosphatidylserine becomes expressed on
present per platelet require glycogen as their source of en- the outer membrane surface.41 The binding of annexin V,
ergy for metabolism.8 In the resting platelet, approximately which has a high affinity for anionic phospholipids, can be
15% ATP (energy) production is generated by glycolysis used to measure this loss of membrane integrity using flow
and 85% by oxygen consumption through the tricarboxylic cytometry.41 Flow cytometry is also used to measure the
acid (TCA) cycle.19 In the activated state, about half the platelet degranulation process during storage by detecting
ATP production in platelets occurs through the glycolytic the surface expression of CD62P or CD63.41 Measurement
pathway, increasing the rate of lactate production.8 Platelets of specific platelet α granules such as β-thromboglobulin
circulate in an inactivated state and require minimal stim- and platelet factor 4 can also assess platelet degranulation
ulation for activation, ensuring their immediate availability during storage. Generally, the quality-control measure-
for hemostasis. ments required by various accreditation organizations for
platelet concentrates include platelet concentrate volume,
platelet count, pH of the unit, and residual leukocyte count
The Platelet Storage Lesion if claims of leukoreduction are made.42 In addition, imme-
diately before distribution to hospitals, a visual inspection
Basic Concepts is made that often includes an assessment of platelet
Platelet storage still presents one of the major challenges swirl.41 The absence of platelet swirling is associated
to the blood bank because of the limitations of storing with the loss of membrane integrity during storage, result-
platelets. In the United States, which has a storage limit of ing in the loss of discoid shape with irreversible sphering.
5 days, approximately 30% of the platelet inventory is dis- Box 1–4 lists the in vitro platelet assays that have been
carded either by the blood supplier or the hospital blood correlated with in vivo survival.
bank.41 The two main reasons for the 5-day shelf-life for
platelets is bacterial contamination at incubation of 22°C
Clinical Use of Platelets
and the loss of platelet quality during storage (known as
the platelet storage lesion). During storage, a varying de- Platelet components are effectively used to treat bleeding as-
gree of platelet activation occurs that results in release of sociated with thrombocytopenia, a marked decrease in
some intracellular granules and a decline in ATP and ADP. platelet number. The efficacy of the transfused platelet con-
This platelet activation often results in temporary aggrega- centrates is usually estimated from the corrected count
tion of platelets into large sheets that must be allowed to increment (CCI) of platelets measured after transfusion. It
rest for the aggregation to be reversed, especially when the should be noted that the CCI does not evaluate or assess
platelet concentrates (PCs) are prepared with the platelet- function of the transfused platelets.43
rich-plasma (PRP) method. Platelets are also transfused prophylactically to increase
The reduced oxygen tension (pO2) in the plastic platelet the circulating platelet count in hematology-oncology
storage container results in the platelets increasing the rate thrombocytopenic patients to prevent bleeding secondary
of glycolysis to compensate for the decrease in ATP regen-
eration from the oxidative (TCA) metabolism. This in-
creases glucose consumption and causes an increase in BOX 1–4
lactic acid that must be buffered. This results in a fall in
pH. During the storage of PCs in plasma, the principal
In Vitro Platelet Assays Correlated With In Vivo
buffer is bicarbonate. When the bicarbonate buffers are de- Survival
pleted during PC storage, the pH rapidly falls to less than • pH
6.2, which is associated with a loss of platelet viability. In • Shape change
addition, when pH falls below 6.2, the platelets swell and • Hypotonic shock response
there is a disk-to-sphere transformation in morphology that • Lactate production
is associated with a loss of membrane integrity. The • pO2
2682_Ch01_001-025 28/05/12 12:22 PM Page 16
to drug and radiation therapy. Platelets are also utilized in is irreversibly spherical. This structural change is considered
some instances to treat other disorders in which platelets to be the factor responsible for the deleterious effects of cold
are qualitatively or quantitatively defective because of storage. When stored even for several hours at 4°C, platelets
genetic reasons. do not return to their disk shape upon rewarming. This loss
In the 1950s and 1960s, platelet transfusions were given of shape is probably a result of microtubule disassembly.
as freshly drawn whole blood or platelet-rich plasma. Circu- Based on many follow-up studies, platelets are currently
latory overload quickly developed as a major complication stored at room temperature.
of this method of administering platelets. Since the 1970s, These studies provided an understanding of the factors
platelets have been prepared from whole blood as concen- that influenced the retention of platelet viability and the
trates in which the volume per unit is near 50 mL in contrast parameters that needed to be considered to optimize stor-
to the 250- to 300-mL volume of platelet-rich plasma units. age conditions. One factor identified as necessary was the
Today, platelets are prepared as concentrates from whole need to agitate platelet components during storage,
blood and increasingly by apheresis. The 2007 National although initially the rationale for agitation was not under-
Blood Collection and Utilization Survey found that only stood.48,49 Subsequently, agitation has been shown to facil-
17% of platelet doses in the United States were whole blood itate oxygen transfer into the platelet bag and oxygen
derived (WBD). Platelets still remain as the primary means consumption by the platelets. The positive role for oxygen
of treating thrombocytopenia, even though therapeutic has been associated with the maintenance of platelet com-
responsiveness varies according to patient conditions and ponent pH.50 Maintaining pH was determined to be a key
undefined consequences of platelet storage conditions.44,45 parameter for retaining platelet viability in vivo when
(See Chapter 13 for the methods for preparing platelet platelets were stored at 20°C to 24°C.
concentrates.) Although storage itself was associated with a small
reduction in post-infusion platelet viability, an enhanced
Current Conditions for Platelet Preservation loss was observed when the pH was reduced from initial
(Platelet Storage) levels of near 7 to the range of 6.8 to 6.5, with a marked
loss when the pH was reduced to levels below 6.49 A pH of
Basic Concepts 6 was initially the standard for maintaining satisfactory
Platelet concentrates prepared from whole blood and viability. The standard was subsequently changed to
apheresis components are routinely stored at 20°C to 24°C, 6.2 with the availability of additional data. As pH was re-
with continuous agitation for up to 5 days. FDA standards duced from 6.8 to 6.2, the platelets progressively changed
define the expiration time as midnight of day 5. Primarily shape from disks to spheres. This change is irreversible
flatbed and circular agitators are in use. There are a number when the pH falls to less than 6.2.19
of containers in use for 5-day storage of WBD and apheresis In the 1970s, when WBD platelets were initially stored
platelets. In the United States, platelets are being stored in as concentrates, a major problem was a marked reduction
a 100% plasma medium, unless a platelet additive solution in pH in many concentrates. This limited the storage period
is used (see section on platelet additives on page 19). Al- to 3 days. The containers being used for storage were iden-
though platelets can be stored at 1°C to 6°C for 48 hours,46 tified as being responsible for the fall in pH because of their
it does not appear that this is a routine practice. limiting gas transfer properties for oxygen and carbon diox-
ide. Carbon dioxide buildup from aerobic respiration and
as the end product of plasma bicarbonate depletion also
History of Platelet Storage: Rationale influenced the fall in pH. The gas transport properties of a
for Current Conditions container are known to reflect the container material, the
gas permeability of the wall of the plastic container, the
surface area of the container available for gas exchange, and
Advanced Concepts
the thickness of the container. Insufficient agitation may
The conditions utilized to store platelets have evolved since also be a factor responsible for pH reduction because
the 1960s as key parameters that influence the retention of agitation facilitates gas transport into the containers.
platelet properties. Initially, platelets were stored at 1°C to
6°C, based on the successful storage of RBCs at this
temperature range. A key study in 1969 by Murphy and Storage in Second-Generation Containers
Gardner showed that cold storage at 1°C to 6°C resulted in
a marked reduction in platelet in vivo viability, manifested Understanding the factors that led to the reduction in pH in
as a reduction in in vivo life span, after only 18 hours of first-generation platelet containers resulted in the development
storage.47 This study also identified for the first time that of second-generation containers, starting around 1982. The
20°C to 24°C (room temperature) should be the preferred second-generation containers, with increased gas transport
range, based on viability results. properties (allowing increased oxygen transport and carbon
The reduction in viability at 1°C to 6°C was associated dioxide escape), are available and are being utilized for storing
with conversion of the normal discoid shape to a form that platelets for 5 days without pH substantially falling. Such con-
tainers are in use for WBD PCs and apheresis components.
2682_Ch01_001-025 28/05/12 12:22 PM Page 17
Chapter 1 Red Blood Cell and Platelet Preservation: Historical Perspectives and Current Trends 17
Containers for platelet storage were originally constructed rate of infused radiolabeled platelets to normal individuals
from polyvinyl chloride (PVC) plastic containing a phthalate whose donation provided the platelets.
plasticizer. The second-generation containers are con- The observation of the swirling phenomenon caused by
structed in some cases with PVC and in other cases with discoid platelets when placed in front of a light source has
polyolefin plastic. For most PVC containers, alternative plas- been used to obtain a semiqualitative evaluation of the re-
ticizers (trimellitate and citrate based) have been used to in- tention of platelet viability properties in stored units.53 The
crease gas transport. The nominal volumes of the containers extent of shape change and the hypotonic shock response in
are 300 to 400 mL and 1 to 1.5 L for WBD platelet concen- in vitro tests appears to provide some indication about the
trates and apheresis components, respectively. The size of retention of platelet viability properties.54 Function is de-
the containers for apheresis components reflects the in- fined as the ability of viable platelets to respond to vascular
creased number of platelets that are being stored and hence damage in promoting hemostasis. Clinical assessment of he-
the need for a larger surface area to provide adequate gas mostasis is being increasingly used.
transport properties for maintaining pH levels near the initial The maintenance of pH during storage at 20°C to 24°C
level of 7 even after 5 days of storage. Box 1–5 lists factors has been associated with the retention of post-transfusion
that should be considered when using 5-day platelet storage platelet viability and has been the key issue that has been
containers. addressed to improve conditions for storage at this temper-
ature. There is also the issue of retaining platelet function
Storing Platelets Without Agitation during storage. Historically, room temperature storage has
for Limited Times been thought to be associated with a reduction in platelet
functional properties. However, the vast transfusion experi-
Although platelet components should be stored with contin- ence with room temperature platelets worldwide indicates
uous agitation, there are data that suggest that platelet prop- that such platelets have satisfactory function. As has been
erties, based on in vitro studies, are retained when agitation suggested many times over the last 30 years, it is possible
is discontinued for up to 24 hours during a 5-day storage pe- that room temperature–stored platelets undergo a rejuvena-
riod.51,52 This is probably related to the retention of satisfac- tion of the processes that provide for satisfactory function
tory oxygen levels with the second-generation containers upon introduction into the circulation.55,56
when agitation is discontinued, as occurs by necessity when The better functionality of cold-stored platelets, based on
platelets are shipped over long distances by, for example, some studies, especially ones conducted in the 1970s, may
overnight courier. have reflected an undesirable activation of platelet processes
as a result of storing platelets at a temperature range of 1°C
Measurement of Viability and Functional to 6°C. Activation is a prerequisite for platelet function in
Properties of Stored Platelets hemostasis. During storage, it takes different forms. Even
with storage at 20°C to 24°C, there is some activation, as
Viability indicates the capacity of platelets to circulate after in-
judged by the release of granular proteins such as p-selectin
fusion without premature removal or destruction. Platelets
(CD62) and platelet factor 4 and granular adenine nu-
have a life span of 8 to 10 days after release from megakary-
cleotides. There are some data that suggest that specific in-
ocytes. Storage causes a reduction in this parameter, even
hibitors of the activation processes may have a beneficial
when pH is maintained. Platelet viability of stored platelets is
influence during storage.57
determined by measuring pretransfusion and post-transfusion
Table 1–14 summarizes platelet changes during storage
platelet counts (1 hour and/or 24 hours) and expressing the
(the platelet storage lesion). It should be noted that except for
difference based on the number of platelets transfused (cor-
change in pH, the effect of in vitro changes on post-transfusion
rected count increment) or by determining the disappearance
platelet survival and function is unknown, and some of the
changes may be reversible upon transfusion.58
Table 1–14 The Platelet Storage Lesion however, false-negative test results have been documented.
With both culture systems, the need to delay sampling and
CHARACTERISTIC CHANGE OBSERVED
the requirement for incubation delay entry of the platelet
Lactate Increased products into inventory. Box 1–6 lists the disadvantages as-
pH Decreased sociated with the use of culture methods for the detection
of bacterial contamination of platelets.
ATP Decreased
The third bacterial detection method approved by the
Morphology scores Decreased FDA, Scansystem, is a laser-based, scanning cytometry
change from discoid
method. In the United States, 100% of apheresis platelets are
to spherical (loss of
swirling effect) tested by the collection facility using culture-based assays.63
Because screening individual units of WBD platelets by these
Degranulation Increased
(β-thromboglobulin, methods is time-consuming, expensive, and uses a signifi-
platelet factor 4) cant amount of the product, less sensitive methods, such as
gram staining and dipstick tests for pH and glucose, were
Platelet activation markers Increased
(P-selectin [CD62P] or CD63) initially used for screening. Since these methods have a sen-
sitivity of about only 50%, many transfusion services chose
Platelet aggregation Drop in responses to some agonists
not to transfuse WBD platelets. This practice made it difficult
for some blood banks to meet the demand for apheresis
platelets, and WBD platelets became underutilized.
In November 2009, the FDA approved the first rapid test
platelets is the most common infectious complication of to detect bacteria in WBD platelets—the Pan Genera
transfusion.60 A large-scale study at American Red Cross Detection (PGD) test (Verax Biomedical). The PDG test,
(ARC) regional blood centers from 2004 to 2006 detected which was previously approved by the FDA for testing
bacteria in 186 out of 1,004,206 donations for a contami- leukocyte-reduced platelets as an adjunct to culture, is an
nation rate of approximately 1 in 5,400.61 Although the immunoassay that detects lipoteichoic acids on gram-
occurrence of patient sepsis is much lower, particularly positive bacteria and lipopolysaccharides on gram-negative
troublesome is the fact that some septic episodes have led bacteria. Both aerobes and anaerobes are detected. The test
to patient deaths. An estimated 10% to 40% of patients can be performed on pools of up to 6 units of WBD
transfused with a bacterially contaminated platelet unit platelets. A sample of only 500 µl is required. Following
develop life-threatening sepsis.60 As a result, in 2002 the pretreatment, the sample is loaded into a disposable plastic
College of American Pathologists (CAP) added a require- cartridge with built-in controls that turn from yellow to
ment for laboratories to have a method to screen platelets blue-violet when the test is ready to be read, in approxi-
for bacterial contamination, and AABB introduced a similar mately 20 minutes. A pink-colored bar in either the gram-
requirement in 2004. positive or gram-negative test window indicates a positive
result. The manufacturer states that the system has a speci-
ficity of 99.8% and can detect bacteria at 103 to 105 colony-
forming units (CFU) per milliliter.64 The PGD test can be
Advanced Concepts performed by transfusion services just prior to release of
There are three commercial systems approved by the FDA platelet products. The optimum time for sampling is at least
for screening platelets for bacterial contamination: 72 hours after collection.
BacT/ALERT (bioMérieux), eBDS (Pall Corp.), and Scan- With the availability of this rapid and sensitive
system (Hemosystem). BacT/ALERT and eBDS are culture- method for screening WBD platelets, AABB issued
based systems. As the level of bacteria in the platelets at the Interim Standard 5.1.5.1.1, which prohibits the use of the
time of collection can be low, samples are not taken until less-sensitive methods (microscopy, pH, glucose) after
after at least 24 hours of storage. This provides time for any January 31, 2011.65 Transfusion services must either
bacteria present to replicate to detectable levels. BacT/
ALERT measures bacteria by detecting a change in carbon-
dioxide levels associated with bacterial growth.62 This
system provides continuous monitoring of the platelet BOX 1–6
sample–containing culture bottles, which are held for the
shelf-life of the platelet unit or until a positive reaction is
Disadvantages of Culture Methods for Detection
detected. The eBDS system measures the oxygen content of Bacterial Contamination of Platelets
of the air within the sample pouch following incubation for • Product loss due to sampling
18 to 30 hours. A decrease in oxygen level indicates the pres- • Delay in product release, further reducing already short shelf-life
ence of bacteria. BacT/ALERT and eBDS are the most widely • False-negative results
used systems for screening platelets in the United States, and • Cost
studies have documented good sensitivity and specificity; • Logistical problems of culturing WBD platelets
2682_Ch01_001-025 28/05/12 12:22 PM Page 19
Chapter 1 Red Blood Cell and Platelet Preservation: Historical Perspectives and Current Trends 19
obtain their platelets from a collection facility that per- Current Trends in Platelet Preservation
forms an approved test for bacterial contamination, or Research
they must perform an approved test themselves.66 At this
time, the approved tests are bacterial culture or the Verax Advanced Concepts
PGD test.
Research and development in platelet preservation is being
The practice of screening platelets for bacterial contam-
pursued in many directions, including the following:
ination has reduced, but not eliminated, the transfusion of
contaminated platelet products. False-negative cultures can 1. Development of methods that would allow platelets to
occur when bacteria are present in low numbers and when be stored for 7 days
the pathogen is a slow-growing organism. The American 2. Development of additive solutions, also termed synthetic
Red Cross received reports of 20 septic transfusion reac- media
tions from 2004 to 2006 following transfusion of culture- 3. Development of procedures to reduce and inactivate the
negative platelets. Eighty percent of the septic reactions level of pathogens that may be in platelet units
were due to Staphylococcus spp., and 65% occurred with 4. Development of platelet substitutes
products transfused on day 5 after collection. Three of 5. New approaches for storage of platelets at 1°C to 6°C
these reactions were fatal, for a fatality rate of 1 per 498,711 6. The development of processes to cryopreserve platelets
distributed products.61
Because current bacterial screening methods are not
100% sensitive, they must be supplemented by other pre- Storage for 7 Days at 20°C to 24°C
cautions, such as the donor interview and proper donor
In 1984, the FDA extended platelet storage from 5 to
arm disinfection. Another more recent precaution is the di-
7 days. Reports of septic transfusion reactions increased
version of the first aliquot (about 20 to 30 mL) of collected
following this change, and in 1986 the storage time was
blood into a separate but connected diversion pouch. This
changed back to 5 days. With the implementation of bac-
procedure minimizes the placement of skin plugs, the most
terial screening of platelets and its impact on their avail-
common source of bacterial contamination, into the
able shelf-life, there is renewed interest in being able to
platelet products. The 2007 National Blood Collection and
store platelets for 7 days. In 2005, the FDA approved a
Utilization Survey found that 50.4% of blood collection fa-
study called “Post Approval Surveillance Study of Platelet
cilities used diversion devices for collecting apheresis
Outcomes, Release Tested” (PASSPORT) to collect data on
platelets, and one study found that diversion reduced bac-
the safety of apheresis platelets tested with an FDA-
terial contamination by 40% to 88%.67
approved bacterial detection test and stored for 7 days. The
In view of the ability to test for bacterial contamina-
study was suspended in 2008 because of safety concerns
tion and the use of diversion pouches and sterile docking
when interim data and published studies suggested that
instruments, there is now interest in being able to store
culture at 24 hours after collection may miss up to 50% of
pools of platelets up to the outdate of the individual con-
contaminated apheresis platelet units.70 FDA and industry
centrates. The retention of platelet properties during stor-
representatives discussed modifications to the study proto-
age of pools has been shown in a number of studies.
col that might increase the safety of 7-day platelets and
Traditionally, four to six WBD platelets are pooled into a
allow resumption of the study—for example, increasing the
single bag by the transfusion service just prior to issue.
size of the culture inoculum, performing anaerobic cultures
This facilitates transfusion but reduces the shelf-life of
in addition to aerobic cultures, and performing a second
the platelets to 4 hours, because they are prepared in an
culture at 5 days of storage. Because consensus could not
open system.
be reached, the PASSPORT study was not resumed.70
In 2005, the FDA approved the use of prestorage
Although work toward approval of safe and efficacious
pooled platelets prepared by Acrodose Systems (Pall
7-day platelets is likely to continue, the shelf-life for
Corp.). Acrodose platelets are pooled ABO-matched,
platelets at this time remains 5 days.
leukoreduced WBD platelets that have been cultured and
are ready for transfusion. Because they are produced in a
closed system, they can be stored for 5 days from collec- Storage with Additive Solutions
tion. They provide a therapeutic dose equivalent to
apheresis platelets and at a lower cost,68 but they do Platelet additive solutions (PASs) were first developed in
expose the recipient to multiple donors. A recent study the 1980s71 and have been used in Europe since 1995 to
comparing transfusion reactions from prestorage-pooled replace a large portion of the plasma in platelet suspen-
platelets, apheresis platelets, and poststorage-pooled sions prepared from whole blood by the buffy coat
WBD platelets found no difference in reaction rates method. In 2010, the FDA approved the first PAS for use
among the different products.69 Prestorage-pooled in the United States. This additive, called PAS-C, was ap-
platelets may prove to be a useful adjunct to apheresis proved for storage of apheresis platelets collected by the
platelets, which are often in short supply, and may lead AMICUS Separator System (Fenwal/Baxter) for up to
to improved utilization of WBD platelets. 5 days. Other PASs are under development and may gain
FDA approval in the future.72
2682_Ch01_001-025 28/05/12 12:22 PM Page 20
PASs are designed to support platelets during storage in potentially add an additional level of safety by protecting
reduced amounts of residual plasma. Historically, platelets against unknown and newly emerging pathogens.79,80
have been stored in 100% plasma. With the addition of a PAS, Two PR/PI methods, both using photochemical technolo-
residual plasma can be reduced to 30% to 40%71 gies to target nucleic acids, are approved for use in Europe
(35% with InterSol). One advantage is that this approach pro- but are approved only for clinical trials in the United States.81
vides more plasma for fractionation. In addition, there are The targeting of nucleic acids is possible because platelets,
data indicating that optimal additive solutions may improve like RBCs, do not contain functional nucleic acids. In the
the quality of platelets during storage, reduce adverse effects INTERCEPT system (Cerus Corp.), amotosalen is activated
associated with transfusion of plasma, and promote earlier by ultraviolet (UV) light and binds to the nucleic acid base
detection of bacteria.63,73,74 Box 1–7 lists the advantages of pairs of pathogens, preventing replication.82 This system was
using a platelet additive solution for platelet storage. approved for clinical use in Europe in 2002. The Mirasol PRT
Research is being conducted to improve the additive solu- system (CaridianBCT Biotechnologies) uses riboflavin (vit-
tions in use. Gulliksson suggested that platelets could be amin B2) and UV light to cause irreversible changes to the
stored for at least 18 to 20 days at 20°C to 24°C with an nucleic acids of pathogens.83 Many studies suggest that PR
optimized additive medium based on considerations that of platelets is safe and effective; however, additional studies
indicate that storage could well inhibit platelet aging with the involving larger groups and pediatric patients are needed.80
appropriate environments/medium.75 Platelet additive solu- Some argue that since most patients who receive platelets
tions in use and those being developed contain varying quan- also receive red cells, PR of platelets will be of limited value
tities of citrate, phosphate, potassium, magnesium, and acetate. until there is an equivalent method for red cells.79
Citrate, magnesium, and potassium control platelet activa-
tion.76 Acetate serves as a substrate for aerobic respiration (mi- Development of Platelet Substitutes
tochondrial metabolism) while also providing a way to
maintain pH levels as it reacts with hydrogen ions when it is In view of the short shelf-life of liquid-stored platelet products,
first utilized. Some formulations also contain glucose, which there has been a long-standing interest to develop platelet sub-
seems to maintain pH better beyond day 5. This might give stitute products that maintain hemostatic function. Platelet
glucose-containing PASs an advantage over nonglucose PASs substitutes are in the early stages of development. It is under-
if extended storage of platelets becomes a reality.77 Currently, stood that platelet substitutes may have use only in specific
glucose-containing PASs are not widely used because glucose clinical situations because platelets have a complex biochem-
caramelizes during the steam sterilization process that is istry and physiology. Besides having a long shelf-life, platelet
used.78 When nonglucose PASs are used, at least 20% to substitutes appear to have reduced potential to transmit
35% of the plasma must be retained in order to provide the pathogens as a result of the processing procedures. A number
glucose the platelets need during storage.78 of different approaches have been utilized.84 Apparently, one
approach with the potential for providing clinically useful
Procedures to Reduce and Inactivate Pathogens products is the use of lyophilization.
Two products prepared from human platelets are in pre-
Despite sensitive methods to detect bacteria in platelets, sep- clinical testing. One preparation uses washed platelets
tic transfusion reactions still occur. As for RBCs, procedures treated with paraformaldehyde, with subsequent freezing in
are being developed to treat platelet components to reduce 5% albumin and lyophilization.85 These platelets on rehy-
or inactivate any residual pathogens (bacteria, viruses, para- dration have been reported to have hemostatic effectiveness
sites) that may be present. The term pathogen reduction (PR) in different animal models. A second method involves the
is preferred to pathogen inactivation (PI) because freeze-drying of trehalose-loaded platelets.86 Additional
inactivation may not be complete.79 PR/PI procedures could products that are apparently being developed include
fibrinogen-coated albumin microcapsules and microspheres
and modified RBCs with procoagulant properties as a result
of fibrinogen binding. Fibrinogen is being used because in
BOX 1–7 vivo this protein cross-links activated platelets to form
Advantages of Using Platelet Additive Solutions platelet aggregates as part of the hemostatic process. Two
other approaches include the development of platelet-
• Optimizes platelet storage in vitro derived microparticles that can stop bleeding and liposome-
• Saves plasma for other purposes (e.g., transfusion or based hemostatic products.84
fractionation)
• Facilitates ABO-incompatible platelet transfusions
New Approaches for Storage of Platelets
• Reduces plasma-associated transfusion side effects, such as febrile
and allergic reactions, and may reduce risk of transfusion-related at 1°C to 6°C
acute lung injury (TRALI)
• Improves effectiveness of photochemical pathogen reduction
Although storage of platelets at 1°C to 6°C was discontinued
technologies many years ago, there has been an interest in developing ways
• Potentially improves bacterial detection to overcome the storage lesion that occurs at 1°C to 6°C.87
The rationale for the continuing effort reflects concerns about
2682_Ch01_001-025 28/05/12 12:22 PM Page 21
Chapter 1 Red Blood Cell and Platelet Preservation: Historical Perspectives and Current Trends 21
storing and shipping platelets at 20°C to 24°C, especially the enzymatic galactosylation of cold-stored platelets to modify
chance for bacterial proliferation. Refrigeration would signif- specifically one type of membrane protein. The addition
icantly reduce the risk of bacterial contamination, allowing of uridine diphosphate galactose is the vehicle for the
for longer storage. Many approaches have been attempted modificaiton.89
without success, although early results showed some prom-
ise. The approaches primarily involve adding substances to Frozen Platelets
inhibit cold-induced platelet activation, as this is thought to
be the key storage lesion. Two reports concluded that platelets Although considered a research technique and not licensed by
stored at 1°C to 6°C could conceivably have satisfactory in the FDA, frozen platelets are used occasionally in the United
vivo viability and function if the surface of the platelets were States as autologous transfusions for patients who are refrac-
modified to prevent the enhanced clearance (unsatisfactory tory to allogeneic platelets. Platelets are collected by apheresis,
viability) from circulation.88,89 the cryopreservative dimethyl sulfoxide (DMSO) is added, and
Based on animal studies, it was suggested that cold- the platelets are frozen at –80°C. The frozen platelets can be
induced spherical platelets can remain in the circulation if stored for up to 2 years. Prior to transfusion, the platelets are
abnormal clearance is prevented. Spherical platelets, mani- thawed and centrifuged to remove the DMSO. Although in
fested as a result of cold storage, have been assumed to be a vivo recovery after transfusion is only about 33%, the platelets
trigger for low viability. The specific approach involves the seem to function effectively.5
SUMMARY CHART
Each unit of whole blood collected contains approxi- Research is being conducted to improve on the current
mately 450 mL of blood and 63 mL of anticoagulant- additive solutions.
preservative solution or approximately 500 mL of Research is being conducted to develop procedures to
blood and 70 mL of anticoagulant-preservative reduce or inactivate pathogens.
solution. RBC substitutes under investigation include hemoglo-
A donor can give blood every 8 weeks. bin-based oxygen carriers and perfluorocarbons.
As of 2011, samples from donors of each unit of do- A platelet concentrate should contain a minimum of
nated blood are tested by 10 screening tests for infec- 5.5 ⫻ 1010 platelets (in 90% of the sampled units
tious diseases markers. according to AABB standards) in a volume routinely
Glycolysis generates approximately 90% of the ATP between 45 and 65 mL that is sufficient to maintain a
needed by RBCs, and 10% is provided by the pentose pH of 6.2 or greater at the conclusion of the 5-day
phosphate pathway. storage period.
Seventy-five percent post-transfusion survival of RBCs When platelet concentrates (usually 4 to 6) are pooled
is necessary for a successful transfusion. using an open system, the storage time changes to
ACD, CPD, and CP2D are approved preservative solu- 4 hours. A new method of pooling that uses a closed
tions for storage of RBCs at 1°C to 6°C for 21 days, and system allows the pool to be stored for 5 days from the
CPDA-1 is approved for 35 days. date of collection.
Additive solutions (Adsol, Nutricel, Optisol) are ap- Apheresis components contain 4 to 6 times as many
proved in the United States for RBC storage for platelets as a PC prepared from whole blood. They
42 days. Additive-solution RBCs have been shown to should contain a minimum of 3.0 ⫻ 1011 platelets (in
be appropriate for neonates and pediatric patients. 90% of the sampled units).
RBCs have been traditionally glycerolized and frozen Platelet components are stored for up to 5 days at 20°C
within 6 days of whole blood collection in CPD or to 24°C with continuous agitation. When necessary, as
CPDA-1 and can be stored for 10 years from the date during shipping, platelets can be stored without con-
of freezing. tinuous agitation for up to 24 hours (at 20°C to 24°C)
Rejuvesol is the only FDA-approved rejuvenation so- during a 5-day storage period. Platelets are rarely
lution used in some blood centers to regenerate ATP stored at 1°C to 6°C.
and 2,3-DPG levels before RBC freezing. If a platelet bag is broken or opened, the platelets must
Rejuvenation is used primarily to salvage O-type and be transfused within 4 hours when stored at 20°C to
rare RBC units that are at outdate or with specific 24°C.
anticoagulant-preservative solution up to 3 days past
outdate.
2682_Ch01_001-025 28/05/12 12:22 PM Page 22
Chapter 1 Red Blood Cell and Platelet Preservation: Historical Perspectives and Current Trends 23
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39. Tappenden, J: Artificial blood substitutes. J R Army Med Corps monitoring of platelets. Transfusion Med 13:189–195, 2003.
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Chapter 2
Basic Genetics
Lorraine Caruccio, PhD, MT(ASCP)SBB
OBJECTIVES
1. Explain Mendel’s laws of independent segregation and random assortment, and describe how he developed them.
2. Correlate the concepts of dominant and recessive traits with examples of the inheritance of blood group antigens.
3. Explain the Hardy-Weinberg principle and how it applies to genetic traits.
4. Given the necessary information, solve Hardy-Weinberg problems for any blood group antigen.
5. Determine the inheritance pattern of a given trait by examining the pedigree analysis.
6. Describe the processes of mitosis and meiosis, and outline the differences between them.
7. Distinguish between X-linked and autosomal traits, and describe how each is inherited.
8. Describe in detail the processes of replication, transcription, and translation, including the basic mechanism of each.
9. List the various types of genetic mutations and describe how they can change the function of living cells and organisms.
10. Describe the cell’s different mechanisms for correcting mutations.
11. Identify some of the ways in which genetics can be used in the modern transfusion laboratory, including the necessary back-
ground information for describing modern genetic testing techniques.
12. Describe in general the modern techniques used in the study of genetics.
26
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clearly understood, as genetics is a very dynamic science completely successful without a clear understanding of the
that has its greatest potential in direct applications. Many principles of genetics and the laws of inheritance. The anti-
areas of transfusion medicine rely on an understanding of gens present on all blood cells are expressed as a phenotype,
blood group genetics and on accurate and sensitive methods but it is the genotype of the organism that controls what anti-
of pathogen testing to keep the blood supply safe. Most of gens may be expressed on the cell. For example, genotyping
the antigens in the various blood group systems (i.e., ABO, the donor or recipient DNA using leukocytes can determine
Rh, Kell, Kidd, etc.) generally follow straightforward inher- which antigens may be present on the cells and therefore
itance patterns, usually of a codominant nature. which antibodies can be made against them. This is espe-
Historically, the major focus and role of genetics in cially true when a clear picture of the red cell antigens pres-
blood banking has been more so in population genetics and ent on the red cells of a donor or recipient is not possible or
inheritance patterns, but now cellular and molecular ge- if an antibody screening test gives ambiguous results. Using
netics are equally important. Increasingly, modern genetic this simple example, we see that in modern blood banking,
techniques are playing a role in analyzing the profile of genetics has an important role.
blood donors and recipients, which was once done only
with serologic testing. Transfusion medicine physicians and Population Genetics
technologists should still know classical genetics, such as
interpretation of familial inheritance patterns. In addition, The major areas of population genetics of concern to blood
they must now master modern molecular methods that re- banking include Mendel’s laws of inheritance, the Hardy-
quire a high level of training and skill, such as in restriction Weinberg principle, and inheritance patterns.
mapping, sequencing, polymerase chain reaction (PCR),
and gene array technology. (See Chapter 4, “Concepts in Early Genetics and Mendel’s Laws of Inheritance
Molecular Biology.”)
The Swedish biologist Carolus Linnaeus started the first clas-
In this chapter, a general overview of genetics at three dif-
sification system of living things in the 17th century and
ferent levels (population, concerning genetic traits in large
used the unit of “species” as its principal definition. Deter-
numbers of individuals; cellular, which pertains to the cel-
mining factors that affected which classification group a
lular organization of genetic material; and molecular, based
species would be put into was based on physical traits and
on the biochemistry of genes and the structures that support
observations. There was no attempt made to understand the
them) is provided in some detail. It also gives a brief
underlying reasons for one trait versus another trait occur-
overview of modern molecular techniques. Chapter 4 ex-
ring in one species or another. The amazing diversity of
plains in greater detail the modern testing methods of mo-
species and the processes that might contribute to it were
lecular biology, including recombinant DNA technology,
not investigated further. In 1859, Charles Darwin published
Southern and Northern blotting, restriction fragment length
his epic book On the Origin of Species after many years of in-
polymorphism analysis, PCR techniques, and cloning and
tense study of various and diverse life-forms. Darwin’s am-
sequencing.
bition was to understand the diversity of life and how one
Classic Genetics organism could gain an advantage over another and better
survive in a given environment, which is referred to as “nat-
The science of genetics is one of the most important areas of ural selection.” It created a revolution in the thinking of
modern biology. The understanding of the inheritance of modern biology and is still controversial today.
blood group antigens and the testing for disease markers at The science of genetics found its modern development in
the molecular level, both of which are vitally important in the work of Gregor Mendel. Mendel was an Austrian monk
transfusion medicine, are based on the science of genetics. and mathematician who used sweet pea plants growing in a
Modern genetics is based upon the understanding of the bio- monastery garden to study physical traits in organisms and
chemical and biophysical nature of nucleic acids, including how they are inherited. He determined the physical traits to
deoxyribonucleic acid (DNA), ribonucleic acid (RNA), and be due to factors he called elementen within the cell. In mod-
the various proteins that are part of the chromosomal archi- ern genetics, we know the physical basis of these so-called
tecture. In addition, genetics is concerned with population elementen are genes within the nucleus of the cell. Mendel
studies and epidemiology. The understanding of inheritance chose a good model organism for his observations. He stud-
patterns in which genetic traits are followed and analyzed, ied the inheritance of several readily observable pea plant
as well as the biochemical reactions that result in gene mu- characteristics—notably flower color, seed color, and seed
tations that can give rise to new alleles, are highly important shape—and based his first law of inheritance, the law of in-
in the study of genetics. New alleles can result in new blood dependent or random segregation, on these results.
groups and disease conditions that affect the health of blood The first generation in the study, called the parental,
donors and blood recipients. pure, or P1 generation, consisted of all red or all white flow-
All areas of transfusion medicine are influenced by genet- ers that bred true for many generations. The plants were
ics, including HLA typing, cell processing, parentage studies, either homozygous for red flowers (RR, a dominant trait;
viral testing, and blood services, and these would not be dominant traits are usually written with uppercase letters)
2682_Ch02_026-044 22/05/12 11:38 AM Page 28
or homozygous for white flowers (rr, a recessive trait; reces- phenotype, and using flower color is a good basic model sys-
sive traits are usually written with lowercase letters). When tem to study this.
these plants were crossbred, the second generation, called Unlike the flower color of many types of plants, most
first-filial, or F1, had flowers that were all red. Thus the blood group genes are inherited in a codominant manner. In
dominant trait was the only trait observed. When plants codominance, both alleles are expressed, and their gene
from the F1 generation were crossbred to each other, the products are seen at the phenotypic level. In this case, one
second-filial, or F2, generation, of plants had flowers that gene is not dominant over its allele, and the protein products
were red and white in the ratio of 3:1 (Fig. 2–1). All the of both genes are seen at the phenotypic level. An example
plants from the F1 generation are heterozygous (or hybrid) of this is seen in Figure 2–2 concerning the MNSs blood
for flower color (Rr). The F2 generation has a ratio of three group system, in which a heterozygous MN individual would
red-flowered plants to one white-flowered plant. This is type as both M and N antigen positive. (See Chapter 8,
because the plants that have the R gene, either RR homozy- “Blood Group Terminology and the Other Blood Groups.”)
gous or Rr heterozygous, will have red flowers because the Mendel’s second law is the law of independent assortment
red gene is dominant. Only when the red gene is absent and and states genes for different traits are inherited separately
the white gene occurs in duplicate, as in the rr homozygous from each other. This allows for all possible combinations of
white-flowered plant, will the recessive white gene expres- genes to occur in the offspring. Specifically, if a homozygote
sion be visible as a phenotype. This illustrates Mendel’s first that is dominant for two different characteristics is crossed
law, the law of independent segregation. Therefore, each with a homozygote that is recessive for both characteristics,
gene is passed on to the next generation on its own. Specif- the F1 generation consists of plants whose phenotype is the
ically, Mendel’s first law shows that alleles of genes have no same as that of the dominant parent. However, when the F1
permanent effect on one another when present in the generation is crossed in the F2 generation, two general
same plant but segregate unchanged by passing into classes of offspring are found. One is the parental type; the
different gametes. other is a new phenotype called a reciprocal type and repre-
An intermediate situation can also occur when alleles ex- sents plants with the dominant feature of one plant and the
hibit partial dominance. This is observed when the pheno- recessive feature of another plant. Recombinant types occur
type of a heterozygous organism is a mixture of both in both possible combinations. Mendel formulated this law
homozygous phenotypes seen in the P1 generation. An ex- by doing studies with different types of seeds produced by
ample of this is plants with red and white flowers that have peas and noted that they can be colored green or yellow and
offspring with pink flowers or flowers that have red and textured smooth or wrinkled in any combination. An illus-
white sections. It is important to remember that although tration of independent assortment of Mendel’s second law is
the phenotype does not show dominance or recessive traits, given in Figure 2–3; his system of pea plant seed types are
the F1 generation has the heterozygous genotype of Rr. It is used as the example.
essential to understand how a genotype can influence a Mendel’s laws apply to all sexually reproducing diploid
organisms whether they are microorganisms, insects, plants,
or animals, or people. However, there are exceptions to the
Parental RR rr Mendelian laws of inheritance. If the genes for separate traits
are closely linked on a chromosome, they can be inherited
as a single unit. The expected ratios of progeny in F1 matings
may not be seen if the various traits being studied are linked.
Gametes R r
MN NN NN MM
First-Filial Rr
MN MN NN MN MN
Second-Filial R r
R RR Rr MN
MM MN NN NN
r Rr rr
Gametes RY ry
p p2 pq p2 + 2pq + q2 = 1.0
First-Filial RY Ry rY ry
q pq q2
Second-Filial RY Ry rY ry
Figure 2–4. Common inheritance patterns.
Ry RRYy RRyy RrYy Rryy many mathematical formulations, however, certain ideal sit-
uations and various conditions must be met to use the equa-
rY RrYY RrYy rrYY rrYy tions appropriately. These criteria are outlined in Box 2–1.
In any normal human population, it is almost impossible
ry RrYy Rryy rrYy rryy
to meet these demanding criteria. Although large popula-
Where R = round r = wrinkled tions exist, collecting sample data from a significantly large
enough segment of a population that correctly represents the
Y = yellow y = green members of the population is not always feasible. Also, mat-
Figure 2–3. A schematic illustration of Mendel’s law of independent assortment ing is not always random, and there is mixing of populations
using seed types. on a global scale now that leads to “gene flow” on a constant
basis. Recently, sequencing of the human genome has re-
vealed that gene mutations occur much more commonly
There can also be differences in the gene ratios of progeny than originally thought. Some of these mutations affect the
of F1 matings, if recombination has occurred during the phenotype of an individual, such as loss of enzyme function,
process of meiosis. An example of this in blood banking is and some do not. Despite these drawbacks, Hardy-Weinberg
the MNSs system, in which the MN alleles and the Ss alleles is still one of the best tools for studying inheritance patterns
are physically close on the same chromosome and are there- in human populations and is a cornerstone of population
fore linked. Recombination happens when DNA strands are genetics.
broken and there is exchange of chromosomal material Most of the various genes controlling the inheritance of
followed by activation of DNA repair mechanisms. The ex- blood group antigens can be studied using the Hardy-Weinberg
change of chromosomal material results in new hybrid geno- equations. A relevant example that shows how to use the
types that may or may not be visible at the phenotypic level. Hardy-Weinberg formula is the frequency of the Rh antigen,
Mendel’s laws of inheritance give us an appreciation of D, in a given population. In this simple example, there are
how diverse an organism can be through the variations in its two alleles, D and d. To determine the frequency of each
genetic material. The more complex the genetic material of allele, we count the number of individuals who have the cor-
an organism, including the number of chromosomes and the responding phenotype (remembering that both Dd and DD
number of genes on the chromosomes, the greater the po- will appear as Rh-positive) and divide this number by the
tential uniqueness of any one organism from another organ- total number of alleles. This value is represented by p in the
ism of the same species. Also, the more complex the genetic Hardy-Weinberg equation. Again, counting the alleles lets us
material, the more complex and varied its responses to con- determine the value of q. When p and q are added, they must
ditions in the environment. Therefore, as long as control is equal 1. The ratio of homozygotes and heterozygotes is de-
maintained during cell division and differentiation, organ- termined using the other form of the Hardy-Weinberg equa-
isms with greater genetic diversity and number can have an tion, p2 + 2pq + q2 = 1. If in our example we tested 1,000
advantage over other organisms in a given setting. random blood donors for the D antigen and found that
Hardy-Weinberg Principle
BOX 2–1
G. H. Hardy, a mathematician, and W. Weinberg, a physi-
cian, developed a mathematical formula that allowed the Criteria for Use of the Hardy-Weinberg Formula
study of Mendelian inheritance in great detail. The Hardy- • The population studied must be large.
Weinberg formula—p + q = 1, in which p equals the gene • Mating among all individuals must be random.
frequency of the dominant allele and q is the frequency of • Mutations must not occur in parents or offspring.
the recessive allele—can also be stated p2 + 2pq + q2 = 1 and • There must be no migration, differential fertility, or mortality of
specifically addresses questions about recessive traits and genotypes studied.
how they can be persistent in populations (Fig. 2–4). Like
2682_Ch02_026-044 22/05/12 11:38 AM Page 30
traits, if an individual does not have the trait, he or she can Terminology
be a carrier and can pass it on to offspring. This is why re-
cessive traits seem to skip generations, which is another Remember that it takes two gametes to make a fertilized egg
helpful clue in determining inheritance patterns. with the correct (2N) number of chromosomes in the nucleus
Autosomal-dominant traits are routinely encountered in the of a cell. Therefore, each parent contributes only half (1N) of
blood bank, as most blood group genes are codominant and the inherited genetic information, or genes, to each child. In
are on autosomal chromosomes. They are passed on from one order to be completely healthy, each child must have the cor-
generation to the next and do not skip generations; therefore, rect number of genes and chromosomes (2N), without major
they are usually present in every generation. Finally, unusually mutations affecting necessary biochemical systems.
rare traits that occur in every generation and in much greater The genetic material has a complex pattern of organization
frequency than the general population are often the result of that has been evolving for millions of years to an amazing
matings between related individuals. Table 2–1 provides level of coordination and control. At the smallest level, genes
examples of inheritance patterns in transfusion medicine. are composed of discrete units of DNA arranged in a linear
fashion, similar to a strand of pearls, with structural proteins
Cellular Genetics wrapped around the DNA at specific intervals to pack it into
tightly wound bundles. The DNA is organized at a higher
Organisms may be divided into two major categories: level into chromosomes, with each chromosome being one
prokaryotic, without a defined nucleus, and eukaryotic, with incredibly long strand of duplex (double-stranded) DNA. A
a defined nucleus. Human beings and all other mammals gene is a section, often very large, of DNA along the chromo-
are included in the eukaryotic group, as are birds, reptiles, some. The specific sequence of nucleotides and the location
amphibians, fish, and some fungus species. The nucleus of on the chromosome determines a gene. In addition, each gene
a cell contains most of the genetic material important for has specific and general sequences that occur upstream
replication and is a highly organized complex structure. The (before the start site) and downstream (after the termination
nuclear material is organized into chromatin, consisting of signals) that contribute to how the gene functions. The spe-
nucleic acids and structural proteins, and is defined by stain- cific location of a gene on a chromosome is called a locus
ing patterns. Heterochromatin stains as dark bands, and (plural = loci), and at each locus there may be only one or
achromatin stains as light bands and consists of highly con- several different forms of the gene, which are called alleles.
densed regions that are usually not transcriptionally active. It is important to keep in mind the distinction between
Euchromatin is the swollen form of chromatin in cells, phenotype and genotype. Genotype is the sequence of DNA
which is considered to be more active in the synthesis of that is inherited. The phenotype is anything that is produced
RNA for transcription. by the genotype, including an enzyme to control a blood
Most cellular nuclei contain these different types of group antigen; the length of long bones of the skeleton; the
chromatin. The chromatin material itself, which chiefly curvature of the spine; the ratio of muscle fibers; the level of
comprises long polymers of DNA and various basic pro- hormones produced; and such obvious traits as eye, skin, and
teins called histones, is compressed and coiled to form hair color. Keep in mind that more than one gene can affect
chromosomes during cell division. Each organism has a a particular trait (part of a phenotype), such as the height of
specific number of chromosomes, some as few as 4 and an individual; all relevant genes can be considered as part of
some as many as 50. In humans, there are 46 chromosomes. the genotype for that trait. Depending on the alleles inherited,
These 46 chromosomes are arranged into pairs, with one an organism can be either homozygous or heterozygous for
of each being inherited from each parent. Humans have a specific trait. The presence of two identical alleles results
22 autosomes and one set of sex chromosomes, XX in the in a homozygous genotype (i.e., AA), and the phenotype is
female and XY in the male. This comprises the 2N state of group A blood. On the other hand, the inheritance of different
the cell, which is normal for all human cells except the alleles from each parent gives a heterozygous genotype.
gametes (sex cells). N refers to the number of pairs of Another important concept is that of the “silent” gene, or
chromosomes in a cell. amorph, and the term hemizygous. An amorph is a gene that
does not produce any obvious, easily detectable traits and is
seen only at the phenotypic level when the individual is
Table 2–1 Examples of Inheritance homozygous for the trait. Hemizygous refers to the condition
Patterns in Transfusion when one chromosome has a copy of the gene and the other
Medicine chromosome has that gene deleted or absent.
TYPE OF PATTERN EXAMPLE
Mitosis
Autosomal-dominant In (Lu) suppressor gene
cells is called mitosis. The chromosomes are duplicated, and Table 2–2 Stages of Mitosis and Meiosis
one of each pair is passed to the daughter cells. During the
process of mitosis, quantitatively and qualitatively identical MITOSIS
DNA is delivered to daughter cells formed by cell division. Stage Description
The complex process of mitosis is usually divided into a
1. Interphase (2N) Resting stage between cell divisions; during
series of stages, characterized by the appearance and move- this period, cells are synthesizing RNA and
ment of the chromosomes. The stages are interphase, proteins, and chromatin is uncondensed.
prophase, metaphase, anaphase, and telophase. The different
phases of mitosis include interphase at the beginning, in 2. Prophase (4N) First stage of mitotic cell division.
Chromosomes become visible and
which DNA is in the form of chromatin and is dispersed condense. Each chromosome has two
throughout the nucleus. This is the stage of the DNA when chromatids from duplication of DNA, and
cells are not actively dividing. New DNA is synthesized by a chromatids are linked via the centromere.
process called replication. In the next stage, prophase, the
3. Metaphase (4N) Chromosomes move toward the equator of
chromatin condenses to form chromosomes. In prophase, the the cell and are held in place by micro-
nuclear envelope starts to break down. In the next stage, tubules attached at the mitotic spindle
metaphase, the chromosomes are lined up along the middle apparatus.
of the nucleus and paired with the corresponding chromo-
4. Anaphase (4N) The two sister chromatids separate. Each one
some. In this stage, chromosome preparations are made for migrates to opposite poles of the cell, and
chromosome analysis in cytogenetics. In anaphase, which the diameter of the cell decreases at
occurs next, the cellular spindle apparatus is formed and the equator.
chromosomes are pulled to opposite ends of the cell. The cell
5. Telophase (2N) Chromosomes are at the poles of the cell,
becomes pinched in the middle, and cell division starts to take and the cell membrane divides between the
place. In the last stage, telophase, the cell is pulled apart, two nuclei. The cell divides, and each cell
division is complete, and the chromosomes and cytoplasm are contains a pair of chromosomes identical to
separated into two new daughter cells. The process of mitosis the parent cell.
is illustrated in Figure 2–6 and outlined in Table 2–2.
MEIOSIS
Stage Description
8. Telophase II (N) Each cell separates into two new cells. There
Anaphase 4N are now four (N) cells with a unique genetic
constitution.
Telophase Meiosis
and cell
division
A different process is used to produce the gametes or sex
2N 2N cells. The process is called meiosis and results in four
Figure 2–6. Mitosis leads to two daughter cells having the same number of unique, rather than two identical, daughter cells. The
chromosomes as the parent cell. uniqueness of the daughter cells generated with meiosis
2682_Ch02_026-044 22/05/12 11:38 AM Page 33
Molecular Genetics The four different bases are adenine (A), cytosine (C),
guanine (G), and thymine (T). Adenine and guanine are
The study of genetics at the molecular level requires an un- purines, consisting of double-ring structures. Cytosine and
derstanding of the biochemistry of the molecules involved. thymine are pyrimidines, which are single-ring structures
This includes knowledge of the physical conformation of (Fig. 2–9). The hydrogen bonding in DNA is specific, in
chromosomes as well as the biological and chemical nature which A bonds only to T with two hydrogen bonds, thus
of the polymers of the different nucleic acids and nuclear forming the weaker pairing, and C bonds only with G with
proteins. three hydrogen bonds, forming the stronger pairing. This is
the classic Watson-Crick base pairing that occurs in the
Deoxyribonucleic Acid B-form, right-handed helical structure of DNA. It was
first postulated by James Watson and Francis Crick, at
Deoxyribonucleic acid (DNA) is a masterpiece of architec- Cambridge University in the early 1950s (Fig. 2–10).1 Since
tural evolution and the “backbone” of heredity. Chromatin then, it has been discovered that DNA can also occur in mod-
is actually a type of polymer structure. Chromosomes are ified forms such as Z-DNA, which is a type of left-handed
composed of long, linear strands of DNA tightly coiled helix with a different three-dimensional conformation but
around highly basic proteins called histones (Fig. 2–8). Each that contains the same four nitrogenous bases.2 In addition,
chromosome is a single, extremely long strand of duplex there are some unusual forms of nitrogenous bases that can
DNA. Remember that DNA is a nucleic acid, and therefore be incorporated into DNA templates.
most of the proteins that interact with it have an overall basic The phosphates in the DNA backbone attach to the sugar
pH. This helps to stabilize the overall complex structure. The at the third and fifth carbon atoms. Remember, all atoms in
complex of DNA and histone protein is referred to as a nu- a molecular structure are numbered. The linkage of the
cleosome. The DNA and protein complex is bound together purine or pyrimidine nitrogenous base to the sugar is at car-
so tightly and efficiently that extremely long stretches of bon 1. Therefore, the two DNA strands are antiparallel—that
DNA, several inches in length, can be packaged inside the is, one strand is 5′ to 3′ (pronounced “5 prime to 3 prime”)
nucleus of a cell on a microscopic level. The DNA and his- in one direction and the complementary strand is 5′ to 3′ in
tones are held together by various proteins that keep the the other direction. During transcription, only one strand is
DNA in a very specific helical conformation. This conforma- copied, which is the complementary strand that gives the
tion also protects the DNA from degradation when it is not correct 5′ to 3′ sequence of messenger RNA (mRNA) that
being replicated or transcribed. corresponds to the template, or coding strand, of the DNA
All DNA in human cells is in the form of a two-stranded molecule.
duplex, with one strand in one direction and its complemen- As there are only four different bases used to make up
tary strand in the opposite direction (the strands are said to DNA templates and there are 20 different amino acids that
be antiparallel). DNA is composed of four nitrogenous are used to construct proteins, it is evident that any single
bases, a 5-carbon sugar molecule called deoxyribose and a nucleotide cannot code for any specific amino acid. What
phosphate group. The sugar and phosphate moieties com- has been discovered is that triplets of nucleotides called a
prise the backbone of the DNA molecule, while the nitroge-
nous bases face in to each other and are stabilized by
hydrogen bonding and Van der Waals forces. The backbone Thymine—Adenine
of a DNA molecule is joined by phosphodiester linkages.
Unlike what is observed in proteins with an ␣-helical struc-
ture, there is little bonding forces between bases on the same
strand, which allows DNA to be strong but flexible.
deoxyribose
deoxyribose
DNA
DNA HISTONES
Cytosine—Guanine
deoxyribose
deoxyribose
The double helix has constant width The genetic code is triplet
U C A G
} } }
}
UUU UCU UAU UGU
Phe Tyr Cys
U UUC UCC UAC UGC
Ser
UUA
UUG } Leu
UCA
UCG
UAA
UAG } STOP
UGA STOP
UGG Trp
} } }
}
CUU CCU CAU CGU
His
CUC CCC CAC CGC
Leu Pro Arg
C CUA
CUG
CCA
CCG
CAA
CAG } Gln
CGA
CGG
Figure 2–10. Base pairing in DNA and DNA structure. (From Lewin B: Genes
VIII. Prentice Hall/Pearson Education, New York, 2004, p 6. Reprinted with permis-
sion of the author.) A
AUU
AUC
} } Ile
ACU
ACC
Thr
AAU
AAC } Asn
AGU
AGC } Ser
AUA
AUG Met
ACA
ACG
AAA
AAG } Lys
AGA
AGG
} Arg
codon, such as ATG, code for one specific amino acid. How-
ever, there is a redundancy to the genetic code in that some
}
} } }
GUU GCU GAU GGU
Asp
amino acids have more than one triplet, which codes for G GUC GCC GAC GGC
Val Ala Gly
their addition to the peptide chain formed during transla-
tion. Generally, the more common the amino acid is in pro-
teins, the greater the number of codons it has. There are four
GUA
GUG
GCA
GCG
GAA
GAG } Glu
GGA
GGG
special codons, including the only codon specific for the ini-
Figure 2–11. The genetic code. (From Lewin B: Genes VIII. Prentice Hall/Pearson
tiation of transcription and translation, called the start codon, Education, New York, 2004, p 168. Reprinted with permission of the author.)
and three different codons that are used to stop the addition
of amino acids in the process of peptide synthesis. Because
these three codons cannot be charged with an amino acid,
they are called stop codons and result in termination of the enzymes and other molecules are required at each step. First,
peptide being translated from mRNA. The genetic code is sections of DNA must be uncoiled from its supercoiled (or
illustrated in Figure 2–11. double-twisted) nature, and the two strands must be sepa-
rated and kept apart; this is done by the enzymes DNA
Replication gyrase (opens the supercoils) and DNA helicase (separates
The replication, or copying, of DNA is a complex process the two strands of duplex DNA). These enzymes, using en-
involving numerous enzymes, nucleic acid primers, various ergy derived from ATP hydrolysis, open the DNA molecules
small molecules, and the DNA helix molecule that serves as and keep the strands separate. In the next step, DNA poly-
its own template for the replication process. DNA must be merase III can synthesize a new strand in the 5′ to 3′ direc-
copied before mitosis can occur and must be copied in such tion on the leading strand. Proteins called single-stranded
a way that each daughter cell will have the same amount of binding proteins interact with the open strands of DNA to pre-
DNA and the same sequence. Nearly all DNA replication is vent hydrogen bonding when it is not needed during repli-
done in a bidirectional manner and is semiconservative in cation. DNA polymerase III also proofreads the addition of
nature.3 Specifically, as enzymes involved in the replication new bases to the growing DNA strands and can remove an
process open the double-stranded DNA helix, one strand of incorrectly incorporated base, such as G paired to T.
DNA is copied in a 5′ to 3′ manner, while the other strand is In order for replication to take place on any piece of DNA,
opened partially in sections and is copied 5′ to 3′ in sections there must first be a short oligonucleotide (composed of
as the double-stranded template continuously opens. In ad- RNA) piece that binds to the beginning of the region to be
dition, each newly synthesized DNA strand will be paired replicated. This “primes” the replication process; therefore,
with one of the parent strands. The replication process is these short oligonucleotide sequences are called primers. All
shown in Figure 2–12. DNA is replicated in a 5′ to 3′ manner. Replication of the
In order for DNA to be replicated with the exact copying other parent strand, the lagging strand, is more complicated
of the template and its sequence into a new double-stranded because of this restriction. As the helix is opened, RNA
helix, many enzymes and proteins must participate in the primer sequences are added to the area of the opening fork,
process. DNA replication occurs in specific steps, and certain and the RNA primers are extended in a 5′ to 3′ manner until
2682_Ch02_026-044 22/05/12 11:38 AM Page 36
3’
5’
5’
Leading strand
3’
Lagging strand 3’
5’
Lagging strand synthesis
the polymerase reaches the previously synthesized end. depurination. Some cancer treatments are often based on this
Rather than being replicated in a continuous manner, these principle that the faster-replicating DNA in cancer cells has
replication forks open up all along the lagging strand and are greater damage and puts cancer cells at greater risk for cell
extended in this way. These small regions of newly replicated death than normal cells. Ionizing radiation and strong
DNA are known as Okazaki fragments. The fragments must oxidants such as peroxides can cause single-strand breaks.
be joined together to make a complete and continuous Ultraviolet (UV) radiation can alter thymine bases, resulting
strand. This is accomplished by the two enzymes DNA poly- in thymine dimers. Certain drugs such as the antibiotic
merase I and DNA ligase. The RNA primers are synthesized mitomycin C can form covalent linkages between bases on
and added to the DNA strands by an enzyme called primase, opposite strands; therefore, during replication, separation of
which anneals to the parent strands. After replication of the the strands at that site will not occur correctly, and the
leading and lagging strands is complete, DNA ligase joins resulting daughter strands will have mutations. Nearly all
the phosphodiester bonds of the DNA backbone to complete defects in DNA replication can be corrected by the various
the intact molecule. Isomerase enzymes recoil the DNA; mechanisms used by the cell to maintain DNA integrity.
once this is completed and the DNA is proofread by proof- However, if too many mistakes occur, the repair systems can
reading enzymes, the cell can continue with mitosis and cell be overwhelmed, and mistakes will not be corrected. The
division. cell in which these mutations occur will have an altered DNA
nucleotide sequence and may or may not be viable.
Repair There are several major DNA repair systems. These in-
DNA must be copied exactly or the information it contains clude photoreactivation, excision repair (also referred to as
will be altered, possibly resulting in a decrease in the organ- cut and patch repair), recombinational repair, mismatch re-
ism’s vitality. However, mistakes in the complex process of pair, and SOS repair (Box 2–2). DNA repair systems can rec-
replication do occasionally occur, and a number of efficient ognize mismatched base pairs, missing nucleotides, and
mechanisms have evolved to correct these mistakes. The altered nucleotides in DNA sequences. For example, when
mechanisms can detect the mistakes, or changes, and correct thymine dimers are formed after exposure to UV light, the
the actual DNA sequence. One of the most important mech- photoreactivation enzyme becomes active and enzymatically
anisms for correcting DNA replication errors is the proof- cleaves the thymine dimers. In addition to the photoreacti-
reading ability of DNA polymerases. The proofreading vation system of DNA repair, thymine dimers can be re-
occurs in both the 5′ to 3′ and 3′ to 5′ directions and allows moved by the rather complex process of excision repair,
the polymerase to backtrack on a recently copied DNA where the disrupted section of the DNA is removed. A cut is
strand and remove an incorrect nucleotide and insert the cor-
rect one in its place. In addition to the proofreading ability
of DNA polymerase enzymes, there is a second type of edit-
BOX 2–2
ing called mismatch repair, where an incorrect nucleotide
is removed and the correct one is inserted in its place. DNA Repair Systems
In addition to errors in the primary nucleotide sequence
• Photoreactivation
of DNA molecules, there are other possible alterations that
• Excision repair
can affect the way the sequence information is processed.
• Recombinational repair
Many different chemicals and environmental factors can alter
• Mismatch repair
DNA by modifying it chemically or physically. These include
• SOS repair
alkylating agents, which react with guanine and result in
2682_Ch02_026-044 22/05/12 11:38 AM Page 37
made on one side of the thymine dimer that bulges out from although they change the DNA sequence and the triplet
the rest of the duplex DNA. DNA polymerase I synthesizes codon(s), may not change the amino acid sequence in the
a short replacement strand for the damaged DNA section. corresponding protein because of redundancy in the genetic
The old strand is removed by DNA polymerase I as it moves code. Recall that some of the amino acids have more than
along the DNA. Finally, the old DNA strand is removed, and one codon that represents them. Therefore, if the new codon
the newly formed DNA segment is ligated into place. is for the same amino acid, the mutation will be silent, and
Recombinational repair uses the correct strand of DNA to the protein sequence will be the same. An example of this is
fill in the strand where the error was deleted. Polymerase I the amino acid threonine, which has ACA, ACC, ACG, and
and DNA ligase, then fill in the other strand. Mismatch re- ACU as possible codons. Therefore, a substitution of C, G,
pair is activated when base pairing is incorrect and a bulge or U for A at the third position of the codon would still have
occurs in the duplex DNA. Mismatch repair enzymes are able a peptide with threonine at that position. A type of “silent
to remove the incorrect nucleotides and insert the correct mutation” also occurs when a mutation happens that causes
ones. Methyl groups on adenines are used by the mismatch a change in the peptide sequence, but that part of the peptide
enzyme systems to determine which nucleotide is correct does not seem critical for its function; therefore, no mutation
and which is a mistake. When DNA and cell damage occurs, is seen at the phenotypic level, such as enzyme function or
SOS repair is induced. Damage can be caused by UV radia- cell surface marker. A transition is a type of mutation in
tion, chemical mutagens, and excessive heat, and by such which one purine is substituted for another purine, or one
treatments as exposure to cross-linking agents. There are cer- pyrimidine is substituted for another pyrimidine. When a
tain sections of highly conserved DNA that are activated purine is substituted for a pyrimidine or a pyrimidine for a
when DNA is damaged, and the genes that are part of the purine, it is called a transversion. Examples of DNA muta-
SOS response system must work in a coordinated manner to tions are outlined in Table 2–4.4
repair the damaged DNA through recombination events that Another type of mutation that can have a deleterious
remove the damaged sections and replace them with the cor- effect on the peptide sequence is called a missense point
rect sequences. Repair systems exist and have been studied mutation. A missense mutation results in a change in a
closely in both prokaryotes and eukaryotes. All of these sys- codon, which alters the amino acid in the corresponding
tems are available to maintain the integrity of the DNA. The peptide. These changes cannot be accommodated by the
fact that so many repair systems exist in the cell proves how peptide while still maintaining its function. Typical examples
important the correct structure and sequence of DNA is to of missense mutations include the alterations in the hemo-
the cell. It also shows how complex the DNA molecule is in globin molecule at a single base pair, resulting in different
that many types of mutations can occur, and therefore many types of inherited anemias.5 A very specific type of serious
types of correction processes are present to repair them. mutation, called a nonsense mutation, results when a point
change in one of the nucleotides of a DNA sequence causes
Mutations one of the three possible stop codons to be formed. The three
Although many effective DNA proofreading and repair sys- stop codons are amber (UAG), opal (UGA), and ochre
tems help to keep newly synthesized DNA from having mu- (UAA), and these terminate the reading of the DNA
tations, none of the systems are foolproof and occasional sequence, so the resulting peptide is truncated at its 3′ end.
mutations occur. Once a mutation is introduced into a DNA A more severe type of mutation happens when there is an
coding strand, the information in that strand is now altered. insertion or deletion of one or more (but never multiplicities
It may be altered at the protein level if the mutation encodes of three) nucleotides in the DNA sequence. The result of this
for a different amino acid or a change in reading frame. In
general, a mutation is any change in the structure or se- Table 2–4 Examples of DNA Mutations
quence of DNA, whether it is physical or biochemical. An in Blood Groups
organism is referred to as a mutant if its DNA sequence is
different from that of the parent organism. The original form TRANSITION
of the DNA sequence, and the organism in which it occurs, S antigen s antigen
is called the wild type. The various chemicals and conditions
that can cause mutations are referred to as mutagens. Many Amino acid Methionine Threonine
mutagens are also carcinogens in that the cells in which they mRNA code AUG ACG
occur have an advantage in growth patterns that allows them
to dominate the cells around them. DNA code TAC TGC
There are different types of mutations, and they may have
TRANSVERSION
very different consequences for the organisms in which they
occur. Also, remember that mutations can be spontaneous. N antigen He antigen
If they occur in the germinal tissue, they are passed on from Amino acid Leucine Tryptophan
one generation to the next. The simplest type of mutation is
the point mutation, in which only one nucleotide in the mRNA code UUG UGG
DNA sequence is changed. Point mutations include substi- DNA code AAC ACC
tutions, insertions, and deletions. Certain substitutions,
2682_Ch02_026-044 22/05/12 11:38 AM Page 38
greater study of RNA sequences by making a more stable ver- from each other, depending on the gene they were tran-
sion of it. RNA isolation also requires greater disruption of scribed from. In addition, mRNA undergoes postsynthesis
the cellular material, usually with chaotropic agents such as processing before it can be transferred out of the nucleus and
guanidine thiocyanate, lithium salts, and mechanical means translated.
to disrupt cell membranes, such as douncing or shearing The third major class of RNA is transfer RNA (tRNA); it
with a needle and syringe. Once DNA or RNA has been is involved in bringing amino acids to the mRNA bound on
isolated, they can be stored in low salt buffers at the respec- the ribosome. Each tRNA molecule can be charged with only
tive correct pH at –20°C or lower for DNA and RNA at –80°C one species of amino acid. However, as mentioned, the ge-
for many months to many years. Storage in water is not rec- netic code is redundant, and many amino acids have more
ommended because the nucleic acids are polymers, hydrol- than one type of tRNA that codes for them. In addition,
ysis reactions will degrade them, and water can contain tRNA has considerable internal hydrogen bonding to acquire
contaminates that can degrade nucleic acids over time, even its formal structure. This hydrogen bonding stabilizes tRNA
at low temperatures. Important is the pH of the buffer used as well.
in nucleic acid isolation and storage, as acidic pH can result The fourth major type of RNA includes small RNA mole-
in degradation of DNA and alkaline pH can degrade RNA. cules, which have other various functions within the cell.
These RNA molecules, such as silencing RNA molecules, are
Ribonucleic Acid necessary for proper gene expression and are altered in
amount and type during cellular growth and differentiation.
Ribonucleic acid (RNA) is similar to DNA in structure but
has certain key differences. It has a very different role to play Transcription
in the cell. One of the key structural differences is that unlike Transcription is the cellular process by which DNA is copied
DNA, which is usually a double-stranded helix, RNA occurs into RNA. Although mRNA accounts for only a small percent
most often as a single-stranded structure, although internal of the total RNA inside a eukaryotic cell, it has the extremely
hydrogen bonding occurs frequently, probably to stabilize important role of being a “transportable” and disposable
the RNA molecules. Both DNA and RNA are made up of nu- form of the genetic code. Messenger RNA allows for highly
cleotides, but in place of thymine in DNA there is uracil in efficient processing of the genetic code into the proteins that
RNA. Uracil is very similar to thymine except that it lacks a play nearly all the functional roles within a cell. Transcrip-
methyl group. Another major difference is the substitution tion begins when the enzyme RNA polymerase II binds to
of the sugar ribose for deoxyribose in the backbone struc- the region upstream (to the left of the 5’ start site) of a gene.
ture. The sugar in DNA, deoxyribose, lacks a hydroxyl group Certain DNA short sequences, called consensus sequences,
(-OH) at the 2′ carbon position (thus, the deoxyribonucleic such as the CAAT box and TATA box, are located at specific
acid, or DNA, name). Ribose in RNA has the hydroxyl group sections upstream of the gene to be transcribed; these are
at this carbon position, whereas DNA has hydrogen. used to position RNA polymerase properly so transcription
In eukaryotes, RNA used to transmit genetic information of a gene is started correctly. This region is referred to as the
(stored as DNA) from the nucleus to the cytoplasm is trans- promoter and is important in how and when a gene is
lated into peptides and proteins. DNA is copied into RNA by expressed.
transcription, modified and transported out of the nucleus Promoter regions also contain specific sequences that
to the ribosomes, where it is translated into protein, which allow certain proteins, transcription factors, to bind to
is then modified if necessary for its proper function. There- them preferentially. This allows for certain genes to be
fore, RNA is the “go-between” of DNA, which stores genetic more or less active than other genes. Transcription factors
information and protein, which is the final product of the are very critical to proper cell growth and differentiation.
expression of that genetic information. It is worth mention- Upstream sequences are not part of the mRNA itself and
ing that certain viruses can store genetic information as RNA, are never transcribed; rather, they function to control tran-
whereas others use DNA, either single- or double-stranded; scription of mRNA. In addition to the promoter regions
some viruses use both during the different parts of their in- that can positively or negatively regulate various transcrip-
fectious cycles. tion factors and other transcription-specific protein effec-
In eukaryotes there are four major types of RNA; each has tors, there are regions of DNA sequence called enhancers
a specific function as well as its own corresponding poly- that can affect transcription rates. Unlike promoters, these
merase. The first class of RNA molecule is ribosomal RNA enhancer regions can have such effects without being close
(rRNA), which makes up a large part of the ribosomal struc- to the coding regions of the genes they influence. RNA is
ture on the endoplasmic reticulum in the cytoplasm. It is synthesized in a 5′ to 3′ direction; transcription starts at
here that RNA is translated into peptide. RNA polymerase I the 3′ end of the coding (or template) strand of the DNA
transcribes rRNA. It is the most abundant and consistent duplex after it is opened to two single strands and pro-
form of RNA in the cell. The second class of RNA is messen- ceeds to the 5′ end. In this way, a 5′ to 3′ coding strand is
ger RNA (mRNA); it is this form that is transcribed from generated with U in place of T. The RNA transcript is com-
DNA that encodes specific genes, such as those determining plementary to the template strand and is equivalent to the
the various blood groups. RNA polymerase II transcribes coding strand of the DNA molecule. Transcription is
mRNA. Unlike rRNA, mRNA molecules are very different illustrated in Figure 2–14.
2682_Ch02_026-044 22/05/12 11:38 AM Page 40
5' UACGCGGUACGGUCAAUGCAUCUACCU
Figure 2–14. Transcription of RNA from DNA. (From Lewin B: Genes VIII. Pren-
tice Hall/Pearson Education, New York, 2004, p 241. Reprinted with permission of
the author.)
Peptide bond synthesis involves transfer of with an amino acid can it transport it to the ribosome for
polypeptide to aa-tRNA translation.
During the elongation step of translation, the incoming
tRNA binds to the A site in presence of the elongation factor
called E2F. Again, GTP is hydrolyzed as the energy source
to move the tRNA in the A site to the P site. As the tRNA in
the A site is moved to the P site, the tRNA in the P site is
released back to the cytoplasm to pick up another amino
acid. In this way, tRNA molecules are conserved. The ribo-
somes move down the codons on the mRNA one at a time,
adding a correct amino acid to the growing peptide chain.
Figure 2–16. Peptide bond synthesis in translation. (Lewin B: Genes VIII. Pren-
tice Hall/Pearson Education, New York, 2004, p 137. Reprinted with permission of
As the ribosome moves down the mRNA, it eventually comes
the author.) to one of the three stop codons—UAA, UGA, or UAG—and
translation of that mRNA is finished. Termination factors
help the ribosome units to separate, and the peptide chain
hydrogen bonding that makes sure the correct aa is joined is further processed. Post-translational processing can con-
in the peptide chain by allowing only the correct tRNA sist of glycosylation, the addition of sugar groups, or the
molecule with the right anticodon to bond to the correct removal of leader peptide sequences that are used to traffic
mRNA codon. The second part of the tRNA molecule is at proteins to the cell membrane.
the 3′ hydroxyl end and binds an amino acid. Only one aa Processing can also consist of complicated folding
can bind to one tRNA molecule; specificity is determined by schemes that ensure the protein is in its correct tertiary form,
the 3′ hydroxyl end. According to the recognition region, an such as the formation of disulfide bonds via cysteine aa
aminoacyl synthase enzyme adds the specific aa to the residues. A large family of proteins called heat-shock pro-
correct tRNA molecule. Only when the tRNA is charged teins assists new proteins to be folded correctly by binding
to hydrophobic (water-avoiding) sections of the nascent
protein chain. Hydrogen bonding, van der Waals forces, and
Protein synthesis has 3 stages hydrophilic (water-attracting) regions of proteins also help
to give the new protein its correct three-dimensional confor-
Initiation 40s subunit on mRNA binding site
is joined by 60s subunit, and aminoacyl
mation. After translation is complete, the mRNA is rapidly
tRNA binds next degraded by enzymes (a process that helps to control gene
expression) or attaches to another ribosome, and the entire
translation process starts all over again. See Figure 2–18.
tRNA is an adaptor
Single-
stranded loop
Anticodon-
codon pairing
Figure 2–17. Stages of protein synthesis in translation. (Lewin B: Genes VIII. Figure 2–18 Schematic illustration of tRNA molecule and base pairing. (Lewin
Prentice Hall/Pearson Education, New York, 2004, p 137. Reprinted with permission B: Genes VIII. Prentice Hall/Pearson Education, New York, 2004, p 115. Reprinted
of the author.) with permission of the author.)
2682_Ch02_026-044 22/05/12 11:38 AM Page 42
Common Steps in Modern Genetics 2. There must be a way to visualize and locate the molecular
Techniques species to be studied. This is often done with probes that
act as signals or beacons that will bind specifically to the
Over the past three decades, the knowledge of genetics has molecule being analyzed; for example, DNA, by interac-
advanced exponentially. Understanding the biochemical and tion with parts of the molecule, such as hybridization
biophysical nature of DNA, RNA, and peptides and proteins with the hydrogen bonds of DNA. The probes can be syn-
has allowed techniques to be developed to study these im- thesized with radiolabels, chemiluminescence molecules,
portant molecules in great detail and to define the ways in fluorochromes, or nanoparticles.
which they interact in cells, groups of cells, complex multi- 3. A method to separate out different species and subspecies
cellular organisms, and even in an entire human being. Most being studied, such as different sequences of DNA, must
of the techniques used in molecular biology (the science of be available. An example of this involves running nucleic
studying and manipulating genetic material) are based on acids through gels, binding them to columns, hybridizing
the biochemical and biophysical properties of genes and them to membranes, or binding them to other molecules.
chromosomes. 4. A method to quantify the isolated and studied species has
The following steps are common to most techniques used to be used to get differences in amount as exact as possi-
in molecular biology: ble in the process studied. Changes in optical density
1. Any material that is to be studied must first be isolated when exposed to UV radiation, radioisotope emission lev-
intact without structural damage. This is often done with els, fluorescence energy detection, chemiluminescence
chemicals that interact with one part of the cellular ma- emission that can be detected with optical imaging
terial and not another. The methods to do this can be instruments can all be used to obtain data.
based on pH changes, salt gradients, detergent lysis, and Several techniques used in molecular biology are reviewed
enzyme activation. in Chapter 4.
SUMMARY CHART
Genetics is defined as the study of inheritance or the cannot be passed on to another generation. Some
transmission of characteristics from parents to offspring. mutations are silent and have no consequence on the
It is based on the biochemical structure of chromatin, organism in which they occur and therefore have no
which includes nucleic acids and the structural proteins selective pressure against them in the population.
that constitute the genetic material as well as various en- Transcription is an enzymatic process whereby genetic
zymes that assist in genetic processes such as replication. information in a DNA strand is copied into an mRNA
All living organisms have specific numbers of chromo- complementary strand. Eukaryotic mRNA is modified
somes. Humans have 22 pairs of autosomes and one after it is made by various processing steps, such as the
set of sex chromosomes, females (XX) and males (XY), removal of introns and addition of a poly-A tail to the
giving a total of 46 chromosomes in diploid cells. 3′ end. These processing steps take place in the nu-
Mendel’s law of independent assortment states that fac- cleus of the cell before the mRNA is exported to the
tors for different characteristics are inherited independent cytoplasmic ribosomes for translation.
of each other if they reside on different chromosomes. Translation is the complex process by which mRNA,
Human chromosomes are composed of the genetic which contains a mobile version of the DNA template
material chromatin, a complex of the nucleic acid poly- encoding the genes for an organism, is turned into pro-
mer DNA wrapped around highly basic proteins called teins, which are the functional units of an organism and
histones. The helical structure of DNA allows a lot of in- the cells that it consists of. Translation occurs on the
formation to be packaged in a very small amount of space. ribosomes, and additional steps may be necessary to get
Replication of DNA is semiconservative and is accom- a specific protein into its final correct form, such as pro-
plished via the enzyme DNA polymerase, which pro- teins that require disulfide linkages and proteins that are
duces a complementary duplicate strand of nucleic positioned within the cell membrane. Proteins are made
acid. Therefore, each strand of DNA can act as a tem- of strings of amino acids and are always read in an amino
plate to be copied to make the opposite strand. DNA terminal (left) to carboxyl terminal (right) direction.
has a direction and is always read and written in the 5′ The polymerase chain reaction (PCR) is an in vitro
(left) to 3′ (right) direction unless specified differently method for enzymatic synthesis and amplification of spe-
for certain applications. cific DNA sequences using a pair of primers, usually
Mutation refers to any structural alteration of DNA in short nucleotide sequences, that hybridize to opposite
an organism (mutant) that is caused by a physical or DNA strands and flank the region of interest. Various
chemical agent (mutagen). Mutations can be beneficial modifications have made the PCR reaction more efficient
or deleterious. Some mutations are lethal and therefore and specific and allow more complex analysis.
2682_Ch02_026-044 22/05/12 11:38 AM Page 43
3. Knippers, R, and Ruff, J: The initiation of eukaryotic DNA repli- Clark, DP, and Russell, LD: Molecular Biology Made Simple and
cation. In DNA Replication and the Cell Cycle. Springer-Verlag, Fun, 2nd ed. Cache River Press, St. Louis, MO, 2000.
New York, 1992, pp 2–10. Glick, BR, Pasternak, JJ, and Patten, CL: Molecular Biotechnology:
4. Antonarakis, SE, and Cooper, DN: Human gene mutation: mech- Principles and Applications of Recombinant DNA, 4th ed. ASM
anisms and consequences. In Speicher, MR, Antonarakis, SE, Press, Washington, DC, 2009.
Motulsky, AG (eds): Vogel and Motulsky’s Human Genetics: Harmening, DM: Modern Blood Banking and Transfusion Practices,
Problems and Approaches, 4th ed. Springer-Verlag, Berlin 5th ed. FA Davis, Philadelphia, PA, 2005.
Heidelberg, 2010, pp 319–351. Hillyer, CD, Silberstein, LE, Ness, PM, Anderson, KC, and Roback,
5. Caskey, CT, et al: Triplet repeat mutations in human disease. J: Blood Banking and Transfusion Medicine, Basic Principles and
Science 256:784–789, 1992. Practice. Churchill Livingstone, Philadelphia, PA, 2007.
6. Yamamoto, F, et al: Molecular genetic basis of the histo-blood Lewin, B: Genes VIII. Prentice Hall, New York, 2003.
group ABO system. Nature 345:229, 1990. Quinley, ED: Immunohematology: Principles and Practice, 3rd ed.
7. Le Van Kim, C, et al: Molecular cloning and primary structure Lippincott Williams & Wilkins, Philadelphia, PA, 2010.
of the human blood group RhD polypeptide. Proc Natl Acad Sci Roback, J, Combs, M, Grossman, B, and Hillyer, C: Blood Group
USA 89(22):10929, 1992. Genetics: Technical Manual, 16th ed. American Association of
Blood Banks, Bethesda, MD, 2009.
Bibliography Roback, J, Combs, M, Grossman, B, and Hillyer, C: Molecular Biology
and Immunology in Transfusion Medicine: Technical Manual,
Alberts, B, et al: Molecular Biology of the Cell, 4th ed. Garland 16th ed. American Association of Blood Banks Bethesda, MD,
Science, New York, 2002. 2009.
Calladine, CR, Drew, HR, Luise, BF, and Travers, AA: Understanding Trent, RJ: Molecular Medicine, 3rd ed. Elsevier Academic Press,
DNA, the Molecule and How it Works, 3rd ed. Elsevier, San Diego, Burlington, MA, 2005.
CA, 2004.
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Chapter 3
Fundamentals of Immunology
Lorraine Caruccio, PhD, MT(ASCP)SBB, and Scott Wise, MHA,
MLS(ASCP)SBB
OBJECTIVES
1. Outline the different components of the immune system and identify their functions.
2. Describe the characteristics of the major cells of the immune system and their functions.
3. List the major effector molecules and the roles they play in the immune response.
4. Outline the basic steps of hematopoiesis in the immune system.
5. Explain the function of the major histocompatibility complex (MHC) class I and II molecules.
6. Describe the physical characteristics of immunoglobulins in relation to structure and list the different subtypes.
7. Explain the activation sequences of the three major complement pathways and describe how they come to a common starting
point.
8. List the methods used in the blood bank to detect antibodies and complement bound to red blood cells.
9. Describe the immune response, including antigen-antibody reactions, lymphocyte functions, and host factors that can activate
and suppress the immune system. Continued
45
2682_Ch03_045-076 28/05/12 2:32 PM Page 46
OBJECTIVES—cont’d
10. List the traditional laboratory techniques used in blood bank testing.
11. Identify the various factors that affect agglutination reactions.
12. Describe some of the common diseases that can affect blood bank testing.
Introduction
BOX 3–1
The immune system (IS) is complicated, tightly controlled, Factors Affecting Antigen-Antibody Reactions
and includes tissues, organs, cells, and biological mediators
• Distance
that coordinate to defend a host organism against intrusion
• Antigen-antibody ratio
by a foreign substance or abnormal cells of self-origin.
• pH
Immunity refers to the process by which a host organism
• Temperature
protects itself from attacks by external and internal agents.
• Immunoglobulin type
Immunity also confers protection from nonself and abnormal
self-elements, which are controlled at different levels. The
number of different types of nonself organisms includes
unicellular and multicellular organisms, such as viroids, Both traditional and nontraditional laboratory testing
viruses, bacteria, mycoplasma, fungi, and parasites. Tumor methods are currently utilized in the transfusion service.
cells, which are too old or misshapen to function, and cells These methods focus on either the detection of antigens or
destined for termination within the host must be recognized on the detection of antibodies. This chapter provides an
and eliminated. introduction to the many areas of immunology and how
The response and elimination of organisms and unwanted they impact transfusion medicine. It will include a brief
cells is accomplished through cellular and/or humoral introduction to the biology and biochemistry of the immune
mechanisms. The cellular defense mechanism is mediated system, and the application of testing procedures used in
by various cells of the IS, such as macrophages, T cells, and evaluating the immune response as related to blood trans-
dendritic cells, which function to eliminate viruses, bacteria, fusion. A brief description of immune-mediated diseases
cancer cells, and other cellular pathogens. In the humoral important to transfusion medicine is included at the end of
mechanism, specific antibodies and complement compo- the chapter.
nents are produced in plasma, saliva, and other bodily secre-
tions. These antibodies bind to specific receptor sites Overview of the Immune System
on cells. Complement may also bind to immunoglobulin
molecules that have specific complement receptor sites. The Basic Concepts
extent of activation and the amount of damage that occurs All organisms at all levels of life are challenged constantly
to red blood cells is dependent upon the complement pathway by various factors from their environment and must protect
involved and on several other host factors. against them. The host organism is a rich source of nutrients
Immunoglobulins, or antibodies, have special significance and protection for an organism that is able to avoid the
for transfusion medicine, because antigens present on trans- host’s IS. One of the most fundamental concepts in
fused cells may cause reactions in the recipient and compli- immunology is the idea of self versus nonself and how the
cate therapy. The majority of blood bank testing is focused IS distinguishes between the two. The two terms have now
on the prevention, detection, and identification of blood been broadened in meaning. Self refers to anything that is
group antibodies and on the typing of RBC antigens. Antigen derived from the host genome and the rearrangement of
characteristics, as well as host factors, have an impact on host genes. It includes cells, fluids, molecules, and more
the immune response. Knowledge of these characteristics complex structures of a host organism. Anything put into
enables testing problems to be resolved. Understanding the the host body, even a close genetic match, can be regarded
IS and its responses is essential to understanding the factors as nonself and therefore can be rejected by a response of the
that can affect agglutination reactions between RBCs and IS. Nonself refers to anything physically outside the host,
antibodies and various testing modifications. whether a living organism (parasites, fungus) or nonliving
Detection of alloantibodies or autoantibodies in routine toxin (poison ivy fluid, insect venom). When foreign objects
blood bank testing procedures is of the utmost importance or damaged host cells are detected by the IS, an immune
in providing compatible blood to patients. This detection response occurs. Immune responses occur at different levels
is dependent upon several factors, including binding forces and are either primary (natural, innate) or secondary (adap-
between antigens and antibodies, properties of the antibody tive, acquired). The overall mechanisms and components
itself, and individual host characteristics. Antigen-antibody of each are outlined in Table 3–1 and Table 3–2.
reactions are influenced by a number of factors, including The innate immune response consists of physical barriers,
distance, antigen-antibody ratio, pH, temperature, and biochemical effectors, and immune cells. The first step of
immunoglobulin type (Box 3–1).
2682_Ch03_045-076 28/05/12 2:32 PM Page 47
• Cytokines
response, and IS memory allows resistance to a pathogen that invading organisms by engulfing and digesting them with
was previously encountered. Innate immunity is the imme- vesicle enzymes.
diate line of immune defense. Two major cells that can use phagocytosis to remove
There are two important features of innate immunity. pathogens are the polymorphonuclear cells (which include
First, the innate immune system is nonspecific. The same neutrophils, basophils, and eosinophils) and the mononu-
response is used against invading organisms, no matter what clear cells (which include the monocytes in plasma and the
the source is, as long as the innate IS can recognize them as macrophages in tissues). Various molecules of the IS col-
nonself. Innate immunity is present at birth and does not laborate with the innate IS’s cells. Opsonins are factors that
have to be learned or acquired. Second, it does not need include antibodies and complement components in plasma
modifications to function and is not altered with repeated that coat pathogens and facilitate phagocytosis. When
exposure to the same antigen. Because innate immunity phagocytes ingest foreign cells and destroy them, they can
functions so well as a first line of defense, it was maintained become activated to release soluble polypeptide substances
in the IS of vertebrates during evolution. called cytokines that have various effects on other cells of
Physical and biochemical barriers and various cells the immune and vascular systems (Table 3–3 lists impor-
make up the innate IS. Physical barriers include intact tant cytokines). There are a large number of different
skin, mucous membranes, cilia lining the mucous mem- cytokines; some have unique functions and others have
branes, and cough reflexes. Biochemical barriers of the overlapping functions. Some work together, and some op-
innate system include bactericidal enzymes such as pose the functions of other cytokines. Many are secreted,
lysozyme and RNases, fatty acids, sweat, digestive en- and some are membrane receptors. Cytokines help to reg-
zymes in saliva, stomach acid, and low pH. Innate im- ulate the immune response in terms of specificity, intensity,
mune cells include phagocytic leukocytes and natural and duration.
killer (NK) cells. Phagocytic cells of different types are Another important component of the innate immune sys-
found in most tissues and organs of individuals, including tem is the complement system. Complement has three major
the brain, liver, intestines, lungs, and kidneys. Phagocytes roles in immunity: (1) the final lysis of abnormal and patho-
include circulating monocytes in the blood and peripheral genic cells via the binding of antibody, (2) opsonization and
macrophages (activated monocytes) that can move bet- phagocytosis, and (3) mediation of inflammation. The pro-
ween vessel walls. Phagocytes recognize complex molec- teins of the complement system are enzymes that are nor-
ular structures on the surface of invading cells or in the mally found in the plasma in a proenzyme inactive state.
secretions and fluids of the host body. They remove the Three ways the complement proteins can be activated are the
2682_Ch03_045-076 28/05/12 2:32 PM Page 49
IL-9 CD4, T, MC, BM stroma Proliferation-activated B cells, T cells, mast cells, and hematopoietic precursor
CD4, T, Mf, MC, fibroblasts Growth and differentiation B-cell and T-cell effectors and hemopoietic
precursors
BM stromal cells Proliferation pre-B cells, CD4 cells, CD8 cells, and activated mature T cells
Chemotaxis T cells
T Activates NK cells
Colony-Stimulating Factors
TNF-a Mf, T
TNF-b T
Continued
2682_Ch03_045-076 28/05/12 2:32 PM Page 50
IFN-b Fibroblasts
IFN-g T
Other
TGF-b T, B
LIF T
Adapted from Delves, PJ, Martin, SJ, Burton, DR, Roitt, IM: Roitt’s Essential Immunology, 8th ed. Wiley-Blackwell, Malden, 2011.
APC = antigen-presenting cells; BM = bone marrow; CSIF = cytokine synthesis inhibitory factor, Fc = immunoglobulin Fc receptor for IgE; G-CSF = granulocyte colony-stimulating factor;
GM-CSF = granulocyte monocyte/macrophage colony-stimulating factor; IFN = interferon; IL = interleukin; LIF = leukocyte inhibitory factor; MC = mast cell; M-CSF = monocyte colony-
stimulating factor; MHC = major histocompatibility complex; Mf = macrophage; NK = natural killer cells; PGE2 = prostaglandin E2; T = T lymphocyte; TGF = transforming growth factor;
TNF = tumor necrosis factor.
classic, alternative, and lectin pathways; all have essentially pathogens and has specific responses, depending on the type
the final result of cell lysis and inflammation. The classic of pathogen it encounters.
pathway uses antigen-antibody binding and therefore is a The acquired IS uses antibodies as specific immune
specific activator of complement. The alternative pathway effectors. Antigen specificity and uniqueness determine
activates complement by recognizing polysaccharides and the particular antibody that will bind to it. The antigen-
liposaccharides found on the surfaces of bacteria and tumor antibody complex is a three-dimensional interaction that
cells; therefore, it uses nonspecific methods of activation. The does not allow recognition of near misses. For example,
lectin pathway is activated by mannose binding proteins antibodies against one blood group antigen do not react
bound to macrophages. against another blood group antigen. An antigen that an
Inflammation is also a critical component of the innate antibody is made against is sometimes referred to as its
IS and is familiar to most people when they have a minor antithetical antigen. The fact that acquired immunity has
wound that has redness and warmth at the abrasion site. memory means medical histories of patients that require
Inflammation is initiated by any type of tissue damage, transfusions are absolutely critical. Antibodies do not
whether it be to the skin or to an internal organ. Burns, always remain in plasma at levels observable with serologic
infections, fractures, necrosis, and superficial wounds all testing, and if antigen-positive RBC units are transfused in
elicit an inflammatory response that is characterized by an a sensitized patient, the second antibody response against
increase in blood flow to the wounded area, increased the transfused cell antigens can be more vigorous, result-
blood vessel permeability at the site to allow for greater ing in intravascular RBC hemolysis.
flow of cells, a mobilization of phagocytic cells into the
site, and a possible activation of acute phase and stress Cells and Organs of the Immune
response proteins at the site of tissue damage. Eventually System
the wound is repaired, new tissue grows in place of
the damaged tissue, and inflammation is stopped. Uncon- The different types of immune system cells can be distin-
trolled inflammation can result in unwanted damage guished by the membrane markers they possess. These are
to healthy tissues. The regulation of inflammation is referred to as clusters of differentiation (CD) markers and
tightly controlled and requires signals to effectively turn are detected by immunotyping methods. The immunization
it on and off. process requires many different types of cells and tissues,
Acquired immunity is the other major arm of the host’s including phagocytic granulocytes of the innate system and
IS and is the most highly evolved. It is also the most specific monocytes and macrophages.
and allows the IS to have memory of pathogens it has Lymphocytes are also important in acquired immunity.
encountered previously. The acquired system is present only They are divided into two major types, the T lymphocyte
in vertebrates. The term acquired refers to the fact that the (T cell) and the B lymphocyte (B cell). The T lymphocyte
immunity is acquired via specific contact with a pathogen or matures in the thymus gland and is responsible for making
aberrant cell. The term adaptive refers to the ability to adapt cytokines and destroying virally infected host cells. B lym-
to and destroy new complex pathogens, although it must phocytes mature in the bone marrow, and when stimulated
first react to them through complex recognition processes. by an antigen, evolve into plasma cells that secrete antibody.
Acquired immunity is specific in recognition of new Natural killer cells are a type of lymphocyte that plays a role
2682_Ch03_045-076 28/05/12 2:32 PM Page 51
in immune protection against viruses. Dendritic cells are that remains in circulation in the plasma, body secretions,
present throughout many systems of the body and are respon- and lymphatics. Antibodies can neutralize toxic substances
sible for antigen processing. Macrophages can also process and antigens that are encountered by binding to them and
antigens. T and B cells communicate with each other and are therefore preventing them from interacting with the host.
both necessary for antibody production. B cells undergo gene When the antigenic site is nonreactive because of antibody
rearrangement in order to have the correct antibody made binding, it cannot interact with host cells to infect them or
that can react with the correct antigen. damage them. Also, binding of antigen by antibody brings
T cells also have receptors that undergo gene rearrange- about opsonization, which aids in the direct killing of
ment. The receptors on the cell membranes of T and B lym- pathogens by cell lysis. When complement is activated by an
phocytes allow them to recognize foreign substances. antigen-antibody complex, the pathogen can be destroyed.
Lymphocytes recognize only one specific antigen, which is Pathogens can be destroyed intra- or extravascularly. Anti-
determined by the genetic programming of that lymphocyte. bodies are one of the most important components of the IS
Because there are so many different antigens that pathogens and blood bank testing procedures.
can carry, the IS has adapted to recognize millions of differ-
ent antigens, with a specific antibody that will match only T Cells
one particular antigen. Therefore, if a foreign antigen is pres- T cells are another very important component of the cellular
ent, only a small percentage of the host’s antibodies and
part of acquired immunity. One of the major differences of
T-cell receptors will recognize it. In cases where an antigen
cellular immunity compared to humoral immunity is that
is recognized by more than one antibody, a process of clonal
T cells recognize antigens that are internalized within a host
selection happens, in which the different cells that recognize
cell. The antigens are then processed and presented on the
the different epitopes of the antigen are expanded. In trans-
host cell surface in small peptide fragments. T cell–mediated
fusion medicine, this plays a role in selecting the best anti-
immunity is involved in the response against fungal and viral
body reagents for testing, as not all epitopes of a complex
infections, intracellular parasites, tissue grafts, and tumors.
antigen, including blood group antigens, will be equally
T-cell receptors do not recognize foreign antigen on their
reactive.
own, as B cells do; they require help in the form of cell mem-
The maturation of B and T cells occurs after an antigen is
brane proteins known as major histocompatibility complex
encountered. Both B and T cells mature into what are called
(MHC) molecules. Therefore, there is a certain level of re-
effector cells, which are the functional units of the IS. The
striction placed on the acquired cellular branch of the IS, as
final effect that B and T cells can cause is the elimination of
determined by the inherited MHC molecules on host cells.
pathogens and foreign cells. Immune memory is also acquired The MHC genes determine the human leukocyte antigens
when lymphocytes mature into memory cells after antigenic (HLA) present on leukocytes and other cells; they have been
stimulation; this allows the IS to recognize antigens from pre- known for many years to cause rejection of tissue grafts.
vious encounters. Once an infection is cleared, some of the
cells that can recognize the antigen remain in circulation. The
lymphocytes are permanently set to respond to the same anti-
Advanced Concepts
gen again by the process of memory. The second time this
antigen is encountered, memory allows for a more effective There are two major classes of MHC genes and antigens:
and rapid immune response. Memory cells can persist for the MHC class I and MHC class II. MHC class I antigens are
lifetime of the host. It is one of the reasons that some immu- found on most nucleated cells in the body, and MHC class
nizations with vaccines do not have to be repeated, except on II antigens are found on most antigen presenting cells. In
a cautionary basis to keep the immunization active. humans, MHC class I genes code for the HLA-A, HLA-B,
and HLA-C antigens; whereas MHC class II genes code for
HLA-DR, HLA-DQ, and HLA-DC antigens. MHC classes I
B Cells
and II are important in the recognition of foreign sub-
Antibodies can be secreted or membrane-bound. Antibodies stances and the immune reactions against them.
are secreted by mature B cells called plasma cells and bind to There are two major functions of T cells. The first major
antigens in a specific manner. The antigens are usually in function is to produce immune mediating substances such
soluble form in the plasma. The receptor on the B cell that as cytokines, which influence many immune functions
recognizes the antigen is a membrane-bound antibody. When throughout the body. The second major function is to kill
the receptor antibody on the B cell reacts with a specific anti- cells that contain foreign antigen. When T cells are acti-
gen and recognizes it, the B cell is activated to divide. The vated by antigen, they start to secrete cytokines and change
cells produced from this rapid division mature into plasma their cellular interactions (see Table 3–3). T cells are also
cells and memory B cells. Memory B cells have antibody on grouped into two major categories with two major func-
their surfaces that is of the same conformation as that of the tions: T helper (TH) cells and T cytotoxic (TC) cells.
B cell from which they were derived. TH cells are also distinguished by the membrane marker
Plasma cells are antibody factories that make large CD4; TC cells are distinguished by the marker CD8. TH
amounts of one specific type of antibody in a soluble form cells are further grouped into TH1 and TH2 and respond to
2682_Ch03_045-076 28/05/12 2:32 PM Page 52
different cytokines. TH cells have the ability to recognize always possible. It usually takes days to months for all the
antigen, along with MHC class II molecules, and provide aspects of an efficient immune response to happen from
help to B cells to evolve into plasma cells and make anti- the moment an antigen is first encountered. The lag phase,
bodies. TH cells therefore determine which antigens be- until an appropriate immune response occurs, is called the
come IS targets, as well as which immune mechanisms will latency, preseroconversion, or window period. It is dur-
be used against them. TH cells aid in the proliferation of ing this time that antibody cannot be detected with sero-
immune cells after they encounter antigen. logic testing. During the latency period, however, T and B
cells are very active in processing antigen and initiating
the primary response to the antigen. The first antibodies
Antigen-Presenting Cells made against the new antigen are different from the
antibodies of the secondary response. The primary antibodies
There are several types of leukocytes that function as antigen- are the immunoglobulin M (IgM) subclass, whereas the
presenting cells (APCs), including macrophages, neutrophils, antibodies of the secondary response are the immunoglob-
and some B cells. In addition to leukocytes, there are ulin G (IgG) subclass and have a different structure.
specialized immune cells capable of antigen presentation. Figure 3–1 depicts the primary and secondary antibody
These include the different types of dendritic cells present responses.
in the skin (Langerhans cells), nervous tissue (glial cells), After the antigen is cleared, memory cells are stored in
lymph nodes, spleen, intestines, liver (Kupffer cells), bone immune organs of the host. When the same antigen is en-
(osteoclasts), and thymus. These APCs first phagocytize the countered again, the memory cells are activated and produce
foreign antigen, process it internally, and then with the help a stronger and more rapid response. IgG antibodies are
of MHC molecules, present short peptide sequences of the formed during the secondary response and are made in great
antigen on their cell membranes. TH cells can then recognize quantities, and although IgM is made during the primary re-
the antigen in the context of MHC presentation and respond sponse, there is a period when it overlaps with the produc-
to it by the appropriate immune reaction. tion of IgG antibodies at the beginning of the secondary
response. IgG secondary antibodies have a higher avidity for
Immune System Organs antigen and can be produced by much lower concentrations
of antigen. Secondary antibodies can usually be measured
Basic Concepts within 1 to 2 days. For example, a primary antibody may re-
The organs of the IS are divided into primary and second- quire more than 100-fold excess of antigen to initiate the first
ary systems. The primary lymphoid organs are the thymus response. Many of the antigens that stimulate the primary
and bone marrow, where immune cells differentiate and response have multiple repeating epitopes such as polysac-
mature. The secondary lymphoid organs include the lymph charides and therefore are good immune stimulators at lower
nodes and spleen, in which immune cells interact with each concentrations.
other and process antigens (Table 3–4).
Cell Lineages and Markers
IgM
IgM
Negative
Inductive period phase
IgM
IgG
IgG
Figure 3–1. Schematic representation of "Memory" cells
primary and secondary antibody responses.
Note the enhanced antibody production and
expanded antibody-producing cell population
during the secondary antibody response. (From
Herscowitz, HB: Immunophysiology. In Bellanti, JA
[ed]: Immunology III. WB Saunders, Philadelphia, "Memory" cells
1985, p 117, with permission.)
and maturing stem cells into the many different cells of the staining of their granules. The neutrophils stain a faint pur-
IS. Growth factors also allow progenitor stem cells to ple or neutral color (neutral granules), the eosinophils a
reproduce and differentiate. The cells of the myeloid line- reddish orange color (acidic granules), and the basophils a
age consist of phagocytic cells such as the monocytes and bluish black color (basic granules). The main role of these
macrophages, often referred to as the mononuclear phago- cells is phagocytosis; they function primarily in acute
cytic system (MPS); the granulocytes or polymorphonuclear inflammatory responses and have enzymes that allow them
cells (PMNs), the neutrophils, eosinophils, and the basophils; to destroy engulfed pathogens. All three types of granulo-
and the APCs such as the dendritic cells in the skin and cytes possess receptors for the FC portion of IgG (CD16)
liver. Erythrocytes and platelets originate from this system. and complement receptors C5a, CR1(CD35), and
The lymphoid lineage consists of the various subpopula- CR3(CD11b). Additionally, eosinophils possess low-affinity
tions of lymphoid cells, the T cells, B cells, and NK cells. FC receptors for IgE and therefore play a critical role in al-
The myeloid-monocyte precursors originate in the bone lergic reactions and inflammation in parasitic infections.
marrow and then differentiate into circulating blood mono- Basophils and mast cells (a type of tissue basophil) possess
cytes. When monocytes encounter antigen, they can differ- high-affinity FC immunoglobulin E (IgE) receptors, are
entiate into tissue macrophages. Phagocytic cells process powerful effectors of inflammation and allergic reactions,
antigens for acquired immunity and can directly kill many and can cause the release of localized histamine.
pathogens such as bacteria and fungi as part of innate Lymphocytic cells are generated in the thymus or bone
immunity. MPS cells present antigen to lymphocytes and marrow and travel through the circulatory system to the
interact with other immune cells via cell membrane recep- lymph nodes and spleen, where they mature and differen-
tors. The FC part of the antibody receptor and the comple- tiate. In a mature adult, the lymphocytes account for about
ment receptor CR1 are used by phagocytes during 20% to 30% of the circulating leukocytes. In primary
opsonization.1 When these cells are functioning as APCs, organs, these cells acquire receptors that enable them to
they express the MHC class II molecules on their mem- interact with antigens and to differentiate between self
branes and lack the FC receptor. Granulocytes originate in and nonself antigens, a very critical part of lymphocyte
the bone marrow. They are the predominant leukocyte in maturation and the acquired IS in general.
the circulation of the mature adult (60% to 70%). In the secondary organs, immune cells are provided
Granulocytes all have granules in their cytoplasm with a highly interactive environment in which immune
and are of three types, distinguished by the hematologic responses are exchanged and made specific. Remember,
2682_Ch03_045-076 28/05/12 2:32 PM Page 54
there are two primary classifications of lymphocytes, T and There are two main cytokine types: lymphokines, which are
B cells, and these similar-looking cells can be distinguished produced by lymphocytes, and monokines, which are pro-
by the presence of specific cell markers by means of using duced by monocytes and macrophages. Cytokines function
sophisticated immunologic methods of testing, such as flow in a complex manner by regulating growth, mobility, and
cytometry (discussed later in this chapter). Specific to the differentiation of leukocytes. One cytokine may act by itself
T cell is the T-cell receptor (TCR), which is in proximity to or together with other cytokines. Other cytokines oppose
and usually identified with the CD3 complex on the T-cell the actions of one or more cytokines and function to quan-
membrane. It associates in cell-to-cell contacts and interacts titatively increase or decrease a particular immune reaction.
with both antigenic determinants and MHC proteins. In Some cytokines are synergistic and need each other to have
addition to CD3 on T cells, there is the separate CD2 marker, their full effect. The effects of cytokines can be in the imme-
which is involved in cell adhesion. It has the unique ability diate area of their release, or they can travel through the
to bind with sheep erythrocytes in vitro.2 Remember that TH plasma to affect distant cells and tissues. There is often
cells have the CD4 marker and recognize antigen together significant overlap in how cytokines function.
with the MHC class II molecules, and TC cells possess CD8 Major classes of cytokines include interleukins (IL), inter-
markers and interact with MHC class I molecules.3 The ferons (IFN), tumor necrosis factors (TNF), and colony-
ratios of T cells with CD4 versus CD8-positive cells can be stimulating factors (CSF). Each class has several members
a marker for particular diseases. One of the defining features (see Table 3–3). Cytokines act by binding to specific target
of AIDS is a reversal of the typical CD4 to CD8 ratio, which cell receptors. When cytokines bind to their receptors on
helped to explain some of the pathology of this illness. cells, the number of receptors is often increased as the cell
There are also compounds referred to as superantigens, is stimulated. Internal cellular signaling pathways become
which can stimulate multiple T cells, causing them to activated. The cell is no longer in a resting state and can have
release large amounts of cytokines. Certain bacteria toxins a new function. After cytokine binding, both the receptor
are superantigens and can lead to lethal reactions in the and the cytokine become internalized, which induces the
host if the immune system is overstimulated. target cell to grow and differentiate. Differentiated cells have
B cells are defined by the presence of immunoglobulin on specialized functions such as secreting antibodies or produc-
their surface; however, they also possess MHC class II anti- ing enzymes. Immune cells and other host cells respond to
gens (antigen presentation); the complement receptors CD35 cytokines and can react with chemoattraction, as well as
and CD21; FC receptors for IgG; and CD19, CD20, and antiviral, antiproliferation, and immunomodulation processes.
CD22 markers, which are the CD markers used to identify Cytokines fine-tune the IS and also function as critical cell
B cells. Membrane-bound immunoglobulin may act as an activators.
antigen receptor for binding simple structural antigens or In addition to the cytokines, other mediator substances
antigens with multiple repeating determinants (referred to as of the IS, including chemokines, immunoglobulins, com-
T cell–independent antigens, meaning they do not require the plement proteins, kinins, clotting factors, acute phase pro-
intervention of T-cell help). When T cell–dependent antigens teins, stress-associated proteins, and the fibrinolytic
(structurally complex and unique substances) are encoun- system, are cellular products that can have powerful cellular
tered, B cells require the intervention of T cells to assist in effects. Cytokines typically communicate between cells
the production of antibody. When B cells become activated, through the plasma. Chemokines are attractant molecules
they mature and develop into plasma cells, which produce that interact between cells, immunoglobulins, and com-
and secrete large quantities of soluble Ig into tissue or plasma. plement proteins and are important in destroying
The third major class of lymphocytes, the NK cells, are pathogens. Acute phase proteins and fibrinolytic proteins
sometimes referred to as third population cells because they play a role in inflammation, a process that recruits appro-
originate in the bone marrow from a developmental line dis- priate cells to an immune site and modifies the vascular
tinct from those of T and B lymphocytes. They are also referred system. In addition to inflammation and cell-to-cell
to as large granular lymphocytes. Unlike B cells, NK cells do communication, some cytokines are immunosuppressive.
not have surface Ig or secrete Ig, nor do they have antigen re- One of the important chemokine receptors in blood bank-
ceptors like the TCR of T cells. NK cells have the CD56 and ing is the Duffy antigen group present on RBCs. The lack
CD16 markers and do not require the presence of an MHC of these antigens can prevent certain types of malaria
marker to respond to an antigen. They are thymus-indepen- parasites from infecting the host. The Duffy antigen may
dent and are able to lyse virally infected cells and tumor also modulate immune response by acting as a sink for
cells directly in a process known as antibody-dependent cell- extra cytokines of the IL-8 family.
mediated cytotoxicity (ADCC) by anchoring immunoglobu-
lin to the cell surface membrane through an FC receptor. Immune System Genetics
important because not all transfusion recipients make factor B. The genes for MHC classes I through III molecules
antibodies to alloantigens on transfused RBCs. Responders are located on the short arm of chromosome 6 and are
are people who have a tendency based on their inheritance highly polymorphic in nature with multiple alleles.
to make antibodies. The terms high responders and low
responders describe individual responses to antigen chal-
Characteristics of Immunoglobulins
lenges. When antigenic stimulation occurs, one B cell forms
an antibody with a single specificity. During the lifetime of Immunoglobulin (Ig), also called antibody, is a complex
the cell, the cell can switch to make a different isotypic class protein produced by plasma cells, with specificity to antigens
of antibody that has the same antigen specificity. Isotype (or immunogens), that stimulate their production. An Ig is
switching requires further DNA rearrangement in mature a specific self protein produced by the host in response to a
B cells. Class switching is dependent on antigenic stimula- specific foreign, nonself protein, or other complex molecule
tion and on the presence of cytokines released by T cells; not tolerated by the host. Immunoglobulins make up a high
this reflects the further adaptation of the immune response. percentage of the total proteins in disseminated body fluids,
Isotype switching is seen in blood banking when antibodies about 20% in a normal individual. Antibodies bind antigen,
react at different temperatures and phases. fix complement, facilitate phagocytosis, and neutralize toxic
substances in the circulation. Thus, antibodies have multiple
functions, some more highly specialized and specific than
others. Immunoglobulins are classified according to the
Advanced Concepts molecular structure of their heavy chains. The five classifi-
The major histocompatibility complex (MHC) is the cations are IgA (α [alpha] heavy chain), IgD (δ [delta] heavy
region of the genome that encodes the human leukocyte chain), IgE (ε [epsilon] heavy chain), IgG (γ [gamma] heavy
antigen (HLA) proteins. (Refer to Chapter 21, “The HLA chain), and IgM (µ [mu] heavy chain).
System.”) The MHC is critical in immune recognition and Table 3–5 illustrates some of the various differences in
regulation of antigen presentation in cell-to-cell interac- the classes of immunoglobulins, such as molecular weight,
tions, transplantation, paternity testing, and specific HLA percentage in serum, valency (number of antigen-binding
patterns. It also correlates with susceptibility to certain dis- sites), carbohydrate content, half-life in the blood, and
eases. HLA molecules are categorized into two classes: MHC whether they exist as monomers or multimers. IgG is the
classes I and II. Class I molecules are found on all nucleated most concentrated in serum, comprising approximately 80%
cells except trophoblasts and sperm, and they play a key of the total serum Ig; next is IgA, at about 13% (although it
role in cytotoxic T-cell function. Class II molecules are is the major Ig found in body secretions); IgM is 6%; IgD is
found on antigen-presenting cells such as B lymphocytes, 1%; and IgE is the least common, present at less than 1%.4
activated T cells, and the various dendritic cells. Class II
molecules on APCs are essential for presenting processed Immunoglobulin Structure
antigen to CD4 T cells and are necessary for T-cell functions
and B-cell help. In addition, there are class III molecules All classes and subclasses of immunoglobulins (Igs) have a
that encode complement components such as C2, C4, and common biochemical structural configuration with similarities
and are probably derived from a common evolutionary
molecular structure with well-defined function. In Figure 3–2, immunoglobulins are known as the variable regions, because
the basic Ig structural unit is composed of four polypeptide they are structured according to the great variation in
chains: two identical light chains (molecular weights of antibody specificity. Structurally and functionally, the Fab
approximately 22,500 daltons) and two identical heavy fragments encompass the portions of the Ig from the hinge
chains (molecular weights from approximately 50,000 to region to the amino terminal end and are the regions respon-
75,000 daltons). In Figure 3–3, covalent disulfide bonding sible for binding antigen (see Fig. 3–2).
holds the light and heavy chains together, and the covalent The domains of immunoglobulins are the regions of the
disulfide linkages in Ig molecules provide greater structural light and heavy chains that are folded into compact globular
strength than do hydrogen bonding and van der Waals loop structures (see Fig. 3–3). The domains are held together
forces. However, they limit the flexibility of the Ig mole- by intrachain covalent disulfide bonds; the V region of the
cule. The heavy chains are also interconnected by disulfide domain specifies the variable region, and the C is the constant
linkages in the hinge region of the molecule. Although region. The domains are also specified according to light and
there are five types of heavy chains, there are only two types heavy chains. Looking at one half of an Ig molecule, one
of light chains: κ (kappa) and λ (lambda). Both types are domain (VL) is the variable region, one domain (CL) is in the
found in all classes of immunoglobulins, regardless of constant region of each light chain, and one variable domain
heavy chain classification. (VH) is on each heavy chain. The number of domains is
Ig molecules are proteins and therefore have two terminal determined by the isotype. There are three constant domains,
regions: the amino (-NH2) terminal and the carboxyl CH1 to CH3, on the heavy chains of IgA, IgD, and IgG, and
(-COOH) terminal. The carboxyl region of all heavy chains four constant domains, CH1 to CH4, on the heavy chains of
has a relatively constant amino acid sequence and is named IgE and IgM. The antigen-binding, or idiotypic, regions
the constant region. Like the heavy chain, the light chain also (which distinguish one V domain from all other V domains)
has a constant region. Due to certain common Ig structures, are located within the three-dimensional structures formed by
enzyme treatment yields specific cleavage products of defined the VL and VH domains together. Certain heavy chain domains
molecular weight and structure. The enzyme papain splits are associated with particular biological properties of
the antibody molecule at the hinge region to give three immunoglobulins, especially those of IgG and IgM, and
fragments: one crystallizable FC fragment and two antigen- include complement fixation. They are identified with the CH2
binding fragments, Fab. The FC fragment encompasses that domain and the CH3 domains, which serve as attachment sites
portion of the Ig molecule from the carboxyl region to the for the FC receptor of monocytes and macrophages.
hinge region and is responsible for complement fixation
and monocyte binding by FC receptors on cells. The FC frag- Immunoglobulins Significant for Blood Banking
ment on IgG antibody only is also responsible for placental
transfer. In contrast to the carboxyl terminal regions, the All immunoglobulins can be significant for transfusion med-
amino terminal regions of both light and heavy chains of icine; however, IgG, IgM, and IgA have the most significance
A
Bi ntig Figure 3–2. Schematic representation of
nd e basic immunoglobulin structure. The inset
Si in n
te g
2
H2
NH
N
Heavy Chain
Hinge Region Variable region
Light Chain
Variable region
S S
Heavy Chain
S Constant region
S S
S S S Light Chain
Constant region
b
Fa
Fa
b
COOH
COOH
S
SS
S
SS
Fc
2682_Ch03_045-076 28/05/12 2:32 PM Page 57
Heavy Chain
Light Chain
Hinge Region
Complement (C1q)
Attachment Site
for the blood bank. Most clinically significant antibodies disease of the newborn (HDN), because antibodies can be
that react at body temperature (37°C) are IgG isotype and formed in response against alloantigens on fetal RBCs that
are capable of destroying transfused antigen-positive RBCs, enter the mother’s circulation, usually during delivery.
causing anemia and transfusion reactions of various severi- HDN can be fatal. It is one area of medicine where
ties. IgM antibodies are most commonly encountered as immunohematology has provided prevention and treat-
naturally occurring antibodies in the ABO system and are ment. Antibody screening, Rh typing, and passive anti-D
believed to be produced in response to commonly occurring antibody have prevented HDN from developing in D-negative
antigens like intestinal flora and pollen grains. (Refer to mothers who give birth to D-positive babies.
Chapter 6, “The ABO Blood Group System.”) Other blood IgG has the greatest number of subclasses: IgG1, IgG2,
groups such as Lewis, Ii, P, and MNS may also produce IgM IgG3, and IgG4, and all four are easily separated by elec-
antibodies, which usually react best at ambient temperature trophoresis. The small differences in the chemical structure
(22°C to 24°C). The primary testing problem encountered
with IgM antibodies is that they can interfere with the
detection of clinically significant IgG antibodies by masking
their reactivity. Unlike IgG, IgM exists in both monomeric J Chain
and polymeric forms (as pentamers) containing a J (joining)
chain (Fig. 3–4).
Advanced Concepts
The pentameric form can be dissociated through cleavage
of covalent bonds interconnecting the monomeric subunits
and the J chain by chemical treatment with sulfhydryl
reducing reagents such as β-2-mercaptoethanol (2-ME) or
dithiothreitol (DTT). These reagents can distinguish a
mixture of IgM and IgG antibodies, because only IgM is
removed by the use of these compounds; therefore, the
removal allows unexpected IgG antibodies to be detected.
IgM
IgG antibodies are significant in transfusion medicine, Monomer
because they are the class of immunoglobulins that are
made in response to transfusion with nonself antigens on
blood products. IgG antibodies are important in hemolytic Figure 3–4. Schematic representation of the pentameric configuration of the
IgM immunoglobulin.
2682_Ch03_045-076 28/05/12 2:32 PM Page 58
within the constant regions of the gamma heavy chains des- in nearly all body fluids. IgA is important in immunohe-
ignate the various subclasses, and the number of disulfide matology, because about 30% of anti-A and anti-B antibodies
bonds between the two heavy chains in the hinge region of are of the IgA class (the remaining percentages are IgM
the molecule constitutes one of the main differences and IgG).6 Also, anti-IgA antibodies can cause severe
between subclasses. Functional differences between the anaphylaxis if IgA are transfused in plasma products to
subclasses include the ability to fix complement and cross patients who are deficient in IgA. Another reason for the
the placenta (Table 3–6). IgG blood group antibodies of a importance of IgA is that it can increase the effect of IgG-
single specificity are not necessarily one specific subclass; induced RBC hemolysis.7
all four subclasses may be present, or one may predomi- IgE is normally found only in monomeric form in trace
nate. For example, the antibodies to the Rh system antigens concentrations in serum, about 0.004% of total immunoglob-
are mostly of the IgG1 and IgG3 subclasses, whereas anti-K ulins, and is important in allergic reactions. The FC portion
(Kell), and anti-Fy (Duffy) antibodies are usually of the of the IgE molecule attaches to basophils and mast cells and
IgG1 subclass. Anti-Jk (Kidd) antibodies are mainly IgG3 facilitates histamine release when an allergen binds to the
and may account for the unusual nature of these different Fab portion of the molecule and cross-links with a second
antibodies with regard to testing and clinical significance. molecule on the cell surface. Histamine is critical for bringing
The purpose for the existence of biological differences in about an allergic reaction. Although hemolytic transfusion
subclass expression is still not completely understood. reactions are not caused by IgE, urticaria may occur because
Especially important in blood banking, severe HDN has of the presence of IgE antibodies. Because IgE causes trans-
been most often associated with IgG1 antibodies.5 fusion reactions by release of histamines, patients who have
Like IgM, IgA exists in two main forms—a monomer several allergic reactions to blood products can be pretreated
and polymer form—as dimers or trimers composed of two with antihistamines to counteract the response when receiv-
or three identical monomers, respectively, joined by a J ing blood products.2 IgD, present as less than 1% of serum
chain. IgA is located in different parts of the IS, depending immunoglobulins, appears to have functions that deal
on subclass. Serum IgA is found in both monomeric and primarily with maturation of B cells into plasma cells. IgD is
polymeric forms; however, secretory IgA is usually found usually bound to the membrane of immature B cells. There-
in the mucosal tissues of the body. Its polymer form fore, IgD may be necessary for regulatory roles during B-cell
acquires a glycoprotein secretory component as it passes differentiation and antibody production but is probably the
through epithelial cell walls of mucosal tissues and appears least significant for blood banking.8
Immunoglobulin Variations
Table 3–6 Biological Properties of IgG
Subclasses There are three main types of antibody-inherited variation:
CHARACTERISTIC IgG1 IgG2 IgG3 IgG4 isotype, allotype, and idiotype. Isotype variation (or class
variation) refers to variants present in all members of a species,
Proportion of total 65–70 23–28 4–7 3–4 including the different heavy and light chains and the different
serum IgG (%)
subclasses. All humans have the same Ig classes and subclasses.
Complement fixation ++ + +++ 0 Allotypic variation is present primarily in the constant region;
(classic pathway) not all variants occur in all members of a species. Idiotypic
Binding to macrophage +++ ++ +++ ±
variation, which determines the antigen-binding specificity (or
FC receptors complementary determining regions, [CDRs]) of antibodies
and T-cell receptors, is found only in the variable (and hyper-
Ability to cross placenta + ± + + variable) regions and is specific for each antibody molecule.
Dominant antibody The allotypes of a growing fetus may cause the maternal
activities: IS to become immunized to paternal allotypic determinants on
fetal immunoglobulins.4 Just as alloimmunization can occur dur-
• Anti-Rh ++ 0 + ±
ing pregnancy, it can occur from transfusions, especially in pa-
• Anti-factor VII 0 0 0 + tients who have received multiple transfusions of blood, plasma,
or gamma globulin. Idiotypes can be seen as nonself because
• Anti-dextran 0 + 0 0
they are often present at concentrations too low to induce self-
• Anti-Kell + 0 0 0 tolerance. Idiotypes are determined by the antigens that react
with them and exist in a type of equilibrium with anti-idiotypic
• Anti-Duffy + 0 0 0
antibodies. The presence of antigen disrupts this equilibrium
• Anti-platelet 0 0 + 0 and may be important in controlling an immune response.
Biological 21 21 7–8 21
half-life (days) Immunoglobulin FC Receptors
0 = negative reactivity; ± = weak (or unusual) reactivity; + = slight (or usual) reactivity; Macrophages and monocytes have receptors for the attach-
++ = moderate (or more common) reactivity; +++ = strong reactivity. ment of IgG and can bind the CH3 domain of the FC portion.
2682_Ch03_045-076 28/05/12 2:32 PM Page 59
Only the IgG1 and IgG3 subclasses are capable of attachment Classical Complement Pathway
to phagocytic receptors. This is one way that incompatible
RBCs coated with IgG antibody are removed by phagocytosis. The activation of the classical complement pathway is initi-
The other phagocytic cells with FC receptors include neu- ated when antibody binds to antigen. This allows the binding
trophils, NK cells, and mature B cells.4,9 of the complement protein C1 to the FC fragment of an IgM,
IgG1, or IgG3 subclass antibody. Complement activation by
IgG antibody depends on concentration of cell surface anti-
Complement System
gen and antigen clustering, in addition to antibody avidity
and concentration. IgM is large and has FC monomers close
Basic Concepts to each other on one immunoglobulin molecule; therefore,
The complement system, or complement, is a complex only one IgM molecule is necessary to activate complement.
group of over 20 circulating and cell membrane proteins The C1 component is actually a complex composed of three
that have a multitude of functions within the immune C1 subunits, C1q, C1r, and C1s, which are stabilized by cal-
response. Primary roles include direct lysis of cells, bacteria, cium ions. Without calcium present, there is no stabilization
and enveloped viruses as well as assisting with opsoniza- of the C1q, r, s complex, and complement is not activated.
tion to facilitate phagocytosis. Another role is production In the C1 complex bound to antigen antibody, C1q is
of peptide fragment split products, which play roles in responsible for catalyzing the C1r to generate activated C1s.
inflammatory responses such as increased vascular perme- Activated C1s is a serine-type protease. The C1q, r, s com-
ability, smooth muscle contraction, chemotaxis, migration, plex (actually as a C1q[r, s]2 unit) acts on C2 and C4 to form
and adherence. Complement components circulate in in- C4b2a. C4b2a uses component C3 as a natural substrate. By-
active form as proenzymes, with the exception of factor D products that result from the activation of the classical
of the alternate pathway. The complement proteins are ac- sequence include C3a; C4b, which binds to the cell surface;
tivated in a cascade of events through three main pathways: C4a, which stays in the medium and has modest anaphyla-
the classical, alternative, and lectin pathways. The three toxin activity; and C3b, which attaches to the microbial
pathways converge at the activation of the component surface. Note that complement fragments that have the b
C3. The classical pathway is activated by the binding of an type are usually bound to membranes, whereas the a fragments
antigen with an IgM, IgG1, or IgG3 antibody. The alternative often have anaphylatoxic activity.
pathway is activated by high molecular weight molecules Human RBCs have CR1 receptors for C4d and C3b, and
with repeating units found on the surfaces of target cells. some of the cleavage products will attach to the RBC mem-
The lectin pathway is activated by attachment of plasma brane. Eventually these fragments are further degraded to
mannose-binding lectin (MBL) to microbes. MBL in turn C4d and C3d through the action of C4BP, factor H (which
activates proteins of the classical pathway. binds C3b), and factor I (which degrades both C4b and
C3b). Some of C3b binds to the C4b2a complex, and the
resulting C4b2a3b complex functions as a C5 convertase.
The C5 convertase then acts on C5 to produce C5a (a strong
Advanced Concepts stimulator of anaphylatoxins) and C5b, which binds to the
Complement components are sequentially numbered C1 cell membrane and recruits C6, C7, C8, and C9 to the cell
through C9, but this refers to their discovery date, not to membrane. When C5b along with C6, C7, C8, and C9 are
their activation sequence. The four unique serum proteins bound, the membrane attack complex forms; this causes cell
of the alternative pathway are designated by letters: factor B, lysis. Figure 3–5 shows the sequential activation of the com-
factor D, factor P (properdin), and IF (initiating factor). plement system via the classical and alternative pathways.
Some activation pathways require Ca2+ and Mg2+ cations as Figure 3–6 illustrates the mechanism of antibody-dependent
cofactors for certain components. In certain nomenclature, cell-mediated cytotoxicity (ADCC).
complement components that are active are designated by a
short bar placed over the appropriate number or letter. The Alternative Complement Pathway
cleavage products of complement proteins are distinguished
from parent molecules by suffixes such as C3a and C3b. The The alternative pathway is older in evolution and allows
complement system is able to modulate its own reactions by complement to be activated without acquired immunity. The
inhibitory proteins such as C1 inhibitor (C1INH), factor alternative pathway is activated by surface contacts with
H, factor I, C4-binding protein (C4BP), anaphylatoxin inac- complex molecules and artificial surfaces such as dialysis
tivator, anaphylatoxin inhibitor, membrane attack complex membranes and dextran polymers. There are four important
(MAC) inhibitor, and C3 nephritic factor (NF). This regula- proteins in this pathway: factor D, factor B, properdin, and
tion of complement is important so that complement pro- C3. In this pathway, complement component factor D is
teins do not destroy healthy host cells and inflammation is analogous to C1s in the classical pathway; factor B is analo-
controlled. The evaluation of C3b and C3d complement gous to C2; and the cleavage product C3b is analogous to
components in transfusion medicine testing procedures is C4. Also, factor C3bBbP is analogous to C4b2a, and
useful in the investigation of hemolytic transfusion reactions C3b2BbP is analogous to C4b2a3b in the classical pathway.
and autoimmune hemolytic anemias. Activation of the alternative pathway requires that a C3b
molecule be bound to the surface of a target cell. Small
2682_Ch03_045-076 28/05/12 2:32 PM Page 60
C4b, 2C
MEMBRANE
ATTACK
MECHANISM
C5b67
C5b6789
Figure 3–5. Schematic diagram illustrating the
sequential activation of the complement system
CELL LYSIS via the classical and alternative pathways.
amounts of C3b are generated continuously, owing to the properdin (P), yielding C3bBbP. This complex cleaves C3
spontaneous hydrolytic cleavage of the C3 molecule. When into additional C3a and C3b. C3b, therefore, acts as a posi-
C3b encounters normal cells, it is rapidly eliminated through tive feedback mechanism for driving the alternative pathway.
the combined interactions of factors H and I. The accumu- The C3b2BbP complex acts as a C5 convertase and initiates
lation of C3b on microbial cell surfaces is associated with the later steps of complement activation (see Fig. 3–5).
attachment of C3b to factor B. The complex of C3b and
factor B is then acted on by factor D. As a result of this Lectin Complement Pathway
action, factor B is cleaved, yielding a cleavage product known
as Bb. The C3bBb complex is stabilized by the presence of The third pathway of activation, the lectin pathway, is acti-
vated by the attachment of MBL to microbes. The subsequent
reactions are the same as those of the classical pathway.
Remember that all three methods of activation lead to a final
IgG Antibody
common pathway for complement activation and membrane
attack complex (MAC) formation.
Lysed Target Cell
Membrane Attack Complex
to the C5b678 complex, a small transmembrane channel or by several biochemical and physical characteristics of the
pore is formed in the lipid bilayer of the cell membrane, and immunogen. Properties such as size, complexity, conforma-
this allows for osmotic lysis and subsequent death of the cell. tion, charge, accessibility, solubility, digestibility, and bio-
chemical composition influence the amount and type of
Binding of Complement by RBC Antibodies immune response (Box 3–2).
Molecules that are too small cannot stimulate antibody
production. Immunogens having a molecular weight (MW)
Basic Concepts less than 10,000 daltons (D), for example, are called haptens
Disruption in the activation of either complement pathway and usually do not elicit an immune response on their own;
can result in damage to the host’s cells. Transfusion medi- however, coupled with a carrier protein having a MW greater
cine specialists are concerned with the formation of anti- than 10,000 D, they can produce a reaction. Antibodies and
bodies with complement capacity that can bring about the cellular responses are very specific for an antigen’s physical
destruction of RBCs. Recall that in order to initiate activa- conformation as opposed to its linear sequence. Overall
tion, C1 molecules bind with two adjacent Ig FC regions. charge is important, as antibody response is also formed to
A pentameric IgM molecule provides two FC regions side the net charge of a molecule, whether it is positive, negative,
by side, thereby binding complement. A monomeric IgG or neutral. Obviously, an antigen must be seen by the IS, and
molecule, on the other hand, binds C1q less efficiently, and so the accessibility of epitopes influences the immune
two IgG molecules are needed in close proximity to bind response. Also, antigenic substances that are less soluble are
complement. For example, as many as 800 IgG anti-A less likely to elicit an immune response. The biochemical
molecules may need to attach to one adult group A RBC to composition of the stimulus plays a role in immune stimu-
bind a single C1 molecule, so there is little complement lation. Remember that RBC antigens are very diverse in
activation by IgG anti-A immunoglobulins.4 structure and composition and may be proteins (such as the
Antibodies against the Rh antigens usually do not bind Rh, M, and N blood group substances) or glycolipids (such
complement due to the low level of Rh antigens on RBC as the ABH, Lewis, Ii, and P blood group substances).
surfaces. Antibodies to the Lewis blood group system are Human leukocyte antigens (HLAs) are glycoproteins.
generally IgM, and they can activate complement but rarely Because of these differences in structure, conformation, and
cause hemolytic transfusion reactions due to their low molecular nature, not all blood group substances are equally
optimal reactivity temperature. Therefore, in blood banking, immunogenic in vivo (Table 3–7).
with the exception of the ABO system, only a few antibodies
activate the complement sequence that leads to complement Characteristics of Blood Group
mediated intravascular hemolysis. However, extravascular Antibodies
hemolysis usually occurs as a result of antibody coating
of RBCs, and the split products of complement activation There are many different and important characteristics of
can stimulate the reticuloendothelial system and cause blood group antibodies, such as whether they are polyclonal
anaphylatoxic effects. or monoclonal, naturally occurring or immune, and alloan-
Antibody-coated RBCs, either self or nonself, are removed tibodies or autoantibodies.
by cells of the mononuclear phagocyte system and by the
cells lining the hepatic and splenic sinusoids. These phago- Polyclonal and Monoclonal Antibodies
cytic cells are able to clear antibody-coated RBCs because
they have cell surface receptors for complement CR1 (C3b) In laboratory testing, there are two types of antibody (reagent
and Ig FC receptors (see Fig. 3–6). IgM-coated RBCs antibodies are called antisera) that are available for use; they
are not eliminated through FC receptor–mediated phago- are manufactured differently and have different properties.
cytosis, but if erythrocytes are coated with IgG and com- An antigen usually consists of numerous epitopes, and
plement, they will be cleared rapidly from circulation it is the epitopes and not the entire antigen that a B cell is
by monocytes and macrophages. If RBCs are coated with
only C3b, they may not be cleared but only sequestered
temporarily. BOX 3–2
Table 3–7 Relative Immunogenicity of today are monoclonal in nature or are a blend of monoclonal
Different Blood Group Antigens antisera.
BLOOD GROUP BLOOD GROUP IMMUNO- Naturally Occurring and Immune Antibodies
ANTIGEN SYSTEM GENICITY (%)*
There are two types of antibodies that concern blood bank-
D (Rho) Rh 50
ing: one is naturally occurring and the other is immune.
K Kell 5 Both are produced in reaction to encountered antigens. RBC
antibodies are considered naturally occurring when they
c (hr’) Rh 2.05
are found in the serum of individuals who have never been
E (rh’’) Rh 1.69 previously exposed to RBC antigens by transfusion, injec-
tion, or pregnancy. These antibodies are probably produced
k Kell 1.50
in response to substances in the environment that resemble
e (hr’’) Rh 0.56 RBC antigens such as pollen grains and bacteria membranes.
The common occurrence of naturally occurring antibod-
Fya Duffy 0.23
ies suggests that their antigens are widely found in nature
C (rh’) Rh 0.11 and have a repetitive complex pattern. Most naturally occur-
ring antibodies are IgM cold agglutinins, which react best at
Jka Kidd 0.07
room temperature or lower, activate complement, and may
S MNSs 0.04 be hemolytic when active at 37°C. In blood banking, the
common naturally occurring antibodies react with antigens
Jkb Kidd 0.03
of the ABH, Hh, Ii, Lewis, MN, and P blood group systems.
s MNSs 0.03 Some naturally occurring antibodies found in normal serum
are manufactured without a known environmental stimu-
Adapted from Kaushansky, K, et al: Williams Hematology, 8th ed. McGraw-Hill Professional, lus.10 In contrast to natural antibodies, RBC antibodies are
New York, 2010.
*Percentage of transfusion recipients lacking the blood group antigen (in the first column) considered immune when found in the serum of individuals
who are likely to be sensitized to a single transfusion of red cells containing that antigen. who have been transfused or who are pregnant. These anti-
gens have a molecular makeup that is unique to human
RBCs. Most immune RBC antibodies are IgG antibodies that
stimulated to produce antibody against. Therefore, these react best at 37°C and require the use of antihuman globulin
different epitopes on a single antigen induce the proliferation sera (Coombs’ sera) for detection. The most common
of a variety of B-cell clones, resulting in a heterogeneous immune antibodies encountered in testing include those that
population of serum antibodies. These antibodies are referred react with the Rh, Kell, Duffy, Kidd, and Ss blood group
to as polyclonal or serum antibodies and are produced in systems.10
response to a single antigen with more than one epitope.
In vivo, the polyclonal nature of antibodies improves the Unexpected Antibodies
immune response with respect to quality and quantity. Anti-
bodies against more than one epitope are needed to give Naturally occurring anti-A and anti-B antibodies are rou-
immunity against an entire antigen, such as a pathogen. tinely detected in human serum and depend on the blood
However, this diversity is not optimal in the laboratory, and type of the individual. Blood group A has anti-B; blood group
in vitro reagents produced by animals or humans can give B has anti-A; blood group O has both and anti-A, B. Blood
confusing test results. Consistency and reliability are needed group AB has neither antibody present in their serum. These
in laboratory testing, and polyclonal sera can vary in anti- naturally occurring antibodies, or isoagglutinins, are signif-
body concentration from person to person and animal to icant and useful in blood typing. They are easily detected by
animal. Sera from the same animal can also vary somewhat, use of A and B reagent RBCs with a direct agglutination tech-
depending on the animal’s overall condition. nique. In normal, healthy individuals, anti-A and anti-B are
Individual sera also differ in the serologic properties of generally the only RBC antibodies expected to be found in a
the antibody molecules they contain, the epitopes they rec- serum sample. People with an IS that does not function
ognize, and the presence of additional nonspecific or cross- normally may not have the expected naturally occurring
reacting antibodies. One way to avoid this problem is to use antibodies. All other antibodies directed against RBC anti-
monoclonal antibodies produced by isolating individual gens are considered unexpected and must be detected and
B cells from a polyclonal population and propagating them identified before blood can be safely transfused, no matter
in cell culture with hybridoma technology. The supernatant their reaction strength or profile.
from the cell culture contains antibody from a single type of The reactivity of unexpected antibodies is highly varied and
B cell, clonally expanded, and therefore has the same variable unpredictable, as they may be either isotype IgM or IgG; rarely,
region and a single epitope specificity. This results in a mon- both may be present in the same sample. These antibodies
oclonal antibody suspension. Monoclonal antibodies are may be able to hemolyze, agglutinate, or sensitize RBCs. Some
preferred in testing because they are highly specific, well antibodies require special reagents to enhance their reactivity
characterized, and uniformly reactive. Most reagents used and detection, especially if more than one antibody occurs in
2682_Ch03_045-076 28/05/12 2:32 PM Page 63
the sample. Due to the enormous polymorphism of the human Intermolecular Binding Forces
population, a diversity of RBC alleles and antigens exist,
requiring a variety of standardized immunologic techniques Intermolecular binding forces such as hydrogen bonding,
and reagents for their detection and identification. electrostatic forces, van der Waals forces, and hydrophobic
The in vitro analysis of unexpected antibodies involves bonds are all involved in antigen-antibody binding reactions.
the use of antibody screening procedures to optimize antigen- Stronger covalent bonds are not involved in this reaction,
antibody reactions. The majority of these procedures include although they are important for the intramolecular confor-
reacting unknown serum from a donor or patient sample mation of the antibody molecule. Hydrogen bonds result
with known reagent cells at various amounts of time, tem- from weak dipole forces between hydrogen atoms bonded to
perature, and media. All routine blood bank testing requires oxygen or nitrogen atoms in which there is incomplete trans-
the use of samples for both expected and unexpected fer of the electronic energy to the more electronegative oxy-
antibodies. gen or nitrogen. When two atoms with dipoles come close
to each other, there is a weak attraction between them.
Alloantibodies and Autoantibodies Although hydrogen bonds are singularly weak, many of
them together can be strong. They are found in complex
Antibodies can be either alloreactive or autoreactive. Alloan- molecules throughout the human body.
tibodies are produced after exposure to genetically different, Electrostatic forces result from weak charges on atoms in
or nonself, antigens, such as different RBC antigens after molecules that have either a positive or negative overall
transfusion. Transfused components may elicit the formation charge (like charges repel and unlike charges attract). This
of alloantibodies against antigens (red cell, white cell, and is seen in the formation of salt bridges. Van der Waals forces
platelets) not present in the recipient. Autoantibodies are are a type of weak interaction between atoms in larger
produced in response to self-antigens. They can cause reac- molecules. Hydrophobic (water-avoiding) bonds result from
tions in the recipient if they have a specificity that is com- the overlap of hydrophobic amino acids in proteins. The
mon to the transfused blood. Some autoantibodies do not hydrophobic amino acids bury themselves together to avoid
have a detectable specificity. water and salts in solution. The repulsion of these amino
Autoantibodies can react at different temperatures, and acids to prevent contact with water and aqueous solutions
cold or warm autoantibodies may both be present. Patients can be very strong collectively.
with autoantibodies frequently have autoimmune diseases In addition, there are hydrophilic (water-loving) bonds
and may require considerable numbers of blood products that allow for the overlap of amino acids that are attracted
and special techniques to find compatible units. A potentially to water. Hydrophobic and hydrophilic bonds repel each
serious problem for blood bankers is transfused patients who other, and these repulsive forces also play a role in the
have alloantibodies that are no longer detectable in the formation of the antigen-antibody bond. All of these bonds
patient’s plasma or serum. If these individuals are transfused affect the total conformation and strength of antigen-antibody
with the immunizing antigen again, they will make a reactions and IS molecules.
stronger immune response against those RBC antigens,
which can cause severe and possibly fatal transfusion reac- Antibody Properties
tions. These recipients will then have a positive autocontrol
or direct antiglobulin test (DAT), which may also be referred There are many important terms that refer to the properties
to as the direct Coombs’ test. This is why the previous of antibody reactions. The first is antibody affinity; it is often
history of any patient is a critical part of the testing. Autoan- defined as the strength of a single antigen-antibody bond
tibodies can be removed from RBCs by special adsorption produced by the summation of attractive and repulsive
and elution techniques and then tested against reagent RBCs. forces. The second term is avidity, which is used to express
Remember that in order to transfuse blood safely, the identity the binding strength of a multivalent antigen with antisera
of antibodies should be determined and recorded. produced in an immunized individual. Avidity, therefore, is
a measure of the functional affinity of an antiserum for the
Characteristics of Antigen-Antibody whole antigen and is sometimes referred to as a combination
Reactions of affinities. Avidity can be important in blood banking
because high-titer, low-avidity antibodies exhibit low antigen-
There are many complex properties of antigen and antibody binding capacity but still show reactivity at high serum
reactions that influence serologic and other testing methods dilutions.11
involving antibodies. The antigen-binding site of the antibody The specificity of an antiserum (or antibody) is one of its
molecule is uniquely designed to recognize a corresponding most important characteristics and is related to its relative
antigen; this antibody amino acid sequence cannot be changed avidity for antigen. Antibody specificity can be further
without altering its specificity. The extent of the reciprocal classified as a specific reaction, cross-reaction, or no reaction
relationship, also called the fit between the antigen and its (Fig. 3–7). A specific reaction implies reaction between
binding site on the antibody, is often referred to as a lock and similar epitopes. A cross-reaction results when certain epitopes
key mechanism. Factors influencing antigen-antibody reac- of one antigen are shared by another antigen and the same
tions include intermolecular binding forces, antibody proper- antibody can react with both antigens. No reaction occurs
ties, host factors, and tolerance. when there are no shared epitopes.
2682_Ch03_045-076 28/05/12 2:32 PM Page 64
Tolerance
In addition, there is the valency of an antibody, which is
the number of antigen-binding sites on an antibody molecule
Advanced Concepts
or the number of antibody binding sites on an antigen. Anti-
gens are usually multivalent, but antibodies are usually Tolerance is defined as the lack of an immune response
bivalent. IgM and IgA antibodies can have higher valences or an active immunosuppressive response. It has been
due to the multimeric nature of some of their structures. observed post-transfusion for many years, but its mech-
Because the biochemistry controlling the forces of antibody anism is still unclear. Tolerance can be either naturally
binding are well understood, the influence of these forces occurring or experimentally induced. Exposure to an
can be manipulated by using various reagents and methods antigen during fetal life usually produces tolerance to
to enhance the reactivity of certain RBC antigens with anti- that antigen. An example of this type of tolerance is
bodies. When more than one antibody is present in a blood found in the chimera, an individual who receives an in
bank sample, it is often necessary to use special techniques utero cross-transfusion of ABO-incompatible blood from
to identify all the antibodies. These techniques and reagents a dizygotic (fraternal) twin.2 The chimera does not
were developed with an understanding of antibody reactivity. produce antibodies against the A and B antigen of the
twin, and the ABO group of such an individual may
Host Factors appear as a testing discrepancy.
The induction of tolerance is used to prevent D-negative
Various host factors play a key role in an individual’s immune mothers from developing anti-D antibodies after delivering
response (Box 3–3). These factors are important in the host’s Rh-positive infants. When a D-negative woman gives birth
overall immune response and in various specific immune to a D-positive infant, she is exposed to D-positive RBCs,
reactions. Each individual’s immune system is unique and most of which occurs during delivery. Approximately 50%
determines how that individual is able to resist disease. Host to 70% of Rh-negative mothers develop anti-D antibodies
factors include nutritional status, hormones, genetic inheri- on first exposure to D-positive cells. These antibodies can
tance, age, race, sex, physical activity level, environmental result in HDN upon later pregnancies with a D-positive
exposure, and the occurrence of disease or injury. It is also fetus. Use of passive immunization to prevent the forma-
believed that immune function decreases as age increases; this tion of these antibodies can prevent HDN. This is accom-
may be one reason why so many diseases such as cancer and plished by administering IgG Rh-immune globulin (RHIG)
autoimmune conditions are seen at a later time in life. A de- within 48 to 72 hours after the birth of the infant. About
crease in antibody levels in older individuals may result in false- 25% to 30% of D-negative individuals are nonresponders
negative reactions, especially in reverse ABO blood typing. and do not produce anti-D antibodies, even when subjected
to repeated exposure to D-positive cells. Because it is
not known who will respond, it is recommended that all
D-negative mothers who deliver D-positive newborns
BOX 3–3
receive RHIG to prevent immunization.
Host Factors: Properties of the Host That Influence
Immune Response
Detection of RBC Antigen-Antibody
• Nutritional status
Reactions
• Hormones
• Genetics
• Age Basic Concepts
• Race Various factors influence detection of RBC antigen-antibody
• Exercise level reactions. These include having a correct sample and the
• Disease proper reagents performing the correct test and under-
• Injury standing how the test should be done and interpreted.
2682_Ch03_045-076 28/05/12 2:32 PM Page 65
Blood Samples Required for Testing hemagglutination (a special type of agglutination), precip-
itation, agglutination inhibition, and hemolysis. Other
One of the most important steps in obtaining the correct and
techniques that quantify antigen or antibody with the use
valid results of an analytical or diagnostic test is to have the
of a radioisotope, enzyme, or fluorescent label—such as
correct sample. Different tests in the blood bank may require
radioimmunoassay (RIA), enzyme-linked immunosorbent
different samples. Some tests require the use of serum to
assay (ELISA) or enzyme immunoassay (EIA), Western
ensure that adequate amounts of viable complement are
blotting (WB), and immunofluorescence (IF)—may be
available for fixation by blood group antibodies. Serum is
used in automated or semiautomated blood banking instru-
obtained when no anticoagulant is used in the sample col-
mentation.2 These techniques are very important in testing
lection tube. The sample clots and is centrifuged to separate
for viral pathogens in donor units and patient samples.
the clotted cells and the liquid serum fraction. An anticoag-
Recently, blood bank automated methods for the typing of
ulated sample would not be conducive to complement acti-
whole blood units and patient samples have become more
vation studies, because anticoagulants bind divalent Ca2+
popular and may become routine in the near future.
and Mg2+ ions and inhibit complement activity.
In most transfusion laboratory testing, hemagglutination
The commonly used anticoagulant ethylenediaminete-
reactions are the major technique used. Hemagglutination
traacetic acid (EDTA) at a ratio of 2 mg to 1 mL of serum
methods for the analysis of blood group antigen-antibody
will totally inhibit complement activation by binding cal-
responses and typing for ABO, Rh, and other blood group
cium and, to a lesser extent, magnesium. Another anticoag-
antigens is accomplished by red cell agglutination reac-
ulant, sodium heparin, inhibits the cleavage of C4. These
tions. Agglutination is a straightforward process and can be
problems can be avoided by using serum instead of plasma
shown to develop in two stages. In the first stage, sensitiza-
for blood bank procedures that require fresh complement.
tion, antigen binding to the antibody occurs. Epitopes on
Currently, however, plasma is used routinely in place of
the surfaces of RBC membranes combine with the antigen-
serum. Years of testing with both samples have shown that
combining sites (Fab region) on the variable regions of the
for most tests, plasma is comparable to serum. Plasma sam-
immunoglobulin heavy and light chains. Antigen and anti-
ples are preferred for DAT and elution studies because they
body are held together by various noncovalent bonds, and
lack fibrin strands, which can cause false-positives. Serum
no visible agglutination is seen at this stage.
should be removed as soon as possible from a clotted blood
In the second stage, a lattice-type structure composed
sample. If testing cannot begin immediately, then serum
of multiple antigen-antibody bridges between RBC antigens
should be removed and stored at 4°C for no longer than
and antibodies is formed. A network of these bridges forms,
48 hours. After this time, serum should be frozen at –50°C
and visible agglutination is present during this stage. The
or lower to retain complement activity. The complement
development of an insoluble antigen-antibody complex, re-
system may become activated during storage of preserved
sulting from the mixing of equivalent amounts of soluble
RBC products. For example, in citrate phosphate dextrose
antigen and antibody, is known as a precipitation reaction.
adenine (CPDA-1)–preserved RBCs, activation of the alter-
Agglutination inhibition is a method in which a positive
nate pathway can be caused by contact of plasma C3 with
reaction is the opposite of what is normally observed in
plastic surfaces of blood bags. This may cause hemolysis that
agglutination. Agglutination is inhibited when an antigen-
will become visible as the RBCs settle upon storage.12
antibody reaction has previously occurred in a test system.
The antigen and antibody cannot bind because another
Traditional Laboratory Testing substrate has been added to the reaction mixture and
Methods blocks the formation of the agglutinates. Inhibition reac-
tions are used in secretory studies to determine whether
The various complex immunologic responses that are
soluble ABO substances are present in body fluids and
important to blood banking have been studied by a number
secretions. The secreted substrate can combine with the
of immunologic methods. These in vitro testing methods
antibody and block RBC antigen-antibody reactions. People
were developed with an understanding of the biochemistry
who have blood group antibodies in their body fluids are
and biophysics of the IS. Many of the methods used today
called secretors.
are modifications of previous blood bank techniques or more
Hemolysis represents a strong positive result and indi-
generalized immunologic testing methods that have been
cates that an antigen-antibody reaction has occurred in
adapted to the needs of immunohematology laboratories.
which complement has been fixed and RBC lysis occurs.
The routine methods used today have to be highly efficient
The Lewis blood group antibodies anti-Lea and anti-Leb
to meet clinical laboratory requirements.
may be regarded as clinically significant if hemolysis occurs
in their in vitro testing reactions.
RIA, ELISA (or EIA), WB, and IF techniques are immuno-
Advanced Concepts logic methods based on detection and quantification of
Many different types of tests are available, and it is impor- antigen or antibody by the use of a radioisotope, enzyme,
tant to be familiar with the different purposes of each. In or fluorescent labels. Either the antigen or the antibody can
vitro testing for the detection of antigens or antibodies may be labeled in these tests. These techniques measure the
be accomplished by commonly used techniques, including interaction of the binding of antigen with antibody and
2682_Ch03_045-076 28/05/12 2:32 PM Page 66
are extremely sensitive. Most of these techniques employ agglutinate more efficiently. Having RBCs in closer physical
reagents that use either antigen or antibody, which is bound proximity allows for an increase in antigen-antibody lattice
in a solid or liquid phase in a variety of reaction systems formation, which results in enhanced agglutination.
ranging from plastic tubes or plates to microscopic particles
or beads. These methods use a separation system and wash- Antigen-Antibody Ratio
ing steps to isolate bound and free fractions and a detection
Antigen and antibody have optimal concentrations; in ideal
system to measure the amount of antigen-antibody inter-
reactive conditions, an equivalent amount of antigen and
action. The values of unknown samples are then calculated
antibody binds. Any deviation from this decreases the
from the values of standards of known concentration.
efficiency of the reaction and a loss of the zone of equiva-
lence between antigen and antibody ratio that is necessary
Factors That Influence Agglutination for agglutination reactions to occur. An excess of unbound
Reactions immunoglobulin leads to a prozone effect, and a surplus
of antigen leads to a postzone effect (Fig. 3–8). In either
situation, the lattice formation and subsequent agglutination
Basic Concepts may not occur, which can give false-negative results. If this
Various factors can influence reactivity of antigen-antibody is suspected, simple steps can be taken to correct it. If the
RBC agglutination reactions. Typical of most biochemical problem is excessive antibody, the plasma or serum may be
reaction systems, agglutination reactions are influenced by diluted with the appropriate buffer. The problem of excessive
the concentration of the reactants (antigen and antibody) antigen can be solved by increasing the serum-to-cell ratio,
and by factors such as pH, temperature, and ionic strength. which tends to increase the number of antibodies available
The surface charge, antibody isotype, RBC antigen dosage, to bind with each RBC. Therefore, antigen-antibody test
and the use of various enhancement media, antihuman systems can be manipulated to overcome the effects of
globulin reagents, and enzymes are all important in antigen- excessive antigen or antibody.
antibody reactions. The most important factors are discussed Another reason antigen amount may be altered is due to
in the following sections. weak expression of antigen on RBCs (dosage effect). Weak
expression occurs as a result of the inheritance of genotypes
Centrifugation that give rise to heterozygous expression of RBC antigens
and resultant weaker phenotypes. For example, M-positive
Centrifugation is an effective way to enhance agglutination RBCs from an individual having the genotype MM (homozy-
reactions because it decreases reaction time by increasing the gous) have more M antigen sites than M-positive cells from
gravitational forces on the reactants and bringing reactants an individual having the MN (heterozygous) genotype. In
closer together. High-speed centrifugation is one of the most the Kidd system, Jka homozygous inheritance has greater
efficient methods used in blood banking. Under the right RBC antigen expression than Jka heterozygous. The same is
centrifugation conditions, sensitized RBCs overcome their true for Jkb homozygous and heterozygous expression. In
natural repulsive effect (zeta potential) for each other and the Rh system, the C, c, E, and e antigens also show dosage
Equivalence
Antibody Excess (Optimum Proportions of Antigen Excess
(Prozone) Antigen and Antibody) (Postzone)
2682_Ch03_045-076 28/05/12 2:32 PM Page 67
effects. Dosage effect is sometimes used when preparing efficient as IgG in agglutination reactions. This allows it to
reagent RBCs for testing. easily bridge the distance between two RBCs.13
Another factor that contributes to the difference in reac-
Effect of pH tivity between the IgM and IgG molecules is the number of
antigen-combining sites on each type of immunoglobulin.
The ideal pH of a test system for antigen-antibody reactions The IgM molecule has the potential to bind 10 separate anti-
ranges between 6.5 and 7.5, which is similar to the pH of gen sites; however, an IgG molecule has only 2 binding sites
normal plasma or serum. Exceptions include some anti-M per molecule, which implies that an IgG molecule would
and some Pr(Sp1) group antibodies that show stronger reac- have to bind 2 RBCs with only 1 binding site on each cell.13
tivity below pH 6.5.13 Acidification of the test serum may aid IgM molecules rarely bind 10 sites, owing to the size and
in distinguishing anti-M and anti-Pr(Sp1) antibodies from spacing of antigens in relation to the size and configuration
other antibodies. of the IgM molecule. Typically, when the IgM molecule
attaches to two RBCs, probably 2 or 3 antigen-combining
Temperature sites attach to each RBC. Agglutination reactions involve
more than one immunoglobulin molecule, and these conditions
Different isotypes of antibodies may exhibit optimal reactiv-
are multiplied many times in order to represent the aggluti-
ity at different temperatures. IgM antibodies usually react
nation reaction. Because of the basic differences in the nature
optimally at ambient temperatures or below 22°C at the
of reactivity between IgM and IgG antibodies, different sero-
immediate spin (IS) phase of testing, whereas IgG antibod-
logic systems must be used to optimally detect both classes
ies usually require 37°C incubation and react optimally at
of clinically significant antibodies. An overview of the sero-
the antihuman globulin (AHG) phase of testing. Because
logic methods traditionally used for antibody detection in
clinically significant antibodies may be in both these tem-
the blood bank laboratory is outlined in Table 3–8.
perature ranges, it is important to do testing with a range of
temperatures (Fig. 3–9).
Enhancement Media
Immunoglobulin Type
Agglutination reactions for IgM antibodies and their cor-
Examples of IgM antibodies that have importance in blood responding RBC antigens are easily accomplished in saline
banking include those against the ABH, Ii, MN, Lewis (Lea, medium, as these antibodies usually do not need enhance-
Leb), Lutheran (Lua), and P blood group antigens. Important ment or modifications to react strongly with antigens.
IgG antibodies are those directed against Ss, Kell (Kk, Jsa, However, detection of IgM antibodies may not have the
Jsb, Kpa, Kpb), Rh (DCEce), Lutheran (Lub), Duffy (Fya, Fyb), same clinical significance as the detection of most IgG
and Kidd (Jka, Jkb) antigens (see Fig. 3–8). IgM antibodies antibodies, because IgG antibodies react best at 37°C and
are generally capable of agglutinating RBCs suspended in a are generally responsible for hemolytic transfusion reactions
0.85% to 0.90% saline medium. The IgM antibody is 160 Å and HDN. To discover the presence of IgG antibodies,
larger than an IgG molecule and approximately 750 times as there are many enhancement techniques or potentiators
available (Table 3–9).
One of the key ways to enhance the detection of IgG an-
tibodies is to increase their reactivity. Many of the commer-
cially available enhancement media accomplish this by
Types of Antibodies reducing the zeta potential of RBC membranes. The net neg-
Reaction Phases ative charge surrounding RBCs (and most other human
cells) in a cationic media is part of the force that repels RBCs
from each other and is due to sialic acid molecules on the
surface of RBCs. Most acids have a negative charge, and the
Immediate IgM large concentration of these molecules on RBCs creates a
Spin Phase “zone” of negative charge around the RBC. This zone is pro-
tective and keeps RBCs from adhering to each other in the
peripheral blood. A potential is created because of the ionic
Antiglobulin IgG cloud of cations (positively charged ions) that are attracted
Phase (37ºC) to the zone of negative charges on the RBC membrane
(Fig. 3–10).13 This potential around the RBC is called the
zeta potential and is an expression of the difference in elec-
trostatic charges at the RBC surface and the surrounding
cations. IgM and IgG antibodies have differences in how they
react to the same zeta potential. Reducing the zeta potential
Figure 3–9. Types of antibodies and reaction phases. (From Kutt, SM, Larison, allows the more positively charged antibodies to get closer
PJ, and Kessler, LA: Solving Antibody Problems, Special Techniques. Ortho Diagnos- to the negatively charged RBCs and therefore increases RBC
tic Systems, Raritan, NJ, 1992, p 10, with permission.) agglutination by IgG molecules.
2682_Ch03_045-076 28/05/12 2:32 PM Page 68
Table 3–8 Serologic Systems Used in Traditional Laboratory Methods for Red Cell Antibody
Detection
Ig CLASS PURPOSE AND TESTS THAT TYPE OF ANTIBODIES
COMMONLY MECHANISM USE SEROLOGIC COMMONLY
REACTION PHASE DETECTED OF REACTION SYSTEM DETECTED
Immediate spin IgM IgM antibodies react ABO reverse testing Expected ABO
best at cold alloantibodies
temperatures.
Autocontrol
Antibody
screening/identification
Autocontrol
*All additives should be added after the IS phase immediately before 37°C incubation
LISS = low ionic strength solutions; PEG = polyethylene glycol
often used because they result in an increased rate of alloantibodies, whereas PEG produces very specific reac-
antibody uptake during sensitization and a decreased tions with reduction in false-positive or nonspecific reac-
reaction incubation time (from 30 to 60 minutes to 5 to tions. PEG is considered to be more effective than albumin,
15 minutes as compared with protein potentiators such as LISS, or polybrene for detection of weak antibodies. These
albumin).13,14 However, they can result in false-positive reagents have been used in automated and manual testing
reactions and may require testing to be repeated with systems.
albumin.
No Agglutination Erythrocyte
Ionic Cloud
IgG
14 nm
25 nm
35 nm
especially in cases in which there are multiple antibodies Blood bank reagents can be either polyclonal or mono-
present in a sample. clonal in source. In the indirect antiglobulin test, the same
Enzymes used in the detection and identification of blood AHG reagents are used to detect antibodies or complement
group antibodies include ficin (isolated from fig plants), that have attached to RBC membranes but with a prior
papain (from papaya), trypsin (from pig stomach), and incubation step with serum (or plasma). If the antibodies
bromelin (from pineapple). It is thought that treating RBCs present in serum cannot cause RBC agglutination but only
with enzymes results in the release of sialic acid from the sensitize the RBCs, then the AHG reagents will allow for
membrane with a subsequent decrease in the negative agglutination to occur by cross-linking the antibodies on the
charges and zeta potential of the RBCs. It has also been sug- RBCs. The use of AHG reagents allows blood bank testing
gested that enzyme treatment removes hydrophilic glycopro- to be more sensitive. AHG is one of the most important
teins from the membrane of RBCs, causing the membrane reagents, and the development of AHG is one of the
to become more hydrophobic, which would allow RBCs milestones in blood bank testing (see Tables 3–8 and 3–9).
to come closer together. Also, because of the removal of
glycoproteins from the membrane, antibody molecules may
no longer be subject to steric hindrance from reacting with Chemical Reduction of IgG and IgM Molecules
RBC antigens. The use of enzymes provides enhanced
antibody reactivity to Rh, Kidd, P1, Lewis, and I antigens
and destroys or decreases reactivity to Fya, Fyb, M, N, and S Advanced Concepts
antigens.14 There are special reagents available that can be used to help
identify the different antibodies present in a mixture of al-
loantibodies or alloantibodies occurring with autoantibod-
Antihuman Globulin Reagents
ies. The reagents generally act on covalent sulfhydryl bonds
and facilitate antibody identification by removal of either
Basic Concepts IgG or IgM antibodies. Dithiothreitol (DTT) and -2-
When RBCs become coated with antibody or complement mercaptoethanol (2-ME) are thiol-reducing agents that
or both but do not agglutinate in regular testing, special break the disulfide bonds of the J (joining) chain of the IgM
reagents are needed to produce agglutination. The direct molecule but leave the IgG molecule intact.10 Another
antihuman globulin (AHG) test is designed to determine if reagent, ZZAP, which consists of a thiol reagent plus a pro-
RBCs are coated with antibody or complement or both. teolytic enzyme, causes the dissociation of IgG molecules
Polyspecific AHG can determine if RBCs have been sensi- from the surface of sensitized RBCs and alters the surface
tized with IgG antibody or complement (components C3b antigens of the RBC.2 Chemical reduction of the disulfide
or C3d) or both. Monospecific AHG reagents react only bond of the IgG molecule is also used to produce chemically
with RBCs sensitized with IgG or complement.14 Refer to modified reagents that react with RBCs in saline. Sulfhydryl
Chapter 5, “The Antiglobulin Test.” compounds reduce the strong but less flexible covalent
2682_Ch03_045-076 28/05/12 2:32 PM Page 71
disulfide bonds in the hinge region of the IgG molecule, Nontraditional Laboratory Methods
allowing the Fab portions more flexibility in facilitating
agglutination reactions.13 Advanced Concepts
Sophisticated testing methods used in immunology
Monoclonal Versus Polyclonal research laboratories have become more commonplace in
Reagents transfusion laboratories. These technologies are discussed
in more detail in Chapter 12, “Other Technologies and
Automation.”
Basic Concepts
Traditional polyclonal antisera reagents have been pro-
duced by immunizing donors with small amounts of RBCs Flow Cytometry
positive for an antigen that they lack and then collecting
the serum and isolating antibodies against that antigen. Some of the most important techniques to study immuno-
AHG reagents were originally made by injecting animals logic reactions and systems are fluorescence-assisted cell
(usually rabbits) with human globulin components and sorting (FACS) and flow cytometry. Flow cytometry makes
then collecting the antihuman antibodies. One antigen can use of antibodies that are tagged with a fluorescent dye.
have a number of different epitopes, and polyclonal Fluorescence occurs when the compound absorbs light energy
reagents are directed against multiple epitopes found on of one wavelength and emits light of a different wavelength.
the original antigen used to stimulate antibody production. Cells that are coated with fluorescent-labeled antibody emit
(Refer to Chapter 5.) a brightly fluorescent color of a specific wavelength. Fluo-
Monoclonal reagents are directed against specific epi- rescent dyes are used as markers in immunologic reactions,
topes and therefore are a potential solution. They are made and a fluorochrome-labeled antibody can be visualized by an
by hybridoma technology with spleen lymphocytes from instrument that detects emitted light. There are many differ-
immunized mice that are fused with rapidly proliferating ent fluorochromes available that have various colors, and
myeloma cells. The spleen lymphocytes have single epitope they can be coupled to nearly any antibody with minimal
specificity; the myeloma cells (a type of immortalized, cul- problems with spectral overlap.
tured cell) make vast amounts of antibody. After extensive Flow cytometry can be used to obtain quantitative and
screening and testing, these very efficient hybrid cells are qualitative data on cell populations and to sort different cell
selected and cultured to produce lines of immortal cell populations. There are direct and indirect procedures for
clones that make a lot of one type of antibody that reacts labeling with fluorochromes. In a direct procedure, the spe-
with one specific epitope. Monoclonal reagents do not use cific antibody, called the primary antibody, is directly conju-
human donors and therefore do not use a human source gated with a fluorescent dye and reacts with a specific
for reagent purposes. antigen. Indirect procedures need a secondary antibody that
Monoclonal reagents have several important advantages is conjugated to a fluorochrome that reacts with an unlabeled
over polyclonal reagents. Because monoclonal reagents are specific primary antibody that has reacted with antigen.
produced from immortal clones maintained in vitro, no Indirect methods often require more complex analysis
batch variation exists, and nearly unlimited high titers of because these reactions have higher amounts of nonspeci-
antibodies can be produced. Also, the immortal clones can ficity. The secondary antibody is usually made against the
be kept growing in in vitro culture for years without loss species of the primary antibody.
of production and are therefore cost-efficient. Monoclonal The principle of flow cytometry is based on the scattering
antibodies react very specifically and often have higher of light as cells are bathed in a fluid stream through which a
affinities. For these reasons, monoclonal reagents are not laser beam enters. The cells move into a chamber one at a
subject to cross-reactivity and interference from nonspecific time and absorb light from the laser. If labeled antibody is
reactions, and they can react strongly with the small quan- bound to the cell, then the light emitted is different from
tities of antigen in some antigen subgroups; therefore, AHG light emitted by cells without antibody. The flow cytometer
phase testing may not be needed. instrument makes the distinction between different wave-
Unfortunately, there are some disadvantages of mono- lengths of light. The light signal is amplified and analyzed.
clonal antisera use, such as overspecificity. The fact that There are a number of components to the flow cytometry
complement may not be fixed in the antigen-antibody reac- system that include: one or more lasers for cell illumination,
tion may cause false-negative results; problems with over- photodetectors for signal detection, a possible cell sorting
sensitivity may cause false-positive results. Some of the device, and a computer to manage the data.15 Fluorescence
disadvantages of monoclonal reagents may be overcome by is used in place of hemagglutination as the endpoint of the
using blends of different monoclonal reagents or by using reaction. Immunofluorescent antibodies and flow cytometry
polyclonal reagents and monoclonal reagents together.2 have been used to quantify fetomaternal hemorrhage, iden-
Monoclonal antisera have generally replaced most of the tify transfused cells and follow their survival in recipients,
polyclonal antisera and have been used for HLA typing, measure low levels of cell-bound IgG, and distinguish
AHG testing, and RBC and lymphocyte phenotyping. homozygous from heterozygous expression of blood group
antigens.2
2682_Ch03_045-076 28/05/12 2:32 PM Page 72
BOX 3–4
Ammann, AJ: Mechanisms of immunodeficiency. In Stites, DP, Terr, AI, and Parslow, TG (eds): Basic and Clinical Immunology, 8th ed. Appleton & Lange, Norwalk, 1994, with
permission.
2682_Ch03_045-076 28/05/12 2:32 PM Page 73
other drug-induced antibodies can lead to hemolytic reac- at an antigen present on fetal cells but absent from maternal
tions through type III hypersensitivity. The type IV reaction cells. The mother is exposed to fetal RBCs as a result of
involves only T cell–mediated responses and their cytokines fetomaternal transfer of cells during pregnancy or childbirth.
and can be fatal if untreated. The most important type IV Maternal memory cells can cause a stronger response during
reaction is graft-versus-host, of which there is also more than a second pregnancy if the fetus is positive for the sensitizing
one type. Immunocompromised and immunosuppressed pa- antigens. IgG1, IgG3, and IgG4 are capable of crossing
tients must receive irradiated blood products so that T lym- the placenta and attaching to fetal RBCs, whereas IgG2
phocytes do not engraft and attack the host tissues. Type IV and IgM are not. Severe HDFN is most often associated
reactions are a problem for bone marrow transplant and stem with IgG1 antibodies and may require exchange transfusion.
cell transfusion recipients. (Refer to Chapter 16, “Adverse Antigens of the ABO, Rh, and other blood group systems
Effects of Blood Transfusion.”) such as Kell have been shown to cause HDFN. (Refer
to Chapter 19, “Hemolytic Disease of the Fetus and Newborn
Monoclonal and Polyclonal Gammopathies [HDFN].”)
Plasma cell neoplasms result in proliferation of abnormal im- Blood Product Transfusions and the
munoglobulin from either a single B-cell clone (monoclonal Immune System
gammopathies) or multiple clones (in polyclonal gam-
mopathies) and may be a specific isotype or only light or The immune system may undergo transient depression
heavy chain molecules. Increased serum viscosity is a result following the administration of blood and blood products.
of these diseases and can interfere with testing. The increased This is referred to as transfusion-related immunomodula-
concentrations of serum proteins can cause nonspecific tion (TRIM). With a weakened immune system, there is an
aggregation (as opposed to agglutination) of erythrocytes increased risk of an individual developing infections or
called rouleaux, which is seen as a stacking of RBCs. It often cancer. The exact mechanism(s) causing TRIM are not
occurs in multiple-myeloma patients. If rouleaux is sus- known and have not been fully elucidated, but three major
pected, saline replacement technique may be needed to mechanisms seem to have emerged: clonal deletion, induc-
distinguish true cell agglutination from nonspecific aggre- tion of anergy, and immune suppression. In clonal deletion,
gation. Roleaux primarily causes problems in the ABO alloreactive lymphocytes are inactivated and removed, thus
reverse grouping, antibody screening, and compatibility test- preventing a potential graft rejection (as in kidney organ).
ing procedures. However, excess immunoglobulin coating With induction of anergy, there is an unresponsiveness of
red cells can cause spontaneous aggregation to occur also. the immune system most likely due to a failure of T cells
to respond. In immune suppression, responding cells
Autoimmune Disease appear to be inhibited by some sort of cellular mechanism
or cytokine.
Autoantibodies are produced against the host’s own cells Immune suppression can be due to a number of factors.
and tissues. It is unknown why this loss of tolerance to self- Certain cytokines, including interleukins and some growth
antigens occurs, but there are many possible explanations factors, can decrease immune responsiveness. Suppression
such as aberrant antigen presentation, failure to obtain of some IS components is critical at certain times, so the
clonal deletion, anti-idiotypic network breakdown, and IS does not become overactivated and attack host cells. T
cross-reactivity between self and nonself antigens. Autoimmune and B cells that recognize host cells and might destroy
hemolytic anemias are an important problem in testing and them must be removed before they can develop into
transfusion. They may produce antibodies that cause RBC mature lymphocytes. Specific suppressor cells have recently
destruction and anemia and result in antibody- or complement- been isolated. They have unique CD profiles and play a
sensitized RBCs. The direct antiglobulin test should be done role in modulating lymphocyte function. Immune suppres-
to detect sensitized RBCs and determine if the cells are sion can also occur when there is too little immunoglobu-
coated with antibody or complement. Special procedures lin made or when too many T and B cells are lost through
such as elution or chemical treatment to remove antibody severe infection, extreme immune stimulation, or IS organ
from cells may be required to prepare RBCs for antigen failure.
typing. Serum autoantibodies may interfere with testing for It is believed that some constituents of cellular blood
clinically significant alloantibodies. Special reagents and products may be responsible for TRIM. These constituents
procedures to denature immunoglobulins may be required include allogeneic mononuclear cells, chemicals released by
to remove autoantibodies from serum so they do not inter- allogenic mononuclear cells as they age, and soluble HLA
fere with the testing. (Refer to Chapter 20, “Autoimmune peptides I that circulate in plasma. Any decision to transfuse
Hemolytic Anemias.”) blood products must be approached with caution. Obviously,
the best way to avoid TRIM is not to transfuse at all. If trans-
Hemolytic Disease of the Newborn fusion is required, the patient’s immune status must be taken
into consideration. The use of leukoreduction filters may
Hemolytic disease of the fetus and newborn (HDFN) can help to reduce the risk for TRIM. (Refer to Chapter 15,
result when the maternal IS produces an antibody directed “Transfusion Therapy.”)
2682_Ch03_045-076 28/05/12 2:32 PM Page 74
SUMMARY CHART
The immune system interacts with other host systems Membrane-bound antibodies are manufactured by
and maintains the host equilibrium. The two major B cells and inserted into their cell membranes, where
roles for the IS are: they act as antigen receptors.
• Protection from pathogens and foreign substances Stimulated B cells differentiate into plasma cells to
• Removal of abnormal and damaged host cells secrete humoral immunoglobulin; B cells receive
The two major branches of the IS are: T-cell help for antibody production and for immuno-
• Innate, or natural—the nonspecific primitive IS logic memory; a single B cell clone manufactures Ig of
• Acquired, or adaptive—the specific, evolved IS a single specificity for a specific antigen for its entire
The two major arms of the acquired IS are: cell lifetime.
• Humoral, mediated by B cells and antibody The primary, or original, immune response occurs after
production the first exposure to an antigen. The secondary, or
• Cellular, mediated by T cells and lymphokines anamnestic, immune response happens after a second
The basic mechanisms used by the IS are: exposure with the same specific antigen.
• Recognition of self and nonself organisms, cells, and Complement consists of a large group of different
tissues enzymatic proteins (convertases/esterases) that circulate
• Removal of unwanted organisms, cells, and tissues in an inactive proenzyme form. Once the cascade is
(self or nonself) started, they activate each other in a sequence to form
• Repair of damaged host tissues products that are involved in optimizing phagocytosis
The acquired IS demonstrates diversity and uniqueness: and cell lysis.
• Individual B and T cells have vast arrays of unique Complement can be activated through three pathways:
membrane molecules that can have configurations • The classical pathway is initiated by antigen-
to match nearly any antigen in the environment. antibody complexes and requires C1q for activa-
• Each individual lymphocyte has one unique recep- tion to proceed.
tor per cell that recognizes one epitope. • The alternative pathway is activated by certain
• Antibodies and T-cell receptors recognize and react macromolecules on the cell walls of bacteria, fungi,
only with the antigen that matches and fits their parasites, and tumor cells and requires C3b, serum
specific configuration. factors B, D, properdin, and initiating factor.
• Selected T and B cells can remain dormant and later • The lectin pathway is activated by binding of MBL
respond more rigorously upon second exposure of to microbes.
a previously recognized antigen. All three pathways meet at a common point in the
The acquired IS demonstrates tolerance: this indicates cascade and result in the formation of the membrane
that immune responses against the host are either attack complex to remove unwanted cells.
removed or downregulated. There are five classes (or isotypes) of immunoglobu-
There are three types of lymphocytes: T cells, B cells, lins, all of which have a basic four-chain protein struc-
and NK cells. ture consisting of two identical light chains and two
T cells (or lymphocytes) have the TCR, which is usually identical heavy chains. Disulfide (covalent) bonds link
associated with the CD3 complex, and T cells require each light chain to a heavy chain and link the two
APCs to respond to antigens. heavy chains to each other.
There are two well-characterized subpopulations of Antibody molecules are isotypic (based on heavy chain
T cells distinguished by CD markers—T helper (TH, subtype), allotypic (based on one heavy chain muta-
CD4-positive) and T cytotoxic (Tc, CD8-positive) tion), or idiotypic (based on hypervariable and variable
lymphocytes. regions of light and heavy chains) and are reflected in
TH lymphocytes have CD4 markers on their cell mem- the Ig sequences.
branes, provide B-cell help to stimulate the immune Blood group antibodies may be characterized by such
response, release lymphokines when stimulated, and factors as epitope and variable region diversity (mon-
recognize antigens in association with MHC class II oclonal or polyclonal), mode of sensitization (naturally
molecules. occurring or immune), expected or unexpected pres-
TC lymphocytes have the CD8 marker on their mem- ence in routine serum samples, isotype class (IgM,
branes and can eliminate specific target cells without IgG, or, rarely, IgA), activity (warm or cold reactive
the help of antibody (cytotoxicity). or both, agglutinating or sensitizing), clinical signifi-
cance, alloantibody or autoantibody specificity, serum
B lymphocytes (or cells) make up about 5% to 15% of
titer, and chemical reactivity (influence of enzymes,
circulating lymphocytes and are characterized by their
sensitivity to pH, DTT, or 2-ME reagents).
membrane-bound antibodies (or immunoglobulins)
2682_Ch03_045-076 28/05/12 2:32 PM Page 75
17. Which of the following refers to a state of equilibrium 3. Sunshine, G, and Coico, R: Immunology: A Short Course,
in antigen-antibody reactions? 6th ed. John Wiley & Sons, Hoboken, 2009.
4. Stites, DP, Parslow, TG, Imboden, JB, and Terr, AI (eds):
a. Postzone Medical Immunology, 10th ed. McGraw-Hill, New York, 2001.
b. Prozone 5. Nance, SJ, Arndt, PA, and Garratty, G: Correlation of IgG
c. Zone of equivalence subclass with the severity of hemolytic disease of the newborn.
d. Endzone Transfusion 30:381, 1990.
6. Rieben, R, et al: Antibodies to histo-blood group substances A
18. Which one of the following properties of antibodies is and B: Agglutination titers, Ig class, and IgG subclasses in
NOT dependent on the structure of the heavy chain con- healthy persons of different age categories. Transfusion 31:607,
1991.
stant region? 7. Sokol, RJ, et al: Red cell autoantibodies, multiple immunoglob-
a. Ability to cross the placenta ulin classes, and autoimmune hemolysis. Transfusion 30:714,
b. Isotype (class) 1990.
c. Ability to fix complement 8. Kerr, WG, Hendershot, LM, and Burrows, PD: Regulation of
IgM and IgD expression in human B-lineage cells. J Immunol
d. Affinity for antigen
146:3314, 1991.
19. Molecules that promote the update of bacteria for 9. Klein, HG, and Anstee, DJ (eds): Red cell antibodies against
self-antigens, bound antigens and induced antigens. In Molli-
phagocytosis are: son’s Blood Transfusion in Clinical Medicine, 11th ed. Wiley-
a. Opsonins Blackwell, Oxford, UK, 2007, pp 253–298.
b. Cytokines 10. Klein, HG, and Anstee, DJ (eds): ABO, Lewis and P groups and
c. Haptens Ii antigens. In Mollison’s Blood Transfusion in Clinical Medi-
cine, 11th ed. Wiley-Blackwell, Oxford, UK, 2007, pp 114–162.
d. Isotypes
11. Turgeon, ML: Fundamentals of Immunohematology, 2nd ed.
20. Select the term that describes the unique confirmation Lippincott Williams & Wilkins, Philadelphia, 2003.
12. Schleuning, M, et al: Complement activation during storage of
of the antigen that allows recognition by a correspon- blood under normal blood bank conditions: Effects of pro-
ding antibody: teinase inhibitors and leukocyte depletion. Blood 79:3071,
a. Immunogen 1992.
b. Epitope 13. Issitt, PD, and Anstee, DJ: Applied Blood Group Serology,
4th ed. Montgomery Scientific, Durham, NC, 1998, pp 34–35.
c. Avidity
14. Kutt, SM, et al: Rh Blood Group System Antigens, Antibodies,
d. Clone Nomenclature, and Testing. Ortho Diagnostic Systems, Raritan,
NJ, 1990, pp 13–14.
21. Which of the following terms refers to the net negative 15. Stevens, CD: Clinical Immunology and Serology: A Laboratory
charge surrounding red blood cells? Perspective, 3rd ed. FA Davis Company, Philadelphia, 2009.
a. Dielectric constant 16. Ammann, AJ: Mechanisms of immunodeficiency. In Stites, DP,
b. Van der Waals forces Terr, AI, and Parslow, TG (eds): Basic and Clinical Immunology,
8th ed. Appleton & Lange, Norwalk, 1994.
c. Hydrogen bonding
d. Zeta potential
References
1. Waytes, AT, et al: Pre-ligation of CR1 enhances IgG-dependent
phagocytosis by cultured human monocytes. J Immunol
146:2694, 1991.
2. Roback, J, Combs, M, Grossman, B, and Hillyer, C: Technical
Manual, 16th ed. CD-ROM. American Association of Blood
Banks, Bethesda, MD, 2009.
2682_Ch04_077-100 22/05/12 11:40 AM Page 77
Chapter 4
Concepts in Molecular Biology
Maria P. Bettinotti, PhD, dip. ABHI; Lorraine Caruccio, PhD, MT(ASCP)SBB;
and Barbara Kraj, MS, MLS(ASCP)CM
OBJECTIVES
1. Refer to the contributions by Griffith, Avery, Hershey, and Chase, and explain how DNA was proven to be the carrier of genetic
information.
2. Explain the significance of Chargaff’s rules and x-ray diffraction studies by Wilkins and Franklin in Watson and Crick’s discovery
of the DNA double helix structure.
3. Describe DNA features fundamental in the design of molecular assays and define the applicable terms: complementarity,
hydrogen bonding, melting point, denaturation, annealing, and polarity.
4. Explain what the central dogma of molecular biology is and how it expanded after the discovery of reverse transcriptase enzyme.
5. Explain what the recombinant DNA technology is and describe the tools used in molecular cloning: restriction endonucleases,
vectors, and host cells.
6. Define gene expression and explain how it may be studied and used in manufacturing of recombinant proteins.
7. Summarize the procedures of plasmid DNA isolation and gel electrophoresis.
8. Explain the principles of the polymerase chain reaction as an in vitro molecular procedure mimicking the process of semicon-
servative DNA replication occurring in vivo.
9. Discuss the differences between classic PCR, reverse-transcriptase, and real-time PCR.
10. Describe the principle of transcription-mediated amplification.
11. Describe Sanger’s sequencing dideoxy chain termination method.
12. Recognize the differences between immunoblotting and hybridization methods: Southern and Northern analysis, microarrays,
and fluorescent in situ hybridization.
13. Explain major principles of methods used in studying gene polymorphisms: RFLP, VNTR, SSP, and SSOP.
14. Explain how nucleic acid testing (NAT) in donor blood improves the process of screening for infectious disease.
15. Describe the applications of RBC molecular antigen typing.
77
2682_Ch04_077-100 22/05/12 11:40 AM Page 78
(E. coli) cultures were infected in parallel with the two different phages. (3) Phages
In 1950, Erwin Chargaff of Columbia University reported were dislodged from bacteria by treatment with a blender. (4) After centrifugation,
results of DNA quantitative analysis that became known as 35S (proteins) were detected only in the supernatant of the first culture and 32P
Chargaff’s rules.5 Later, Maurice Wilkins and Rosalind (DNA) only in the pellet of the second culture. The phages remained in the super-
natant and the bacteria in the pellet. From the bacterial pellet, a new generation of
Franklin produced x-ray diffraction photographs of DNA
viruses could be raised.
that suggested a helical structure. Based on these clues, in
1953, James Watson and Francis Crick published a seminal
in which they proposed that the DNA molecule was a double Consistent with x-ray diffraction, about 10 base pairs were
α helix.6 DNA was a helical ladder, the rails of which were stacked on top of each other at each turn of a helix. Adenine
built from alternating units of deoxyribose and phosphate. always paired by two hydrogen bonds with thymine; guanine
Each rung of the ladder was composed of a pair of nitrogen- always paired by three hydrogen bonds with cytosine. Due
containing nucleotides (a base pair) held together by hydrogen to this complementarity resulting from formation of nonco-
bonds. valent hydrogen bonds, the nucleotide alphabet of one-half
The double helix consisted of two strands of nucleotides of the DNA helix determined the alphabet of the other half.
that ran in opposite directions (they were antiparallel), des- Chemistry and dynamics of hydrogen bonding is pivotal
ignated by polarity (directionality) labels 3’ and 5’, which in understanding the fundamentals of molecular testing
refer to the number assigned by convention to the deoxyri- methods, as many procedures are based on the processes
bose carbon atom linked to either the hydroxyl or phosphate of DNA strands’ separation (denaturation) and annealing
group (see Chapter 2 and Fig. 2–10). The 3’ and 5’ ends (renaturation). These processes occur at different tempera-
determine the direction of DNA replication and transcription. tures, depending on the total number of hydrogen bonds in
2682_Ch04_077-100 22/05/12 11:40 AM Page 80
the molecule, which determines the DNA melting point of individuals with and without sickle cell anemia had
(Tm). The value of Tm is necessary in calculations used to different mobility in an electric field (electrophoretic mobil-
choose temperatures for polymerase chain reaction or other ity).9 This indicated that hemoglobin from the patients had
hybridization-based assays.7 DNA melting point has nothing a different electric charge; therefore, amino acid composition
to do with the conversion of solid state into liquid, as the was different from normal hemoglobin. Carriers of the disease
term could imply. Rather, it is the temperature at which half had both types of hemoglobin.
of all hydrogen bonds in the molecule are broken, while the In 1957, Ingram identified that substituting valine for glu-
other half are still intact. In other words, the Tm determines tamic acid in the sixth amino acid position from the NH2 end
how much energy is needed to keep the strands of the helix of the β-globin chain was the cause of the defective hemo-
apart. Temperatures above the Tm promote the separation globin.10 Thus, a direct link was established between a gene
of the strands, while temperatures below Tm keep the mutation and the structure of a protein. Concurrent research
strands together. showed that proteins were synthesized in the cytoplasm in
The Watson-Crick model and the chemistry of hydrogen eukaryotic cells. These studies established that the microsomal
bonding reveal how DNA could replicate. During replication, fraction, which we now call ribosomes, was the major cellular
the hydrogen bonds break, the strands separate, and each component required for protein synthesis.11
one functions as a template for the synthesis of another com- Because DNA was in the nucleus, separated from the
plementary half molecule. In this enzymatic process, two cytoplasm by the nuclear membrane, an intermediary molecule
identical DNA molecules are generated, each containing the had to be responsible for conveying the genetic information
original strand and a new complementary strand. Because from the nucleus to the cytoplasm. Ribonucleic acid (RNA)
each daughter double helix contains an “old” strand and a was a good candidate for this role. Its structure suggested
newly synthesized strand, this model of replication is called that RNA could be produced based on a DNA template, and
semiconservative. The mechanism of DNA replication was RNA was located mainly in the cytoplasm where protein
studied using cellular components to reconstruct, in vitro, synthesis occurred.
the biochemical reactions that occur within the cell. In Experimental evidence supporting the role of RNA as the
this way, the whole process was dissected so that molecular intermediary molecule came from the studies on bacterial
testing procedures could be developed that mimicked the viruses (bacteriophages). When bacteriophage T4 infects
natural process that occurred in vivo. In eukaryotes, DNA E. coli, the synthesis of bacterial RNA stops, and the only
polymerase III is able to form a new strand using deoxyri- new RNA synthesized is transcribed from T4 DNA. Using
bonucleoside triphosphates (dNTPs) as substrates, double- radioactive labeling, Sidney Brenner, François Jacob, and
stranded DNA as template, and magnesium as an enzyme Matthew Meselson showed that the newly synthesized
cofactor. This enzyme can add nucleotides onto the hydroxyl T4 RNA associated with bacterial ribosomes, unlike ribosomal
group (OH) at the 3’ end of an existing nucleic acid RNA.12 These RNA molecules, which served as templates for
fragment. Both strands are synthesized at the same time8 protein synthesis, were given the name messenger RNAs
(see Fig. 2–12). (mRNAs).
The examples of techniques mimicking DNA replication But how could nucleotides direct the incorporation of an
are DNA sequencing and polymerase chain reaction (PCR), amino acid into a protein? The experimental answer to this
described later in this chapter. question came again from studying cell-free extracts. In these
experiments, a third type of RNA molecule, which we now
Expression of Genetic Information call transfer RNA (tRNA), was isolated. These were short
molecules, 70 to 80 nucleotides long, and some displayed
Genes act by determining the sequence of amino acids and the remarkable characteristic of having amino acids cova-
therefore the structure of proteins. Knowledge of DNA struc- lently attached to their 3’ end. The enzymes responsible for
ture revealed that the genetic information must be specified attaching a specific amino acid to a specific tRNA were also
by the order of the four nitrogen-containing bases (A, C, G, isolated from cell-free extracts and were given the name
and T). Proteins are polymers of 20 different amino acids, aminoacyl tRNA synthetases. Sequencing of several tRNAs
the sequence of which determines their structure and func- showed that they all had a common three-dimensional struc-
tion. But how could the sequence of nucleotides determine ture, but a unique three-nucleotide-long sequence was
the sequence of amino acids? always present in a loop region. This sequence, which we
The discovery of the molecular basis of sickle cell anemia now call anticodon, provided the specificity for each tRNA,
provided the first experimental evidence that the nucleotide carrying its specific amino acid, to align with the correspon-
sequence of a DNA molecule indeed determined the amino ding codon (triplet) in the mRNA.8 (Refer to Chapter 2 and
acid sequence of a protein. Sickle cell anemia is a genetic dis- Figs. 2–15 through 2–18 for more information on the
ease inherited according to an autosomal-recessive pattern. process of translation.)
Patients with this disease have two copies of the mutated The next question was which triplet of nucleotides of the
beta globin chain gene located on chromosome 11. Individ- mRNA corresponded to which amino acid; in other words,
uals with only one mutated copy of the gene show mild or it was time to decipher the genetic code. By 1966, Marshall
no clinical manifestation and are called carriers. In 1949, Nirenberg and Har Gobind Khorana had cracked the code.
Linus Pauling and Vernon Ingram showed that the hemoglobin Nirenberg used cell-free extracts containing ribosomes,
2682_Ch04_077-100 22/05/12 11:40 AM Page 81
amino acids, tRNAs, and aminoacyl tRNA synthetases, and transcription is an important tool in molecular biology tech-
added synthetic mRNA polymer containing only uracil niques. Natural or recombinant enzymes with reverse tran-
(-UUUUUU-). He was able to produce in vitro a polymer scriptase activity can be used to generate DNA copies of any
peptide containing only the amino acid phenylalanine. In RNA molecule. Using reverse transcriptase mRNAs can be
this way, he showed that UUU was the codon for phenylala- transcribed to its complementary DNA (cDNA) and studied
nine.13 During the next few years, all possible mRNA com- by recombinant DNA techniques. For example, infectious
binations were tried. The 61 different triplets were assigned disease screening assays designed to detect HIV in donors’
to their corresponding amino acids, and 3 triplets were found blood and other specimens start with the process of reverse
to code for a stop signal (refer to Fig. 2–11). The genetic transcription, because HIV viruses are retroviruses and their
code is called degenerate because one amino acid may be genetic material consists of RNA. (Refer to Chapter 18,
encoded by more than one nucleotide triplet. This serves as “Transfusion-Transmitted Diseases.”) The cDNA synthesized
a mechanism that protects the code from devastating effects during reverse transcription is subsequently amplified in
of mutations occurring in the third or second base of the polymerase chain reaction, which allows for creation
triplet. In most cases, the mutation results in amino acid of enough copies of genetic material to analyze (refer to
change only when it affects the first base of the triplet. “Reverse Transcriptase PCR” section in this chapter).
Scientists have added further disclaimers to the central
The Central Dogma of Molecular dogma:
Biology, Expanded
• Genes are not “fixed.” Some of them do not have a desig-
Deciphering the genetic code confirmed the central dogma nated chromosomal locus and function as transposable
of molecular biology. The genetic material is DNA. DNA is genetic elements in the eukaryotic genome. Another
self-replicating and is transcribed into mRNA, which in turn mechanism of physical rearrangement is in vivo DNA
serves as a template for the synthesis of proteins. Although recombination. The examples are the immunoglobulin
the central dogma remains true, the knowledge acquired in chain and T-cell receptor (TCR) gene rearrangements.
the following years has refined and enlarged it, a process that • DNA sequences and protein amino acid sequences do not
continues now and into the future. exactly correspond to one another. In many organisms,
A different method of information flow in biological sys- including humans, coding sequences (exons) are inter-
tems was discovered. A particular group of animal RNA rupted by noncoding sequences (introns) that are excised
viruses called tumor viruses can cause cancer in infected from the immature RNA shortly after transcription. This
animals. In the early 1960s, Howard Temin discovered that introduces the possibility of alternative splicing to create
replication of these viruses required DNA synthesis in the different proteins from the same gene. Also, different read-
host cells. He formulated the hypothesis that RNA tumor ing frames can generate different mRNAs and therefore
viruses, subsequently called retroviruses, replicated via the different proteins. Viruses frequently use this last mecha-
synthesis of a DNA intermediate or provirus. In 1970, Temin nism. In vitro reverse transcription procedures will result
and David Baltimore showed independently that these in cDNA that corresponds only to sequences originally
viruses contain an enzyme that catalyzes the synthesis of present within exons. Hence, cDNA derived from very
DNA using RNA as a template.14,15 long genes is a much shorter representation of the gene
The presence of viral DNA in replicating host cells was (Fig. 4–3). Scientists have discovered further roles for
also demonstrated. The synthesis of DNA from RNA, now RNA. Some RNA molecules display catalytic activity; by
called reverse transcription, was thus definitely established RNA interference, small RNA molecules help to regulate
as a mode of biological information transfer. In vitro reverse gene expression.16
Figure 4–3. In eukaryotes, the coding sequences (exons) are inter- Promoter Exon 1 Intron 1 Exon 2 Intron 2 Exon 3
rupted by noncoding sequences (introns). Newly transcribed RNA 5′ 3′
(pre-mRNA) is considered heterogenous RNA (hnRNA) and loses the
3′ 5′
introns in the process of splicing. The mature mRNA is modified
DNA
by polyadenylation of the 3’ end and addition of 7-methylguanine
“cap” at the 5’ end, and it contains only the exons. In vitro reverse Transcription
transcription procedures will result in cDNA that corresponds only to
sequences originally present within exons. Hence, cDNA is a shorter
representation of the gene. (Modified from Buckingham, L, and Flaws, Pre-mRNA 5′ 3′
ML: Molecular Diagnostics. Fundamentals, Methods and Clinical
Processing
Applications. F.A. Davis Company, Philadelphia, PA, 2007.)
mRNA 7 Me G AAAAA
5′ 3′
Reverse transcription
cDNA
2682_Ch04_077-100 22/05/12 11:40 AM Page 82
Recombinant DNA one organism can be cut and pasted into a carrier DNA mole-
cule or vector (e.g., a plasmid or bacteriophage). The new DNA
The classic experiments in molecular biology, which we have molecule, which is a “recombinant” of the original DNA with
described, used simple and rapidly replicating organisms (bac- the vector DNA, can be introduced using various techniques
teria and viruses) as study models. However, the genomes of into another, usually simpler, host organism. Because the genetic
eukaryotes are much more complicated, and scientists had to code is almost universal, the host organism treats the gene as
explore new ways to isolate and study individual genes of these its own and replicates this “transgene” along with its own
organisms. The big breakthrough came with the development DNA. This technique is called molecular cloning. The ABO
of recombinant DNA technology. A fragment of DNA from gene was cloned by Yamamoto’s group in 199017 (Table 4–1).
1924 Bernstein, applying the Hardy-Weinberg principle, proposes that the ABO antigens are coded by one gene.
1944 Avery, MacLeod, and McCarty prove DNA as the carrier of inheritance (following Griffith’s transformation in 1928).
1953 Watson and Crick decipher DNA structure based on Chargaff’s rules (A + G = T + C and T = A and C = G) and x-ray studies by
R. Franklin (Nobel ‘62 with Wilkins).
1970 Smith and Wilcox isolate the first restriction enzyme from H. influenzae (RFLP method developed in 1978).
Temin and Baltimore discover enzyme reverse transcriptase.
1975 Maxam and Gilbert invent the first DNA sequencing method. Sanger improves it in 1977.
1977–78 First demonstration of a molecular basis for blood group polymorphisms: two aa differences in glycophorin A identified as the basis
for M and N blood groups (several researchers).
1981 First demonstration of a molecular basis for rare blood group polymorphisms: basis for Mg and Mc rare blood group variants
identified (H. Furthmayr and O. O. Blumenfeld groups).
1986 Kary Mullis publishes polymerase chain reaction (PCR) to amplify DNA fragments using thermostable Taq enzyme.
1987 CD55 (DAF) responsible for the Cromer system and glycophorin B are cloned.
1991 RHD and RHAG genes of the Rh system and KEL gene are cloned.
1992 Higuchi and others at Roche Molecular Systems and Chiron demonstrate real-time PCR (simultaneous amplification and detection).
1994 Genes responsible for the Lutheran system, Colton blood group antigens, and Kidd (JK) system are cloned.
1996 S. Iwamoto and colleagues identify promoter mutations as responsible for the Duffy a-b phenotype.
1999 The Blood Group Antigen Gene Mutation Database (BGMUT) is set up by O. Bloomenfeld and S. K. Patnaik. Moved in 2006 to
National Center for Biotechnology Information site at www.ncbi.nlm.nih.gov/gv/mhc/xslcgi.cgi?cmd=bgmut/home.
2004 Consortium for Blood Group Antigens established with the purpose to “provide education for laboratories involved in DNA/RNA
testing”; initiative by NYBC (M. Reid).
First International Workshop of Molecular Blood Group Genotyping (ISBT initiative).
2005 Microarray-based blood group typing is described by multiple researchers (Transfusion, vol. 45).
Cloning is the reproduction of daughter cells from one Numerous restriction endonucleases have been isolated
single cell by fission or mitotic division, giving rise to a pop- from different bacteria. These enzymes have been classified
ulation of genetically identical clones. Successive divisions as type I, II, and III according to their mechanism of action.
of the host cell create a population of clones containing the Only the type II enzymes are relevant for our description
DNA fragment of interest. The gene or genes of interest of molecular cloning.
can be studied using the techniques available for the host In 1970, Hamilton Smith and Kent Wilcox at Johns
organism. These techniques have allowed detailed molecular Hopkins University isolated the first type II restriction
studies of the structure and function of eukaryotic genes and endonuclease. Type II restriction enzymes are extremely
genomes.18 useful for the molecular biologist; they cut DNA at a
precise position within its recognition sequence and have
The Coding Sequence of a Gene no modifying activity.20 More than 200 restriction endonu-
cleases, covering more than 100 different recognition sites,
In humans and most other eukaryotes, the coding part of a are available commercially. The name given to each
gene consists of exons, which in the genomic DNA alternate enzyme reflects its origin. For example, Smith and
with noncoding introns. Newly transcribed RNA (pre-mRNA) Wilcox’s endonuclease was called HindII because it was ob-
is considered heterogenous RNA (hnRNA) and loses the tained from Haemophilus influenzae strain Rd. The number
introns in the process of splicing. The mature mRNA contains II indicates that it was the second endonuclease to be iden-
only the exons, the coding sequence of nucleotides that tified in these bacteria. Restriction endonucleases usually
directs the process of translation into the amino acid recognize sequences 4 to 8 nucleotides in length. The
sequence of the corresponding protein.19 recognition sequences are palindromic; that is, they read
Both the genomic DNA and the coding sequence of a gene the same in the 5’ to 3’ direction on both strands of the
can be cloned. First, the mRNA is reverse transcribed in vitro double helix. Some enzymes cut both strands in the middle
to its cDNA, using either synthetic or retroviral enzyme of the target sequence, generating blunt ends. Some enzymes
reverse transcriptase. Because eukaryotic mRNAs end with cut both strands off the center, generating staggered ends.
a poly-A tail, a short synthetic oligo-dT nucleotide can be For example enzyme EcoRI, isolated from E. coli, recog-
used as a universal primer starting the cDNA synthesis (this nizes this sequence:
process may also be achieved using the random hexamers
or sequence-specific primers, depending on the application). 5’GAATTC3’
The final product is a hybrid double strand of mRNA and 3’CTTAAG5’
cDNA. The mRNA is degraded, and a DNA strand comple- EcoRI cuts after the first G in both strands, generating
mentary to the cDNA is synthesized using DNA polymerase. fragments with the following ends:
The cDNA, which has the information for the coding gene
sequence, can be subjected to the same techniques as 5’G AATTC3’
genomic DNA.18 3’CTTAA G5’
Table 4–2 Examples of Type II Restriction chain reaction. For example, the PCR-RFLP method was used
Endonucleases to discriminate between RHD+ and RHD genotypes, based
on the different number of Pst I enzyme restriction sites in
ENZYME RECOGNITION SEQUENCE the region flanking the RHD gene (so called Rhesus box).21
AluI AG↓CT
Gel Electrophoresis
AosI TGC↓GCA
The restriction patterns mentioned in the previous section
ApyI CC↓(A,T)GG may be examined by gel electrophoresis (Fig. 4–4), another
technique fundamental for DNA cloning. It allows not only
AsuI G↓GNCC
for visual analysis of DNA fragments’ length, but also for its
AsuII TT↓CGAA isolation and purification for further applications.18
The word electrophoresis means “to carry with electric-
AvrII C↓CTAGG
ity.” Agarose gel electrophoresis is a simple and rapid method
BalI TGG↓CCA of separating DNA fragments. The DNA sample is intro-
duced into a well in a gel immersed in buffer and is exposed
BamHI G↓GATCC
to electric current. The DNA molecules run at different
BclI T↓GATCA speeds according to their size. After staining with a fluores-
cent dye, bands of DNA of specific length can be seen under
BglII A↓GATCT
ultraviolet (UV) light and isolated from the gel. In solution,
BstEII G↓GTNACC at a neutral pH, DNA is negatively charged because of its
phosphate backbone. When placed in an electric field, DNA
BstNI CC↓(A,T)GG
molecules are attracted toward the positive pole (anode) and
BstXI CCANNNNN↓NTGG repelled from the negative pole (cathode).
To make a gel, molten agarose (a seaweed extract) is
ClaI AT↓CGAT
poured into a mold where a plastic comb is suspended. As it
DdeI C↓TNAG cools, the agarose hardens to form a porous gel slab with pre-
formed wells. The gel has a consistency similar to gelatin and
EcoRI G↓AATTC
is placed in a tank full of buffer and with electrodes at
EcoRII ↓CC(A,T)GG opposing ends. The samples containing the DNA fragments
are pipetted into the wells, after mixing them with a loading
Fnu4HI GC↓NGC
solution of high density, such as sucrose or glycerol. The
FnuDII CG↓CG dense solution helps the DNA sink when loaded into the
wells and allows for monitoring of the progress of elec-
HaeI (A,T)GG↓CC(T,A)
trophoresis due to presence of bromophenol blue dye (a
HaeII PuGCGC↓Py tracer). When electric current (80 to 120 V) is applied to
electrodes on the tank, the DNA fragments migrate toward
HaeIII GG↓CC
the anode. The porous gel matrix acts as a sieve through
HhaI GCG↓C which larger molecules move with more difficulty than
smaller ones. The distance run by a DNA fragment is
HincII GTPy↓PuAC
inversely proportional to its molecular weight and therefore
HindII GTPy↓PuAC to its length or number of nucleotides.
DNA bands can be detected in the gel upon staining with
HindIII A↓AGCTT
a fluorescent dye, such as ethidium bromide (EB) or SYBR
HinfI G↓ANTC Green. The gel may be soaked in a diluted fluorescent dye
solution, or, alternatively, the electrophoresis is run with a
HpaI GTT↓AAC
gel and buffer containing the dye. EB is a planar molecule,
HpaII C↓CGG which intercalates between the stacked nucleotides of the
DNA helix. This feature makes it a mutagenic factor, which
MboI ↓GATC
calls for cautious handling of gels, equipment exposed to this
MstI TGC↓GCA agent, and waste.
SYBR Green dye isn’t mutagenic, and many facilities have
NotI GC↓GGCCGC
adopted this dye in their staining procedures to assure labo-
PstI CTGCA↓G ratory safety.22 When the gel is exposed to ultraviolet light
of 300 nm, the stained DNA fragments are seen as fluorescent
RsaI GT↓AC
orange (EB) or green (SYBR) bands and can be documented
SacI GAGCT↓C by photography. Each band of DNA is formed by millions of
DNA molecules of equal length, which is established by com-
SacII CCGC↓GG
parison with standard bands of size marker reagents. Gel
2682_Ch04_077-100 22/05/12 11:40 AM Page 85
Cathode Anode
(–) (+)
D
N
EB
UV A
GEL
Figure 4–4. Agarose gel electrophoresis. L
Negatively charged DNA molecules migrate
E
toward the positive pole. The agarose gel acts as
a molecular sieve: The shorter the DNA mole- N
Direction of DNA Migration G
cule, the longer the distance of migration. DNA
bands are detected by exposure to ultraviolet T
light (UV) after staining with the fluorescent dye H
ethidium bromide (EB) or SYBR Green. DNA Length
electrophoresis, in low melting point agarose, is used in (“sticky”) ends. Otherwise, the procedure is called “blunt
molecular cloning to isolate and purify the vector and insert, end cloning.” The circular DNA of the plasmid is cut (or
which will be used to form a recombinant DNA vector. linearized) with a chosen restriction enzyme. The DNA
fragment to be inserted is cut with the same enzyme. The
Vectors sticky ends of the linearized plasmid can form hydrogen
A vector is a DNA molecule of known nucleotide sequence bonds with the complementary nucleotides in the overhang
that is used to carry a foreign DNA fragment into a host of the DNA fragment to be inserted (hence called an insert).
organism. Vectors can be used to produce large quantities of Both fragments are run in a preparative, low-melting-point
a target DNA fragment. They can also be used to get a foreign agarose gel. The bands corresponding to the expected size
gene expressed in a host cell to study the function of the of the linearized plasmid and insert are cut out, and the
gene or to manufacture large quantities of the encoded purified DNA fragments are isolated. Both fragments are
protein product.18 “pasted” together with DNA ligase, which forms phospho-
diester bonds between adjacent nucleotides (Fig. 4–5).
Plasmids. Plasmids are the simplest kind of vectors. They are Typical plasmid vectors consist of DNA 2 to 4 kb long
bacterial, circular genetic elements that replicate independ- and can accommodate inserts up to 15 kb long. Several
ently from the chromosome. The plasmid vectors used in other vectors are available for cloning fragments of DNA of
molecular cloning are “designer plasmids,” modified to make different sizes.
perfect recombinant DNA carriers. A vast selection of plas-
mids are commercially available that are useful for different Other Vectors. Lambda () vectors contain the part of the bac-
purposes, such as DNA sequencing, protein expression in teriophage’s genome necessary for lytic replication in E. coli
bacteria, and protein expression in mammalian cells. and one or more restriction endonuclease sites for insertion
All plasmids contain an origin of replication. Plasmid of the DNA fragment of interest. They can accommodate
vectors can be under “stringent” control of replication, in foreign DNA 5 to 14 kb long. The recombinant DNA is
which case they replicate only once per cell division. By con- “packaged” into viral particles and used to infect E. coli.
trast, the “relaxed” plasmids replicate autonomously and can
grow as hundreds of copies per cell. Relaxed plasmids are Cosmids are vectors that can accept DNA 28 to 45 kb long.
used to amplify large amounts of cloned DNA. Naturally They are useful for producing large-insert genomic libraries.
occurring plasmids contribute significantly to bacterial Cosmids have a small region of bacteriophage necessary
genetic diversity by encoding functions, such as resistance for packaging viral DNA into particles. The linear genomic
to certain antibiotics, which provides the bacterium with a DNA fragment is inserted into the vector and packaged into
competitive advantage over antibiotic-sensitive strains. Plas- bacteriophage particles. After infecting the E. coli cells, the
mids used as vectors also contain genes that encode for
antibiotic resistance. When bacteria grow in a culture
medium with the given antibiotic, only host cells containing BOX 4–1
the plasmid will survive. Thus, a pure population of bacteria
can be obtained. Examples of antibiotics used for bacterial Antibiotics Used in Bacterial Cloning
cloning are listed in Box 4–1. • Ampicillin
Cloning a DNA fragment requires inserting it into the • Carbenicillin
plasmid vector. Plasmids are designed to contain one or more • Chloramphenicol
cloning sites, also called polylinkers, which are a series of • Hydromycin B
recognition sequences for different restriction endonucleases. • Kanamycin
Most commonly, these are recognition sites for enzymes that • Tetracycline
cut both strands off the center, which generates cohesive
2682_Ch04_077-100 22/05/12 11:40 AM Page 86
Amp
Plasmid diluted enough so that each cell will give rise to an individual
r
Or
i colony. Plates are incubated at 37°C. After 12 to 24 hours
(at the maximum rate, cells divide every 22 minutes), visible
5'AATT 3' 5'AATT 3'
colonies of identical daughter cells appear. Individual
Linearized
Insert colonies are picked and further propagated in liquid culture
3' TTAA5' 3' Plasmid TTAA5'
medium with antibiotic. The incubation proceeds with con-
2 Agarose Gel Electrophoresis tinuous shaking to maintain the cells in suspension and to
(–) promote aeration and production of cell mass that may be used
for efficient isolation of vector DNA containing the insert.
t
Targe T
Gene AATAA Plasmid Isolation
TT
TT ATT
(+)
A
ri
r
1¬¬¬Transform E. coli with recombinant plasmid probe, such as a cDNA or genomic clone or a PCR prod-
Bacterial uct (see the “Detection of Nucleic Acids and Proteins”
Chromosome section).
As seen in the section on vectors, different vectors are
Recombinant useful for isolating DNA fragments of different sizes. For
Plasmid example, the Human Genome Project used BAC vectors to
Am
O
r
Recombinant Proteins Used in Transfusion Medicine An alternative to cloning for isolating large amounts of a
single DNA fragment or gene is the polymerase chain reac-
• Factor VIII tion (PCR), which was developed by Kary Mullis at Cetus
• Factor IX Corporation in 1985.26 As opposed to cloning, which is
• Factor VII performed in a living cell, PCR is carried out completely in
• Factor XIII vitro. Provided that some sequence of the DNA molecule is
• Thrombopoietin known, PCR can be used to amplify a defined segment of
• G-CSF DNA several million times.
• GM-CSF In PCR, DNA (or cDNA) is replicated in the test tube or
• Epoetin α on a microtiter-like plate, replacing the steps that normally
• Interferon-α occur within a cell by adding synthetic or recombinant
reagents and by cyclic changes in the reaction temperature.
G-CSF = granulocyte colony stimulating factor; GM-CSF = granulocyte
macrophage colony stimulating factor The role of the enzyme primase, which in vivo generates the
primers to which DNA polymerase attaches the successive
nucleotides, is replaced by synthetic oligonucleotides called
forward and reverse primers. These primers are usually
Gene Therapy 15 to 20 nucleotides long and flank the target region to be
Gene therapy is the introduction of new genetic material amplified. In other words, the primers are designed to anneal
into the cells of an organism for therapeutic purposes. In to complementary DNA sequences at the 3’ end of each
the United States, only somatic gene therapy is allowed in strand of the DNA fragment (Fig. 4–7).
humans—that is, the treated cells are somatic and not The product obtained (the amplicon) will have the
germline cells. The obvious clinical use of gene therapy is sequence of the template bracketed by the primers. The two
the treatment of inherited diseases. In the first clinical trial primers are mixed with a DNA sample containing the target
of that kind, the gene coding for adenosine deaminase was sequence; thermostable DNA polymerase; the four deoxyri-
introduced into lymphocytes of children suffering from bonucleoside triphosphates (dNTPs); and magnesium,
severe combined immunodeficiency disease.25 However, which acts as enzyme cofactor. Thermus aquaticus, a bac-
several current clinical trials are focused on oncology. terium that grew in hot springs, was the original source of
These treatments seek to enhance the host antitumor heat-resistant polymerase, consequently named Taq.
responses by overexpression of cytokines or by genetic Currently, numerous recombinant enzymes are on the
alteration of the tumor cell. market. The thermostability of the enzyme is of utmost
One approach is the use of viral vectors, such as retro- importance because the first step of the PCR is heat denatu-
viruses, adenoviruses, herpesviruses, and lentiviruses. ration. Applying high temperatures breaks the hydrogen
Most human clinical trials to date have used retroviral bonds between the complementary nucleotides and plays the
vectors. However, the use of these vectors carries serious role of the helicases and gyrases that unwind the DNA so
potential risks that must be weighed against the severity that the DNA polymerase complex can reach the template.
of the underlying illness. The exogenous gene may cause At 94°C, the double helix is denatured into single strands.
disease if overexpressed or expressed in certain cell types. In the second step, annealing of the primers to the target
Contaminants may be introduced during vector manufac- sequences flanking the fragment of interest is achieved by
ture. Also of concern is the potential for recombination cooling to a temperature of 50° to 60°C, which promotes
of gene therapy vectors with human endogenous sequences. renaturation (reestablishment of hydrogen bonds). The exact
The search for better vectors is under way in animal optimum temperature of annealing is determined by the
models and holds promise for a safer and more effective number of G and C residues in the primers: The more GC,
gene therapy. the more hydrogen bonds to break and the higher melting
Region under Figure 4–7. The FORWARD primer always anneals to the minus
Sense (plus) strand investigation strand (also called antisense strand). It is complementary to the 3’ end
Template DNA of the minus strand (and identical to the 5’ end of the plus strand).
5′ 3′ The REVERSE primer always hybridizes with the plus strand (also called
G A A T CG T CG AGC T GC T AGC T T T G T T CG A the sense strand). It is complementary to the 3’ end of the plus strand
G A A A C A A and is identical to the 5’ end of the minus strand. (Modified from Buck-
Forward 3′ 5′
Primer ingham, L, and Flaws, ML: Molecular Diagnostics. Fundamentals,
Primer
Reverse Methods and Clinical Applications. F.A. Davis Company. Philadelphia,
5′ 3′ PA, 2007.)
A T CG T C
C T T AGC AGC T CG A CG A T CG A A A C A AGC T
3′ 5′
Template DNA
Antisense (minus) strand PCR
2682_Ch04_077-100 22/05/12 11:40 AM Page 89
point Tm of the molecule. Typically, the optimum annealing during PCR is not indefinite; therefore, the theoretical
temperature is 5 degrees Celsius below the Tm. number of product molecules is not reached. After a certain
The third step, primer extension by DNA polymerase, is number of cycles, the accumulation of product reaches a
done at 72°C. This is the optimal temperature for the poly- plateau, the level of which depends on the initial number of
merase, which incorporates nucleotides to the 3’ OH end of target sequences, the quantity of reagents, and the efficiency
the primers using the target DNA as template. The cycles of of extension.
DNA synthesis, which consist of denaturation, annealing, Aside from cloning, the PCR has numerous applications
and extension, are repeated simply by repeating the cycle of in immunohematology—for example, various modifications
temperatures. The reaction is carried out in a programmable of this method have been used in the detection of infectious
heating and cooling machine called a thermocycler. The agents in donors’ blood.27,28 (Refer to Chapter 18.)
instrument contains a heating block built from materials
characterized by high thermal conductivity (e.g., silver) that DNA Sequencing
allow for rapid changes of temperatures and shortens time
between the steps of each cycle. Figure 4–8 shows a Individual fragments of DNA can be obtained in sufficient
schematic PCR reaction. In each cycle, an original template amount for determining their nucleotide sequence either
strand is copied to generate a complementary strand, which by molecular cloning or by PCR. Current methods of DNA
begins at the 5’ end of the primer and ends wherever the Taq sequencing are rapid and efficient. The easiest way of deter-
or other thermostable polymerase ceases to function. After mining an amino acid sequence within a protein is the
the second cycle, DNA is synthesized, using the newly sequencing of a cloned gene, as the coding sequence of a
copied strands as templates. In this case, synthesis stops gene can be unequivocally translated into the amino acid
when it reaches the end of the molecule defined by the sequence of its encoded protein.
primer. By the end of the third cycle, a new blunt-ended Modern sequencing is based on the chain termination
double-stranded product is formed, with its 5’ and 3’ ends method developed in 1977 by Fred Sanger at the Medical
precisely coinciding with the primers. Research Council’s Laboratory of Molecular Biology in Cam-
These blunt-ended fragments accumulate exponentially bridge, England.29 Just like the PCR, the Sanger sequencing
during subsequent rounds of amplification, while the origi- method is a controlled in vitro process mimicking DNA syn-
nal DNA that was outside the region flanked by the primers thesis. When a short synthetic primer (complementary to the
isn’t amplified. Thus, the majority of fragments in the later beginning of the fragment to be sequenced) is hybridized to a
PCR cycles have both ends defined. A single DNA molecule single-stranded template in the presence of the four dNTPs,
amplified through 30 cycles of replication would theoreti- DNA polymerase can synthesize a new strand of DNA com-
cally yield 230 (approximately 1 billion) progeny molecules. plementary to the template. The addition of an incoming
In reality, the exponential phase of product accumulation nucleotide occurs via the 3’ hydroxyl group in the deoxyribose
necessary to form the phosphodiester linkage. The principle which is carried out in a thermocycler, contains a DNA
of Sanger method is based on including small amounts of template, a primer, DNA polymerase, the four dNTPs, and
modified dNTPs that are lacking that hydroxyl group into the four ddNTPs, each labeled with a different fluorescent dye.
reaction mix. These modified dNTPs are 2’, 3’-dideoxyribonu- The reaction is heated to 94°C, which causes DNA denatu-
cleoside triphosphates. They are designated by a double d ration to a single strand, followed by cooling to 65°C to allow
(ddNTPs). Upon incorporation into the newly synthesized annealing of the primer and its extension by enzymatic
DNA, they cause an immediate chain termination (hence addition of nucleotides. Several cycles are repeated.
the name of the method), as no new phosphodiester linkage Working from the primer, the polymerase adds dNTPs or
with the following nucleotide is possible. Originally, Sanger ddNTPs that are complementary to the DNA template. The
sequencing required using radioisotopes and laborious casting ratio of dNTPs to ddNTPs is adjusted so that a ddNTP is
of polyacrylamide electrophoretic gels in which the DNA frag- incorporated into the elongating DNA chain, approximately
ments were separated by size in denaturing conditions created once in every 100 nucleotides. Each time a ddNTP is incor-
by the presence of urea. porated, synthesis stops and a DNA fragment of a discrete
Currently, most laboratories are using the automated size, labeled fluorescently according to its last nucleotide, is
fluorescent cycle sequencing method (Fig. 4–9). The reaction, produced. At the end of 20 to 30 cycles of the reaction,
millions of copies of the template DNA sequence are termi-
nated at each nucleotide so a mixture of fragments is
Template Taq Polymerase obtained, each longer by one nucleotide than the previous
5' ACTGCGGCTACGCCCGTATGCG 3' dNTP's one. These fragments are separated by size by capillary gel
Primer (5' ACTGCGG 3') electrophoresis. The fluorescent labels are detected as the
3' TGACGCCGATGCGGGCATACGC 5'
+ ddTTP terminated fragments pass a laser aimed at the capillary.
O
94 C Denature ddATP When the fluorescent terminators are struck by the laser
d dCTP beam, each emits a colored light of a characteristic wave-
5' ACTGCGGCTACGCCCGTATGCG 3'
3' TGACGCCGATGCGGGCATACGC 5' ddGTP length, which is collected by the detector and interpreted by
the computer software as A (green), T (red), C (blue), or G
65OC Anneal and Extend (yellow). The final output is an electropherogram showing
colored peaks corresponding to each nucleotide position.
5' ACTGCGG 3'
3' TGACGCCGATGCGGGCATACGC 5'
Detection of Nucleic Acids
5' ACTGCGG 3' and Proteins
5' 3'
5' 3' The concepts of molecular cloning, creating expression DNA
5' 3' libraries and gene therapy, are fundamental in contemporary
5' 3' molecular research and development setting. Yet cloning may
5' 3' seem esoteric to a clinical laboratorian, presented with the
5' 3'
task of identifying a specific mutation or confirming the
5' 3'
presence of a pathogen. In the following paragraphs, some
5' 3'
5' 3'
classical and newer methods for detecting specific nucleic
5' 3' acids and proteins will be described.
5' 3'
5' 3' Nucleic Acid Hybridization
5' 3'
5' 3' Base pairing between complementary strands of DNA or
RNA allows the specific detection of nucleic acid sequences.
25 Cycles Double-stranded nucleic acids denature at high temperature
(90° to 100°C) and renature when cooled to form double-
C T A C G C C C G T A T G C G
stranded molecules as dictated by complementary base
pairing. This process of renaturation is called nucleic acid
hybridization. Nucleic acid hybrids can be formed between
two strands of DNA, two strands of RNA, or one strand of
RNA and one of DNA. DNA or RNA sequences complemen-
Figure 4–9. DNA sequencing: automated fluorescent cycle method. The double-
stranded DNA template is denatured by heating at 94°C. Cooling to 65°C allows tary to any purified DNA fragment can be detected by
the annealing of the oligonucleotide primer. In the presence of the four dNTPs nucleic acid hybridization. The DNA fragment—for exam-
and the four fluorescently labeled ddNTPs, Taq polymerase incorporates nucleotides ple, a cloned gene or a PCR product—is labeled using
into the growing chain, following the template sequence in a 5’ to 3’ direction. Once radioactive, fluorescent, or chemiluminescent tags. The
in approximately 100 nucleotides, a ddNTP will be incorporated and synthesis will
labeled DNA is used as a probe for hybridization with any
stop. A fragment of DNA is generated that contains a fluorescent tag in its last nu-
cleotide. After capillary gel electrophoresis, the fluorescent labels are detected as DNA or RNA complementary sequence. The resulting hybrid
the terminated fragments pass a laser aimed at the capillary. The final output is an double strand will be labeled and can easily be detected by
electropherogram showing colored peaks corresponding to each nucleotide position. autoradiography or digital imaging.
2682_Ch04_077-100 22/05/12 11:40 AM Page 91
Figure 4–10. Southern blotting. (1) The DNA restriction 1 Agarose gel
digest is submitted to agarose gel electrophoresis. (2) The electrophoresis
DNA fragments are transferred from the gel to a filter mem- Gel
brane by flow of buffer with negative pressure under vac- Cathode 2 Blotting with transfer buffer Filter
uum. (3) The filter is hybridized with a labeled probe, and (–)
the labeled DNA bands are detected by digital imaging. Sealing Mask
Sealing Frame
Porous Support
Vacuum
Filter
2682_Ch04_077-100 22/05/12 11:40 AM Page 92
GenomeLab SNPStream by Beckman Coulter is another concurrent detection of HIV, HCV, and HBV (Roche Cobas
example of microarray technology that was customized TaqScreen MPX).37
for RBC antigen typing at the Genome Quebec Innovation
Center, Canada.33 Transcription Mediated Amplification
Other FDA approved tests for HIV, HCV, HBV, and West Nile
Fluorescent In Situ Hybridization virus in donors’ blood are performed by means of another
In this technique, scientists use fluorescent probes to detect method developed by Gen-Probe: transcription mediated
homologous DNA or RNA sequences in chromosomes or amplification (TMA). The starting material in this assay is
intact cells. In this case, the hybridization of the probe to RNA. The RNA serves as a template for a reverse transcrip-
specific cells or subcellular structures is determined by direct tase to synthesize a complementary DNA, which is bound to
microscopic examination. In 1995, this method was used to the RNA by hydrogen bonds to form a RNA/DNA hybrid.
localize the ABO gene to chromosome 9.34 The DNA is never amplified in the assay. After enzymatic
removal of the RNA from the hybrid, the DNA is transcribed
PCR-Based and Other Amplification Techniques to hundreds of RNA molecules. These multiple RNA mole-
cules go through another reverse transcription process to
Amplification of DNA by PCR allows the detection of even sin- produce more RNA/DNA hybrids, and the whole process is
gle copies of DNA molecules. By contrast, Southern blotting repeated many times. The detection of the pathogen (if its
has a detection limit of around 100,000 copies. The specificity RNA is present in the sample) occurs via hybridization
of the PCR amplification depends on the primers that hybridize protection assay (HPA) technology. In this technology,
to complementary sequences of the template molecule span- ssDNA probes labeled with chemiluminescent molecules are
ning the target DNA fragment. With carefully chosen primers, added to form hybrids with the amplified RNA molecules
PCR can be used to selectively amplify DNA molecules from produced during TMA. Light is emitted upon hybridization
complex mixtures, such as genomic DNA from cell extracts. of the probes to the RNA and captured by a luminometer.
Nucleic acid testing (NAT) is rapidly becoming a stan-
Real-Time PCR
dard method in blood banking. It allows the detection of
In real-time PCR, conducted in a special type of thermocy- pathogens before the appearance of a testable immune
cler (LightCycler), the product formed during each cycle of response, such as screening of antibodies. Narrowing the
amplification is detected by fluorescence at the same time preseroconversion window (during which donors can be
that it is produced. In addition to primers, the real-time re- infected but do not yet test positive by serologic methods)
action mixture contains DNA probes that are complementary helps to enhance the safety of blood products.38,39 Since NAT
to the region between the primers (called TaqMan or molec- implementation, the average seroconversion window for HIV
ular beacons or FRET probes), labeled with fluorophores has been shortened to 11 days (as compared to 22 days when
that emit fluorescent light when binding to the newly using serologic testing and 16 days when performing the
synthesized amplicon. If the assay is used to detect polymor- standard confirmatory p24 antigen testing). The average
phism, a post-PCR monitoring of amplicon/probe separation seroconversion window for HCV has been shortened to 10
upon slow increase of temperature is performed. This process days (as compared to the original 82 days when serologic
is called melting curve analysis and allows for determination testing is used). Consequently, the residual risk of HIV
of homo- or heterozygosity. A classical example of melting transmission decreased from 9.7 to 1.2 incidents per
curve analysis is the detection of factor V Leiden mutation million donations, and the residual risk of HCV transmission
in patients with this inherited coagulopathy.35 decreased from 2 to 1 incident per million donations. A sig-
Real-time PCR allows the quantification of specific mRNAs nificant progress in testing for the transfusion-transmitted
in complex mixtures and is extremely useful in the study of pathogens in donor units was the ability of the NAT assays
gene expression when combined with reverse transcription. to detect these pathogens concurrently. In 2008, the Gen-
Probe assay, initially approved for detection of HIV-1 and
Reverse Transcriptase PCR
HCV only, was also approved for the detection of HBV.36
Single copies of RNA can be detected by adding a step of
cDNA synthesis by reverse transcription (RT), prior to the Antibodies as Probes for Proteins
PCR amplification (RT-PCR). By RT-PCR, cDNA molecules
can be specifically amplified from total RNA obtained from Gene expression at the protein level can be studied by using
cell extracts, tissue sections, or plasma. The high sensitivity labeled antibodies as probes. In particular, monoclonal antibod-
of PCR-based DNA and RNA detection techniques makes ies are widely used in the technique called immunoblotting.40
them valuable for early detection of transfusion-transmitted This technique is also known as Western blotting by analogy
viruses, such as HIV and hepatitis B and C. The first RT-PCR to Southern and Northern blotting. Proteins in cell extracts are
assays developed by National Genetics Institute (UltraQual separated by polyacrylamide gel electrophoresis in the presence
HIV-1) and by Roche (COBAS AmpliScreen HIV-1) were of the ionic detergent SDS. In SDS-PAGE electrophoresis, each
designed to detect these pathogens in donors’ blood protein binds many detergent molecules, which causes its
and were approved by the FDA in 2001 and 2003.28,36 denaturation and provides a negative charge. Proteins will
Real-time RT-PCR is employed in the multiplex assay for migrate to the anode; the rate of migration depends on their
2682_Ch04_077-100 22/05/12 11:40 AM Page 93
size. The proteins are then transferred from the gel onto a filter
membrane. The protein/antigen of interest is detected by incu- DRw52 LRS PROBE
bation of the membrane with a specific labeled antibody. An
example of Western blot is the HIV p24 confirmatory testing.
HLA typing for allogeneic hematopoietic stem cell trans- Overlaps between the fragment sequences were used to
plant (HSCT). assemble the whole genome. This draft encompassed about
90% of the euchromatin portion of the human genome. Con-
DNA Profiling or “Fingerprinting” tinuing work by researchers around the world rendered a
high-quality human genome sequence in 2003—50 years
Minisatellites and microsatellites are regions of DNA inter- after the Nature paper in which Watson and Crick described
spersed in the human genome formed by variable number the structure of DNA. This wealth of information, which
of tandem repeats (VNTRs) of short nucleotide sequences. is constantly updated, can be accessed through the National
The variability depends on the number of repeat units Center for Biotechnology Information (NCBI) website
contained within each “satellite.” Minisatellites or variable (www.ncbi.nlm.nih.gov).42
tandem repeats are composed of repeated units ranging in In its first 50 years, molecular biology has been primarily
size from 9 to 80 bp. Microsatellites or short tandem repeats an analytical science, concerned mainly with reducing
(STRs) contain repeat units of 2 to 5 nucleotides. biological problems to the level of individual genes. This
The polymorphism of these loci in the human population approach has been extraordinarily successful in finding the
is so high that there is a very low probability that two indi- key genes involved in cellular replication and development
viduals will have the same number of repeats. When several and in identifying the genes involved in genetic disorders.
loci are analyzed simultaneously, the probability of finding The challenge for the next few decades is going to be under-
two individuals in the human population with the same standing the synchronized activity of numerous genes during
polymorphic pattern is extremely low. The independence of key biological events, such as development and memory, and
inheritance of the different loci tested is ensured by choosing understanding the multiple genes presumably involved
loci on different chromosomes. The pattern of polymor- in complex diseases such as cancer and diabetes. Another
phism (the differences in number of repeats or length) can complication is the fact that several different proteins can
be determined by RFLP or PCR analysis. In 1997, the Federal be produced from a single gene by alternative RNA splicing
Bureau of Investigation recommended a panel of 13 STRs, and that additional forms are created by post-translational
plus an XY marker, for criminal investigations. With this modifications.
number of independently inherited polymorphisms, the To solve these complex issues, biologists are turning to
probability of even the most common combinations is less more synthetic approaches that allow them to examine the
than 1 in 10 billion. Thus, modern DNA testing can uniquely coordinated expression of multiple genes. Experiments per-
identify each person, hence the name DNA fingerprinting. formed using DNA and protein chips are starting to show
DNA profiling is used in forensic applications, paternity how hundreds or thousands of genes are expressed coordi-
testing, and to follow chimerism after HSCT. nately in response to different developmental and environmen-
tal stimuli. Analysis of these data is one of the applications
Systems Biology of bioinformatics, a discipline that uses computer algorithms
to manage and analyze large-scale experiments.
The molecular techniques developed in recent years and The functions of genes implicated in a specific pathway
older techniques, still valuable in the study of nucleic acids can be further explored by reverse genetics. Genes can be
and the cellular processes they are involved in, keep improv- “knocked out” or “knocked in” as wild type or mutated into
ing and expanding. Many are being automated and minia- the germline of a mouse or other animal model.43,44 The
turized. While it is necessary to have the exact sequence of resulting knockout transgenic animal can be analyzed for
DNA that is of interest (e.g., if it is associated with an inher- metabolic or behavioral changes, which can give clues to the
ited disease), it is equally important to understand how that mechanism of function of the gene under study. A novel way
DNA is expressed inside of a living cell. As technology is of studying specific gene function is RNA interference, by
developed, it will be interesting to see what new methods which small synthetic RNA molecules can be used to silence
become available and occur in common use. Of particular a specific target gene.16 This new biology that is emerging
interest to biotechnology is the development of nanotechnol- is called systems biology, and it requires integration of knowl-
ogy and nanostring technology applications, high throughput edge from experiments done in vitro, in vivo, and in silico
DNA systems, and computational bioinformatics in which (in a computer).
complex models of expression and regulation (such as
proteasomes) are being assembled and analyzed. Red Cell Genotyping
Some of the most exciting recent advances in molecular
biology have been the result of analyzing the complete Molecular RBC antigen typing could be of tremendous
nucleotide sequence of the human genome. A draft sequence advantage in situations where serologic testing is impossi-
of the human genome was published in 2001 by two inde- ble or inconclusive. Currently considered as supplemental
pendent teams of researchers.24,41 The public effort was led rather than routine practice, it has the potential to revolu-
by the International Human Genome Project, which used tionize the way blood cell inventory may be searched for
BAC libraries (briefly described in the “Tools for DNA multiple antigen-negative units. The knowledge of theoretical
Cloning” section). The other group, led by Craig Venter of principles and practical skills to perform red cell genotyp-
Celera Genomics, used a “shotgun” approach in which small ing are becoming indispensable in contemporary blood
fragments of genomic DNA were cloned and sequenced. banking.
2682_Ch04_077-100 22/05/12 11:40 AM Page 95
Genetic Basis of Blood Groups or the extracellular domain of a membrane protein. Other
mechanisms of polymorphism generation are gene deletion,
Early important events in the molecular biology of trans- single nucleotide deletion, insertion, and intergenic recom-
fusion medicine includes the cloning and sequencing bination. Clinically significant blood group systems are
of the gene that codes for the MN polymorphism, GYPA, listed in Table 4–3.
in 1986, followed by the ABO gene in 1990 (review
Table 4–1). Now the genes for all the blood group systems Clinical Applications of Red Cell Genotyping
have been identified and localized in specific chromosomal
regions.23 Also known are the alleles that code for the There are several important applications for red cell geno-
blood group polymorphisms, the structure of the gene typing in use today.50,51 These methods help to confirm sero-
products, and in many cases their function. This knowl- logic testing. In cases where serology is not possible or not
edge has allowed the development of red cell group typing sensitive enough or where discrepancies occur, genotyping
at the DNA level. Molecular genotyping is being increas- provides results that can be used to obtain a blood type on a
ingly used in clinical blood banking, where it does not donor or patient. Applications of genotyping include fetal
replace but complements traditional phenotyping. Molec- DNA typing, blood group typing of donors for alloimmu-
ular biology provides information to help make decisions nized patients, screening blood donors to locate rare blood
in transfusion medicine, such as obtaining compatible group phenotypes, screening blood inventory for antigen-
blood in cases of massive transfusion or transplantation. negative units, determining the frequency of blood group
The field of molecular biology is constantly changing and polymorphisms in a given population, determining zygosity
improving. Methods become more efficient and less expensive. for the fathers of fetuses at risk for hemolytic disease of
As greater numbers of populations are analyzed, different the fetus and newborn (HDFN), and blood group typing of
alleles are discovered and added to databases that are patients with autoimmune hemolytic anemia and other
updated frequently. diseases.
A committee of the International Society of Blood Trans-
Fetal DNA Typing
fusion (ISBT) is responsible for the nomenclature of red
cell surface antigens. A monograph published in 2004 Initially, ABO and Rh typing should be done on a pregnant
described the ISBT terminology for red cell surface antigens mother and should include an antibody screening. If
and genes and tabulated a complete classification.45 Since the woman is D negative and has a negative antibody
then, two updates have been published.46,47 The full current screen using IgG AHG techniques, she is a candidate for
classification can be found at the ISBT/IBGRL website RhIG. If the antibody screening is positive, then the
(http://ibgrl.blood.co.uk/).48 antibody specificity must be determined. If the father
Most of the blood group systems are coded by variants has the corresponding antigen, then his zygosity, either
of a single gene. There are four exceptions: Rh; Xg; heterozygous or homozygous, should be established. If
Chido/Rodgers, which have two genes each; and MNS, the father is heterozygous, then the genotype of the fetus
which has three. As an example, the Rh system consists of can be determined using PCR testing of amniocentesis
two genes: D and CE. These genes have 97% homology, samples, chorionic villus sampling, or cordocentesis
which can add difficulty in molecular methods for genotyp- samples.
ing this system. Table 4–3 enumerates the blood group PCR has a very high sensitivity, approaching 100%, and a
systems, their genes with the chromosomal location, and the low false-negative rate, so it is an excellent method. DNA
corresponding antigens. probes are known for most if not all genes playing a role in
HDFN. As an alternate approach, the typing for the D anti-
Molecular Basis of Blood Group Polymorphism gen at the molecular level can be done on maternal samples
as early as the second trimester. Fetal DNA in maternal
According to the Blood Group Antigen Gene Mutation Data samples is derived from apoptotic syncytiotrophoblasts; it
base (dbRBC) as of July 7, 2010, there were 30 blood group increases in concentration throughout pregnancy and is
systems, 39 genes, and 1,165 alleles known in the human cleared soon after delivery.
population. The dbRBC contains a complete collection of Maternal-only, sample DNA testing will likely become
genes and alleles and provides comprehensive information more routine in the near future, as it has many advan-
on blood groups antigens.49 It is frequently updated and pro- tages. All DNA-based information allows earlier interven-
vides DNA sequences and alignments. tion in cases where HDFN is suspected.52 (Refer to
The systems ABO, P, Lewis, H, I, and MNS comprise Chapter 19, “Hemolytic Disease of the Fetus and Newborn
antigens that are carbohydrates on glycoproteins or glyco- [HDFN].”) In addition to HDFN, there is risk to the
lipids. The genes that constitute these systems code for developing fetus in cases of platelet and neutrophil anti-
glycosyltransferases that catalyze oligosaccharide chain gen incompatibility, resulting in fetal and neonatal
synthesis. For the rest of the blood group systems, the anti- alloimmune thrombocytopenia (FNAIT) and neonatal
gens are direct consequences of amino acid variation in the alloimmune neutropenia (NAN). Genotyping can help
protein sequence. Most blood group variants are the result resolve these cases and determine if there is an associated
of one or more single nucleotide polymorphisms (SNPs) risk due to maternal platelet and neutrophil antigens and
encoding amino acid substitutions in a glycosyltransferase HLA type.
2682_Ch04_077-100 22/05/12 11:40 AM Page 96
Table 4–3 Blood Group Systems with Number of Antigens, Coding Gene(s), and Chromosomal
Location
ISBT SYSTEM ISBT SYSTEM GENE CHROMOSOMAL NO. OF
NO. NAME SYMBOL NAME(S) LOCATION ALLELES
003 P P1 P1 22q11.2–qter 1
019 Kx XK XK Xp21.1 1
020 Gerbich GE GYPC 2q14.3 8
Extensive Blood Group Typing of Donors Determining the Frequency of Blood Group
for Alloimmunized Patients Polymorphisms in a Population
Patients with multiple antibodies may require extensive test- Genotyping databases can be used to predict the possibility
ing to find compatible blood for transfusion. It is now possible of finding a specific allele in a given population once a
to genotype potential donors and the patient to prevent further similar population has been analyzed at the genotype level.
immunization and to characterize the donor at a more specific Because most blood group alleles are the result of a single
level.53 DNA testing can be done in place of extensive family nucleotide polymorphism, screening of blood samples for
studies to determine the genotype. Sequencing of the entire allele frequency can be relatively easy and done by simple
gene of interest, often required to obtain sufficient informa- DNA-based methods. Several different methods employed
tion, may be labor-intensive. The amount of work may be less- for this include RFLP and SSP-PCR (single or multiplex),
ened if exon sequencing is done first and if RFLPs can be done real-time quantitative PCR, sequencing and microarray tech-
on certain areas of the gene at the beginning of the study. nology using DNA probes on chips.
Focused sequencing with comparisons to antigenic profiles
can also be used. Having extensive information on the Blood Group Typing of Patients With Autoimmune
patients’ and the potential donors’ genotypes allows transfu- Hemolytic Anemia and Other Diseases
sion to take place in a safer environment. Knowing the anti- Patients with autoimmune hemolytic anemia and other con-
gens that the patient does not have based on genotyping ditions such as sickle cell anemia can have trouble obtaining
prevents further alloantibodies from being made from the compatible donor units. They are often sensitized to many
transfusion of blood that is positive for these antigens. Because antigens and have a rapid turnover of red cells, requiring
red cells have lost their nuclei, white cells can be used for frequent transfusion. Because they can have a complex pop-
genotyping and other normal adult diploid cells. ulation of red cells, with autologous cells mixed with trans-
fused cells from more than one donor, genotyping methods
Screening of Blood Donors to Find Rare are an excellent way to type these donors. A small amount
Blood Group Phenotypes of patient white cells or cheek cells can be used to determine
Using DNA-based methods to screen blood donors allows for the genotype using standard DNA-based methods, and the
a high-throughput system screening in comparison with serol- subsequent patient phenotype can be determined from the
ogy-based methods. Blood group antisera often are not readily genotype.
available for rare blood group phenotypes or are prohibitively Although crossmatching may not provide totally com-
expensive to use on a large scale. Genotyping with PCR tech- patible results, especially with autoimmune diseases, at
niques allows for the probes (or primers) to be synthesized at least alloantigens can be ruled out when transfusion is
low cost and used in DNA-based methods, as long as informa- required, thereby preventing further alloimmunization in
tion about the sequence of the gene is known. Different alleles an already-difficult-to-transfuse patient. In addition to
can be tested at the same time in the multiplex PCR assays or saving the time required for repeated adsorptions, elu-
on microarrays. Once known, the genotype information can tions, and extensive phenotyping in these patients and in
be put into a database and does not require repeated testing, donor units, the information obtained by DNA-based
which saves considerable time and money. A rare donor profile methods is highly accurate and easy to obtain. It is a
can be generated, linking potential donors to potential recipi- permanent record of the patient’s genetic profile, and it is
ents for better recruitment in cases of emergency. For patients necessary information in cases where transplant may be
requiring donor units with rare phenotypes, the costs and time considered. It is also a permanent record of the donor’s
involved in genotyping such donor units are acceptable. profile when performed.
SUMMARY CHART
The central dogma of molecular biology: DNA → RNA The structure of DNA determines its function. The
→ protein. sequence of nucleotides of one strand determines the
Proteins have structural, enzymatic, and gene-regulatory sequence of its complementary strand, the basis for
function. Through these mechanisms, the genotype of the semiconservative way of replication.
a cell is translated into its phenotype. A gene is transcribed into precursor RNA, and the
DNA is the genetic material. spliced mRNA is translated into the amino acid
DNA is a double helix, consisting of two antiparallel sequence of the coded protein. The sequence of
strands of stacked nucleotides paired through hydro- mRNA unequivocally determines the sequence of the
gen bonds. Adenine (A) always pairs with thymine (T), protein.
and cytosine (C) always pairs with guanine (G).
Continued
2682_Ch04_077-100 22/05/12 11:40 AM Page 98
SUMMARY CHART—cont’d
Recombinant DNA is the DNA of one organism “cut Genomic and cDNA libraries are collections of clones
and pasted” into a carrier vector. The foreign gene containing the genetic material of a cell.
introduced in a host organism is functional because the Base pairing between complementary strands of DNA or
genetic code is universal. RNA (hybridization with a labeled probe) is used to de-
By DNA cloning, recombinant genes of complex animals, tect specific nucleic acid sequences in complex mixtures.
such as humans, are introduced into simple organisms, Southern blotting, Northern blotting, and dot blotting
such as bacteria, and other model organisms, such as are hybridization-based techniques for nucleic acid
mice, allowing structural and functional studies. sequence-specific recognition.
Restriction endonucleases, bacterial enzymes that rec- The polymerase chain reaction (PCR) is an in vitro
ognize and cut specific DNA sequences, are fundamen- method for DNA amplification.
tal tools for DNA cloning. Molecular polymorphism is studied at the genetic
Gel electrophoresis separates nucleic acids by size. level by DNA typing. Methods for DNA typing
The most common host cell is the bacterium E. coli. relevant for transfusion medicine are restriction
Plasmids, the most commonly used vectors, are inde- fragment length polymorphism, allele-specific oligonu-
pendently replicating circular DNA molecules modified cleotide probe hybridization, allele-specific PCR
to provide the host cell with resistance to antibiotics amplification, DNA sequencing, and DNA profiling
(selectable marker) and one or more restriction enzyme (DNA fingerprinting).
sites for inserting the recombinant gene. PCR and TMA are used for the early detection of
By reverse transcription, mRNA is transcribed into transfusion-transmitted pathogens.
complementary DNA (cDNA). Other therapeutic uses of molecular biology are gene
Automated fluorescent DNA sequencing based on the therapy and the clinical use of recombinant proteins,
Sanger dideoxy, chain termination method is a stan- such as interferons, coagulation factors, and growth
dard laboratory procedure. factors.
DNA sequencing of a cloned cDNA corresponding to Molecular RBC antigen typing is used to either confirm
a given gene is the easiest way to determine the amino serologic testing or in cases where serology is not possible
acid sequence of a protein. or is not sensitive enough or where discrepancies occur.
9. RFLP and SSP are techniques used for: 12. Brenner, S, Jacob, F, and Meselson, M: An unstable intermediate
carrying information from genes to ribosomes for protein
a. Protein isolation synthesis. Nature 190:576–581, 1961.
b. RNA isolation 13. Nirenberg, M, and Leder, P: RNA codewords and protein
c. DNA typing synthesis. Science 145:1399–1407, 1964.
d. Protein typing 14. Temin, HM, and Mizutani, S: RNA-dependent DNA polymerase
in virions of Rous sarcoma virus. Nature 226:1211–1213, 1970.
10. Recombinant DNA techniques: 15. Baltimore, D: RNA-dependent DNA polymerase in virions of
a. Are not used in a clinical setting RNA tumour viruses. Nature 226:1209–1211, 1970.
16. Sharp, PA: RNA interference—2001. Genes Dev 15:485–490,
b. Are useful research tools 2001.
c. Are not used in blood banking 17. Yamamoto, F, et al: Cloning and characterization of DNA
d. Are useful only for research complementary to human Histo-blood group A transferase
mRNA. J Biol Chem 265:1146–1151, 1990.
11. Transcription mediated amplification: 18. Sambrook, J, and Russell, DW: Molecular Cloning: A Labora-
a. Requires thermostable DNA polymerase tory Manual, 3rd ed. Cold Spring Harbor Laboratory Press,
b. Is an isothermal procedure Cold Spring Harbor, NY, 2001.
19. Alberts, B, et al: Molecular biology of the cell, 4th ed. Garland
c. Is an obsolete method currently replaced by SSOP Publishing, New York, 2002.
d. Utilizes probes labeled with fluorescent tags 20. Nathans, D, and Smith, HO: Restriction endonucleases in the
analysis and restructuring of DNA molecules. Ann Rev
12. Preseroconversion window: Biochem 44:273–293, 1975.
a. Is the time when donors can be infected but do not 21. Wagner FF, and Flegel, WA: RHD gene deletion occurred in the
yet test positive by serologic methods Rhesus box. Blood 95:3662–3668, 2000.
b. May be narrowed by using molecular methods 22. Kirsanov, KI, Lesovaya, EA, Yakubovskaya, MG, and Belitsky,
GA: SYBR Gold and SYBR Green II are not mutagenic in the
c. Refers mainly to viral pathogens
Ames test. Mutat Res 699(1-2):1–4, 2010.
d. All of the above 23. Logdberg, L, Reid, M, and Zelinski, T: Human blood group
genes 2010: Chromosomal locations and cloning strategies
13. Red blood cell molecular antigen typing is useful in all revisited. Transfus Med Rev 25(1):36–46, 2011.
listed situations except: 24. International Human Genome Sequencing Consortium: Initial
a. In screening RBC inventory for antigen-negative units sequencing and analysis of the human genome. Nature
b. When reagent antibodies are weak or unavailable 409:860–921, 2001.
25. Gaspar, HB: Bone marrow transplantation and alternatives for
c. In quantitative gene expression analysis
adenosine deaminase deficiency. Immunol Allergy Clin North
d. When resolving ABO discrepancies Am 30(2):221–236, 2010.
26. Saiki, RK, Gelfand, DH, Stoffel, S, Scharf R, Higuchi, R, Horn,
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with a thermostable DNA polymerase. Science 239:487–491,
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oxyribonucleic acid fraction isolated from Pneumococcus type infectious disease testing using molecular methods. Advance
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171:737–738, 1953. 32. Hashmi, G, et al: A flexible array format for large-scale,
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disease. Science 110(2865):543–548, 1949. 34. Bennett, EP, et al: Genomic cloning of the human histo-blood
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Nature 180:326–328, 1957. 35. Lyondagger, E, Millsondagger, A, Phan, T, and Wittwer, CT:
11. Keller, EB, Zamecnik, PC, and Loftfield, RB: The role of micro- Detection and Identification of base alterations within the
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36. U.S. Food and Drug Administration: Complete list of donor 46. Daniels, GL, et al. International Society of Blood Transfusion
screening assays for infectious agents and HIV diagnostic Committee on terminology for red cell surface antigens: Cape
assays. Available at www.fda.gov/cber/products/testkits.htm. Town report. Vox Sanguinis 92:250–253, 2007.
37. Wiedmann, M, Kluwick, S, Walter, M, Fauchald, G, Howe, J, 47. Daniels GL, et al. International Society of Blood Transfusion
Bronold, M, et al: HIV-1, HCV and HBV seronegative window Committee on terminology for red blood cell surface antigens:
reduction by the new Roche cobas TaqScreen MPX test in Macao report. Vox Sanguinis 96:153–156, 2009.
seroconverting donors. J Clin Virol 39(4):282–287, 2007. 48. ISBT/IBGRL website: http://ibgrl.blood.co.uk/.
38. Glynn, SA, Kleinman, SH, Wright, DJ, and Busch, MP: Inter- 49. Blumenfeld, OO, and Patnaik, SK. Allelic genes of blood group
national application of the incidence rate/window period antigens: A source of human mutations and cSNPs documented
model. Transfusion 42:966–972, 2002. in the Blood Group Antigen Gene Mutation Database. Hum
39. Dodd, RY, Notari, EP, Stramer, S: Current prevalence and inci- Mutat 23(1):8–16, 2004.
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Transfusion 42:975–979, 2002. 51. Westhoff, CM: The potential of blood group genotyping
40. Kohler, G, and Milstein, C: Continuous cultures of fused cells for transfusion medicine practice. Immunohematology 24(4):
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495–497, 1975. 52. Finning, K, Martin, P, Summers, J, and Daniels, G: Fetal
41. Venter, JC, et al: The sequence of the human genome. Science genotyping for the K (Kell) and Rh C, c, and E blood groups
291:1304–1351, 2001. on cell-free fetal DNA in maternal plasma. Transfusion
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44. Bronson, SK, and Smithies, O: Altering mice by homologous Historical landmarks in the field of study of blood group
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Chapter 5
The Antiglobulin Test
Ralph E. B. Green, BAppSc, FAIMLS, MACE and Dwane A. Klostermann,
MSTM, MT(ASCP)SBB
OBJECTIVES
1. State the principle of the antiglobulin test.
2. Differentiate monoclonal from polyclonal and monospecific from polyspecific antihuman globulin (AHG) reagents.
3. Describe the preparation of monoclonal and polyclonal AHG reagents.
4. List the antibody requirements for AHG reagents.
5. Explain the use of polyspecific versus monospecific AHG in the indirect antiglobulin test (IAT).
6. List the advantages and disadvantages of anticomplement activity in polyspecific AHG.
7. Compare and contrast the IAT and the direct antiglobulin test (DAT).
8. Explain the principle and applications of red blood cell sensitization.
9. Explain the reasons for the procedural steps in the DAT and IAT.
10. Interpret the results of a DAT and IAT panel.
11. Identify the factors that affect the antiglobulin test.
Continued
101
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OBJECTIVES—cont’d
12. Formulate the steps necessary to resolve errors associated with the performance of the antiglobulin test.
13. Describe the multiple methodologies utilized in AHG testing.
14. State the advantages and disadvantages of various methodologies utilized for DAT and IAT testing based on the needs of the
transfusion service department.
Introduction to blood group serology, the principle of the test had in fact
been described by Moreschi4 in 1908. Moreschi’s studies
The antihuman globulin test, which is also referred to as the used rabbit antigoat serum to agglutinate rabbit RBCs that
Coombs’ test, is based on the principle that antihuman glob- were sensitized with low nonagglutinating doses of goat
ulins (AHGs) obtained from immunized nonhuman species antirabbit RBC serum.
bind to human globulins such as IgG or complement, either Coombs’ procedure involved the injection of human
free in serum or attached to antigens on red blood cells serum into rabbits to produce antihuman serum (described
(RBCs). The antihuman globulin test (AGT) is an essential in detail later in this chapter). After absorption to remove
testing methodology when it comes to transfusion medicine; heterospecific antibodies and after dilution to avoid prozone,
without its use, patients’ well-being would be negatively the AHG serum still retained sufficient antibody activity to
impacted. permit cross-linking of adjacent RBCs sensitized with IgG
There are two major types of blood group antibodies: IgM antibodies. The cross-linking of sensitized RBCs by AHG
and IgG. Because of their large pentamer structure, IgM produced hemagglutination, indicating that the RBCs had
antibodies bind to corresponding antigen and directly agglu- been sensitized by an antibody that had reacted with an
tinate RBCs suspended in saline. Some IgG antibodies are antigen present on the cell surface.
termed nonagglutinating, or incomplete antibodies, because Early AHG reagents were prepared using a crude globulin
their monomer structure is too small to directly agglutinate fraction as the immunogen. In 1947, Coombs and Mourant
sensitized RBCs (refer back to the ionic cloud concept demonstrated that the antibody activity that detected Rh
schematic found in Chapter 3, “Fundamentals of Immunol- antibodies was associated with the anti–gamma globulin
ogy”). Adding AHG that contains anti-IgG to RBCs sensi- fraction in the reagent. In 1951, Dacie5 presented the first
tized with IgG antibodies allows for hemagglutination of indication that there might be another antibody activity pres-
these sensitized cells. We use an anti-antibody to observe the ent that influenced the final reaction. He observed that
formation of an Ag/Ab complex that would otherwise go different reaction patterns were obtained when dilutions of
undetected. Some blood group antibodies have the ability to AHG were used to test cells sensitized with warm as com-
bind complement to the RBC membrane. In such cases, an pared with cold antibodies. In 1957, Dacie and coworkers6
anticomplement component can be added to the AHG published data showing that the reactivity of AHG to cells
reagent, rendering it polyspecific. Antiglobulin tests detect sensitized with warm antibodies resulted from anti–gamma
IgG or complement-sensitized RBCs. globulin activity, whereas anti–nongamma globulin activity
was responsible for the activity of cells sensitized by cold
History of the Antiglobulin Test antibodies. The nongamma globulin component was shown
to be beta globulin and had specificity for complement. Later
Before the antiglobulin test was developed, only IgM anti- studies7,8 revealed that the complement activity was a result
bodies were detected. The introduction of the AGT permitted of C3 and C4.
the detection of nonagglutinating IgG antibodies and led to The antiglobulin test can be used to detect RBCs sensi-
the discovery and characterization of many new blood group tized with IgG alloantibodies, IgG autoantibodies, and com-
systems. plement components. Sensitization can occur either in vivo
In 1945, Coombs and associates1 described the use of the or in vitro. The use of AHG to detect in vitro sensitization
antiglobulin test for the detection of weak and nonaggluti- of RBCs is a two-stage technique referred to as the indirect
nating Rh antibodies in serum. In 1946, Coombs and antiglobulin test (IAT). In vivo sensitization is detected by
coworkers2 described the use of AHG to detect in vivo a one-stage procedure called the direct antiglobulin test
sensitization of the RBCs (later called the direct antiglobulin (DAT). The IAT and DAT still remain the most common
test [DAT]) of babies suffering from hemolytic disease of the procedures performed in blood group serology.9
newborn (HDN). Although the test was initially of great
value in the investigation of Rh HDN, its versatility for Antihuman Globulin Reagents
detecting other IgG blood group antibodies soon became
evident. Several AHG reagents have been defined by the Food and
The first of the Kell blood group system antibodies3 and Drug Administration (FDA) Center for Biologics Evaluation
the associated antigen were reported only weeks after and Research (CBER). These are listed in Table 5–1 and are
Coombs had described the test. Although Coombs and asso- described in the following paragraphs. Antihuman globulin
ciates1 were instrumental in introducing the antiglobulin test reagents can be polyspecific or monospecific.
2682_Ch05_101-118 22/05/12 11:41 AM Page 103
1. Rabbit polyclonal Contains anti-lgG and anti-C3d (may contain other anticomplement and other
anti-immunoglobulin antibodies)
2. Rabbit/murine monoclonal blend Contains a blend of rabbit polyclonal antihuman IgG and murine monoclonal anti-C3b
and anti-C3d.
Monospecific Anti-IgG
1. Rabbit polyclonal Contains anti-IgG with no anticomplement activity (not necessarily gamma-chain specific)
2. IgG heavy-chain specific Contains only antibodies reactive against human gamma chains
Anticomplement
Rabbit polyclonal
1. Anti-C3d and anti-C3b Contains only antibodies reactive against the designated complement
Data from Tyler, V (ed): Technical Manual, 12th ed. American Association of Blood Banks, Bethesda, MD, 1996.
Figure 5–2. Preparation of monoclonal AHG reagents. The production Hybridoma Technology
of antibody is longer lasting than the polyclonal source, as the hybridoma Monoclonal Antihuman Globulin
can live indefinitely. A monoclonal blend may be manufactured by blending
monoclonal anti-C3b, monoclonal anti-C3d, and monoclonal anti-IgG. Mono- Complement IgG
specific antihuman globulin reagents can be manufactured by conventional
or hybridoma technology. Mouse is injected with
purified human antigen
Antibody-producing
lymphocytes collected
from spleen
Myeloma Myeloma
cell cell
Fusion with
myeloma cell to
form hybridoma
Clones grown in
Monoclonal tissue culture to Monoclonal
Anti-C3b,d,g yield monoclonal Anti-IgG
antibodies
AHG Polyspecific
Monoclonal Blend
2682_Ch05_101-118 22/05/12 11:41 AM Page 106
Therefore, anti-IgG activity must be present in the AHG longer than 15 minutes, the number of C3c determinants falls
reagent. Anti-IgM and anti-IgA activity may be present, but rapidly because C3c is split off the C3bi molecule. This finding
neither is essential. The presence of anti–light chain activity further supports the use of anti-C3d in international reference
allows detection of all immunoglobulin classes. reagents by the Joint Working Party.
The final degradation step has been shown to occur in
Anticomplement vivo27 and is a common occurrence in both warm and cold
autoimmune hemolytic anemias. Engelfriet and others28 have
Some antibodies “fix” complement components to the RBC also shown that degradation of C3b to C3d can occur in vitro,
membrane after complexing of the antibody with its corre- providing that the incubation period is greater than 1 hour. In
sponding antigen. These antibodies are listed in Table 5–2 1976, Garratty and Petz29 confirmed the need for anti-C3d
and are described in more detail in Chapters 6 through 9. activity in AHG for use in the DAT. They also confirmed Engel-
The terms most, some, and rare in the table refer to antibodies friet’s observation that, given sufficient time, cell-bound C3b
that bind complement the vast majority of the time, that could be degraded to C3d in vitro. Refer back to Table 5–1 for
show variability in their ability to bind complement, and a detailed listing of all AHG reagents’ components.
that rarely bind complement, respectively. These membrane-
bound complement components can be detected by the Use of Polyspecific Versus
anticomplement activity in AHG. Monospecific AHG in the IAT
As a result of studies published during the 1960s19–22 indi-
cating the need for anticomplement activity in AHG to allow As previously stated, polyspecific AHG contains both anti-
the IAT to detect antibodies, polyspecific reagent was intro- IgG activity and anti-C3 activity. There is considerable debate
duced. Evidence was also presented showing that the presence among blood bankers over the use of monospecific anti-IgG
of anticomplement activity would enhance the reactions of versus polyspecific AHG for routine antibody detection and
clinically significant antibodies (e.g., anti-Fya and anti-K).19 pretransfusion testing. Because most clinically significant
During complement activation, C3 and C4 are split into antibodies detected during antibody screening are IgG, the
two components. C3b and C4b bind to the RBC membrane, most important function of polyspecific AHG is the detection
whereas C3a and C4a pass into the fluid phase. Further degra- of IgG antibodies.
dation of membrane-bound C3b and C4b occurs by removing There have been numerous reports of clinically significant
C3c and C4c to leave C3d and C4d firmly attached to the RBC RBC alloantibodies that were undetectable with monospecific
membrane.23–25 The ISBT/ICSH Joint Working Party26 consid- anti-IgG but were detected with the anticomplement compo-
ered anti-C3c to be the most important anticomplement com- nent of AHG.30 Unfortunately, polyspecific AHG has also been
ponent because of its limited capacity to cause nonspecific associated with unwanted positive reactions that are not caused
reactions. However, when RBCs are incubated with serum for by clinically significant antibodies. To investigate these vari-
ables, Petz and coworkers31 examined 39,436 sera comparing
monospecific anti-IgG with polyspecific AHG. They also com-
Table 5–2 Antibodies Capable of Binding pared the albumin technique with low ionic strength solutions
Complement (LISS)-suspended RBCs. Four Jka antibodies were detected only
with polyspecific anti-IgG using albumin or LISS-suspended
MOST SOME RARE RBCs. An additional anti-Jka was detected only with polyspecific
ABO Xga D AHG when using LISS but not with albumin. Also, five anti-
bodies of anti-Kell, anti-Jka, and Fya specificities were detected
Lea LKE P1 when using LISS, but not albumin, with both polyspecific AHG
Leb Lan Lua and anti-IgG. Their results concluded that some clinically
significant antibodies are detected with the anticomplement
Jka Lub component of AHG but not with anti-IgG. This is especially
Jkb Kell true for anti-Jka, a complement-binding IgG antibody often
associated with delayed hemolytic transfusion reactions.
Sc1 Fya Petz and others31 also determined the number of false-
Co3 Fyb positive reactions obtained when using polyspecific AHG
versus anti-IgG with LISS and albumin. False-positive reac-
Ge2 Coa tions were defined as those caused by antibodies with no
Ge3 Cob definable specificity or by antibodies considered to be clini-
cally insignificant because of optimum reactivity at cold
Ii Dia temperatures (anti-I, anti-H, anti-P1, anti-M). Of the unwanted
P S positive reactions, 93% were caused by C3 on the cells. The
authors emphasize that, if the first step in evaluating
PP1Pk S a weakly positive AHG reaction is to repeat using the
Vel Yta prewarmed technique, although not recommended, about
60% of the false-positive weak reactions become negative.
2682_Ch05_101-118 22/05/12 11:41 AM Page 107
In a 3-year study, Howard and associates32 found eight Principle and Application
patients whose antibodies were detected primarily or
solely by AHG containing anticomplement activity. Seven The direct antiglobulin test (DAT) detects in vivo sensitiza-
of these antibodies had anti-Jka or anti-Jkb specificity. tion of RBCs with IgG or complement components. Clinical
Some of them could be detected using homozygous Jka or conditions that can result in in vivo coating of RBCs with
Jkb cells and an AHG containing only anti-IgG activity. antibody or complement are:
Two of the anti-Jka antibodies were associated with delayed • Hemolytic disease of the newborn (HDN)
hemolytic transfusion reactions. The complement-only • Hemolytic transfusion reaction (HTR)
Kidd antibodies represented 23% of all Kidd antibodies • Autoimmune and drug-induced hemolytic anemia (AIHA)
detected during the study. The authors concluded that
they would continue to use polyspecific AHG reagent for Table 5–3 lists the clinical application and in vivo sensi-
routine compatibility testing. tization detected for each situation.
In summary, one must balance the advantage of detecting The DAT is not a required test in routine pretransfusion
clinically significant complement-only antibodies with the protocols. Eder34 tested the clinical utility of the DAT at a
disadvantages resulting from using antiglobulin serum con- large tertiary care hospital in Philadelphia. A retrospective
taining anticomplement activity.30 Deciding to use the AHG study was performed from 1999 to 2002. DATs with anti-IgG
reagent for indirect tests is the prerogative of the individual were performed on 15,662 pretransfusion patient samples;
blood bank. Many blood banks have begun using monospe- 15% were positive. Subsequent eluate testing revealed 76%
cific anti-IgG for routine pretransfusion testing, citing cost were nonreactive, 9% were pan reactive, and 12% passively
containment measures necessitated by the high number of acquired ABO or D antibodies. Only one case demonstrated
repeats versus the rarity of complement-only detected an RBC antibody in the eluate that was not detected in the
antibodies such as anti-Jka. Milam33 states that rare clinical serum, concluding that even in a tertiary care setting, the
transfusion intolerance, when using monospecific anti-IgG routine DAT is inefficient, yielding a positive predictive value
over polyspecific AHG reagents to screen for unexpected of 0.16%. Judd and coworkers revealed similar findings on
antibodies and to test for blood group compatibility, offers 65,049 blood samples in a 29-month period, where only
reliability without interference from common and clinically 5.5% of samples resulted in a positive DAT.35
insignificant IgM-complement fixing antibodies.
DAT Panel
Principles of the Antiglobulin Test
Initial DATs include testing one drop of a 3% to 5% suspen-
The antiglobulin test is based on the following simple sion of washed RBCs with polyspecific (anti-IgG, anti-C3d)
principles:10 reagent (please refer to the DAT procedure on DavisPlus
website). Positive results are monitored by a DAT panel using
• Antibody molecules and complement components are monospecific anti-IgG and anti-C3d to determine the spe-
globulins. cific type of protein sensitizing the cell. In an effort to save
• Injecting an animal with human globulin stimulates the valuable tech time, some institutions run polyspecific and
animal to produce antibody to the foreign protein (i.e., monospecific reagents at one time as well as a saline control.
AHG). Serologic tests employ a variety of AHG reagents The saline control serves to detect spontaneous agglutination
reactive with various human globulins, including anti-IgG of cells or reactions occurring without the addition of AHG
antibody to the C3d component of human complement, reagents. In warm AIHA, including drug-induced hemolytic
and polyspecific reagents that contain both anti-IgG and anemia, the RBCs may be coated with IgG or C3d, or both.
anti-C3d activity. Patterns of reactivity and the type of protein sensitization in
AHG reacts with human globulin molecules, either bound AIHA are summarized in Table 5–4.
to RBCs or free in serum. Washed RBCs coated with human In a transfusion reaction workup, the DAT may demon-
globulin are agglutinated by AHG. strate IgG or C3d, or both, depending on the nature and
specificity of the recipient’s antibody. In the investigation
The complete procedures for the direct and indirect of HDN, testing for complement proteins is not necessary
antihuman globulin tests can be found in Procedures inasmuch as the protein sensitizing the newborn RBCs is
5-1 and 5-2 on the textbook’s companion website.
Figure 5–3 illustrates in vitro sensitization detected in the Table 5–3 Direct Antiglobulin Test
IAT and in vivo sensitization detected by the DAT.
CLINICAL IN VIVO
APPLICATION SENSITIZATION
Direct Antiglobulin Test
HDN Maternal antibody coating fetal RBCs
Described above are the overall principles of the antiglobulin
test. Figure 5–3 depicts two variations of the AGT, the DAT HTR Recipient antibody coating donor RBCs
and the IAT. The following section details the DAT’s principle, AIHA Autoantibody coating individual’s RBCs
application, construct, and evaluation.
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Figure 5–3. The antihuman globulin (AHG) test. (A) Indirect AHG (IAT), with sensitization occurring in vitro, looking for unknown antibody in the patient’s serum/plasma.
(Note: If the IAT was identifying RBC antigens, the RBCs would be from the patient and the antibody would be the known reagent). (B) Direct AHG (DAT) with sensitization
already occurring in vivo. (Note: The polyspecific AHG reagent is a mixture of anti-IgG and anti-C3d.)
presumed to be maternal IgG. Problems can arise in accurate Table 5–4 DAT Panel: Patterns of Reactivity
D typing in the case of a newborn with a positive DAT. If in Autoimmune Hemolytic
the DAT is positive due to IgG and the immediate spin for
Anemia*
D typing is negative, a test for weak D cannot be performed.
The same is true for a patient with AIHA due to a warm IgG ANTI-IgG ANTI-C3d TYPE OF AIHA
antibody coating the patient cells. The antibody must be
+ + WAIHA
removed from the RBCs for accurate phenotyping. Other
techniques can be used to remove antibody from the patient’s + — WAIHA
RBCs. These include chloroquine diphosphate, EDTA-
— + CAS; PCH, WAIHA
glycine, and murine monoclonal antibodies.
+ + Mixed-type AIHA (cold and warm)
Evaluation of a Positive DAT Data from Roback, JD, Combs, MR, Grossman, BJ, and Hillyer, CD (eds): Technical Manual,
16th ed. AABB, Bethesda, MD, 2008.
Clinical consideration should dictate the extent to which *The DAT with monospecific antiglobulin reagents is helpful in classifying AIHAs. Other
a positive DAT is evaluated. Interpreting the significance procedures and studies are necessary to diagnose and characterize which form of
autoimmune disease is present.
of a positive DAT requires knowledge of the patient’s CAS = cold agglutinin syndrome; PCH = paroxysmal cold hemoglobinuria; WAIHA = warm
diagnosis, drug therapy, and recent transfusion history. A autoimmune hemolytic anemia
2682_Ch05_101-118 22/05/12 11:41 AM Page 109
positive DAT may occur without clinical manifestations of • Is the patient receiving intravenous immune globulin
immune-mediated hemolysis. Table 5–5 outlines the in vivo (IVIG) or intravenous Rh immune globulin (IV RhIG)?
phenomena that may be associated with a positive DAT. • Has the patient received a marrow or other organ
The AABB Technical Manual states that “a positive DAT transplant?
alone is not diagnostic. The interpretation of the significance
of this positive result requires knowledge of the patient’s di- Indirect Antiglobulin Test
agnosis; recent drug, pregnancy, and transfusion history; and
The IAT is performed to determine in vitro sensitization of
information on the presence of acquired or unexplained he-
RBCs and is used in the following situations:
molytic anemia.”36 Answering the following questions before
investigating a positive DAT for patients other than neonates • Detection of incomplete (nonagglutinating) antibodies to
will help determine what further testing is appropriate: potential donor RBCs (compatibility testing) or to screening
cells (antibody screen) in serum
• Is there evidence of in vivo hemolysis?
• Determination of RBC phenotype using known antisera
• Has the patient been transfused recently? If so, did the
(e.g., Kell typing, weak D testing)
patient receive blood products or components containing
• Titration of incomplete antibodies
ABO-incompatible plasma?
• Does the patient’s serum contain unexpected antibodies? Table 5–6 lists the IATs and the in vitro sensitization
• Is the patient receiving any drugs? detected for each application. For in vitro antigen-antibody
• Is the patient receiving antilymphocyte globulin or antithy- reactions, the IAT tasks are listed and explained in
mocyte globulin? Table 5–7.
Transfusion 1. Recipient alloantibody and donor antigen Alloantibodies in the recipient of a recent transfusion
that react with antigen on donor RBC
2. Donor antibody and recipient antigen Antibodies present in donor plasma that react with
antigen on a transfusion recipient’s RBCs
Drug induced 1. Type I (hapten-dependent Ab) Drug binds covalently to membrane proteins and
stimulates hapten-dependent Ab
2. Type II (autoantibody) Drug induces autoantibody specific for RBC mem-
brane proteins through unknown mechanism; Ab
reacts with normal RBCs in the absence of drug.
3. Type III (drug-dependent Ab) Drug induces Ab that binds to RBC only when drug is
present in soluble form, unknown mech; Ab reacts
with normal RBCs when soluble drug is present.
Autoimmune hemolytic 1. WAIHA (IgG and/or C3) Autoantibody reacts with patient’s RBCs in vivo.
anemia
Modified from Roback, JD, Combs, MR, Grossman, BJ, and Hillyer, CD (eds): Technical Manual, 16th ed. AABB, Bethesda, MD, 2008.
AIHA = autoimmune hemolytic anemia; CAS = cold agglutinin syndrome; PCH = paroxysmal cold hemoglobinuria.
2682_Ch05_101-118 22/05/12 11:41 AM Page 110
Antibody Antibody Antibody reacting Increasing the ratio of serum to cells increases the sensitivity
identification panel with panel cells of the test system. Generally, a minimum ratio of 40:1 should
be the target, and this can be achieved by using 2 drops
Antibody Rh antibody Antibody and selected
titration titer Rh cells of serum and 1 drop of a 5% volume of solute per volume
of solution (v/v) suspension of cells.34 When using cells
RBC phenotype RBC antigen Specific antisera + RBCs suspended in saline, it is often advantageous to increase the
detection to detect antigen
ratio of serum to cells in an effort to detect weak antibodies
(ex: weak
D, K, Fy) (e.g., 4 drops of serum with 1 drop of a 3% [v/v] cell
suspension will give a ratio of 133:1).
Reaction Medium
The DAT does not require the incubation phase because
of the antigen-antibody complexes formed in vivo. Reaction mediums include albumin, LISS, and polyethylene
glycol.
Factors Affecting the Antiglobulin Test
Albumin
The DAT can detect a level of 100 to 500 IgG molecules per
The macromolecules of albumin allow antibody-coated cells
RBC and 400 to 1,100 molecules of C3d per RBC.28
to come into closer contact with each other so that aggregation
For the IAT, there must be between 100 and 200 IgG or
occurs. In 1965, Stroup and MacIlroy37 reported on the
C3 molecules on the cell to obtain a positive reaction. The
creased sensitivity of the IAT if albumin was incorporated into
number of IgG molecules that sensitize an RBC and the rate
the reaction medium. Their reaction mixture, consisting of
at which sensitization occurs can be influenced by several
2 drops of serum, 2 drops of 22% (w/v) bovine albumin, and
factors, including:
1 drop of 3% to 5% (v/v) cells, was shown to provide the same
• Ratio of serum to cells sensitivity at 30 minutes of incubation as a 60-minute saline-
• Reaction medium only test. The use of albumin does not seem to provide any
advantage over LISS techniques and adds to the cost of the
test.37 Petz and coworkers31 also showed that an albumin tech-
Table 5–7 Tasks and Purposes of the nique may miss several clinically significant antibodies. There-
Indirect Antiglobulin Test fore, it is rarely, if ever, used as an IAT media for routine tests.
TASK PURPOSE
Low Ionic Strength Solutions
Incubate RBCs with antisera Allows time for antibody molecule Low ionic strength solutions (LISS) enhance antibody
attachment to RBC antigen uptake and allow incubation times to be decreased—from
Perform a minimum of Removes free globulin molecules 30 to 60 minutes to 10 to 15 minutes—by reducing the zeta
three saline washes potential surrounding an RBC. Some LISS also contain
macromolecular substances. The LISS technique, introduced
Add antiglobulin reagent Forms RBC agglutinates
(RBC Ag + Ab + anti-IgG) by Low and Messeter,38 has critical requirements with
respect to the serum-to-cell ratio. Moore and Mollison39
Centrifuge Accelerates agglutination by showed that optimum reaction conditions were obtained
bringing cells closer together using 2 drops of serum and 2 drops of a 3% (v/v) suspension
Examine for agglutination Interprets test as positive or of cells in LISS. Increasing the serum-to-cell ratio increased
negative the ionic strength of the reaction mixture, leading to a
decrease in sensitivity and counteracting the shortened
Grade agglutination Determines the strength of
reactions reaction incubation time of the test. A LISS medium may be achieved
by either suspending RBCs in LISS or using a LISS additive
Add antibody-coated Checks for neutralization of reagent, with the latter being more common practice.
RBCs to negative reactions antisera by free globulin
molecules (Coombs’ control cells Polyethylene Glycol
are D-positive RBCs coated with
anti-D) Polyethylene glycol (PEG) is a water-soluble linear polymer
and is used as an additive to increase antibody uptake. Its
2682_Ch05_101-118 22/05/12 11:41 AM Page 111
action is to remove water molecules surrounding the RBC Saline for Washing
(the water of hydration theory), thereby effectively concen-
trating antibody. Anti-IgG is the AHG reagent of choice with Ideally, the saline used for washing should be fresh or
PEG testing to avoid false-positive reactions.10 Because PEG buffered to a pH of 7.2 to 7.4. Saline stored for long periods
may cause aggregation of RBCs, reading for agglutination in plastic containers has been shown to decrease in pH,
following 37°C incubation in the IAT is omitted. Several which may increase the rate of antibody elution during the
investigators40 compared the performance of PEG as an washing process, yielding a false-negative result.44 One of
enhancement media with that of LISS. Findings indicated the contributors (dk) has seen this phenomenon occur in
that PEG increases the detection of clinically significant working student labs when expiration dates are ignored to
antibodies while decreasing detection of clinically insignificant conserve resources. Changes in pH may have important
antibodies. Barrett and associates41 reported that as PEG has implications when monoclonal AHG is used, inasmuch as
been used for pretransfusion antibody screening, 6,353 RBC monoclonal antibodies have been shown to have narrow pH
components have been transfused without any reported ranges for optimum reactivity. Significant levels of bacterial
acute or delayed HTRs. contamination in saline have been reported;45 this situation
can contribute to false-positive results.
Temperature
Addition of AHG
The rate of reaction for the majority of IgG antibodies is
optimal at 37°C; therefore, this is the usual incubation AHG should be added to the cells immediately after washing
temperature for the IAT. This is also the optimum temper- to minimize the chance of antibody eluting from the cell and
ature for complement activation. subsequently neutralizing the AHG reagent. The volume of
AHG added should be as indicated by the manufacturers.
Incubation Time However, Voak and associates46 have shown that adding two
volumes of AHG may overcome washing problems when low
For cells suspended in saline, incubation times may vary levels of serum contamination remain. These authors indi-
between 30 and 120 minutes. The majority of clinically cated that the neutralization of AHG is a problem only with
significant antibodies can be detected after 30 minutes of free IgG left in serum following inadequate saline washings
incubation, and extended incubation times are usually not and not with residual serum complement components. The
necessary. If a LISS or PEG technique is being used,38,39 complement fragments free in serum are not the same as the
incubation times may be shortened to 10 to 15 minutes. complement fragments bound to RBCs, and therefore resid-
With these shortened times, it is essential that tubes be ual serum does not contain C3b and C3d to neutralize the
incubated at a temperature of 37°C. Extended incubation anti-C3b and anti-C3d in AHG reagent.
(i.e., up to 40 minutes) in the LISS technique has been
shown to cause antibody to elute from the RBCs, decreasing Centrifugation for Reading
the sensitivity of the test.42 However, this could not be
confirmed by Voak and coworkers.43 Centrifugation of the cell pellet for reading of hemaggluti-
nation along with the method used for resuspending the cells
Washing of RBCs is a crucial step in the technique. The CBER-recommended
method for the evaluation of AHG uses 1,000 relative
When both the DAT and IAT are performed, RBCs must centrifugal forces (RCFs) for 20 seconds, although the
be saline-washed a minimum of three times before adding technique described in this chapter suggests 500 RCFs for
AHG reagent. Washing the RBCs removes free unbound 15 to 20 seconds. The use of higher RCFs yields more
serum globulins. Inadequate washing may result in a false- sensitive results; however, depending on how the pellet is
negative reaction because of neutralization of the AHG resuspended, it may give weak false-positive results because
reagent by residual unbound serum globulins. The wash- of inadequate resuspension or may give a negative result if
ing phase of a DAT and IAT becomes one of the most resuspension is too vigorous. The optimum centrifugation
important steps in testing. The wash phase can be conditions should be determined for each centrifuge.
controlled using check cells, or group O cells sensitized
with IgG. Sources of Error
Washing should be performed immediately after being
removed from the incubator and in as short a time as possible Some of the most common sources of error associated with
to minimize the elution of low-affinity antibodies. The cell the performance of the AHG test have been outlined in the
pellet should be completely resuspended before adding the previous section. Box 5–1 lists reasons for false-negative and
next saline wash. All saline should be discarded after the false-positive AHG reactions. An anticoagulant such as
final wash, because residual saline dilutes the AHG reagent EDTA should be used to collect blood samples for the DAT
and therefore decreases the sensitivity of the test. Centrifu- to avoid the in vitro complement attachment associated with
gation at each wash should be sufficient to provide a firm refrigerated clotted specimens.47
cell pellet and therefore minimize the possible loss of cells All negative antiglobulin test reactions must be checked
with each discard of saline. by the addition of IgG-sensitized cells. Adding IgG-coated
2682_Ch05_101-118 22/05/12 11:41 AM Page 112
BOX 5–1
Modified from Roback, JD, Combs, MR, Grossman, BJ, and Hillyer, CD (eds): Technical Manual, 16th ed. AABB, Bethesda, MD, 2008.
RBCs to negative test reactions should demonstrate hemag- by the attached antibody. A high ionic strength solution is
glutination of these RBCs with the anti-IgG in the AHG then added to reverse the rouleaux; however, if agglutination
reagent. If no hemagglutination follows the addition of IgG- is present, it will remain. The test can be carried through
coated RBCs, the test result is invalid and the test must be to an AHG technique if required. If this is performed, a
repeated. The most common technical errors that result in monospecific anti-IgG reagent must be used, because the
failure to demonstrate hemagglutination after the addition low ionic conditions cause considerable amounts of C4 and
of IgG-coated RBCs are inadequate washing, nonreactive C3 to coat the cells and would give false-positive reactions
AHG reagent, and failure to add AHG reagent. While most if a polyspecific reagent were used.
blood banks do not check monospecific anti-C3d reactivity The antiglobulin test has also been performed using
with the addition of C3d-coated RBCs to negative reactions, microplates. Crawford and colleagues49 used microplates for
these cells are available and may also be produced in-house.47 a number of different grouping procedures, including the
IAT. Microplate technology is used increasingly in blood
Modified and Automated Antiglobulin group serology, and many techniques are being adapted for
Test Techniques it. Redman and associates50 have adapted the LIP technique
for use in microplates. Although their report does not
Modifications to the antiglobulin test technique (LISS, PEG, include the use of an AHG phase, this additional step could
and albumin) have just been described; however, additional easily be included.
modifications may be used in special circumstances, includ- It should also be mentioned that polybrene has a low
ing the low-iconic polybrene technique, enzyme-linked sensitivity to detection of anti-Jka and –Jkb and the Jka anti-
antiglobulin test, solid phase technology, and gel test. gen.51 Therefore, the potential exists for a clinically signifi-
cant anti-Kidd antibody to be missed using the polybrene
Low Ionic Polybrene Technique method of enhancement for IAT.
In 1980, Lalezari and Jiang48 reported on the adaptation of Enzyme-Linked Antiglobulin Test
the automated low ionic polybrene (LIP) technique for use
as a manual procedure. The technique relies on low ionic In the enzyme-linked antiglobulin test (ELAT), an RBC
conditions to rapidly sensitize cells with antibody. Polybrene, suspension is added to a microtiter well and washed with
a potent rouleaux-forming reagent, is added to allow the sen- saline. AHG, which has been labeled with an enzyme, is
sitized cells to approach each other and permit cross-linking added. The enzyme-labeled AHG will bind to IgG-sensitized
2682_Ch05_101-118 22/05/12 11:41 AM Page 113
RBCs. Excess antibody is removed, and enzyme substrate is For the DAT, 50 µL of a 0.8% RBC suspension in LISS
added. The amount of color produced is measured spec- solution (ID-Diluent 2) is added to the top of each microtube
trophotometrically and is proportional to the amount of of the LISS/Coombs ID cards. The cards are centrifuged at
antibody present. The optical density is usually measured at 910 rpm for 10 minutes.55 In the case of a positive reaction,
405 nm. The number of IgG molecules per RBC can also be monospecific reagents (anti-IgG, anti-C3d) can be used in
determined from this procedure. the gel test. For a detailed description of this technology, see
Chapter 12.
Solid Phase Technology
Comparison of AHG Methodologies
Solid-phase technology may be used for performing
antiglobulin tests. Several different techniques have been Transfusion service departments typically work to detect all
reported using either test tubes51b or microplates.52,53 With clinically significant antibodies, both DAT and IAT types, and
the availability of microplate readers, this modification lends none of the clinically insignificant antibodies such as warm
itself to the introduction of semiautomation. Direct and and cold-reacting autoantibodies. A common question that
indirect tests can be performed using solid-phase methodology. arises in these departments is which detection method
In the former, antibody is attached to a microplate well, and should be employed to reach such goals. This section briefly
RBCs are added. If antibody is specific for antigen on RBCs, compares various AHG methods for DAT and IAT testing.
the bottom of the well will be covered with suspension; if no Table 5–8 outlines some of the advantages and disadvantages
such specificity occurs, RBCs will settle to the bottom of the in various AHG testing methodologies.
well. In the latter, known RBCs are bound to a well that has
been treated with glutaraldehyde or poly L-lysine. Test serum DAT Methods
is added to RBC-coated wells, and if antibody in serum is
specific for antigen on fixed RBCs, a positive reaction occurs There have been numerous studies comparing the tube and
as previously described. For a detailed description of gel test when performing DATs. The main difference in the
this technology, see Chapter 12, “Other Technologies and two techniques is that the former requires washing, and the
Automation.” latter omits a washing stage, resulting in discrepant results
between the two methods. Multiple studies56–60 concluded
Gel Test that a gel technique used for detecting in vivo sensitized RBCs
was more sensitive than the conventional tube technique.
The gel test is a process that detects RBC antigen-antibody However, a comparison study using solid-phase methodology
reactions by means of using a chamber filled with polyacry- for DAT has not been reported. The gel method, although
lamide gel. The gel acts as a trap; free unagglutinated RBCs more sensitive, isn’t necessarily more sensitive only to clini-
form pellets in the bottom of the tube, whereas agglutinated cally significant antibodies. Blood banks should be aware of
RBCs are trapped in the tube for hours. Therefore, negative the differences in the DAT when using the popular gel test
reactions appear as pellets in the bottom of the microtube, over the tube technique. Additional comparative studies will
and positive reactions are fixed in the gel. add to the current body of knowledge.
There are three different types of gel tests: neutral, spe-
cific, and antiglobulin. A neutral gel does not contain any IAT Methods
specific reagent and acts only by its property of trapping
agglutinates. The main applications of neutral gel tests are There has been a downward trend in conventional tube test-
antibody screening and identification with enzyme-treated ing technique in AHG testing, with a gain in popularity for
or untreated RBCs and reverse ABO typing. Specific gel tests gel technology.61 From a timing and sensitivity perspective,
use a specific reagent incorporated into the gel and are useful the conventional tube test using saline is the least preferred
for antigen determination. method.61 The most popular tube medium is LISS followed
The gel low ionic antiglobulin test (GLIAT) is a valuable by PEG.62 Although the LISS and PEG methods are more
application of the gel test and may be used for the IAT or the sensitive than the saline tube method, they are the most
DAT. AHG reagent is incorporated into the gel. For example, labor-intense methods requiring the most skilled staff. One
in an IAT gel, 50 µL of a 0.8% RBC suspension is pipetted advantage they have over gel and solid phase is the lowered
onto a gel containing AHG, serum is added, and the tube is cost of required reagents. Bunker and colleagues63 concluded
centrifuged after a period of incubation. At the beginning of in their study that the PEG IAT method was the most cost-
centrifugation, the RBCs tend to pass through the gel, but effective pretransfusion antibody screening technique when
the medium in which they are suspended remains above. compared to the solid red cell adherence assay.
This results in separation between the RBCs and the medium Gel technology’s introduction into the transfusion service
without a washing phase. RBCs come in contact with AHG department in the last decade has greatly benefitted the trend
in the upper part of the gel, and the positive and negative in laboratories to use generalist bench techs rather than
reactions are separated. The detection of unexpected anti- department-focused techs. Gel technology is less labor-
bodies by GLIAT compares favorably with conventional intensive than conventional tube testing and incorporates
AHG methods and provides a safe, reliable, and easy-to-read standardization both procedurally and in endpoint grading.
AHG test.54 This standardization cannot be duplicated using tube testing.
2682_Ch05_101-118 22/05/12 11:41 AM Page 114
Solid phase • No need for check cells • Increased sensitivity for all Abs
• Stable endpoints • Detects unwanted Abs
• Small test volume • Warm auto Abs enhanced
• Enhanced anti-D • Increased costs
• Increased sensitivity for all Abs • Increased need for additional instrumentation
• Ability to be automated
Ab = antibody; CTT = conventional tube testing; LISS = low ionic strength saline; PEG = polyethylene glycol
Gel methods for IAT testing have been shown to be more Over the coming years, the changes in blood bank tech-
sensitive than saline, LISS, and PEG at detecting passively nology, along with the changes in emphasis on the importance
acquired anti-D in postnatal patients.64 Weisbach and asso- of crossmatching versus antibody screening, will probably
ciates65 have also shown that labs using the gel method are further modify the role of the antiglobulin test. Method-
more likely to detect weak antibody reactivity than the LISS dependent antibodies have been documented repeatedly
tube method. An advantage to using the gel technology in a in AHG method comparison studies. Some antibodies will
high-volume lab is its ability to operate as an automated system. be detectable using only one specific method of testing and
A few disadvantages to using the gel method are the increased will be negative in all others. However, AHG methods are
cost of instrumentation and reagents, the need to stay profi- comparable in regards to sensitivity and specificity.
cient with alternative methods for backup, and the increased When deciding which method to use in day-to-day
likelihood of detecting unwanted autoantibodies. operations, it is important to consider time, resources, fa-
Solid-phase technology can be incorporated as a manual cilities and staffing, and the cost of overall testing and how
or automated method for use in IAT. Like gel technology, these factors will impact patient care. Current research
solid-phase technology is more likely to detect weakly reac- is under way to utilize molecular diagnostic methods, par-
tive antibodies than the LISS tube method.65 However, with ticularly PCR, to identify RBC genotypes in place of the
a higher sensitivity comes the detection of more nonspecific serologic phenotyping AHG methods. At present, however,
reactivity.66,67 Thus, introducing additional costs required to serologic AHG testing methods remain the most important
investigate positive screening results. Garozzo and colleagues68 in the blood bank for detecting clinically significant
showed that the solid-phase method is even more sensitive antibodies to RBCs and RBC antigens and for detecting
at detecting the anti-D than the gel method. immune hemolysis.
2682_Ch05_101-118 22/05/12 11:41 AM Page 115
SUMMARY CHART
The antiglobulin test is used to detect RBCs sensitized The IAT detects in vitro sensitization of RBCs and
by IgG alloantibodies, IgG autoantibodies, and com- can be applied to compatibility testing, antibody
plement components. screen, antibody identification, RBC phenotyping, and
AHG reagents containing anti-IgG are needed for titration studies.
the detection of IgG antibodies because the IgG A positive DAT is followed by a DAT panel using
monomeric structure is too small to directly aggluti- monospecific anti-IgG and anti-C3d to determine the
nate sensitized RBCs. specific type of protein sensitizing the RBC.
Polyspecific AHG sera contain antibodies to human EDTA should be used to collect blood samples for
IgG and the C3d component of human complement. the DAT to avoid in vitro complement attachment
Monospecific AHG sera contain only one antibody associated with refrigerated clotted specimens.
specificity: either anti-IgG or antibody to anti–C3b-C3d. There are multiple sources or error that can be intro-
Classic AHG sera (polyclonal) are prepared by injecting duced into the AHG procedure.
human globulins into rabbits, and an immune stimulus LISS, PEG, polybrene, and albumin can all be used as
triggers production of antibody to human serum. enhancement media for AHG testing, with each having
Hybridoma technology is used to produce monoclonal their own advantages and disadvantages.
antiglobulin serum. Conventional tube testing, gel technology, enzyme-
The DAT detects in vivo sensitization of RBCs with linked technology, and solid-phase testing are available
IgG or complement components. Clinical conditions methods to use in AHG testing.
that can result in a positive DAT include HDN, HTR, Method-dependent antibodies do exist and should be
and AIHA. evaluated on a case-by-case basis.
2682_Ch05_101-118 22/05/12 11:41 AM Page 116
16. A 27-year-old group O mother has just given birth to a 23. Lachman, PJ, and Muller-Eberhard, HJ: The demonstration in
beautiful, group A baby girl. Since the mother has IgG human serum of “conglutinogen-activating-factor” and its
effect on the third component of complement. J Immunol
anti-A in her plasma, it is likely that the baby is experi- 100:691, 1968.
encing some in vivo red cell destruction. Which of the 24. Muller-Eberhard, HJ: Chemistry and reaction mechanisms of
following methods and tests would be most effective at complement. Adv Immunol 8:1, 1968.
detecting the anti-A on the baby’s RBCs? 25. Cooper, NR: Isolation and analysis of mechanisms of action of
an inactivator of C4b in normal human serum. J Exp Med
a. DAT using common tube technique
141:890, 1975.
b. DAT using gel 26. Case, J, et al: International reference reagents: Antihuman glob-
c. IAT using common tube technique ulin, an ISBT/ICSH Joint Working Party Report. Vox Sang
d. IAT using gel 77:121, 1999.
27. Brown, DL, et al: The in vivo behaviour of complement-coated
red cells: Studies in C6-deficient, Ce-depleted and normal
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5. Dacie, JF: Differences in the behaviour of sensitized red cells 30. Petz, LD, et al: Clinical Practice of Transfusion Medicine, 3rd
to agglutination by antiglobulin sera. Lancet ii:954, 1951. ed. Churchill Livingstone, New York, 1996, p 207.
6. Dacie, JV, et al: “Incomplete” cold antibodies: Role of comple- 31. Petz, LD, et al: Compatibility testing. Transfusion 21:633,
ment in sensitization to antiglobulin serum by potentially 1981.
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6:412, 1963. 33. Milam JD: Laboratory medicine parameter: Utilizing monospe-
8. Jenkins, GC, et al: Role of C4 in the antiglobulin reaction. cific antihuman globulin to test blood group compatibility. Am
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45:367, 1983. 39. Moore, HC, and Mollison, PL: Use of a low-ionic strength
14. Holt, PDJ, et al: NBTS/BRIC 8: A monoclonal anti-C3d anti- medium in manual tests for antibody detection. Transfusion
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Blackwell Scientific, Oxford, 1983, p 502. 41. Barrett, V, et al: Analysis of the routine use of polyethylene
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antiglobulin test. Transfusion 1:9, 1961. Sci 37:107, 1980.
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1962. 45. Green, C, et al: Quality assurance of physiological saline used
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48. Lalezari, P, and Jiang, RF: The manual polybrene test: A simple 61. Casina, TS: In search of the Holy Grail: Comparison of anti-
and rapid procedure for detection of red cell antibodies. Trans- body screening methods. Immunohematology 22(4):196–202,
fusion 20:206, 1980. 2006.
49. Crawford, MN, et al: Microplate system for routine use in blood 62. Shulman, IA, Maffei, LM, and Downes, KA: North American
bank laboratories. Transfusion 10:258, 1970. pretransfusion testing practices, 2001–2004: Results from the
50. Redman, M, et al: Typing of red cells on microplates by College of American Pathologists Interlaboratory Comparison
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51. Liu, JC, Wang, Y, Liu, FP, and He, YS: The manual polybrene 129(8):984–989, 2005.
test has limited sensitivities for detecting the Kidd blood group 63. Bunker, ML, Thomas, CL, and Geyer, SJ: Optimizing pretrans-
system. Scand J Clin Lab Investig 69:7, 797–800, 2009. fusion antibody detection and identification: A parallel, blinded
52. Moore, HH: Automated reading of red cell antibody identifica- comparison of tube PEG, solid-phase, and automated methods.
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24:218, 1984. 64. Klostermann, DA, Puca, KE, Scott, EA, and Johnson, ST: Com-
53. Plapp, FV, et al: A solid phase antibody screen. Am J Clin parison of methods for detection of antenatal anti-D. Transfu-
Pathol 82:719, 1984. sion 46(9S):148A, 2006.
54. Lapierre, Y, et al: The gel test: A new way to detect red cell 65. Weisbach, V, et al: Comparison of the performance of micro-
antigen-antibody reactions. Transfusion 30:2, 1990. tube column systems and solid-phase systems and the tube
55. Tissot, JD, et al: The direct antiglobulin test: Still a place for low-ionic-strength solution additive indirect antiglobulin test
the tube technique? Vox Sang 77:223, 1999. in the detection of red cell alloantibodies. Transfusion Med
56. Chuansumrit, A, et al: The benefit of the direct antiglobulin 16(4):276–284, 2006.
test using gel technique in ABO hemolytic disease of the new- 66. Yamada, C, Serrano-Rahman, L, Vasovic, LV, Mohandas, K, and
born. Southeast Asian J Trop Med Pub Health 28:428, 1997. Uehlinger, J: Antibody identification using both automated
57. Lai, M, et al: Clinically significant autoimmune hemolytic solid-phase red cell adherence assay and a tube polyethylene
anemia with a negative direct antiglobulin test by routine glycol antiglobulin method. Transfusion 48(8):1693–1698,
tube test and positive by column agglutination method. 2008.
Immunohematology 18:109, 2002. 67. Dwyre, DM, Erickson, Y, Heintz, M, Elbert, C, and Strauss, RG:
58. Mitek, JF, et al: The value of the gel test and ELAT in autoim- Comparative sensitivity of solid phase versus PEG enhance-
mune haemolytic anaemia. Clin Lab Haem 17:311, 1995. ment assays for detection and identification of RBC antibodies.
59. Das, SS, Chaudhary, R, and Khetan, D: A comparison of Transfus Apher Sci 35(1):19–23, 2006.
conventional tube test and gel technique in evaluation of direct 68. Garozzo, G, et al: A comparison of two automated methods for
antiglobulin test. Hematology 12(2):175–178, 2007. the detection and identification of red blood cell alloantibodies.
60. Novaretti, MC, Jens, E, Pagliarini, T, Bonifacio, SL, Dorlhiac- Blood Transfusion 5(1):33–40, 2007.
Llacer, PE, and Chamone, DA: Comparison of conventional tube
test technique and gel microcolumn assay for direct antiglobulin
test: Large study. J Clin Lab Anal 18(5):255–258, 2004.
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Chapter 6
The ABO Blood Group System
Denise M. Harmening, PhD, MT(ASCP), CLS (NCA); Glenda Forneris, MHS,
MT(ASCP)SBB; Barbara J. Tubby MSEd, BS, MT(ASCP)SBB
OBJECTIVES
1. Describe the reciprocal relationships between ABO antigens and antibodies for blood types O, A, B, and AB.
2. Identify the frequencies of the four major blood types in the white, black, Hispanic, and Asian populations.
3. Explain the effect of age on the production of ABO isoagglutinins.
4. Describe the immunoglobulin classes of ABO antibodies in group O, A, and B individuals.
5. Predict the ABO phenotypes and genotypes of offspring from various ABO matings.
6. Explain the formation of H, A, and B antigens on the red blood cells (RBCs) from precursor substance to immunodominant
sugars.
7. Describe the formation of H, A, and B soluble substances.
8. Explain the principle of the hemagglutination inhibition assay for the determination of secretor status.
9. Describe the qualitative and quantitative differences between the A1 and A2 phenotypes.
10. Describe the reactivity of Ulex europaeus with the various ABO groups.
11. Describe the characteristics of the weak subgroups of A (A3, Ax, Aend, Am, Ay, Ael).
12. Describe the characteristics of the Bombay phenotypes.
13. Explain the effects of disease on the expression of ABH antigens and antibodies.
14. Interpret the results from an ABO typing and resolve any discrepancies, if present.
Introduction ABO type may result in immediate lysis of donor RBCs. This
produces a very severe, if not fatal, transfusion reaction in the
The ABO system is the most important of all blood groups in patient. Testing to detect ABO incompatibility between a
transfusion practice. It is the only blood group system in donor and potential transfusion recipient is the foundation
which individuals have antibodies in their serum to antigens on which all other pretransfusion testing is based.
that are absent from their RBCs. This occurs without any Even today, transfusion of the wrong ABO group remains
exposure to RBCs through transfusion or pregnancy. Due to the leading cause of death in hemolytic transfusion reaction
the presence of these antibodies, transfusion of an incompatible fatalities reported to the FDA; however, transfusion-related
119
2682_Ch06_119-148 28/05/12 12:26 PM Page 120
Table 6–1 Transfusion-Related Fatalities membrane. In 1901, Landsteiner drew blood from himself
by Complication, FY 2009 and five associates, separated the cells and serum, and then
mixed each cell sample with each serum.2 He was inadver-
COMPLICATION NUMBER FY09 tently the first individual to perform forward and reverse
TRALI 13* 30% grouping. Forward grouping (front type) is defined as using
known sources of commercial antisera (anti-A, anti-B) to
HTR (non-ABO) 8 18% detect antigens on an individual’s RBCs. Figure 6–1 outlines
HTR (ABO) 4 9% the steps of performing the forward grouping for ABO (see
color insert following page 128), and Table 6–2 lists the
Microbial infection 5 11% results of the forward grouping procedure.
TACO 12 27% Reverse grouping (back type) is defined as detecting ABO
antibodies in the patient’s serum by using known reagent
Anaphylaxis 1 2% RBCs, namely A1 and B cells. Figure 6–2 outlines the steps of
Other 1** 2% performing the reverse ABO grouping (see color insert follow-
ing page 128), and Table 6–3 summarizes the results of the
TOTALS 44 100% procedures. Table 6–4 lists the characteristics of the routine
reagents used for ABO testing in the blood bank laboratory.
*In FY 2007, the review committee began using the Canadian Consensus Conference criteria5,6
for evaluating TRALI cases; these numbers include both “TRALI” and “possible TRALI” cases. ABO forward and reverse grouping tests must be per-
**Other: Hypotensive Reaction7 Key: HTR = hemolytic transfusion reaction; TACO = transfusion- formed on all donors and patients.3 ABO grouping is the
associated circulatory overload; TRALI = transfusion-related acute lung injury
Data from 2009, F. R. (n.d.): Vaccines, Blood & Biologics; U.S. Food and Drug Administration.
Fatalities Reported to FDA Following Blood Collection and Transfusion: Annual Summary for
Fiscal Year 2009. Retrieved March 22, 2010, from www.fda.gov/BiologicsBloodVaccines/ Table 6–2 ABO Forward Grouping:
SafetyAvailability/ReportaProblem/TransfusionDonationFatalities/ucm204763.htm. Principle—Detection of Antigens
on Patient’s RBCs With Known
acute lung injury (TRALI) was the most frequent cause of death Commercial Antisera
in fiscal year (FY) 20091 (Table 6–1). In FY 2009, there were PATIENT PATIENT INTERPRETATION
four reports of fatal hemolytic transfusion reactions due to ABO RBCS WITH RBCS WITH OF BLOOD
incompatible blood transfusions (Box 6–1 lists the causes in ANTI-A ANTI-B GROUP
each of these four cases).1 This chapter presents the ABO blood
group system and discusses the biochemistry, properties, and 0 0 O
characteristics of ABO antigens and antibodies. In addition, 4+ 0 A
weak subgroups and common discrepancies will be introduced
to provide a working knowledge for routine ABO testing. 0 4+ B
4+ 4+ AB
Historical Perspective and Routine
ABO Testing + = visual agglutination
0 = negative
Note: Reaction gradings vary from patient to patient.
Karl Landsteiner truly opened the doors of blood banking
with his discovery of the first human blood group system,
ABO. This marked the beginning of the concept of individual
uniqueness defined by the RBC antigens present on the RBC
Table 6–3 ABO Reverse Grouping:
Principle—Detection of ABO
Antibodies (Isoagglutinins) in
BOX 6–1
Serum of Patient With Known
Commercial RBCs
Causes of Fatal Hemolytic Transfusion Reactions Due
PATIENT PATIENT
to ABO Incompatible Blood Transfusions in FY 2009 SERUM WITH SERUM WITH INTERPRETATION
• Case 1: Recipient identification error at the time of transfusion REAGENT REAGENT OF BLOOD
(nursing error) A1 CELLS B CELLS GROUP
• Case 2: Patient sample labels switched (phlebotomist error)
4+ 4+ O
• Case 3: Sample collected from incorrect patient (phlebotomist
error) 0 3+ A
• Case 4: Patient sample mistyped (lab error)
3+ 0 B
Data from 2009, F. R. (n.d.): Vaccines, Blood & Biologics; U.S. Food and Drug
Administration. Fatalities Reported to FDA Following Blood Collection and 0 0 AB
Transfusion: Annual Summary for Fiscal Year 2009. Retrieved March 22, 2010,
from www.fda.gov/BiologicsBloodVaccines/SafetyAvailability/ReportaProblem/ + = visual agglutination
TransfusionDonationFatalities/ucm204763.htm. 0 = negative
Note: Reaction gradings vary from patient to patient.
2682_Ch06_119-148 28/05/12 12:26 PM Page 121
*General rule: Always drop clear solutions first and RBCs second to make sure you have added both a source of antibody and antigen.
most frequently performed test in the blood bank. There is grouping is unique to the ABO blood group system. The
always an inverse reciprocal relationship between the regular occurrence of anti-A and/or anti-B in persons lacking
forward and reverse type; thus, one serves as a check on the the corresponding antigen(s) serves as a confirmation of re-
other. For example, if the individual has A antigens only on sults in ABO grouping. Table 6–5 summarizes the forward
their red cells, there will be an “expected” naturally occurring and reverse grouping for the common ABO blood groups.
anti-B antibody in their serum since they lack the B antigen. The frequency of these blood groups in the white and
It has been postulated that bacteria, pollen particles, and black populations is outlined in Table 6–6.4 Group O and A
other substances present in nature are chemically similar to are the most common blood types, and blood group AB is
A and B antigens. Bacteria are widespread in the environ- the rarest. However, frequencies of ABO groups differ in a
ment, which constantly exposes individuals to A-like and few selected populations and ethnic groups (Table 6–7).4 For
B-like antigens. This exposure serves as a source of stimula- example, group B is found twice as frequently in blacks and
tion of anti-A and anti-B. All other defined blood group sys- Asians as in whites. In addition, there is a significant
tems do not regularly have in their serum expected decrease in the distribution of group A in these two ethnic
“naturally occurring” antibodies to antigens they lack on populations in comparison to whites. It has been reported
their RBCs. Antibody production in most other blood group that subgroup A2 is rarely found in Asians.5
systems requires the introduction of foreign RBCs by
transfusion or pregnancy, although some individuals can ABO Antibodies
occasionally have antibodies present that are not related to
the introduction of foreign RBCs. (These antibodies are usu- Individuals normally produce antibodies directed against the
ally of the IgM type and are not consistently present or ex- A and/or B antigen(s) absent from their RBCs. These antibodies
pected in everyone’s serum.) Therefore, performance of serum have been described as naturally occurring because they are
O 0 0 No A or B antigen 4+ 4+ A and B
A 4+ 0 A 0 2+ B
B 0 4+ B 3+ 0 A
AB 3+ 3+ A and B 0 0 No A or B antibodies
Table 6–6 Frequency of ABO Blood from group O individuals contains not only anti-A and anti-B
Groups in the United States* but also anti-A,B, which reacts with A and B cells. Anti-A,B
antibody activity, originally thought to be just a mixture of
RACE anti-A and anti-B, cannot be separated into a pure specificity
Blood Group Whites Blacks when adsorbed with either A or B cells.7 For example, if group
O serum is adsorbed with A or B cells, the antibody eluted will
O 45% 50% react with both A and B cells.7 Anti-A,B antibody is not a com-
A 40% 26% bination of anti-A and anti-B but is a separate “cross-reacting”
antibody that is usually IgG in nature.7 Knowing the amount
B 11% 20% of IgG anti-A, anti-B, or anti-A,B in a woman’s serum some-
AB 4% 4% times allows prediction or diagnosis of hemolytic disease
of the fetus and newborn (HDFN) caused by ABO incompat-
*Percentages rounded to the nearest whole number ibility.8 Often cord blood samples from babies of group O
Data from Garratty, G, Glynn, SA, and McEntire, R: ABO and Rh (D) phenotype frequencies of
mothers are examined for possible ABO HDFN (see Chapter
different racial/ethnic groups in the United States. Transfusion 44:703–706, 2004.
19, “Hemolytic Disease of the Fetus and Newborn [HDFN]”).
Both immunoglobulin classes of ABO antibodies react prefer-
produced without any exposure to RBCs. The ABO antibod- entially at room temperature (20°C to 24°C) or below and
ies are predominantly IgM, and they activate complement efficiently activate complement at 37°C.9
and react at room temperature or colder.5 ABO antibodies Testing RBCs with reagent anti-A,B is not required as a
produce strong direct agglutination reactions during ABO routine part of ABO testing.10 However, some believe that
testing. The production of ABO antibodies is initiated at anti-A,B is more effective at detecting weakly expressed A
birth, but titers are generally too low for detection until the and B antigens than reagent anti-A or anti-B. However, the
individual is 3 to 6 months of age.6 Therefore, most antibod- production and use of monoclonal antisera have made anti-
ies found in cord blood serum are of maternal origin. Results A and anti-B reagents much more sensitive, to the point
of serum ABO testing before 3 to 6 months of age cannot where weak A and B antigens can be detected routinely.
be considered valid because some or all of the antibodies Therefore, anti-A,B reagent is not usually used in routine
present may be IgG maternal antibodies that have crossed ABO red cell testing on patient samples. It is still routinely
the placenta. As a result, it is logical to perform only forward used when performing ABO confirmation of blood donors,
grouping on cord blood from newborn infants. because it is more economical to use one reagent (anti-A,B)
Antibody production peaks when an individual is between than to use two reagents (anti-A and anti-B) to verify group O
5 and 10 years of age and declines later in life.6 Elderly people donor units.3 Reagent anti-A,B can be prepared using
usually have lower levels of anti-A and anti-B; therefore, anti- blended monoclonal anti-A and anti-B; polyclonal human
bodies may be undetectable in the reverse grouping (see the anti-A,B; or a blend of monoclonal anti-A, anti-B, and anti-
“ABO Discrepancies” section later in this chapter). ABO anti- A,B.3 Consult the manufacturer’s package insert to determine
bodies can cause rapid intravascular hemolysis if the wrong if a reagent anti-A,B reacts with a specific weak A phenotype.
ABO group is transfused; this can result in the patient dying.1
Although anti-A (from a group B individual) and anti-B Inheritance of the ABO Blood Groups
(from a group A individual) contains predominantly IgM
antibody, there may be small quantities of IgG present.5 Serum The theory for the inheritance of the ABO blood groups was
first described by Bernstein in 1924. He demonstrated that
an individual inherits one ABO gene from each parent and
Table 6–7 ABO Phenotype Frequencies that these two genes determine which ABO antigens are
of Ethnic Groups in the United present on the RBC membrane. The inheritance of ABO
genes, therefore, follows simple Mendelian genetics. ABO,
States
like most other blood group systems, is codominant in
U.S. FREQUENCIES (%) (ROUNDED TO THE NEAREST expression.9 (For a review of genetics, see Chapter 2, “Basic
WHOLE NUMBER) Genetics.”) One position, or locus, on each chromosome 9
Phenotype Whites Blacks Hispanic* Asian** is occupied by an A, B, or O gene.11,12 The O gene is consid-
ered an amorph, as no detectable antigen is produced
O 45 50 56 40 in response to the inheritance of this gene. Therefore, the
A 40 26 31 28 group O phenotype is an autosomal recessive trait with the
inheritance of two O genes that are nonfunctional.
B 11 20 10 25 The designations group A and B refer to phenotypes,
AB 4 4 3 7 whereas AA, BO, and OO denote genotypes. In the case of an
O individual, both phenotype and genotype are the same,
*Hispanic includes Mexican, Puerto Rican, Cuban, and other Hispanics. because that individual would have to be homozygous for
**Asian includes Chinese, Filipino, Indian, Japanese, Korean, and Vietnamese.
Data from Garratty, G, Glynn, SA, and McEntire, R: ABO and Rh (D) phenotype frequencies of
the O gene. An individual who has the phenotype A (or B)
different racial/ethnic groups in the United States. Transfusion 44:703–706, 2004. can have the genotype AA or AO (or BB or BO). Box 6–2 lists
2682_Ch06_119-148 28/05/12 12:26 PM Page 123
AO ⫻ BO AB (AB) or A (AO) or
Formation of A, B, and H Red Cell Antigens B (BO) or O (OO)
The formation of ABH antigens results from the interaction of A⫻O AA ⫻ OO A (AO)
genes at three separate loci (ABO, Hh, and Se). These genes AO ⫻ OO A (AO) or O (OO)
do not actually code for the production of antigens but rather
produce specific glycosyltransferases that add sugars to a A ⫻ AB AA ⫻ AB AB (AB) or A (AA)
basic precursor substance (Table 6–9). A, B, and H antigens AO ⫻ AB AB (AB) or A (AA or AO)
are formed from the same basic precursor material (called a or B (BO)
paragloboside or glycan) to which sugars are attached in re-
B⫻O BB ⫻ OO B (BO)
sponse to specific enzyme transferases elicited by an inherited
gene.11–13 BO ⫻ OO B (BO) or O (OO)
The H antigen is actually the precursor structure on
B ⫻ AB BB ⫻ AB AB (AB) or B (BB)
which A and B antigens are made. Inheritance of the H gene
results in the formation of the H antigen. The H and Se genes BO ⫻ AB AB (AB) or B (BB or BO)
are closely linked and located on chromosome 19, in contrast or A (AO)
to ABO genes, which are located on chromosome 9. The H AB ⫻ O AB ⫻ OO A (AO) or B (BO)
and Se genes are not part of the ABO system; however, their
inheritance does influence A and B antigen expression. The
H gene must be inherited to form the ABO antigens on the with adult cells. The expression of A and B antigens on the
RBCs, and the Se gene must be inherited to form the ABO RBCs is fully developed by 2 to 4 years of age and remains
antigens in secretions. The precursor substance on erythrocytes constant throughout life.8 In addition to age, the phenotypic
is referred to as type 2. This means that the terminal galac- expression of ABH antigens may vary with race, genetic
tose on the precursor substance is attached to the interaction, and disease states.15
N-acetylglucosamine in a beta 1 → 4 linkage (Fig. 6–3). A
type 1 precursor substance refers to a beta 1 → 3 linkage Interaction of Hh and ABO Genes
between galactose and N-acetylglucosamine, which will be
described below. ABH antigens on the RBC are constructed Individuals who are blood group O inherit at least one
on oligosaccharide chains of a type 2 precursor substance.14 H gene (genotype HH or Hh) and two O genes. The
The ABH antigens develop early in fetal life but do not in- H gene elicits the production of an enzyme called
crease much in strength during the gestational period. The α-2-L-fucosyltransferase, which transfers the sugar L-fucose
RBCs of the newborn have been estimated to carry anywhere to an oligosaccharide chain on the terminal galactose of
from 25% to 50% of the number of antigenic sites found on type 2 chains.13 The sugars that occupy the terminal positions
the adult RBC.10 As a result, reactions of newborn RBCs with of this precursor chain and confer blood group specificity are
ABO reagent antisera are frequently weaker than reactions called the immunodominant sugars. Therefore, L-fucose is
2682_Ch06_119-148 28/05/12 12:26 PM Page 124
H α-2-L-fucosyltransferase L-fucose H
A α-3-N-acetylgalactosaminyltransferase N-acetyl-D-galactosamine A
B α-3-D-galactosyltransferase D-galactose B
the sugar responsible for H specificity (blood group O; In the formation of blood group A, the A gene
Fig. 6–4). The O gene at the ABO locus, which is sometimes (AA or AO) codes for the production of α-3-N-acetylgalac-
referred to as an amorph, does not elicit the production of a tosaminyltransferase, which transfers an N-acetyl-D-galac-
catalytically active polypeptide transferase; therefore, the H tosamine(GalNAc) sugar to the H substance. This sugar is
substance remains unmodified.13 Consequently, the O blood responsible for A specificity (blood group A; Fig. 6–5). The
group has the highest concentration of H antigen. The H A-specific immunodominant sugar is linked to a type 2
substance (L-fucose) must be formed for the other sugars to precursor substance that now contains H substance through
be attached in response to an inherited A and/or B gene. the action of the H gene.
The H gene is present in more than 99.99% of the random The A gene tends to elicit higher concentrations of trans-
population. Its allele, “h,” is quite rare, and the genotype hh is ferase than the B gene. This leads to the conversion of
extremely rare. The term Bombay has been used to refer to the practically all of the H antigen on the RBC to A antigen sites.
phenotype that lacks normal expression of the ABH antigens As many as 810,000 to 1,170,000 antigen sites exist on an
because of the inheritance of the hh genotype. The hh genotype A1 adult RBC in response to inherited genes.5 Individuals
does not elicit the production of α-2-L-fucosyltransferase. who are blood group B inherit a B gene (BB or BO) that codes
Therefore, L-fucose is not added to the type 2 chain, and H for the production of α-3-D-galactosyltransferase, which
substance is not expressed on the RBC. Even though Bombay attaches D-galactose (Gal) sugar to the H substance previ-
(hh) individuals may inherit ABO genes, normal expression, as ously placed on the type 2 precursor substance through the
reflected in the formation of A, B, or H antigens, does not action of the H gene.14 This sugar is responsible for B speci-
occur. (See “The Bombay Phenotypes” section.) ficity (blood group B; Fig. 6–6). Anywhere from 610,000 to
(6) CH2OH
5 0 GAL FUC Fucose:
Immunodominant
4 D-galactose 1
sugar responsible
GLNAC
3 2
"H" antigen for "H" specificity
0 (6) CH2OH
"1 4 Linkage" 5 0 GAL
4 N-acetylglucosamine 1
GL Protein
3 2
Type-2 precursor chain NHCOCH3
Ceramide
D-galactose
Glucose
Spectrin
HH L-fucosyl
( Precursor
Structure )
P.S.
Hh transferase
"H" antigen
(genotype inherited)
Figure 6–3. Type 2 precursor chain. Figure 6–4. Formation of the H antigen.
2682_Ch06_119-148 28/05/12 12:26 PM Page 125
ar
s ug
nt )
en i na m ine
g
a nti dom tosa
"A u no a lac
(i mm lg
ety
ac
N-
e
feras
a ns
l tr
i ny
s am
lacto
/AO l ga
AA ety
ac
"H structure" N- "B antigen"
Type-2-precursor chain HH / Hh
BB/BO
(immunodominant (immunodominant
(Precursor structure) L-fucosyl transferase D-galactosyl transferase
sugar L-fucose) sugar galactose)
Aa
nd
N- B
ge
ga ace ne
t
lac yl s
tos ga
yl lac
hh tra ost
ns am
fer in
as yl
es an
d "A
B
su an
ga tig
rs en
an N- "(
dg ac im
Precursor structure unchanged ala ety mu
cto lg no
se ala do
(Bombay phenotype) ) cto mina
sa nt
mi
ne
Comparison of A, B, and H Antigens on RBCs with Table 6–11 ABH Substance in the Saliva
A, B, and H Soluble Substances of Secretors (SeSe or Sese)*
The formation of soluble A, B, and H substances is the same SUBSTANCES IN SALIVA
as that described for the formation of A, B, and H antigens ABO Group A B H
on the RBCs, except for a few minor distinctions that are
compared in Table 6–10. O None None ↑↑
In the past, tests for ABH secretion have been used to A ↑↑ None ↑
establish the true ABO group of an individual whose RBC
antigens are poorly developed. The demonstration of A, B, B None ↑↑ ↑
and H substances in saliva is evidence for the inheritance of AB ↑↑ ↑↑ ↑
an A gene, B gene, H gene, and Se gene. The term secretor
refers only to secretion of A, B, and H soluble antigens * Nonsecretors (sese) have no ABH substances in saliva.
in body fluids. The glycoprotein-soluble substances (or ↑↑ and ↑, respectively, represent the concentration of ABH substances in saliva.
antigens) normally found in the saliva of secretors are listed
in Table 6–11. Both ABH red cell antigens and ABH soluble
Adding the same immunodominant sugars to the type
substances are formed due to the attachment of an immu-
1 and 3 chains in the body secretions allow for A, B, and H
nodominant sugar to an oligosaccharide chain. Although
soluble substances to be made in body secretions. Box 6–3
several types of oligosaccharide chains exist, types 1 and
summarizes the body fluids in which ABH-soluble sub-
3 are primarily associated with body secretions, while types
stances can be found.
2 and 4 are associated with the red cell membrane.22 It is
interesting to note that types 1 and 2 are more abundant, The procedure for determining the secretor status
and they differ only in the linkage position of galactose (Gal) (saliva studies) can be found as Procedure 6-1 on
to N-acetylglucosamine (GlcNAc); namely type 1 has a the textbook’s companion website.
beta 1→3 linkage and type 2 has a beta 1→4 linkage.22
Adding specific immunodominant sugars to the type 2 and
ABO Subgroups
4 chains leads to formation of A, B, and H antigens on the
red cell membrane, with the majority being present on type The original reports of most ABO subgroups were made
2 chains. before the availability of the monoclonal typing reagents
currently used in routine ABO grouping. ABO subgroups
represent phenotypes that show weaker variable serologic
Table 6–10 Comparison of ABH Antigens reactivity with the commonly used human polyclonal anti-
on RBCs with A, B, and H A, anti-B, and anti-A,B reagents.
Soluble Substances
A Subgroups
ABH ANTIGENS A, B, AND H SOLUBLE
ON RBCS SUBSTANCES
Basic Concepts
RBC antigens can be Secreted substances are
glycolipids, glycoproteins, glycoproteins.
In 1911, von Dungern described two different A antigens
or glycosphingolipids. based on reactions between group A RBCs and anti-A and
anti-A1.23 Group A RBCs that react with both anti-A and
RBC antigens are Secreted substances are primarily anti-A1 are classified as A1, whereas those that react with
synthesized only on synthesized on type 1 precursor
type 2 precursor chains. chains.12
anti-A and not anti-A1 are classified as A2 (Table 6–12 and
Figs. 6–9 and 6–10). RBCs from A1 and A2 individuals react
Type 2 chain refers to a Type 1 chain refers to a beta-1→3
beta 1→4 linkage in which linkage in which the number one
the number one carbon carbon of the galactose is attached
of the galactose is to the number three carbon of the BOX 6–3
attached to the number N-acetylglucosamine sugar of the
four carbon of the precursor substance.
Fluids in Which A, B, and H Substances can be
N-acetylglucosamine Detected in Secretors
sugar of the precursor • Saliva
substance.
• Tears
The enzyme produced The enzyme produced by the Se • Urine
by the H gene gene (α-2-L-fucosyltransferase) • Digestive juices
(α-2-L-fucosyltransferase) preferentially acts on type 1 chains • Bile
acts primarily on type in secretory tissues.
• Milk
2 chains, which are
prevalent on the • Amniotic fluid
RBC membrane. • Pathological fluids: pleural, peritoneal, pericardial, ovarian cyst
2682_Ch06_119-148 28/05/12 12:26 PM Page 128
Table 6–12 A1 Versus A2 Phenotypes gene elicits production of high concentrations of the
enzyme α-3-N-acetylgalactosaminyltransferase, which con-
REACTIONS OF PATIENT’S RBCS WITH
verts almost all of the H precursor structure to A1 antigens
Blood Anti-A Reagent Anti-A1 Lectin on the RBCs. The very potent gene A1 creates from 810,000
Group (anti-A plus anti-A1) Reagent to 1,170,000 antigen sites on the adult RBC; whereas
A1 + + inheriting an A2 gene, results in the production of only
240,000 to 290,000 antigen sites on the adult A2 RBC.10
A2 + 0 The immunodominant sugar on both A1 and A2 RBCs is
N-acetyl-D-galactosamine.
+ = positive (agglutination)
0 = negative (no agglutination) Qualitative differences also exist, since 1% to 8% of A2
individuals produce anti-A1 in their serum, and 22% to 35%
of A2B individuals produce anti-A1.8 This antibody can cause
discrepancies between forward and reverse ABO testing
equally strong with current reagent monoclonal anti-A in and incompatibilities in crossmatches with A1 or A1B cells.
ABO forward typing tests.2 Because anti-A1 is a naturally occurring IgM cold-reacting
The A subgroups are generally more common than B sub- antibody, it is unlikely to cause a transfusion reaction because
groups. The weaker serologic reactivity of ABO subgroups it usually reacts only at temperatures well below 37°C. It is
is attributed to the decreased number of A and B antigen sites considered clinically significant if it is reactive at 37°C.
on their red cells. Classification into A1 and A2 phenotypes It is now known through ABO genotyping that polymor-
accounts for 99% of all group A individuals. The cells of phism at the ABO locus results in subgroup alleles such as
approximately 80% of all group A (or AB) individuals are A1 the A2 allele, which is characterized by a single-base substi-
(or A1B), and the remaining 20% are A2 (or A2B) or weaker tution at nucleotide 467 and a single-base substitution at
subgroups. The differences between A1 and A2 are both nucleotide 1059. These substitutions alter the active site of
quantitative and qualitative (Table 6–13). the coding region and subsequently change the specificity of
The production of both types of antigens is a result of the A glycosyltransferase.24 It should be noted that in routine
an inherited gene at the ABO locus. Inheritance of an A1 forward grouping, the reagent anti-A strongly agglutinates
A1 A2
A1 A1 A + +
A2 A + 0
A1 A2
A1 A1 + +
A2 A + 0
2682_Ch06_119-148 28/05/12 12:26 PM Page 129
Table 6–13 Quantitative and Qualitative Weak subgroups of the A antigen will often have an
Differences of Subgroups A1 inverse reciprocal relationship between the amount of H
and A2 antigen on the RBC and the amount of A antigens formed
(i.e., the more A antigen formed, the less H antigen expressed
QUANTITATIVE QUALITATIVE on the RBC). The H antigen on the RBCs of A1 and A1B
• ↓ Number of antigen • Differences in the precursor individuals is so well hidden by N-acetyl-D-galactosamine
sites oligosaccharide chains that anti-H is occasionally found in the serum. This anti-H
• ↓ Amount of • Subtle differences in transferase is a naturally occurring IgM cold agglutinin that reacts best
transferase enzyme enzymes below room temperature. As can be expected, this antibody
is formed in response to a natural substance and reacts
• ↓ Amount of • Formation of anti-A1, in a most strongly with cells of group O individuals (which
branching percentage of some subgroups
have the greatest amount of H substance on their RBCs).
Anti-H reacts weakly with the RBCs of A1B individuals
(which contain small amounts of H substance). It is an insignif-
both A1 and A2 phenotypes. Differentiation of A1 and A2 phe- icant antibody in terms of transfusion purposes, because it has
notypes can be determined by using a reagent made from the no reactivity at body temperature (37°C). However, high-
seeds of the plant Dolichos biflorus, which serves as a source titered anti-H may react at room temperature and present a
of anti-A1. This reagent is known as anti-A1 lectin. problem in antibody screening procedures, because reagent
Lectins are seed extracts that agglutinate human cells screening cells are group O (see Chapter 9, “Detection and Iden-
with some degree of specificity. This reagent agglutinates tification of Antibodies”). This high-titered anti-H may also
A1 (or A1B) cells but does not agglutinate A2 (or A2B cells). present a problem with compatibility testing (see Chapter 10,
Box 6–4 lists the lectins used in blood banking. The charac- “Pretransfusion Testing”). Anti-H lectin from the extract of the
teristics of the A1 and A2 phenotypes are presented in plant Ulex europaeus closely parallels the reactions of human
Table 6–14. Because the A2 glycosyltransferase activity is anti-H. Both antisera agglutinate RBCs of group O and A2 and
weaker in adding the immunodominant sugar to the H anti- react very weakly or not at all with groups A1 and A1B.10 Group
gen precursor, A2 red cells will show increased reactivity in B cells give reactions of variable strength (Fig. 6–11).
comparison to A1 red cells with the reagent anti-H lectin.
H antigen is found in greatest concentration on the RBCs
of group O individuals. H antigen may not be detectable in
group A1 individuals, because in the presence of the A1 gene, Advanced Concepts
almost all of the H antigen is converted to A1 antigen by plac- The discussion thus far has presented a basic overview of the
ing the large N-acetyl-D-galactosamine sugar on the H sub- two major ABO subgroups, A1 and A2. A more plausible, yet
stance. Because of the presence of so many A1 antigens, the H more detailed, theory of ABO subgroups has been proposed
antigen on A1 and A1B RBCs may be hidden and therefore may by the identification of four different forms of H antigens,
not be available to react with anti-H antisera. In the presence two of which are unbranched straight chains (H1, H2)
of an A2 gene, only some of the H antigen is converted to A and two of which are complex branched chains (H3, H4;
antigens, and the remaining H antigen is detectable on the cell. Fig. 6–12).22,25 The antigens H1 through H4 correspond to
BOX 6–4
Lectins Used in Blood Banking O > A2 > B > A2B > A1 > A1B
• Dolichos biflorus—agglutinates A1 or A1B
• Bandeiraea simplicifolia—agglutinates B cells greatest least
amount of amount of
• Ulex europaeus—agglutinates O cells (H specificity) and other ABO H H
blood groups depending on the amount of H antigen available.
Figure 6-11. Reactivity of anti-H antisera or anti-H lectin with ABO blood groups.
GALNAC GALNAC
GlcNac GlcNac
Aa
H1
GAL GAL
Glc GlcNac Ab
H2
GAL
Ceramide
Glc
RBC
Ceramide
H1
RBC
GALNAC GALNAC H2
GlcNac Ad
Ceramide H4
RBC GAL
Glc
H3
Ceramide
Denotes immunodominant
sugar for H antigen RBC
Denotes immunodominant
sugar for A antigen
H4
the precursor structures on which the A enzyme can act to As stated previously, most group A infants appear to be
convert H antigen to blood group A active glycolipids. Al- A2 at birth, with subsequent development to A1 a few
though the chains differ in length and complexity of branch- months later. Newborns have a deficiency of the branched
ing, the terminal sugars giving rise to their antigenic H3 and H4 antigens and therefore the Ac and Ad antigens as
specificity are identical. Studies on the chemical and physical well, possibly accounting for the A2 phenotype. Adult cells
characteristics of the A1 and A2 enzyme transferases have contain a higher concentration of branched H3 and H4
demonstrated that these two enzymes are different qualita- structures and therefore Ac and Ad determinants of the
tively.13,14 Straight chain H1 and H2 glycolipids can be con- A antigen in A1 individuals.26
verted to Aa and Ab antigens, respectively, by both A1 and A2
enzymes, with the A2 enzyme being less efficient. The more
complex branched H3 and H4 structures can be converted to Weak A Subgroups
Ac and Ad antigens by A1 enzyme and only very poorly by A2
enzyme.25 As a result, more unconverted H antigens (specif- Basic Concepts
ically H3 and H4) are available on group A2 RBCs, and only Subgroups weaker than A2 occur infrequently and are most
Aa and Ab determinants are formed from H1 and H2 structures.25 often recognized through an ABO discrepancy (unexpected
On the RBCs of some A2 individuals, Ac is extremely low reactions in the forward and reverse grouping). These
and Ad is completely lacking (Box 6–5). These are the indi- subgroups of A make up 1% of those encountered in the
viduals in whom one would likely find anti-A1 in the serum. laboratory and therefore are mainly of academic interest.27
This anti-A1 antibody could really be an antibody to Ac and Characteristics of weak A subgroups include:
Ad determinants, which these A2 individuals lack. Also, in
22% to 35% of A2B individuals, anti-A1 can be found in the • Decreased number of A antigen sites per RBC (resulting
serum. Since the B enzyme transferase is usually more efficient in weak or no agglutination with human polyclonal
than the A enzyme in converting H structures to the appro- anti-A)
priate antigen, A2 enzymes would probably fail completely • Varying degrees of agglutination by human anti-A,B8
when paired with a B enzyme. As a result, A2B individuals • Increased variability in the detectability of H antigen,
would be far more likely to lack Ac and Ad components with resulting in strong reactions with anti-H
subsequent production of anti-Ac and anti-Ad (anti-A1). • Presence or absence of anti-A1 in the serum
Secretor studies, adsorption-elution tests, and molecular
testing can be utilized to subdivide A individuals into A3,
Ax, Aend, etc.28 (Table 6–15).
BOX 6–5
Occasionally, weak subgroups of A may present practical
Structural Characteristics of A1 and A2 RBCs problems; for example, if an Ax donor was mistyped as a
• A2 RBCs: Predominantly Aa and Ab and unconverted H3 and H4 group O and was transfused to a group O patient. This is
antigen sites potentially dangerous because the group O patient pos-
• A1 red cells: Aa, Ab, Ac, and Ad determinants and no unconverted sesses anti-A,B, which agglutinates and lyses Ax RBCs,
H3 and H4 antigen sites causing rapid intravascular hemolysis.
A3 Aend Ax Am Ay Ael
ABO DISCREPANCY
Forward and Reverse testing
do not match as expected.
Note: The initial testing was
performed using patient’s RBCs If an error in specimen collection
suspended in serum or plasma. and identification is suspected.
body production or cannot produce the ABO antibodies. centrifugation, the serum-cell mixtures can be incubated at
Common populations with discrepancies in this group are: 4°C for 15 minutes. An auto control and O cell control must
always be tested concurrently with the reverse typing when
• Newborns (the production of ABO antibodies is not
trying to solve the discrepancy, since the lower temperature
detectable until 4 to 6 months of age)
of testing will most likely enhance the reactivity of other
• Elderly patients (the production of ABO antibodies is
commonly occurring cold agglutinins (such as anti-I)
depressed)
that react with all adult RBCs (see Chapter 8). Table 6–17
• Patients with a leukemia (e.g., chronic lymphocytic
shows a type of discrepancy that may be seen with weak or
leukemia) or lymphoma (e.g., malignant lymphoma)
missing antibodies.
demonstrating hypogammaglobulinemia
The red cell results present a group O individual and
• Patients using immunosuppressive drugs that yield
the serum results present an AB individual. Since serum
hypogammaglobulinemia
problems are more common, it is more likely that the serum
• Patients with congenital or acquired agammaglobulinemia
immunoglobulins are decreased.
or immunodeficiency diseases
• Patients with bone marrow or stem cell transplantations Group II Discrepancies
(patients develop hypogammaglobulinemia from therapy
Group II discrepancies are associated with unexpected reac-
and start producing a different RBC population from that
tions in the forward grouping due to weakly reacting or miss-
of the transplanted bone marrow)
ing antigens. This group of discrepancies is probably the least
• Patients whose existing ABO antibodies may have been
frequently encountered. Some of the causes of discrepancies
diluted by plasma transfusion or exchange transfusion
in this group include:
• ABO subgroups
• Subgroups of A (or B) may be present (see the “ABO
Resolution of Common Group I Discrepancies Subgroups” section)
Obtaining the patient’s history may resolve this type of dis- • Leukemias may yield weakened A or B antigens (Table 6–18),
crepancy, such as a newborn sample that would not have and Hodgkin’s disease has been reported in some cases to
ABO antibodies in the serum until the child was 4 to mimic the depression of antigens found in leukemia.
6 months of age. If the history indicates an elderly individ- • The “acquired B” phenomenon will show weak reactions
ual, or the diagnosis indicates hypogammaglobulinemia, with anti-B antisera and is most often associated with diseases
then the best way to resolve this discrepancy is to enhance of the digestive tract (e.g., cancer of the colon). Table 6–19
the weak or missing reaction in the serum. This is usually shows the ABO testing results of an acquired B phenomenon.
performed by incubating the patient serum with reagent A1
and B cells at room temperature for approximately 15 to Resolution of Common Group II Discrepancies
30 minutes or by adding one or two drops more plasma The agglutination of weakly reactive antigens with the
or serum to the test. If there is still no reaction after reagent antisera can be enhanced by incubating the test mix-
Table 6–17 Example of ABO Discrepancy Seen With Weak or Missing Antibodies
FORWARD GROUPING REACTION REVERSE GROUPING REACTION
OF PATIENT’S CELLS WITH OF PATIENT’S SERUM WITH
Anti-A Anti-B A1 Cells B Cells
Patient 0 0 0 0
Patient’s probable group: O (elderly patient or newborn)
Note: The absence of agglutination with reagent cells in the reverse type is because the production of ABO antibodies can be weak or absent in the elderly.
Resolution: (1) Check age of the patient. (2) Increase incubation time to 30 minutes (not appropriate for newborn sample). (3) Lower the temperature to 4°C for 15 minutes (include O cells and
an autocontrol).
A +mf 0 0 3+
B 0 ±/+ 4+ 0
Note: Weak reactivity with anti-A and anti-B is because the disease, leukemia, has resulted in the weakened expression of the corresponding antigen.
2682_Ch06_119-148 28/05/12 12:26 PM Page 139
Patient 4+ 2+ 0 4+
Patient’s probable group: A
Note: Patient RBCs have acquired a B-like antigen that reacts with reagent anti-B and is associated with cancer of the colon or other diseases of the digestive tract.
Resolution: (1) Acidify Anti-B reagent to a pH of 6. (2) Run DAT (refer to Chapter 5, “The Antiglobulin Test”). (3) Run autocontrol.
ture at room temperature for up to 30 minutes, which will Rare Group II Discrepancies
increase the association of the antibody with the RBC
antigen. If it is still negative, incubate the text mixture at Advanced Concepts
4°C for 15 to 30 minutes. Include group O and autologous Weakly reactive or missing reactions in RBC grouping may be
cells as controls. RBCs can also be pretreated with enzymes due to excess amounts of blood group–specific soluble (BGSS)
and retested with reagent antisera. substances present in the plasma, which sometimes occurs
The acquired B antigen arises when bacterial enzymes with certain diseases, such as carcinoma of the stomach and
modify the immunodominant blood group A sugar pancreas. Excess amounts of BGSS substances will neutralize
(N-acetyl-D-galactosamine) into D-galactosamine, which is the reagent anti-A or anti-B, leaving no unbound antibody to
sufficiently similar to the group B sugar (D-galactose) and react with the patient cells. This yields a false-negative or weak
cross-reacts with anti-B antisera. This pseudo-B antigen is reaction in the forward grouping. Washing the patient cells free
formed at the expense of the A1 antigen and disappears after of the BGSS substances with saline should alleviate the prob-
recovery.39 The reaction of the appropriate antiserum with lem, resulting in correlating forward and reverse groupings.
these acquired antigens demonstrates a weak reaction, often Antibodies to low-incidence antigens in reagent anti-A or
yielding a mixed-field appearance (see Table 6–19). anti-B may also result in weakly reactive or missing reactions
Blood group reagents of a monoclonal anti-B clone (ES4) in RBC grouping (Table 6–20). It is impossible for manufac-
strongly agglutinate cells with the acquired B antigen. The turers to screen reagent antisera against all known RBC anti-
pH of reagents containing ES4 has been lowered; conse- gens. It has been reported (although rarely) that this additional
quently, only those cells with the strongest examples of antibody in the reagent antisera has reacted with the corre-
acquired B antigen react with the antisera. Testing the sponding low-incidence antigen present on the patient’s RBCs.
patient’s serum or plasma against autologous RBCs gives a This gives an unexpected reaction of the patient’s cells with
negative reaction, because the anti-B in the serum does not anti-A or anti-B, or both, mimicking the presence of a weak
agglutinate the patient’s RBCs with the acquired B antigen. antigen. The best way to resolve this discrepancy is by repeat-
The acquired B antigen is also not agglutinated when ing the forward type, using antisera with a different lot num-
reacted with anti-B that has a pH greater than 8.5 or ber. If the cause of the discrepancy is a low-incidence antibody
less than 6.40 Secretor studies can be performed when trying in the reagent antisera reacting with a low-incidence antigen
to characterize the acquired B phenomenon. If the patient on the patient’s cells, the antibody probably will not be present
is in fact a secretor, only the A substance is secreted in in a different lot number of reagent. This is only seen when
the acquired B phenomenon. Treating RBCs with acetic human source antiserum is used. Most ABO reagents in use
anhydride reacetylates the surface molecules, then markedly today are monoclonal antibodies, and these reagents are free
decreases the reactivity of the cells tested with anti-B. The of contaminating antibodies to low-incidence antigens.
reactivity of normal B cells is not affected by treatment with Chimerism is defined as the presence of two cell popu-
acetic anhydride.24 lations in a single individual (Table 6–21). It was discovered
Table 6–20 Example of ABO Discrepancy Caused by Low-Incidence Antibodies in the Reagent
Antisera
FORWARD GROUPING REACTION OF REVERSE GROUPING REACTION OF
PATIENT CELLS WITH PATIENT SERUM WITH
Anti-A Anti-B A1 Cells B Cells
Patient 0 1+ 4+ 4+
Note: Reaction with anti-B in the forward type is due to agglutination between a low-incidence antibody in reagent anti-B and the corresponding antigen on the patient’s cells.
Resolution: Use a different lot number for reagent Anti-B
2682_Ch06_119-148 28/05/12 12:26 PM Page 140
in twins (born to a group O mother and group B father) • Plasma expanders, such as dextran and polyvinylpyrrolidone
who had a mixture of both B and O cells instead of the ex- • Wharton’s jelly in cord blood samples
pected group of either B or O. Detecting a separate cell pop- • Table 6–22 shows an example of ABO discrepancy caused
ulation may be easy or difficult, depending on what by rouleaux formation.
percentage of cells of the minor population are present.
Reactions from chimerism are typically mixed field. Resolution of Common Group III Discrepancies
True chimerism, which occurs in twins, is rarely found, Rouleaux is a stacking of erythrocytes that adhere in a coin-
and the two cell populations will exist throughout the lives like fashion, giving the appearance of agglutination. It can be
of the individuals. In utero exchange of blood occurs observed on microscopic examination (Fig. 6–15). Cell
because of vascular anastomosis. As a result, two cell grouping can usually be accomplished by washing the
populations emerge, both of which are recognized as self, patient’s RBCs several times with saline. Performing a saline
and the individuals do not make anti-A or anti-B. There- replacement technique will free the cells in the case of
fore, expected isoagglutinins are not present in the reverse rouleaux formation in the reverse type. In this procedure,
grouping, depending on the percentage of the population serum is removed and replaced by an equal volume of saline.
of red cells that exist in each twin. If the patient or donor In true agglutination, RBC clumping will still remain after the
has no history of a twin, then the chimera may be due to addition of saline. Rouleaux can be a nuisance in the labora-
dispermy (two sperm fertilizing one egg) and indicates tory, since it is an in vitro problem observed during laboratory
mosaicism. More commonly, artificial chimeras occur,
which yield mixed cell populations as a result of:
• Blood transfusions (e.g., group O cells given to an A or
B patient)
• Transplanted bone marrows or peripheral blood stem
cells of a different ABO type
• Exchange transfusions
• Fetal-maternal bleeding
Patient 4+ 4+ 2+ 2+
Note: Agglutination with A1 and B cells in reverse type is due to rouleaux formation as a result of increased serum protein or plasma abnormalities.
Resolution: (1) Microscopic examination (2) Saline replacement technique (3) Wash cells with saline three times (4) Run antibody screen (refer to Chapter 9)
2682_Ch06_119-148 28/05/12 12:26 PM Page 141
testing. It is not an in vivo problem for the patient. (Refer to • Unexpected ABO isoagglutinins
companion website for the saline replacement procedure.) • Unexpected non-ABO alloantibodies
Cord blood samples received in the laboratory can
also pose a problem in ABO testing, since cord cells may be Resolution of Common Group IV Discrepancies
contaminated with a substance called Wharton’s jelly, which Potent cold autoantibodies can cause spontaneous agglutina-
may cause the red cells to aggregate. Washing cord cells six tion of the patient’s cells. These cells often yield a positive
to eight times with saline should alleviate spontaneous direct Coombs’ or antiglobulin test (see Chapter 20, “Autoim-
rouleaux due to Wharton’s jelly. This substance is a viscous mune Hemolytic Anemias”). If the antibody in the serum reacts
mucopolysaccharide material present on cord blood cells, with all adult cells—for example, anti-I—the reagent A1 and B
and thorough washing should result in an accurate ABO cells used in the reverse grouping also agglutinate.
grouping. However, because testing is usually not performed To resolve this discrepancy, the patient’s RBCs could be
on cord serum (because the antibodies detected are usually incubated at 37°C for a short period, then washed with saline
of maternal origin), reverse grouping may still not correlate at 37°C three times and retyped. If this is not successful in
with the RBC forward grouping. resolving the forward type, the patient’s RBCs can be treated
Group IV Discrepancies with 0.01 M dithiothreitol (DTT) to disperse IgM-related ag-
glutination. As for the serum, the reagent RBCs and serum can
These discrepancies between forward and reverse groupings
be warmed to 37°C, then mixed, tested, and read at 37°C. The
are due to miscellaneous problems and have the following
test can be converted to the antihuman globulin phase if nec-
causes:
essary. Weakly reactive anti-A or anti-B may not react at 37°C,
• Cold reactive autoantibodies in which RBCs are so heavily which is outside their optimum thermal range. If the reverse
coated with antibody that they spontaneously agglutinate, typing is still negative (and a positive result was expected), a
independent of the specificity of the reagent antibody cold autoabsorption (patient cells with patient serum) could
(Fig. 6–16 and Table 6–23). be performed to remove the cold autoantibody from the
• Patient has circulating RBCs of more than one ABO group serum. The absorbed serum can then be used to repeat the
due to RBC transfusion or marrow/stem cell transplant serum typing at room temperature. (Refer to Chapter 9 for
cold autoadsorption and alloadsorption with rabbit erythro-
cyte stroma [REST] for the removal of cold autoantibodies.)
Unexpected ABO isoagglutinins in the patient’s serum react
at room temperature with the corresponding antigen present
on the reagent cells (Table 6–24). Examples of this type of ABO
discrepancy include A2 and A2B individuals, who can produce
naturally occurring anti-A1, or A1 and A1B, individuals who
may produce naturally occurring anti-H. (Refer to the previous
sections on ABO subgroups.) Serum grouping can be repeated
using at least three examples of A1, A2, B cells; O cells; and an
autologous control (patient’s serum mixed with patient’s
RBCs).3 The specificity of the antibody can be determined by
examining the pattern of reactivity (e.g., if the antibody agglu-
tinates only A1 cells, it can most likely be identified as anti-A1).
The patient’s RBCs can be tested with Dolichos biflorus to
confirm the presence of the ABO subgroup. Dolichos biflorus
will agglutinate cells of the A1 but not the A2 phenotype.
Unexpected alloantibodies in the patient’s serum other
than ABO isoagglutinins (e.g., anti-M) may cause a discrep-
Figure 6–16. Autoagglutination in a patient with cold agglutinin disease. ancy in the reverse grouping (Table 6–25). Reverse grouping
Patient 2+ 4+ 4+ 2+
Note: Reaction with anti-A in forward type is due to spontaneous agglutination of antibody coated cells; reaction with B cells in reverse type is due to cold autoantibody (e.g., anti-I) reacting with I
antigen on B cells.
Resolution: (1) Wash patient cells with warm saline and retest; (2) Run DAT and autocontrol; (3) Run antibody screen (refer to Chapter 9)
2682_Ch06_119-148 28/05/12 12:26 PM Page 142
Patient 4+ 4+ 1+ 0
Note: Reactions with patient serum are due to anti-A1 agglutinating A1 reagent red cells.
Resolution: (1) Test cells with anti-A1 lectin; (2) Test serum with A1, A2, and O cells; (3) Run an autocontrol
Patient 4+ 4+ 1+ 1+
Note: Reactions with patient serum are due to non-ABO alloantibody agglutinating an antigen other than A1 and B on reagent red cells.
Resolution: (1) Run an antibody screen and panel (refer to Chapter 9)
cells possess other antigens in addition to A1 and B, and it is RBCs with the cis-AB phenotype (a rare occurrence)
possible that other unexpected antibodies present in the pa- express a weakly reactive A antigen (analogous to A2
tient’s serum will react with these cells. In this situation, an cells) and a weak B antigen.4 The B antigen usually yields
antibody identification panel should be performed with the a weaker reaction with the anti-B from random donors,
patient’s serum. Once the unexpected alloantibodies are with mixed-field agglutination typical of subgroup B3
identified, reagent A1 and B cells negative for the correspon-
ding antigen can be used in the reverse grouping.
reported in several cases. Weak anti-B (present in the ABO locus is normal. There have been other examples
the serum of most cis-AB individuals) leads to an ABO of cis-ABs that do not fit the above scenario. In these
discrepancy in the reverse grouping. The serum of most examples, there was a point mutation at the ABO locus,
cis-AB individuals contains a weak anti-B, which reacts and an enzyme was produced that was capable of transfer-
with all ordinary B RBCs, yet not with cis-AB RBCs. A ring both A-specific and B-specific sugars to the precursor
and B transferase levels are lower than those found in molecule.41 Many families have been reported in other parts
ordinary group AB sera.4 of the world, with a high incidence of cis-AB being found
Various hypotheses have been offered to explain the in Japan.
cis-AB phenotype. Many favor an unequal crossing over Table 6–26 provides some examples of serologic reac-
between the A and B gene with gene fusion and the forma- tions involving ABO discrepancies, with possible causes
tion of a new gene. However, the banding pattern of the and resolution steps. Figure 6–18 provides a simplified
distal end of the long arm of chromosome 9 representing summary of ABO discrepancies.
*AutoAbsorption should not be performed on patient’s cells that have been transfused within the last 3 months.
RT = room temperature
2682_Ch06_119-148 28/05/12 12:26 PM Page 145
Forward Reverse
(RBCs) (plasma)
Passively
Acquired Antibody
(i.e. plasma exchange,
Figure 6–18. Simplified Summary of ABO mismatched
Discrepancies platelets)
SUMMARY CHART
ABO frequencies: group O, 45%; group A, 40%; group Group O persons have the greatest amount of H
B, 11%; group AB, 4%. substance; group A1B persons contain the least amount
ABO blood group system has naturally occurring of H substance.
antibodies that are primarily IgM. Approximately 80% of the individuals inherit the A
ABO genes, like those of most other blood groups, are gene phenotype as A1; the remaining 20% phenotype
inherited in a codominant manner. as A2 or weaker subgroups.
ABH-soluble antigens are secreted by tissue cells and Approximately 1% to 8% of A2 persons produce anti-
are found in all body secretions. The antigens secreted A1 in their serum.
depend on the person’s ABO group. Glycoproteins in secretions are formed on type
ABO reverse grouping is omitted from cord blood test- 1 precursor chains.
ing on newborns, because their antibody titer levels The ABH antigens on RBCs are formed on type 2
are generally too low for detection. precursor chains.
ABO RBC antigens can be glycolipids, glycoproteins, Forward and reverse grouping normally yield strong
or glycosphingolipids; ABO-secreted substances are (3+ to 4+) reactions.
glycoproteins. Group A persons have anti-B in their serum; group B
L-fucose is the immunodominant sugar responsible for persons have anti-A in their serum; group AB persons
H specificity. have neither anti-A nor anti-B in their serum; group O
N-acetylgalactosamine is the immunodominant sugar persons contain both anti-A and anti-B in their serum.
responsible for A specificity. Approximately 78% of the random population inherit
D-galactose is the immunodominant sugar responsible the Se gene and are termed secretors; the remaining
for B specificity. 22% inherit the se gene and are termed nonsecretors.
The hh genotype is known as the Bombay phenotype, The Se gene codes for the production of L-fucosyltrans-
or Oh, and lacks normal expression of the ABH antigens. ferase.
2. The major immunoglobulin class(es) of anti-B in a group Patient Cells With Patient Serum With
A individual is (are):
Anti-A Anti-B Anti-A1 A1 cells B cells
a. IgM
b. IgG 4+ 4+ Neg 2+ Neg
c. IgM and IgG
d. IgM and IgA The reactions above may be seen in a patient who is:
3. What are the possible ABO phenotypes of the offspring a. A1 with acquired B
from the mating of a group A to a group B individual? b. A2B with anti-A1
c. AB with increased concentrations of protein in the
a. O, A, B
serum
b. A, B
d. AB with an autoantibody
c. A, B, AB
d. O, A, B, AB
2682_Ch06_119-148 28/05/12 12:26 PM Page 147
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44. Cho, D, Yazer, MH, Shin, M, et al: Dispermic chimerism in revisited: a review and update. Immunohematology, vol
a blood donor with apparent B3 blood group and mosaic 47, 25(2):48–59, 2009.
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Chapter 7
The Rh Blood Group System
Susan T. Johnson, MSTM, MT(ASCP)SBB and Merilyn Wiler, MA, MT(ASCP)SBB
OBJECTIVES
Explain the derivation of the term Rh.
1. Differentiate Rh from LW blood group systems.
2. Compare and contrast the Fisher-Race and Wiener theories of Rh inheritance.
3. Translate the five major Rh antigens, haplotypes, and predicted haplotypes, from one nomenclature to another, including
Fisher-Race, Wiener, Rosenfield, and ISBT.
4. Define the basic biochemical structure of Rh.
5. Compare and contrast the genetic pathways for the regulator type of Rhnull and the amorphic Rhnull.
6. Describe and differentiate five mechanisms that result in weakened expression of D on red blood cells.
7. List one instance in which the weak-D status of an individual must be determined.
8. List and differentiate four types of Rh typing reagents, and provide two advantages of each type.
9. Define three characteristics of Rh antibodies.
10. Describe three symptoms associated with an Rh hemolytic transfusion reaction.
11. Compare and contrast Rhnull and Rhmod and describe the role of RhAG in Rh antigen expression.
12. List four Rh antigens (excluding DCcEe), and give two classic characteristics of each antigen.
13. Determine the most probable genotype of an individual when given the individual’s red blood cell typing results, haplotype
frequencies, and ethnicity.
149
2682_Ch07_149-171 22/05/12 11:43 AM Page 150
Table 7–1 Frequency of Common Rh It is essential to remember that d does not represent an anti-
Antigens in Caucasians gen but simply represents the absence of D antigen. C, c, E,
and e represent actual antigens recognized by specific anti-
ANTIGEN GENE FREQUENCY (%) bodies. There has never been an antibody that recognizes
D 85 d antigen, supporting the fact that d antigen does not exist.
Further discussion on the absence of D follows later in the
No D (absence of D) 15 chapter. For many students and working laboratory scientists,
C 70 the Fisher-Race terminology represents the easiest way to
think about the five major Rh system antigens, but it has
E 30 shortcomings. Many antigens assigned to the Rh blood group
c 80 system were given names using a variety of terminologies. In
addition, as the number of Rh antigens continues to grow, the
e 98 original Fisher-Race terminology is becoming too limiting.
In very rare instances, an individual may fail to express
any allelic antigen at one or both Rh loci; that is, a person
may lack E and e, or all CcEe antigens. The probable geno-
absence of D antigen; however, the term continues to be type for the Rh (D)-positive person exhibiting a deletion
utilized with Fisher-Race terminology as a placeholder. The phenotype such as these is written DC- or Dc-, or D–. A
phenotype (antigens expressed on the RBC detected by typ- deletion of Cc with Ee has not been reported. The person
ing) of a given RBC is defined by the presence of D, C, c, E, expressing no Rh antigens on the RBC is said to be Rhnull,
and e expression. The gene frequency in the Caucasian pop- and the phenotype may be written as —/—. Weakened
ulation for each Rh antigen is given in Table 7–1, and the Rh expression of all Rh antigens of an individual has also been
haplotype (the complement of genes inherited from either reported. These individuals are said to have the Rhmod
parent) frequencies are given in Table 7–2. Notice how the phenotype. Placing parenthesis around (D), (C), and (e)
frequencies vary with ethnic background. indicates weakened antigen expression.
According to the Fisher-Race theory, each person inherits
a set of Rh genes from each parent (i.e., one D or d, one C Wiener: Rh-Hr Terminology
or c, and one E or e) (see Fig. 7–1). Because Rh genes were
thought to be codominant, each inherited gene expresses In his early work defining the Rh antigens, Wiener9 believed
its corresponding antigen on the RBC. The combination there was one gene responsible for defining Rh that produced
of maternal and paternal haplotypes determines one’s an agglutinogen containing a series of blood factors. Accord-
genotype (the Rh genes inherited from each parent) and ing to Weiner, this Rh gene produced at least three factors
dictates one’s phenotype. An individual’s Rh phenotype is within an agglutinogen (Fig. 7–2). The agglutinogen may be
reported as DCE rather than CDE because Fisher postulated considered the phenotypic expression of the haplotype. Each
that the C/c locus lies between the D/d and E/e loci. This factor is an antigen recognized by an antibody. Antibodies
information is based on frequencies of the various gene can recognize single or multiple factors (antigens).
combinations. Table 7–3 lists the major agglutinogens and their respective
factors, along with the shorthand term that has come to repre-
sent each agglutinogen. Wiener’s terminology is complex and
Table 7–2 Fisher-Race Haplotypes of the unwieldy; nevertheless, many blood bankers use modified
Rh System Wiener terminology interchangeably with other nomencla-
tures. This terminology allows one to convey Rh antigens
PREVALENCE (%)
inherited on one chromosome or haplotype and makes it easier
Haplotype White Black Asian to discuss a genotype. A medical laboratory scientist conveying
DCe 42 17 70 a probable genotype to a coworker would have to say DcE/DcE.
However, R2R2 is much easier to verbally communicate.
dce 37 26 3
DcE 14 11 21
Agglutinogen Rh Factor
Dce 4 44 3
Factor Rh0 Rh0
dCe 2 2 2
dcE 1 < 0.01 < 0.01 Rh° gene Factor hr' hr'
Modified from Roback, JD, Combs, MR, Grossman, B, Hillyer, C (eds): Technical Manual, Figure 7–2. Wiener’s agglutinogen theory. Antibody will recognize each factor
16th ed. AABB, Bethesda, MD, 2008. within the agglutinogen.
2682_Ch07_149-171 22/05/12 11:43 AM Page 152
rh rh hr’hr’’ r ce
Fisher-Race nomenclature may be converted to Wiener Standard type is used to describe the gene product or
nomenclature and vice versa. It is important to remember agglutinogen. Subscripts are used with the uppercase
that an agglutinogen in Wiener nomenclature actually rep- R and superscripts with the lowercase r (i.e., R1 or R2 or r⬘).
resents the presence of a single haplotype expressing three Phenotypes of Rhnull and Rhmod are written as stated. The
different antigens (see Table 7–3). When describing an genotype for the Rhnull that arises from an amorphic gene
agglutinogen, the uppercase R denotes the presence of the at both Rh loci is written as 苷 rr and pronounced “little r
original factor, the D antigen. The lowercase r indicates the double bar.”
absence of D antigen. The presence of uppercase C is indicated When referring to the Rh antigens (or blood factors) in
by a 1 or a single prime (⬘). Lowercase c is implied when Wiener nomenclature, the single prime (⬘) refers to either
there is no 1 or ⬘ indicated. (It is assumed that the third anti- C or c and the double prime (⬙) to either E or e. If the r pre-
gen is e). That is, R1 is the same as DCe; r⬘ denotes Ce; and cedes the h (i.e., rh⬘ or rh⬙), this refers to the C or E antigens,
R0 is equivalent to Dce. The presence of E is indicated by the respectively. When the h precedes the r, this refers to either
Arabic number 2 or double prime (⬙). Lowercase e is implied the c (hr⬘) or e (hr⬙) antigen. Rh0 is equivalent to D. In the
when there is no 2 or ⬙ indicated—that is, R2 is the same as Wiener nomenclature, there is no designation for the absence
DcE; r⬙ denotes cE, and r is equivalent to ce. (Again, it is of D antigen. By using these designations, the laboratorian
assumed that a c antigen is present.) When both C and E are should be able to recognize immediately which antigens are
uppercase, the letter z or y is used. Rz denotes DCE, whereas present on the RBCs described. However, it is difficult to use
ry represents CE. See Table 7–4 for a summary of this short- the Wiener nomenclature to adequately describe additional
hand nomenclature. alleles within an agglutinogen. Because of this, many of the
Italics or superscripts are used when describing Rh more recently described antigens of the Rh system have not
genes in the Wiener nomenclature (i.e., R1 or R2, R1 or R2). been given Rh-Hr designations.
R 1 + + 0 0 + R1
r ‘ 0 + 0 0 + r’
R 2 + 0 + + 0 R2
r ‘’ 0 0 + + 0 r’’
R Z + + + 0 0 Rz
r y 0 + + 0 0 ry
R 0 + 0 0 + + R0
r None 0 0 0 + + r
As the Rh blood group system expanded, it became more As the world of blood transfusion began to cooperate and
difficult to assign names to new antigens using existing share data, it became apparent there was a need for a univer-
terminologies. In the early 1960s, Rosenfield and associates10 sal language. The International Society of Blood Transfusion
proposed a system that assigns a number to each antigen of (ISBT) formed the Committee on Terminology for Red Cell
the Rh system in order of its discovery or recognized rela- Surface Antigens. Its mandate was to establish a uniform
tionship to the Rh system (Table 7–5). This system has no nomenclature that is both eye- and machine-readable and is
genetic basis, nor was it proposed based on a theory of Rh in keeping with the genetic basis of blood groups.11 The ISBT
inheritance, but it simply demonstrates the presence or adopted a six-digit number for each authenticated antigen
absence of the antigen on the RBC. A minus sign preceding belonging to a blood group system. The first three numbers
a number designates the absence of the antigen. If an antigen represent the system and the remaining three the antigenic
has not been typed, its number will not appear in the specificity. Number 004 was assigned to the Rh blood group
sequence. An advantage of this nomenclature is that the RBC system, and then each antigen assigned to the Rh system was
phenotype is thus succinctly described. given a unique number to complete the six-digit computer
For the five major antigens, D is assigned Rh1, C is Rh2, number. Table 7–6 provides a listing of these numbers.
E is Rh3, c is Rh4, and e is Rh5. For RBCs that type D + C When referring to individual antigens, an alphanumeric
+ E + c negative, e negative, the Rosenfield designation designation similar to the Rosenfield nomenclature may be
is Rh: 1, 2, 3, –4, –5. If the sample was not tested for used. The alphabetic names formerly used were left unchanged
e, the designation would be Rh: 1, 2, 3, –4. All Rh system but were converted to all uppercase letters (e.g., Rh, Kell
antigens have been assigned a number. became RH, KELL). Therefore, D is RH1, C is RH2, and so
The numeric system is well suited to electronic data pro- forth. (Note: There is no space between the RH and the
cessing. Its use expedites data entry and retrieval. Its primary assigned number.)
limiting factor is that there is a similar nomenclature for The phenotype designation includes the alphabetical
numerous other blood groups such as Kell, Duffy, Kidd, symbol that denotes the blood group, followed by a colon
Lutheran, Scianna, and more. K:1,2 refers to the K and k and then the specificity numbers of the antigens defined.
antigens of the Kell blood group system. Therefore, when A minus sign preceding the number indicates that the
using the Rosenfield nomenclature on the computer, one antigen was tested for but was not present. The phenotype
must use both the alpha (Rh:, K:) and the numeric (1, 2, –3, D + C – E + c + e + or DcE/ce or R2r would be written
etc.) to denote a phenotype. RH:1, –2, 3, 4, 5.
Modified from the Blood Group Antigen Facts Book. Elsevier, San Diego, CA, 2004, p 113–114, with permission.
2682_Ch07_149-171 22/05/12 11:43 AM Page 154
Rh6 ce hr 004006 f
Rh20 VS es 004020
Rh21 CG 004021
Rh24† ET 004024
Rh25*/† 004025
*Rh25 was formerly assigned to the LW antigen. LW is now known as LWa and is no longer considered a member of the Rh system.
†
Obsolete names: Rh13, Rh14, Rh15, Rh16 former classification of partial D types, Rh 24, Rh25 formerly LW, Rh38 formerly Duclos.
When referring to a gene, an allele, or a haplotype, the possible genotypes that can occur with the given test results
symbols are italicized. A haplotype is followed by a space or are also listed, but they are not commonly seen.
an asterisk, and then the numbers of the specificities are Determining probable or predicted genotypes was useful
separated by commas. The R1 haplotype or DCe would be for parentage studies, also known as relationship testing, and
RH 1,2,5 or RH*1,2,5. for population studies. Other molecular methods are proving
more powerful today. Probable genotypes are useful in
Overview of Rh Terminologies predicting the potential for HDFN in offspring of Rh-negative
women with anti-D; however, molecular testing, commonly
Blood bankers must be familiar with Fisher-Race, Wiener, referred to as zygosity testing, can be performed to confirm
Rosenfield, and ISBT nomenclatures and must be able to whether the father possesses one or two copies of the
translate among them when reading about, writing about, or RHD gene.
discussing the Rh blood group system. There are substantial differences in phenotypes and
Tables 7–4, 7–5, and 7–6 summarize the data presented predicted genotypes of various populations. For example,
in this section. These tables also include probable genotypes the phenotype D+C-E-c+e+ is most commonly seen in
based on the antigens found in selected RBC populations. the black population but is considered relatively rare in
Table 7–7 correlates Rh phenotypes with the most probable whites (see Tables 7–5 and 7–7). These differences must be
or predicted genotype in a designated population. Results remembered when trying to locate compatible blood for
of typing do not define genotype, only phenotype. Other recipients with unusual or multiple Rh antibodies.
2682_Ch07_149-171 22/05/12 11:43 AM Page 156
To further emphasize the interchangeable use of the Rh system. Two theories of Rh genetic control were initially
terminologies for the basic antigens, see Table 7–5, which postulated. Wiener hypothesized that a single gene produces
defines common genotypes using the Fisher-Race, Wiener, a single product that contains separately recognizable factors
and Rosenfield nomenclatures. Frequencies listed are for (see Fig 7–2). In contrast, Fisher and Race proposed that the
those found in the Caucasian population. Rh locus contains three distinct genes that control
Finally, as the genetics and biochemistry of the Rh blood production of their respective antigens (see Fig 7–1). Later,
group system have been unraveled, the terminology has Tippett correctly proposed two RH genes, RHD and RHCE,
continued to change. For consistency of use, RHD and that control expression of Rh antigens.12
RHCE, all uppercase and in italics, will be used from this
point forward in the text to indicate genes. RhD, RhCe, RH Genes
RhcE, Rhce, and RhCE will be used to designate proteins on
which the Rh antigens reside. It is now known that only two closely linked genes located on
chromosome 1 control expression of Rh proteins—namely,
Molecular Genetics RHD and RHCE.13–15 The gene RHD codes for the presence or
absence of the RhD protein, and the second gene RHCE codes
Several theories have been described to explain genetically for either RhCe, RhcE, Rhce, or RhCE proteins (Fig. 7–3). RHD
the results of serologic and biochemical studies in the and RHCE genes each have 10 exons and are 97% identical.
2682_Ch07_149-171 22/05/12 11:43 AM Page 157
1 2 3 4 5 6 7 8 9 10 1 2 3 4 5 6 7 8 9 10 1 2 3 4 5 6 7 8 9 10
Figure 7–3. Locus 1 presence of RHD codes for the presence of D (A) or no D (B). Locus 2 codes for Ce, CE, cE, ce. Each gene consists of 10 coding exons.
DCe/dce DcE/dce
Biochemistry
Basic Concepts
The product of RH genes are nonglycosylated proteins. This
means no carbohydrates are attached to the protein. Rh
DCe/DcE DCe/dce dce/DcE dce/dce
antigens reside on transmembrane proteins and are an
Figure 7-4. Example of a normal pattern of Rh inheritance.
2682_Ch07_149-171 22/05/12 11:43 AM Page 158
integral part of the RBC membrane.21 The gene products Table 7–8 Number of D Antigen Sites of
of RHD and RHCE are remarkably similar in that both Cells with Various Phenotypes
encode for proteins composed of 416 amino acids that
NUMBER OF D ANTIGEN
traverse the cell membrane 12 times. Their proteins differ
Rh PHENOTYPE SITES
by only 32 to 35 amino acids.22 Amino acid position 103 is
important in determining C or c expression and position R1r 9,900–14,600
226 differentiates E from e (Fig. 7–5). Only small loops
R0r 12,000–20,000
of Rh proteins are exposed on the surface of the RBC and
provide the conformational requirements for many serologic R2r 14,000–16,600
differences between the Rh blood types.
R1R1 14,500–19,300
R1R2 23,000–31,000
Weak D: Variations of D Antigen The first mechanism that may result in weakened expression
Expression of D antigen was originally described as a position effect or
gene interaction effect.30 The allele carrying RHD is trans
When Rh-positive RBC samples are typed for the D antigen, (or in the opposite haplotype) to the allele carrying C; for
they are expected to show strong positive reactivity with example, Dce/dCe. The Rh antigen on the RBC is normal, but
anti-D reagents. However, some individuals have RBCs that the steric arrangement of the C antigen in relationship to
the D antigen appears to interfere with the expression of D
Cell Exterior antigen. This interference with D expression does not occur
C/c E/e when the C gene is inherited in the cis position to RHD, such
as DCe/dce. It is not possible to serologically distinguish
genetic weak D from the position effect weak D. Molecular
Cell studies would differentiate the two types. Practically speaking,
Membrane this is unnecessary because the D antigen is structurally
complete. These individuals can receive D-positive RBCs
with no adverse effects.
The D antigens expressed appear to be complete but fewer Normal RHD Normal RHCE
in number. On a molecular level, mutations in the RHD gene 1 2 3 4 5 6 7 8 9 10 1 2 3 4 5 6 7 8 9 10
occur, causing changes in amino acids present in the trans-
membrane or intracellular region of the RhD protein, thus
causing conformational changes in the protein. Mutations Partial DVI
for weak D type 1 and 2 are indicated on Figure 7–6. When
these changes occur, normal RhD antigen expression is al- 1 2 3 4 5 6 7 8 9 10 1 2 3 4 5 6 7 8 9 10
tered. Individuals with weak D phenotype rarely make anti-D,
since changes in their RhD protein occur “inside” the red Figure 7–7. One type of partial DVI gene where three exons of RHCE gene are
cell. Mutations in the RHD gene causing this altered inserted into RHD gene.
expression have been categorized into types 1 through 53
and counting. Type 1 through 3 are the most common
mutations found in individuals of European ancestory.25
Today, partial-D antigens can be classified on a molecular
level and are attributed to hybrid genes resulting from portions
Partial D
of the RHD gene being replaced by portions of the RHCE
The third mechanism in which D antigen expression can be gene, as shown in Figure 7–7. The resulting protein contains
weakened is when one or more D epitopes within the entire a portion of RhD and RhCE in various combinations, depend-
D protein is either missing or altered, termed partial D.32 ing on the hybrid gene’s makeup. These protein changes
Some individuals have red cells with partial-D antigen that occur external to the red cell membrane. If an individual
may type weaker than expected or that may not react at all with a hybrid RhD-RhCe-RhD protein is exposed to red
when routine procedures are used with most commercial blood cells possessing normal RhD protein, they will make
anti-D reagents. Others have partial-D types that may show antibody to the portion of the RhD protein they are missing.37
normal typing with reagent anti-D. Anti-D made by individuals expressing partial D can cause
In the early 1950s, several reports33,34 described individ- hemolytic disease of the fetus and newborn (HDFN) or trans-
uals who were typed D-positive but who produced an anti-D fusion reactions, or both. Once anti-D is identified, Rh-negative
that reacted with all D-positive samples except their own. blood should be used for transfusion. The identification of
The formation of alloanti-D by D-positive individuals a person with a partial-D routinely occurs after the person
required explanation. begins producing anti-D unless there are discrepant Rh (D)
Wiener and Unger35 postulated that the D antigen is made typings. This discovery should prompt collection of additional
of antigenic subparts, genetically determined, that could be ab- samples to be sent to an immunohematology reference
sent in rare instances. If an individual lacked one (or more) laboratory for further RhD classification.
pieces, or epitopes, of the total D antigen, alloantibody can be
made to the missing epitope(s) if exposed to RBCs that possess
the complete D antigen. This theory has become well accepted. Advanced Concepts
Tippett and Sanger36 worked with RBCs and sera of With the advent of monoclonal antibodies and the depletion
partial-D individuals to classify these antigens. Their work and deterioration of the available anti-D made by persons
was based on testing anti-D sera from D-positive people, with partial-D phenotypes, Tippett and coworkers38 pursued
with RBCs from other D-positive people who also made the classification of partial-D antigens using monoclonal
anti-D. This led to a method of categorizing partial D. anti-D (MAb-D). Table 7–9 presents a summary of the
Seven categories were recognized, designated by Roman partial-D categories known at the time of the study. Today,
numerals I through VII. Category I is now obsolete, and a a commercial monoclonal anti-D panel is available. Although
few of the categories have been further subdivided. helpful, it does not define all partial D and weak D types
clearly. A combination of serologic typing and molecular
Weak D Partial D analysis are often required to accurately categorize partial-D
types.
Exterior
Del
I + ± + 0 + + + 0
IIIa + + + + + + + +
IIIb + + + + + + + +
IIIc + + + + + + + +
IVa 0 0 0 + + + + 0
IVb 0 0 0 0 + + + 0
Va 0 + + + 0 + + +
VI 0 0 + + 0 0 0 +
VII + + + + + + 0 +
DFR ± ± + + ± ± 0 +
DBT 0 0 0 0 0 ± + 0
R0Har 0 0 0 0 ± ± 0 0
+ = positive reaction; 0 = negative reaction; ± = positive with some antibodies and negative with other antibodies.
gene that alters expression of the RhD protein. This phenotype Rh-positive. However, if paired with a D-deletion, they may
is relatively common in individuals of Asian ethnicity, occur- type D-positive, depending on the anti-D reagent being used.
ring in 10% to 30% of that population.19 It is rare in whites. These individuals should be classified as RhD-negative since
they essentially lack the RhD protein.
D Epitopes on RhCE Protein The Crawford (ceCF) phenotype is found in individuals
of African descent. It results from a specific amino acid
Like RhD proteins that can express some RhCE protein, change in the RHce gene, resulting in an RhD epitope on the
resulting in the partial-D phenotypes, RhCE protein can Rhce protein.29
express RhD epitopes detected by some monoclonal anti-D.
This can cause discrepant RhD type results with historical Detection of Rh Antibodies and
information on a patient or donor. There are rare individuals Antigens
who possess these unusual proteins and when typed with
anti-D will show positive reactivity even though the D
Basic Concepts
epitope is on the RhCE protein. Examples of these unusual
phenotypes include DHAR and ceCF (Crawford). Rh antibodies are fairly straightforward to detect and iden-
R0Har, also known as DHAR, results from a hybrid gene tify as compared to understanding the molecular genetics
RHCE-RHD-RHCE where only a small portion of RHD is and biochemistry of the Rh blood group system. Further
inserted into the RHCE gene (Fig. 7–8).28 If this hybrid gene discussion on detecting antibodies made by individuals to
is paired with a normal RHD gene, the individual will type Rh antigens follows.
with E+e- RBCs versus 2+ positive reactivity with E+e+ Rh Typing Reagents
RBCs. In addition, Rh antibodies are enhanced when testing
with enzyme-treated RBCs. Reagents used to type for D and for the other Rh antigens
Rh antigens are highly immunogenic; the D antigen is may be derived from a variety of sources. The reagents may
the most potent.39 This is not surprising given that most be high-protein-based or low-protein-based, saline-based,
Rh-negative individuals lack the entire RhD protein, resulting chemically modified, monoclonal, or blends of monoclonals.
in a difference of 32 to 35 amino acids.22 Exposure to less The goal is to use a reagent anti-D that will allow for typing
than 0.1 mL of Rh-positive RBCs can stimulate antibody individuals’ RBCs as quickly and accurately as typing for ABO.
production in an Rh-negative person. While the D antigen Saline reactive reagents, which contain IgM immunoglob-
is most immunogenic, c antigen is the next most likely Rh ulin, were the first typing reagents available to test for the
antigen to elicit an immune response, followed by E, C, and D antigen. Saline anti-D has the advantage of being low-
e. It is not uncommon to see several Rh antibodies in a protein-based and can be used to test cells that are already
patient; for example, anti-D and anti-C or anti-c and anti-E coated with IgG antibody, as in patients who have warm
(see Case Study 7-2 and Figure 7-9). autoantibodies binding to their RBCs. If a high-protein-based
IgG1, IgG2, IgG3, and IgG4 subclasses of Rh antibodies reagent was used when typing these antibody-coated RBCs,
have been reported. IgG1 and IgG3 are of the greatest clinical a false-positive reaction would be obtained because the RBCs
significance because the reticuloendothelial system rapidly would agglutinate on their own without the addition of
clears RBCs coated with IgG1 and IgG3 from the circulation. anti-D. The primary disadvantages of saline typing reagents
IgA Rh antibodies have also been reported but are not routinely are their limited availability, cost of production, and lengthy
tested for in the blood bank.35 incubation time. Because saline anti-D is an IgM immunoglob-
As with most blood group antibodies, IgM Rh antibodies ulin, it cannot be used for weak-D typing.
are formed initially, followed by a transition to IgG. Rh anti- In the 1940s, high-protein anti-D reagents were devel-
bodies often persist in the circulation for years. An individual oped that consisted primarily of IgG anti-D. Human plasma
with low-titer Rh antibody may experience an anamnestic containing high-titer D-specific antibody was used as the raw
(secondary) antibody response if exposed to the same sensi- material. Potentiators of bovine albumin and macromolecu-
tizing antigen. Therefore, in the clinical setting, accuracy of lar additives such as dextran or polyvinylpyrrolidone were
D typing is essential, as is the careful checking of patient his- added to the source material to optimize reactivity in
tory to determine whether an Rh antibody has been identi- the standard slide and rapid tube tests to allow for direct
fied previously. Most commonly found Rh antibodies are agglutination of red cells using an IgG anti-D.40 In essence,
considered clinically significant. Therefore, antigen-negative this media causes RBCs to be in closer proximity to each
blood must be provided to any patient with a history of other and allows IgG anti-D to crosslink and cause direct
Rh-antibody sensitization, whether the antibody is currently agglutination. These reagents are commonly referred to as
demonstrable or not. high-protein reagents.
Rh antibodies do not bind complement. For complement However, the presence of potentiators and the higher pro-
to be fixed (or the complement cascade activated), two IgG tein concentration increase the likelihood of false-positive
immunoglobulins must attach to an RBC antigen in close reactions. To assess the validity of the high-protein Rh typing
proximity to each other. Rh antigens (to which the antibody results, a control reagent was manufactured and had to be
would attach) are not situated on the RBC surface this tested in parallel with each Rh test. If the control reacted,
closely. Therefore, when an Rh antibody coats the RBCs, the test result was invalid and had to be repeated using a dif-
intravascular, complement-mediated hemolysis does not ferent technique or reagent anti-D. The major advantages of
occur. RBC destruction resulting from Rh antibodies is high-protein anti-D reagents are reduced incubation time
primarily extravascular. However, a few rare examples of and the ability to perform weak-D testing and slide typing
complement binding Rh antibodies have been reported. with the same reagent. This type of anti-D reagent is also
Because Rh antibodies are primarily IgG and can tra- polyspecific. More than one clone of anti-D is produced by
verse the placenta and because Rh antigens are well devel- immunized human donors and is therefore able to recognize
oped early in fetal life, Rh antibodies formed by multiple epitopes on the RhD protein.
Rh-negative pregnant women cross the placenta and may In the late 1970s, scientists chemically modified the IgG
coat fetal RBCs that carry the corresponding antigen. This anti-D molecule by breaking the disulfide bonds that maintain
results in the fetal cells having a positive direct antiglobu- the antibody’s rigid shape.41 This allows the antibody to relax
lin test; in HDFN, the coated fetal cells are removed pre- and to span the distance between RBCs in a low-protein
maturely from the fetal circulation (see Chapter 19, medium. The chemically modified reagents can be used for
“Hemolytic Disease of the Fetus and Newborn [HDFN]”). both slide and tube testing and do not require a separate, man-
Until the discovery of Rh immune globulin, anti-D was the ufactured Rh control as long as the samples type as A, B, or O.
most frequent cause of HDFN. When samples test AB Rh-positive or when the Rh test is
performed by itself, a separate saline control or 6% to 8%
albumin control must be used to ensure the observed reactions
D > c > E > C > e are true agglutination and not a result of spontaneous agglu-
Figure 7–9. Immunogenicity of common Rh antigens. tination. Fewer false-positive test reactions are obtained
2682_Ch07_149-171 22/05/12 11:43 AM Page 162
because of the lower-protein suspending medium. Because of common causes of false Rh typing results and suggests correc-
its lower-protein base and ready availability, the chemically tive actions that may be taken to obtain an accurate Rh type.
modified anti-D replaced the need for saline (IgM) anti-D
reagents. Clinical Considerations
As monoclonal antibody production became available, Rh
monoclonal antibodies were produced. These reagents are Determining an individual’s RhD type and testing to identify
derived from single clones of antibody-producing cells. The antibodies to Rh antigens has been reviewed. When Rh
antibody-producing cells are hybridized with myeloma cells to antibodies are identified it is important to understand the
increase their reproduction rate, thereby maximizing their clinical implications when Rh incompatibility exists.
antibody-producing capabilities. Because the D antigen is
composed of many epitopes and the monoclonal Rh antibodies Transfusion Reactions
have a narrow specificity, monoclonal anti-D reagents are usu-
ally a combination of monoclonal anti-D reagents from several Rh antigens are highly immunogenic. The D antigen is the
different clones to ensure reactivity with a broad spectrum of most immunogenic antigen outside the ABO system. When
Rh-positive RBCs. Some companies also blend IgM and IgG anti-D is detected, a careful medical history will reveal RBC
anti-D to maximize visualization of reactions at immediate spin exposure through pregnancy or transfusion ofproducts con-
testing and to allow indirect antiglobulin testing for weak D taining RBCs. Circulating antibody appears within 120 days
antigen with the same reagent.42 Monoclonal blends can be of a primary exposure and within 2 to 7 days after a second-
used for slide, tube, microwell, and most automated Rh testing. ary exposure.
Because these reagents are not human-derived, they lack all Rh-mediated hemolytic transfusion reactions, whether
potential for transmitting infectious disease. caused by primary sensitization or secondary immunization,
As with all commercial typing reagents, Rh antigen typing usually result in extravascular destruction of immunoglobulin-
must be performed with strict adherence to manufacturer’s coated RBCs. The transfusion recipient may have an unex-
directions, use of proper controls, and accurate interpretation plained fever, a mild bilirubin elevation, and a decrease in
of test and control results. Table 7–10 summarizes several hemoglobin and haptoglobin. The direct antiglobulin test
1. Cell suspension too 1. Adjust suspension, retype 1. Immunoglobulin-coated cells 1. Use saline-active typing reagent
heavy (in vivo)
2. Cold agglutinins 2. Wash with warm saline, 2. Saline-suspended cells (slide) 2. Use unwashed cells
retype
3. Test incubated too 3. Follow manufacturer’s 3. Failure to follow manufac- 3. Review directions; repeat test
long or drying (slide) instructions precisely turer’s directions precisely
4. Rouleaux 4. Use saline-washed cells, 4. Omission of reagent 4. Always add reagent first and check
retype manufacturer’s directions before adding cells
5. Fibrin interference 5. Use saline-washed cells, 5. Resuspension too vigorous 5. Resuspend all tube tests gently
retype
6. Contaminating 6. Try another manufacturer’s 6. Incorrect reagent selected 6. Read vial label carefully; repeat
low-incidence reagent or use a known serum
antibody in reagent antibody
7. Polyagglutination 7. See chapter on 7. Variant antigen 7. Refer sample for further
polyagglutination investigation
8. Bacterial contamination 8. Open new vial of reagent, 8. Reagent deterioration 8. Open new vial
of reagent vial retype
9. Incorrect reagent 9. Repeat test; read vial
selected label carefully
10. Centrifugation too long 10. Repeat test using shorter 10. Centrifugation too short 10. Repeat test using longer
centrifugation time centrifugation time
11. rpm too high 11. Repeat test using lower rpm 11. rpm too low 11. Repeat testing using higher rpm
2682_Ch07_149-171 22/05/12 11:43 AM Page 163
is usually positive, and the antibody screen may or may not In the second type of Rhnull syndrome (the amorphic
demonstrate circulating antibody. When the direct antiglob- type), there is a mutation in each of the RHCE genes inher-
ulin test indicates that the recipient’s RBCs are coated with ited from each parent and the common deletion of the RHD
IgG, elution studies may be helpful in defining the offending gene found in most D-negative individuals. The RHAG gene
antibody specificity. If antibody is detected in either the is normal.
serum or eluate, subsequent transfusions should lack the It should be noted that Rhnull individuals of either regu-
implicated antigen. It is not unusual for a person with a lator or amorphic type are negative for the high prevalence
single Rh antibody to produce additional Rh antibodies if antigen LW and for FY5, an antigen in the Duffy blood group
further stimulated.39 system. S, s, and U antigens found on glycophorin B may be
depressed.44
Hemolytic Disease of the Fetus and Newborn Individuals with Rhnull syndrome demonstrate a mild
compensated hemolytic anemia,45 reticulocytosis, stomato-
Hemolytic disease of the fetus and newborn (HDFN) is cytosis, a slight-to-moderate decrease in hemoglobin and
briefly described here because of the historic significance of hematocrit levels, an increase in hemoglobin F, a decrease in
the discovery of the Rh system in elucidating its cause; it is serum haptoglobin, and possibly an elevated bilirubin level.
covered in more detail in Chapter 19. As stated previously, The severity of the syndrome is highly variable from indi-
anti-D was discovered in a woman after delivery of a stillborn vidual to individual, even within one family. When transfu-
fetus. The mother required transfusion. The baby’s father sion of individuals with Rhnull syndrome is necessary, only
donated blood for the transfusion, and the mother subse- Rhnull blood can be given.
quently experienced a severe hemolytic transfusion reaction. Individuals of the Rhmod phenotype have a partial sup-
Levine and Stetson1 postulated that the antibody causing the pression of RH gene expression caused by mutations in the
transfusion reaction also crossed the placenta and destroyed RHAG gene. When the resultant RhAG protein is altered,
the RBCs of the fetus, causing its death. The offending anti- normal Rh antigens are also altered, often causing weakened
body was subsequently identified as anti-D.3 expression of the normal Rh and LW antigens. Rhmod RBCs
HDFN caused by Rh antibodies is often severe because exhibit other blood group antigens; however, like Rhnull, S,
the Rh antigens are well developed on fetal cells, and Rh s, and U antigen expression may be depressed.45 Rhmod indi-
antibodies are primarily IgG, which readily cross the viduals exhibit features similar to those with the Rhnull syn-
placenta. drome; however, clinical symptoms are usually less severe
After years of research, a method was developed to pre- and rarely clinically remarkable.46
vent susceptible (D-negative) mothers from forming anti-D,
thus preventing RhD HDFN. Rh-immune globulin, a purified Unusual Phenotypes and Rare Alleles
preparation of IgG anti-D, is given to D-negative woman dur-
ing pregnancy and following delivery of a D-positive fetus.43 Several of the less frequently encountered Rh antigens are
Rh-immune globulin is effective only in preventing RhD described briefly in the following paragraphs. Refer to other
HDFN. No effort has been made to develop immune globulin textbooks for in-depth discussions.28,47–49
products for other Rh antigens (e.g., C, c, E, e). When pres-
ent, Rh HDFN may be severe and may require aggressive Cw
treatment. Refer to Chapter 19 for a more detailed discussion
of HDFN—its etiology, serology, and treatment. Cw was originally considered an allele at the C/c locus.50
Later studies showed that it can be expressed in combi-
Rh Deficiency Syndrome: Rhnull and nation with both C and c and in the absence of either
Rhmod allele. It is now known that the relationship between C/c
and Cw is only phenotypic and that Cw is antithetical to
Rare individuals have Rh deficiency or Rhnull syndrome the high-prevalence antigen MAR.51 Cw results in a single
and fail to express any Rh antigens on the RBC surface. amino acid change most often found on the RhCe protein.
This syndrome is inherited in one of two ways—amorphic Cw is found in about 2% of whites and is very rare in
and regulator. Other rare individuals exhibit a severely blacks.
reduced expression of all Rh antigens, a phenotype called Anti-Cw has been identified in individuals without known
Rhmod. exposure to foreign RBCs and after transfusion or pregnancy.
Individuals who lack all Rh antigens on their RBCs are Anti-Cw may show dosage (i.e., reacting more strongly with
said to have Rhnull syndrome, which can be produced by two cells from individuals who are homozygous for Cw). Because
different genetic mechanisms.37 In the regulator-type Rhnull of the low prevalence of Cw, Cw antigen–negative blood is
syndrome, a mutation occurs in the RHAG gene. This results readily available.
in no RhAG protein expression and subsequently no RhD or
RhCE protein expression on the RBCs, even though these f (ce)
individuals usually have a normal complement of RHD and
RHCE genes. These individuals can pass normal RHD and The f antigen is expressed on the RBC when both c and
RHCE genes to their children. e are present on the same haplotype; it has been called a
2682_Ch07_149-171 22/05/12 11:43 AM Page 164
compound antigen.52 However, f is likely a single entity Rh13, Rh14, Rh15, and Rh16
resulting from conformational changes in the Rhce protein.53
The antigen f was included in a series of these compound Advanced Concepts
antigens, which were previously referred to as cis-products
to indicate that the antigens were on the same haplotype. Rh13, Rh14, Rh15, and Rh16 define four different parts of
It is now known they are expressed on the Rhce protein. the D mosaic, as it was originally described.55 Although
Phenotypically, the following samples appear the same these parts are included in the partial-D categories II to VII
when tested with the five major Rh antisera: D+C+E+c+e+, as defined by Tippett and Sanger,36 they are not directly
resulting in the predicted genotypes of DCE/dce or comparable. These antigens are now obsolete.
DcE/DCe. However, when tested with anti-f, only the
DCE/dce shows positive reactivity, confirming the former Rh17 (Hr0)
genotype.
Anti-f is generally a weakly reactive antibody often Rh17, also known as Hr0, is an antigen present on all RBCs
found with other antibodies. It has been reported to cause with the “common” Rh phenotypes (e.g., R1R1, R2R2, rr).56
HDFN and transfusion reactions. In case of transfusion, In essence, this antibody is directed to the entire protein
f-negative blood should be provided. Anti-f is not available resulting from the RHCE genes. When RBCs phenotype as
as a reagent; however c-negative or e-negative blood may D–, the most potent antibody they make is often one
be provided since all c-negative or e-negative individuals directed against Rh17 (Hr0).
are f-negative.
Rh23, Rh30, and Rh40
rhi (Ce)
Rh23, Rh30, and Rh40 are all low-prevalence antigens
Similar to f, rhi was considered a compound antigen present associated with a specific category of partial-D. These
when C and e are on the RhCe protein.52 A sample with low-prevalence antigens result from the formation of the
the phenotype D +C +E +c +e + can be either DcE/DCe or hybrid proteins seen in individuals with partial-D pheno-
DCE/dce. Anti-rhi shows positive reactivity only with types. Rh23 (also known as Wiel and Dw) is an antigenic
DCe/dce RBCs. marker for category Va partial-D.57 Rh30 (also known as
Antigens cE and CE or Rh22 also exist, but antibodies Goa or Dcor) is a marker for partial DIVa.58 Rh40 (also
produced to these antigens are not commonly seen. known as Tar or Targett) is a marker for partial DVII.59
Rh52 or BARC is associated with some partial-DVI
G types.36
G is an antigen present on most D-positive and all C-positive
RBCs. The antigen results from the amino acid serine at
Rh33 (Har)
position 103 on the RhD, RhCe, and RhCE protein. In The low-prevalence antigen Rh33 is most often found in
antibody identification testing, anti-G reacts as though it whites and is associated with the rare variant haplotype
were a combination of anti-C plus anti-D because all called R0Har.60 R0Har gene codes for normal amounts of c,
C-positive and D-positive cells are G+.54 G was originally reduced amounts of e, reduced f, reduced Hr0, and
described in an rr person who received D+C–E–c+e+ RBCs. reduced amounts of D antigen written as (D)c(e). The D
Subsequently, the recipient produced an antibody that reactions are frequently so weak that the cells are often
appeared to be anti-D plus anti-C, which should be impos- typed as Rh-negative. As previously discussed, R0Har or
sible because the C antigen was not on the transfused DHAR results from a hybrid gene RHCE-RHD-RHCE in
RBCs. Further investigation showed the antibody was which only a small portion of RHD is inserted into the
directed toward D + G, not anti-C. This also explains RHCE gene.
situations in which an Rh-negative individual who has
received Rh-negative blood will look like they made anti-D. Rh32
The Rh-negative blood the patient received was C-positive,
thus G-positive and the antibody produced is actually Rh:32 is a low-prevalence antigen associated with a variant
anti-G, not anti-D and anti-C. =
of the R1[D(C)(e)] haplotype called RN.61 The C antigen and
Anti-G versus anti-D and anti-C is important when eval- e antigen are expressed weakly. The D antigen expression is
uating obstetric patients. If the patient has produced anti-G exaggerated or exalted. This gene has been found primarily
and not anti-D, they are considered a candidate for RhIg. in blacks.
Elaborate adsorption and elution studies, usually performed
in an immunohematology reference laboratory, are valuable Rh43 (Crawford)
in these situations to discriminate anti-D from anti-C from
anti-G. Rh43, also known as the Crawford antigen, is a low-prevalence
For transfusion purposes, it is not necessary to discrimi- antigen on a variant Rhce protein.29 The Crawford (ceCF)
nate anti-D and anti-C from anti-G, as the patient would antigen is of very low prevalence found in individuals of
receive D-C blood regardless if the antibody is –D, –C or –G. African descent.
2682_Ch07_149-171 22/05/12 11:43 AM Page 165
CASE STUDIES
Case 7-1
Two units of blood are ordered for an 89-year-old woman with myelodysplastic syndrome. She had been transfused 3 years
ago and has six children. Routine pretransfusion testing follows.
Anti-A Anti-B Anti-D A1 Cells B Cells Interpretation
0 0 0 4+ 4+
1. What is the patient’s ABO, Rh type?
Rh MNS LU P Lewis Kell Duffy Kidd GEL
D C E c E f M N S s Lua Lub P1 Lea Leb K k Fya Fyb Jka Jkb IAT
1 + + 0 0 + 0 + + + + 0 + + + 0 + + + 0 0 + 2+
2 + 0 + + 0 0 + 0 + 0 0 + + 0 + 0 + + + + 0 2+
2. How would you interpret the results of the antibody detection test/screen?
3. What information can you obtain from your evaluation of the antibody detection test/screen?
4. What additional testing should be performed?
Gel Antibody Identification Panel
Rh MNS Lu P1 Lewis Kell Duffy Kidd
D C E c e f M N S s Lua Lub P1 Lea Leb K k Fya Fyb Jka Jkb IAT
1 + + 0 0 + 0 + + + + 0 + 0 + 0 0 + 0 + 0 + 2+
2 0 0 0 + + + + 0 + 0 0 + + 0 + + 0 + + + 0 0
3 0 0 + + 0 0 0 + 0 + 0 + + 0 + + + + 0 0 + 0
4 + + 0 0 + 0 + 0 + + 0 + 0 0 0 0 + + + + + 2+
5 0 0 + + + + + + 0 + 0 + 0 0 + 0 + 0 + 0 + 0
6 0 + 0 0 + 0 + + 0 + 0 + + 0 + + + + + + + 0
7 + 0 + + 0 0 0 + 0 + 0 + 0 0 + 0 + 0 + + 0 2+
8 0 0 0 + + + + 0 + 0 0 + + 0 + 0 + + 0 0 + 0
9 + + 0 + + + + + 0 + 0 + + + 0 0 + 0 + + 0 2+
10 + 0 + + 0 0 0 + + + 0 + + + 0 0 + + 0 + 0 2+
11 + 0 0 + + + 0 + 0 0 0 + + 0 + 0 + 0 0 + + 2+
AC 0
NOTE: In this case study, antibodies are excluded only if the patient’s serum does not react with panel cells that possess
a double-dose expression of the antigen (from a donor with homozygous expression).
5. What antibody(ies) is(are) present in the patient’s plasma?
Case 7-2
A 25-year-old woman, pregnant with her second child, had routine orders for a “type and screen,” with the
following results:
Anti-A Anti-B Anti-D A1 Cells B Cells Interpretation
0 0 3+ 4+ 4+
1. How would you interpret the results of the ABO, Rh type and antibody detection test (screen)?
Rh MNS LU P Lewis Kell Duffy Kidd IS 37C PEG
D C E c E f M N S s Lua Lub P1 Lea Leb K k Fya Fyb Jka Jkb 0 NH IAT
1 + + 0 0 + 0 + + + + 0 + + + 0 + + + 0 0 + 0 NH 0
2 + 0 + + 0 0 + 0 + 0 0 + + 0 + 0 + + + + 0 0 NH 3+
NH = No hemolysis observed
2682_Ch07_149-171 22/05/12 11:43 AM Page 167
A 29-year-old female, pregnant for the first time, presented to her OB physician in her early first trimester for initial
prenatal care. She had no known prior transfusions.
Forward Reverse
Anti-A Anti-B Anti-D A1 B Interpretation
4+ 0 0 0 4+
1. What is the patient’s ABO, Rh type?
Rh MNS LU P Lewis Kell Duffy Kidd GEL
D C E c E F M N S s Lua Lub P1 Lea Leb K k Fya Fyb Jka Jkb IAT
1 + + 0 0 + 0 + + + + 0 + + + 0 + + + 0 0 + 0
2 + 0 + + 0 0 + 0 + 0 0 + + 0 + 0 + + + + 0 0
Approximately 1 month later, the Transfusion Service received a call from the OB physician, stating the
patient had been previously typed as “A-positive” at another facility and her donor card states “A-positive.” A sample is
sent to the laboratory for repeat ABO, Rh typing.
The patient’s repeat ABO, Rh type showed the same results: A Rh-negative. Because of the reported discrepancy, the
patient’s Rh (D) typing was performed using several different anti-D reagents.
2682_Ch07_149-171 22/05/12 11:43 AM Page 168
SUMMARY CHART
The Rh antibody was so named on the basis of anti- in order of its discovery (Rh1 = D, Rh2 = C, Rh3 = E,
body production by guinea pigs and rabbits when Rh4 = c, Rh5 = e).
transfused with rhesus monkey RBCs. Rh antigens are characterized as nonglycosylated
Historically, Rh was a primary cause of HDFN, erythro- proteins in the RBC membrane.
blastosis fetalis, and a significant cause of hemolytic The most common phenotype in whites is R1r (31%);
transfusion reactions. the most common phenotype in blacks is R0r (23%),
Fisher-Race DCE terminology is based on the theory followed by R0R0 at 19%.
that antigens of the system are produced by three The Rh antigens are inherited as codominant alleles.
closely linked sets of alleles and that each gene is re- A partial-D individual is characterized as lacking one
sponsible for producing a product (or antigen) on the or more pieces or epitopes of the total D antigen and
RBC surface. may produce alloantibody to the missing fraction if
A person who expresses no Rh antigens on the RBC is exposed to RBCs with the complete D antigen.
said to be Rhnull, and the phenotype may be written Blood donor units for transfusion are considered
as –––/–––. Rh-positive if either the D or weak-D test is positive;
In the Wiener Rh-Hr nomenclature, it is postulated if both the D and weak-D tests are negative, blood for
that the gene responsible for defining Rh actually transfusion is considered Rh-negative.
produces an agglutinogen that contains a series of blood Most Rh antibodies are IgG immunoglobulin and react
factors in which each factor is an antigen recognized optimally at 37°C or following antiglobulin testing;
by an antibody. exposure to less than 0.1 mL of Rh-positive RBCs can
It is currently accepted that two closely linked genes stimulate antibody production in an Rh-negative person.
control the expression of Rh; one gene (RHD) codes Rh-mediated hemolytic transfusion reactions usually
for the presence of RhD, and a second gene (RHCE) result in extravascular hemolysis.
codes for the expression of CcEe antigens.
Rh antibodies are IgG and can cross the placenta to
In the Rosenfield alpha/numeric terminology, a num- coat fetal (Rh-positive) RBCs.
ber is assigned to each antigen of the Rh system
25. Roback, JD, Combs, MR, Grossman, B, and Hillyer, C (eds): 47. Daniels, G: Human Blood Groups, 2nd ed. Blackwell Scientific,
Technical Manual, 16th ed. AABB, Bethesda, MD, 2008. Oxford, 2002.
26. Race, RR, Sanger, R, and Lawler, SD: The Rh antigen Du. Ann 48. Reid, MR, and Lomas-Francis, C. The Blood Group Antigen
Eugen Lond 14:171, 1948. Facts Book, 2nd ed. Elsevier, San Diego, 2004.
27. Westhoff, CM: Rh complexities: Serology and DNA typing. 49. Issitt, PD, and Anstee, DJ: Applied Blood Group Serology,
Transfusion 47:17S–22S, 2007. 4th ed. Montgomery Scientific Publications, Durham, NC, 1998.
28. Beckers, EA, Faas, BH, von dem Borne, AE, et al: The R0Har 50. Callendar, ST, and Race, RR: A serological and genetic study of
RH:33 phenotype results from substitution of exon 5 of the multiple antibodies formed in response to blood transfusion
RHCE gene by the corresponding exon of the RHD gene. Br J by a patient with lupus erythematosus diffuses. Ann Eugen
Haematol Mar 92(3):751–757, 1996. Lond 13:102, 1946.
29. Flegel, WA, Wagner, FF, Chen, Q, et al: The RHCE allele ceCF: 51. Sistonen, P, et al: MAR, a novel high-incidence Rh antigen
The molecular basis of Crawford (RH43). Transfusion revealing the existence of an allele sub-system including Cw
46(8):1334–1342, 2006. (Rh8) and Cx (Rh9) with exceptional distribution in the
30. Ceppellini, R, Dunn, LC, and Turri, M: An interaction between Finnish population. Vox Sang 66:287–292, 1994.
alleles at the Rh locus in man which weakens the reactivity of 52. Rosenfield, RE, and Haber, GV: An Rh blood factor, Rh1 (Ce)
the Rh0 factor (D0). Proc Natl Acad Sci 41:283, 1955. and its relationship to hr (ce). Am J Hum Genet 10:474, 1958.
31. Wagner, FF, Gassner, C, Muller, TH, et al: Molecular basis of 53. Chen, YX, Peng, J, Novaretti, M, Reid, ME, and Huang, CH:
weak D phenotypes. Blood 93:385–393, 1999. Deletion of arginine codon 229 in the Rhce gene alters e and f
32. Tippett, P, Lomas-Francis, C, and Wallace, M: The Rh antigen but not c antigen expression. Transfusion Mar;44(3):391–398,
D: Partial D antigens and associated low incidence antigens. 2004.
Vox Sang 70:123–131, 1996. 54. Allen, FH, and Tippett, PA: A new Rh blood type which reveals
33. Shapiro, M: The ABO, MN, P and Rh blood group systems in the Rh antigen G. Vox Sang 3:321, 1958.
South African Bantu: A genetic study. South Afr Med J 25:187, 55. Wiener, AS, and Unger, LJ: Further observations on the blood
1951. factors RhA, RhB, RhC, RhD. Transfusion 2:230, 1962.
34. Argall, CI, Ball, JM, and Trentelman, E: Presence of anti-D 56. Allen, FH, Jr, and Corcoran, PA: Proc 11th Ann Mtg. AABB,
antibody in the serum of Du patient. J Clin Lab Med 41:895, Cincinnati, Abstract, 1958.
1953. 57. Chown, B, et al: The Rh antigen Dw (Wiel). Transfusion 4:169,
35. Wiener, AS, and Unger, LJ: Rh factors related to the Rh0 factor 1964.
as a source of clinical problems. JAMA 169:696, 1959. 58. Lewis, M, et al: Blood group antigen Goa and the Rh system.
36. Tippett, P, and Sanger, R: Observations on subdivisions of the Transfusion 7:440, 1967.
Rh antigen D. Vox Sang 7:9, 1962. 59. Lewis, M, et al: Assignment of the red cell antigen Targett
37. Huang, CH, Liu, P, and Cheng, JG: Molecular biology and (Rh 40) to the Rh blood group systems. Am J Hum Genet
genetics of the Rh blood group system. Semin Hematol 37:150– 31:630, 1979.
165, 2000. 60. Giles, CM, et al: An Rh gene complex which results in a “new”
38. Tippett, P, Lomas-Francis, C, and Wallace, M. The Rh antigen antigen detectable by a specific antibody, anti-Rh 33. Vox Sang
D: Partial D antigens and associated low incidence antigens. 21:289, 1971.
Vox Sang 70:123–131, 1996. 61. Rosenfield, RE, et al: Problems in Rh typing as revealed by a
39. Mollison, PL: Blood Transfusion in Clinical Medicine, 6th ed. single Negro family. Am J Hum Genet 12:147, 1960.
Blackwell Scientific Publications, Oxford, 1988. 62. Issitt, PD: Applied Blood Group Serology, 3rd ed. Montgomery
40. Diamond, LK, and Denton, RC: Rh agglutination in various Scientific Publication, Miami, FL, 1985.
media with particular reference to the value of albumin. J Clin 63. Vege, S, and Westhoff, CM. Molecular characterization of GYPB
Lab Med 30:821, 1945. and RH in donors in the American Rare Donor Program. Im-
41. Romans, DG, et al: Conversion of incomplete antibodies to munohematology 22:143–147, 2006.
direct agglutinins by mild reduction. Proc Natl Acad Sci USA 64. Huang, CH, Chen, Y, and Reid, M: Human D(IIIa) erythro-
74:2531, 1977. cytes: RhD protein is associated with multiple dispersed amino
42. Denomme, GA, Dake, LR, Vilensky, D, Ramyar, L, and Judd, acid variations. Am J Hematol. Jul;55(3):139–145, 1997.
WJ: Rh discrepancies caused by variable reactivity of partial 65. DeNatale, A, et al: A “new” Rh antigen, common in Negroes,
and weak D types with different serologic techniques. Trans- rare in white people. JAMA 159:247, 1955.
fusion 48(3):473–478, 2008. 66. Sanger, R, et al: An Rh antibody specific for V and Rs. Nature
43. Queenan, JT: Modern Management of the Rh Problem, 2nd ed. (Lond) 186:171, 1960.
Harper & Row, New York, 1977. 67. Daniels, GL, Faas, BH, Green, CA, et al: The VS and V blood
44. Schmidt, PJ, et al: Aberrant U blood group accompany Rhnull. group polymorphisms in Africans: A serologic and molecular
Transfusion 7:33, 1967. analysis. Transfusion 38:951–958, 1998.
45. Schmidt, PJ, and Vos, GH: Multiple phenotypic abnormalities 68. Contreras, M, et al: The Rh antigen Evans. Vox Sang 34:208,
associated with Rhnull (—/—). Vox Sang 13:18, 1967. 1978.
46. Chown, B, et al: An unlinked modifier of Rh blood groups: 69. Race, RR, and Sanger, R: Blood Groups in Man, 6th ed. Blackwell
Effects when heterozygous and when homozygous. Am J Hum Scientific, Oxford, 1975.
Genet 24:623, 1972.
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Chapter 8
Blood Group Terminology and the Other
Blood Groups
Regina M. Leger, MSQA, MT(ASCP)SBB, CMQ/OE(ASQ)
172
2682_Ch08_172-215 22/05/12 11:44 AM Page 173
Chapter 8 Blood Group Terminology and the Other Blood Groups 173
OBJECTIVES
Blood Group Terminology
1. Describe how antigens, antibodies, genes, and phenotypes are correctly written.
2. List the four categories for classification of RBC surface blood group antigens used by the ISBT.
3. For each of the blood group systems described in this chapter:
• List the major antigens and common phenotypes
• Describe the serologic characteristics and clinical significance of the antibodies
• Identify null phenotypes
Antigen Characteristics
4. Describe the formation of Lewis antigens and their adsorption onto RBCs.
5. Define the interaction of Lewis genes with ABO, H, and secretor genes.
6. List substances present in secretions and the Lewis phenotypes based on a given genotype.
7. Describe the reciprocal relationship of I antigen to i antigen.
8. List the antigen frequencies for the common antigens K, M, S, s, Fya, Fyb, Jka, Jkb, and P1.
9. Define Kpa, Jsa, and Lua as low-prevalence antigens and Kpb, Jsb, Lub, and I as high-prevalence antigens.
10. Define the association of M and N with glycophorin A (GPA) and S and s with glycophorin B (GPB).
11. Describe the antigen phenotypes S–s–U–, Js(a+), and Fy(a–b–) associated with blacks.
12. Define the null phenotypes Mk, p, Ko, Fy(a–b–), Jk(a–b–), and Lu(a–b–), and describe their role in problem-solving.
13. Compare dominant and recessive forms of the Lu(a–b–) and Jk(a–b–) phenotypes.
14. Explain I, P1, and Lutheran antigens as being poorly expressed on cord RBCs.
15. Describe the phenotypic relationship between LW and Rh.
16. List the Gerbich-negative phenotypes and the antibodies that can be made by each.
17. List the antigens that are denatured by routine blood bank enzymes (M, N, S, s, Fya, Fyb) and antigens whose reactivity with
antibody is enhanced with enzymes (I, i, P1, Jka, Jkb).
18. List which antigens are destroyed by treatment with DTT (dithiothreitol).
19. Explain the prevalence of Dia with South, Central, and North American native populations, and the consequent higher prevalence
of the Di(b–) phenotype in those populations.
Antibody Characteristics
20. List the characteristics of the Lewis antibodies, including clinical significance.
21. Define antibodies to M, N, I, and P1 as being typically non-RBC-induced (“naturally occurring”), cold-reacting agglutinins that
are usually clinically insignificant.
22. Describe antibodies to K, k, S, s, Fya, Fyb, Jka, and Jkb as usually induced by exposure to foreign RBCs (“immune”), antiglobulin-
reactive antibodies that are clinically significant.
23. List the antibody specificities that commonly show dosage (anti-M, -N, -S, -s, -Fya, -Fyb, -Jka and -Jkb).
24. Describe the characteristic reactivity of antibodies to Ch, Rg, JMH, and Knops antigens, and their clinical significance.
25. List the antibodies in the Dombrock system that are clinically significant for transfusion (anti-Doa and anti-Dob).
Chapter 8 Blood Group Terminology and the Other Blood Groups 175
Table 8–1 Examples of Terminology for Genes, Antigens, Antibodies, and Phenotypes
GENE ANTIGEN ANTIBODY PHENOTYPE
System Conventional ISBT Antigen Positive Antigen Negative
negative result, and multiple results are separated by a Transfusion (ISBT) formed the Working Party on Terminology
comma (e.g., Sc:–1,2). Phenotypes of more than one blood for Red Cell Surface Antigens in 1980.2 The numeric system
group system are separated by a semicolon—for example, this group proposed was not intended to replace traditional
S+s+; K–; Fy(a+b–). terminology but rather to enable communication on com-
One must remember that serologic tests determine only puter systems where numbers are necessary. Each antigen is
RBC phenotype, not genotype. A genotype is composed of given a six-digit identification number. The first three digits
the actual genes that an individual has inherited and can represent the system, collection, or series, and the second
be determined only by family or DNA studies. Sometimes three digits identify the antigen. The antigens within the
the genotype can be predicted or inferred by the phenotype. system are numbered sequentially in order of discovery. Each
When based on results from RBC antigen typing, the genotype system also has an alphabetical symbol. For example, using
is a probable interpretation as to which genes the individual the ISBT terminology, the K antigen is 006001 with the first
carries in order to have the observed phenotype. three numbers (006) representing the system and the second
Antibodies are described by their antigen notation with three numbers (001) representing the number assigned
the prefix anti-, including a hyphen before the antigen sym- to K. Antigens can also be written using the system symbol
bol. Use of correct blood group terminology, especially for followed by the antigen number; for instance, KEL1 (the
antibodies identified in a patient’s serum, is very important redundant sinistral zeroes can be omitted). This committee’s
so that correct information is conveyed for patient care. work is ongoing, and the assignment of RBC antigens to
Some examples of correct and incorrect terminology are blood group systems is periodically updated. The first mono-
given in Table 8–2. graph was released in 1990, and updates are published in
Numeric terminology was originally introduced for the Vox Sanguinis3 and on the Working Party’s page of the ISBT
Kell and Rh systems and was subsequently applied to other website (www.isbtweb.org).
systems. To facilitate computer storage and retrieval of blood The ISBT assigns RBC antigens to a system, collection, or
group information and to help standardize blood group low- or high-prevalence series. This chapter uses traditional
system and antigen names, the International Society of Blood terminology, but the ISBT symbol and number are indicated
for the blood group systems and collections discussed.
As defined by the ISBT, a blood group system “consists of
Table 8–2 Examples of Correct and one or more antigens controlled at a single gene locus, or by
two or more very closely linked homologous genes with little
Incorrect Terminology
or no observable recombination between them.”4 Each system
CORRECT INCORRECT is genetically distinct. To date, there are 30 blood group
Fy(a+) Fya+, Fy(a+), Fya+, Fya(+), Duffy a-positive, Duffya+ systems (Table 8–3).3
Collections are antigens that have a biochemical, sero-
Fy(a–b+) Fya–b+, Fya(–)Fyb(+) logic, or genetic relationship but do not meet the criteria
Anti-Fya Anti Fya, anti-Duffy, anti-Duffya for a system.4 Antigens classified as a collection are
assigned a 200 number. Some of the previously established
K Kell (name of system), K1 collections have been made obsolete as criteria have been
Anti-k Anti-Cellano, anti-K2 met to establish a system or incorporate antigens into
an existing system. See Table 8–4 for a listing of the ISBT
M+N– M(+), MM collections.
Ge:–2 Ge2–, Ge:2–, Ge2-negative All remaining RBC antigens that are not associated with
a system or a collection are catalogued into the 700 series of
2682_Ch08_172-215 22/05/12 11:44 AM Page 176
Table 8–3 International Society of Blood Transfusion (ISBT) Blood Group Systems
GENE NAME CHROMOSOMAL
NUMBER NAME SYMBOL ISBT (HGNC) LOCATION NO. OF AGS
011 Yt YT YT (ACHE) 7q 2
012 Xg XG XG (XG) X 2
MIC2 (CD99)
019 Kx XK XK (XK) Xp 1
Chapter 8 Blood Group Terminology and the Other Blood Groups 177
Table 8–4 International Society of Blood low-prevalence antigens or the 901 series of high-prevalence
Transfusion Blood Group antigens. Refer to Table 8–5 for a listing of low- and high-
prevalence antigens recognized by the ISBT. Antigens in
Collections
these series represent those with a prevalence of less
COLLECTION ANTIGEN than 1% or more than 90% of most random populations,
Number Name Symbol Number Symbol respectively. As terminology has evolved, the terms high- and
low-incidence, also previously known as high- and low-
205 Cost COST 205001 Csa frequency, are currently being replaced by the terms high- and
205002 Csb
low-prevalence, reflecting the occurrence of an inherited
207 Ii I 207002 i characteristic at the phenotypic level.5
208 Er ER 208001 Era The Lewis (007) System
208002 Erb
208003 Er3
The Lewis blood group system is unique because the
209 GLOB 209003 LKE Lewis antigens are not intrinsic to RBCs but are on type
209004 PX2 1 glycosphingolipids that are passively adsorbed onto the
RBC membrane from the plasma. The Lewis system was
210 210001 Lec
210002 Led named after one of the first individuals to make the antibody,
reported by Mourant in 1946.6 This antibody, later called
212 Vel VEL 212001 Vel anti-Lea, agglutinated RBCs from about 25% of English peo-
212002 ABTI
ple.7 In 1948, an antibody, later called anti-Leb, was found
213 MN CHO 213001 Hu that reacted with Le(a–) individuals. It was thought that Lea
213002 M1 and Leb were antithetical antigens, but we now know this is
213003 Tm not so because they do not result from alternative alleles of a
213004 Can
single gene. Rather, they result from the interaction of two
213005 Sext
213006 Sj fucosyltransferases encoded by independent genes, Le and Se.
The Lewis blood group system has been assigned the ISBT
Antigens in shaded boxes are discussed in text. system number 007 and the system symbol LE.
Table 8–5 International Society of Blood Transfusion Antigens of Low (700 Series) and High
(901 Series) Prevalence
NUMBER NAME SYMBOL NUMBER NAME SYMBOL
Chapter 8 Blood Group Terminology and the Other Blood Groups 179
Genetics and Biosynthesis Figure 8–1. Precursor type 1 and type 2 chains. The type 1 chain has a beta
1→3 linkage of the terminal galactose (Gal) to the N-acetylglucosamine (GlcNAc)
of the precursor structure. The type 2 chain has a beta 1→4 linkage of the terminal
Advanced Concepts galactose.
Table 8–8 Antigens and Phenotypes common and is frequently found with anti-Lea or anti-Leb.
Resulting From Interaction The antibody is heterogenous and occurs mainly in Le(a–b–)
secretors of group A1, B, or A1B. The antigens now known
of Lewis, Secretor, and
as Lex and Ley are products of FUT3 on type 2 precursor
ABO Genes chains and are not associated with the RBC surface and are
LEWIS, SECRETOR, AND ABO GENES not part of the Lewis blood group.9
Genes Antigens in Secretions RBC Phenotype ALeb and BLeb result from the addition of the A or B
immunodominant sugar, respectively, to type 1H chain in
Le, Se, A/B/H Lea, Leb, A, B, H A, B, H, Le(a – b+) individuals who have at least one Se and one Le allele.
lele, Se, A/B/H A, B, H A, B, H, Le(a – b –) Se converts type 1 chains to type 1H, providing a suitable
acceptor for the A and B carbohydrates (see Fig. 8–2).
Le, sese, A/B/H Lea A, B, H, Le(a+b –)
The P Blood Group: P1PK (003)
lele, sese, A/B/H None A, B, H, Le(a – b –)
and Globoside (028) Systems
Le, sese, hh, A/B Lea Oh, Le(a+b –) and Related (209) Antigens
Le, Se, hh, A/B Lea, Leb, A, B, H A*, B*, Le(a – b+) Traditionally, the P blood group comprised the P, P1, and Pk
antigens and, later, Luke (LKE). The biochemistry and molec-
*para-Bombay
ular genetics, although not completely understood as yet, make
it clear that at least two biosynthetic pathways and genes at dif-
ferent loci are involved in the development and expression of
gastrointestinal tract is thought to be the primary source of
these antigens. Consequently, these antigens cannot be consid-
Lewis glycolipid in plasma.5 Le(a–b–) RBCs incubated with
ered a single blood group system. Currently, in ISBT nomen-
plasma from Le(a+) or Le(b+) individuals can be converted
clature, P1 and Pk are assigned to the P1PK blood group system
to Le(a+b–) or Le(a–b+), respectively. With saliva as
(003, symbol P1PK), P is assigned to the Globoside blood
a source of Lewis substances, Le(a–b–) RBCs cannot be
group system (028, symbol GLOB), and LKE and PX2 are
converted into Lewis-positive phenotypes because Lewis
assigned to the Globoside collection (209, symbol GLOB).
substances in saliva, being glycoproteins, are not adsorbed
The reader will notice the confusing use of GLOB as the sym-
onto the RBC membranes.
bol for both a system and a collection. For simplicity in this
chapter, these antigens will be referred to as the P blood group.
Development of Lewis Antigens The P blood group was introduced in 1927 by Landsteiner
and Levine. In their search for new antigens, they injected
Depending on the genes inherited, Lea and Leb glycoproteins rabbits with human RBCs and produced an antibody, initially
will be present in the saliva of newborns, but Lewis glyco- called anti-P, that divided human RBCs into two groups:
lipids are not detectable in the plasma until about 10 days P+ and P–.7
after birth. As a result, cord blood and RBCs from newborn In 1951, Levine and colleagues10 described anti-Tja (now
infants phenotype as Le(a–b–). Some can be shown to be known as anti-PP1Pk), an antibody to a high-prevalence anti-
weakly Le(a+) when tested with a potent anti-Lea or with gen that Sanger11 later showed was related to the P blood
methods more sensitive than direct agglutination. Lewis anti- group. Because anti-Tja defined an antigen common to P+
gens will start to appear shortly after birth, with Lea developing and P– cells and was made by an apparent P null individual,
first when the Le gene is present. The Lewis fucosyltrans- the original antigen and phenotypes were renamed. Anti-P
ferase is more active than the secretor fucosyltransferase in became anti-P1; the P+ phenotype became P1; the P– phenotype
newborns, so more type 1 chains are available for conversion became P2; and the rare P null individual became p.
to Lea. As the secretor transferase activity increases, convert- The P blood group became more complex in 1959 when
ing type 1 to type 1H, Leb will be detected. In children who Matson and coworkers12 described a new antigen, Pk. This
inherit both Le and Se genes, the transformation can be antigen is expressed on all RBCs except those of the very rare
followed from the Le(a–b–) phenotype at birth to Le(a+b–) p phenotype, but it is not readily detected unless P is absent
after 10 days to Le(a+b+) and finally to Le(a–b+), the true (i.e., in the P1k and P2k phenotypes).
Lewis phenotype, after about 6 years. In contrast, children The phenotypes, antigens, and antibodies associated with
who inherit Le and sese genes phenotype as Le(a–b–) at birth the P blood group are summarized in Table 8–9. There are two
and transform to Le(a+b–) after 10 days; the Le(a+b–) phe- common phenotypes: P1 and P2, and three rare phenotypes: p,
notype persists throughout life. Individuals with lele genes P1k, and P2k. The P1 phenotype describes RBCs that react with
phenotype as Le(a–b–) at birth and for the rest of their lives. anti-P1 and anti-P; the P2 phenotype describes RBCs that do
not react with anti-P1 but do react with anti-P. When RBCs are
Other Lewis Antigens tested only with anti-P1 and not with anti-P, the phenotype
should be written as P1+ (or P1) or P1–. Only when P1– RBCs
Leab is present on all Le(a+b–) and Le(a–b+) RBCs and on are tested and found to be reactive with anti-P should they be
90% of cord RBCs. The antigen was previously known as Lex, designated as phenotype P2. RBCs of the p phenotype do not
but in 1998 the ISBT renamed it Leab.9 Anti-Leab is fairly react with anti-P1, anti-P, or anti-Pk. RBCs of the P1k phenotype
2682_Ch08_172-215 22/05/12 11:44 AM Page 181
Chapter 8 Blood Group Terminology and the Other Blood Groups 181
react with anti-P1 and anti-Pk but not with anti-P. RBCs of the The P1 antigen deteriorates rapidly on storage. When
P2k phenotype react with anti-Pk but not with anti-P1 or anti-P. older RBCs are typed or used as controls for typing reagents
Individuals with the p phenotype (P null) are very rare: or when older RBCs are used to detect anti-P1 in serum,
5.8 in a million. P nulls are slightly more common in Japan, false-negative reactions may result.
North Sweden, and in an Amish group in Ohio.1
The antibodies generally fall into two categories: clinically Anti-P1
insignificant or potently hemolytic.
Anti-P1 is a common, naturally occurring IgM antibody in
the sera of P1– individuals. Anti-P1 is typically a weak, cold-
Basic Concepts reactive saline agglutinin optimally reactive at 4°C and not
seen in routine testing. Stronger examples react at room
The P blood group antigens, like the ABH antigens, are
temperature, and rare examples react at 37°C and bind com-
synthesized by sequential action of glycosyltransferases,
plement, which is detected in the antiglobulin test when
which add sugars to precursor substances. The precursor
polyspecific (anti-IgG plus anti-C3) reagents are used. Anti-
of P1 can also be glycosylated to type 2H chains, which
body activity can be neutralized or inhibited with soluble P1
carry ABH antigens. P1, P, or Pk may be found on RBCs,
substance. If room temperature incubation is not included,
lymphocytes, granulocytes, and monocytes; P can be found
antibody activity can often be bypassed altogether. Examples
on platelets, epithelial cells, and fibroblasts. P and Pk have
of anti-P1 that react only at temperatures below 37°C can be
also been found in plasma as glycosphingolipids and as
considered clinically insignificant.
glycoproteins in hydatid cyst fluid.7 The antigens have not
Because P1 antigen expression on RBCs varies and deterio-
been identified in secretions. RBCs carry approximately
rates during storage, antibodies may react only with RBCs that
14 × 106 copies of globoside, the P structure, per adult RBC
have the strongest expression and give inconclusive patterns
and about 5 × 105 copies of P1.9
of reactivity when antibody identification is performed. When
The P blood group antigens are resistant to treatment
anti-P1 is suspected, incubating tests at room temperature
with ficin and papain, DTT, chloroquine, and glycine-acid
or lower or pretreating test cells with enzymes can enhance
EDTA. Reactivity of the antibodies can be greatly enhanced
reactions to confirm specificity. Providing units that are
by testing with enzyme-treated RBCs.
crossmatch-compatible at 37°C and the antiglobulin phase,
without typing for P1, is an acceptable approach to transfusion.
Rare examples of anti-P1 that react at 37°C can cause in
The P1 Antigen vivo RBC destruction; both immediate and delayed HTRs
have been reported.13 Anti-P1 is usually IgM; IgG forms
The P1 antigen is poorly expressed at birth and may take up are rare. HDFN is not associated with anti-P1, presumably
to 7 years to be fully expressed.7 Antigen strength in adults because the antibody is usually IgM and the antigen is so
varies from one individual to another: RBCs from some poorly developed on fetal RBCs.
P1+ individuals are P1 strong (P1+s) and others are P1 weak
(P1+w). These differences may be controlled genetically or
Biochemistry
may represent homozygous versus heterozygous inheritance
of the gene coding for P1. The strength of P1 can also vary
with race. Blacks have a stronger expression of P1 than Advanced Concepts
whites. The rare dominant gene for the In(lu) type Lu(a–b–) The RBC antigens of the P blood group exist as glycosphin-
RBCs, discussed in the Lutheran section, inhibits the expres- golipids. As with ABH, the antigens result from the sugars
sion of P1 so that P1 individuals who inherit this modifier added sequentially to precursor structures. Biochemical
gene may type serologically as P1–.
2682_Ch08_172-215 22/05/12 11:44 AM Page 182
analyses have shown that the precursor substance for P1 is Soluble P1 substances have potential use in the blood
also a precursor for type 2H chains that carry ABH antigens. bank and are commercially available. When it is necessary
However, the genes responsible for the formation of the P1 to confirm antibody specificity or to identify underlying
and ABH antigens are independent. antibodies, these substances can be used to neutralize anti-P1.
There are two distinct pathways for the synthesis of the
P blood group antigens, as shown in Figure 8–3. The com- Anti-PP1Pk
mon precursor is lactosylceramide (or Gb2, also known as
Originally called anti-Tja, anti-PP1Pk was first described in
ceramide dihexose or CDH). The pathway on the figure’s
the serum of Mrs. Jay, a p individual with adenocarcinoma
left results in the formation of paragloboside and P1. Para-
of the stomach.10 Her tumor cells carried P system antigens,
globoside is also the type 2 precursor for ABH. The path-
and the antibody was credited as having cytotoxic properties
way shown on the figure’s right side leads to the production
that may have helped prevent metastatic growth postsurgery
of the globoside series: Pk, P, and Luke (LKE).
(the T in the Tja refers to tumor).
Anti-PP1Pk is produced by p individuals early in life without
Genetics RBC sensitization and reacts with all RBCs except those of the
p phenotype. Unlike antibodies made by other blood group
The gene encoding the enzyme responsible for the synthe- null phenotypes, the anti-P, anti-P1, and anti-Pk components
sis of Pk, a 4-α-galactosyltransferase (Gb3 or Pk synthase), of anti-PP1Pk are separable through adsorption.7 Components
was cloned independently by three research groups of anti-PP1Pk have been shown to be IgM and IgG.7 They react
in 2000.14 The gene (B3GALNT1) encoding the 3-β-N- over a wide thermal range and efficiently bind complement,
acetylgalactosaminyltransferase (Gb4 synthase) that is re- which makes them potent hemolysins. Anti-PP1Pk has the
sponsible for converting Pk to P was also cloned in 2000.14 potential to cause severe HTRs and HDFN.
Several mutations in both genes have been identified that The antibody is also associated with an increased
result in the p and Pk phenotypes. A polymorphism in the incidence of spontaneous abortions in early pregnancy.
Pk synthase was recently identified that ties the P1 and Pk Although the reason for this is not fully known, it has been
antigens together at the genetic level; consequently, the P suggested that having an IgG anti-P component is an
system (003), to which the P1 antigen was assigned, was important factor. Women with anti-P and anti-PP1Pk and
renamed P1PK.3 a history of multiple abortions have successfully delivered
The P1PK gene (located at chromosome 22q11.2) and the infants after multiple plasmaphereses to reduce their anti-
P gene (located at chromosome 3q26.1) are inherited independ- body level during pregnancy.15
ently. The gene for the synthesis of LKE has not yet been cloned.
Alloanti-P
Other Sources of P1 Antigen and Antibody
In addition to being a component of the anti-PP1Pk in
The discovery of strong anti-P1 in two P1– individuals p individuals (see above), anti-P is found as a naturally
infected with Echinococcus granulosus tapeworms led to the occurring alloantibody in the sera of Pk individuals. Its re-
identification of P1 and Pk substance in hydatid cyst fluid. activity is similar to that of anti-PP1Pk in that it is usually a
This fluid was subsequently used in many of the studies potent hemolysin reacting with all cells except the autocon-
that identified the biochemical structures of the P blood trol and those with the p phenotype. However, it differs from
group. Strong antibodies to P1 have also been found in anti-PP1Pk in that it does not react with cells that have the
patients with fascioliasis (bovine liver fluke disease) and in extremely rare Pk phenotype, and the individual making the
bird handlers. antibody may type P1+. Alloanti-P is rarely seen, but because
it is hemolytic with a wide thermal range of reactivity, it is
very significant in transfusion. IgG class anti-P may occur
Lactosylceramide (Gb2) and has been associated with habitual early abortion.
Chapter 8 Blood Group Terminology and the Other Blood Groups 183
systems but is demonstrable only by the Donath-Landsteiner blood group system status (system number 027, symbol I)
test. The etiology and diagnosis of PCH are more fully and the i antigen remains in the Ii collection (collection
discussed in Chapter 20, “Autoimmune Hemolytic Anemias.” number 207, symbol I). The reader will notice the confusing
use of the same ISBT symbol, capital I, for both the I system
Antibodies to Compound Antigens and Ii collection; in ISBT terminology, the antigen numbers
provide the distinction between the I (027001) and i
Considering the biochemical relationship of the P blood (207002) antigens.
group antigens to ABH and I, it is not surprising that anti-
bodies requiring more than one antigenic determinant have The I and i Antigens
been described, including anti-IP1, -iP1, -ITP1, and -IP. Most
examples are cold-reactive agglutinins.
Basic Concepts
Luke (LKE) Antigen The antigens are best introduced by classic serologic facts.
Both I and i are high-prevalence antigens, but they are
In 1965, Tippett and colleagues16 described an antibody expressed in a reciprocal relationship that is developmen-
in the serum of a patient with Hodgkin’s lymphoma that tally regulated. At birth, infant RBCs are rich in i; I is almost
divided the population into three phenotypes: 84% tested undetectable. During the first 18 months of life, the quantity
Luke+, 14% were weakly positive or Luke(w), and 2% were of i slowly decreases as I increases until adult proportions
Luke–. Although this Mendelian-dominant character segre- are reached; adult RBCs are rich in I and have only trace
gated independently of the P blood group, it was thought to amounts of i antigen.
be phenotypically related because the antibody reacted with There is no true I– or i– phenotype. The strength of I
all RBCs except 2% of P1 and P2 phenotypes and those hav- and i varies from individual to individual, and the relative
ing the rare p and Pk phenotypes. All individuals with the amount detected will depend on the example of anti-I or
p and Pk phenotype are Luke–. anti-i used. Data suggest that i reactivity on RBCs is
inversely proportional to marrow transit time and RBC age
Disease Associations in circulation.
Some people appear not to change their i status after
Several pathological conditions associated with the P blood birth. They become the rare adult i. Adult i RBCs generally
group antigens have been described: parasitic infections are express more i antigen than do cord RBCs. A spectrum of
associated with anti-P1, early abortions with anti-PP1Pk or Ii phenotypes and their characteristic reactivity are shown
anti-P, and PCH with autoanti-P. The P system antigens also in Table 8–10.
serve as receptors for P-fimbriated uropathogenic E. coli—a Treatment of RBCs with ficin and papain enhances
cause of urinary tract infections. The Pk antigen is a receptor reactivity of the I and i antigens with their respective anti-
for shiga toxins, which cause shigella dysentery and E. coli– bodies. The I and i antigens are resistant to treatment with
associated hemolytic uremic syndrome. In addition, P is the DTT and glycine-acid EDTA.
receptor of human parvovirus B19. Recent studies demon-
strate that Pk provides some protection against HIV infection
of peripheral blood mononuclear cells.17
Anti-I
The I (027) System and I Antigen Anti-I is a common autoantibody that can be found in virtu-
ally all sera, although testing at 4°C and/or against enzyme-
The existence of cold agglutinins in the serum of normal
treated RBCs may be required to detect the reactivity.1
individuals and in patients with acquired hemolytic anemia
Consistently strong agglutination with adult RBCs and weak
has long been recognized. In 1956, Wiener and coworkers2,7
or no agglutination with cord or adult i RBCs define its
gave a name to one such agglutinin, calling its antigen I for
classic activity (see Table 8–10).
“individuality.” The antibody reacted with most blood spec-
Autoanti-I, found in the serum of many normal healthy
imens tested. The few nonreactive I– specimens were
individuals, is benign—that is, not associated with in vivo
thought to be from homozygotes for a rare gene producing
the “i” antigen; the I– phenotype in adults is now called
adult i. In 1960, Marsh and Jenkins18 reported finding anti-i,
and the unique relationship between I and i began to unfold. Table 8–10 I and i Antigens
I and i are not antithetical antigens. Rather, they are
PHENOTYPE STRENGTH OF REACTIVITY WITH
branched and linear carbohydrate structures, respectively,
that are formed by the action of glycosyl transferases. The Anti-I Anti-i Anti-IT
gene encoding the transferase that converts i active straight Adult I Strong Weak Weak
chains into I active branched chains has been cloned, and
several mutations responsible for the rare adult i phenotype Cord Weak Strong Strong
have been identified.19 The synthesis of i antigen is not con- Adult i Weak Strong Weakest
trolled by this same gene. Consequently, I has been raised to
2682_Ch08_172-215 22/05/12 11:44 AM Page 184
RBC destruction. It is usually a weak, naturally occurring, Anti-I is not associated with HDFN because the antibody
saline-reactive IgM agglutinin with a titer less than 64 at 4°C. is IgM, and the I antigen is poorly expressed on infant RBCs.
Stronger examples agglutinate test cells at room temperature
and bind complement, which can be detected in the antiglob- Anti-i
ulin test if polyspecific reagents are used. Some examples
Alloanti-i has never been described. Autoanti-i is a fairly rare
may react only with the strongest I+ RBCs and give incon-
antibody that gives strong reactions with cord RBCs and adult
sistent reactions with panel RBCs.
i RBCs and weaker reactions with adult I RBCs. Most exam-
Incubating tests in the cold enhances anti-I reactivity
ples of autoanti-i are IgM and react best with saline-suspended
and helps confirm its identity; albumin and enzyme methods
cells at 4°C. Only very strong examples of autoanti-i are
also enhance anti-I reactivity. Testing enzyme-treated RBCs
detected in routine testing because standard test cells (except
with slightly acidified serum may even promote hemolysis.
cord RBCs) have poor i expression (see Table 8–10).
Occasionally, benign cold autoanti-I can cause problems in
Unlike anti-I, autoanti-i is not seen as a common antibody
pretransfusion testing. Usually, avoiding room temperature
in healthy individuals. Potent examples are associated with
testing and using anti-IgG instead of a polyspecific antihu-
infectious mononucleosis (Epstein-Barr virus infections) and
man globulin help to eliminate detection of cold reactive
some lymphoproliferative disorders. High-titer autoantibod-
antibodies that may bind complement at lower temperatures.
Cold autoadsorption to remove the autoantibody from the ies with a wide thermal range may contribute to hemolysis,
serum may be necessary for stronger examples; cold autoad- but because i expression is generally weak they seldom cause
sorbed plasma or serum can also be used in ABO typing. significant hemolysis. IgG anti-i has also been described and
Pathogenic autoanti-I (e.g., the type associated with cold has been associated with HDFN.1
agglutinin syndrome) typically consists of strong IgM agglu-
tinins with higher titers and a broad thermal range of activity, Biochemistry and Genetics
reacting up to 30° or 32°C. When peripheral circulation
cools in response to low ambient temperatures, these anti- Advanced Concepts
bodies attach in vivo and cause autoagglutination and An early association of I and i to ABH was demonstrated
peripheral vascular occlusion (acrocyanosis) or hemolytic by complex antibodies involving both ABH and Ii specificity
anemia. Refer to Chapter 20 for more information. (see the “Antibodies to Compound Antigens” section). I and
Pathogenic anti-I typically reacts with adult and cord RBCs i antigens are precursors for the synthesis of ABO and
equally well at room temperature and at 4°C, and antibody Lewis antigens, and thus they are internal structures on
specificity may not be apparent unless the serum is diluted these oligosaccharide chains.
or warmed to 30°C or 37°C. Potent cold autoantibodies can ABH and Ii determinants on the RBC membrane are car-
also mask clinically significant underlying alloantibodies and ried on type 2 chains that attach either to proteins or to lipids.
can complicate pretransfusion testing. Procedures to deal See Figure 8–4 for examples of glycolipid structures for i and
with these problems are discussed in Chapters 9 and 20. I antigens. The i antigen activity is defined by at least two re-
The production of autoanti-I may be stimulated by peating N-acetyllactosamine [Gal(β1-4)GlcNAc(β1-3)] units
microorganisms carrying I-like antigen on their surface. in linear form. I antigen activity is associated with a branched
Patients with M. pneumoniae often develop strong cold form of i antigen. The IGnT (also known as GCNT2) gene on
agglutinins with I specificity and can experience a transient chromosome 6p24 encodes the N-acetylglucosaminyltrans-
episode of acute abrupt hemolysis just as the infection begins ferase, which adds GlcNAc to form the branches.19,20
to resolve. Alloanti-I exists as an IgM or IgG antibody in the In summary, fetal, cord, and adult i RBCs carry predom-
serum of most individuals with the adult i phenotype. inantly unbranched chains and have the i phenotype. Normal
Although adult i RBCs are not totally devoid of I, the anti-I adult cells have more branched structures and express
in these cases does not react with autologous RBCs. It has I antigen. The gene responsible for I antigen (IGnT) codes
been traditional to transfuse compatible adult i units to these for the branching enzyme. Family studies show that the
people, although such practice may be unnecessary, especially adult i phenotype is recessive. Heterozygotes (e.g., children
when the antibody is not reactive at 37°C.1 Technologists inheriting I from one parent and i from the other parent)
must be aware that strong autoanti-I can mimic alloanti-I: have intermediate I antigen expression. Several gene
if enough autoantibody and complement are bound to a mutations have been identified that result in the adult
patient’s RBCs, blocking the antigenic sites, they may falsely i phenotype.
type I-negative.
Antigen Structure Figure 8–4. The linear and branched structures carrying
i and I activity.
(None) Gal(1-4)GlcNAc(1-3)Gal(1-4)GlcNAc(1-3)Gal(1-4)Glc-Cer
i Gal(1-4)GlcNAc(1-3)Gal(1-4)GlcNAc(1-3)Gal(1-4)Glc-Cer
Gal(1-4)GlcNAc(1-3)
I Gal(1-4)GlcNAc(1-3)Gal(1-4)Glc-Cer
Gal(1-4)GlcNAc(1-6)
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Chapter 8 Blood Group Terminology and the Other Blood Groups 185
Other Sources of I and i Antigen to react. For example, anti-IA reacts with RBCs that carry
both I and A but will not react with group O, I+, or group A
I and i antigens are found on the membranes of leukocytes adult i RBCs. (Table 8–11 summarizes some common cold
and platelets in addition to RBCs. It is quite likely that the autoantibodies.) Anti-IH is commonly encountered in the
antigens exist on other tissue cells, much like ABH, but this serum of group A1 individuals. Anti-IH reacts stronger with
has not been confirmed. group O and group A2 RBCs than with group A1 RBCs. Anti-
I and i have also been found in the plasma and serum of IH should be suspected when serum from a group A individual
adults and newborns and in saliva, human milk, amniotic directly agglutinates all group O RBCs but is compatible with
fluid, urine, and ovarian cyst fluid. The antigens in secretions most group A donor units.
do not correlate with RBC expression and are thought to
develop under separate genetic control. For example, the Disease Associations
quantity of I antigen in the saliva of adult i individuals and
newborns is quite high. Well-known associations between strong autoantibodies and
disease or microorganisms have already been discussed: anti-
The IT Antigen and Antibody I with cold agglutinin syndrome and M. pneumoniae, and
anti-i with infectious mononucleosis.
In 1965, Curtain and coworkers21 reported a cold agglu- Diseases can also alter the expression of I and i antigens
tinin in Melanesians that did not demonstrate classical I on RBCs. Conditions associated with increased i antigen
or i specificity. In 1966, Booth and colleagues22 confirmed on RBCs include those with shortened marrow maturation
these observations and carefully described the agglutinin’s time or dyserythropoiesis: acute leukemia, hypoplastic ane-
reactivity. This agglutinin reacted strongly with cord RBCs, mia, megaloblastic anemia, sideroblastic anemia, thalassemia,
weakly with normal adult RBCs, and most weakly with sickle cell disease, paroxysmal nocturnal hemoglobinuria
adult i RBCs. They concluded that the agglutinin recog- (PNH), and chronic hemolytic anemia.1,7 Except in some
nizes a transition state of i into I and designated the speci- cases of leukemia, the increase in i on RBCs is not usually
ficity IT (T for “transition”). However, detection of IT on associated with a decrease in I antigen; the expression of I
fetal RBCs ranging in age from 11 to 16 weeks does not antigen can appear normal or sometimes enhanced.
support this hypothesis.23 This benign IgM anti-IT was Chronic dyserythropoietic anemia type II or hereditary
frequently found in two populations: Melanesians and the erythroblastic multinuclearity with a positive acidified
Yanomama Indians in Venezuela. Whether it is associated serum test (HEMPAS) is associated with much greater i
with an organism or parasite in these regions is unknown. activity on RBCs than control cord RBCs. HEMPAS RBCs
Examples of IgM and IgG anti-IT reacting preferentially at are very susceptible to lysis with both anti-i and anti-I, and
37°C have also been found in patients with warm autoim- lysis by anti-I appears to be the result of increased antibody
mune hemolytic anemia, with a special association with uptake and increased sensitivity to complement.1 In Asians,
Hodgkin’s disease.23 the adult i phenotype has been associated with congenital
cataracts.20
Antibodies to Compound Antigens
The MNS (002) System
Many other I-related antibodies have been described: anti-
IA, -IB, -IAB, -IH, -iH, -IP1, -ITP1, -IHLeb, and -iHLeb. Bear- Following the discovery of the ABO blood group system,
ing in mind the close relationship of I to the biochemical Landsteiner and Levine began immunizing rabbits with
structures of ABH, Lewis, and P antigens, it is not surprising human RBCs, hoping to find new antigen specificities.
to find antibodies that recognize compound antigens. These Among the antibodies recovered from these rabbit sera
specificities are not mixtures of separable antibodies; rather, were anti-M and anti-N, both of which were reported in
both antigens must be present on the RBCs for the antibody 1927.7 Data from family studies suggested that M and N were
*Reactions vary with antibody strength; very potent examples may need to be diluted before specificity can be determined.
0 = negative; + = positive
2682_Ch08_172-215 22/05/12 11:44 AM Page 186
antithetical antigens. In 1947, after the implementation of Table 8–12 Prevalence of Common MN
the antiglobulin test, Walsh and Montgomery discovered and Ss Phenotypes
S, a distinct antigen that appeared to be genetically linked
to M and N. Its antithetical partner, s, was discovered in PHENOTYPE WHITES (%) BLACKS (%)
1951. Family studies (and later, molecular genetics) M+N– 28 26
demonstrated the close linkage between the genes control-
ling M, N, and S, s antigens. There is a disequilibrium in M+N+ 50 44
the expression of S and s with M and N. In whites, the com- M–N+ 22 30
mon haplotypes were calculated to appear in the following
order of relative frequency: Ns > Ms > MS > NS.1,7 The S+s– 11 3
prevalence of the common MN and Ss phenotypes are listed S+s+ 44 28
in Table 8–12.
In 1953, an antibody to a high-prevalence antigen, S–s+ 45 69
U (for almost universal distribution), was named by Weiner. S–s–U– 0 <1
The observation by Greenwalt and colleagues24 that all
U– RBCs were also S–s– resulted in the inclusion of U into
the system.
Forty-six antigens have been included in the MNS system,
making it almost equal to Rh in size and complexity S and s Antigens
(Table 8–13). Most of these antigens are of low prevalence
S and s antigens are located on a smaller glycoprotein called gly-
and were discovered in cases of HDFN or incompatible
cophorin B (GPB) that is very similar to GPA (see “Biochem-
crossmatch. Others are high-prevalence antigens. Antibodies
istry” section and Fig. 8–5). S and s are differentiated by the
to these low- and high-prevalence antigens are not com-
amino acid at position 29 on GPB. Methionine defines S,
monly encountered in the blood bank. The genes encoding
whereas threonine defines s. The epitope may also include the
the MNS antigens are located on chromosome 4.
amino acid residues at position 34 and 35 and the carbohydrate
The MNS blood group system has been assigned the ISBT
chain attached to threonine at position 25.7 There are fewer
number 002 (symbol MNS), second after ABO.
copies of GPB (about 200,000) than GPA per RBC.7 In addition,
there are about 1.5 times more copies of GPB on S+s– RBCs
M and N Antigens
than on S–s+ RBCs.1 S and s also are well developed at birth.
S and s antigens are less easily degraded by enzymes
Basic Concepts because the antigens are located farther down the glycoprotein,
The M and N antigens are found on a well-characterized and enzyme-sensitive sites are less accessible. Ficin, papain,
glycoprotein called glycophorin A (GPA), the major RBC bromelin, pronase, and chymotrypsin can destroy S and s
sialic acid–rich glycoprotein (sialoglycoprotein, SGP). The activity, but the amount of degradation may depend on the
M and N antigens are antithetical and differ in their amino strength of the enzyme solution, the length of treatment, and
acid residues at positions 1 and 5 (Fig. 8–5). M is defined the enzyme-to-cell ratio. Trypsin does not destroy the S and
by serine at position 1 and glycine at position 5; N has s antigens, and neither does DTT, AET, chloroquine, or
leucine and glutamic acid at these positions, respectively. glycine-acid EDTA treatment.
The antibody reactivity may also be dependent on adjacent
carbohydrate chains, which are rich in sialic acid. There are
about 106 copies of GPA per RBC.13 The antigens are well
developed at birth. NH
M N
1
Because M and N are located at the outer end of GPA, 1 Ser 1 Leu
they are easily destroyed by the routine blood bank en- 2 Ser 2 Ser
3 Thr 3 Thr NH2
zymes ficin, papain, and bromelin and by the less common 4 Thr 4 Thr
enzymes trypsin and pronase. The antigens are also de- 5 Gly 5 Glu ‘N’ 1
stroyed by ZZAP, a combination of DTT and papain or ficin, 29 S/s ⫽ Met/Thr
but they are not affected by DTT alone, 2-aminoethyliso- 72 U⫽[ 39
thiouronium bromide (AET), α-chymotrypsin, chloro-
quine, or glycine-acid EDTA treatment. Treating RBCs with
Glu ⫽ glutamic acid
neuraminidase, which cleaves sialic acid (also known as Gly ⫽ glycine 96 72
neuraminic acid or NeuNAc), abolishes reactivity with only Leu ⫽ leucine
some examples of antibody. M and N antibodies are hetero- Met ⫽ methionine
Ser ⫽ serine 131
geneous; some may recognize only specific amino acids, Thr ⫽ threonine GPA GPB
but others recognize both amino acids and carbohydrate
⫽ N-linked glycan
chains.
Figure 8–5. Comparison of glycophorin A (GPA) and glycophorin B (GPB).
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Chapter 8 Blood Group Terminology and the Other Blood Groups 187
Chapter 8 Blood Group Terminology and the Other Blood Groups 189
GYPA GYPB and 56, and anti-EnaFR reacts with a ficin-resistant (FR) area
around amino acids 62 to 72.1,7
Misaligned
Although the gene responsible for this phenotype has
been termed En, it is now known that the En(a–) phenotype
has more than one origin. Most often, the En(a–) phenotype
GYPA GYPB results from homozygosity for a rare gene deletion at the
DNA repair GYPA locus; consequently, no GPA is produced, but GPB is
not affected. This type of En(a–) inheritance is called
GYPA GYP(B-A-B) En(a–)Fin, representing the type described in the Finnish
report.29 However, the En(a–) phenotype in the English
report28 probably represents heterozygosity for a hybrid gene
GYPA GYPB along with the very rare Mk gene; this type is often called
Figure 8–7. In a gene conversion event, nucleotides from one strand of DNA
En(a–)UK. Several other En(a–) phenotypes have been
are transferred to the misaligned homologous gene. If this is the coding strand, a reported that result from homozygosity for a variant allele
hybrid glycophorin will be formed; the partner chromosome will be repaired and or inheritance of two dissimilar variant alleles.
carry the naïve (unaltered) GYPA and GYPB genes. Anti-Ena can be confused with two other specificities
that react with all normal RBCs—anti-Pr and anti-Wrb. Anti-Pr
This high-prevalence antigen is found on RBCs of all indi- does not react with enzyme-treated RBCs and can be con-
viduals except about 1% of African Americans (and 1% to fused with anti-EnaFS; anti-Wrb does not react with En(a–)
35% of Africans) who lack GPB because of a partial or RBCs but does react with enzyme-treated RBCs and can be
complete deletion of GYPB. The RBCs usually type confused with anti-EnaFR. Anti-Ena has caused severe HTRs
S–s–U–, and these individuals can make anti-U in response and HDFN. It is extremely difficult and may be impossible
to transfusion or pregnancy. Anti-U is typically IgG and to find units compatible for patients with anti-Ena; siblings
has been reported to cause severe and fatal HTRs and are a potential source of compatible blood if they are also
HDFN. ABO and Rh compatible.
The U antigen is resistant to enzyme treatment; thus, most Mk Phenotype
examples of anti-U react equally well with untreated and
The rare silent gene Mk was named by Metaxas and
enzyme-treated RBCs. However, there are rare examples of
Metaxas-Buhler in 1964 when they found an allele that did
broadly reactive anti-U that do not react with papain-treated
not produce M or N.7 A second family showed that Mk was
RBCs.1
also silent at the Ss locus. Several other Mk heterozygotes
Some examples of anti-U react with apparent U– RBCs,
have been found. In 1979, two related MkMk blood donors
although weakly, by adsorption and elution.7 Such RBCs are
were found in Japan. The RBCs of these individuals typed
said to be U variant (Uvar); these have an altered GPB that
M–N–S–s–U–En(a–)Wr(a–b–), but they had a normal
does not express S or s. There is a strong correlation between
hematologic picture. More individuals have since been iden-
the low-prevalence antigen He, found in about 3% of African
tified, and it is now known that the Mk gene represents a
Americans, and Uvar expression.27
single, near-complete deletion of both GYPA and GYPB7;
Because examples of anti-U are heterogeneous, U– units
thus, MkMk is the null phenotype in the MNS system. The
selected for transfusion must be crossmatched to determine
MkMk genotype is associated with decreased RBC sialic acid
compatibility. Some patients may tolerate Uvar units; others
content but increased glycosylation of RBC membrane
may not. Many examples of anti-U are actually anti-U plus
bands 3 and 4.1.
anti-GPB. If the patient is U– and N–, the antibody may
actually be a potent anti-N plus anti-U, making the search Other Antibodies in the MNS System
for compatible blood more difficult.
Antibodies to antigens other than M, N, S, and s are rarely
En(a–) Phenotype encountered and can usually be grouped into two categories:
In 1969, Darnborough and coworkers28 and Furuhjelm those directed against low-prevalence antigens and those
and colleagues29 described an antibody to the same high- directed against high-prevalence antigens.
prevalence antigen, called Ena (for envelope), which reacted Antibodies to high-prevalence antigens are easily de-
with all RBCs except those of the propositi. In both of these tected with antibody detection RBCs. Antibodies to low-
cases, the En(a–) individuals appeared to be M–N– with prevalence antigens are rarely detected by the antibody
reduced NeuNAc on their RBCs. The RBCs of the two indi- detection test but are seen as an unexpected incompatible
viduals were mutually compatible. crossmatch or an unexplained case of HDFN. Few hospital
Most En(a–) individuals produce anti-Ena, which is an blood banks have the test cells available to identify the
umbrella term for reactivity against various portions of GPA specificity, but enzyme reactivity and MNS antigen typing
unrelated to M or N, but not all antibodies detect the same may offer clues.
portion. Anti-EnaTS recognizes a trypsin-sensitive (TS) When antibodies to low-prevalence antigens are encoun-
area on GPA between amino acids 20 and 39, anti-EnaFS re- tered, it is common practice to transfuse units that are
acts with a ficin-sensitive (FS) area between amino acids 46 crossmatch-compatible at 37°C and in the antiglobulin
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Chapter 8 Blood Group Terminology and the Other Blood Groups 191
phase. Typing sera for MNS antigens other than M and N and The Kell (006) and Kx (019) Systems
S and s are not generally available, so the antigen status of
compatible RBCs can seldom be confirmed. Also, when one The Kell blood group system consists of 32 high-prevalence
antibody to a low-prevalence antigen is found, antibodies and low-prevalence antigens; it was the first blood group
to other low-prevalence antigens are also frequently present system discovered after the introduction of antiglobulin testing.
in the same serum. If the antibody is directed to a high- Anti-K was identified in 1946 in the serum of Mrs. Kelleher.
prevalence antigen, the assistance of an immunohematology The antibody reacted with the RBCs of her newborn infant, her
reference laboratory is generally needed to identify the older daughter, her husband, and about 7% of the random
antibody and obtain appropriate antigen-negative units. population.7 In 1949, anti-k, the high-prevalence antithetical
partner to K, was described. Kell remained a two-antigen sys-
Autoantibodies tem until the antithetical antigens Kpa and Kpb were described
in 1957 and 1958, respectively. Likewise, Jsa (described in
Autoantibodies to M and N have been reported.1 Not all 1958) and Jsb (described in 1963) were found to be antithetical
examples of anti-M in M+ individuals or anti-N in N+ indi- and related to the Kell system. The discovery of the null phe-
viduals are autoantibodies. Many fail to react with the notype in 1957, designated Ko, helped associate many other
patient’s own RBCs. It may be that these individuals have al- antigens with the Kell system. Antibodies that reacted with all
tered GPA and that their antibody is specific for a portion of RBCs except those with the Ko phenotype recognized high-
the common antigen they lack. Autoantibodies to U and Ena prevalence antigens that were phenotypically related.
are more common and may be associated with warm-type The 32 antigens included in the Kell blood group system,
autoimmune hemolytic anemia. designated by the symbol KEL or 006 by the ISBT, are listed in
Table 8–14. In 1961, a numerical notation was proposed for the
Disease Associations Kell system. As new antigens were discovered, some received
only a number for the antigen name. However, the traditional
GPAM may serve as the receptor by which certain pyelonephri- notation persisted and is more useful in conveying antithetical
togenic strains of E. coli gain entry to the urinary tract. The relationships (e.g., K and Jsa). Consequently, the commonly
malaria parasite Plasmodium falciparum appears to use alter- used nomenclature for Kell antigens is a mixture of symbols
native receptors, including GPA and GPB for cell invasion; using letters and numbers. The associated antigen Kx is the only
some of these receptors also involve NeuNAc. antigen in the Kx system, ISBT number 019 and symbol XK.
Chapter 8 Blood Group Terminology and the Other Blood Groups 193
Antibodies to k antigen are seldom encountered. Only the father for the K antigen. If he’s K+, the fetus should be
2 in 1,000 individuals lack k and are capable of developing monitored carefully for signs of HDFN.
the antibody. The likelihood that these few individuals will
receive transfusions and become immunized is even less. Antibodies to Kpa, Jsa, and Other Low-Prevalence
Kell Antigens
Kpa, Kpb, and Kpc Antigens
Antibodies to the low-prevalence Kell antigens are rare
Alleles Kpa and Kpc are low-prevalence mutations of their because so few people are exposed to these antigens. Because
high-prevalence partner Kpb. The Kpa antigen is found in routine antibody detection RBCs do not carry low-prevalence
about 2% of whites. The Kpa gene is associated with suppres- antigens, the antibodies are most often detected through
sion of other Kell antigens on the same molecule, including unexpected incompatible crossmatches or cases of HDFN.
k and Jsb.7 The effect appears to result from a reduced The serologic characteristics and clinical significance
amount of the Kell glycoprotein (produced by the Kpa allele) of these antibodies parallel anti-K. The original anti-Kpa
inserted in the RBC membrane. The Kpc antigen is even was naturally occurring, but most antibodies result from
more rare. transfusion or pregnancy.
transport protein. The absence of Xk results in McLeod Anti-Ku appears to be a single specificity and cannot be
syndrome (see the “McLeod Phenotype and Syndrome” separated into components. Anti-Ku has caused both HDFN
section below). and HTRs.7
Because Ko RBCs are negative for k, Kpb, Jsb, and so
forth, they are very useful in investigating complex antibody
Genetics problems. They can help confirm a Kell system specificity or
rule out other underlying specificities. When Ko RBCs are
The KEL gene, located on chromosome 7 (at 7q34), is not available, they can be made artificially by treating normal
organized into 19 exons of coding sequence. Single base RBCs with DTT, AET, or glycine-acid EDTA.
mutations encoding amino acid substitutions are responsible
for the different Kell antigens. Several different mutations The McLeod Phenotype and Syndrome
(e.g., point, frameshift, or splice site mutations) have been
found that result in the rare null phenotype Ko.7,9 In 1961, Allen and coworkers33 described a young male med-
No Kell haplotype has been shown to code for more than ical student who initially appeared to be Kell null but who
one low-prevalence antigen. People who test positive for two demonstrated weak expression of k, Kpb, and Jsb detectable
low-prevalence Kell antigens have always been found to by adsorption-elution methods. This unusual phenotype was
carry the encoding alleles on opposite chromosomes. For called McLeod, after the student.
example, someone who types Kp(a+) and Js(a+) is geneti- The McLeod phenotype is very rare. All who have it
cally kKpaJsb on one chromosome and kKpbJsa on the other. are male, and inheritance is X-linked through a carrier
The XK gene, which encodes the Kx antigen, is independent mother. McLeod phenotype RBCs lack Kx and another high-
of KEL and is located on the short arm of the X chromosome prevalence antigen, Km, and have marked depression of all
at position Xp21.1.7 other Kell antigens. The weakened expression of the Kell
antigens is designated by a superscript w for “weak”—for ex-
The Kx Antigen ample, K–k+w Kp(a–b+w). The McLeod phenotype has been as-
sociated with several mutations and deletions at the XK locus.
Kx is present on all RBCs except those of the rare McLeod A significant proportion of the RBCs in individuals with the
phenotype (see the “McLeod Phenotype and Syndrome” McLeod phenotype are acanthocytic (having irregular shapes
section below). Ko and Kmod phenotype RBCs have increased and protrusions) with decreased deformability and reduced
Kx antigen.7 When Kell antigens are denatured with AET or in vivo survival. As a result, individuals with the McLeod
DTT, the expression of Kx increases. phenotype have a chronic but often well-compensated
hemolytic anemia characterized by reticulocytosis, bilirubine-
The Ko Phenotype and Anti-Ku(K5) mia, splenomegaly, and reduced serum haptoglobin levels.
Individuals with the McLeod phenotype have a variety of
Ko RBCs lack expression of all Kell antigens. Ko RBCs have
muscle and nerve disorders that, together with the serologic
no membrane abnormality and survive normally in circulation.
and hematologic picture, are collectively known as the
The phenotype is rare; data suggest a frequency of 1:25,000
McLeod syndrome, one of the neuroacanthocytosis syn-
in whites.7
dromes. McLeod individuals develop a slow, progressive
Immunized individuals with the Ko phenotype typically
form of muscular dystrophy between ages 40 and 50 years
make an antibody called anti-Ku (K5) that recognizes the
and cardiomegaly (leading to cardiomyopathy). The associ-
“universal” Kell antigen (Ku) present on all RBCs except Ko.
ated neurological disorder presents initially as areflexia (a
lack of deep tendon reflexes) and progresses to choreiform
Kell movements (well-coordinated but involuntary movements).
COOH These individuals also have elevated serum creatinine phos-
phokinase levels of the MM type (cardiac/skeletal muscle)
and carbonic anhydrase III levels.
Kx In 1971, Giblett and colleagues made an association
between the rare Kell phenotypes, including the McLeod
phenotype, and the rare X-linked chronic granulomatous
S-S
disease (CGD).34 CGD is characterized by the inability of
phagocytes to make NADH oxidase, an enzyme important
in generating H2O2, which is used to kill ingested bacteria.
NH2 Afflicted children can die at an early age from overwhelming
NH2 infections if not treated. Not all males with the McLeod
COOH phenotype have CGD, nor do all patients with CGD have the
McLeod phenotype.
Figure 8–8. Proposed structures for Kell and Kx proteins. The two proteins are
linked through one disulfide bond. The conformation of the large external domain
At one time it was suggested that CGD was caused by a lack
of the Kell glycoprotein is unknown; 15 cysteine residues suggest the presence of of Kx on white blood cells, and several alleles at the XK locus
disulfide bonds and extensive folding. were proposed to explain Kx expression on McLeod RBCs and
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Chapter 8 Blood Group Terminology and the Other Blood Groups 195
CGD white blood cells. More recent data have shown that this infection. Cultures containing the disrupted organism con-
theory is not valid. The XK gene resides on the X chromosome verted K– cells to K+ but bacteria-free filtrates did not.
near deletions associated with CGD, Duchenne muscular
dystrophy, and retinitis pigmentosa, in the Xp21 region. Autoantibodies
The expression of Kx in women who are carriers of the
McLeod phenotype follows the Lyon hypothesis, which Most Kell autoantibodies are directed against undefined
states that in early embryo development, one X chromosome high-prevalence Kell antigens, but identifiable autoantibodies
randomly shuts down in female cells that have two. All cells to K, Kpb, and K13 have been reported. Mimicking specificities
descending from the resulting cell line express only the allele have also been reported, such as when an apparent anti-K is
on the active chromosome. Hence, McLeod carriers exhibit eluted from DAT+ K– RBCs and the anti-K in the eluate can
two RBC populations: one having Kx and normal Kell be adsorbed onto K– RBCs.
antigens, the other having the McLeod phenotype and
acanthocytosis. The percentage of McLeod phenotype RBCs The Duffy (008) System
in carriers varies from 5% to 85%.1
The Duffy blood group system was named for Mr. Duffy, a
McLeod males with CGD make anti-Kx + Km, which reacts
multiply transfused hemophiliac who in 1950 was found to
strongly with Ko RBCs, weaker with normal Kell phenotype
have the first described example of anti-Fya. One year later,
RBCs, and not at all with McLeod phenotype RBCs. Anti-Km
the antibody defining its antithetical antigen, Fyb, was found
is made by McLeod males without CGD. There are rare reports
in the serum of a woman who had had three pregnancies.
of a McLeod male without CGD who has made anti-Kx +
In 1955, Sanger and colleagues37 reported that the major-
Km.35 The expression of Kell antigens on RBCs with common,
ity of African Americans tested were Fy(a–b–). The gene
McLeod, and Ko phenotypes is summarized in Table 8–16.
responsible for this null phenotype was called Fy. FyFy
appeared to be a common genotype in blacks, especially in
Altered Expressions of Kell Antigens Africa; the gene is exceedingly rare in whites.
Weaker-than-normal Kell antigen expression is associated In 1975, it was observed that Fy(a–b–) RBCs resist infec-
with the McLeod phenotype and the suppression by the tion in vitro by the monkey malaria organism Plasmodium
Kpa gene (cis-modified effect) on Kell antigens. Depressed knowlesi. It was later shown that Fy(a–b–) RBCs also resist
Kell antigens are also seen on RBCs with the rare Gerbich- infection by P. vivax (one of the organisms causing malaria
negative phenotypes Ge: –2, –3, 4 and Ge: –2, –3, –4. The in humans).37 This discovery provides an explanation for
phenotypic relationship between Gerbich and Kell is not un- the predominance of the Fy(a–b–) phenotype in persons
derstood. The umbrella term Kmod is used to describe other originating from West Africa.
phenotypes with very weak Kell expression, often requiring Antibodies to other antigens in the Duffy blood group
adsorption-elution tests for detection. As a group, these system, Fy3, Fy5, are rarely encountered. RBCs that are
RBCs have a reduced amount of Kell glycoprotein and Fy(a–b–) are also Fy: –3, –5. Fy5 is also not present on Rhnull
enhanced Kx expression. Some Kmod individuals make an RBCs, regardless of the Fya or Fyb status of those RBCs. The
antibody that resembles anti-Ku but does not react with Duffy blood group system is designated by the symbol FY or
other Kmod RBCs (unlike anti-Ku made by Ko individuals). 008 by the ISBT.
Patients with autoimmune hemolytic anemia, in which
the autoantibody is directed against a Kell antigen, may have Fya and Fyb Antigens
depressed expression of that antigen. Antigen strength
returns to normal when the anemia resolves and the DAT Basic Concepts
becomes negative. This phenomenon appears to be more The Duffy antigens most important in routine blood bank
common in the Kell system than in others.1 serology are Fya and Fyb. They can be identified on fetal
Finally, RBCs may appear to acquire Kell antigens. RBCs as early as 6 weeks gestational age and are well
McGinnis and coworkers36 described a K– patient who developed at birth. There are about 13,000 to 14,000 Fya
acquired a K-like antigen during a Streptococcus faecium
Table 8–16 Expression of Kell Antigens on RBCs With Common, Ko, and McLeod Phenotypes
PHENOTYPE RBC ANTIGEN EXPRESSION POSSIBLE ANTIBODY
Kell Antigens Km Kx
or Fyb sites on Fy(a+b–) and Fy(a–b+) RBCs, respectively; Anti-Fya and anti-Fyb have been associated with acute and
there are half that number of Fya sites on Fy(a+b+) RBCs.7 delayed HTRs. Once the antibody is identified, Fy(a–) or
The antigens have not been found on platelets, lymphocytes, Fy(b–) blood must be given; finding such units in a random
monocytes, or granulocytes, but they have been identified population is not difficult. For example, one in three random
in other body tissues, including brain, colon, endothelium, units of blood is Fy(a–) and one in five random units of
lung, spleen, thyroid, thymus, and kidney cells.9 The preva- blood is Fy(b–). Anti-Fya and anti-Fyb are associated with
lence of the common phenotypes in the Duffy system are HDFN that ranges from mild to severe.
given in Table 8–17. The disparity in distribution in different Rare autoantibodies with mimicking Fya and Fyb speci-
races is notable. ficity have been reported—for example, anti-Fyb that can be
Fya and Fyb antigens are destroyed by common proteolytic adsorbed onto and eluted from Fy(a+b–) RBCs. Issitt and
enzymes, such as ficin, papain, bromelin, and chymotrypsin, Anstee1 suggest that these may represent alloantibodies with
and by ZZAP (which contains either papain or ficin in “sloppy” specificity made early in an immune response.
addition to DTT); they are not affected by DTT alone, AET,
or glycine-acid EDTA treatment. Neuraminidase may reduce Biochemistry
the molecular weight of Fya and Fyb, but it does not destroy
antigenic activity and neither does purified trypsin. Advanced Concepts
Enzymes, membrane solubilization methods, immunoblot-
ting, radiolabeling, and amino acid sequencing have all
Anti-Fya and Anti-Fyb been used to study the biochemistry of Duffy antigens.1
Duffy antigens reside on a glycoprotein of 336 amino acids
Anti-Fya is a common antibody and is found as a single
that has a relative mass of 36 kD and two N-glycosylation
specificity or in a mixture of antibodies. Anti-Fya occurs
sites (Fig. 8–9).38 The glycoprotein is predicted to traverse
three times less frequently than anti-K. Anti-Fyb is 20 times
the cell membrane seven times and has two predicted disul-
less common than anti-Fya and often occurs in combination
fide bridges.
with other antibodies. The antibodies are usually IgG and
The amino acid at position 42 on the Duffy glycoprotein
react best at the antiglobulin phase. Rare examples of
defines the Fya and Fyb polymorphism: Fya has glycine, and
anti-Fya and anti-Fyb bind complement. A few examples are
Fyb has aspartic acid. The Fy3 epitope, as defined by mon-
saline agglutinins. Antibody activity is enhanced in a low
oclonal antibody, is on the third extracellular loop, and Fy6
ionic strength medium. Because anti-Fya and anti-Fyb do not
appears to involve amino acids 19 through 25.38
react with enzyme-treated RBCs, this is a helpful technique
The Duffy glycoprotein is a member of the superfamily
when multiple antibodies are present.
of chemokine receptors and is known as the Duffy antigen
Some examples of anti-Fya and anti-Fyb show dosage, react-
receptor for chemokines (DARC). Thus, in addition to
ing more strongly with RBCs that have a double dose than RBCs
being a receptor for the malaria parasite P. vivax, the
from heterozygotes. It must be remembered that some reagent
Duffy glycoprotein binds a variety of proinflammatory
RBCs that appear to be from homozygotes (and have a double
cytokines.
dose of either Fya or Fyb) may actually be from heterozygotes if
they are from black donors; a silent allele, Fy, is commonly
found in blacks. For example, Fy(a+b–) RBCs will have a Genetics
double dose of Fya if they are from a white FyaFya donor but
will have a single dose of Fya if they are from a black donor who In 1968, the Duffy gene was linked to a visible, inherited
is genetically FyaFy. Additional phenotypic markers commonly abnormality of chromosome 1, thus becoming the first human
found in black donors can give a clue to the possible presence gene to be assigned to a specific chromosome. The gene is
of the silent Fy allele: Ro, S–s–, V+VS+, Js(a+), Le(a–b–). located near the centromere on the long arm of chromosome 1
at position 1q23.2. The Fy locus is syntenic to the Rh locus,
which is located near the tip of the short arm; that is, they
Table 8–17 Prevalence of Common Duffy
Phenotypes
Fy6 Fy3
Fya/Fyb
[
AFRICAN NH2
[
Fy(a+b–) 17 9 90.8
Fy(a+b+) 49 1 8.9
Fy(a–b–) Very rare 68 0 Figure 8–9. Proposed structure for the Duffy protein. Disulfide bonds probably
link the NH2 terminal domain and the third loop and the first and second loop.
2682_Ch08_172-215 22/05/12 11:44 AM Page 197
Chapter 8 Blood Group Terminology and the Other Blood Groups 197
are on the same chromosome, but they are far enough apart in that it reacted with the cells from an Fy(a–b–)Fy:–3
that linkage cannot be demonstrated and serologically they white female, but it did not react with Fy(a+) or Fy(b+)
appear to segregate independently. Rhnull RBCs and reacted only weakly with Fy(a+) or Fy(b+)
There are three common alleles at the Fy locus: Fya, Fyb, D__ RBCs.
and Fy. Fya and Fyb encode the antithetical antigens Fya and Sometimes, sera containing anti-Fy5 also contain anti-
Fyb, respectively, and Fy is a silent allele and is the major Fya. Several examples of anti-Fy5 have been reported in mul-
allele in blacks. The Fy gene in Fy(a–b–) blacks is an Fyb tiply transfused Fy(a–b–) sickle cell patients with a mixture
variant with a change in the promoter region of the gene, of other antibodies.
which disrupts the binding site for mRNA transcription in The molecular structure of Fy5 is not known, but it
the RBC.9 Consequently, Fy(a–b–) blacks do not express Fyb appears to be the result of interaction between the Rh com-
on their RBCs but express Fyb in other tissues. The presence plex and the Duffy glycoprotein. People who are Fy(a–b–)
of Fyb in tissues presumably precludes the recognition of Fyb or Rhnull do not make Fy5 antigen and are at risk of making
as foreign; thus, no anti-Fyb is made by these individuals. A the antibody, although few do. Like Fy3, Fy5 is not destroyed
molecular analysis of Fy(a–b–) whites revealed different by enzymes.
mutations. These individuals carry no Duffy protein on their
RBCs or on other tissues and thus can form anti-Fyb and The Kidd (009) System
anti-Fy3.
Typing for Duffy antigens has been performed on the The Kidd blood group is a simple and straightforward sys-
RBCs of chimpanzees, gorillas, and old- and new-world tem consisting of only three antigens. In 1951, Allen and
monkeys. The results suggest that Fy3 developed first, then colleagues41 reported finding an antibody in the serum of
Fyb, and that Fya arose during human evolution.9 Mrs. Kidd, whose infant had HDFN. The antibody, named
anti-Jka, reacted with 77% of Bostonians. Its antithetical
Fyx partner, Jkb, was found 2 years later. The null phenotype
Jk(a–b–) was described in 1959. The propositus made an
Fyx was described in 1965 as a new allele at the Fy locus. antibody to a high-prevalence antigen called Jk3, which
It does not produce a distinct antigen but rather is an in- is present on any RBC positive for Jk a or Jkb. No other
herited weak form of Fyb that reacts with some examples antigens associated with the Kidd system have been
of anti-Fyb. Fyx has been described in white populations. described.
Individuals with Fyx may type Fy(b–), but their RBCs ad- The Kidd system is designated by the symbol JK or 009
sorb and elute anti-Fyb. They also have depressed expres- by the ISBT. It has special significance to routine blood bank-
sion of their Fy3 and Fy5 antigens. The decreased ing because of its antibodies, which can be difficult to detect
expression of Fyb due to Fyx appears to be related to a and are a common cause of HTRs.
reduced amount of Duffy glycoprotein on the surface of
RBCs.39 There is no anti-Fyx. Jka and Jkb Antigens
Table 8–18 Prevalence of Kidd Phenotypes intravascular hemolysis has been noted in severe reactions,
coated RBCs more often are removed extravascularly. The rate
WHITES BLACKS ASIANS of clearance of incompatible RBCs can vary but is usually rapid.
PHENOTYPE (%) (%) (%) Contrary to their hemolytic reputation in transfusion,
Jk(a+b–) 28 57 23 most Kidd antibodies are only rarely associated with severe
cases of HDFN.42
Jk(a+b+) 49 34 50
Jk(a–b+) 23 9 27 Biochemistry
Jk(a–b–) Exceedingly Exceedingly 0.9 Polynesians
rare rare Advanced Concepts
Heaton and McLoughlin43 reported in 1982 that Jk(a–b–)
RBCs resist lysis in 2M urea, a solution commonly used to
lyse RBCs in a sample before it is used in some automated
Anti-Jka and Anti-Jkb platelet-counting instruments. Urea crosses the RBC mem-
brane, causing an osmotic imbalance and an influx of
Kidd antibodies have a notorious reputation in the blood water, which rapidly lyses normal cells. With Jk(a+) or
bank. They demonstrate dosage, are often weak, and are Jk(b+) RBCs, lysis in 2M urea occurs within 1 minute; with
found in combination with other antibodies, all of which Jk(a–b–) cells, lysis is delayed by 30 minutes.7
make them difficult to detect. The predicted Kidd glycoprotein has 389 amino acids with
Anti-Jka is more frequently encountered than anti-Jkb, but 10 membrane-spanning domains and two N-glycosylation
neither antibody is common. The antibodies are usually IgG sites, one of which is extracellular on the third extracellular
(antiglobulin reactive) but may also be partly IgM and are loop (Fig. 8–10). The glycoprotein is a urea transporter.
made in response to pregnancy or transfusion.
The ability of Kidd antibodies to show dosage can con-
found inexperienced serologists. Many anti-Jka and anti-Jkb Genetics
react more strongly with RBCs that carry a double dose of
the respective antigen and may not react with Jk(a+b+) The Jk locus is on chromosome 18 at position 18q12.3. The
RBCs. An anti-Jka that reacts only with Jk(a+b–) RBCs can gene SLC14A1 (for solute carrier family 14, member 1) is a
give inconclusive panel results and appear compatible with member of the urea transporter gene family. The gene is
Jk(a+b+) cells. Readers are urged to rule out anti-Jka and organized into 11 exons. The Jka/Jkb polymorphism is asso-
anti-Jkb only with Jk(a+b–) and Jk(a–b+) panel cells, respec- ciated with an amino acid substitution at position 280, pre-
tively, and to type all crossmatch-compatible units with com- dicted to be located on the fourth extracellular loop of the
mercial antisera. To ensure that antisera can indeed detect glycoprotein. Molecular studies have demonstrated the silent
weak expressions of the antigen, Jk(a+b+) RBCs should be Jk allele can arise from mutations in both the Jka and Jkb
tested in parallel as the positive control. alleles. Jka and Jkb are inherited as codominant alleles.
Antibody reactivity can also be enhanced by using LISS or
PEG (to promote IgG attachment), by using four drops of Jk(a–b–) Phenotype and the Recessive Allele, Jk
serum instead of two (to increase the antibody-to-antigen ratio),
People with the null Jk(a–b–) phenotype lack Jka, Jkb, and
or by using enzymes such as ficin or papain. In vitro hemolysis
the common antigen Jk3. Although very rare, the Jk(a–b–)
can sometimes be observed with enzyme-treated RBCs if serum
phenotype is most abundant among Polynesians, and it has
is tested; antigen dose may influence this hemolytic activity.1
also been identified in Filipinos, Indonesians, Chinese, and
Many examples of the Kidd antibodies bind complement.
Japanese.1 The null phenotype has also been reported in sev-
Rare examples are detected only by the complement they
eral European families (Finnish, French, Swiss, and English)
bind (i.e., they are nonreactive in antiglobulin tests using
and in the Mato Grosso Indians of Brazil. The delayed lysis
anti-IgG reagents). Testing serum (rather than plasma)
and using polyspecific reagents with both anti-IgG and anti-
complement can be helpful in these situations.1
The titer of anti-Jka or anti-Jkb quickly declines in vivo. Jka/Jkb
A strong antibody identified following a transfusion reaction
may be undetectable in a few weeks or months.1 This con-
firms the need to check blood bank records for previously
identified antibodies before a patient is transfused. It is
equally important to inform the patient that he or she has
such an antibody and to provide a wallet card that notes the
specificity in case the patient is transfused elsewhere. NH2 COOH
The decline in antibody reactivity and the difficulty in Figure 8–10. Proposed structure for Kidd protein. One of two proposed N-glycans
detecting Kidd antibodies are reasons why they are a common is extracellular and is located on the third extracellular loop; the Jka/Jkb polymorphism
cause of HTRs, especially of the delayed type. Although is located on the fourth extracellular loop.
2682_Ch08_172-215 22/05/12 11:44 AM Page 199
Chapter 8 Blood Group Terminology and the Other Blood Groups 199
of Jk(a–b–) RBCs in 2M urea has proved an easy way to made anti-c, anti-N, the first example of anti-CW, and anti-
screen families and populations for this rare phenotype. Levay, now known as Kpc!) The new antibody was named
No clinical abnormalities have been associated with Lutheran for the donor; the donor’s last name was Lutteran
the Jk(a–b–) phenotype to date. Several unrelated Jk(a–b–) but the donor blood sample was incorrectly labeled.2 In
individuals had normal blood urea nitrogen, creatinine, and 1956, Cutbush and Chanarin45 described anti-Lub, which
serum electrolytes, but studies on two individuals with defined the antithetical partner to Lua.
this phenotype showed a marked defect in their ability to The blood group system appeared complete until 1961,
concentrate urine. when Crawford and colleagues46 described the first Lu(a–b–)
Family studies show that most Jk(a–b–) nulls are homozy- phenotype. Unlike most null phenotypes at the time, this one
gous for the rare “silent” allele Jk. Parents of JkJk offspring demonstrated dominant inheritance. In 1963, the Lu(a–b–)
and children of JkJk parents type Jk(a+b–) or Jk(a–b+) but phenotype inherited as a recessive silent allele was described.
never Jk(a+b+), because they are genetically JkaJk or JkbJk. Twenty antigens are part of the Lutheran system, num-
Their RBCs also demonstrate a single dose of Jka or Jkb bered through Lu22; some antigens are also known by other
antigen in titration studies. common names. Two numbers (Lu10 and Lu15) are obso-
lete. Most of these antigens are high prevalence; four sets
Jk(a–b–) Phenotype and the Dominant In(Jk) Allele of antigens are antithetical. Many of the antigens were
associated with the Lutheran system when their correspon-
Another genetic explanation for the Jk(a–b–) phenotype is ding antibodies were nonreactive with the rare Lu(a–b–)
association with a dominant gene called In(Jk), for “in- RBCs. All are summarized in Table 8–19. The ISBT designa-
hibitor,” that shows a dominant pattern of inheritance within tion of the Lutheran blood group system is LU or 005.
a Japanese family analogous to the inhibitor gene responsible
for the dominant type Lu(a–b–) phenotype in the Lutheran
blood group system.7,9 Dominant type Jk(a–b–) RBCs adsorb Basic Concepts
and elute anti-Jk3 and anti-Jka or anti-Jkb (depending on
Blood bankers seldom deal with the serology of the
which genes were inherited), indicating that the antigens are
Lutheran blood group system. The antigens are either high
expressed but only very weakly. Individuals with the domi-
prevalence, so only a few people lack the antigen and can
nant type Jk(a–b–) phenotype do not make anti-Jk3. Family
make an alloantibody, or very low prevalence, so that only
studies show that the In(Jk) gene does not reside at the Jk
a few people are ever exposed. Consequently, the antibodies
locus. The molecular basis is unknown.
are seen infrequently, and there are not much data on the
clinical significance of Lutheran antibodies.
Anti-Jk3
Although the antigens have been detected on fetal RBCs
Alloanti-Jk3 is an IgG antiglobulin-reactive antibody that looks as early as 10 to 12 weeks of gestation, they are poorly de-
like an inseparable anti-JkaJkb. Because panel cells are Jk(a+) or veloped at birth. As a result, HDFN is rare and only mild.9
Jk(b+), anti-Jk3 reacts with all RBCs tested except the autocon- Lutheran antigens have not been detected on platelets, lym-
trol. Most blood banks do not have the rare cells needed to con- phocytes, monocytes, or granulocytes. However, Lutheran
firm anti-Jk3; however, they can easily determine its most glycoprotein is widely distributed in tissues: brain, lung,
probable specificity by means of antigen typing. The individual pancreas, placenta, skeletal muscle, and hepatocytes (espe-
making the antibody will type Jk(a–b–). Like other Kidd anti- cially fetal hepatic epithelial cells).7 The presence of Lutheran
bodies, anti-Jk3 reacts optimally by an antiglobulin test, and the glycoprotein on placental tissue may result in adsorption
reactivity is enhanced with enzyme pretreatment of the RBCs. of maternal antibodies to Lutheran antigens, thus decreasing
Anti-Jk3 has been associated with severe immediate and the likelihood of HDFN.9
delayed HTRs and with mild HDFN. Compatible units are Lutheran antigens are resistant to the enzymes ficin and
best found by typing siblings or searching the rare donor files. papain and to glycine-acid EDTA treatment but are destroyed
by treatment with the enzymes trypsin and α-chymotrypsin.
Most Lutheran antibodies do not react with RBCs treated
Autoantibodies
with the sulfhydryl reagents DTT and AET.
Autoantibodies with Kidd specificity (anti-Jka, anti-Jkb, and
anti-Jk3) are rare, but they have been associated with
autoimmune hemolytic anemia.1 As with other blood Lua and Lub Antigens
groups, Kidd autoantibodies may have mimicking specificity
or may be associated with depressed antigen expression. Lua and Lub are antigens produced by allelic codominant
genes. The prevalence of common phenotypes are listed in
The Lutheran (005) System Table 8–20. Most individuals are Lu(b+); 8% of whites and
5% of blacks are Lu(a+).9
In 1945, anti-Lua was found (and described in detail a year Lutheran antigen expression is variable from one individ-
later) in the serum of a patient with lupus erythematosus, ual to another. The number of Lub sites per RBC is low,
following the transfusion of a unit of blood carrying the estimated to be from 1,640 to 4,070 on Lu(a–b+) RBCs and
corresponding low-prevalence antigen.44 (This patient also from 850 to 1,820 on Lu(a+b+) RBCs.7
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Chapter 8 Blood Group Terminology and the Other Blood Groups 201
Biochemistry Lu and the Se gene (FUT2) was the first example of autosomal
linkage described in humans.
Advanced Concepts
Lu(a–b–) Phenotypes
The Lutheran antigens are located on a type 1 transmembrane
protein. The protein exists in two forms as a result of alter- Three genetic explanations for the Lu(a–b–) phenotype have
native RNA splicing: the longer Lu glycoprotein and the been described. These are summarized in Table 8–21.
shorter basal cell adhesion molecule (B-CAM). The longer
85-kD protein contains 597 amino acids with five extracel- Dominant Type Lu(a–b–)
lular domains, a hydrophobic transmembrane domain of The first Lu(a–b–) family study was reported by the proposi-
19 amino acids, and a cytoplasmic domain of 59 amino acids tus herself.46 Because the phenotype was seen in successive
(Fig. 8–11). The smaller isoform (78 kD) is identical except generations in 50% of her family members and others, and
for a shorter cytoplasmic domain of 59 amino acids. The because null individuals passed normal Lutheran genes to
external portion consists of five disulfide-bonded domains. their offspring, the expression of Lutheran was thought to
The Lutheran glycoproteins belong to the immunoglobulin be suppressed by a rare dominant regulator gene later called
superfamily of proteins; the repeating extracellular domains In(Lu) for “inhibitor of Lutheran.” Recently, mutations in the
are homologous to immunoglobulin variable or constant gene for Erythroid Krüppel-like Factor (EKLF), a transcrip-
domains. The Lutheran proteins are multifunctional adhesion tion factor, were shown to be associated with the In(Lu)
molecules that bind laminin, notably in sickle cell disease.47 phenotype in 21 of 24 In(Lu) individuals studied.49 In all
The molecular basis for the four pairs of antithetical anti- cases, the mutated EKLF allele occurred in the presence of a
gens and several of the high-prevalence antigens has been normal EKLF allele. The authors of this study concluded
determined through the creation of Lutheran glycoprotein that the In(Lu) phenotypeis caused by inheritance of a loss-
mutants and subsequent sequencing of the exons encoding of-function mutation on one allele of EKLF.
the extracellular domains. For most of the antigens, expres- Dominant-type Lu(a–b–) RBCs carry trace amounts of
sion was caused by a single nucleotide polymorphism, result- Lutheran antigens as shown by adsorption-elution studies.
ing in single amino acid changes on the protein level.48 For example, the RBCs of a person with the dominant type
Lu(a–b–) who inherited two normal Lub genes will type
Lu(a–b–) with routine methods but will adsorb and elute
Genetics anti-Lub. Because individuals with the dominant type
The Lu gene is located on chromosome 19 at position Lu(a–b–) RBCs have normal Lutheran antigens, they do not
19q13.2, along with genes that govern expression of several make anti-Lu3.
blood group antigens (H, Se, Le, LW, Oka) . A linkage between In addition to reduced expression of Lutheran antigens,
dominant type Lu(a–b–) RBCs also can have reduced expres-
sion of CD44 and a weak expression of P1, i, AnWj, MER2,
NH2
and Inb blood group antigens.
Lua/Lub
Lu21 Recessive Type Lu(a–b–)
Lu5
Lu17 V V In some families, the Lu(a–b–) phenotype demonstrates
Lu12 recessive inheritance, the result of having two rare silent
Lu4
Lu8/Lu14 V V
alleles LuLu at the Lutheran locus. The parents and offspring
Lu16 of these nulls may type Lu(a–b–), but dosage studies and
titers show them to carry a single dose of Lub.
Lu20 C C
Lu6/Lu9 Unlike the dominant type, people with recessive Lu(a–b–)
RBCs truly lack all Lutheran antigens (i.e., they have the null
Lu7 C C phenotype) and can make an inseparable anti-Luab called
anti-Lu3. They also have normal antigen expression of P1, i,
Lu13
and the other antigens that are weakened with the dominant
Aua/Aub C C type. This distinction emphasizes the importance of testing
an antibody against recessive Lu(a–b–) RBCs before defining
the specificity phenotypically related to Lutheran.
Different inactivating mutations were recently reported for
three individuals with the recessive Lu(a–b–) type.50 In all three
individuals the mutations would result in a truncated glycopro-
Lu B-CAM tein that would not be integrated in the RBC surface membrane.
Figure 8–11. The two structures encoded by the Lu gene: the longer Lu Recessive X-Linked Inhibitor Type
glycoprotein and the shorter basal cell adhesion molecule (B-CAM). The five
extracellular disulfide-bonded domains are homologous to immunoglobulin variable An Lu(a–b–) phenotype in a large Australian family did not
(V) or constant (C) domains. fit either the dominant or recessive inheritance patterns. All
2682_Ch08_172-215 22/05/12 11:44 AM Page 202
Lu(a–b–) family members were male and carried trace located on the last (seventh) extracellular loop of the
amounts of Lub detected by adsorption-elution. The pattern protein. The Di(a–b–) phenotype has not been reported.
of inheritance suggested an X-borne inhibitor to Lutheran. Wra, a low-prevalence antigen, and the antithetical, high-
The researchers proposed calling the locus XS, XS1 being the prevalence Wrb are associated with an amino acid substitu-
common allele and XS2 the rare inhibitor that suppresses in tion on the fourth external loop, close to the insertion point
a hemizygous state. There have been no other families of the protein into the RBC membrane. However, expression
reported with this rare X-linked Lu(a–b–) phenotype.9 of Wrb requires the presence of both band 3 and a normal
GPA of the MNS blood group system. GPA-deficient RBCs
Anti-Lu3 are Wr(a–b–).7
Localization of Dia and Dib antigens to band 3 enabled
Anti-Lu3 is a rare antibody that reacts with all RBCs except many low-prevalence antigens to be assigned to the Diego
Lu(a–b–) RBCs. The antibody looks like inseparable anti- system: Wda, Rba, WARR, ELO, Wu, Bpa, Moa, Hga, Vga, Swa,
Luab and recognizes a common antigen, Lu3, that is present BOW, NFLD, Jna, KREP, Tra, Fra, and Sw1.
whenever Lua or Lub is present (much like the Jk3 associa- Diego antigens are expressed on RBCs of newborns. The
tion with Jka and Jkb). Anti-Lu3 is usually antiglobulin- antigens are resistant to treatment with ficin and papain,
reactive. This antibody is made only by individuals with the DTT, and glycine-acid EDTA, with the exception of Bpa,
recessive type of Lu(a–b–). which is sensitive to papain.7,9
Diego system antibodies are sometimes IgM, but are usually
The Diego (010) System IgG, reactive in the indirect antiglobulin test. Both anti-Dia and
anti-Dib have caused HTRs and HDFN.7,9 Anti-Wra is a rela-
The Diego system is composed of 22 antigens: three sets tively common antibody in donors and patients; some are di-
of independent pairs of antithetical antigens—Dia/Dib, rectly agglutinating, but most require the indirect antiglobulin
Wra/Wrb, and Wu/DISK—and 17 low-prevalence antigens.3,7 test to be detected. Anti-Wra has caused severe HTRs. Only a
The system was named after the first antibody maker in a few examples of alloanti-Wrb in individuals with Wr(a–b–)
Venezuelan family during an investigation of HDFN. The RBCs have been described, so information about clinical sig-
Diego system is designated DI and number 010 by the ISBT. nificance is insufficient. Autoanti-Wrb is relatively common in
The Diego antigens are carried on band 3, a major integral the serum of patients with warm autoimmune hemolytic
RBC membrane glycoprotein with about 1 million copies per anemia. Little or no data are available on the clinical signifi-
RBC. Band 3 is also known as the red cell anion exchanger cance of antibodies to the low-prevalence Diego antigens, with
(AE1) or solute carrier family-4. anion exchanger, member 1 the exception of anti-ELO, which has caused severe HDFN.7
(SLC4A1). The protein crosses the membrane multiple
times, and both the amino- and carboxyl-terminal domains The Yt (011) System
are in the cytoplasm. A large N-glycan on the fourth external
loop carries over half the RBC A, B, H, and I blood group Two antigens make up the Yt system, which was named in
antigens. The long amino-terminal domain of band 3 inter- 1956 for the first antibody maker and used the last letter “t”
acts with ankyrin and protein 4.2 of the membrane skeleton. in the patient’s last name, which was Cartwright.51 Appar-
The gene encoding band 3 and the Diego antigens, SLC4A1, ently “why T” became “Yt.”9 Yta is the high-prevalence anti-
consists of 20 exons and is located at chromosome 17q21-q22. gen in all populations; Ytb is the low-prevalence antigen
Reported in 1955, anti-Dia had caused HDFN in a found in about 8% of whites and 21% to 26% of Israelis, but
Venezuelan baby. Anti-Dib was described 2 years later. Dia is it is not found in Japanese.7,9 Three phenotypes are observed:
rare in most populations but is polymorphic in people of the common Yt(a+b–) and Yt(a+b+) and the rare Yt(a–b+).
Mongoloid ancestry. In South American Indians, the preva- The Yt(a–b–) phenotype has not been reported. The Yt
lence of Dia can be as high as 54%.9 Dia is also present in system has the ISBT designation YT and system number 011.
the North and Central American native populations but is The Yt antigens are antithetical and represent an amino
surprisingly rare in Canada and among the Alaskan Inuit.7 acid substitution on the glycosylphosphatidylinositol (GPI)-
The prevalence of Dib is generally greater than 99% but is linked RBC glycoprotein acetylcholinesterase (AChE). AChE
96% in Native Americans. The Dia/Dib polymorphism is is an important enzyme participating in neurotransmission,
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Chapter 8 Blood Group Terminology and the Other Blood Groups 203
but the function of RBC-bound AChE is not known. The named after the first antibody maker. In 1962, a new high-
gene is located at chromosome 7q22. prevalence antigen was named Sm; 1 year later, a new low-
Yt antigens are variably sensitive to ficin and papain, are prevalence antigen, Bua was found. After it was confirmed
sensitive to DTT, and are resistant to glycine-acid EDTA that these two antigens were antithetical, the Scianna blood
treatment. The antigens are developed at birth but are group system was established in 1974 and the two antigens
expressed more weakly on cord RBCs than on adult RBCs, were renamed Sc1 and Sc2, respectively. The prevalence
and are absent from RBCs of people with paroxysmal noc- of Sc2 in Northern Europeans is 1% but is higher in the
turnal hemoglobinuria (PNH) III.7 Mennonite population.
Anti-Yta and anti-Ytb are IgG and are stimulated by preg- In 1980, an individual in the Marshall Islands in the South
nancy or transfusion. Anti-Yta is not an uncommon antibody, Pacific was found with the Sc:–1,–2 phenotype. He had made
so it appears that Yta is reasonably immunogenic. However, an antibody to an unknown high-prevalence antibody. The
Ytb appears to be a poor immunogen, as the antibody is rare. antibody was named anti-Sc3; separable anti-Sc1 and anti-
Yt antibodies have not caused HDFN. Some examples of Sc2 were not identified. The very rare Sc:–1,–2,–3 phenotype
anti-Yta have been shown to be clinically significant for is the Scianna null type. Three examples of anti-Sc3, nonre-
transfusion while others have not.7 active with Sc:–1,–2, were found to be incompatible with the
RBCs of the other anti-Sc3 makers, indicating the existence
The Xg (012) System of additional high-prevalence antigens.53
The SC gene is located on chromosome 1 at 1p34. The
Anti-Xga was discovered in 1962 in the serum of a multiply product of the gene is a protein called erythroid membrane-
transfused man. The antibody detected an antigen with a associated protein (ERMAP), which is an RBC adhesion
higher prevalence in females than in males. Family studies protein. Once location of Scianna to ERMAP was made, other
were used to confirm the antigen Xga expression was con- antigens were assigned to the system. The low-prevalence
trolled by an X-linked gene. The antigen was named after antigen Rd became Sc4. Sc5 (STAR), Sc6 (SCER), and Sc7
the X chromosome and g for “Grand Rapids,” where the (SCAN) are all high-prevalence antigens.54
patient was treated.9 The Scianna antigens are resistant to ficin and papain but
The Xg system is designated by the symbol XG and num- are slightly weakened by DTT treatment. The antigens are
ber 012. There are two antigens in the Xg system: Xga and expressed on cord RBCs.
CD99. CD99 is also known as 12E7 and MIC2. The gene Alloantibodies to Scianna antigens are rare and little is
encoding Xga is located on the X chromosome at Xp22.3. known about their clinical significance. They are usually IgG
The gene responsible for CD99, MIC2, is located at Xp22.2. and reactive in the antiglobulin test. None have been
CD99 became part of the Xg system because the MIC2 and reported to cause a severe HTR. Only mild HDFN has been
XG genes are adjacent and homologous. Xga has a pheno- reported, except for one severe case for anti-Sc4, for which
typic relationship to CD99: all Xg(a+) individuals have the baby required exchange transfusion. Autoantibodies to
a high expression of CD99 on their RBCs and all Xg(a–) Sc1 and Sc3 have been reported.
females have a low expression of CD99, but 68% of Xg(a–)
males have a high expression and 32% have a low expression The Dombrock (014) System
of CD99.7 Both Xga and CD99 escape X chromosome inacti-
vation. The Xg glycoprotein crosses the RBC membrane The Dombrock blood group system, designated by the ISBT
once, with the amino terminus directed externally. There are with the symbol DO and number 014, was named for the first
approximately 9,000 copies of Xga per RBC.9 antibody maker, Mrs. Dombrock, found in 1965. Anti-Dob,
The prevalence of Xga is 66% in males and 89% in which recognizes the antithetical antigen, was identified in
females. Because males have only one X chromosome, 1973. The prevalence of the three resulting phenotypes,
Xg(a+) males are hemizygotes. Females, having two X chro- Do(a+b–), Do(a+b+), and Do(a–b+), varies in different popu-
mosomes, can be homozygotes or heterozygotes. However, lations. In whites, they are 18%, 49%, and 33%, respectively.
homozygosity for the gene does not directly correlate to The high-prevalence antigens Gya and Hy were both
RBC antigen strength.52 Cord RBCs express Xga weakly. described in 1967. RBCs from whites who are Gy(a–) were
Weak expression of Xga is seen on RBCs from some adult found to be Hy–, but Hy– RBCs from blacks were weakly
females, but weak expression on RBCs from adult males Gy(a+). The high-prevalence antigen Joa was described in
is rare.9 The antigen is sensitive to ficin and papain but 1972, and the phenotypic relationship to Gya and Hy was
resistant to DTT treatment. later shown: Gy(a–) RBCs or Hy– RBCs are also Jo(a–). In
Anti-Xga is usually IgG; some examples are naturally 1995, it was reported that Gy(a–) RBCs were also
occurring. Anti-Xga has not been implicated in HDFN or as Do(a–b–).55 The Gy(a–) phenotype is the Dombrock null.
a cause of HTRs. Two CD99– Japanese individuals have been Two additional high-prevalence antigens, DOYA and DOMR,
found with alloanti-CD99. were recently added to the system.3 The Hy– and Jo(a–)
phenotypes are not found in whites and are rare in blacks.
The Scianna (013) System The Dombrock antigens are carried on a mono-ADP-
ribosyltransferase 4 (ART4) attached to the RBC membrane
The Scianna blood group system, ISBT symbol SC and num- by a GPI anchor. The gene encoding the Dombrock glyco-
ber 013, currently consists of seven antigens. The system is protein is located at chromosome 12p12.3.
2682_Ch08_172-215 22/05/12 11:44 AM Page 204
The Dombrock antigens are resistant to ficin, papain, Landsteiner and Wiener reported that an antibody produced
and glycine-acid EDTA, and are sensitive to 0.2 M DTT in rabbits (and later, guinea pigs) after injection with RBCs
treatment. The antigens are present on cord RBCs, but are of rhesus monkeys reacted with 85% of human RBCs.57 This
absent from PNH III RBCs. The Doa and Dob antigens antibody was called anti-Rh for anti-rhesus. The reactivity
are considered to be poor immunogens and the antibodies of anti-Rh was similar to the human antibody reported by
are rarely found as single specificities; Gya, however, is Levine and Stetson in 1939 in a woman who delivered a
highly immunogenic.9 stillborn infant and had a transfusion reaction to the blood
Anti-Doa and anti-Dob have caused delayed HTRs but donated by her husband.58 Both sera identified the same
no clinical HDFN. The Dombrock antibodies are usually population of Rh+ and Rh– RBCs. The two antibodies were
IgG, reacting optimally with enzyme-treated RBCs. These later shown to be different in several studies; in the 1960s,
antibodies are usually weakly reactive and disappear, both the human anti-Rh was renamed anti-D (but called anti-Rho
factors making them difficult to identify. by some workers) and the rabbit anti-Rh was called anti-LW
in honor of Landsteiner and Wiener. Examples of human
The Colton (015) System anti-LW were subsequently described.
There are three LW antigens: LWa, LWab, and LWb. The
The Colton blood group system, ISBT symbol CO and
first two, LWa and LWab, are common, high-prevalence
number 015, consists of four antigens. The system was
antigens, and LWb is of low prevalence, found in less than
named in 1967 for the first antibody maker; it should have
1% of most Europeans but in 6% of Finns.9 LWab was origi-
been named Calton, but the handwriting on the tube was
nally defined by the antibody made by an individual with an
misread!9 The high- and low-prevalence antithetical anti-
inherited LW(a–b–) phenotype. Terminology for LW anti-
gens are Coa and Cob, respectively. The Cob antigen is pres-
gens has evolved as more information became available. The
ent in about 10% of most populations.9 The third antigen,
ISBT has used LW5, LW6, and LW7 as the antigen numbers
Co3, is present on all RBCs except those of the very rare
for LWa, LWab, and LWb, respectively, to prevent confusion
Co(a–b–) phenotype. Co4, a high-prevalence antigen, has
with obsolete terminology that used LW1-LW4 to designate
been identified on RBCs from two individuals with the
phenotypes. The null phenotype is LW(a–b–); in one indi-
Co(a–b–) phenotype.3
vidual who made anti-LWab (Mrs. Big), this phenotype
The Colton antigens are carried on an integral membrane
resulted from a 10 base pair deletion in exon 1 of an LWa
protein, aquaporin 1 (AQP1), which accounts for 80% of
gene, which introduced a premature stop codon. Rhnull RBCs
water readsorption in the kidneys.9 The glycoprotein crosses
also type LW(a–b–) and are considered to be the only true
the RBC membrane multiple times. The gene (AQP1) is
LW– RBCs because they fail to elicit the formation of
located at chromosome 7p14. The Colton antigens are
anti-LW in animals, whereas injection of Mrs. Big’s LW(a–b–)
expressed on RBCs of newborns and are resistant to treat-
RBCs into guinea pigs caused the formation of anti-LW. The
ment with ficin and papain, chloroquine, and DTT.
LW phenotypes are shown in Table 8–22.
Antibodies are usually IgG and are enhanced with enzyme-
As was shown by the similarity in reactivity of the original
treated RBCs. Anti-Coa is often seen as a single specificity and
animal anti-Rh and the human anti-Rh (later called anti-D),
has been reported to cause HTRs and HDFN.56 Anti-Cob ap-
there is a phenotypic relationship between the D antigen and
pears more often with other specificities but has also caused
anti-LW. Anti-LW usually reacts strongly with D+ RBCs,
HTRs and mild HDFN.9 Anti-Co3, which reacts with all Co(a+)
weakly (and sometimes not at all) with D– RBCs from adults,
and Co(b+) RBCs, has been reported to cause severe HDFN.1
and not at all with Rhnull RBCs. A weak anti-LW may react
The Landsteiner-Wiener (016) System only with D+ RBCs and may appear to be anti-D unless
enhancement techniques are used. This is because there
The Landsteiner-Wiener blood group system, ISBT symbol are more LW antigen sites on D+ RBCs from adults than on
LW and number 016, had its origins along with the discovery D– RBCs.9 However, anti-LW reacts equally well with cord
of the D antigen of the Rh blood group system. In 1940, RBCs regardless of their D type. Distinguishing anti-LW
LW(a+b+) + + + 3% 6%
LW(a–b–) Big – – –
LW(a–b–) Rhnull – – –
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Chapter 8 Blood Group Terminology and the Other Blood Groups 205
from anti-D is most easily accomplished by testing DTT- a system in 1990, designated by the ISBT as GE and number
treated D+ RBCs: the D antigen is not denatured by DTT, so 020. There are currently six high-prevalence Gerbich antigens
anti-D would still be detected; however, LW antigen is (Ge2, Ge3, Ge4, GEPL, GEAT, and GETI) and five low-
destroyed by DTT, so anti-LW would no longer react. LW prevalence antigens (Wb, Lsa, Ana, Dha, and GEIS). The anti-
antigens are resistant to treatment of RBCs with enzymes and gens are carried on sialoglycoprotein structures GPC and
glycine-acid EDTA. GPD. The glycoproteins help to maintain the RBC mem-
The structure that carries the LW antigens is a glycopro- brane integrity through interaction with protein band 4.1,
tein known as intracellular adhesion molecule 4 (ICAM-4), and because they are rich in sialic acid, they contribute to
a member of the immunoglobulin superfamily. The LW the net negative charge of the RBC membrane (as do GPA
glycoprotein is part of the band 3/Rh macrocomplex59; Rhnull and GPB of the MNS system). There are about 135,000 copies
RBCs lack the LW glycoprotein. The LW gene is located on of GPC and 50,000 copies of GPD per RBC.9 GPC and GPD
chromosome 19 at 19p13.3. are both encoded by the GYPC gene, located on chromosome
LW antigens may be depressed during pregnancy and in 2 at 2q14.3.
some diseases, such as lymphoma and leukemia.9 Autoanti- There are three Gerbich-negative phenotypes in which the
LW made by these patients can appear to be an alloantibody; RBCs lack one or more of the high-prevalence antigens:
as the antigen strength returns the antibody diminishes. Ge:–2,3,4 (Yus type), Ge:–2,–3,4 (Gerbich type), and
Autoanti-LW is also common in serum from patients with Ge:–2,–3,–4 (Leach type). The Leach type is the Gerbich null
warm autoimmune hemolytic anemia. No anti-LW has been phenotype. These are summarized in Table 8–23. Outside of
shown to cause serious HDFN or transfusion reactions. Papua, New Guinea, the Gerbich-negative phenotypes are
Many patients with anti-LW have successfully been transfused very rare, but they have been found in diverse populations.
with D– RBCs. Only two examples of alloanti-LWab have The Yus phenotype has been found in Mexicans, Israelis, and
been described, the antibodies of the only two known others but has not been found in Papua, New Guinea and
propositi with an inherited LW(a–b–) phenotype.7 other Melanesians.9 The Gerbich phenotype is polymorphic
in certain areas of Papua, New Guinea and has also been
The Chido/Rodgers (017) System found among Europeans, Africans, Native Americans, Japanese,
and Polynesians.9
The Chido/Rodgers blood group system, designated by the Gerbich antigens are expressed at birth. RBCs of the
ISBT with symbol CH/RG and number 017, was named after Gerbich or Leach phenotypes have weak expression of Kell
the first two antibody producers, Ch for Chido and Rg for blood group antigens, and some anti-Vel fail to react with
Rodgers. These two antibodies were described in 1967 and Ge:–2,–3,4 RBCs.9 Gerbich antigens are resistant to treat-
1976, respectively. Serologically, they were both characterized ment with DTT and glycine-acid EDTA; Ge2 and Ge4 are
as nebulous because antigen strength on different samples ficin and papain sensitive, but Ge3 is ficin resistant.
of RBCs was variable.1 It was also appreciated that both Some Gerbich antibodies may be IgM, but most are IgG.
anti-Ch and anti-Rg could be neutralized by plasma. Gerbich antibodies are sometimes clinically significant for
Ch and Rg antigens are not intrinsic to the RBC membrane. transfusion and sometimes not. Gerbich antibodies can
Rather, they are on the fourth component of complement be eluted from DAT+ cord bloods, but only three cases of
(C4), and are adsorbed onto RBCs from plasma.1 The C4 serious HDFN due to anti-Ge3 have been reported, and two
glycoprotein has two isoforms: C4B carries the Ch antigens were children of the same mother.60,61 In these cases, the
and C4A expresses Rg antigens. Genes at two closely linked severe anemia was late onset, after birth, associated with
loci located at chromosome 6p21.3 encode these isoforms. inhibition of erythroid cell growth; in one case there was also
The Chido/Rodgers system consists of nine antigens: Ch1 early onset of hemolysis.
to Ch6, Rg1, and Rg2 are all high prevalence; WH has a Anti-Ge2 is the most common of the Gerbich antibodies.7
prevalence of about 15%.9 Differentiation of the determinants It is the antibody made by the Ge:–2,3,4 phenotype individ-
is not made for routine serology. uals, but it is also the more common antibody made by the
Ch is present in 96% to 98% of most populations.1,9 Rg is Ge:–2,–3,4 phenotype and Ge:–2,–3,–4 phenotype individuals.
present in 97% to 98% of most populations.1,9 The antigens Anti-Ge3 is less frequently made by the Ge:–2,–3,4 and
are destroyed by ficin and papain but are resistant to treat- Ge:–2,–3,–4 phenotype individuals. Anti-Ge4 is a very rare
ment with DTT and glycine-acid EDTA.9 antibody.
Anti-Ch and anti-Rg are usually IgG and react weakly,
often to moderate or high titration endpoints. Neutralization
of anti-Ch and anti-Rg with pooled plasma is often used as
part of the identification of these antibodies in a patient’s Table 8–23 Gerbich-Negative Phenotypes
serum. Anti-Ch and anti-Rg are clinically insignificant for
PHENOTYPE TYPE ANTIBODY
transfusion.1
Ge: –2, 3, 4 Yus Anti-Ge2
The Gerbich (020) System
Ge: –2, –3, 4 Gerbich Anti-Ge2 or anti-Ge3
The Gerbich blood group system was named in 1960 after Ge: –2, –3, –4 Leach type Anti-Ge2 or anti-Ge3
Mrs. Gerbich, the first antibody producer. Gerbich became
2682_Ch08_172-215 22/05/12 11:44 AM Page 206
The Cromer (021) System reactions, are not neutralized by pooled normal serum
(unlike anti-Ch and anti-Rg), and are difficult to adsorb and
In 1965, an antibody was found in a black prenatal patient, elute.64 Antibody reactivity is enhanced with longer incuba-
Mrs. Cromer, that reacted with all RBCs except her own and tion (e.g., 1 hour, at 37°C). Weak and variable reactivity is due
two siblings. It was originally thought her antibody recog- to variable expression of CR1 on different samples of RBCs.
nized an antigen antithetical to Goa of the Rh blood group The “Helgeson phenotype” represents the serologic null phe-
system. The antibody was named anti-Cra in 1975.7,9 notype for the Knops blood group; these RBCs type Kn(a–b–),
The antigens of the Cromer system are carried on decay McC(a–), Sl(a–), and Yk(a–) because of the low copy number
accelerating factor (DAF, CD55), a complement regulatory of CR1, but they are not truly devoid of Knops antigens.9,64
protein. The CD55 gene is located at chromosome 1q32. The The antibodies are usually IgG, reactive in the antiglobu-
glycoprotein encoded by the gene is attached to the RBC lin test, but are clinically insignificant for both transfusion
membrane through a GPI linkage. PNH III RBCs are defi- and HDFN. Of the Knops antibodies, anti-Kna is frequently
cient in DAF so they also lack Cromer antigens. found in multiply transfused individuals and multispecific
The Cromer system has 16 antigens: 13 high-prevalence sera; anti-Sla is more frequently found in blacks.
antigens and 3 low-prevalence antigens.62,63 All these anti- CR1 binds the complement component fragments C3b
gens are absent from Inab phenotype RBCs, the Cromer and C4b and processes immune complexes for transportation
null phenotype, which is very rare. The Cr(a–) phenotype to the liver and spleen and subsequent clearance from the
is typically found in blacks and is not found in whites. circulation.7,64 CR1 has also been identified as a receptor for
Three Cromer antigens, Tca, Tcb, and Tcc, are antithetical; several pathogenic organisms.
Tca is the high-prevalence antigen and the other two are
low prevalence. The Dra antigen is of high prevalence; The Indian (023) System
Dr(a–) RBCs have weakened expression of all other high-
prevalence Cromer antigens due to a markedly reduced The Indian blood group system was named because the first
copy number of DAF. In(a+) individuals were from India. There are now four anti-
Cromer system antigens are resistant to treatment with gens in the system, designated IN and number 023 by the ISBT.
ficin and papain but are destroyed by α-chymotrypsin, which The antigen Ina was reported in 1973 and is present on RBCs
is used to distinguish specificities in this system from other of 4% of Indians, 11% of Iranians, and 12% of Arabs.9 Inb is
blood group antibodies. Cromer antigens are weakened with the antithetical high-prevalence antigen. These and two other
DTT treatment and are resistant to glycine-acid EDTA. high-prevalence antigens are located on CD44, an adhesion
Antibodies in the Cromer system are usually IgG, but do molecule. The gene encoding CD44 is located at chromosome
not cause HDFN. DAF is strongly expressed on placental 11p13. The extremely rare In(a–b–) phenotype has been found
tissue and will adsorb Cromer antibodies.9,63 Anti-Cra and in only one individual who presented with congenital dysery-
anti-Tca have been implicated in HTRs, but other examples thropoietic anemia and whose RBCs also typed Co(a–b–).65
of these specificities have not caused clinical reactions after CD44 is reduced on RBCs of dominant type Lu(a–b–)
transfusion of incompatible units.62 Anti-IFC is the antibody individuals. Ina and Inb are weakly expressed on cord RBCs
made by individuals with the Cromer null Inab phenotype. and are sensitive to treatment with ficin, papain, and DTT
but are resistant to glycine-acid EDTA.9
The Knops (022) System Antibodies are usually IgG and reactive in the antiglobu-
lin test and they do not bind complement. Positive DATs
There are nine antigens in the Knops blood group system, but no clinical HDFN have been reported for anti-Ina and
designated by the ISBT as KN and number 022. The system anti-Inb; decreased cell survival with anti-Ina and an imme-
was established when the antigens were shown to be located diate HTR due to anti-Inb have been reported.9
on complement receptor 1 (CR1) and was named after
Mrs. Knops, the first antibody maker. The gene is located at The Ok (024) System
chromosome 1q32.
With the exception of the low-prevalence antigens Knb Currently, there are three high-prevalence antigens in the Ok
and McCb, the Knops antigens have a prevalence of more system, designated by the ISBT symbol OK and number 024.
than 90% in most populations; however, ethnic differences Anti-Oka was identified in 1979 and was named after the
exist. Sla is present on RBCs of only about 60% of African antibody maker, Mrs. Kobutso. Because Ko was already in
Americans. Among West Africans, Sla has a prevalence use, the first two letters were switched to Ok.2 Her parents
of 30% to 38%, and KCAM has a prevalence of only 20%.64 were cousins from a small Japanese island. Two of three sib-
The antithetical pairs of antigens are Kna and Knb, McCa lings of the proposita were also Ok(a–).7 RBCs from 400 in-
and McCb, and Sla and Vil. Knops antigens are weakly dividuals from the same island were all Ok(a+).1 Two
expressed on cord RBCs and weaken upon storage of adult additional antigens, OKGV and OKVM, were recently added
RBCs (e.g., older units of blood). The antigens are weakened to the system.
by treatment with ficin and papain and are destroyed by The OK antigens are carried on CD147, a member of
DTT; the antigens are resistant to glycine-acid EDTA.9 the immunoglobulin superfamily that mainly functions
Serologically, these antigens have been grouped together as receptors and adhesion molecules. The gene locus is at
because their corresponding antibodies demonstrate variable chromosome 19p13.3.
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Chapter 8 Blood Group Terminology and the Other Blood Groups 207
Oka is well developed on RBCs from newborns and is for the first antibody maker, John Milton Hagen. The system
resistant to treatment with ficin and papain, DTT, and (ISBT symbol JMH and number 026) was established after it
glycine-acid EDTA.9 was shown that the JMH protein is the GPI-linked glycopro-
The original anti-Oka was IgG, reactive in the antiglobulin tein CD108 and the gene SEMA7A was cloned. The gene is
test. At least one other example of the antibody has been located at chromosome 15q24.1.
found. The antibody caused reduced survival of 51Cr-labeled Five other antigens were recently added to the system,
Ok(a+) RBCs injected into the original antibody maker.1 JMH2 through JMH6; these are JMH variants associated with
Anti-Oka has not been reported to cause HDFN. amino acid substitutions in the protein.68 JMH1 represents
the antigen recognized by antibodies made by individuals
The Raph (025) System lacking the JMH protein.
Most examples of anti-JMH are not found in patients
The only antigen in the Raph system is MER2, which was who lack the JMH protein or who have one of the variant
originally defined by two monoclonal antibodies; it has since JMH phenotypes. Rather, the patient’s JMH– status is
been recognized by human polyclonal antibodies. The antigen acquired and can be transient.1 It is widely accepted that
name is derived from monoclonal, and Eleanor Roosevelt, JMH levels decline during the later years of life, sometimes
the laboratory where the antibody was produced. When to the point of not being detected serologically.1 Once JMH
MER2 was raised to system status, the system was named expression is reduced, anti-JMH can be made. In some
Raph for the first patient to make the alloanti-MER2. MER2 cases with anti-JMH, the DAT is positive and some JMH is
is encoded by a gene located at chromosome 11p15. detected on the patient’s RBCs. This autoanti-JMH with a
Three examples of alloanti-MER2 were found in Jews positive DAT has never been associated with autoimmune
originating from India and living in Israel; two were related. RBC destruction.1
All three had end-stage renal disease. A fourth example of JMH is weakly expressed on cord RBCs and is destroyed
anti-MER2 was found in a healthy Turkish blood donor. by treating RBCs with ficin and papain, and DTT; the antigen
Blood samples from these four individuals were studied. It is resistant to treatment with glycine-acid EDTA.9
was shown that MER2 is located on CD151, a tetraspanin, Anti-JMH is usually IgG (predominantly IgG4 in ac-
which appears to be essential for the assembly of basement quired JMH-negative people).9 The antibodies are often
membranes in the kidney and skin.66 high titer but weakly reactive, even when tested without
The polymorphism identified by the monoclonal antibod- dilution, and they are not neutralized with pooled plasma.
ies indicated that 8% of the English blood donor population JMH antibodies are generally considered clinically insignif-
is MER2–. Subsequent studies suggest the antigen-negative icant. Rare examples of alloanti-JMH in individuals whose
status represents the low end of antigen expression on RBCs express variant forms of CD108 may be clinically
RBCs.66 MER2 is abundant on platelets and is expressed on significant.1
erythroid precursors of individuals with either MER2+ or
MER2– RBCs. MER2 expression decreases over time with The Gill (029) System
increasing maturation of erythroid cells.66 It has been sug-
gested that most people whose RBCs type MER2– have Anti-GIL was first identified in 1980, but the antigen was
MER2 expressed on other cells and tissues and will not make not raised to system status until 2002 when it was shown
anti-MER2, and those individuals who have made alloanti- that GIL is genetically discrete from all other blood group
MER2 are true MER2–. Those individuals who have made systems. The ISBT Gill system symbol is GIL and number
anti-MER2 showed mutations in CD151. In the patients with 029. There is only one antigen, GIL.
renal disease, a truncated CD151 protein would be predicted, This antigen is found on the glycerol transporter aqua-
but in patients without renal disease, mutations would result porin 3 (AQP3), a member of the major intrinsic protein
in an altered CD151 that appears to be present and func- family of water channels. AQP3 is located at chromosome
tional at the cell surface.66,67 9p13. The GILnull phenotype results from a frameshift and a
The antigen is resistant to treatment with ficin and papain premature stop codon.
but is sensitive to treatment with trypsin, α-chymotrypsin, Reactivity with anti-GIL is enhanced with ficin and
pronase, and AET. papain treatment of RBCs; the antigen is resistant to DTT
Little is known about the clinical significance of anti- and glycine-acid EDTA treatment.
MER2, but one patient with the antibody showed signs of a RBCs of two babies born to mothers with anti-GIL have
transfusion reaction after transfusion with 3 units. As 8% of had a positive DAT but no clinical HDFN.7 One example
the population is expected to be MER2–, it would be prudent of anti-GIL was found following a hemolytic transfusion
to transfuse with crossmatch compatible units.67 reaction.
The John Milton Hagen (026) System The RH-Associated Glycoprotein (030)
System
JMH is a high-prevalence antigen. Numerous examples of
anti-JMH have been seen, especially in patients 50 years and Rh-associated glycoprotein is the newest blood group
older. In 1978, a large number of samples with this antibody system (IBST symbol RHAG and number 030).3 The Rh-
were characterized and the antibody was named anti-JMH associated glycoprotein (RhAG) does not have Rh blood
2682_Ch08_172-215 22/05/12 11:44 AM Page 208
group antigens; however, its presence in a complex with the cases of no to mild HDFN, the mothers had not been trans-
Rh proteins is essential for Rh antigen expression. Absence fused.71 Some patients with anti-Jra have received Jr(a+)
of RhAG due to inactivating mutations in RHAG results in incompatible units for transfusion and have had no ill effect,
the Rhnull phenotype; some missense mutations in RHAG re- but in other cases, incompatible transfusions have resulted
sult in the Rhmod phenotype.69 Unlike the RhD and RhCcEe in HTRs.72
proteins, RhAG is glycosylated on the first extracellular loop.
RhAG is encoded by RHAG, located at chromosome 6p11-21. Sda
Two antigens have been definitively assigned to the RHAG
system: Duclos (RHAG1), previously 901013 in the high- The Sda antigen is a high-prevalence antigen named for Sid,
prevalence series, and Ola (RHAG2), previously 700043 in who was the head of the maintenance department at the
the low-prevalence series. Two antigens, DSLK (for Duclos- Lister Institute in London. His RBCs had been used for many
like) and RHAG4, have been provisionally assigned to the years as a panel donor, and they reacted strongly with exam-
system. ples of a new antibody. The soluble form of Sda is Tamm-
Horsfall glycoprotein found in urine. The antigen is not
expressed on RBCs of newborns but is in their saliva, urine,
Miscellaneous Antigens
and meconium. The strength of Sda on adult RBCs varies and
Vel is markedly reduced in pregnancy. The antigen is found on
91% of RBC samples, and Sda substance is found in 96% of
Vel, a high-prevalence antigen, is in the Vel collection, along urine samples. Only 4% of people are Sd(a–).9 Strong examples
with another high-prevalence antigen ABTI. Anti-Vel was of Sda are noted as Sd(a++).
first described in 1952 and was named after the first antibody Anti-Sda can naturally occur (i.e., without known stimu-
maker. Anti-Vel is characterized by its ability to activate com- lation by transfusion or pregnancy) in the sera of individuals
plement and cause in vitro and in vivo hemolysis. The anti- who are Sd(a–). Anti-Sda is usually an IgM agglutinin that is
body is most often IgG but can be IgM, and it has caused reactive at room temperature, but it can be detected in the
severe, immediate HTRs.1,7 Anti-Vel has also caused one case indirect antiglobulin test and does not react with cord RBCs.
of severe HDFN in which the mother had previously been Reactivity is described as small, refractile (shiny) aggluti-
transfused.9 nates in a sea of free RBCs. Because the soluble antigen is
Vel antigen expression is weak on cord RBCs and can be present in urine of Sd(a+) individuals, neutralization of the
variable on RBCs of different adult individuals. RBCs with refractile agglutinates by urine is a technique used to identify
weak expression of Vel can be mistyped as Vel–. This is one anti-Sda. The Sda antigen is resistant to treatment with ficin,
of the reasons why anti-Vel can be a difficult antibody to papain, DTT, and glycine-acid EDTA. Reactivity of the anti-
work with and identify. Reactivity with anti-Vel can be body is enhanced with enzyme-treated RBCs. Anti-Sda is
enhanced with enzyme-treated RBCs. The antigen is also generally considered clinically insignificant for transfusion,
resistant to glycine-acid EDTA and DTT treatment, though though there are two reports of transfusion reactions associ-
some examples of anti-Vel do not react with DTT-treated ated with the transfusion of Sd(a++) RBCs.
RBCs.70
Applications to Routine Blood Banking
Ata
The major blood group systems outside of ABO and Rh
Anti-Ata was first described in 1967 in the serum of a black become important only after patients develop unexpected
woman named Mrs. Augustine.1 Ata is a high-prevalence anti- antibodies. Then a fundamental knowledge of antibody
gen, and all At(a–) individuals have been black. The antigen characteristics, clinical significance, and antigen frequency
is fully developed at birth and is resistant to treatment with is needed to help confirm antibody specificity and to select
ficin and papain, DTT, and glycine-acid EDTA. The antibody appropriate units for transfusion.
is usually IgG, reactive in the antiglobulin test. Anti-Ata has Only a few antibody specificities are commonly seen: M, P1,
caused severe HTRs and one reported mild case of HDFN.7,9 and I antibodies react at room temperature and are considered
clinically insignificant; K, S, s, Fya, Fyb, Jka, and Jkb antibodies
Jra react in the antiglobulin phase and are clinically significant.
These and selected others are summarized in Table 8–24.
Jra is a high-prevalence antigen in most populations; the Not all antibody problems are easily solved; panel reactions
Jr(a–) phenotype is found more commonly in Japanese. The are sometimes inconclusive. As described in this chapter, the
first anti-Jra was described in 1970; several examples have existence of silent, regulator, and inhibitor genes can affect
since been found. The antigen is fully developed at birth and antigen expression. Hopefully the reader will find that the
is resistant to treatment with ficin and papain, DTT, and information in this chapter provides a starting point for sero-
glycine-acid EDTA. logic problem-solving. For further detailed information about
Anti-Jra is usually IgG. Clinical significance of anti-Jra is the RBC antigens and antibodies described here, see Issitt and
not well established since it is a rare antibody. One fatal case Anstee1 and Daniels.7 Resolution of antibody problems involv-
of HDFN was recently reported; in this case, the mother had ing the unusual specificities described here may require the
previously been massively transfused, whereas in previous assistance of an immunohematology reference laboratory.
2682_Ch08_172-215 22/05/12 11:44 AM Page 209
Chapter 8 Blood Group Terminology and the Other Blood Groups 209
69 B
3B
PP1Pk† Most Some Some Enhance Most Most Yes Mild < 0.1
P‡ Most Some Some Enhance Most Some Yes Mild— < 0.1
severe
80 B
77 B
57 B
Lub Few Few Most Variable effect Some No Yes Mild 0.15
B = blacks; HDFN = hemolytic disease of the newborn; HTR = hemolytic transfusion reactions; ≤ RT = room temperature or colder; W = whites.
*Usually clinically insignificant
†Potent hemolysins may be associated with early abortions
‡Associated with severe delayed HTR
§Provided IgM is present
2682_Ch08_172-215 22/05/12 11:44 AM Page 210
SUMMARY CHART
Blood Group Terminology RBCs except those of other p individuals. Antibodies
The ISBT terminology for RBC surface antigens provides may be a mixture of IgM and IgG, efficiently bind com-
a standardized numeric system for naming authenti- plement, may demonstrate in vitro hemolysis, and can
cated antigens that is suitable for electronic data pro- cause severe HTRs. Anti-PP1Pk is associated with
cessing equipment. This terminology was not intended spontaneous abortions.
to replace conventional terminology. Alloanti-P is found as a naturally occurring alloantibody
In the ISBT classification, RBC antigens are assigned in the sera of Pk individuals and is clinically significant.
a six-digit identification number: The first three Autoanti-P is most often the specificity associated with
digits represent the system, collection, or series, the cold-reactive IgG autoantibody in patients with
and the second three digits identify the antigen. All paroxysmal cold hemoglobinuria (PCH).
antigens are catalogued into one of the following The autoanti-P of PCH usually does not react by routine
four groups: tests but is demonstrable as a biphasic hemolysin only
• A blood group system if controlled by a single gene in the Donath-Landsteiner test.
locus, or by two or more closely linked genes
• A collection if shown to share a biochemical, sero- The I and i Antigens
logic, or genetic relationship I and i antigens are not antithetical; they have a recip-
• The high-prevalence series (901) if found in more rocal relationship.
than 90% of most populations Most adult RBCs are rich in I and have only trace
• The low-prevalence series (700) if found in less amounts of i antigen.
than 1% of most populations At birth, infant RBCs are rich in i; I is almost unde-
Lewis Blood Group tectable; over the next 18 months of development, the
infant’s RBCs will convert from i to I antigen.
Lewis blood group antigens are not synthesized by the
Anti-I is typically a benign, weak, naturally occurring,
RBCs. These antigens are adsorbed from plasma onto
saline-reactive IgM autoagglutinin, usually detectable
the RBC membrane.
only at 4°C.
The Le gene codes for L-fucosyltransferase, which adds
Pathogenic anti-I is typically a strong cold autoagglu-
L-fucose to type 1 chains.
tinin that demonstrates high-titer reactivity at 4°C and
The Le gene is needed for the expression of Lea sub- reacts over a wide thermal range (up to 30°–32°C).
stance, and Le and Se genes are needed to form Leb
substance.
Patients with M. pneumoniae infections may develop
strong cold agglutinins with autoanti-I specificity.
The lele genotype is more common among blacks
Anti-i is a rare IgM agglutinin that reacts optimally
than among whites and results in the Le(a–b–)
at 4°C; potent examples may be associated with infec-
phenotype.
tious mononucleosis.
Lewis antigens are poorly expressed at birth.
Lewis antibodies are generally IgM (naturally occurring) The MNS Blood Group System
made by Le(a–b–) individuals. Anti-M and anti-N are cold-reactive saline agglutinins
Lewis antibodies are frequently encountered in pregnant that do not bind complement or react with enzyme-
women. treated cells; both anti-M and anti-N may demonstrate
Lewis antibodies are not considered significant for dosage.
transfusion medicine. Anti-S and anti-s are IgG antibodies, reactive at 37°C
and the antiglobulin phase. They may bind complement
The P Blood Group and have been associated with HDFN and HTRs.
The P blood group consists of the biochemically The S–s–U– phenotype is found in blacks.
related antigens P, P1, Pk and LKE. Anti-U is usually an IgG antibody and has been asso-
P1 antigen expression is variable; P1 antigen is poorly ciated with HTRs and HDFN.
developed at birth.
The Kell Blood Group System
Anti-P1 is a common naturally occurring IgM anti-
body in the sera of P1– individuals; it is usually The Kell blood group antigens are well developed at
a weak, cold-reactive saline agglutinin and can birth and are not destroyed by enzymes.
be neutralized with soluble P1 substance found in The Kell blood group antigens are destroyed by DTT,
hydatid cyst fluid. ZZAP, and glycine-acid EDTA.
Anti-PP1Pk is produced by the rare p individuals early Excluding ABO, the K antigen is rated second only to
in life without RBC sensitization and reacts with all D antigen in immunogenicity.
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Chapter 8 Blood Group Terminology and the Other Blood Groups 211
SUMMARY CHART—cont’d
The k antigen is high prevalence. Other Blood Groups and Antigens
Anti-K is usually an IgG antibody reactive in the The Diego system antigens are located on a major RBC
antiglobulin phase and is made in response to preg- protein, band 3, also known as the RBC anion ex-
nancy or transfusion of RBCs; it has been implicated changer (AE1).
in severe HTRs and HDFN.
Anti-Dia, anti-Dib, and anti-Wra are generally consid-
The McLeod phenotype, affecting only males, is de- ered to be clinically significant; all have caused severe
scribed as a rare phenotype with decreased Kell system HTRs and HDFN. Anti-Wra is a relatively common
antigen expression. The McLeod syndrome includes antibody.
the clinical manifestations of abnormal RBC morphol-
ogy, compensated hemolytic anemia, and neurological
Wrb expression requires the presence of a normal GPA
(MNS system); alloanti-Wrb is extremely rare.
and muscular abnormalities. Some males with the
McLeod phenotype also have the X-linked chronic Anti-Yt a is a fairly common antibody to a high-
granulomatous disease. prevalence antigen that is sometimes clinically signif-
icant and sometimes insignificant.
The Duffy Blood Group System The Xga antigen is found on the short arm of the
Fya and Fyb antigens are destroyed by enzymes and X chromosome and is of higher prevalence in females
ZZAP; they are well developed at birth. The Fy(a–b–) (89%) than in males (66%). Although it is usually IgG,
phenotype is prevalent in blacks but virtually nonex- anti-Xga has not been implicated in HDFN or as a
istent in whites. cause of HTRs.
Fy(a–b–) RBCs resist infection by the malaria organism Antibodies to Scianna system antigens are rare and
P. vivax. little is known about their clinical significance. The
Anti-Fya and anti-Fyb are usually IgG antibodies and rare null phenotype, Sc:–1,–2,–3, has been observed in
react optimally at the antiglobulin phase of testing; the Marshall Islands and New Guinea.
both antibodies have been implicated in delayed HTRs In addition to the Doa and Dob antigens, the Gya, Hy,
and HDFN. Joa antigens are assigned to the Dombrock system.
Anti-Doa and anti-Dob have caused HTRs but no
The Kidd Blood Group System clinical HDFN; these antibodies are usually weak and
Anti-Jka and anti-Jkb may demonstrate dosage, are difficult to identify.
often weak, and found in combination with other The Colton system is composed of the antithetical Coa
antibodies; both are typically IgG and reactive in the and Cob antigens as well as the high-prevalence Co3
antiglobulin test. antigen; the antigens are carried on aquaporin 1, a red
Kidd system antibodies may bind complement and are cell water channel. The Colton antibodies have caused
made in response to foreign RBC exposure during HTRs and HDFN.
pregnancy or transfusion. LW has a phenotypic relationship with the D antigen;
Kidd system antibodies are a common cause of delayed Rhnull RBCs type LW(a–b–).
HTRs. Anti-LW reacts strongly with D+ RBCs and can look
Kidd system antibody reactivity is enhanced with like anti-D. DTT treatment of test RBCs will distin-
enzymes, LISS, and PEG. guish between these two antibodies because the LW
antigen is denatured by DTT, but the D antigen is not.
The Lutheran Blood Group System In other words, anti-LW does not react with DTT-
Lua and Lub are antigens produced by codominant treated D+ RBCs but anti-D does.
alleles; they are poorly developed at birth. The antigens in the Chido/Rodgers system are located
Anti-Lua may be a naturally occurring saline agglutinin on the complement fragments C4B and C4A, respec-
that reacts optimally at room temperature. tively, that are adsorbed onto RBCs from plasma.
Anti-Lub is usually an IgG antibody reactive at the The clinically insignificant anti-Ch and anti-Rg react
antiglobulin phase; it is usually produced in response to weakly, often to moderate or high-titer endpoints in
foreign RBC exposure during pregnancy or transfusion. the antiglobulin test and may be tentatively identified
The Lu(a–b–) phenotype is rare and may result by plasma inhibition methods.
from three different genetic backgrounds; only indi- Gerbich-negative phenotypes are very rare outside of
viduals with the recessive type Lu(a–b–) can make Papua, New Guinea.
anti-Lu3.
Continued
2682_Ch08_172-215 22/05/12 11:44 AM Page 212
SUMMARY CHART—cont’d
Gerbich antibodies are sometimes clinically significant JMH antibodies most often occur in individuals with
for transfusion and sometimes insignificant. Only acquired JMH– status. Anti-JMH in these individuals
three cases of serious HDFN due to anti-Ge3 have been is not clinically significant.
reported. Anti-Vel is most often IgG but can be IgM, and has
The Cromer antigens are carried on the decay acceler- caused severe immediate HTRs and HDFN. When
ating factor and are distributed in body fluids and on serum is tested, anti-Vel characteristically causes in
RBCs, WBCs, platelets, and placental tissue. vitro hemolysis.
The rare anti-Cra and anti-Tca have been found only in Anti-Ata has been found only in blacks; the antibody
black individuals; some examples have caused HTRs. is usually IgG and has caused severe HTRs.
The Knops antigens are located on complement Anti-Jra is found more commonly in Japanese, but
receptor 1 (CR1). Knops antibodies are clinically in- clinical significance is not well established, since it
significant and have weak and “nebulous” reactivity at is a rare antibody; it has caused HTRs and a fatal case
the antiglobulin phase; they are not inhibited by plasma. of HDFN.
The Ina antigen is more prevalent in Arab and Iranian Anti-Sda has characteristic shiny and refractile agglu-
populations, with Ina and Inb antigen expression tinates under the microscope and is inhibited with
being depressed on the dominant type Lu(a–b–) urine from Sd(a+) individuals.
RBCs.
Chapter 8 Blood Group Terminology and the Other Blood Groups 213
9. Which of the following genotypes would explain RBCs 17. Which of the following Duffy phenotypes is prevalent
typed as group A Le(a+b–)? in blacks but virtually nonexistent in whites?
a. A/O Lele HH Sese a. Fy(a+b+)
b. A/A Lele HH sese b. Fy(a–b+)
c. A/O LeLe hh SeSe c. Fy(a–b–)
d. A/A LeLe hh sese d. Fy(a+b–)
10. Anti-LebH will not react or will react more weakly with 18. Antibody detection cells will not routinely detect which
which of the following RBCs? antibody specificity?
a. Group O Le(b+) a. Anti-M
b. Group A2 Le(b+) b. Anti-Kpa
c. Group A1 Le(b+) c. Anti-Fya
d. None of the above d. Anti-Lub
11. Which of the following best describes MN antigens and 19. Antibodies to antigens in which of the following blood
antibodies? groups are known for showing dosage?
a. Well developed at birth, susceptible to enzymes, a. I
generally saline reactive b. P
b. Not well developed at birth, susceptible to enzymes, c. Kidd
generally saline reactive d. Lutheran
c. Well developed at birth, not susceptible to enzymes,
20. Which antibody is most commonly associated with
generally saline reactive
delayed hemolytic transfusion reactions?
d. Well developed at birth, susceptible to enzymes,
generally antiglobulin reactive a. Anti-s
b. Anti-k
12. Which autoantibody specificity is found in patients with c. Anti-Lua
paroxysmal cold hemoglobinuria? d. Anti-Jka
a. Anti-I
21. Anti-U will not react with which of the following RBCs?
b. Anti-i
c. Anti-P a. M+N+S+s–
d. Anti-P1 b. M+N–S–s–
c. M–N+S–s+
13. Which of the following is the most common antibody d. M+N–S+s+
seen in the blood bank after ABO and Rh antibodies?
22. A patient with an M. pneumoniae infection will most
a. Anti-Fya
likely develop a cold autoantibody with specificity to
b. Anti-k
which antigen?
c. Anti-Jsa
d. Anti-K a. I
b. i
14. Which blood group system is associated with resistance c. P
to P. vivax malaria? d. P1
a. P
23. Which antigen is destroyed by enzymes?
b. Kell
c. Duffy a. P1
d. Kidd b. Jsa
c. Fya
15. The null Ko RBC can be artificially prepared by which d. Jka
of the following treatments?
24. The antibody to this high-prevalence antigen demon-
a. Ficin and DTT
strates mixed-field agglutination that appears shiny and
b. Ficin and glycine-acid EDTA
refractile under the microscope:
c. DTT and glycine-acid EDTA
d. Glycine-acid EDTA and sialidase a. Vel
b. JMH
16. Which antibody does not fit with the others with respect c. Jra
to optimum phase of reactivity? d. Sda
a. Anti-S
b. Anti-P1
c. Anti-Fya
d. Anti-Jkb
2682_Ch08_172-215 22/05/12 11:44 AM Page 214
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48. Karamatic Crew, V, Green, C, and Daniels, G: Molecular bases 68. Seltsam, A, Strigens, S, Levene, C, et al: The molecular diversity
of the antigens of the Lutheran blood group system. Transfu- of Sema7A, the semaphorin that carries the JHM blood group
sion 43:1729–1737, 2003. antigens. Transfusion 47:133–146, 2007.
49. Singleton, BK, Burton, NM, Green, C, et al: Mutations in 69. Tilley, L, Green, C, Poole, J, et al: A new blood group system,
EKLF/KLF1 form the molecular basis of the rare blood group RHAG: Three antigens resulting from amino acid substitutions
In(Lu) phenotype. Blood 112:2081–2088, 2008. in the Rh-associated glycoprotein. Vox Sang 98:151–159, 2010.
50. Karamatic Crew, V, Mallinson, G, Green, C, et al: Different in- 70. Rainer, T, Israel, B, Caglioti, S, et al: The effects of dithiothre-
activating mutations in the LU genes of three individuals with itol-treated red blood cells with anti-Vel [abstract]. Transfusion
the Lutheran-null phenotype. Transfusion 47:492–498, 2007. 44:122A, 2004.
51. Eaton, BR, Morton, JA, Pickles, MM, et al: A new antibody, 71. Peyrard, T, Pham, B, Arnaud, L, et al: Fatal hemolytic disease
anti-Yta, characterizing a blood-group antigen of high inci- of the fetus and newborn associated with anti-Jra. Transfusion
dence. Br J Haematol 2:333–341, 1956. 48:1906–1911, 2008.
52. Byrne, KM, and Byrne, PC: Review: Other blood group systems— 72. Kwon, MY, Su, L, Arndt, PA, et al: Clinical significance of anti-
Diego, Yt, Xg, Scianna, Dombrock, Colton, Landsteiner-Wiener, Jra: Report of two cases and review of the literature. Transfusion
and Indian. Immunohematology 20:50–58,2004. 44:197–201, 2004.
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Chapter 9
Detection and Identification of Antibodies
Kathleen S. Trudell, MLS (ASCP)CM, SBBCM
OBJECTIVES
1. Differentiate between the following antibodies: expected and unexpected, immune, naturally occurring, passive, autoantibody
and alloantibody, warm and cold.
2. Explain what factors make an antibody clinically significant.
3. Describe which patient populations require an antibody screen.
4. Select appropriate cells to include in a screen cell set.
5. Describe the impact of various enhancement media on antibody detection.
6. Compare and contrast antibody detection methods.
7. Interpret the result of an antibody screen.
8. List the limitations of the antibody screen.
9. Interpret the results of an antibody identification panel.
10. Summarize the exclusion and inclusion methods.
11. Correlate knowledge of the serologic characteristics of commonly encountered antibodies with antibody identification panel
findings.
12. Identify the situations in which additional panel cells should be tested and select appropriate cells.
13. Describe antigen-typing techniques.
14. Calculate the number of red blood cell (RBC) units that must be antigen-tested to fulfill a physician’s request for crossmatch.
15. Explain the principles behind enzyme and neutralization techniques.
16. Given a patient’s history and initial results, choose the correct method for performing an adsorption.
17. Describe elution methods and give an example of when each would be used.
18. List three chemicals used in performing a partial elution and the advantages of use.
216
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OBJECTIVES—cont’d
19. Outline the procedure for determining the antibody titer level, including reporting of results.
20. Given a patient scenario, identify additional steps that could be employed to resolve a complex antibody identification problem,
including investigating a warm autoantibody.
Introduction for Blood Banks and Transfusion Services requires the use of
an antibody screen to detect clinically significant antibodies
The detection of antibodies directed against red blood cell in both the blood donor and the intended recipient as part
antigens is critical in pretransfusion compatibility testing. It of pretransfusion compatibility testing.3 The donors may be
is one of the principle tools for investigating potential used as a source of antigen-typing sera and antigen-negative
hemolytic transfusion reactions and immune hemolytic RBC units. An antibody screen may be included when eval-
anemias. In addition, it aids in detecting and monitoring uating the compatibility of hematopoietic progenitor cell
patients who are at risk of delivering infants with hemolytic (HPC) and bone marrow donors with the intended trans-
disease of the fetus and newborn (HDFN). plant recipient.4 Antibodies detected in these donors may in-
The focus of antibody detection methods is on “irregular” dicate that additional processing steps are required to reduce
or “unexpected” antibodies, as opposed to the “expected” the plasma in the product. An antibody screen is included
antibodies of the ABO system. The unexpected antibodies in standard prenatal testing for obstetric patients to evaluate
of primary importance are the immune alloantibodies, the risk of HDFN in the fetus and to assess the mother’s
which are produced in response to red blood cell (RBC) candidacy for Rh-immune globulin (RHIG) prophylaxis.5
stimulation through transfusion, transplantation, or preg-
nancy. Other unexpected antibodies may be “naturally oc- Tube Method
curring” (i.e., produced without RBC stimulation). Naturally
occurring antibodies may form as a result of exposure to The traditional method for detecting antibodies is an indi-
environmental sources, such as pollen, fungus, and bacteria, rect antiglobulin test performed in a test tube. RBC reagents,
which have structures similar to some RBC antigens. Anti- enhancement reagents, and AHG reagents are used to sensi-
bodies produced in one individual and then transmitted tize the reagent RBCs with the patient’s antibodies, followed
to another via plasma-containing blood components or by formation of visible RBC agglutinates.
derivatives such as intravenous immunoglobulin (IVIG)
Method Overview
are known as passively acquired antibodies, a third cate-
gory of unexpected antibody. The presence of naturally In the test tube method, the patient’s serum or plasma
occurring and passive antibodies may complicate the de- is mixed with RBCs that have known antigen content
tection and identification of immune, clinically significant (Fig. 9–1). The test may include an immediate spin phase to
antibodies. detect antibodies reacting at room temperature. This phase
Clinically significant antibodies are those that cause is not required and may lead to the detection of clinically
decreased survival of RBCs possessing the target antigen. insignificant cold antibodies.
These antibodies are typically IgG antibodies that react at The test must include a 37°C incubation phase during
37°C or that react in the antihuman globulin (AHG) phase which IgG molecules sensitize any RBCs that possess the tar-
of the indirect antiglobulin test. Autoantibodies may also get antigen, coating those RBCs with antibody. Enhancement
complicate the detection of clinically significant antibodies. media may be added to increase the degree of sensitization.
Autoantibodies are directed against antigens expressed on Depending on the enhancement added, the tubes may be
one’s own RBCs. Because they react with all RBCs tested, au- centrifuged and observed for hemolysis and agglutination
toantibodies may mask the presence of clinically significant following incubation. To observe for hemolysis, the tube is
alloantibodies. carefully removed from the centrifuge so as not to dislodge
After detection, an antibody identification panel is per- the RBC button. The supernatant is observed for pink or red
formed to determine the specificity of the antibody (or anti- discoloration. To observe for agglutination, the tube is gently
bodies) present. Once identified, the antibody’s clinical tilted or rolled to dislodge the cell button. The degree of
significance can be ascertained and the appropriate transfu- reactivity is graded as 0 (no agglutination present) to w+
sion considerations put into place. This chapter describes (agglutination barely visible to the naked eye) to 4+ (one
antibody detection and identification methods and the reso- solid agglutinate). See Color Plate 1.
lution of complex antibody cases. The degree of agglutination should be judged only after
all of the RBCs have been dislodged from the bottom of the
Antibody Screen test tube. The tubes are then washed with 0.9% saline a min-
imum of three times to remove all antibodies that remain
Only a small percentage of the population (between 0.2% unbound. AHG reagent, also known as Coombs’ serum, is
and 2%) has detectable RBC antibodies.1,2 However, certain added to each tube. The tubes are centrifuged and examined
populations require careful screening. The AABB’s Standards for hemolysis and agglutination. In this phase, hemolysis
2682_Ch09_216-240 22/05/12 11:45 AM Page 218
2 drops Add enhancement reagent Wash 3 to 4 times Add 2 drops Confirm negative
patient’s plasma (optional) with normal saline AHG reagent reactions with Coomb’s
⫹ Incubate at 37°C Unbound antibodies Sensitized RBCs control cells
1 drop If antibodies present, removed cross-linked by
screen cell reagent RBC sensitization occurs antibodies in AHG
Figure 9–1. Steps for performing the tube antibody screen test.
may appear as a loss of cell button mass. If the RBCs are should be one cell that is positive for each of the following
coated with IgG antibodies, the anti-IgG antibody in the antigens: D, C, c, E, e, K, k, Fya, Fyb, Jka, Jkb, Lea, Leb, P1,
AHG reagent will create a bridge between sensitized RBCs, M, N, S, and s. Other antigens will be expressed as well. Each
resulting in observable agglutination. If there are no antibod- set of screen cells will be accompanied by an antigen profile
ies directed against any of the antigens present on the screen sheet, detailing which antigens are present in each vial of
cell RBCs, the RBCs will not be sensitized, and there will be cells. These profiles are lot-specific and should not be inter-
no agglutination. Again, depending on the enhancement changed. Figure 9–2 shows an example of a three-cell profile.
media used, the agglutination reactions may be observed Ideally, there will be homozygous expression of many of the
macroscopically only or may include a microscopic exami- antigens within the screen cell set, allowing for detection of
nation. All negative tests will have Coombs’ control cells antibodies that show dosage. A cell with homozygous antigen
(also known as check cells) added to confirm the negative expression is from an individual who inherited only one allele
result. at a given genetic locus. Therefore, the cell surface has a
“double dose” of that antigen. A cell with heterozygous antigen
RBC Reagents expression is from a person who inherited two different alleles
The RBC reagents used in the antibody screen come from at a locus. The alleles “share” the available antigen sites on the
group O individuals who have been typed for the most com- cell surface. In Figure 9–3, the pair of chromosomes on the left
mon, and the most significant, RBC antigens. Group O cells both possess the same allele, giving rise to the “homozygous”
are used so that anti-A and anti-B will not interfere in the RBC. The pair of chromosomes on the right each possess a
detection of antibodies to other blood group systems. The different allele, resulting in the “heterozygous” cell.
RBCs are suspended at a concentration between 2% and 5% Certain antibodies, such as those of the Kidd system,
in a preservative diluent, which maintains the integrity of may be detected only when tested against RBCs expressing
the antigens and prevents hemolysis. The screen cells are one allele (homozygous). Antibodies that react more
packaged in sets of two or three cell suspensions, each hav- strongly with cells having homozygous antigen expression
ing a unique combination of antigens. Within the set, there are said to show dosage. Box 9–1 lists the antibodies that
Enhancement Reagents
Various enhancement reagents, or potentiators, may be
added to the cell/serum mixture before the 37°C incubation
phase to increase the sensitivity of the test system. These
reagents may also allow for a shortened incubation time.
BOX 9–1
Polyethylene Glycol. Polyethylene glycol (PEG) in a LISS of 0.8%. With this technique, the patient’s serum or plasma
solution removes water from the test system, thereby specimen and screen cells are added to a reaction chamber
concentrating any antibodies present. This increases the that sits above the gel. There are up to six chamber/gel
degree of RBC sensitization. PEG can cause nonspecific microtubules contained in a plastic card, about the size of a
aggregation of cells, so centrifugation after the 37°C incuba- credit card. The card is incubated at 37°C for 15 minutes to
tion is not performed. Generally, PEG test systems are more 1 hour,10 thus allowing sensitization to occur. The card is
sensitive than LISS, albumin, or saline systems. However, in then centrifuged for 10 minutes. During this time, the RBCs
patients with elevated levels of plasma protein, such as in are forced out of the reaction chamber down into the gel.
multiple myeloma, PEG is not appropriate for use due to The gel contains anti-IgG. If sensitization occurred, the anti-
increased precipitation of proteins.6 IgG will react with the antibody-coated RBCs, resulting in
agglutination. The agglutinated cells will be trapped within
AHG Reagents the gel because of the action of the anti-IgG and because the
Adding AHG reagent allows for the agglutination of incom- agglutinates are too large to pass through the spaces between
plete antibodies. Polyspecific AHG reagent (also called gel particles. If no agglutination occurred, the RBCs will
polyvalent or broad spectrum Coombs’ serum) contains anti- form a pellet at the bottom of the microtubule (Fig. 9–5).
bodies to both IgG and complement components, either C3 There are numerous advantages to the gel technique. It is
and C4 or C3b and C3d (refer to Chapter 5, “The Antiglob- reported to be as sensitive as the PEG tube test method.11,12
ulin Test”). It has been suggested that antibodies to the C3 The omission of the washing and Coombs’ control steps
components, especially C3d, are more desirable in the results in fewer hands-on steps for the technologist to per-
reagent, as these are more abundant on the RBC surface dur- form. Reactions are stable for up to 24 hours and may be
ing complement activation and lead to fewer false-positive captured electronically, leading to standardized grading of
reactions.7 The presence of anticomplement in the AHG reactions and facilitating review by a supervisor. Mixed-field
reagent may lead to the detection of clinically insignificant reactions may be more apparent in gel. One of the greatest
antibodies. Relatively few examples of clinically significant advantages is the ability to automate many of the pipetting
antibodies, most notably Jka, react with complement alone.8 and reading steps, thereby allowing increased productivity.
As AABB’s Standards requires only the detection of clinically Disadvantages include the need for incubators and cen-
significant antibodies when performing donor antibody trifuges that can accommodate the gel cards.
screening and pretransfusion testing in the recipient,9 many
technologists use monospecific AHG reagent containing Solid Phase Adherence Method
anti-IgG only. This also avoids time-consuming investiga-
tions of insignificant antibodies. A third method that is commonly used to perform the anti-
Any test that is negative after adding the AHG reagent body screen is solid phase adherence (refer to Chapter 12).
should be controlled by adding Coombs’ control cells. These One example of this method is Immucor’s Capture-R. With
are Rh-positive RBCs that have been coated with anti-D. In Capture-R, RBC antigens coat microtiter wells rather than
a negative antiglobulin test, these antibody-coated cells will being present on intact RBCs (Fig. 9–6A). The patient’s
react with the anti-IgG in the AHG reagent that remains free, serum or plasma is added to each well in the screen cell set
resulting in visible agglutination. The addition of the along with LISS. Incubation at 37°C allows any antibodies
Coombs’ control cells proves that adequate washing was present to react with the antigens (Fig. 9–6B). The wells are
performed to remove unbound antibodies before the AHG then washed to remove unbound antibodies. Rather than tra-
reagent was added, that the AHG reagent was added to the ditional AHG reagent, indicator red blood cells that have
test tube, and that the AHG reagent was working properly.
If the Coombs’ control cells fail to agglutinate, the antibody
screen must be repeated from the beginning. Reaction
Use of the tube test remains popular, due to the flexibility Chamber
of the test system, use of commonly available laboratory
equipment, and relative low cost. The disadvantages include Gel
the instability of the reactions and subjective nature of grad-
ing by the technologist, the amount of hands-on time for
the technologist, and problems related to the failure of the
washing phase to remove all unbound antibody.
Gel Method
The antibody screen may also be performed using a Positive Negative reaction.
microtubule filled with a dextran acrylamide gel (refer to reaction. Cells in pellet at
Cells trapped bottom of
Chapter 12, “Other Technologies and Automation”). The
in Gel. microtubule.
screen cells used for this technique meet the same criteria as
for the tube test but are suspended in LISS to a concentration Figure 9–5. The gel test system.
2682_Ch09_216-240 22/05/12 11:45 AM Page 221
been coated with anti-IgG are added. The wells are then A method currently being investigated is erythrocyte-
centrifuged for several minutes. If sensitization occurred, the magnetized technology (EMT). In this method, microtiter
indicator cells react with the antibodies bound to the anti- wells are coated with anti-IgG. Paramagnetic polymer beads
gens coating the microtiter well, forming a diffuse pattern in have been adsorbed onto the screen cells by the manufac-
the well (Fig. 9–6C). If no sensitization occurred (a negative turer. These screen cells are incubated in the microtiter wells
reaction), the indicator cells form a pellet in the bottom of along with the patient’s plasma at 37°C for 20 minutes.13 A
the well (Fig. 9–6D). high-density liquid separates the screen cell/plasma mixture
The solid phase adherence test has been successfully from the anti-IgG until the microtiter plate is placed on a
automated. Such instruments may perform pipetting steps magnetized shaker. At that point, the magnet “pulls” the
and determine the degree of reactivity by taking multiple screen cells through the high-density liquid. Screen cells that
readings of light transmission through each well. Other have been sensitized with antibody will react with the anti-
advantages include a smaller sample size (when compared IgG coating the well, yielding a diffuse pattern throughout
with the tube test), making it ideal in a pediatric setting, and the well. Screen cells that have not been coated with anti-
a LISS reagent that changes color when added to serum or body will form a pellet at the bottom of the well. Unbound
plasma. This ensures that an adequate sample is present in antibodies will remain in a layer above the high-density
the test system. Among the disadvantages is the need for liquid, eliminating the wash step that is necessary in solid
careful pipetting when performing the test manually, due to phase adherence.
the small sample and reagent volume. An inadequate volume
of indicator cells may result in a pattern similar to that of a Interpretation
weak positive reaction. Interpretations made by automated
Agglutination or hemolysis at any stage of testing is a posi-
instruments should be carefully evaluated when the speci-
tive test result, indicating the need for antibody identification
men is hemolyzed, icteric, or lipemic, as this may interfere
studies. However, evaluation of the antibody screen results
with the light measurement used to determine reactivity.
(and autologous control, if tested at this time) can provide
Staff should be carefully trained to visually interpret results
clues and give direction for the identification and resolution
if automation is not used. Those who have primarily used
of the antibody or antibodies. The investigator should con-
the tube test may interpret the diffuse positive pattern as a
sider the following questions:
negative reaction and the dense pellet of the negative reac-
tion as a positive (4+) reaction. Incubators, washers, and 1. In what phase(s) did the reaction(s) occur?
centrifuges that can hold the microtiter wells are among the Antibodies of the IgM class react best at room temper-
special equipment needed for this method. ature or lower and are capable of causing agglutination
A C
B
D
Figure 9–6. The solid phase test system. (A) illustrates the microtiter well coated with RBC antigens. (B) The patient’s antibody has attached to the RBC antigens in the
microtiter well. (C and D) The figure on the left is a detailed side view of what is taking place in the well, on a cellular level, and the figure on the right is the view looking
down into the well, as the technologist would view it for grading purposes. C illustrates the reaction between the patient’s antibodies and the indicator cells. In D, when no
patient antibody is present, the indicator cells form a pellet at the bottom of the well.
2682_Ch09_216-240 22/05/12 11:45 AM Page 222
of saline-suspended RBCs (immediate spin reaction). • Rouleaux does not interfere with the AHG phase of
Antibodies of the IgG class react best at the AHG phase. testing because the patient’s serum is washed away
Of the commonly encountered antibodies, anti-N, anti-I, prior to the addition of the AHG reagent.
and anti-P1 are frequently IgM, whereas those directed • Unlike agglutination, rouleaux is dispersed by adding
against Rh, Kell, Kidd, Duffy, and Ss antigens are usually one to three drops of saline to the test tube.
IgG. Lewis and M antibodies may be IgG, IgM, or a mix-
ture of both. Limitations
2. Is the autologous control negative or positive?
The autologous control is the patient’s RBCs tested Screening reagents and methods are designed to detect as
against the patient’s serum or plasma in the same manner many clinically significant antibodies as possible and to
as the antibody screen. A positive antibody screen and a avoid detecting those antibodies that are insignificant. When
negative autologous control indicate that an alloantibody using a three-cell screen set, a negative result with all three
has been detected. A positive autologous control may in- cells gives the technologist 95% confidence that there are no
dicate the presence of autoantibodies or antibodies to clinically significant antibodies are present.
medications. If the patient has been recently transfused Despite this, there are limitations to the antibody screen.
(i.e., in the previous 3 months), the positive autologous The screen will not detect antibodies when the antibody titer
control may be caused by alloantibody coating circulating has dropped below the level of sensitivity for the screening
donor RBCs. Evaluation of samples with positive autolo- method employed. One study, which reviewed antibodies
gous control or direct antiglobulin test (DAT) results is detected over a 20-year period, showed that 26% of antibod-
often complex and may require a great deal of time and ies became undetectable over time, with a median time of
experience on the part of the investigator. Some technol- 7 months.14 Another study of antibodies formed in response
ogists choose to omit the autologous control when per- to transfusion found that two-thirds of antibodies were no
forming the antibody screen and include it only when longer detectable 5 years after formation.15 The screen also
performing antibody identification studies. cannot detect antibodies directed against low-prevalence
3. Did more than one screen cell sample react? If so, did antigens that are not present on any of the RBCs in the screen
they react at the same strength and phase? cell set. Antibodies showing dosage may not be detected if
More than one screen cell sample may be positive when none of the screen cells have homozygous expression of the
the patient has multiple antibodies, when a single anti- target antigen.
body’s target antigen is found on more than one screen Several factors may influence the sensitivity of the anti-
cell, or when the patient’s serum contains an autoantibody. body screen. These include cell-to-serum ratio, temperature
A single antibody specificity should be suspected when all and phase of reactivity, length of incubation, and pH.
screen cells yielding a positive reaction react at the same
phase and strength. Multiple antibodies are most likely Cell-to-Serum Ratio
when screen cells react at different phases or strengths, When antibody is present in the test system in excess
and autoantibodies should be suspected when the autol- (when compared to antigen concentration), false-negative
ogous control is positive. Figure 9–7 provides several ex- reactions occur as a result of prozone. When the antigen
amples of antibody screen results with possible causes. is in excess, false-negative reactions occur due to postzone.
4. Is hemolysis or mixed-field agglutination present? A ratio of two drops of serum to one drop of the RBC sus-
Certain antibodies, such as anti-Lea, anti-Leb, anti-PP1Pk, pension typically gives the proper balance between antigen
and anti-Vel, are known to cause in vitro hemolysis. and antibody to allow sensitization and agglutination to
Mixed-field agglutination is associated with anti-Sda and occur. (This ratio may be altered, depending on the test
Lutheran antibodies. method employed.) Occasionally, when an antibody is
5. Are the cells truly agglutinated, or is rouleaux present? weak, the amount of serum in the test system may be in-
Serum from patients with altered albumin-to-globulin creased to four to ten drops, providing more antibodies
ratios (e.g., patients with multiple myeloma) or from to react with the available antigens. This should be done
those who have received high-molecular-weight plasma only when potentiators have not been included in the test
expanders (e.g., dextran) may cause nonspecific aggre- system.
gation of RBCs, known as rouleaux. Rouleaux is not a
significant finding in antibody screening tests but is Temperature and Phase of Reactivity
easily confused with antibody-mediated agglutination. The optimal temperature at which an antibody reacts can
Knowing the following characteristics of rouleaux helps provide useful clues to antibody identity. When performing
in differentiating between rouleaux and agglutination: pretransfusion compatibility testing, the focus is on clini-
• Cells have a “stacked coin” appearance when viewed cally significant antibodies, which generally react at 37°C
microscopically (see Color Plate 2). or with the anti-IgG in the AHG reagent. Technologists may
• Rouleaux is observed in all tests containing the omit the immediate spin and room temperature phases to
patient’s serum, including the autologous control and limit the detection of insignificant cold antibodies. Should
the reverse ABO grouping. it be necessary to identify an antibody reacting at room
2682_Ch09_216-240 22/05/12 11:45 AM Page 223
temperature, it may be useful to incubate the screen at 18°C place. A saline environment may require an incubation
or 4°C to enhance reactivity. In such cases, an autocontrol time of 30 minutes to 1 hour, whereas potentiators may
should be included to aid in the detection of common cold shorten the incubation time to as little as 10 minutes.
autoantibodies, such as anti-I or anti-IH. Table 9–2 sum-
marizes the optimal phase of reactivity for some of the most pH
common antibodies. Most antibodies react best at a neutral pH between 6.8
and 7.2;16 however, some examples of anti-M demonstrate
Length of Incubation enhanced reactivity at a pH of 6.5.17 Acidifying the test sys-
Antigen-antibody reactions are in dynamic equilibrium. If tem may aid in distinguishing anti-M from other antibodies.
too little contact time is allowed, too few RBCs will be sen-
sitized to be detected by routine methods. If the incubation Antibody Identification
time is allowed to continue for too long, bound antibody
may begin to dissociate from the RBCs. Incubation time is Once an antibody has been detected, additional testing is
dependent on the medium in which the reaction takes necessary to identify the antibody and determine its clinical
2682_Ch09_216-240 22/05/12 11:45 AM Page 224
M, N Duffy
P1 Kidd
Lua S,s
Lub
Xga
significance. The patient’s serum or plasma is tested against results must be interpreted carefully when the patient has
additional RBCs possessing known antigens. The test method recently received a transfusion, because positive reactions
used should be as sensitive as that used for detection. may be caused by the presence of donor RBCs remaining in
the patient’s circulation. Positive reactions caused by donor
Patient History RBCs usually show mixed-field agglutination, but this de-
pends on how recently the transfusion was given and how
Information concerning the patient’s age, sex, race, diagno- much blood was transfused.
sis, transfusion and pregnancy history, medications, and
intravenous solutions may provide valuable clues in anti- Reagents
body identification studies, particularly in complex cases.
Knowing the patient’s race may be valuable, as some anti- An antibody identification panel is a collection of 11 to 20
bodies are associated with a particular race. For example, group O RBCs with various antigen expression. The pattern
anti-U is more frequently associated with persons of African of antigen expression should be diverse so that it will be pos-
descent because most U-negative individuals are found in sible to distinguish one antibody from another and should
this population. include cells with homozygous expression of Rh, Duffy,
Transfusion and pregnancy history are also helpful, as pa- Kidd, and MNSs antigens. A profile sheet specifying the anti-
tients who have been exposed to “non-self” RBCs via trans- gens on each cell and providing a place to record reactions
fusion or pregnancy are more likely to have produced accompanies each panel (Fig. 9–8). As with the screen cells,
immune antibodies. Naturally occurring antibodies (e.g., the profile sheet is lot-specific and should not be inter-
anti-M, anti-Leb) should be suspected in patients with no changed with that of another panel. The profile sheet will
transfusion or pregnancy history. Medications such as IVIG, often indicate the presence of rare cells, which are positive
RhIG, and antilymphocyte globulin may passively transfer for low-prevalence antigens or negative for high-prevalence
antibodies such as anti-A or anti-B, anti-D, and antispecies antigens.
antibodies, respectively. This will result in the presence of
an unexpected serum antibody that is likely to confound the Exclusion
interpretation of antibody identification.
The patient’s history is especially important when the When interpreting panel results, the first step is to exclude
autologous control or DAT is positive. Certain infectious and antibodies that could not be responsible for the reactivity
autoimmune disorders are associated with production of seen. To perform exclusions or “rule-outs,” the RBCs that
RBC autoantibodies, and some medications are known to gave a negative reaction in all phases of testing are examined.
cause positive DATs. Furthermore, in a patient transfused The antigens on these negatively reacting cells probably will
within the past 3 months, a positive DAT result may indicate not be the antibody’s target. Generally, it is advisable to per-
a delayed hemolytic transfusion reaction. form this rule-out technique only if there is homozygous
Information regarding recent transfusions is also impor- expression of the antigen on the cell. This avoids excluding
tant when antigen-typing the patient’s RBCs. Antigen-typing a weak antibody that is showing dosage. Exceptions are
2682_Ch09_216-240 22/05/12 11:45 AM Page 225
Donor Cell
number D C c E e Cw K k Kpa Kpb Jsa Jsb Fya Fyb Jka Jkb Lea Leb P1 M N S s Lua Lub Xga
R1R1 1 + + 0 0 + 0 0 + 0 + 0 + + + + 0 0 + + + + + + 0 + +
R1r 2 + + + 0 + + + + 0 + 0 + + 0 0 + 0 + 0 + 0 + + 0 + +
R1R1 3 + + 0 0 + 0 0 + 0 + 0 + 0 + + + 0 + + 0 + 0 + 0 + +
R2R2 4 + 0 + + 0 0 0 + 0 + 0 + + 0 + + 0 + + + 0 + 0 0 + +
r’r 5 0 + + 0 + 0 0 + 0 + 0 + 0 0 0 + + 0 + + 0 + + 0 + 0
r’’r’’ 6 0 0 + + 0 0 0 + 0 + 0 + + 0 0 + 0 0 + 0 + 0 + 0 + +
rr K 7 0 0 + 0 + 0 + + 0 + 0 + + 0 0 + 0 + + 0 + 0 + 0 + +
rr 8 0 0 + 0 + 0 0 + 0 + 0 + 0 + + 0 0 + 0 + 0 + 0 0 + +
rr 9 0 0 + 0 + 0 0 + 0 + 0 + + 0 + 0 0 + + + + 0 + 0 + 0
R1r 10 + + + 0 + 0 0 + 0 + 0 + + 0 + + 0 + + + 0 0 + 0 + +
R0 11 + 0 + 0 + 0 0 + 0 + 0 + + + 0 + 0 + + + 0 0 + 0 + +
Patient
Cells
Figure 9–8. Antibody identification profile sheet: + indicates the antigen is present on the cell; 0 indicates the antigen is not present.
made for low-prevalence antigens that are rarely expressed of reactivity matches a pattern of antigen-positive cells
homozygously, such as K, Kpa, Jsa, and Lua. In the sample (inclusion technique). Evaluation of panel results should be
panel shown in Figure 9–9, cell numbers 1, 3, 4, 6, 7, 10, carried out in a logical step-by-step method to ensure proper
and 11 reacted positively and cannot be used for exclusions. identification and to avoid missing antibody specificities that
Cell number 2 reacted negatively and can be used to exclude may be masked by other antibodies. A logical approach to
D, C, e, Cw, K, Kpb, Jsb, Fyb, Jka, P1, M, S, Lub, and Xga. Cell antibody identification is outlined below, using a series of
number 5 can be used to exclude k, Jkb, and Leb. Cell num- questions and the example illustrated in Figure 9–9.
ber 8 is used to rule out c, Lea, and Lua, and cell number 9
1. In what phase(s) and at what strength(s) did the posi-
eliminates N and s. This leaves anti-E, anti-Kpa, anti-Jsa, and
tive reactions occur? Do all of the positive cells react to
anti-Fya as possible antibodies present.
the same degree? Carefully grading the observed reac-
Evaluation of Panel Results tions may aid in antibody identification. The strength of
the reaction does not indicate the significance of the
After each negatively reacting cell has been evaluated, the antibody, only the amount of antibody available to par-
remaining antigens should be examined to see if the pattern ticipate in the reaction. A stronger reaction may be due
Donor Cell
D C c E e Cw K k Kpa Kpb Jsa Jsb Fya Fyb Jka Jkb Lea Leb P1 M N S s Lua Lub Xga IS 37 AHG CC
number
R1r 1 + + + 0 + 0 0 + 0 + 0 + + + + + 0 + + + + + + 0 + + 0 0 2+
R1R1 2 + + 0 0 + + + + 0 + 0 + 0 + + 0 0 0 + + 0 + 0 0 + + 0 0 0 3+
R2R2 3 + 0 + + 0 0 0 + 0 + 0 + + 0 0 + 0 + + 0 + 0 + 0 + + 0 0 3+
R0r 4 + 0 + 0 + 0 0 + 0 + 0 + + 0 + + + 0 0 + 0 + + 0 + 0 0 0 3+
r’r 5 0 + + 0 + 0 0 + 0 + 0 + 0 0 0 + 0 + + + 0 + + 0 + 0 0 0 0 3+
r’’r 6 0 0 + + + 0 0 + 0 + 0 + + + 0 + 0 + + 0 + 0 + 0 + + 0 0 2+
rr K 7 0 0 + 0 + 0 + + 0 + 0 + + + + + 0 + 0 + + 0 + 0 + + 0 0 2+
rr 8 0 0 + 0 + 0 0 + 0 + 0 + 0 + + 0 + 0 + + + + 0 + + 0 0 0 0 3+
r’r” 9 0 + + + + 0 0 + 0 + 0 + 0 + + 0 0 + + 0 + 0 + 0 + + 0 0 0 3+
rr 10 0 0 + 0 + 0 0 + 0 + 0 + + 0 + + 0 + + + 0 + 0 0 + + 0 0 3+
R1r 11 + + + 0 + 0 0 + 0 + 0 + + + 0 + 0 + + + + + + 0 + + 0 0 2+
Patient 0 0 0 3+
Cells
to dosage (cells with homozygous antigen expression 5. Is the autologous control (last row in panel antigen pro-
reacting more strongly than cells with heterozygous anti- file) positive or negative? In the example shown, the auto-
gen expression). Different reaction strengths could also control is negative, indicating that the positive reactions are
indicate the presence of more than one antibody. A cell caused by alloantibody, not by autoantibody. The presence
that possesses more than one of the target antigens may of autoantibodies may mask the presence of alloantibodies,
react more strongly than a cell possessing only one of the and complicates the process of antibody identification. Res-
target antigens. A third possibility is an antigen with vari- olution of autoantibody problems is discussed briefly in the
able expression. I, P1, Lea, Leb, Vel, Ch/Rg, and Sda anti- case studies at the end of this chapter.
gens are expressed more strongly on some RBCs than on 6. Is there sufficient evidence to prove the suspected anti-
others; antibodies to these antigens may react more body? Conclusive antibody identification requires testing
strongly with one panel cell than another. the patient’s serum with enough antigen-positive and anti-
2. Do all of the positive cells react at the same phase, or gen-negative RBC samples to ensure that the pattern of
do any react at different or multiple phases? Reactions reactivity is not the result of chance alone. Testing the
of certain cells at one phase and different cells at another patient’s serum with at least three antigen-positive and three
phase may indicate the presence of multiple antibodies. antigen-negative cells (also known as the 3 and 3 rule) will
Cells that react at multiple phases may also be a sign of result in a probability (P) value of 0.05.18 A P value is a sta-
an antibody showing dosage, with the cells having tistical measure of the probability that a certain set of
homozygous antigen expression reacting at an earlier events will happen by random chance. A P value of 0.05
phase than those with heterozygous antigen expression. or less is required for identification results to be considered
Phase of reactivity may be helpful in establishing the valid, and it means that there is a 5% (1 in 20) chance that
clinical significance of an antibody. IgM antibodies, which the observed pattern occurred for reasons other than a spe-
are usually not significant, most often react at the imme- cific antibody reacting with its corresponding antigen.
diate spin phase, room temperature or colder. Clinically Stated another way, it means that the interpretation of the
significant IgG antibodies are most often detected during data will be correct 95% of the time. When multiple anti-
the AHG phase. Some potent IgG antibodies, such as D, bodies are present, the 3 and 3 rule must be applied to each
E, and K, may become evident following the 37°C incu- specificity separately. For example, if anti-K and anti-E are
bation phase. In the case presented in Figure 9–9, all both suspected, the 3 and 3 rule would be fulfilled if three
reactions occurred in the AHG phase with strengths of K–E– cells reacted negatively, three K+E– cells reacted pos-
both 2+ and 3+. Multiple antibodies or a single antibody itively, and three K–E+ cells reacted positively. Other re-
showing dosage should be considered. searchers have derived formulas where P 0.05 is fulfilled
3. Does the serum reactivity match any of the remaining with two positive cells and three negative cells19 or two
specificities? When a single alloantibody is present, the positive cells and two negative cells.20
pattern of reactivity usually matches a pattern of antigen Testing of RBCs selected from other panels is necessary
expression exactly. In our example, the serum reactivity when inadequate numbers of antigen-positive or antigen-
matches the Fya pattern exactly. The serum gave uniform negative cells have been tested initially. In our example,
positive results with all Fya-positive cells (1, 3, 4, 6, 7, the patient’s serum reacted with seven Fya-positive cells
10, and 11) and negative results with all of the Fya- (1, 3, 4, 6, 7, 10, and 11) but did not react with four
negative cells (2, 5, 8, and 9). The reason for the variation Fya-negative cells (2, 5, 8, and 9). As a result, additional
in strength in the AHG phase is due to dosage; all cells cells need not be tested to increase the confidence level
yielding 2+ reactions were heterozygous for Fya, and all to 95%, and the identification of anti-Fya is conclusive.
cells yielding 3+ reactions were homozygous for Fya. 7. Does the patient lack the antigen corresponding to the
4. Are all commonly encountered RBC antibodies ex- antibody? Individuals cannot make alloantibodies to anti-
cluded? As previously mentioned, in this case anti-E, gens that they possess on their own RBCs; therefore, the
anti-Kpa, and anti-Jsa were not ruled out. None of the last step in identification studies is to test the patient’s
RBCs on this panel were positive for Kpa or Jsa, so they RBCs for the corresponding antigen. A negative result is
cannot be the cause of the observed reactions. These two expected, providing additional evidence that identification
antigens are characterized as low prevalence, occurring results are correct. If the patient’s RBCs are positive for
in less than 5% of the population. Antibodies to low- the corresponding antigen, misidentification of the anti-
prevalence antigens are uncommon because the chance body or a false-positive typing are the most likely expla-
of being exposed to the antigen is low; therefore, it may nations. Antigen typing can also help resolve complex
not be necessary to pursue additional testing to exclude cases, as it may eliminate possible specificities. For exam-
these specificities. In contrast, if a commonly encoun- ple, an R1R1, K-negative, Fy(a–b+), Jk(a+b+), M+N+S+s+
tered antibody is not ruled out, it is important to test patient could form only anti-c, anti-E, anti-K, or anti-Fya.
selected cells that will rule out the presence of the anti- It is not practical to do extended typing on all patients
body. In this example, additional testing should be per- with antibodies; however, judicious use of this procedure
formed to exclude anti-E. A negative result when testing can be useful, especially in patients who chronically re-
an E+ e– Fya- RBC sample would exclude anti-E (see the ceive transfusions and are at risk for alloimmunization,
“Selected Cell Panels” section). such as patients with sickle cell disease or thalassemia.
2682_Ch09_216-240 22/05/12 11:45 AM Page 227
Phenotyping may be complicated when a patient has a single-nucleotide polymorphisms (SNPs) that give rise to
positive DAT or has been transfused in the last 3 months. various red blood cell antigens.26–29 Advantages of molec-
When the DAT is positive due to IgG coating the RBCs, ular testing include the ability to screen for multiple anti-
typing reagents employing the indirect antiglobulin test gens at one time and to screen large numbers of donors
(IAT) may give invalid results. The coating antibody in a relatively short time. There is no interference from a
blocks the antigen sites, preventing the typing serum from recent transfusion or positive DAT on the RBCs. In addi-
reacting. The AHG reagent will react with the coating tion, there are no limitations due to the lack of rare anti-
antibody, yielding false-positive results. Removal of the sera or the expense in purchasing these reagents. Several
antibody coating (elution) will be necessary to get an of these methods have been automated to reduce the
accurate phenotype. Two reagents useful in stripping an- amount of hands-on time required of the technologist.
tibody from the RBC surface, while leaving the membrane
intact to allow phenotyping, are chloroquine diphosphate Additional Techniques for Resolving
and acid glycine/EDTA.21 In the chloroquine diphosphate Antibody Identification
method, washed RBCs are incubated with the reagent at
room temperature for 30 minutes to 2 hours. The cells are There may be times when the initial antibody identification
washed to remove the chloroquine diphosphate. When panel does not reveal a clear-cut specificity. When multiple
the treated cells yield a negative DAT, they may then be specificities remain following the exclusion and inclusion
phenotyped. Rh antigens may show diminished reactivity process, additional testing is necessary.
with this method. Acid glycine/EDTA (EGA) is a rapid
method for removing antibody. Kell antigens are denatured Selected Cell Panels
when using this method, so patient cells cannot be reliably Perhaps the simplest step to take is to test additional
typed for these antigens. cells from a different panel. The cells selected for testing
In cases where the coating antibody resists elution, an should have minimal overlap in the antigens they possess.
absorption method has been described.22 RBCs to be pheno- Figure 9–10 shows the results of an initial panel in which
typed are incubated with diluted antiserum. Following anti-E, anti-Kpa, anti-Jsa, anti-Fya, and anti-Jkb are not
incubation, the cell/antiserum mixture is centrifuged. The excluded. The lower section of the figure shows a selected
supernatant is harvested and tested against a cell with het- cell panel that could be used to differentiate between those
erozygous antigen expression. If the patient’s RBCs are pos- antibodies. Anti-Jkb appears to be present in the sample.
itive for the target antigen, the antibody will have been Anti-Fya is eliminated by selected cell number 1, and anti-
absorbed from the diluted antiserum, and the supernatant E is eliminated by selected cell number 3. Finding cells
will react negatively. If the patient’s RBCs are negative for the that are positive for low-prevalence antigens such as Cw,
target antigen, the antibody will remain in the antiserum, Kpa, Jsa, and Lua may not be possible in many cases.
and the supernatant will be positive when tested against the Selected cell panels are also useful when a patient has a
heterozygous cell. known antibody and the technologist is attempting to deter-
Recently transfused patients present a different chal- mine if additional antibodies are present. Figure 9–11 is an
lenge. Mixed-field reactions are common when phenotyp- example of a selected cell panel for a patient with a history
ing these patients; the donor cells that stimulated antibody of anti-Fya. As it is not necessary to demonstrate the presence
formation react with the typing serum, whereas the of anti-Fya, the panel cells selected are each negative for Fya
patient’s autologous cells do not react. To get an accurate but possess examples of other common, significant antigens.
phenotype of the patient’s autologous cells, reticulocyte In this case, the remaining major clinically significant anti-
typing can be performed. For this technique, the patient’s body specificities have been excluded.
RBCs are drawn into microhematocrit tubes and cen-
trifuged. Because reticulocytes are less dense than mature Enzymes
RBCs, the patient’s reticulocytes should be at the top of
the RBC layer. These cells can be harvested and used for When it appears that multiple antibodies may be present in
antigen typing.23 a sample, treating the panel cells with enzymes may help sep-
Positive and negative control cells should be tested with arate the specificities and allow for identification. Ficin is
each antisera used for antigen typing, on the day of use. The commonly used to treat RBCs; however, papain, bromelin,
positive control cell should have heterozygous antigen or trypsin may also be used. Enzymes modify the RBC sur-
expression to ensure that the antiserum has the sensitivity face by removing sialic acid residues and by denaturing or
to detect small quantities of the antigen. The negative control removing glycoproteins. The effect is to destroy certain anti-
cell should lack the target antigen to confirm reactivity with gens and enhance expression of others. Table 9–3 reviews
only the target antigen. how enzymes affect some common antigens.
Antigen typing is routinely performed using the tube, Enzymes may be utilized in place of enhancement media,
solid phase adherence,24 or gel methods.25 Flow cytome- such as LISS or PEG, in a one-step enzyme test method. A
try has been used to detect minute quantities of antigens. second, more sensitive method uses enzymes to treat the
Various molecular methods based on polymerase chain panel RBCs first, and then the antibody identification panel
reaction (PCR) are being used to examine DNA for the is performed using the treated cells. Because enzymes
2682_Ch09_216-240 22/05/12 11:45 AM Page 228
Donor Cell
D C c E e Cw K k Kpa Kpb Jsa Jsb Fya Fyb Jka Jkb Lea Leb P1 M N S s Lua Lub Xga IS 37 AHG CC
number
R1r 1 + + + 0 + 0 0 + 0 + 0 + + + + + 0 + + + + + + 0 + + 0 0 1+
R1R1 2 + + 0 0 + + + + 0 + 0 + 0 + + 0 0 0 + + 0 + 0 0 + + 0 0 0 3+
R2R2 3 + 0 + + 0 0 0 + 0 + 0 + + 0 + + 0 + + 0 + 0 + 0 + + 0 0 1+
R0r 4 + 0 + 0 + 0 0 + 0 + 0 + + 0 + + + 0 0 + 0 + + 0 + 0 0 0 1+
r’r 5 0 + + 0 + 0 0 + 0 + 0 + 0 0 + 0 0 + + + 0 + + 0 + 0 0 0 0 3+
r”r 6 0 0 + + + 0 0 + 0 + 0 + + + 0 + 0 + + 0 + 0 + 0 + + 0 0 2+
rr K 7 0 0 + 0 + 0 + + 0 + 0 + + + + + 0 + 0 + + 0 + 0 + + 0 0 1+
rr 8 0 0 + 0 + 0 0 + 0 + 0 + 0 + + 0 + 0 + + + + 0 + + 0 0 0 0 3+
r’r” 9 0 + + + + 0 0 + 0 + 0 + 0 + + 0 0 + + 0 + 0 + 0 + + 0 0 0 3+
rr 10 0 0 + 0 + 0 0 + 0 + 0 + + 0 + + 0 + + + 0 + 0 0 + + 0 0 1+
R1r 11 + + + 0 + 0 0 + 0 + 0 + + + 0 + 0 + + + + + + 0 + + 0 0 2+
Patient 0 0 0 3+
Cells
R1R1 SSI + + 0 0 + 0 + + 0 + 0 + + 0 + 0 0 + + 0 + + + 0 + 0 0 0 0 3+
rr SS2 0 0 + 0 + 0 0 + 0 + 0 + + + 0 + + 0 + 0 + + 0 0 + + 0 0 3+
R2R2 SS3 + 0 + + 0 0 0 + 0 + 0 + 0 + + 0 0 + 0 + 0 + 0 0 + 0 0 0 0 3+
1 + + 0 0 + 0 0 + 0 + 0 + 0 + + 0 0 + + + + + 0 0 + + 0 2+
2 + + 0 0 + + 0 + + + 0 + 0 + + + 0 + + + 0 + 0 0 + 0 0 2+
3 + 0 + + 0 0 + + 0 + 0 + 0 + 0 + 0 0 0 0 + 0 + 0 + + 0 2+
4 0 + + 0 + 0 0 + 0 + 0 + 0 0 + + + 0 + + 0 + + 0 + + 0 2+
Patient
0 2+
Cells
Figure 9–11. Selected cell panel for a patient with known antibody.
Table 9–3 The Effect of Proteolytic destroy some antigens, not all specificities can be excluded
Enzymes on Select Antigen- using the enzyme panel alone. When possible, the reactivity
of cells on the enzyme treated panel should be compared to
Antibody Reactions
the reactivity of the same cells before enzyme treatment. Ob-
ENHANCED INACTIVATED serving which cells reacted positively in the untreated panel
Rh Duffy but did not react (or gave weaker reactions) with the treated
panel will aid the technologist in identification. Similarly, ob-
Kidd MNS servation of cells that reacted more strongly or at an earlier
Lewis Xga phase in the enzyme panel than in the untreated panel may
lead to identification.
P1
Neutralization
I
be used to neutralize antibodies in serum, allowing for Table 9–4 Sources of Substances for
separation of antibodies or confirmation that a particular Neutralization of Certain
antibody is present. The patient’s serum is first incubated
Antibodies
with the neutralizing substance, allowing the soluble
antigens in that substance to bind with the antibody. An SOURCE OF NEUTRALIZING
antibody identification panel is performed using the ANTIBODY SUBSTANCE
treated serum. The neutralizing substance inhibits reac- Anti-P1 Hydatid cyst fluid, pigeon drop-
tions between the antibody and panel RBCs. Use of a con- pings, turtledoves’ egg whites
trol (saline and serum) is necessary to prove that the loss
Anti-Lewis Plasma or serum, saliva
of reactivity is due to neutralization and not to dilution of
antibody strength by the added substance. In Figure 9–12, Anti-Chido, anti-Rodgers Serum (contains complement)
positive reactions are seen in cells 1 through 4, 7, and 8.
Anti-Sda Urine
When the serum is incubated with Lewis’ substance, pos-
itive reactions are seen in only cells 3, 4, and 7, which Anti-I Human breast milk
matches the pattern of anti-Kell. Anti-Leb activity has been
inhibited by Lewis’ substance. This technique is helpful
when multiple antibodies are suspected. Table 9–4 lists
some of the antibodies that can be neutralized and the by centrifugation. The absorbed serum is tested against an
source of corresponding neutralizing substance. Lewis and RBC panel for the presence of unabsorbed alloantibodies. The
P1 substances are available commercially. adsorbent is typically composed of RBCs but may be another
antigen-bearing substance.
Adsorption
Commercial Reagents for Adsorption
Antibodies may be removed from serum by adding the target Human platelet concentrate is used to adsorb Bg-like anti-
antigen and allowing the antibody to bind to the antigen, in a bodies from serum. The HLA antigens present on platelets
manner similar to the neutralization technique. In the bind the HLA-related Bg antibodies,30 leaving other speci-
adsorption method, the antigen-antibody complex is com- ficities in the serum. Antibody identification can be per-
posed of solid precipitates and is removed from the test system formed on the adsorbed serum.
Control: Serum +
Serum + Lewis
Rh MNS Lutheran P1 Lewis Kell Duffy Kidd Albumin Saline Substance
CELL D C E c e f V C w
M N S s Lu a
Lu b
P1 a
Le Le b
K k Fy a
Fy b
Jk a
Jk b
37 C AHG AHG AHG
1. r’r-2 0 + 0 + + + 0 0 + + 0 + 0 + 0 0 + 0 + + 0 + + 0 2+ 2+ 0
w
2. R1 R1 -1 + + 0 0 + 0 0 + + + + 0 + + + 0 + 0 + 0 + + 0 0 2+ 2+ 0
3. R1R1-6 + + 0 0 + 0 0 0 0 + 0 + 0 + + + 0 + + 0 + + 0 0 2+ 2+ 2+
4. R2R2-8 + 0 + + 0 0 0 0 + + + + 0 + + 0 + + 0 0 + 0 + 0 2+ 2+ 2+
5. r’’r-3 0 0 + + + + 0 0 + + + 0 0 + + + 0 0 + 0 + 0 + 0 0 0 0
6. rr-32 0 0 0 + + + + 0 + 0 + 0 0 + + 0 0 0 + + + + 0 0 0 0 0
7. rr-10 0 0 0 + + + 0 0 + + + + 0 + 0 0 + + + 0 + + + 0 2+ 2+ 2+
8. rr-12 0 0 0 + + + 0 0 0 + 0 + 0 + + 0 + 0 + + 0 0 + 0 2+ 2+ 0
9. Ro-4 + 0 0 + + + 0 0 + 0 0 + 0 + 0 + 0 0 + 0 0 0 + 0 0 0 0
Cord cell / / / / / / / / / / / / / / / 0 0 / / / / / /
Patient 0 0 0 0
Poly negative
IgG
C3
Figure 9–12. Neutralization using Lewis’ substance in a serum containing anti-Leb and anti-Kell.
2682_Ch09_216-240 22/05/12 11:45 AM Page 230
Rabbit erythrocyte stroma (RESt) performs a similar begins to adsorb onto the patient’s RBC and is removed from
function with some cold-reacting autoantibodies. RESt the serum. In the last set of figures, the autoantibody has once
possesses I, H, and IH-like structures. Incubating the again coated the patient’s RBC, leaving only the alloantibody
patient’s serum at 4°C with RESt will remove these in- in the serum. The serum is separated from the autologous
significant antibodies, which may interfere with the detec- RBCs, and an antibody identification panel is performed to
tion of clinically significant warm-reacting antibodies. reveal any alloantibodies that had previously been masked by
Most other antibody specificities remain unaffected by the autoantibody. Figure 9–14 shows an example of an anti-
RESt adsorption.31 However, RESt also possesses struc- body identification panel before and after warm autoadsorp-
tures similar to B and P1 antigens. Because RESt may tion. Anti-K is apparent in the postadsorption panel.
absorb anti-B, reverse grouping and crossmatching with
RESt-adsorbed serum is not recommended. Homologous Adsorption
When a patient is so anemic that there are not enough au-
Autoadsorption tologous RBCs available to perform an adequate number of
Autoantibodies are commonly removed through adsorption adsorptions or when a patient has been recently transfused
techniques. Perhaps the simplest method is adsorption using (donor RBCs in the specimen may adsorb alloantibodies),
the patient’s own RBCs. The autologous cells are first washed homologous or differential adsorptions may be employed
thoroughly to remove unbound antibody. They may then in place of autoadsorption. For homologous adsorption, the
be treated to remove any autoantibody coating the RBCs. patient is phenotyped, and then phenotypically matched
Next, the cells are incubated with the patient’s serum for up RBCs are used for the adsorption in place of autologous cells.
to 1 hour. Temperature of incubation depends on the thermal If an exact match cannot be made, the focus is on finding
range of the autoantibody being removed, generally 4°C for cells that lack the antigens to which the patient may form
cold-reacting autoantibodies and 37°C for warm-reacting au- antibodies. For example, if the patient types as R1R1, K–,
toantibodies. The sample is inspected for signs of agglutina- Fya+, Fyb+, Jka–, Jkb+, S+, s–, then he or she may form anti-
tion throughout the incubation period. If agglutination E, anti-c, anti-K, anti-Jka, and anti-s. The homologous donor
appears, all the RBC binding sites are saturated with autoan- cells used for adsorption must be negative for E, c, K, Jka,
tibody. The serum is harvested and incubated with a new and s antigens in order for those antibodies to remain in the
aliquot of autologous RBCs. When no agglutination is appar- adsorbed serum.
ent during incubation, the harvested serum is tested against
the patient’s RBCs. If no reactivity is observed, the absorption Differential Adsorption
is complete. However, if a reaction is observed, autoantibody When phenotyping the patient is difficult because of a pos-
remains in the serum, and further absorption is necessary. It itive DAT or recent transfusion, differential absorption may
is not unusual for three to six aliquots of RBCs to be used for be performed. For this method, the patient’s serum sample
autoadsorption. With some powerful warm autoantibodies, is divided into a minimum of three aliquots. Each aliquot is
the technologist may be unable to remove the autoantibody adsorbed using a different cell. One cell is usually R1R1, one
completely and must settle for diminished reactivity. is usually R2R2, and the third is usually rr. Among the three,
Figure 9–13 illustrates the steps in performing an autoad- one must be negative for K, another negative for Jka, and the
sorption. In the first set of figures, the patient’s RBC is coated third negative for Jkb. The cells are treated with an enzyme
with autoantibody. Autoantibody can be seen free in the to render them negative for antigens of the Duffy and MNSs
serum, along with an alloantibody. The cells are washed and systems. Following adsorption, antibody identification
then treated to remove the autoantibody. They are incubated panels are performed separately on each aliquot, and the re-
with the patient’s serum (middle figure). The autoantibody activities are compared to reveal underlying alloantibodies.
R1R1 1 + + 0 0 + 0 0 + 0 + 0 + + 0 + + + 0 + + + + + 0 + + 2+ 0 3+
R1R1 2 + + 0 0 + + + + 0 + 0 + + + 0 + 0 + + + 0 + + 0 + + 3+ 2+
R2R2 3 + 0 + + 0 0 0 + 0 + 0 + 0 + 0 + 0 + + + + + 0 0 + + 2+ 0 3+
R0r 4 + 0 + 0 + 0 0 + 0 + 0 + 0 0 + + 0 + + + 0 + 0 0 + 0 2+ 0 3+
r’r 5 0 + + 0 + 0 0 + 0 + 0 + + 0 + 0 0 0 0 0 + 0 + 0 + 0 2+ 0 3+
r”r 6 0 0 + + + 0 + + 0 + 0 + + + + + 0 + + + + + + 0 + + 3+ 2+
rr 7 0 0 + 0 + 0 0 + 0 + 0 + + + + + + 0 0 + + 0 + 0 + + 2+ 0 3+
rr 8 0 0 + 0 + 0 0 + 0 + 0 + 0 + 0 + 0 + + + 0 0 + 0 + + 2+ 0 3+
rr 9 0 0 + 0 + 0 0 + 0 + 0 + 0 + + 0 0 + 0 + + + + 0 + 0 2+ 0 3+
rr 10 0 0 + 0 + 0 + + 0 + 0 + 0 + + + 0 + + 0 + 0 + 0 + + 3+ 2+
R0r 11 + 0 + 0 + 0 0 + 0 + 0 + + + 0 + 0 + + 0 + 0 + 0 + + 2+ 0 3+
Patient 2+ 0 3+
Cells
See Chapter 20, “Autoimmune Hemolytic Anemias” for a intact, is useful to prepare RBCs for phenotyping and to use
more complete discussion. in autoadsorption procedures. Chloroquine diphosphate,
Adsorption may also be performed when multiple alloan- EGA, and ZZAP are examples of chemicals used for these
tibodies are present in order to separate the specificities. The purposes.
adsorbing cell must be antigen-positive for one suspected
specificity but negative for others. Following adsorption, the Temperature-Dependent Methods
serum is tested to see which, if any, additional alloantibodies
The simplest elution methods involve changing the temper-
have been unmasked.
ature of the antigen-antibody environment. Heat may be
used to remove antibody. After washing with saline, coated
Direct Antiglobulin Test and Elution
RBCs are suspended in an equal volume of saline or albumin.
Techniques
The gentle heat method, performed at 45°C, allows for
Detection of antibodies coating RBCs is valuable when antibody removal while leaving the RBC intact.32 Elution
investigating suspected hemolytic transfusion reactions, performed at 56°C is a total elution method, allowing for
HDFN, and autoimmune and drug-induced hemolytic ane- antibody identification.33 The Lui freeze method34 also per-
mias. The direct antiglobulin test (DAT) is used to detect in forms a total elution. With this method, washed, coated
vivo sensitization of RBCs. In the tube method, the patient’s RBCs suspended in saline or albumin are frozen at –18°C
RBCs are washed thoroughly to remove any unbound anti- or colder until solid. The mixture is then thawed rapidly,
body, and then AHG reagent is added. If IgG antibodies causing the RBCs to burst, freeing the bound antibody.
or complement are coating the RBCs, agglutination will be Temperature-dependent elutions are best at detecting IgG
observed. If neither is present, no agglutination will be ob- antibodies directed against antigens of the ABO system.
served. Coombs’ control cells are added to validate the neg-
ative test. The DAT may also be performed using solid phase pH
adherence and gel methods. A common and relatively quick and easy method for total
When IgG antibodies are detected, the next step is to dis- elution in order to detect non-ABO antibodies is acid elu-
sociate the antibodies from the RBC surface to allow for iden- tion.35,36 In this method, the washed antibody-coated cells
tification. Elution techniques are used to release, are mixed with a glycine acid solution at a pH of 3. The
concentrate, and purify antibodies. The methods used to re- antigen-antibody bond is disrupted, and the antibody is
move the antibody change the thermodynamics of the envi- released into the acidic supernatant. The supernatant is har-
ronment, change the attractive forces between antigen and vested, and the pH is neutralized so that antibody identifi-
antibody, or change the structure of the RBC surface. The cation testing can take place. Citric acid and digitonin acid
antibody is then freed into a solution known as an eluate. are also used in similar methods.
The eluate may be tested against an RBC panel to identify
the antibody. A total elution, in which antibody is released Organic Solvents
and the RBC antigens are destroyed, is usually necessary
when performing antibody identification. Partial elution, in Several organic solvents have been used in total elution
which antibody is removed but RBC antigens remain methods, including dichloromethane, xylene, and ether.
2682_Ch09_216-240 22/05/12 11:45 AM Page 232
These solvents act on the lipids in the RBC membrane to reduce When performing the antibody titer, careful preparation
surface tension and lead to the reversal of the van der Waals of dilutions is necessary. Contamination from a tube with a
forces that hold antigens and antibodies together.37,38 Organic higher antibody concentration can lead to falsely elevated
eluates are very potent as compared with the temperature- titer-level results. Changing pipette tips between each tube
dependent eluates and are best for detecting non-ABO anti- when preparing the dilutions and working or reading from
bodies. However, these procedures are time-consuming, and the most diluted tube to the least diluted can help avoid this
the chemicals pose several health and safety hazards, as they problem.
may be carcinogenic or flammable. Organic solvents are rarely The phenotype of the RBCs selected for use in testing
used in the clinical laboratory. should be consistent throughout the series of titer-level stud-
The most critical step in preparing any eluate is the original ies. If a cell with homozygous antigen expression was used
washing, which is used to remove unbound immunoglobu- for the initial titer, then all subsequent titer specimens should
lins. If allowed to remain in the test system, these antibodies be tested against a homozygous cell. The method used must
will contaminate the final eluate and yield false-positive re- also be consistent. It has been reported that titers using the
sults. As a control, the last wash supernatant should be tested gel method are more sensitive than those using the tube
in parallel with the eluate to detect the presence of unbound method and therefore result in a higher titer level.39 The tech-
antibody. The last wash should be nonreactive, or the eluate nologist must make the test systems as identical as possible
results will be invalid. in order to make valid comparisons between samples.
Titer-level studies are useful in monitoring the obstetric
Antibody Titration patient who has an IgG antibody that may cause HDFN. An
increase in antibody titer level during pregnancy suggests
Once an antibody is identified, it is sometimes useful that the fetus is antigen-positive and therefore at risk of de-
to quantify the amount that is present. While techniques veloping HDFN. An increasing titer level may indicate the
employing flow cytometry, radioimmunoassay, or enzyme- need for intrauterine exchange transfusion. An antibody titer
linked immunoassay may give more precise results, these may also be used to help differentiate immune anti-D from
methods are not readily available in every laboratory. Per- passively acquired anti-D due to RhIG administration. The
forming an antibody titration can help determine antibody titer level in RhIG is rarely above 4.40
concentration levels. Twofold serial dilutions of serum Performing a titer is one way to confirm the presence of
containing an antibody are prepared and tested against a antibodies that have previously been known as HTLA (high
suspension of RBCs that possesses the target antigen. The titer, low avidity). These antibodies, directed against high-
titer level is the reciprocal of the greatest dilution in which prevalence antigens, are observed at the AHG phase of testing
agglutination of 1+ or greater is observed. A score may also with weakly positive reactions. The weak reactions persist
be assigned, based on the strength of reactivity. Each reaction through extensive dilutions (as high as 2048).41 Examples of
is given a value, and the score is determined by adding up these antibodies include anti-Ch, anti-Rg, anti-Csa, anti-Yka,
the individual values. anti-Kna, anti-McCa, and anti-JMH. These antibodies usually
After determining the initial titer, the specimen should be are not clinically significant but may mask significant
frozen. When new specimens are submitted for titer deter- antibodies.
minations, the initial titer specimen should be tested in par-
allel to control variability among technologists’ technique Providing Compatible Blood Products
(e.g., pipetting, grading reactions) and the relative strength
of the target antigen on the cells used in testing. A compar- The relative difficulty in providing compatible blood products
ison of the current specimen’s results and the initial speci- is determined by the frequency of the antigen in the popula-
men’s current results should be made. A fourfold or greater tion and by the clinical significance of the antibody. If the an-
increase in titer (reactivity in two or more additional tubes) tibody does not cause decreased survival of antigen-positive
or an increase in score of 10 or more is considered to be sig- RBCs, then use of random blood products that are crossmatch-
nificant. Table 9–5 shows an example of titer-level results compatible is acceptable. Examples of such antibodies include
that indicate a significant increase in antibody levels. anti-M, anti-N, anti-P1, anti-Lea, and anti-Leb.42
Score 8 8 5 0 0 0 0 Score 21
Score 10 10 8 8 5 0 0 Score 41
When the patient sample contains clinically significant an- calculation would be 2/(0.70 × 0.20) = 14.3. Fourteen or
tibodies or the patient has a history of clinically significant an- 15 units would have to be antigen-typed for E and c in order
tibodies, units for transfusion must be antigen-negative. The to find two units that are negative for both antigens.
crossmatch technique must demonstrate compatibility at the In certain cases, knowing the donor’s race is helpful when
AHG phase.43 If the patient sample is plentiful, the technolo- selecting units for antigen testing because the frequency of
gist may choose to crossmatch units, then antigen-type those some antigens varies between races. For example, when
that are crossmatch-compatible. If sample quantity is limited searching for units that are Fya-negative, it may be prudent
or if the antibody is no longer detectable in the serum, units to screen black donors, as 90% will be negative for Fya com-
should be antigen-typed first, then crossmatched. pared with only 34% of whites.
Knowing the frequency of the antigen in the population Some transfusion medicine experts have proposed
is helpful when determining the number of units that must that certain populations, particularly sickle cell and beta-
be antigen-typed to find a sufficient number to fill the cross- thalassemia patients, receive units that are phenotypically
match request. The number of units requested is divided by matched.44,45 These patients, who are transfused repeatedly,
the frequency of antigen-negative individuals. For example, seem more likely to make alloantibodies than the general
if a crossmatch for two units of RBCs is ordered on a patient population; if they are immunized, it may be difficult to find
whose serum contains anti-E, the calculation would be compatible blood. When the number of antigens that must
2 (units requested)/0.70 (the frequency of E-negative indi- be negative makes it difficult to find suitable units, rare-
viduals). The result is 2.8, meaning that three units would donor registries may be consulted. These registries maintain
need to be typed for E antigen in order to find two E-negative lists of donors and may provide frozen units of rare pheno-
units. When multiple specificities are present, the frequen- types. Another approach is to transfuse units that are phe-
cies of antigen negative are multiplied together. If E-negative, notypically matched for Rh antigens and K, as these antigens
c-negative units were required in the above example, the are the most immunogenic.46
R1r 1 + + + 0 + 0 0 + 0 + 0 + + + + + 0 + + + + + + 0 + + 1+ 0 3+
R1R1 2 + + 0 0 + + + + 0 + 0 + 0 + + 0 0 0 + + 0 + 0 0 + + 3+ 2+
R2R2 3 + 0 + + 0 0 0 + 0 + 0 + + 0 0 + 0 + + 0 + 0 + 0 + + 0 3+ 0 3+
R0r 4 + 0 + 0 + 0 0 + 0 + 0 + + 0 + + + 0 0 + 0 + + 0 + 0 0 3+ 0 3+
r’r 5 0 + + 0 + 0 0 + 0 + 0 + 0 0 0 + 0 + + + 0 + + 0 + 0 0 3+ 0 3+
r”r 6 0 0 + + + 0 0 + 0 + 0 + + + 0 + 0 + + 0 + 0 + 0 + + 1+ 0 3+
rr K 7 0 0 + 0 + 0 + + 0 + 0 + + + + + 0 + 0 + + 0 + 0 + + 2+ 2+
rr 8 0 0 + 0 + 0 0 + 0 + 0 + 0 + + 0 + 0 + + + + 0 + + 0 2+ 0 3+
r’r’’ 9 0 + + + + 0 0 + 0 + 0 + 0 + + 0 0 + + 0 + 0 + 0 + + 2+ 0 3+
rr 10 0 0 + 0 + 0 0 + 0 + 0 + + 0 + + 0 + + + 0 + 0 0 + + 0 3+ 0 3+
R1r 11 + + + 0 + 0 0 + 0 + 0 + + + 0 + 0 + + + + + + 0 + + 1+ 0 3+
Patient 0 3+ 0 3+
Cells
3. Comparing the original reactions to those of the en- Autologous donations should be encouraged. Other
zyme panel, which antibodies appear to be present in sources of antigen-negative blood may include family
this specimen? members and the rare donor registry. Fortunately, be-
4. What additional testing could be performed to elimi- cause these antigens do occur so frequently, it is rare to
nate anti-C? find a patient with an antibody to one of them. Refer to
Figure 9–16.
Case 9-2
1. What antibody appears to be present in this specimen?
Antibody to a High-Prevalence Antigen 2. What additional testing needs to be performed to con-
firm this identification?
High-prevalence antigens are those that are present in al-
3. What additional patient information would be useful
most all individuals (98% or more). Suspect an antibody
when resolving a case such as this?
to a high-prevalence antigen when all or most screen and
panel cells are positive, with reactions in the same phase Case 9-3
and at the same strength, along with a negative autocon-
trol (Fig. 9–16). Panel cells that are negative for these Antibody to a Low-Prevalence Antigen
high-prevalence antigens are usually indicated on the
Low-prevalence antigens are present in less than
antigen profile sheet or may be indicated on the manu-
10% of the population. Antibodies to these antigens are
facturer’s extended typing list, which usually accompanies
uncommon because exposure to the antigen is rare. The
the panel set.
antibody screen will most likely be negative; therefore, no
Among the antibodies included in this category are the
panel will have been performed. Antibodies to these anti-
so-called HTLA. Although these antibodies are usually
gens should be suspected when an antiglobulin cross-
not clinically significant themselves, up to 25% of patients
match is incompatible and other reasons for reactivity,
with an HTLA antibody also make clinically significant
such as ABO incompatibility or positive donor DAT, have
antibodies.47 It is not necessary to determine the speci-
been eliminated. These antibodies may also be suspected
ficity of the HTLA antibody, but removing these antibod-
when an infant has a positive DAT and there is no known
ies is usually necessary to identify any underlying
blood group discrepancy between mother and infant.
alloantibodies. Some HTLA antibodies, notably anti-Ch
Because these antigens are infrequent, finding antigen-
and anti-Rg, may be neutralized by normal serum, which
negative units for crossmatch is usually not difficult. Refer
contains complement. Routine blood bank enzymes will
to Figure 9–17.
destroy anti-Ch, anti-Rg, and anti-JMH, whereas anti-Kna
and anti-McCa are destroyed by dithiothreitol (DTT). 1. What low-prevalence antigen is found on cell 4?
Finding compatible blood for patients with an anti- 2. What additional testing should be performed to con-
body to a high-prevalence antigen may be a challenge. firm the identity of the antibody?
R1R1 1 + + 0 0 + 0 0 + 0 + 0 + + 0 + + + 0 + + + + + 0 + + 2+
R1R1 2 + + 0 0 + + + + 0 + 0 + + + 0 + 0 + + + 0 + + 0 + + 2+
R2R2 3 + 0 + + 0 0 0 + 0 + 0 + 0 + 0 + 0 + + + + + 0 0 + + 2+
R0 r 4 + 0 + 0 + 0 0 + 0 + 0 + 0 0 + + 0 + + + 0 + 0 0 + 0 2+
r’r 5 0 + + 0 + 0 0 + 0 + 0 + + 0 + 0 0 0 0 0 + 0 + 0 + 0 2+
r”r 6 0 0 + + + 0 + + 0 + 0 + + + + + 0 + + + + + + 0 + + 2+
rr 7 0 0 + 0 + 0 0 + 0 + 0 + + + + + + 0 0 + + 0 + 0 + + 2+
Cob +
rr 8 0 0 + 0 + 0 0 + 0 + 0 + 0 + 0 + 0 + + + 0 0 + 0 + + 2+
rr 9 0 0 + 0 + 0 0 + 0 + 0 + 0 + + 0 0 + 0 + + + + 0 + 0 2+
r
rr
Yt(a) – 10 0 0 + 0 + 0 + + 0 + 0 + 0 + + + 0 + + 0 + 0 + 0 + + 0 3+
R0 r 11 + 0 + 0 + 0 0 + 0 + 0 + + + 0 + 0 + + 0 + 0 + 0 + + 2+
Patient 0 3+
Cells
Donor Cell
D C c E e Cw K k Kpa Kpb Jsa Jsb Fya Fyb Jka Jkb Lea Leb P1 M N S s Lua Lub Xga IS 37 AHG CC
number
R1R1 1 + + 0 0 + 0 0 + 0 + 0 + + 0 + + + 0 + + + + + 0 + + 2+ W+ 0 3+
R1R1 2 + + 0 0 + + + + 0 + 0 + + + 0 + 0 + + + 0 + + 0 + + 2+ W+ 0 3+
R2R2 3 + 0 + + 0 0 0 + 0 + 0 + 0 + 0 + 0 + + + + + 0 0 + + 2+ W+ 0 3+
R0r 4 + 0 + 0 + 0 0 + 0 + 0 + 0 0 + + 0 + + + 0 + 0 0 + 0 2+ W+ 0 3+
r’r 5 0 + + 0 + 0 0 + 0 + 0 + + 0 + 0 0 0 0 0 + 0 + 0 + 0 2+ W+ 0 3+
r”r 6 0 0 + + + 0 + + 0 + 0 + + + + + 0 + + + + + + 0 + + 2+ W+ 0 3+
rr 7 0 0 + 0 + 0 0 + 0 + 0 + + + + + + 0 0 + + 0 + 0 + + 2+ W+ 0 3+
rr 8 0 0 + 0 + 0 0 + 0 + 0 + 0 + 0 + 0 + + + 0 0 + 0 + + 2+ W+ 0 3+
rr 9 0 0 + 0 + 0 0 + 0 + 0 + 0 + + 0 0 + 0 + + + + 0 + 0 2+ 1+ 0 3+
rr 10 0 0 + 0 + 0 + + 0 + 0 + 0 + + + 0 + + 0 + 0 + 0 + + 2+ 1+ 0 3+
R0r 11 + 0 + 0 + 0 0 + 0 + 0 + + + 0 + 0 + + 0 + 0 + 0 + + 2+ 1+ 0 3
I neg Cord 0 0 0 3+
Cell
Patient 2+ 1+ 0 3+
Cells
SUMMARY CHART
The purpose of the antibody screen is to detect unexpected Screen cells are commercially prepared group O red
antibodies, which may be found in approximately 0.2% blood cell suspensions obtained from individual
to 2% of the general population. The antibodies may be donors who are phenotyped for the most commonly
classified as immune (the result of RBC stimulation in the encountered and clinically important RBC antigens.
patient), passive (transferred to the patient through blood RBCs from a homozygous individual have a double
products or derivatives), or naturally occurring (the result dose of a single antigen, which results from the inher-
of environmental factors). Antibodies may also be itance of two genes that code for the same antigen,
classified as alloantibodies, directed at foreign antigens, whereas heterozygous individuals carry only a single
or autoantibodies, directed at one’s own antigens. dose each of two different antigens. (Each gene codes
A clinically significant antibody is one that results in for a different antigen.)
the shortened survival of RBCs possessing the target Antibodies in the Kidd, Duffy, Lutheran, Rh, and MNSs
antigen. Clinically significant antibodies are IgG blood group systems show dosage and yield stronger
antibodies that react best at 37°C or in the AHG reactions against RBCs with homozygous expression
phase of testing. They are known to cause hemolytic of their corresponding antigen.
transfusion reactions and HDFN.
2682_Ch09_216-240 22/05/12 11:45 AM Page 237
SUMMARY CHART—cont’d
Enhancement reagents, such as LISS and PEG, least three antigen-negative cells (i.e., reagent cells that
are solutions added to serum and cell mixtures in the do not express the corresponding antigen), and the
IAT to promote antigen-antibody binding or aggluti- patient’s RBCs phenotype negative for the correspon-
nation. ding antigen.
Coombs’ control cells are RBCs coated with human The DAT detects RBCs that were sensitized with anti-
IgG antibody, which are added to all AHG-negative body in vivo. Elution methods are used to free anti-
tube tests to ensure that there was an adequate washing body from the cell surface to allow for identification.
step performed and that the AHG reagent is present The calculation used to determine the number of ran-
and functional in the test system. dom donor units that should be antigen typed in order
Gel and solid phase adherence methods are alterna- to provide the requested number of antigen-negative
tives to tube testing. These methods may be automated RBC units for patients with an antibody involves
to increase efficiency. dividing the number of antigen-negative units requested
The antibody exclusion method rules out possible an- by the frequency of antigen-negative individuals in the
tibodies based on antigens that are present on nega- donor population.
tively reacting cells. The relative quantity of an RBC antibody can be deter-
Conclusive antibody identification is achieved when the mined by testing serial twofold dilutions of serum
serum containing the antibody is reactive with at least against antigen-positive RBCs; the reciprocal of the
three antigen-positive cells (i.e., reagent cells that highest serum dilution showing agglutination is the
express the corresponding antigen), negative with at antibody titer.
6. Patient JM appears to have a warm autoantibody. She was 9. What additional cells need to be tested to be 95% confi-
transfused 2 weeks ago. What would be the next step per- dent that the identification is correct?
formed to identify any alloantibodies that might be in her a. Three e-negative cells that react negatively and one
serum? additional e-positive cell that reacts positively
a. Acid elution b. One additional E-positive cell to react positively and
b. Warm autoadsorption using autologous cells one additional K-positive cell to react positively
c. Warm differential adsorption c. Two Jkb homozygous positive cells to react posi-
d. RESt™ adsorption tively and one Jkb heterozygous positive cell to react
negatively
7. What is the titer and score for this prenatal anti-D titer?
d. No additional cells are needed
(Refer to Figure 9–20.)
a. Titer = 64; score = 52 10. Using the panel in Figure 9–21, select cells that would
b. Titer = 1:32; score = 15 make appropriate controls when typing for the C antigen.
c. Titer = 64; score = 21 a. Cell number 1 for the positive control and cell number
d. Titer = 32; score = 52 2 for the negative control
b. Cell number 1 for the positive control and cell number
For Questions 8 through 10, refer to Figure 9–21.
6 for the negative control
8. Select the antibody(ies) most likely responsible for the c. Cell number 2 for the positive control and cell number
reactions observed: 4 for the negative control
a. Anti-E and anti-K d. Cell number 4 for the positive control and cell number
b. Anti-Fya 5 for the negative control
c. Anti-e
d. Anti-Jkb
Donor Cell
D C c E e Cw K k Kpa Kpb Jsa Jsb Fya Fyb Jka Jkb Lea Leb P1 M N S s Lua Lub Xga IS 37 AHG CC
number
R1R1 1 + + 0 0 + 0 0 + 0 + 0 + + + + 0 0 + + + + + + 0 + + 0 0 0 3+
R1r 2 + + + 0 + + + + 0 + 0 + + 0 0 + 0 + 0 + 0 + + 0 + + 0 0 3+
R1R1 3 + + 0 0 + 0 0 + 0 + 0 + 0 + + + 0 + + 0 + 0 + 0 + + 0 0 0 3+
R2R2 4 + 0 + + 0 0 0 + 0 + 0 + + 0 + + 0 + + + 0 + 0 0 + + 0 2+ 3+
r’r 5 0 + + 0 + 0 0 + 0 + 0 + 0 0 0 + + 0 + + 0 + + 0 + 0 0 0 0 3+
r”r’’ 6 0 0 + + 0 0 0 + 0 + 0 + + 0 0 + 0 0 + 0 + 0 + 0 + + 0 2+ 3+
rr K 7 0 0 + 0 + 0 + + 0 + 0 + + 0 0 + 0 + + 0 + 0 + 0 + + 0 0 3+
rr 8 0 0 + 0 + 0 0 + 0 + 0 + 0 + + 0 0 + 0 + 0 + 0 0 + + 0 0 0 3+
rr 9 0 0 + 0 + 0 0 + 0 + 0 + + 0 + 0 0 + + + + 0 + + + 0 0 0 0 3+
R1r 10 + + + 0 + 0 0 + 0 + 0 + + 0 + + 0 + + + 0 0 + 0 + + 0 0 0 3+
R0 11 + 0 + 0 + 0 0 + 0 + 0 + + + 0 + 0 + + + 0 0 + 0 + + 0 0 0 3+
Patient 0 0 0 3+
Cells
11. Which of the following methods may be employed to 11. Ciavarella, D, Pate, L, and Sorenson, E: Serologic comparisons
remove IgG antibodies that are coating a patient’s red of antibody detection and titration methods: LISS, PeG, gel
(abstract). Transfusion 42:108S, 2002.
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a. Adsorption Uehlinger, J: Antibody identification using both automated
b. Elution solid-phase red cell adherence assay and a tube polyethylene
c. Neutralization glycol antiglobulin method. Transfusion 48:1693, 2008.
13. Bouix, O, Ferrera, V, Delamaire, M, Redersdorff, JC, and Roubi-
d. Titration
net, F: Erythrocyte-magnetized technology: An original and
12. A technologist has decided to test an enzyme-treated innovative method for blood group serology. Transfusion
48:1878, 2008.
panel of RBCs against a patient’s serum. Which of the 14. Schonewille, H, Haack, HL, and van Zijl, AM: RBC antibody
following antibody pairs could be separated using this persistence. Transfusion 40:1127, 2000.
technique? 15. Tormey, CA, and Stack, G: The persistence and evanescence
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b. Anti-S and anti-Fya
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ciation of Blood Banks, Arlington, VA, 1988, p 25.
13. An antibody demonstrates weak reactivity at the AHG 17. Issitt, PD, and Anstee, DJ: Applied Blood Group Serology,
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39. Ciavarella, D, Pate, L, and Sorenson, E: Serologic comparisons 48. Sanguin, J, Angus, N, and Sutton, DM: Repeated serological
of antibody detection and titration methods: LISS, PeG, gel testing of patients with warm autoimmune hemolytic anemia
(abstract) Transfusion 42:108S, 2002. may not be necessary (abstract). Transfusion 41:106S, 2001.
40. Roback, J (ed): Technical Manual, 16th ed. American Associa- 49. Judd, WJ: Investigation and management of immune hemoly-
tion of Blood Banks, Bethesda, MD, 2008, p 633. sis: Autoantibodies and drugs. In Wallace, ME, and Levitt, JS
41. Schulman, IA, and Petz, LD: Red cell compatibility testing: (eds): Current Applications and Interpretations of the Direct
Clinical significance and laboratory methods. In Petz, LD, Antiglobulin Test. American Association of Blood Banks,
Swisher, SN, and Kleinman, S (eds): Clinical Practice of Trans- Arlington, VA, 1988, p 47.
fusion Medicine, 3rd ed. Churchill Livingstone, New York, 50. Cheng, CK, Wong, ML, and Lee, AW: PEG adsorption of au-
1996, p 199. toantibodies and detection of alloantibodies in warm autoim-
42. Roback, J (ed): Technical Manual, 16th ed. American Associa- mune hemolytic anemia. Transfusion 41:13, 2001.
tion of Blood Banks, Bethesda, MD, 2008, p 454. 51. Leger, RM, and Garratty, G: Evaluation of methods for de-
43. Standards for Blood Banks and Transfusion Services, 26th ed. tecting alloantibodies underlying warm autoantibodies.
American Association of Blood Banks, Bethesda, MD, 2009. Transfusion 39:11, 1999.
2682_Ch10_241-259 22/05/12 11:45 AM Page 241
Chapter 10
Pretransfusion Testing
William B. Zundel, MS, MLSCM, SBB
OBJECTIVES
1. Describe appropriate methods for proper patient identification in sample collection.
2. Outline the procedure for testing donor and patient specimens.
3. Select appropriate donor units based on availability, presence, or absence of unexpected alloantibody in the patient.
4. Compare and contrast crossmatch procedures.
5. Resolve incompatibilities in the crossmatch.
6. Explain compatibility testing procedures and protocols in special circumstances.
7. State the limitations of compatibility testing procedures.
8. Describe a scheme for effective blood utilization.
9. List the steps necessary to reidentify the patient before transfusion.
10. Discuss future issues of compatibility testing.
241
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of the recipient’s own RBCs. Table 10–1 outlines the steps responsible for identifying the patient and collecting
in pretransfusion testing and associated AABB standards recipient blood must adhere to the strict standards set
from the AABB’s Technical Manual, 16th edition.1 Strict ad- forth to ensure recipient safety and acceptable survival
herence to and application of each parameter of pretransfu- rates.
sion testing is imperative in managing safe blood transfusion
therapy. Each parameter must also be considered in any com- Positive Recipient Identification
prehensive review of the process used to select blood for a
recipient. A major cause of transfusion-associated fatalities is clerical
Pretransfusion testing cannot guarantee normal survival error resulting in incorrect ABO groupings and transfusion
of transfused RBCs in the recipient’s circulation. The poten- of ABO incompatible blood. In a study of transfusion errors
tial benefits of RBC transfusion should always be weighed in New York State over a 10-year period, 47% of errors in-
against the potential risks any time this form of therapy is volved misidentification of the patient or the blood at bed-
considered. Although adverse responses to transfusion can- side.2 Clerical error is the greatest threat to safe transfusion
not always be avoided, results are much more likely to be fa- therapy. The most common cause of error is misidentifica-
vorable if pretransfusion testing is carefully performed and tion of the recipient. Examples include misidentification of
if results of laboratory testing show no incompatibility the recipient when the blood sample is drawn, mix-up of
between donor and recipient. samples during handling in the laboratory, and misidenti-
The Clinical Laboratory Improvement Amendments of fication of the recipient when the transfusion is given.
1988 (CLIA ‘88) gives the United States federal govern- Exact procedures for proper identification of the recipient,
ment authority to regulate pretransfusion testing. Testing recipient sample, and donor unit must be established and
regulated via CLIA ‘88 includes ABO group, Rh type, utilized by all staff responsible for each aspect of transfu-
antibody detection and identification, and crossmatch sion therapy.
testing. To prevent collecting samples from the wrong patient, a
facility-generated recipient ID wristband must always be
Identification, Collection, and Preparation compared with the blood requisition form (blood request
of Samples form). The blood request form must state the recipient’s
full name and unique hospital identification number.3
Pretransfusion testing begins and ends with the proper Other information such as age, date of birth, address, sex,
identification and collection of the patient sample. Those and name of requesting physician can be used to further
Reprinted with permission from Roback, J (ed): Technical Manual, 16th ed. American Association of Blood Banks, Bethesda, MD, 2008.
2682_Ch10_241-259 22/05/12 11:45 AM Page 243
prior to collection and cannot be avoided. In these circum- Patient RBCs can be obtained from either clotted or anti-
stances, care must be taken to note the extent of hemolysis coagulated samples. They can be washed with physiological
present in the patient serum or plasma and observe for any saline before use to remove plasma or serum, which may
increases during each stage of testing. interfere with some testing procedures. A 2% to 5% saline
Serum or plasma may be used for pretransfusion test- suspension of RBCs is used for most serologic testing proce-
ing. Disadvantages to using plasma include the possible dures; however, the manufacturer’s directions should be
formation of small fibrin clots, which may be difficult to consulted for the proper cell concentration to use for typing
distinguish from true agglutination. Also, plasma antico- tests performed with licensed reagents.
agulants may inactivate complement so that some antibod-
A method for preparing a button of washed RBCs
ies may not be detected. Having said this, more transfusion
suitable for performing one test is outlined in
services are using plasma today than ever before due to
Procedure 10-1 on the textbook’s companion website.
the ease of handling. About 10 mL of blood is usually
sufficient for all testing procedures if there are no known
serologic problems. Collection of Donor Samples
Tubes must be labeled before they leave the recipient’s
bedside. If imprinted labels are used, they must be compared Samples for donor testing must be collected at the same
with the recipient’s wristband and requisition form before time as the full donor unit. Depending on the method used
the tubes are used. Labels must be attached to the tubes at for testing, clotted or anticoagulated pilot samples are
the bedside in a tamper-proof manner that will make removal obtained. The donor information and medical history card,
and reattachment impossible. All writing must be legible and the pilot samples for processing, and the collection bag
indelible, and each tube must be labeled with the patient’s must be labeled with the same unique number before start-
full name, unique identification number, and date of sample ing the phlebotomy, and the numbers must be verified
collection.5 The phlebotomist must initial or sign the label again immediately after collection.8 The donor unit identi-
and add additional pertinent information as required by the fication number is used to identify all records of testing and
facility’s SOP. eventual disposition of all component parts of the unit of
To avoid contamination with materials that may cause blood. More detailed information on donor samples can be
confusing serologic results, blood samples should not be found in Chapter 13, “Donor Screening and Component
taken from intravenous (IV) tubing lines. Venous samples Preparation.”
are to be drawn only from below the infusion site, not above RBCs for donor pretransfusion testing can be prepared
it. For example, if a patient has an IV line in the antecubital from the segmented tubing through which the donor blood
area of the arm, any vein below the angle of the arm to the was collected. The tubing or segment is attached to the col-
hand can be used. If a sample must be taken from an IV line, lection bag, and each segment is imprinted with the same
the line should be disconnected for 5 to 10 minutes, the first number. These numbers are different from the donor unit
10 mL of blood drawn should be discarded, and then the identification number but nonetheless are a positive means
sample should be obtained. of sampling a given unit of blood.
When a specimen is received in the laboratory, blood Donor RBCs can be obtained from the segments in many
bank personnel must confirm that the information on the ways that permit several procedures to be performed from
sample and requisition form agree. All discrepancies must the same segment. One technique that works well for sam-
be resolved before the sample is accepted, and if any doubt pling is using a lancet to make a tiny hole in the segment
exists, a new sample must be drawn. Receipt of an unlabeled through which a single drop of blood can be expressed and
specimen requires that a new sample be obtained.6 then disposing the lancet in a biohazard sharps container.
Recipient samples should be tested as soon as possible The hole is essentially self-sealing, so the rest of the blood
after collection. If using a serum sample, the recipient serum in the segment remains uncontaminated. Another technique
should be separated from the RBCs as soon as possible after is to cut the RBC end of the segment tubing with scissors
the sample has clotted. If testing cannot be performed imme- and use an applicator stick to remove cells or squeeze the
diately, samples should be kept at 1°C to 6°C. tubing to express a drop.
As noted in Chapter 3, “Fundamentals of Immunology,” Commercially manufactured segment puncture devices
recent pregnancy or transfusion indicates an opportunity eliminate the need for scissors and lancets, thus enabling
for a humoral immune response. Antibody production occurs the dispensing of the RBCs into a test tube in one motion.
over a predictable range of time, which varies from patient The segment may be stored with the cut end down in
to patient. Specimens used in pretransfusion testing should a properly labeled test tube and covered or stoppered
ideally be collected during the critical phases of the immune to minimize contamination and maintain RBC integrity.
response. In an attempt to capture this important time for The contents of the segment should not be emptied into
each patient, serum obtained from samples fewer than a test tube for storage because of the increased risk of
72 hours after collection must be used for antibody screening contamination. Regardless of the method used to harvest
and crossmatch testing if the patient was pregnant or received cells from a segment, it is important that engineering
RBC products by transfusion within the last 3 months, or if or work practice controls be used to eliminate or minimize
these histories are unknown.7 aerosol production when the segment is cut or opened.
2682_Ch10_241-259 22/05/12 11:45 AM Page 245
Refer to Chapter 25, “Transfusion Safety and Federal initiated. A new sample should be collected from the patient,
Regulatory Requirements” for additional information on if necessary, to resolve the problem.
safety procedures. ABO, Rh, and unexpected antibody screening test results
Both donor and recipient samples must be stored for a should be included in the record. Notations concerning
minimum of 7 days following transfusion.9 The samples unusual serologic reactions and the identity of unexpected
should be stoppered, carefully labeled, and refrigerated at antibodies in the patient’s serum should also be included in
1°C to 6°C. They should be adequate in volume so they can the record. This may be the most important information.
be reevaluated if the patient experiences any adverse reaction Sometimes an unexpected antibody can drop below detectable
to the transfusion. levels in a patient’s serum, and previous records are the only
source of information regarding its presence, identity, and
Compatibility Testing Protocols clinical significance.
ABO, Rh grouping, and antibody screening of the patient’s
Compatibility testing refers to the serologic aspect of
serum can be performed in advance of or at the same time
pretransfusion testing. It includes every serologic facet,
as the crossmatch. If the patient has had a transfusion or has
beginning with donor blood and ending with the recipient
been pregnant within the last 3 months or if the history is
blood sample.
unavailable or uncertain, the sample must be obtained from
the patient within 3 days of the scheduled transfusion.15 An
Testing the Donor Sample accurate medical history, including information on medica-
According to the Code of Federal Regulations10 and the AABB tions, recent blood transfusions, and previous pregnancies,
Standards for Blood Banks and Transfusion Services,11 ABO may help to explain unusual results.
grouping and Rh typing (including a test for weak D) and ABO Grouping
tests intended to prevent disease transmission must be per-
formed on a sample of donor blood taken at the time of col- Determining the patient’s correct ABO group is the most
lection. AABB Standards requires a screening test for critical pretransfusion serologic test. ABO grouping can
unexpected antibodies to RBC antigens on samples from be performed on slides or in tubes, using solid-phase RBC
donors who have a history of transfusion or pregnancy.12 adherence or column gel technology. Testing is performed
Testing is performed by the facility collecting the donor unit, in a manner similar to that described in Chapter 6, “The
and results must be clearly indicated on all product labels ABO Blood Group System,” using potent licensed reagents
appearing on the unit. according to the manufacturer’s directions. If the ABO
AABB Standards13 also requires that the transfusing facility forward and reverse grouping results do not agree, addi-
confirm the ABO cell grouping on all units and Rh typing tional testing must be conducted to resolve the discrep-
on units labeled Rh-negative. Repeat weak-D testing is not ancy. Useful information on resolving ABO grouping
required. The transfusing facility is not required to repeat discrepancies has also been presented in Chapter 6. If the
any other testing procedure on donor blood. The sample patient’s ABO group cannot be satisfactorily determined
used for this testing must be obtained from an attached seg- and immediate transfusion is required, group O–packed
ment on the donor unit. RBCs should be used.
All testing must be performed using in-date licensed Rh Typing
reagents according to manufacturers’ directions and protocol
established in the facility’s written SOP. A detailed explana- Rh typing is performed using anti-D blood typing reagents.
tion of the processing of donor blood can be found in Tube or slide tests should be performed according to the
Chapter 13. manufacturer’s directions for the reagent, which may
include the use of a suitable diluent control. When indi-
Testing the Patient Sample cated, these controls must be run in parallel when Rh typing
tests are performed on patient samples to avoid incorrectly
A record must be maintained of all results obtained in testing designating Rh-negative patients as Rh-positive. If the dilu-
patient samples. Some large transfusion services keep this ent control is positive, the result of the Rh typing test is
information on a computerized retrieval system for ready invalid. In such a case, a direct antiglobulin test (DAT)
access. However, when computer records are not available, should be performed on the patient’s RBCs to determine
these transfusion services must have another system that whether uptake of autoantibodies or alloantibodies (if the
permits retrieval of patient testing results.14 recipient has been recently transfused) is responsible for
Ideally, the same unique identification number should be the positive control. If the DAT is positive, accurate
assigned each time a patient is admitted to a health-care fa- Rh typing can sometimes be performed using saline-active
cility for treatment. The number can then be used as a or chemically modified Rh blood typing serum with an ap-
method for positive identification by comparing results of propriate diluent or 8% albumin control. If the Rh type of
previous and current testing. Verification of previous results the recipient cannot be determined and transfusion is
helps establish that the current samples were collected from essential, Rh-negative blood should be given. Currently,
the correct individual. Any discrepancies between previous most monoclonal or monoclonal blend anti-D reagents are
and current results must be resolved before transfusion is room temperature–reactive and do not require the use of
2682_Ch10_241-259 22/05/12 11:45 AM Page 246
a control. See Chapter 7, “The Rh Blood Group System,” Table 10–2 Clinical Significance of
for more in-depth discussion. 37°C-Reactive Antibodies
The test for weak D is unnecessary when testing trans-
fusion recipients.16 Individuals typing as Rh-negative in VERY UNUSUAL
direct testing should receive Rh-negative blood, and those USUALLY* (IF EVER)† SOMETIMES
typing as Rh-positive in direct testing should receive ABO Bg (HLA) Cartwright (e.g., Yta)‡
Rh-positive blood. There are those in the blood bank com-
Rh Ch/Rg (complement C4) Lutheran (e.g., Lub+)‡
munity who prefer complete Rh typing of all recipients to
conserve Rh-negative blood for Rh-negative patients. Kidd Leb Gerbich‡
Female patients whose RBCs type as weak D are considered
Duffy JMH Dombrock‡
Rh-positive and may receive Rh-positive blood during
transfusion. S, s, U Xga M, N‡
Some patients who type as Rh-positive, whether by
P Lea
direct or indirect testing, may produce anti-D following
transfusion of Rh-positive RBC components. This occurs Vel
rarely and does not justify the routine transfusion of
LW
Rh-negative blood to these Rh-positive patients until the
antibody is detected. Ii
Antibody Screening H
The recipient’s serum or plasma must be tested for clinically Ata
significant unexpected antibodies. The object of the anti-
Inb
body screening test is to detect as many clinically significant
unexpected antibodies as possible. In general, “clinically Mia
significant unexpected antibody” refers to antibodies that
Csa
are reactive at 37°C or in the antihuman globulin test and
are known to have caused a transfusion reaction or unac- * These antibodies usually cause obvious clinical symptoms and decreased RBC survival.
ceptably short survival of transfused RBCs. Table 10–2 Sometimes no obvious clinical symptoms occur.
outlines these antibodies and their clinical significance as † These antibodies rarely (if ever) cause clinically obvious symptoms, but there are some
data to suggest that some unusual examples of Bg,36,39–41 anti-Kn/Mc/Yk,36,42 and JMH36,43
“usually,” “rarely,” or “sometimes” causing obvious clinical cause shortened RBC survival.
symptoms.17 ‡ These antibodies rarely cause acute severe HTR, but when they are “clinically significant,”
The incidence of unexpected alloantibodies depends on they may cause obvious clinical symptoms (e.g., jaundice); they more often cause only short-
ened RBC survival.
the fact that antibody formation is the result of exposure to Reprinted with permission from Garratty, G: Evaluating the clinical significance of blood
a foreign RBC antigen and the patient’s ability to respond to group alloantibodies that are causing problems in pretransfusion testing. Vox Sanguinis 74:
that exposure. This occurs by allogeneic transfusion of RBCs, 285–290, 1998. Table 1, p 289.
pregnancy, or transplantation. Therefore, the incidence of
unexpected antibodies in the general patient population is
low: 1.64% in one large study18 and 0.78% in another.19 It Selection of Appropriate Donor Units
follows, then, that the more frequently a patient is exposed
to foreign RBC antigens, the more likely that patient will pro- In almost all cases, the first choice for transfusion is blood
duce unexpected alloantibodies. This is evidenced by a study and blood components of the patient’s own ABO and
of multiply transfused sickle cell patients in which 29% of Rh group. This is defined as ABO group–specific. When
pediatric and 47% of adult patients developed clinically sig- blood and blood components of the patient’s ABO blood
nificant alloantibodies.20 group are not available or some other reason precludes their
Detection of unexpected antibodies is important for the use, units selected must lack any antigen against which the
selection of donor RBCs that will have the best survival rate recipient has a clinically significant antibody. However, it is
in the patient’s circulation and reduce the risk of hemolytic completely acceptable to use blood and blood components
transfusion reaction. Refer to Chapter 9, “Detection and that do not contain all of the antigens carried on the patient’s
Identification of Antibodies,” for a complete discussion of own RBCs (e.g., group A– or B–packed RBCs can be safely
the detection and identification of unexpected clinically sig- given to a group AB recipient).
nificant alloantibodies. When a recipient must be given blood of a different ABO
Antibody screening tests should demonstrate the presence group, only packed RBCs can be given. Whole blood cannot
of all potentially clinically significant alloantibodies in the be administered in these situations because incompatible,
recipient’s serum or plasma and indicate the need for further preformed ABO antibodies are present in the whole-blood
studies. All antibodies encountered in the screening test plasma. For example, group A whole blood cannot be trans-
must be identified to determine potential clinical significance fused into a group AB recipient, because the plasma of the
and to decide whether there is a need to select antigen-negative group A whole blood has anti-B antibodies present. Group
units for transfusion. O packed RBCs can be safely used for all patients; however,
2682_Ch10_241-259 22/05/12 11:45 AM Page 247
conservation of a limited supply of group O blood should 37°C or whose antibodies react well only with panel cells
dictate its use for recipients of other ABO types only in carrying homozygous expression of the corresponding anti-
special circumstances. If ABO group–specific blood is not gens. It must also be used for patients whose serum no
available or is in low supply, alternative blood groups are longer exhibits demonstrable in vitro reactivity, but that pre-
chosen, as summarized in Table 10–3. viously was known to contain clinically significant IgG
Rh-negative blood can be given to Rh-positive patients; antibodies such as anti-Jka, anti-K1, or anti-E.
however, good inventory management should conserve this Donor units should be selected so that the RBCs are of ap-
limited resource for use in Rh-negative recipients. But if the propriate age for the patient’s needs and will not expire before
Rh-negative unit is near expiration, the unit should be given use. For efficient inventory management, units that will def-
rather than wasted. Rh-positive blood should not be given to initely be transfused should be selected from units close to
Rh-negative female patients of childbearing age. Transfusion their expiration date based on the needs of the recipient.
of Rh-negative male patients and female patients beyond Before compatibility testing, donor units should be ex-
menopause with Rh-positive blood is acceptable as long as amined visually for unusual appearance, correct labeling,
no preformed anti-D is demonstrable in their sera. About 80% and hermetic seal integrity. Donor units showing abnormal
of Rh-negative patients who receive 200 mL or more of Rh- color, turbidity, clots, incomplete or improper labeling
positive blood respond to such a transfusion by producing information, or leakage of any sort should be returned to
anti-D.21 However, this outcome must sometimes be weighed the collecting facility.
against the alternatives of not transfusing at all if the supply
of Rh-negative blood has been exhausted. If the formation of Crossmatch Testing
anti-D is unlikely to be of great significance (e.g., in an
Rh-negative elderly surgical patient), many technologists feel The crossmatch test has traditionally meant the testing of
the use of Rh-positive blood is judicious. In these situations, the patient’s serum with the donor RBCs, including an
approval by or notification of the blood bank’s medical direc- antiglobulin phase or simply an immediate spin phase to
tor is necessary, according to the laboratory’s SOP. confirm ABO compatibility. The terms compatibility test and
When an unexpected antibody is found in the patient’s crossmatch are sometimes used interchangeably, but they
serum during an antibody screening test, donor units should be clearly differentiated. A crossmatch is only one
selected at random are crossmatched with the patient’s part of pretransfusion testing (see Table 10–1).
serum and shall include incubation at 37°C and the AHG Originally the serologic crossmatch preceded antibody
test.22 This should not be assumed to be the standard, screening as part of pretransfusion compatibility testing to
although this may help identify the unexpected antibody. If check for unexpected alloantibodies. Considering that over
a clinically significant antibody is identified, the serologically 99% of clinically significant unexpected antibodies in pa-
compatible units are then phenotyped with commercial tients’ sera can be detected by adequate antibody screening
antiserum to verify that they are antigen-negative for the cor- procedures, many blood banks abbreviate or even eliminate
responding antibody. For example, if a recipient has an the serologic crossmatch. What, then, is the value of per-
anti-K1 antibody, the crossmatch-compatible donor RBC forming serologic crossmatching between patient and donor
unit is tested with commercial anti-K1 antisera for the pres- samples? Two main functions of the serologic crossmatch
ence of the K1 antigen. test can be cited:
There is no need to provide antigen-negative RBCs for
1. It is a final check of ABO compatibility between donor
patients whose sera contain antibodies that are reactive only
and patient.
below 37°C, because these antibodies are incapable of causing
2. It may detect the presence of an antibody in the patient’s
significant RBC destruction in vivo.
serum that will react with antigens on the donor RBCs but
Potent examples of IgG, warm reactive antibodies in
that was not detected in antibody screening because the
patients’ serum can also be used to select suitable donor
corresponding antigen was lacking from the screening cells.
units by direct crossmatch testing. Commercially prepared
typing reagents must be used to select blood for patients The current AABB Standards23 state that tests to detect
whose serum contains weak examples of antibodies active at ABO incompatibility are sufficient if no clinically significant
Table 10–3 Suggested ABO Group Selection Order for Transfusion of RBCs
RECIPIENT ABO GROUP 1st CHOICE 2nd CHOICE 3rd CHOICE 4th CHOICE
AB AB A B O
A A O
B B O
O O
Reprinted with permission from AABB Technical Manual, 14th ed, p 454, Table 21.1 Suggested ABO Group Selection Order for Transfusion of RBCs.
2682_Ch10_241-259 22/05/12 11:45 AM Page 248
antibodies were detected in the antibody screening process rouleaux is observed, or when infants’ specimens are
and if no historical record exists of clinically significant tested. Adding ethylenediaminetetraacetic to the test
unexpected antibodies being detected. system reportedly eliminates some of the false-positive
Elimination of advanced crossmatch testing—for patients reactions, thus improving the sensitivity of the immediate
undergoing surgical procedures, in which blood is unlikely spin crossmatch.32
to be used—has been implemented successfully in many
facilities. This is accomplished by using the “type and Antiglobulin Crossmatch
screen” in conjunction with the maximum surgical blood
order schedule (MSBOS) approach or the abbreviated cross- The antiglobulin crossmatch procedure begins in the same
match. This leads to the next generation of crossmatch manner as the immediate spin crossmatch, continues to a 37°C
decisions: the computer crossmatch. incubation, and finishes with an antiglobulin test. Several
enhancement media may be applied to boost antigen-antibody
Serologic Crossmatch Tests reactions. These may include albumin, low ionic strength
solution (LISS), polyethylene glycol, and polybrene (as dis-
The serologic crossmatch test consists of mixing the recipi- cussed in Chapter 5, “The Antiglobulin Test”). For greatest sen-
ent’s serum with donor RBCs. Several procedures can be sitivity, an antihuman globulin (AHG) reagent containing both
used for serologic crossmatch testing, including the imme- anti-IgG and anticomplement may be selected for the final
diate spin and antiglobulin crossmatch. The objective of test- phase of this crossmatch method. However, many laboratories
ing is to select donor units that can provide maximal benefit routinely use monospecific anti-IgG AHG reagents.
to the patient, which should be kept in mind when develop- An autocontrol, consisting of the patient’s own cells and
ing the test protocol. Crossmatch methods can generally be serum, may be tested in parallel with the crossmatch test.
categorized by the test phase in which the procedure ends. Although current AABB Standards no longer requires an auto-
control, some technologists still find it useful. Perkins33 calcu-
A sample procedure for a one-tube crossmatch test
lated the predictive value of a positive autocontrol (3.6%) when
is given in Procedure 10-2 on the textbook’s com-
the antibody screen was negative and decided to continue using
panion website.
the autocontrol in pretransfusion testing. Results of the auto-
control help clarify possible explanations for positive results in
Immediate Spin Crossmatch the crossmatches and are discussed later in this chapter.
means of a logical system that allows them to be easily transfusion of non-ABO-specific blood products (e.g.,
recalled; actual observations and interpretations must be platelets) or after organ (e.g., liver) or bone marrow
recorded. All work should be signed or initialed by the tech- transplantation. Checking the serum grouping result to
nologist performing the test. If an incompatibility is found, confirm the presence of an unexpected reaction with A1
the record should clearly show the location of results of the cells or checking the patient’s transfusion and transplant
follow-up studies, and additional testing should be performed. histories is helpful when investigating these cases.
3. An autoantibody in the patient’s serum reacting with the
Resolution of Incompatibilities in the Serologic corresponding antigen on donor RBCs. The autocontrol
Crossmatch tube will be positive. The antibody screening test and tests
of the patient’s serum with donor cells will show positive
The primary objective of the crossmatch test is to detect the results. Most autoantibodies have specificity for antigens
presence of antibodies in the recipient’s serum, including of relatively high incidence. Panel adsorption and elution
anti-A and anti-B, that could destroy transfused RBCs. A pos- studies are important to assess whether underlying alloan-
itive result in the crossmatch test requires explanation, and tibodies are also present. Techniques for management of
the recipient should not receive a transfusion until the cause patients with autoantibodies include, among other tests,
of the incompatibility has been determined. When the cross- autoadsorption of the patient’s serum to remove autoan-
match test result is positive, the results of the autocontrol tibody activity. Compatibility testing could then be
and antibody screening test should be reviewed to identify performed using the autoabsorbed serum. Chapter 20,
patterns that may help determine the cause of the problem. “Autoimmune Hemolytic Anemias” provides further dis-
cussion of autoantibodies and their serologic activity.
Causes of Positive Results in the 4. Prior coating of the donor RBCs with protein, resulting
Serologic Crossmatch in a positive antihuman globulin test. If one isolated pos-
itive result is obtained, a DAT should be performed on
A positive result in the serologic crossmatch test may be
the donor’s RBCs. Donor cells that demonstrate a positive
caused by any of the following:
DAT will be incompatible with all recipients tested in the
1. Incorrect ABO grouping of the patient or donor. ABO AHG phase, because the cells are already coated with im-
grouping should be immediately repeated, especially if munoglobulin or complement.
strong incompatibility is observed in a reading taken after 5. Abnormalities in the patient’s serum.
immediate spin. Samples that bear undisputable identity • Imbalance of the normal ratio of albumin and gamma
with the original patient sample and the donor bag globulin (A/G ratio), as in diseases such as multiple
should be used for retesting. myeloma and macroglobulinemia, may cause RBCs to
2. An alloantibody in the patient’s serum reacting with the stick together on their flat sides, giving the appearance
corresponding antigen on donor RBCs. The autocontrol of stacks of coins when viewed microscopically. This is
tube will be negative unless the patient has been recently called rouleaux formation (see Color Plate 2). This prop-
transfused with incompatible RBCs. If the antibody erty of the serum will affect all tests, including the auto-
screening test is positive, antibody identification panel control. Strong rouleaux may mimic true agglutination.
studies should allow identification of antibody specificity, Rouleaux are usually strongest after 37°C incubation but
which then permits selection of units lacking the antigens do not persist through washing before the AHG test.
for compatibility testing. Chapter 9 provides further dis-
Problems with rouleaux can often be resolved
cussion of antibody detection and identification and has
using the saline replacement technique (see
examples for study.
Procedure 10-3 on the textbook’s companion
• If RBCs of all donors tested are incompatible with the
website).35
patient’s serum and the antibody screening test is pos-
itive, suspect either an antibody directed against an • The presence of high-molecular-weight dextrans or
antigen of high incidence or multiple antibodies in the other plasma expanders may cause false-positive results
patient’s serum. Consult a reference laboratory if you in pretransfusion testing. However, Bartholomew36
are unable to identify the specificity. Note: If the patient raised doubt that the use of dextran interferes with pre-
has ABO-compatible siblings, the siblings may lack the transfusion testing. In scenarios in which plasma
antigen(s) to which the patient has been sensitized and expanders interfere, all tests, including the autocontrol,
may be excellent potential donors in an emergency. are generally affected equally. Saline replacement may
• If the antibody screening test is negative and only one be useful to resolve the problem.
donor unit is incompatible, an antibody in the patient’s • An antibody against additives in the albumin reagents
serum may be directed against an antigen of relatively may cause false-positive results in compatibility tests.
low incidence that is present on that donor’s RBCs. Rarely, a patient’s serum reacts against the albumin in
• If the antibody screening test is negative, the patient’s testing reagents. This occurs when the patient has anti-
serum may contain either naturally occurring (e.g., bodies to the stabilizing substances, such as caprylate,
anti-A1) or passively acquired ABO agglutinins. Passive added to the albumin reagents.37 Thus, caprylate-free
acquisition of anti-A, anti-B, or anti-A,B may occur after albumin solutions should be used in testing.
2682_Ch10_241-259 22/05/12 11:45 AM Page 250
6. Contaminants in the test system. Dirty glassware, bacte- serologic immediate spin test. Many believe that the computer
rial contamination of samples, chemical or other contam- crossmatch is safer than the immediate spin because of the
inants in saline, and fibrin clots may produce false-positive integrity of the computer software to detect ABO incompati-
compatibility test results. Refer to Table 10–4 for sugges- bility between the sample submitted for pretransfusion testing
tions on investigating causes of pretransfusion tests. and the donor unit.39 The computer crossmatch compares re-
cent ABO serologic results and interpretations on file for both
Computer Crossmatch the donor and the recipient being matched and determines
compatibility based on this comparison. Butch and colleagues40
A report by Judd38 indicated that an electronic (computer) provide an excellent model of the computer crossmatch SOP.
crossmatch to detect ABO incompatibilities was as safe as the Also helpful is the “Computer Crossmatch” (Electronic Based
• Rouleaux formation.
• Antibody reacts only with red cells having strong expression of a particular antigen (e.g., dosage) or variation in antigen strength (e.g., P1).
• Antibodies demonstrating dosage and donor red cells are from heterozygotes (i.e., expressing a single dose of antigen).
• Alloantibody(ies)
Positive Antibody Screen, Incompatible Crossmatches, Positive Autocontrol, Negative Direct Antiglobulin Test
• Rouleaux formation.
• Rouleaux formation.
Testing for the Compatibility between the Donor’s Cell Type two concordant patient ABO/Rh types must be on file or the
and the Recipient’s Serum or Plasma Type) Draft Guidance, computer crossmatch is not permissible.43
CBER June 2007. Additional benefits of using the computer One of the ABO determinations must be done on the cur-
crossmatch include annual savings, reduced sample require- rent sample. Having a previous ABO result on file may serve
ments, reduced handling of biological materials, and elimina- as the second occasion. When there are no results on file,
tion of false reactions associated with the immediate spin testing of the same sample by a second technologist, if pos-
crossmatch. Although the advantages outweigh the disadvan- sible, or testing of a second current sample is indicated. The
tages, a compilation of the College of American Pathologists’ computer system must have logic to notify the user of the
interlaboratory comparison program survey data41 indicates following: discrepancies between the donor ABO group and
that as of 2004, only 2.1% of labs participating in the CAP Rh type on the unit label, and those determined by blood
survey use a computer crossmatch. group confirmatory tests, and ABO incompatibility between
Figure 10–4 is a flowchart that illustrates the necessary the recipient and the donor unit. Additionally, the computer
steps for a computer crossmatch to meet the AABB Stan- system must be validated to show that it can detect data
dards.42 The AABB Standards specifies that the computer cross- entry discrepancies and ABO incompatibilities between
match can be used only for the purpose of detecting an ABO patient and donor.44
incompatibility between the donor unit and the recipient
sample that was submitted for pretransfusion testing. Current Pretransfusion Testing in Special
testing for unexpected antibodies must be nonreactive, and Circumstances
there must not be any history of such antibodies. Also, at least
As with all things, there are special circumstances that just
don’t fit into the rigid regulatory world of pretransfusion test-
ing. There are circumstances associated with emergency trans-
fusion needs that take precedence over standard protocols such
Type & Screen Blood
Requested Requested as waiving pretransfusion testing in trauma situations. This is
just one of the following special circumstances discussed.
Store
Sample Emergencies
Tested
Obtain In-Date Blood component needs sometimes exceed pretransfusion
Do ABO/Rh &
Antibody Screen Sample No
Sample
Yes
testing requirements. In other words, the recipient may require
Available? the transfusion of RBC components prior to the completion
of pretransfusion testing. Several approaches can be utilized
in these circumstances. Some laboratories use an “emergency”
pretransfusion testing procedure that employs a shortened
incubation time, often with addition of LISS, to accelerate
Identify Significant antigen–antibody reactions. Others maintain that regular
Detected Antibodies
Present. Now Yes
procedures should be used in all circumstances and that blood
Antibodies
per SOP or in Past? should be issued before completing the standard pretransfu-
sion testing procedure, if necessary. They believe that there is
greater danger in using an unfamiliar procedure under pres-
No sure than in releasing blood without completed testing.
Although both lines of reasoning have merit, the ideal com-
Select
Antigen-negative promise may be to develop regular testing procedures that are
Units Per SOP & do most efficient so that they can be used in emergency and rou-
IAT Crossmatch tine situations alike. Whatever the approach, the protocol for
More than
one ABO/Rh No
handling emergencies must be decided in advance of the sit-
Yes
on File? uation and be familiar to all staff in the transfusion service.
Adequate pretransfusion samples should be collected before
transfusion of any donor blood, if possible, so that pretrans-
Repeat ABO/Rh fusion testing, if necessary, can subsequently be performed.
If blood must be issued in an emergency, the patient’s
ABO and Rh group should be determined so that ABO group–
No Yes Resolve
specific blood can be given. In extreme emergencies, when
Do Electronic Conflict?
Crossmatch per SOP there is no time to obtain and test a pretransfusion sample,
group O Rh-negative packed cells can be used. If the patient
is Rh-negative and large amounts of blood are likely to be
Figure 10–4. Flowchart for the electronic crossmatch that meets the require-
ments set forth by the AABB. (Reprinted with permission from Judd, JW:
needed, a decision should be made rapidly whether inventory
Requirements for the electronic crossmatch. Vox Sanguinis 74:409–417, 1998, allows and the situation demands transfusion of Rh-negative
Figure 1, p 410.) blood. Conversion to Rh-positive is best made immediately if
2682_Ch10_241-259 22/05/12 11:45 AM Page 252
the patient is a man or is a woman beyond childbearing age. (e.g., anti-K1, anti-Jka). Crossmatch testing is performed using
Injections of Rh immunoglobulin to prevent formation of the mother’s serum sample.
anti-D may sometimes be appropriate after the crisis has been
resolved. This product is discussed in detail in Chapter 19, Neonatal Transfusions
“Hemolytic Disease of the Fetus and Newborn.”
Accurate records of all units issued in the emergency must Blood for an exchange or regular transfusion of a neonate
be maintained. A conspicuous tie tag or label must be placed (younger than 4 months of age) should be compatible with
on each unit that indicates compatibility testing was not any maternal antibodies that have entered the infant’s circu-
completed before release of the unit, and the physician must lation and are reactive at 37°C or AHG. Blood of the infant’s
sign a waiver authorizing and accepting responsibility for ABO and Rh group can be used, as long as the ABO and Rh
using blood products prior to completion of pretransfusion groups are not involved in the fetomaternal incompatibility.
testing, according to the Code of Federal Regulations.45 Sub- An initial pretransfusion specimen from the infant must
sequent pretransfusion testing should be completed accord- be typed for ABO and Rh groups (only anti-A and anti-B
ing to the chosen protocol, and any incompatible result reagents are required for ABO grouping, omitting the testing
should be reported immediately to the recipient’s physician of the infant’s serum with reagent RBCs).46 Antibody detec-
and the blood bank medical director. tion testing can be performed using the maternal serum,
the infant’s serum (e.g., cord serum), or an eluate prepared
Transfusion of Non-Group-Specific Blood from the infant’s RBCs. In addition, when cells selected
for transfusion are not group O, the infant’s serum or plasma
When donor units of an ABO group other than the recipient’s must be tested to demonstrate the absence of anti-A
own type have been transfused, such as giving a group A re- (using A1 cells) and anti-B. This testing must include an
cipient large volumes of group O RBCs, testing the recipient’s antiglobulin phase.47
serum in a freshly drawn sample for the presence of unex- It is unnecessary to repeat these pretransfusion tests during
pected anti-A or anti-B must be performed prior to giving any any one hospital admission, provided that the infant received
additional RBC transfusions. When serum from the freshly only ABO-compatible and Rh-compatible transfusions and had
drawn sample is compatible with donor RBCs of the recipi- no unexpected antibodies in the serum or plasma.48 The pres-
ent’s own ABO group, ABO group–specific blood may be ence of clinically significant antibodies, including anti-A and
given for the transfusion. If the serologic crossmatch reveals anti-B, indicates that cells lacking the corresponding antigen
incompatibility, additional transfusions should be of the must be selected for transfusion until the antibody is no longer
alternative blood group. demonstrable in the infant’s serum.49 A crossmatch does not
For example, if a group B patient has been given a large have to be performed in these situations. Box 10–1 summarizes
number of units of group O packed cells, anti-B may be pres-
ent in adequate amounts to result in a positive reaction in
an immediate spin crossmatch. Group O units should there-
fore be used for any additional transfusions. BOX 10–1
Compatibility Testing for Transfusion of Plasma Compatibility Tests for Infants Once per Admission
Products Routine
ABO
Compatibility testing procedures are not required for trans- Rh
fusion of plasma products. However, for transfusion of large Antibody screen
volumes of plasma and plasma products, a crossmatch test • Using maternal serum or
between the donor plasma and patient RBCs may be per- • Using infant’s serum, especially when:
formed, although current Standards does not require a cross- • No maternal specimen is available
match test. The primary purpose for testing is to detect ABO • Mother has clinically insignificant antibodies or
incompatibility between donor and patient; therefore, an • Using infant’s eluate
immediate spin crossmatch is sufficient. Additional
IAT using infant serum and A1 or B cells
Intrauterine Transfusions
• Cells can be reagent or donor (i.e., major crossmatch)
Blood for intrauterine transfusion must be compatible with • Must be done if non-group O cells will be transfused
maternal antibodies capable of crossing the placenta. If the • Antigen typing donor unit
ABO and Rh groups of the fetus have been determined, group- • While infant antibody screen is positive
specific blood could be given provided that there is no feto- • Donor units must lack antigen corresponding to antibody
maternal ABO or Rh incompatibility. If the ABO and Rh Every 3 Days
groups of the fetus are not known, then group O Rh-negative Same tests as above when:
RBCs should be selected for the intrauterine transfusion. The • ABO- or Rh-incompatible units are transfused or
group O Rh-negative cells must lack any antigens against • Unexpected antibodies are demonstrating via antibody screen
which the mother’s serum contains unexpected antibodies
2682_Ch10_241-259 22/05/12 11:45 AM Page 253
the compatibility tests for infants (younger than 4 months old) unexpected antibodies and tests designed to prevent disease
and how frequently they must be performed. transmission are not required when the blood will be used
For both intrauterine and infant (younger than 4 months) within the collecting facility.53 These units must be labeled
transfusions, blood should be as fresh as possible and no “For autologous use only.”54 According to AABB Standards,
older than 7 days. Refer to Chapters 15 and19 for additional the pretransfusion testing and identification of the recipient
information on neonatal transfusion. and the blood sample are required and must conform to the
protocols mentioned earlier in this chapter. However, tests
Massive Transfusions for unexpected antibodies in the recipient’s serum or plasma
and a crossmatch test are optional.55
When the amount of whole blood or packed cell compo-
nents infused within 24 hours approaches or exceeds the pa- Limitations of Compatibility Testing
tient’s total blood volume, the compatibility testing Procedures
procedure may be shortened or eliminated at the discretion
of the transfusion service physician following written policy As mentioned in the introduction to this chapter, no current
guidelines.50 The current technical manual uses the follow- testing procedure can guarantee the fate of a unit of blood
ing guideline: “Massive transfusion is arbitrarily defined that is to be transfused. Even a compatible crossmatch
either as the administration of 8 to 10 RBC units to an adult cannot guarantee that the transfused RBCs will survive nor-
patient in less than 24 hours, or as the acute administration mally in the recipient. Despite careful and meticulous in
of 4 to 5 RBC units in 1 hour.” If the patient is known to vitro testing, some compatible units will be hemolyzed
have a clinically significant unexpected antibody, all infused in the patient. In some cases, even limited survival of donor
units should be tested for and lack the corresponding cells may help to maintain a patient until the patient
antigen, if time permits. The antibody in the patient’s serum can begin to produce his or her own cells. Certainly no
may not be demonstrable because of dilution with large patient should be denied a transfusion if he or she needs
volumes of plasma and other fluids. However, a rapid rise in one to survive, and donor cells that appear incompatible by
antibody titer level and subsequent destruction of donor in vitro testing procedures may, in fact, survive quite well
RBCs may occur if antigen-positive units are infused. The in vivo. Refer to Table 10–5 for suggestions on streamlining
transfusion service physician may decide that it is better to pretransfusion testing processes.
give antigen-untested units than to hold up transfusion by In vivo compatibility can be determined using donor
waiting for the test results. The rationale is that it is impor- RBCs labeled with radioactive chromium (51Cr) or tech-
tant to give the patient a chance to survive and then to treat netium (99mTc) to measure the likelihood of successful trans-
the immune-mediated anemia induced by massive transfu- fusion when standard in vitro testing procedures are
sion of antigen-untested units. Again, the physician must inconclusive.56,57 If a transfusion is needed to save a patient’s
sign a waiver of testing or a release form for all untested units life and all units are incompatible and if the 51Cr studies
for transfusion. indicate adequate survival of donor RBCs, then transfusion
of an in vitro incompatible unit may need to be considered.
Specimens with Prolonged Clotting Time This decision should be made in consultation with the blood
bank medical director and the patient’s physician. The blood
Testing difficulties may be observed in blood samples should be transfused slowly, and the patient should be mon-
from patients who have prolonged clotting times caused itored carefully.58
by coagulation abnormalities associated with disease
or medications (such as heparin). A fibrin clot may form Blood Inventory Management
spontaneously when partially clotted serum is added
to saline-suspended screening or donor RBCs. Complete Many blood bankers are acutely aware of the need to use
coagulation of these samples can often be accelerated blood efficiently due to limited blood supplies and increasing
by adding thrombin. One drop of thrombin, 50 U/mL, demands for blood. Technologists have observed that there
to 1 mL of plasma (or the amount of dry thrombin that have been many surgical procedures, such as dilatation and
will adhere to the end of an applicator stick) is usually curettage and cholecystectomy, for which blood was rou-
sufficient to induce clotting.51 A small amount of prota- tinely ordered but rarely used. Blood bankers also pointed
mine sulfate can be added to counteract the effects of out that for many other surgical procedures, more units were
heparin in samples of blood collected from patients on this being ordered than were used.
anticoagulant.52 The Maximum Surgical Blood Order Schedule (MSBOS)
was developed to promote more efficient utilization of blood.
Preoperative Autologous Blood The goal of the MSBOS is to establish realistic blood ordering
levels for certain procedures. Because variation exists in the
Autologous transfusion refers to the removal and storage of surgical requirements of institutions, the standard blood
blood or components from a donor for the donor’s possible orders should be based on the transfusion pattern of each
use at a later time, usually during or after an elective surgical institution and should be agreed upon by the staff surgeons,
procedure. The ABO and Rh groups of the units must be anesthesiologists, and the blood bank medical director. (See
determined by the facility collecting the blood. Tests for Chapter 24, “Utilization Management.”)
2682_Ch10_241-259 22/05/12 11:46 AM Page 254
Type ABO, Rh A specimen has been collected; Antibody detection testing is not per-
patient’s ABO/Rh are known. formed.
Type and hold
Type and screen ABO, Rh, antibody detection Most of the pretransfusion test- Does not include crossmatch.
test/identification ing has been performed; com-
patible blood can be provided
in most situations.
Type and crossmatch ABO, Rh, antibody detection Routine pretransfusion testing Units are removed from inventory and
test/identification, RBC unit has been performed; compati- may not be available for use by other
selection or phenotyping, ble blood can be provided in patients in a timely manner.
crossmatch most situations.
Reprinted with permission from Roback, J (ed): Technical Manual, 16th ed. American Association of Blood Banks, Bethesda, MD, 2008.
Utilization of a type and screen policy is another Reidentification of the Patient Before
method to manage blood inventory levels efficiently and Transfusion
to reduce blood banking operating costs.59–61 The type
and screen method involves testing the recipient’s blood Reestablishing the intended recipient’s identity and the
sample for ABO, Rh, and unexpected antibodies. The selected donor product is the final step in the transfusion
specimen is refrigerated and kept available for immediate process. The same careful approach used to properly iden-
crossmatching if the need arises. If transfusion becomes tify the patient before sample collection must now be ap-
necessary, then an immediate spin or computer crossmatch plied to verify that the recipient is indeed the same person
may be performed. The blood bank must ensure that the who provided the initial blood sample for testing. In addi-
appropriate donor blood is available in case it is needed. tion, the actual product and accompanying record of testing
The type and screen policy does not apply to patients with must be verified as relating to the same donor unit number.
existing clinically significant unexpected alloantibodies, The bedside check just prior to blood administration is
because donor blood lacking the corresponding antigens the most critical step for preventing mistransfusion.62
must be available and should be fully crossmatched prior Mistransfusion, in which the wrong blood is transfused
to surgery or transfusion. to the recipient, is the single most frequent error result-
A type and screen policy applies to a recipient who does ing in ABO-incompatible transfusions and is one of the
not have any clinically significant unexpected alloantibodies leading causes of morbidity and death resulting from blood
present or any abnormal serologic results in the ABO or transfusion.63
Rh testing. If blood is needed quickly, the blood bank is then After pretransfusion testing is completed, two records
prepared to perform the immediate spin (or computer) cross- must be prepared. A statement of compatibility must be
match and release blood of the same ABO and Rh group as retained as part of the patient’s permanent medical record
that of the recipient before releasing the unit to the hospital if the blood is transfused, and a label or tie tag must be
floor. Once the blood is issued, both a 37°C incubation and attached to the unit stating the intended recipient’s identity,
AHG crossmatch can be performed using the same tube em- the results of pretransfusion testing, and the donor unit
ployed for the immediate spin crossmatch, if the antiglobulin number.64 This identification must remain on the donor unit
crossmatch is the standard protocol used by the laboratory. throughout the transfusion.
If either the 37°C incubation or AHG phase of testing is pos- The original blood transfusion request form can be used
itive, the patient’s physician is notified immediately, and the conveniently to accomplish one or both of these record-
transfusion of the unit of blood is stopped. keeping requirements. Some facilities use a multipart form
The application of the type and screen in combination to record the history of pretransfusion testing and trans-
with the MSBOS can greatly enhance the effectiveness of fusion of the unit. Useful information might include the
the blood inventory management program in a health-care initials or signature of the phlebotomist taking the sample,
facility by minimizing the crossmatch-to-transfusion ratio, donor numbers, results of pretransfusion testing, initials
reducing blood bank personnel workload, and using the or signature of the technologist performing the testing, and
blood inventory efficiently. signatures of the persons who verify the recipient’s identity
2682_Ch10_241-259 22/05/12 11:46 AM Page 255
before transfusion and those (two) who start the transfu- if the unit was allowed to warm above 10°C or to cool
sion. One copy of the form can be placed on the recipient’s below 1°C.68
chart after the transfusion is completed, and the other
is returned to the blood bank, if desired, for filing. The last The Future of Pretransfusion Testing
copy of the form might be printed on card stock and per-
forated so that it can be easily removed and attached to Automation with pretransfusion testing instruments, such
the donor unit in the laboratory. The most important as continuous-flow and batch analyzers, has streamlined
feature of this system is that the patient’s nameplate im- compatibility testing, especially in large blood centers. Two
pression rather than a handwritten transcription indicates of the most successful approaches have used microplates to
all forms used to identify the patient-donor combination. perform either liquid agglutination tests or solid-phase RBC
Most facilities now use computer-generated labels to adherence tests. In addition to microplates is the column
attach to each form. agglutination technology, whether using gel or glass beads
Recognizing the complexity of the process, other useful in the column to capture agglutinates.69,70 These methods
systems are emerging throughout the transfusion center and provide efficient and economic compatibility tests for pro-
throughout health care as a whole, including for adminis- cessing large numbers of donor specimens. Similar innova-
tering patient medications. Machine-readable, bar-code tions are emerging to streamline blood banking testing in
patient–blood unit identification is ideally suited to bedside hospital transfusion services.
check requirements and has been recently reported to The use of column gel technology is on the rise in hospital
significantly improve transfusion practice.65 In addition to and transfusion services. Utilization of the column gel
bar codes on patient identification wristbands, radio fre- method for antibody detection has risen dramatically from
quency identification devices (RFID) are being integrated. 26.1% in 2001 to 42% in 2004.71 The gel test is sensitive for
RFID consists of at least two parts: an integrated circuit for both antigen testing and antibody detection and identifica-
storing, processing, and sending information (i.e., patient tion and ABO and Rh typing.72,73 Advantages of the gel
number, ABO group, Rh type, etc.) and an antenna or re- system include standardizing pipetting of reagents and spec-
ceiver to collect or transmit the information. For example, imens, reading of agglutination reactions, reviewing stable
on the patient wristband is the RFID or bar code that can reaction endpoints up to 24 hours, and significantly reducing
now be used for identification purposes at every stage of specimen volume. Disadvantages include longer turnaround
pretransfusion testing. time for ABO determinations and less sensitive detection of
Whatever system is used, the information should be ABO antibodies in patient serum or plasma when compared
verified at least twice before the blood product is trans- with the tube method.74
fused. A copy of the original blood requisition form, placed The emergence of molecular biology techniques in blood
on the recipient’s chart after samples are collected, can be bank testing is just now beginning. Nucleic acid amplifica-
used as the request for release of the units from the blood tion techniques, often based on polymerase chain reaction,
bank. This allows another check of the nameplate impres- have demonstrated application in blood typing75 and in
sions on all forms. Before blood is taken from the blood screening blood for hepatitis C. This testing process goes
bank to the patient treatment area, the following records beyond detecting antigenic determinants on the RBC mem-
must be checked: ABO and Rh results, clinically significant brane and goes directly into the genetic foundation of those
unexpected antibodies, and adverse reactions to transfu- antigens. Molecular testing is currently one of the hottest
sion.66 In addition, the person releasing and the person topics in pretransfusion testing.76–78
accepting the units should verify agreement between the As a result of the French requirement to perform a final
donor numbers and ABO and Rh groups on the compati- check of the ABO compatibility of the recipient and donor,
bility form and on the products themselves. The unit several methods are available, including:
should also be inspected visually for any abnormalities in
• A card with four columns containing reagent anti-A and
appearance, indicating contamination. If any abnormality
anti-B (two for recipient and two for donor)
is seen, the unit should not be issued unless specifically
• A card with six wells, two for recipient and donor samples,
authorized by the medical director.67
and four with dried anti-A and anti-B for testing of the re-
Before transfusion is initiated, a reliable professional
cipient and donor.79
(preferably two) must once again verify identity of the
patient and donor products. A system of positive patient Preparing for clinical care in space, National Aeronautics
identification by comparison of wristband identification and and Space Administration (NASA) scientists showed that
compatibility forms must be followed strictly. This is the ABO and Coombs-sensitized standard blood grouping tests
most critical check and yet the most fallible, because the can be performed under microgravity. This was done using
transfusion may take place in the operating suite or emer- a closed self-operating system that automatically performed
gency room where the person responsible for identification the tests and fixed the results onto filter paper for analysis
may be involved with many other duties as well. on Earth. Agglutinates were smaller than usual; however,
If a unit is returned to the blood bank for any reason reaction endpoints were clear.80 Although these researchers
within the specified time for that laboratory, it should noted that additional experiments in space were needed
not be reissued if the container closure was opened or to confirm and quantify their results, these preliminary
2682_Ch10_241-259 22/05/12 11:46 AM Page 256
SUMMARY CHART
Most fatal transfusion reactions are caused by clerical corresponding antigen on the donor RBCs, donor hav-
errors. ing a positive DAT, abnormalities in patient serum
Samples and forms must contain patient’s full name such as increased protein concentration (rouleaux) or
and unique identity number. contaminants in test system.
Writing must be legible and indelible. Emergencies:
Date of collection must be written on sample. • May have to select uncrossmatched, group O, Rh-
negative packed RBCs.
Sample must be collected within 3 days of scheduled
• May want to give uncrossmatched, group O, Rh-
transfusion.
positive packed RBCs if male patient or female
A blood bank specimen must have two unique patient patient is beyond childbearing years.
identifiers—the date of collection and initials or sig- • May be able to provide type-specific, uncross-
nature of the person who collected the sample. matched RBCs.
Confirm blood type of donor. Plasma products units:
Check patient records (history) for results of previous • No compatibility testing required.
tests. Perform ABO grouping, Rh typing, and antibody Transfusion to fetus:
screening on patient. • Compatibility testing performed using mother’s
Select donor unit based on ABO group and Rh type of sample.
patient; further consider presence of antibodies in pa- • Donor unit must lack antigen against maternal
tient by antigen-typing donor unit for the correspon- antibody.
ding antigen. • Group O Rh-negative donor selected when fetal
Perform immediate spin or antiglobulin crossmatch type is unknown or when type is known but is not
based on current or historical serologic results. compatible with mother’s type.
Electronic crossmatch can replace immediate spin Transfusion to infant:
crossmatch when two blood types are on file for the • Maternal sample can be used for compatibility testing.
patient and antibody screen is negative. • Initial sample from infant typed for ABO (front
Positive results in the crossmatch may be caused by type) and Rh.
incorrect ABO grouping of patient or donor, alloanti- • Donor unit selected should be compatible with
body or autoantibody in patient reacting with the both mother and baby.
2682_Ch10_241-259 22/05/12 11:46 AM Page 257
13. A crossmatch is positive at AHG phase with polyspecific 21. Mollison, PL: Blood Transfusion in Clinical Medicine, 9th ed.
AHG reagent but is negative with monospecific anti-IgG Blackwell Scientific, Oxford, England, 1993, p 225.
22. Roback, J (ed): Technical Manual, 16th ed. American Associa-
AHG reagent. This may indicate the antibody: tion of Blood Banks, Bethesda, MD, 2008, p 453.
a. Is a weak anti-D 23. Standards, op cit, p 35, 5.15.1.1.
b. Is a clinically insignificant Lewis antibody 24. Roback, J. (ed): Technical Manual, 16th ed. American Associ-
c. Can cause decreased survival of transfused RBCs ation of Blood Banks, Bethesda, MD, 2008, p 453
25. Alexander, D, and Henry, JB: Immediate spin crossmatch in
d. Is a Duffy antibody
routine use: A growing trend in compatibility testing for red
14. The emergency room requests 6 units of packed RBCs cell transfusion therapy. Vox Sang 70:48, 1996.
26. Walker, RH: On the safety of the abbreviated crossmatch.
for a trauma patient prior to collection of the patient’s In Polesky, HF, and Walker, RH (eds): Safety in Transfusion
specimen. The most appropriate course of action is to: Practices; CAP Conference, Aspen, 1980. College of American
a. Release group O RBCs to ER with trauma patient Pathologists, Skokie, IL, 1982, p 75.
identification on each unit sent 27. Shulman, IA, et al: Experience with the routine use of an
abbreviated crossmatch. Am J Clin Pathol 82:178, 1984.
b. Refuse to release units until you get a patient sample
28. Dodsworth, H, and Dudley, HAF: Increased efficiency of
c. Indicate necessity for signed patient waiver for transfusion practice in routine surgery using pre-operative
incomplete pretransfusion testing antibody screening and selective ordering with an abbreviated
d. Explain need of patient’s ABO group prior to issuing crossmatch. Br J Surg 72:102, 1985.
blood 29. Garratty, G: Abbreviated pretransfusion testing. Transfusion
26:217, 1986.
15. Which is not an example of the most common form of 30. Freidberg, RC, et al: Type and screen completion for scheduled
error associated with fatal transfusion reactions? surgical procedures. Arch Pathol Lab Med 127, 2003.
31. Judd, WJ: Are there better ways than the crossmatch to demon-
a. Phlebotomist labels patient A tubes with patient B strate ABO incompatibility? Transfusion 31:192, 1991.
information 32. Shulman, IA, and Calderon, C: Effect of delayed centrifuga-
b. Technologist enters results of patient A testing into tion or reading on the detection of ABO incompatibility by
patient B field the immediate-spin crossmatch. Transfusion 31:197, 1991.
33. Perkins, JT, et al: The relative utility of the autologous control
c. Wrong RBC unit is tagged for transfusion
and the antiglobulin test phase of the crossmatch. Transfusion
d. Antibody below detectable levels during pretransfusion 30:503, 1990.
testing 34. CFR, op cit, part 606, section 160.
35. Green, TS: Rouleaux and autoantibodies (or things that go
References bump in the night). In Treacy, M (ed): Pre-Transfusion Testing
for the 80s. American Association of Blood Banks, Washington,
1. Roback, J (ed): Technical Manual, 16th ed, American Association DC, 1980, p 93.
of Blood Banks, Bethesda, MD, 2008. 36. Bartholomew, JR, et al: A prospective study of the effects of
2. Linden, JV, et al: Transfusion error in New York State: An dextran administration on compatibility testing. Transfusion
analysis of 10 years experience. Transfusion 40:1207, 2000. 26:431, 1986.
3. Carson, TH: Standards for Blood Banks and Transfusion 37. Golde, DW, et al: Serum agglutinins to commercially pre-
Services, 26th ed. American Association of Blood Banks, pared albumin. In Weisz-Carrington, P: Principles of Clinical
Bethesda, MD, 2009, p 32, 5.11.2. Immunohematology. Year Book Medical, Chicago, 1986,
4. Ibid, p 3, 2.1.2 p 214.
5. Ibid, pp 32, 33, 5.11.2–4. 38. Judd, JW: Requirements for the electronic crossmatch. Vox
6. Ibid, p 33, 5.11.2.3, 5.11.3. Sang 74:409, 1998.
7. Ibid, p 34, 5.13.3.2. 39. Ibid.
8. Ibid, p 21, 5.6.3. 40. Butch, SH, et al: Electronic verification of donor-recipient com-
9. Ibid, p 33, 5.11.4. patibility: The computer crossmatch. Transfusion 34:105,
10. Code of Federal Regulations (CFR), Title 21, Food and Drugs. 1994.
Office of the Federal Register, National Archives and Records Serv- 41. Shulrman, IA, et al: North American Pretransfusion Testing
ice, General Services Administration, Part 610, section 40, revised Practices, 2001–2004. Results from the College of American
June 11, 2001, and Part 640, section 5 revised August 6, 2001. Pathologists Interlaboratory Comparison Program Survey Data,
11. Standards, op cit, pp 29–31, 5.8, and pp 33, 34, 5.13. 2001–2004, p 986.
12. Ibid, p 29, 5.8.3.1. 42. Judd, JW: Requirements for the electronic crossmatch. Vox
13. Ibid, p 33, 5.12. Sang 74:409, 1998.
14. Ibid, p 63, 6.2.5. 43. Ibid p 36, 5.15.2.
15. Ibid, p 34, 5.13.3.2 44. Standards, op cit p 36, 5.15.2.1–5.
16. Ibid, p 33, 5.13.2. 45. CFR, op cit, part 606, section 160.
17. Garratty, G: Evaluating the clinical significance of blood group 46. Standards, op cit, p 37, 5.16.
alloantibodies that are causing problems in pretransfusion 47. Ibid.
testing. Vox Sang 74:285, 1998. 48. Ibid.
18. Giblett, ER: Blood group alloantibodies: An assessment to some 49. Ibid.
laboratory practices. Transfusion 17:299, 1977. 50. Ibid, p 38, 5.17.5.
19. Spielmann, W, and Seidl, S: Prevalence of irregular red cell 51. Roback, J (ed): Technical Manual, 16th ed. American Associa-
antibodies and their significance in blood transfusion and tion of Blood Banks, Bethesda, MD, 2008, p 441.
antenatal care. Vox Sang 26:551, 1974. 52. Ibid.
20. Aygun B, et al: Clinical significance of RBC alloantibodies and 53. Standards, op cit, pp 30, 31, 5.8.5.
autoantibodies in sickle cell patients who received transfusion. 54. Ibid, p 45, 5.1.6A.
Transfusion 42:37, 2002. 55. Ibid, p 29, 5.8
2682_Ch10_241-259 22/05/12 11:46 AM Page 259
56. Garratty, G: Evaluating the clinical significance of blood group Pathologists Interlaboratory Comparison Program Survey Data,
alloantibodies that are causing problems in pretransfusion 2001–2004, p 986.
testing. Vox Sang 74:285, 1998. 72. Weisbach, V, et al: Comparison of the performance of four
57. Marcus CS, et al: Radiolabeled red cell viability II. 99mTc microtube column agglutination systems in the detection of
and 111In for measuring the viability of heterologous red cells red cell alloantibodies. Transfusion 39:1045, 1999.
in vivo. Transfusion 7:420, 1986. 73. Dittmar, K, et al: Comparison of DATs using traditional tube
58. Garratty, G: Evaluating the clinical significance of blood group agglutination to gel column and affinity column procedures.
alloantibodies that are causing problems in pretransfusion Transfusion 41:1258, 2001.
testing. Vox Sang 74:285, 1998. 74. Langston, JL, et al: Evaluation of the gel system for ABO group-
59. Shulman, IA, et al: Experience with a cost-effective crossmatch ing and D typing. Transfusion 39:300, 1999.
protocol. JAMA 254:93, 1985. 75. Anstee, DJ: Red cell genotyping and the future of pretransfu-
60. Davis, SP, et al: Maximizing the benefits of type and screen by sion testing. Blood 114(2): 2009.
continued surveillance of transfusion practice. Am J Med 76. Reid, ME: Overview of molecular methods in immunohema-
Technol 49:579, 1983. tology. Transfusion 47(suppl): 2007.
61. Issitt, PD: Applied Blood Group Serology, 4th ed. Montgomery 77. Klapper, E, et al: Toward extended phenotype matching: A new
Scientific, Durham, NC, 1998, pp 892, 893. operational paradigm for the transfusion service. Transfusion
62. Dzik, WH: Emily Cooley lecture 2002: Transfusion safety in 50: 2010.
the hospital. Transfusion, 43:181–190, 2003. 78. Hillyer, D, et al: Integrating molecular technologies for the red
63. Dzik, WH: New technology for transfusion safety. Br J Haematol, blood cell typing and compatibility testing into blood centers
136:181–190, 2006. and transfusion services. Transfus Med Rev 22(2): 2008, pp
64. Standards, op cit, pp 11, 12, 5.1.6.3. 117–132.
65. Ohsaka, A, et al: Causes of failure of a barcode-based pretrans- 79. Migeot, V, et al: Reliability of bedside ABO testing before trans-
fusion check at the bedside: Experience in a university hospital. fusion. Transfusion 42:1348, 2002.
Transfusion Medicine, 18:216–222, 2008. 80. Morehead, RT, et al: Erythrocyte agglutination in microgravity.
66. Ibid, pp 39, 40, 5.18. Aviat Space Environ Med 60:235, 1989.
67. Ibid. 81. Stussi, G, et al: Isotype-specific detection of ABO blood group
68. Ibid. antibodies using a novel flow cytometric method. Br J Haematol,
69. Sandler, SG: A fully automated blood typing system for hospital 130:954–963, 2005.
transfusion services. ABS2000 Study Group. Transfusion 82. Roback, JD, et al: An automatable format for accurate immuno-
40:201, 2000. hematology testing by flow cytometry. Transfusion 43:918,
70. Morelati, R, et al: Evaluation of a new automated instrument 2003.
for pretransfusion testing. Transfusion 38:959, 1998. 83. Dzik, WH: New technology for transfusion safety. Br J Haematol,
71. Shulrman, IA, et al: North American Pretransfusion Testing 136:181–190, 2006.
Practices, 2001–2004. Results from the College of American
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Chapter 11
Overview of the Routine Blood Bank
Laboratory
Darlene M. Homkes, MT(ASCP); Cara L. Calvo, MS, MT(ASCP)SH, CLS(NCA);
and Burlin Sherrick, MT(ASCP)SBB
OBJECTIVES
1. Describe a modern blood bank laboratory in terms of its operations, personnel, facilities, and equipment.
2. Explain the purpose of the following equipment used in a blood bank laboratory: apheresis machine, flatbed agitator, sterile
docking device, and leukoreduction filter.
3. Summarize blood bank laboratory services in terms of policies, procedures, and tests performed.
4. Compare and contrast testing performed on donor units and recipient blood samples.
5. Apply knowledge of immunohematology theory and skills developed in classroom practice to actual work situations in a blood
bank laboratory.
personnel. However, the blood bank laboratory also functions Table 11–1 Blood Bank Areas and
as a component of the heavily regulated health-care industry. Functions
This means that the blood bank laboratory also operates under
constraint of laws administered by various governmental AREA FUNCTIONS
agencies and in accordance with standards established by Component preparation • Separation of whole blood into
nonregulatory accrediting organizations such as the AABB.3,4 and storage packed RBCs, plasma, platelets, and
(Refer to Chapter 25, “Transfusion Safety and Federal Regula- cryoprecipitate
tory Requirements” for additional information.)
• Storage of blood products at
Blood bank laboratories are located in a variety of settings, appropriate temperatures
and the extent of services that a blood bank offers varies accord-
ingly. For example, hospital blood banks may choose to collect • Apheresis procedures
and process blood components for their own use, or they may Donor processing • Donor units tested for:
choose to contract to receive blood products from a blood
center. For ease of discussion, it is assumed that the blood bank – ABO and Rh
is divided into distinct areas, each with its own purpose and – Antibody screen
function: component preparation and storage, donor process-
ing, product labeling, main laboratory, and reference laboratory – Serologic test for syphilis
areas (Table 11–1). With the exception of very large transfusion – Transfusion-transmitted viruses
services, these areas are not physically separated but overlap
within the overall structure of the blood bank laboratory. Product labeling • RBCs and any other components are
labeled
Component Preparation and Storage • Products are stored at their proper
temperatures
Depending on the needs of a hospital, blood components
may be acquired from external sources such as the American Main laboratory • Patient samples tested for:
Red Cross or other regional blood centers, or components
– ABO and Rh
may be processed in-house.
– Antibody screen
Blood Banks With Collection Facilities
– Crossmatch
Blood banks that collect their own units of whole blood can
use their blood resources more efficiently by separating them – DAT
into a variety of components, including packed red blood
– Prenatal evaluation
cells (RBCs), platelets, fresh frozen plasma (FFP), frozen
plasma prepared within 24 hours of collection (FP24), and – Postpartum evaluation
cryoprecipitate (Table 11–2; see also see Chapter 13, “Donor
– Cord blood studies
Screening and Component Preparation”).
After collection, whole blood is allowed to cool to room • Issue blood products
temperature (20°C to 24°C) if platelets are to be prepared
Reference laboratory • Resolution of:
from the unit, or cooled to 1°C to 10°C if the unit is to be
processed for frozen plasma. FFP must be prepared within – ABO and Rh discrepancies
8 hours of collection of the whole blood unit, whereas FP24
– Antibody identification
may be prepared up to 24 hours following collection of the
whole blood unit. Platelets may be prepared from whole – Positive DAT
blood stored at room temperature for up to 24 hours after
– Warm autoantibodies
the whole blood unit is collected.4
The blood is centrifuged to pack the red cells using large, – Cold autoantibodies
floor-model temperature-controlled centrifuges, and the
– Transfusion reactions
components (packed cells, plasma, platelets) are separated
(Fig. 11–1). Additionally, through a controlled thawing
process, the frozen plasma can be further manipulated to
yield cryoprecipitate. Blood Banks Without Collection Facilities
Some blood components may also be prepared through a Blood banks that depend on an outside source for their blood
procedure known as apheresis. A donor’s blood is removed, an- supplies usually receive their products in component form.
ticoagulated, and transported directly to an apheresis machine. However, situations do arise in which products must be
The blood is separated into specific components (platelets, modified (Fig. 11–2). For example, a patient who is deficient
plasma, granulocytes, red blood cells) by centrifugation within in IgA may require washed RBCs. Automated cell washers
the machine, and the desired component(s) is removed. The are used to prepare this product. A unit of blood is introduced
remaining portion of the blood is returned to the donor. into a sterile disposable bowl that has tubing connected to a
(Apheresis is described in detail in Chapter 14, “Apheresis.”) normal saline solution. A portion of this saline is added
2682_Ch11_260-272 22/05/12 11:46 AM Page 262
Product Labeling
Figure 11–1. Processing of whole blood units from collection to labeling. Figure 11–3. Automated cell washers such as this may be used to prepare
Note that component preparation and donor processing may occur concurrently. washed RBCs or to deglycerolize frozen RBCs. (Courtesy National Institutes of
FP24 = frozen plasma prepared within 24 hours of collection. Health, Bethesda, MD.)
2682_Ch11_260-272 22/05/12 11:46 AM Page 263
Figure 11–5. Platelet rotators prevent the formation of platelet aggregates and
optimize the exchange of gases required for platelet survival.
BOX 11–1
purpose. Box 11–2 lists the required tests that are performed
on blood after donation. Blood processing centers utilize
the testing methods that will provide the safest blood prod-
uct for patients. For instance, nucleic acid amplification
testing (NAT) is being used to detect HIV-1 and HCV, and
this technology is under investigation for detecting other
infectious disease agents.1
Automation has greatly increased the efficiency and pro-
ductivity of this section of the blood bank. Description of
these tests and of the automation is provided in Chapter 13.
Product Labeling
Prenatal Evaluation. Accurate serologic testing of obstetric Requests for Other Blood Components
patients is an essential component in the prevention and When components containing large amounts of RBCs
treatment of hemolytic disease of the fetus and newborn (e.g., granulocyte concentrates) are requested, pretransfusion
(HDFN). Maternal blood samples are evaluated during preg- testing is identical to that performed for RBC requests. There
nancy to determine the ABO group and Rh type of the are no such requirements for platelets or FFP.
mother and the presence of serum antibodies that can
potentially cause HDFN. If the woman is classified as Rh-D Issue of Blood Products
negative, she may be a candidate for antenatal Rh-immune After all pretransfusion testing has been completed, blood
globulin unless her serum contains actively acquired anti-D. components may be released for transfusion to the desig-
Weak D testing is not required as part of a prenatal evalua- nated recipient. It is essential that all serologic discrepancies
tion. It is not possible to differentiate weak D from partial D be resolved before issuing blood products, except in extreme
serologically. Since patients who are partial-D positive may emergency. The individual who obtains the blood product
still develop anti-D, many hospital blood banks consider all from the blood bank for delivery to the nursing unit is
prenatal patients who test D-negative initially as “negative” usually required to present a written request for the blood
without further testing. (See Chapter 7, “The Rh Blood product containing a minimum of two patient identifiers.
Group System.”) The individual in the blood bank who will issue the blood
If the result of the antibody screen is positive, the antibody product inspects the unit for any abnormal appearance
must be identified. Serial titrations may be performed during (Box 11–3) and verifies that all required transfusion forms
the course of the pregnancy if the antibody is considered and labels are complete and that they adequately identify the
potentially harmful to the fetus. The obstetrician uses these transfusion recipient. If another individual is responsible for
laboratory results together with other methods for evaluating delivering the blood product to the appropriate location, he
the fetal condition and the need for clinical intervention (see or she may also verify that all information is complete. A
Chapter 19, “Hemolytic Disease of the Fetus and Newborn”). computer crossmatch may also be performed at this time,
and if there are no discrepancies, the component can be
Postpartum Evaluation. All women admitted for delivery are
released for transfusion. Some form of documentation is
required to be tested to determine their Rh status. Weak-D used to record the transaction.
testing is not required. If the mother is Rh-negative and her Some component preparation or modification may be
baby is Rh-positive, the maternal sample is further evaluated necessary before the issue of blood products. FFP may be
to detect a feto-maternal hemorrhage (FMH) in excess of thawed in a constant-temperature (30°C to 37°C) water bath,
30 mL of whole blood. (One 300-µg dose of RhIg prevents individual platelet concentrates may be pooled into a single
maternal Rh immunization from exposure of up to 30 mL of bag for ease of transfusion, and a packed RBC may need to
fetal whole blood.4) Commercial kits utilizing rosetting tech- be irradiated (Fig. 11–9). These modifications may shorten
niques are commonly used for this purpose. Once an FMH the expiration date of the products and must be reflected on
in excess of 30 mL of whole blood has been detected, quan- the unit itself and in the computer system.
tification is performed using a Kleihauer-Betke test, flow
cytometry, or enzyme-linked antiglobulin test. This may be Reference Laboratory
performed in the blood bank or in a separate laboratory. If
HDFN is suspected, an antibody screen is performed on the Whether it is an entity separate from the main laboratory or,
mother’s serum, and if the result is positive, attempts are as in most cases, an integrated part, the reference laboratory
made to identify the antibody. is the problem-solving section of the transfusion service. The
reference laboratory’s goal is to ensure that any discrepancies
Cord Blood Studies. Protocols to evaluate cord blood speci- detected in routine testing are resolved in an accurate and
mens can vary widely from blood bank to blood bank. Cord time-efficient manner. Other chapters in this book discuss
blood from infants born to Rh-negative mothers is tested in depth the various problems encountered in serologic testing
for D and for weak D to determine the mother’s candidacy and the strategies for resolving them. Here is a brief summary
for RhIg prophylaxis. ABO and Rh typing and direct
antiglobulin testing (DAT) are performed on cord samples
from infants born to women with clinically significant BOX 11–3
antibodies. Additional testing may also be performed. Many
blood banks follow published guidelines stating that Reasons to Quarantine Blood Components Before
beyond these circumstances, routine testing of cord blood Shipment or Transfusion
is not necessary unless the clinical situation warrants it. If • Plasma of RBC unit is brown, red, murky, or purple
an infant develops symptoms that suggest HDFN, a full • Zone of hemolysis is above RBC mass
cord blood study is performed that may include ABO and • Inadequate sealing of RBC segments in tubing
Rh typing (including a test for weak D) and DAT. If the • Hemolysis of RBCs
DAT result is positive, an eluate and subsequent antibody • Grossly lipemic units
identification are performed. Note that only forward testing • Unusual cloudy or turbid appearance of platelet unit
is performed in the ABO test.
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Antibody Identification
A positive antibody screen in the absence of a positive auto-
control or DAT result indicates possible alloantibody immu-
nization by means of blood transfusion or pregnancy (see
Chapter 9, “Detection and Identification of Antibodies”). To
establish the identity and clinical significance of an antibody
and to provide appropriate blood for transfusion, antibody
investigation may include:
• Antibody identification panels using various enhancement
media (albumin, low ionic strength solution, polyethylene
glycol) and test systems (such as tubes, gel, or solid phase
technologies)
• Antibody identification panels pretreated with reagents such
as enzymes, dithiothreitol (DTT), or 2-aminoethyliso-
thiouronium bromide
• Neutralization using such substances as plasma, saliva,
urine, or human milk
• Antigen typing
• Titration
• Adsorption and elution studies
• Treatment of serum with DTT or 2-mercaptoethanol
• IgG subclassing
• Monocyte monolayer assay
• Use of rare sera and cells
Figure 11–9. Irradiators are used to prevent lymphocytes in RBC products from
causing graft-versus-host disease in susceptible patient populations. (Courtesy Positive DAT
National Institutes of Health, Bethesda, MD.) A positive DAT may be the result of an immune reaction to
a drug, a disease state, or a delayed hemolytic transfusion
of some situations and common methods used in their reaction (see Chapter 16, “Adverse Effects of Blood Transfu-
investigation. It should be noted that before any serologic sion”). The investigation of a positive DAT may include:
testing is performed, special testing should always include • Use of monospecific reagents (anti-IgG, anti-C3)
an investigation of patient’s diagnosis, age, pregnancy, drug, • Elution techniques
and transfusion history. • Antibody identification
ABO Discrepancies • Removal of cell-bound antibody using chloroquine
diphosphate
All inconsistencies between forward and reverse grouping • RBC phenotyping
must be resolved before an ABO interpretation can be made • Drug studies
(see Chapter 6, “The ABO Blood Group System”). ABO inves- • Cell separation techniques
tigations may include:
• Variations in incubation times and temperatures Warm Autoantibodies
• Testing with A2 cells or anti-A1 lectin In addition to causing a positive DAT, the presence of
• Room temperature antibody identification a warm autoantibody in a patient’s serum may mask
• Adsorption and elution using human sources of anti-A or the presence of clinically significant alloantibodies (see
anti-B Chapter 20, “Autoimmune Hemolytic Anemias”). Tests
• Autoadsorption performed in the investigation of warm autoantibodies
• Removal of RBC-bound cold autoantibodies may include:
• Secretor studies • Removal of RBC-bound autoantibody followed by serum
Rh Discrepancies autoadsorption
• Heterologous or differential serum adsorptions
Problems in Rh typing may occur because of certain clinical
• Elution techniques
conditions or inherited characteristics (see Chapter 7). Rh
• Autoantibody identification
investigations may include:
• Reticulocyte enrichment or other cell separation techniques
• Rh phenotyping
• Adsorption and elution using Rh antisera Cold Autoantibodies
• Isolation of cell populations Potent cold autoantibodies may cause discrepancies in ABO
• Use of rare antisera and RBCs testing and a positive DAT result and may mask the presence
2682_Ch11_260-272 22/05/12 11:46 AM Page 268
Transfusion Reactions
Any adverse reactions to transfusion must be investigated
to determine if the reaction is antibody-mediated (see
Chapter 16). The extent of transfusion reaction investigations
varies widely, depending on the policies established by a Figure 11–10. A gel card is loaded into a centrifuge. The gel test employs the
particular blood bank and the result of initial testing. At a principle of controlled centrifugation of RBCs through a dextran-acrylamide
minimum, an investigation of a suspected hemolytic reaction gel/reagent matrix contained in a specially designed microtube.
to transfusion should include an ABO, Rh, DAT, and visual
checkfor hemolysis using a post-transfusion sample.3 If there
is strong evidence of an antibody-mediated transfusion procedures outlining the training program for new hires in
reaction, further investigation may include: their institution. In all cases, the blood bank laboratory must
have a qualified director to manage the personnel and the
• Elution followed by antibody identification operations. However, the director may delegate to a qualified
• Use of more sensitive techniques for antibody detection supervisor the day-to-day work-related activities that include
in serum and eluates, including enzymes, polybrene, poly- managing personnel and reporting test results.
ethylene glycol, or enzyme-linked antiglobulin test
• Cell separation techniques Standard Operating Procedures
Figure 11–11. Sample Blood Administration Observation Form. This form is used to document direct observations of transfusions. Audits such as this are performed to
verify compliance with transfusion process regulations and policies.
2682_Ch11_260-272 22/05/12 11:46 AM Page 270
things as presence of a signed patient consent form, a properly The blood bank supervisor will usually prepare a monthly
documented order to transfuse, proper patient identification, or quarterly blood bank report listing such things as the
a correct patient and unit verification process was followed, number of physicians whose crossmatch versus transfusion
and other process items as determined by hospital policy. (C:T) ratio exceeds the hospital’s target, the number of trans-
A report should be prepared from this audit data, usually fusion reaction investigations, patients transfused who may
quarterly, and this report should be presented to the blood not meet hospital transfusion triggers, or any other parameter
bank medical director and the hospital patient safety or risk the blood bank medical director deems necessary to monitor
management team for evaluation (Fig. 11–12). See Chapter (Fig. 11–13). This report is given to the blood bank medical
23 for more information. director and often a medical review committee for evaluation.
YEAR
Quarter # # Failure description #
Audits Failures Transfusions
1
2
3
4
Figure 11–12. Sample Blood Audit Summary Form. This form is used to summarize the data collected from transfusion process observations over a period of time,
most often quarterly. This form is then presented for review by the medical director or transfusion oversight committee.
2682_Ch11_260-272 22/05/12 11:46 AM Page 271
Figure 11–13. Sample Blood Utilization Report. This report helps the blood bank monitor effective use of blood products, including the number of physicians whose
C:T ratio exceeds the hospital standard, and possible inappropriate transfusions. This report is typically reviewed by the blood bank medical director and a medical peer
review committee.
SUMMARY CHART
The goal of every blood bank laboratory is to provide Blood and blood components must be stored and
quality service and safe blood products. shipped under conditions that ensure their viability.
All blood bank laboratory operations are regulated Every donor unit is tested to determine ABO group and
by law. Rh type, including weak D when indicated. Units are
Personnel employed by blood bank laboratories must also tested for selected viral markers.
be qualified by education, training, or experience. The type of Rh-negative units must be confirmed because
A primary source of operational information is the SOP of potential sensitization that may occur if Rh-positive
manual. blood is transfused to an Rh-negative recipient.
Hemapheresis is a process that uses an apheresis Manual tube procedures are being replaced by emerg-
machine to selectively remove components from donor ing technologies, including gel-based methods and
blood. automation.
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Chapter 12
Other Technologies and Automation
Phyllis S. Walker, MS, MT(ASCP)SBB and Denise M. Harmening,
PhD, MT(ASCP)
OBJECTIVES
1. Explain the principles of gel, solid-phase red cell adherence (SPRCA), protein A, and enzyme-linked immunosorbent assay
(ELISA) technology.
2. Describe the test reactions and how the results are interpreted for each technology.
3. List the advantages and disadvantages of each technology.
4. Describe the automated equipment that is available for each technology.
5. Compare the gel and SPRCA technologies in terms of equipment, test reactions, procedures, sensitivity and specificity, and
quality control.
Introduction Blood banks and transfusion services are the last areas
of the clinical laboratory to move to automation. Chem-
Previous chapters have discussed ABO and Rh typing, direct istry, hematology, and immunology have been using
antiglobulin testing (DAT), and antibody detection and automation for many years, but blood services have been
identification procedures based on routine test tube testing hampered by the complexity of the testing and the subjec-
techniques. In response to the pressures of current good tivity of the test interpretation. In recent years, new pres-
manufacturing practices, two other technologies, the gel test sure has been applied to this area of the laboratory.
and solid-phase assays, have emerged to provide accurate, Personnel shortages, turnaround time requirements, and
reproducible blood bank testing. These technologies provide the need for cost containment because of increased
the increased safety offered by plasticware and reduce bio- managed care and greater regulatory demands have pro-
hazardous waste. In addition, these technologies have been vided the incentive for blood services to seek automation.
automated, decreasing the opportunities for human error By using walk-away automation, laboratory personnel
and freeing laboratory personnel to perform other tasks. Sur- are able to perform multiple tasks simultaneously. Auto-
veys indicate that there is an ongoing trend toward adopting mated equipment provides the level of quality assurance
these other technologies in clinical laboratories.1,2 This required by current regulatory standards. Bar-coding reduces
chapter presents the history, applications, basic principles, identification errors by providing accurate patient and
and advantages and disadvantages of each technology, and reagent identification, and standardized techniques reduce
describes currently available automated equipment. testing errors.
273
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Gel Technology
In 1985, Dr. Yves Lapierre of Lyon, France, developed the
gel test.3 Using various media, including gelatin, acrylamide
gel, and glass beads, Dr. Lapierre investigated ways to trap
Gel & Reagent
red blood cell (RBC) agglutinates during standardized sedi-
mentation or centrifugation. Gel particles were found to
be the ideal material for trapping the agglutinates, and this Figure 12–1. Gel microtube. Microtubes filled with gel containing anti-IgG are
discovery led to a patented process for separating aggluti- used for compatibility testing, antibody detection, and identification.
nated red blood cells. In addition, it was discovered that
antiglobulin testing could be performed without multiple
saline washes to remove unbound immunoglobulin and that through a dextran-acrylamide gel that contains predispensed
antiglobulin control cells were not needed to confirm the reagents. Each microtube is composed of an upper reaction
presence of antiglobulin reagent in negative tests. Compared chamber that is wider than the tube itself and a long, narrow
with traditional tube technology, the gel test provides a more portion referred to as the column (Fig. 12–1). In the gel
stable endpoint and more reproducible results. This improve- test, a plastic card with microtubes is used instead of
ment reduces the variability associated with the physical test tubes (Fig. 12–2). A gel card measures approximately
resuspension of RBC buttons after centrifugation and the 5 × 7 centimeters and consists of six microtubes. Each
subsequent interpretation of hemagglutination reactions. microtube contains predispensed gel, diluent, and reagents,
if applicable. Measured volumes of serum or plasma or
History RBCs are dispensed into the microtube’s reaction chamber
(Fig. 12–3). If appropriate, the card is incubated (Fig. 12–4)
In 1988, Dr. Lapierre worked with DiaMed A.G. to develop and and then centrifuged (Fig. 12–5).
produce the gel test in Europe. In September 1994, the FDA The reaction chamber is where RBCs may be sensitized
granted Micro Typing Systems (MTS) a license to manufacture (antigen-antibody binding) during incubation. The column
and distribute an antiglobulin anti-IgG gel card and a buffered of each microtube contains dextran-acrylamide gel particles
gel card in the United States. In January 1995, Ortho Diag- suspended in a diluent with reagents added, if applicable.
nostic Systems Inc. (ODSI) and MTS signed an agreement The shape and length of the column provides a large surface
giving ODSI exclusive rights to distribute the gel test in North area for prolonged contact of the RBCs with the gel particles
America. In March 2002, Ortho Clinical Diagnostics fully during centrifugation.
acquired MTS and became known as Micro Typing Systems.
This gel-based test is named the ID-Micro Typing System.4
Applications
In the United States, the FDA has approved gel technology for
ABO forward and reverse grouping, Rh typing, direct antiglob-
ulin testing, antibody screening, identifying antibodies, and
compatibility testing. The ABO blood grouping card has gels
that contain anti-A, anti-B, and anti-A,B for forward grouping.
Microtubes with buffered gel are used for ABO reverse group-
ing.5 The Rh typing card uses microtubes filled with gel con-
taining anti-D.6 The Rh phenotype card has gels that contain
anti-D, anti-C, anti-E, anti-c, anti-e, and a control.7 Microtubes
filled with gel containing anti-IgG are used for compatibility
testing, antibody detection, and identification.8
Principle
The gel test, which is performed in a specially designed Figure 12–2. The gel card. Each measures approximately 5 x 7 centimeters and
microtube, is based on the controlled centrifugation of RBCs consists of six microtubes (instead of test tubes).
2682_Ch12_273-288 22/05/12 11:47 AM Page 275
Figure 12–7. Gel technology reaction grading chart. (Courtesy of Ortho Clinical Diagnostics, Raritan, New Jersey.)
Advantages and Disadvantages These factors combine to produce more objective, consistent,
and reproducible interpretation of the test results.
Gel technology, which is applicable to a broad range of blood Because the gel technology offers objective, consistent
bank tests, offers several advantages over routine tube test- results, it is ideally suited to individuals who have been
ing.9 Standardization is one of the major advantages, since cross-trained to work in the blood bank. Other advantages
there is no tube shaking to resuspend the RBC button. Tube- include the decreased sample volume needed for testing and
shaking technique varies among technologists, which results the enhanced sensitivity and specificity of the results. Finally,
in variations in reading, grading, and interpreting the test. gel technology offers improved productivity when compared
The gel technique provides stable, well-defined endpoints of with traditional tube testing.10
the agglutination reaction that can be observed or reviewed The major disadvantages of the gel technology are the
for up to 3 days. It includes simple standardized procedures, sample restrictions and the need for special equipment.
no wash step, and no need for antiglobulin control cells. Hemolyzed or grossly icteric blood samples should not be
2682_Ch12_273-288 22/05/12 11:47 AM Page 277
used, because the results may be difficult to interpret visually solid-phase serologic assays. In 1978, Rosenfield and cowork-
in the ID Micro Typing System.5 Grossly lipemic samples ers13 were the first to apply the principle of solid-phase
containing particulates that clog the gel, as indicated by dif- immunoassay to RBC typing and antibody screening tests.
fuse blotches of RBCs, must be clarified by centrifugation Solid-phase technology relevant to serologic testing in
or filtration and retested. Rouleaux caused by serum or blood centers and transfusion services include solid-phase
plasma with abnormally high concentrations of protein red cell adherence (SPRCA), solid-phase protein A, and
(such as in patients with multiple myeloma or Waldenström’s solid-phase enzyme-linked immunosorbent assay (ELISA).
macroglobulinemia or from patients who have received
plasma expanders of high molecular weight) may produce Solid-Phase Red Cell Adherence
hazy reactions or false-positive results.5 Special incubators
and centrifuges are needed to accommodate the microtube History
cards, and a specific pipette must be used to measure and In 1984, Plapp and coworkers reported using solid-phase
dispense the 25 µL of plasma or serum and the 50 µL of 0.8% red cell adherence (SPRCA) for detecting RBC antigens
RBCs into the reaction chambers of the microtubes. Finally, and antibodies.14,15 The first SPRCA assays to be devel-
the calibration on the special pipette must be checked on a oped commercially were manufactured by Immucor under
regular schedule.11,12 the trade name of Capture for the detection of RBC and
platelet antibodies. Currently, Capture immunoassays are
Automation available to detect antibodies to RBCs, platelets, and
cytomegalovirus.16
Automated equipment for serologic testing using the gel test
is being performed successfully for ABO and Rh testing, Applications
antibody screening, compatibility testing, and antibody SPRCA assays are either first- or second-generation tests. In
identification in both the United States and Europe. The first-generation tests, the user adds the target antigen
ORTHO ProVue™ Analyzer (Fig. 12–8) is a walk-away (RBCs, platelets, or platelet proteins, etc.) to the microplate
instrument with a capacity for testing 48 samples and wells before starting the test. In second-generation tests, the
16 reagents. Instrument safety features include a bar-code manufacturer binds the target antigen (RBC, platelet glyco-
tracking system and three cameras that record sample, proteins, or CMV) to the microplate test wells during the
reagent, and card identification. A camera in the instrument manufacturing process.
performs image analysis and uses a mathematical algorithm First- and second-generation SPRCA assays are currently
to interpret the results. approved by the FDA for antibody screening, antibody iden-
tification, weak-D testing, IgG autologous control, and com-
Solid-Phase Technology patibility testing. Second-generation assays use microwells
that are preloaded with antibody screening cells, in either a
Solid-phase immunoassays have been used for many years in
set of two-cells (I and II), a set of three cells, a set of four
immunology and chemistry laboratories. In these test systems
cells, or a pool of two cells. The two- and four-cell sets are
(immunoassays), one of the test reactants (either antigen or
recommended for antibody detection in transfusion recipi-
antibody) is bound to a solid support (usually a microtiter
ents. Pooled cells are used for donor antibody detection
well) before the test is started. The ability of plastics, such as
when increased sensitivity is undesirable. Reagent panels of
polystyrene, to absorb proteins from solution and to bind
preloaded cells are also available for RBC antibody identifi-
them irreversibly makes plastic microplate wells ideal for
cation. When it is desirable to test selected cells, intact
reagent RBCs are added by the user to chemically modified
microwells (i.e., a first-generation test).
Principle
SPRCA assays may be performed with either plasma or
serum, but plasma is preferable. If a clotted sample is incom-
pletely clotted, the serum is difficult to remove during the
wash cycle, which allows residual unbound serum to clot and
make the endpoint of the test unreadable. For best results,
the manufacturer recommends adding a pH-stabilizing buffer
to the isotonic saline that is used to wash the microplates. A
suitable buffer is available from the manufacturer.17
Capture technology offers both first- and second-generation
SPRCA tests, which are performed in microplate wells—in
either full 96-well U-bottomed plates or in 1 × 8 or 2 × 8 U-
bottomed strips.18 To perform second-generation tests, patient
Figure 12–8. ORTHO ProVue™ Analyzer. (Courtesy of Ortho Clinical Diagnostics, serum or plasma and low ionic strength saline (LISS) are added
Raritan, New Jersey.) to microwells that are coated with the target antigen, and the
2682_Ch12_273-288 22/05/12 11:47 AM Page 278
wells are incubated at 37°C, to allow time for possible antibod- Select, which identifies RBC antibodies, and Capture-P,
ies to attach to the antigens in the well. After incubation, the which detects platelet antibodies, are examples of first-
wells are washed with pH-buffered isotonic saline to remove generation solid-phase tests, whereas Capture-R Ready-
unbound serum proteins. Next, anti-IgG-coated indicator Screen, Capture-R Ready-ID, and Capture-P Ready Screen
RBCs are added, and the microwells are centrifuged. Centrifu- are second-generation tests.18
gation forces the indicator RBCs into close contact with IgG The laboratory equipment that is needed to perform
antibodies from the test serum or plasma that are bound to the SPRCA testing includes a microplate centrifuge capable
immobilized target antigen (i.e., the sandwich technique). If of holding 96-well microplates or strip wells, a microplate
antibody is attached to the antigen during the incubation incubator, and a microplate washer. Also, an illuminated
phase, the indicator cells form a monolayer of RBCs. If no surface for reading manual microplates is very desirable.
antibody is present, nothing is attached to the antigen, and the (Fig. 12–11).
indicator cells form a clearly delineated button at the center of
the microplate well during centrifugation (Fig. 12–9). Advantages and Disadvantages
Figure 12–10 compares test results from solid-phase Standardization is the major advantage of SPRCA technology.
technology with traditional tube testing reactions. Positive SPRCA provides stable, well-defined endpoints of the reaction.
tests show adherence of indicator RBCs to part or all of the Objective, consistent, reproducible test results facilitate tech-
well bottom, depending on the reaction’s strength. Capture-R nologist training or cross-training. SPRCA is convenient
4⫹
Incubate
YY
Y
Y
YY Y
Y
YY
YYY
Y
Y
Y Y Y Y
Y
Y
Y
Y
YY Y Y Y
YYYY
3⫹
Wash
YY
YYY
Y
Y
YY
Y
Y
YY Y Y Y
YYYY
2⫹
Centrifuge
Side view 1⫹
Top view
Automation
Full automation of the solid-phase technology is available from
Immucor using an FDA-cleared, walk-away instrument called
Galileo Echo™19 (Fig. 12–12). Echo™ is the third-generation
instrument for automating the Capture solid-phase technology.
Echo™ can be continuously loaded and unloaded and can
hold 20 samples, 16 reagents, and 32 microstrips. The tests
that are available on Echo™ include ABO/Rh type, donor con-
firmation–ABO retype, weak D, RBC phenotype, antibody
screen (three-cell), antibody identification (three panels),
DAT (IgG only), and an IgG crossmatch. Echo™ provides
multiple quality control features, including bar-code readers
to ensure proper identification of samples and reagents, liq-
uid level sensors, clot detectors, controlled temperature set-
tings, and incubation timers. A fourth-generation instrument
called Galileo Neo is currently awaiting FDA clearance.20
This instrument will allow 224 samples to be loaded at once.
History
In 1992, Biotest AG developed a test in Europe for detecting
IgG antibodies, using microplate wells that are coated with
protein A. Protein A is a component of the cell wall of
Staphylococcus aureus that has a very high affinity for the FC
portion of most immunoglobulin classes.21 In 2005, the FDA
approved the distribution of the Solidscreen II assay in the
United States, and in 2010, Bio-Rad Laboratories acquired
Figure 12–10. Comparison of traditional tube test reactions with solid-phase
this test from Biotest AG. Solidscreen II and the automated
reactions.
equipment for performing the assay, TANGO™ optimo, are
because no predilution of reagents is required. It is possible currently available from Bio-Rad Laboratories.
to test hemolyzed, lipemic, or icteric samples, and the enhanced Applications
sensitivity makes detecting weak alloantibodies easier. Also,
the Immucor Capture technology includes a LISS reagent that Solidscreen II is an assay that uses traditional antiglobulin tech-
changes color when serum or plasma is added; this safety nique to detect and identify red cell antibodies, perform com-
feature ensures that the patient sample is added to the test patibility tests, detect IgG on patients’ red blood cells, and type
system when manual testing is performed. red cells for weak D and partial D antigens (DVI and DVII).22
The major disadvantage of SPRCA is the need for special-
ized equipment—a microplate centrifuge, a 37°C incubator
for microplates, and a light source for reading the final
results. In addition, the increased sensitivity may also be a
Figure 12–11. Equipment for manual SPRCA technology. (Courtesy of Immucor, Figure 12–12. Galileo Echo™ instrument. (Courtesy of Immucor, Norcross,
Norcross, Georgia.) Georgia.)
2682_Ch12_273-288 22/05/12 11:47 AM Page 280
Applications
The solid-phase ELISA assay is used by Gen-Probe GTi
Diagnostics in two product lines, MACE® (MACE1 and
Figure 12–13. TANGO™ optimo instrument. (Courtesy of Bio-Rad Laboratories, MACE2) and PAK® (PAK1, PAK2-LE, PAK12, PAK12G, and
Hercules, California.) PAKPLUS®), to screen and identify platelet antibodies.25
2682_Ch12_273-288 22/05/12 11:47 AM Page 281
Y Y Y YYY Y Y Y Y
Incubate at 37°C
Y Y Y Y
Y
Y Y
Y
Y
YY Y
Y
YY
YY
YYY Y
YY
YY
Y Y YY YY
YY Y YY
Figure 12–14. Solidscreen II test procedure. (Courtesy of Bio-Rad Laboratories, Hercules, California.)
The MACE® products provide well-characterized mono- serum or plasma is incubated with intact platelets to allow
clonal antibodies immobilized in microwells that are used antibody, if present, to bind to the platelet glycoproteins.
to capture glycoproteins from platelets supplied by the Unbound antibodies are washed from the platelets, and the
user.26 The MACE® products allow a user to detect and antibody-sensitized platelets are solubilized by the addition
differentiate between antibodies that bind to platelet-specific of a lysis buffer that contains a nonionic detergent. Next,
glycoproteins (IIb/IIIa, Ib/IX, Ia/IIa, and IV) and class I HLA. the platelet lysate containing soluble glycoproteins is
The MACE® kits provide a test that can be used for either transferred to the microwells. This allows the platelet
antibody identification, if previously typed platelets are used, glycoproteins (sensitized or unsensitized with patient
or a platelet crossmatch, if potential donor platelets are used. antibody) to be captured by immobilized monoclonal
The PAK® products provide well-characterized platelet antibodies. Control samples are handled similarly. After a
glycoproteins that are either immobilized directly or cap- brief incubation, unbound glycoproteins are washed away.
tured by monoclonal antibodies to wells of a microwell An alkaline phosphatase labeled AHG reagent (anti-IgG)
plate.27 The available formats allow a user to detect and dif- is added to the wells and incubated. The unbound anti-IgG
ferentiate between antibodies that bind to platelet-specific gly- is washed away, and the substrate PNPP (p-nitrophenyl
coproteins (IIb/IIIa, Ib/IX, Ia/IIa, and IV) and class I HLA. phosphate) is added.
PAKAUTO® is designed to detect platelet autoantibodies Next, a 30-minute incubation period allows the conju-
eluted from a patient’s platelets using elution methods gated anti-IgG to produce a color change in the substrate.
already well established in the blood bank. An elution is first After the incubation period, the reaction is stopped by a
performed on washed patient platelets. The eluate and a sodium hydroxide solution. The optical density of the color
serum sample are tested side by side against the platelet gly- that develops is measured in a spectrophotometer at 405 or
coproteins Ib/IX, Ia/IIa, and IIb/IIIa, which are individually 410 nm using a reference filter of 490 nm. A positive result
presented in different wells of the ELISA assay. Unlike the indicates the presence of glycoprotein-specific antibody in
PAIgG assay (flow assay using intact platelets), which detects the patient’s plasma. The amount of color change (strength
all antibodies bound to a platelet’s surface, PAKAUTO® de- of the reaction) depends on the amount of conjugated anti-
tects only platelet-specific autoantibodies. This is useful in the IgG that was bound to the platelets, which depends on the
diagnosis of autoimmune thrombocytopenic purpura (AITP). amount of antibody in the patient’s serum.
The solid-phase ELISA test (PAK®) is used to detect and
Principle differentiate between antibodies that bind to platelet-specific
The solid-phase ELISA test (MACE®) is used primarily for glycoproteins (IIb/IIIa, Ib/IX, Ia/IIa, and IV) and class I
compatibility testing.26 To perform MACE® tests, patient HLA.27 (PAK® tests may be used to test for alloantibody in
2682_Ch12_273-288 22/05/12 11:47 AM Page 282
GPIIb/IIIa
antibodies are then washed away. Then, similar to the pro- HPA-3a/3a A
HPA-4a/
cedure described above, an alkaline phosphatase labeled HPA-1b/1b
AHG reagent (anti-IgG/A/M) is added to the wells and HPA-3b/3b B
HPA-4a/
incubated. The unbound AHG reagent is washed away and
GPIa/IIa
the substrate PNPP (p-nitrophenyl phosphate) is added. HPA-5b/5b C
After a 30-minute incubation period, the reaction is stopped
HPA-5a/5a D
by a sodium hydroxide solution. Finally, the optical density
of the color that develops is measured in a spectrophotometer.
GPIb/IX E
Figure 12–15 illustrates the results of an antibody test
on two samples, as well as a positive and negative control.
GPIV F
Sample 1 reacts with platelet alloantigen HPA-5b/5b,
suggesting the sample contains antibody to HPA-5b antigens.
HLA Class I G
Sample 2 reacts with HLA class I antigens.
Table 12–1 Procedural Steps of Gel and Solid-Phase Red Cell Adherence Technologies
PROCEDURAL STEPS GEL TEST SOLID-PHASE RED CELL ADHERENCE TEST
ID-MTS Capture-R Select Capture-R Ready Screen, Capture-R Ready-ID
Capture solid-phase; ID-MTS gel test; RBC = red blood cells; N/A = not applicable.
2682_Ch12_273-288 22/05/12 11:47 AM Page 283
Rh Yes Yes
LISS
Media LISS Enzyme
DTT
Minimum tests 6 8
Specificity
DTT = dithiothreitol; IgG = immunoglobulin G; IgM = immunoglobulin M; N/A = not applicable; ↑ = increased; ↓ = decreased; LISS = low ionic strength saline; neg = negative control; SPRCA =
solid-phase red cell adherence; +s = strong positive control; +w = weak positive control.
2682_Ch12_273-288 22/05/12 11:47 AM Page 284
SUMMARY CHART
Advantages of gel and solid-phase technologies over • Solid-phase technology is currently approved for
routine tube testing are: antibody screening, antibody identification, and
• Standardization: There is no tube shaking or resus- compatibility testing.
pension of an RBC button to cause subjectivity in • Advantages of solid-phase technology include the
the interpretation of the test. ease of use, because no predilution of reagents is
• Stability: There are well-defined endpoints of the required, and the ability to test hemolyzed, lipemic,
reaction. or icteric samples. Enhanced sensitivity increases
• Decreased sample volume needed for testing the detection of weak alloantibodies.
• Enhanced sensitivity and specificity • The major disadvantage of solid-phase technology
Gel test points to remember: is the need to purchase special equipment: a cen-
• The principle of the gel test is hemagglutination. trifuge that can spin microplates, a 37°C incubator
• In the gel test, RBCs and serum or plasma are for microplates, and a light source for reading the
allowed to incubate together in a reaction chamber. final results.
• Following incubation, controlled centrifugation Solid-phase protein A technology points to remember:
drives the RBCs through a specially designed mi- • IgG antibodies are captured in microwells that are
crotube filled with beads of dextran-acrylamide gel. coated with protein A.
• Agglutinated cells remain at the top of the tube or • Solidscreen II, which uses solid-phase protein A
are trapped in the gel, depending on the size of the technology, is an assay that uses traditional
agglutinates. antiglobulin technique.
• Unagglutinated cells move through the gel to the • Solid-phase protein A testing is available only in
bottom of the tube. the United States as an automated technology on
• The gel test reactions are stable for observation or the TANGO optimo instrument.
review for 2 to 3 days. Solid-phase immunosorbent assay (ELISA) points to
• Gel technology is currently approved for ABO remember:
forward and reverse grouping, Rh typing, DAT, • The MACE products provide well-characterized
antibody screening, antibody identification, and monoclonal antibodies immobilized in microwells
compatibility testing. that are used to capture glycoproteins from
• The major disadvantage of the gel technology is the platelets supplied by the user.
need to purchase special equipment: a centrifuge • MACE is used primarily for compatibility testing.
to accommodate the microtube cards used for test- • The PAK products provide well-characterized
ing and a pipette for dispensing plasma or serum platelet glycoproteins that are either immobilized
and RBC suspensions into the reaction chambers directly or captured by monoclonal antibodies to
of the microtubes. wells of a microwell plate.
SPRCA assay points to remember: • PAK is used to detect and differentiate between
• The principle of SPRCA is based on solid-phase antibodies that bind to platelet-specific glycoproteins
technology. (IIb/IIIa, Ib/IX, Ia/IIa, and IV) and class I HLA,
• In SPRCA tests, the target antigen is affixed to the primarily for compatibility testing.
bottom of the microplate wells. Luminex-based assay points to remember:
• If the test plasma contains antibodies to the anti- • Luminex-based assay is a bead-based assay that
gen, they attach to the fixed antigen, and indicator uses florescence and flow cytometry to test for
cells detect the attached antibodies by forming a platelet/HLA antibodies.
monolayer of RBCs. • Luminex assay uses a mixture of 100 different
• If the test plasma contains no antibodies to the colored beads, each bead coated with a different
antigen, there is no attachment to the fixed anti- protein.
gen, and the indicator cells form a clearly delin- • The flow cytometer distinguishes between each
eated button at the center of the microplate well. of the 100 beads by the amount and color of the
• Solid-phase reactions are stable for observation or internal dyes.
review for 2 days.
2682_Ch12_273-288 22/05/12 11:47 AM Page 286
23. TANGO optimo instrument (solid phase protein A automation), 30. Garozzo, G, Licitra, V, Criscione, R, et al: A comparison of two
product brochure, Biotest AG, 2009. automated methods for the detection and identification of red
24. Heintz, M, Bahl, M, Mann, J, Yohannes, M, and Levitt, J: blood cell alloantibodies. Blood Transfus 5:33–40, 2007.
Evaluation of blood group antisera and antibody screening with 31. Yamada, C, Serrano-Rahman, L, Vasovic, LV, et al: Antibody
the TANGOTM automated blood bank analyzer. Transfusion identification using both automated solid-phase red cell adher-
44:125A, 2004. ence assay and a tube polyethylene glycol antiglobulin method.
25. ELISA products designed to detect antibodies reactive with Transfusion 48:1693–1698, 2008.
platelet glycoproteins. Product brochure, GTI Diagnostics, 32. Combs, MR, and Bredehoeft, SJ: Selecting an acceptable and
Waukesha, WI, 2006. safe antibody detection test can present a dilemma. Immuno-
26. MACE1. Package insert, GTI Diagnostics, Waukesha, WI, 2008. hematol 17:86–89, 2001.
27. PAK1. Package insert, GTI Diagnostics, Waukesha, WI, 2007. 33. Callahan, DL, Kennedy, MS, Ranalli, MA, et al: Delayed hemolytic
28. Weisbach, V, Kohnhäuser, T, Zimmermann, R, et al: Comparison transfusion reaction caused by Jkb antibody detected by only
of the performance of microtube column systems and solid- solid phase technique. Transfusion 40:113S, 2000.
phase systems and the tube low-ionic-strength solution additive 34. Barker, JM, Scillian, J, Spindler, BJ, and Cruz, MC: A delayed
indirect antiglobulin test in the detection of red cell alloanti- transfusion reaction due to anti-Fya not detected by LISS-tube
bodies. Trans Med 16:276–284, 2006. or gel techniques. Transfusion 44:119A, 2004.
29. Ramsey, G, Sumugod, RD, Garland, FD, et al: Automated 35. Casina, TS: In search of the holy grail: Comparison of
solid- phase RBC antibody screening in a transfusion service. antibody screening methods. Immunohematol 22:196–202,
Transfusion 45:123A, 2005. 2006.
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Chapter 13
Donor Screening and Component Preparation
Patricia A. Wright, BA, MT(ASCP)SBB, and Virginia C. Hughes, PhD(ABD),
MT(ASCP)SBB, CLS(NCA)I
OBJECTIVES
1. Identify the organizations that regulate or accredit the immunohematology laboratory.
2. State the minimum acceptable levels for the following tests in allogeneic and autologous donation:
• Weight
• Temperature
• Pulse
Continued
289
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OBJECTIVES—cont’d
• Blood pressure
• Hemoglobin
• Hematocrit
3. Differentiate between acceptable donation and permanent deferral given various medical conditions.
4. Differentiate among the four different types of autologous donations.
5. Describe the procedure for a whole blood donation, including arm preparation, blood collection, and postphlebotomy care
instructions for the donor.
6. Differentiate among mild, moderate, and severe donor reactions and list recommended treatments for each.
7. List the tests required for allogeneic, autologous, and apheresis donation.
8. State the acceptable interval of donation for allogeneic donors.
9. State the acceptable interval of donation for apheresis donors.
10. List the labeling criteria for a unit of allogeneic and autologous blood.
11. Identify the storage conditions, shelf life, quality-control requirements, and indications for use for the following:
• Whole blood
• Red blood cells, including irradiated, leukoreduced, and saline washed
• Random and apheresis platelets, including irradiated, leukoreduced, and washed
• Pooled random platelets
• Frozen, deglycerolized RBCs
• Fresh frozen plasma and plasma frozen within 24 hours
• Liquid plasma
• Cryoprecipitate and pre-pooled cryoprecipitate
• Granulocyte concentrates
• Factor concentrates, including activated factor VII, VIII, IX, and XIII concentrates
• Rho(D) immunoglobulin
• Normal serum albumin
• Immune serum globulin
• Plasma protein fraction
• Antithrombin III concentrates
requirements of CMS (Centers for Medicare and Medicaid restriction; however, each donor-patient must be evalu-
Services) and the CLIA ’88 (Clinical Laboratory Improve- ated by the blood bank medical director.
ment Amendments of 1988). The mission of the AABB is • Consent to donate: Donors should be informed of the
to establish and provide the highest standard of care for pa- procedure for donating blood and its potential risks. They
tients and donors in all aspects of transfusion medicine. must also be given educational materials informing them
The AABB has published books on transfusion medicine of the signs and symptoms associated with HIV (human
throughout its existence; two resources that are vital to immunodeficiency virus) infection and AIDS (acquired
donor screening procedures are AABB Standards for Blood immune deficiency syndrome), behaviors that put them
Banks and Transfusion Services and AABB Technical Manual. at high risk of infection, and a caution not to donate
The specific guidelines for donor screening and component blood as a means of getting an HIV test. At some point in
preparation are discussed in these publications and will be the interview process, donors must sign a statement doc-
referred to throughout this chapter. umenting that they have given consent to the donation.
This is typically done at the end of the donor history
College of American Pathologists questionnaire. (Blood donor education materials are
shown in Fig. 13–2.)
The College of American Pathologists (CAP) also provides • Additional information: The following additional infor-
a voluntary inspection and accreditation program for its mation may be helpful in some cases:
member institutions. Since most transfusion services are part • The name of the patient for whom the blood is intended
of the clinical laboratory departments in a hospital, they are (directed donation)
included in a CAP inspection. As with the AABB inspections, • Race of the donor for unique phenotypes
CAP inspections are approved by CMS and meet the CLIA • Cytomegalovirus (CMV) status (some patient groups,
requirements. such as neonates, require CMV-negative blood in cer-
tain circumstances)
Donor Screening
Donor screening encompasses the medical history require- Medical History Questionnaire
ments for the donor, the (mini) physical examination, and
Obtaining an accurate medical history of the donor is
serologic testing of the donor blood. Any one of these areas
essential to ensure protection of the donor and benefit to
may preclude a potential donor from the donation process.
the recipient. A standardized medical history questionnaire
The medical history information and physical examination
was developed by a task force that included representatives
are designed to answer two questions: (1) Will a donation
from the AABB, the FDA, and the blood and plasma indus-
of approximately 450 mL of whole blood at this time be
try (Fig. 13–3). The questionnaire was designed to be
harmful to the donor? (2) Could blood drawn from this
self-administered by the donor but if preferred may be ad-
donor at this time potentially transmit a disease to the
ministered by a trained donor historian.3 Self-administered
recipient?
questionnaires must be reviewed by trained personnel
before completing the screening process and prior to col-
Registration lecting blood. The interviewer should be familiar with
As outlined in AABB Standards, blood collection facilities the questions, and the interview should be conducted in a
must confirm donor identity and link the donor to existing secluded area of the blood center or donor site. The ques-
donor records.1 Most facilities require a photographic tions are designed so that a simple “yes” or “no” can be
identification such as a driver’s license, passport, or school answered but elaborated if indicated. The medical history
identification card. In addition, to prevent an ineligible is conducted on the same day as the donation. The currently
donor from donating again, every donor must be checked approved version of the Donor History Questionnaire
against a permanent record of previously deferred donors.2 (DHQ) can be downloaded from the FDA website.
The following is a list of information used by the collection
facility in the registration process and is kept on record as Medical History Questions
a single donation record form (Fig. 13–1) or electronically: The following is a summary of the DHQ questions with
elaboration and explanation. For more in-depth informa-
• Name (first, last, MI)
tion on the questionnaire and interpretations of the ques-
• Date and time of donation
tions, please refer to the AABB Technical Manual and the
• Address
FDA website.
• Telephone
• Gender • Are you feeling healthy and well today? Donors should
• Age or date of birth: The minimum age for an allogeneic appear to be in good health without obvious signs or
donation is between 16 and 17 years, depending on in- symptoms of colds, flu, or other illness.
dividual state requirements (see applicable state laws). • Are you currently taking an antibiotic or taking any
There is no upper age limit. For autologous donation other medication for an infection? Donors currently tak-
(donating blood to be used for oneself), there is no age ing antibiotics for an infection or for prophylaxis after
Text continued on page 296
2682_Ch13_289-330 22/05/12 11:48 AM Page 292
CPDA-1: 1
REMARKS: Fenwal Donormatic
Additive: 2
POSITIVE ANTIBODIES: mL
POSITIVE ANTIGENS:
ABO Prescreening (First Time Donor) LAB CMV: INITIALS LAB TYPE INITIALS
0 1 A B
5
DISCONNECT: END TIME:
Figure 13–1. Single donation record form. (LifeSouth Blood Center, Montgomery, AL, with permission.)
2682_Ch13_289-330 22/05/12 11:48 AM Page 293
Figure 13–2. Blood Donor Educational Materials. (U.S. Food and Drug Administration, “Guidance for industry: Implementation of acceptable full-length Donor History
Questionnaire and accompanying materials for use in screening donors of blood and blood components,” October 2006.)
2682_Ch13_289-330 22/05/12 11:48 AM Page 294
Figure 13–3. Donor History Questionnaire. (U.S. Food and Drug Administration, “Guidance for industry: Implementation of acceptable full-length Donor History
Questionnaire and accompanying materials for use in screening donors of blood and blood components,” October 2006.)
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dental surgery may be deferred temporarily until the • In the past 48 hours, have you taken aspirin or anything
donor has completed the prescribed antibiotic regimen with aspirin in it? Donors who have taken piroxicam, as-
and the infection has cleared up. Donors who have taken pirin, or anything with aspirin in it within 3 days of do-
tetracyclines or other antibiotics used to treat acne are nation may not be a suitable donor for platelet pheresis;
acceptable for donation. All drugs and medications must these medications inhibit platelet function. There is no re-
be cleared by the blood collection facility or blood bank striction for whole blood donation.
medical director. If the donor’s response is a “yes” the in- • In the past 6 weeks, have you been pregnant or are you
terviewer must investigate further. Types and descriptions pregnant now? Female donors should be temporarily
of deferrals are listed in Box 13–1. deferred for 6 weeks following termination of pregnancy.
• Are you now taking or have you ever taken any med- Exceptions can be made by the blood bank medical
ications on the Medication Deferral List (Fig. 13–4)? director for an autologous donation if complications
The Medication Deferral List was developed along with are anticipated at delivery. A first-trimester or second-
the DHQ and is recommended to be used in conjunction trimester abortion or miscarriage is not cause for defer-
with the DHQ. It can be found on the FDA website ral. A 12-month deferral would apply if the woman
along with other donor history documents. Each facil- received a transfusion during her pregnancy.
ity’s medical director is responsible for determining if • In the past 8 weeks, have you donated blood, platelets,
any other medications require a deferral. Most centers or plasma? In the past 16 weeks, have you donated a
have a predetermined list of medications and their de- double unit of red cells using an apheresis machine? The
ferral requirements. time interval between allogeneic whole blood donations
• Have you read the educational materials (see Fig. 13–2)? is 8 weeks or 56 days. If the prospective donor has partic-
Prior to beginning the Donor History Questionnaire, ipated in an apheresis donation (platelets, leukocytes,
prospective donors must be provided information about granulocytes), at least 48 hours must pass before donating
the collection procedure and any risks involved. They whole blood.1 (FDA limits plateletpheresis procedures to
must also be informed (in a language they can under- no more than 24 in a calendar year.) Infrequent plasma
stand) about high-risk behavior related to the AIDS virus apheresis requires a 4-week deferral. The deferral time
and must be given the opportunity to ask questions re- for a double red cell unit apheresis is 16 weeks due to the
garding any aspect of the collection procedure. The donor additional volume of red cells that are donated.
needs to acknowledge that they have read and understand • In the past 8 weeks, have you had any vaccinations or
all of the material. This is generally done by having the other shots? Have you had contact with someone who
donor sign a statement indicating they have read and un- had a smallpox vaccination? If a potential donor has re-
derstand the materials, they have answered the health his- ceived a live attenuated or bacterial vaccine such as
tory questions honestly, and they have been informed of measles (rubeola), mumps, oral polio, typhoid, or yellow
the risks of donation and give their consent to the dona- fever, there is a 2-week deferral; if the donor has received
tion. This meets the requirements for documentation of a live attenuated vaccine for German measles (rubella) or
informed consent. chickenpox, there is a 4-week deferral.1 However, there is
no deferral for toxoids or killed or synthetic viral, bacter-
ial, or rickettsial vaccines such as diphtheria, hepatitis A,
hepatitis B, influenza, Lyme disease, pneumococcal poly-
BOX 13–1
saccharide, polio injection (Salk), anthrax, cholera,
Types of Deferral pertussis, plague, paratyphoid, rabies, Rocky Mountain
Temporary Deferral: Prospective donor is unable to donate blood spotted fever, tetanus, or typhoid injection, if the donor is
for a limited period of time. symptom-free and afebrile.2 Deferral for smallpox vacci-
EXAMPLE: Donor has received a blood transfusion; defer for nation is 14 to 21 days or until the scab has fallen off. In
12 months from date of transfusion. Donor received vaccination for addition, donors who have been in close contact with
yellow fever; defer for 2 weeks from date of vaccination. someone who was recently vaccinated are at risk of possi-
Indefinite Deferral: Prospective donor is unable to donate
blood for someone else for an unspecified period of time due to ble infection as well.4 The FDA defines close contact as
current regulatory requirements. This donor would not be able to exposure to the vaccination site or bandages, clothing,
donate blood until the current requirement changes. These donors towels, or bedding that have been in contact with the vac-
may be eligible to donate autologous blood. cination site. Determine if the donor developed any active
EXAMPLE: Donor states they have lived in England for 1 year in Vaccinia virus infection symptoms (new rash or skin sores
1989; defer indefinitely.
Permanent Deferral: Prospective donor will never be eligible since the time of contact7) from the vaccine.
to donate blood for someone else. These donors may be eligible to • In the past 12 months, have you had a blood transfusion;
donate autologous blood. Some permanent deferrals may result a transplant such as organ, tissue, or bone marrow; or a
from the testing performed on a previous donation. graft such as bone or skin? Donors who during the pre-
EXAMPLE: Donor states that he or she has hepatitis C; defer ceding 12 months have received a transfusion of blood or
permanently.
From: Donor History Questionnaire user brochure, FDA its components or other human tissues (organ, tissue,
May 2008. Available at www.fda.gov. bone marrow transplant, or bone or skin graft) known to
be possible sources of blood-borne pathogens should be
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Accutane© (Amnesteem, Claravis, Sotret, isotretinoin) − usually given for severe acne
Growth Hormone from Human Pituitary Glands − used usually for children with delayed or impaired growth
Plavix (clopidogrel) and Ticlid (ticlopidine) – inhibits platelet function; used to reduce the chance for heart attack and stroke.
Experimental Medication or Unlicensed (Experimental) Vaccine – usually associated with a research protocol
IF YOU WOULD LIKE TO KNOW WHY THESE MEDICINES AFFECT YOU AS A BLOOD DONOR, PLEASE KEEP READING:
• If you have taken or are taking Proscar, Avodart, Propecia, Accutane, Soriatane, or Tegison, these medications can cause
birth defects. Your donated blood could contain high enough levels to damage the unborn baby if transfused to a pregnant woman.
Once the medication has been cleared from your blood, you may donate again. Following the last dose, the deferral period is one
month Proscar, Propecia and Accutane, six months for Avodart and three years for Soriatane. Tegison is a permanent deferral.
• Growth hormone from human pituitary glands was prescribed for children with delayed or impaired growth. The hormone was
obtained from human pituitary glands, which are found in the brain. Some people who took this hormone developed a rare nervous
system condition called Creutzfeldt-Jakob Disease (CJD, for short). The deferral is permanent.
• Insulin from cows (bovine, or beef, insulin) is an injected material used to treat diabetes. If this insulin was imported into the
US from countries in which “Mad Cow Disease” has been found, it could contain material from infected cattle. There is concern
that "Mad Cow Disease" is transmitted by transfusion. The deferral is indefinite.
• Hepatitis B Immune Globulin (HBIG) is an injected material used to prevent infection following an exposure to hepatitis B. HBIG
does not prevent hepatitis B infection in every case, therefore persons who have received HBIG must wait 12 months to donate
blood to be sure they were not infected since hepatitis B can be transmitted through transfusion to a patient.
• Feldene is a non-steroidal anti-inflammatory drug that can affect platelet function. A donor taking Feldene will not be able to
donate platelets for 2 days; however, its use will not affect whole blood donations.
• Plavix and Ticlid are medications that can decrease the chance of a heart attack or stroke in individuals at risk for these
conditions. Since these medications can affect platelets, anyone taking Plavix or Ticlid will not be able to donate platelets for
14 days after the last dose. Use of either medication will not prohibit whole blood donations.
• Experimental Medication or Unlicensed (Experimental) Vaccine is usually associated with a research protocol and the effect
on blood donation is unknown. Deferral is one year unless otherwise indicated by Medical Director.
Figure 13–4. Medication Deferral List. (U.S. Food and Drug Administration, “Guidance for industry: Implementation of acceptable full-length Donor History Questionnaire
and accompanying materials for use in screening donors of blood and blood components,” October 2006.)
deferred for 12 months from the time of receiving the Skin-penetrating injuries from instruments, equipment,
blood product or graft. needles, and so on, that are nonsterile and contaminated
• In the past 12 months, have you come in contact with with blood or body fluids other than the donor’s own are
someone else’s blood or had an accidental needle-stick also cause for deferral. This includes tattoos, permanent
injury? Had a tattoo? Had ear or body piercing? Deferral makeup, and ear and body piercings unless applied by a
is 12 months due to exposure to substances known to be state-regulated organization where sterile needles and ink
sources of blood-borne pathogens. Exposure is assumed are not reused.2
if the blood came in contact with an open wound, any bro- • In the past 12 months, have you had sexual contact
ken skin, or mucous membranes (nose, mouth, eyes, etc.). with anyone who has HIV/AIDS or has had a positive
2682_Ch13_289-330 22/05/12 11:48 AM Page 298
test for HIV/AIDS? Deferral is for 12 months from the risk of exposure to malaria, Creutzfeldt-Jakob disease
time of sexual contact with a person with clinical or lab- (CJD or vCJD), and more recently leishmaniasis.
oratory evidence of HIV infection or who is at high risk • Malaria: Travelers to areas the CDC (Centers for Dis-
for infection. ease Control and Prevention) considers to be endemic
• In the past 12 months, have you had sexual contact with for malaria should be deferred for 1 year following
a prostitute or anyone else who takes money or drugs or departure from the endemic area, provided the donor
other payment for sex? Persons who have engaged in sex has showed no signs or symptoms of malaria infection,
with such people are deferred from donating blood or its with or without antimalarial prophylactic drug therapy.
components for 12 months from the time of sexual contact. Immigrants, refugees, citizens, or persons who have
• In the past 12 months, have you had sex with anyone resided in the endemic area for at least 5 consecutive
who has ever used a needle to take drugs or steroids years should be deferred for 3 years from the time of
or anything not prescribed by their doctor? In the past departure from the area provided they have remained
12 months, have you ever had sex with anyone who symptom free.
has hemophilia or has used clotting factor concen- • CJD and vCJD: Creutzfeldt-Jakob disease and variant
trates? The FDA mandates persons who have had sex Creutzfeldt-Jakob disease are members of a group of
with any person who is a past or present IV drug user neurological disorders known as the transmissible
should be deferred for 12 months; additionally, persons spongiform encephalopathies or prion diseases, which
who have had sex with any person with hemophilia or affect sheep, cows, and humans. CJD results in progres-
related blood disorder who has received factor concen- sive dementia and spongiform alterations in the brain
trates should be deferred for 12 months. and is rapidly fatal. CJD may be transmitted by corneal
• Female donors: Have you had sexual contact with a male transplants, human dura mater grafts, pituitary-derived
who has ever had sexual contact with another male? human growth hormone, and neurosurgical instru-
Women who have had sex with men who have had ments.12 Donors who are at higher risk for CJD or vCJD
sex with another man, even once since 1977, should be should be indefinitely deferred.
deferred for 12 months.5 There is no tangible evidence • Leishmaniasis: Leishmania spp. are intracellular proto-
of HIV being transmitted by close contact (living in the zoan parasites that cause leishmaniasis. They are
same house, working with, shaking hands, kissing, etc.); endemic tropical and subtropical areas in the Middle
therefore, potential donors who meet the definition of East, Mediterranean coast, Africa, Central and South
being in close contact with someone with AIDS or an America, and Asia. In 2003, with the deployment of
HIV-positive individual need not be deferred.6 military and National Guard troops in Iraq, a 12-month
• In the past 12 months, have you had sexual contact with deferral from the date of departure from the area was in-
a person who has hepatitis? Have you lived with a person stituted for any military personnel stationed in Iraq and
who has hepatitis? Sexual contact or living with a person any civilian and contract personnel who have visited the
(“close contact”) who has acute or chronic hepatitis B country. This same deferral was in place between 1990
(test positive for HBsAg or HBV) or who has symptomatic and 1993 as a result of Operation Desert Storm.
hepatitis C or other hepatitis virus requires a 12-month • From 1980 through 1996:
deferral following discontinuation of the “close contact.” • Did you spend time that adds up to 3 months or more
FDA defines “living with” as residing in the same dwelling in the United Kingdom? (Review list of countries in
(house, apartment, or dormitory).3 the UK.) FDA recommendations state that potential
• In the past 12 months, have you been treated for syphilis donors who have spent 3 or more months, cumulatively,
or gonorrhea? Prospective donors with a history of syphilis in the UK from 1980 and 1996 are to be deferred indef-
or gonorrhea, of treatment for either, or of a reactive initely (Box 13–2).
screening test for syphilis, or where no confirmatory test • Were you a member of the U.S. military, a civilian
was performed, should be deferred for 12 months after military employee, or a dependent of a member of
completion of therapy. The agent that causes syphilis, the U.S. military? U.S. military personnel and their
Treponema pallidum, may live for 1 to 5 days in cold storage dependents and civilian military employees who spent
so that only a fresh unit of RBCs may transmit infection. a total of 6 months or more at a U.S. military base
At room temperature, however, this agent thrives very well, in Europe (United Kingdom, Belgium, Netherlands,
placing platelet concentrates above RBCs as potentially Germany) from 1980 through 1990 or who were at a
supporting transfusion-transmitted syphilis. To date, base in Spain, Portugal, Turkey, Italy, or Greece from
though, only three cases of transfusion-transmitted syphilis 1980 through 1996 are deferred indefinitely.
have been documented.7–10 • From 1980 to the present, did you:
• Have you been in juvenile detention, lockup, or prison • Spend time that adds up to 5 years or more in Europe?
for more than 72 hours? Deferral is 12 months from the (Review the list of countries in Europe; see Box 13–2.)
last date of the incarceration.11 Individuals who have spent at least 5 years, cumula-
• In the past 3 years, have you been outside of the tively, in Europe between 1980 and the present should
United States or Canada? This question is general and be indefinitely deferred as donors of whole blood,
is used to detect a donor who may have an increased blood components, or source leukocytes intended for
2682_Ch13_289-330 22/05/12 11:48 AM Page 299
BOX 13–2
European Countries with BSE or Increased Risk of BSE (CJD and vCJD; used for deferral of donors based on
geographic risk of BSE)
Albania United Kingdom
Greece Denmark
Romania Netherlands
Austria Federal Republic of Yugoslavia
Hungary Finland
Slovak Republic Norway
Belgium France
Republic of Ireland Poland
Slovenia Germany
Bosnia-Herzegovina Portugal
Italy Countries Included in the United Kingdom
Spain
England
Bulgaria
Northern Ireland
Liechtenstein
Scotland
Sweden
Wales
Croatia
the Isle of Man
Luxembourg
the Channel Islands
Switzerland
Gibraltar
Czech Republic
Falkland Islands
Macedonia
Data from Guidance for industry revised preventive measures to reduce the possible risk of transmission of Creutzfeldt-Jakob disease (CJD) and variant Creutzfeldt-Jakob disease
(vCJD) by blood and blood products. FDA, Rockville, MD, May 2010.
transfusion. However, they may donate source plasma receive clotting factor concentrates should be deferred
if not otherwise deferred.12 from donating blood or components unless otherwise
• Receive a blood transfusion in the United Kingdom or approved by the medical director, because there is a
France? (Review the list of countries in the UK; see high risk of bleeding following venipuncture. Receipt
Box 13–2.) Those who have received a transfusion of of clotting factor concentrates, like receipt of blood
blood, platelets, plasma, cryoprecipitate, or granulo- transfusions, requires a 12-month deferral.
cytes in the UK or France since 1980 should be indefi- • Had hepatitis? Donors with a history of hepatitis
nitely deferred as a blood donor. after their 11th birthday or a confirmed positive test for
• From 1977 to the present, have you: hepatitis B surface antigen (HBsAg) or a repeatedly reac-
• Received money, drugs, or other payment for sex? Men tive test for anti-HBc should be indefinitely deferred.
or women who engage in sex for money or drugs since Donor suitability with regard to the age restriction for
1977 are permanently deferred. hepatitis can be assessed by the medical director; recol-
• Male Donors: Have you had sexual contact with an- lections of symptoms, diagnoses, and laboratory data can
other male, even once? Male-to-male sexual contact, be helpful in deciding if the donor is suitable for whole
even once, since 1977 is cause for permanent deferral. blood donation. The FDA stipulates that a history of an
• Have you ever: elevated alanine aminotransferase or a reactive test for
• Had a positive test for HIV/AIDS virus? Indefinite de- antibodies to hepatitis A virus or to hepatitis B surface
ferral for any person with clinical or laboratory diagno- antigen should not exclude a potential donor without ad-
sis of HIV infection. ditional clinical evidence of viral hepatitis. If viral hepatitis
• Used needles to take drugs, steroids, or anything not before the age of 11 years is suspected, the donor should
prescribed by your doctor? Donors’ arms should be be deferred temporarily until the circumstances are inves-
checked for evidence of scars or punctures indicating tigated and medical opinion concludes there is no history
addiction to self-injected drugs or for presence of any or diagnosis of viral hepatitis after age 11.
skin lesions in the venipuncture site. Donors with evi- • Had malaria? Prospective donors with a history of
dence of past or present nonprescription drug use malaria are deferred for 3 years following treatment and
should be indefinitely deferred. being asymptomatic.
• Used clotting factor concentrates? Prospective donors • Had Chagas’ disease? Had babesiosis? A history of
with hemophilia or other bleeding disorders and who Chagas’ disease or babesiosis is cause for indefinite
2682_Ch13_289-330 22/05/12 11:48 AM Page 300
deferral. Chagas’ disease, also known as American • Been in Africa? HIV-1 group O virus is endemic to the
trypanosomiasis, is caused by the protozoan parasite central and west regions of Africa. Anyone who was
Trypanosoma cruzi. The vector responsible for trans- born or lived in any of the African countries on the FDA
mitting the parasite is the hematophagous bug list of countries with HIV-1 group O risk (Box 13–3)
belonging to the family Reduviidae. Transmission since 1977 should be deferred indefinitely. In addition, if
occurs when mucous membranes or breaks in the the prospective donor has traveled to any of the countries
skin are contaminated with the feces of infected on the list since 1977 and received a blood transfusion
hematophagous bugs.19 Chagas’ disease is endemic or other medical treatment with a blood product, they
in parts of Central and South America and Mexico, should be deferred indefinitely.15
where an estimated 16 to 18 million persons are in- • Had sexual contact with anyone who was born in or
fected with T. cruzi. Approximately 25,000 to 100,000 lived in Africa? The FDA recommends indefinite de-
Latin American immigrants living in the United States ferral for any prospective donor who indicates having
are infected with T. cruzi. Chagas’ disease may be had sex with a person who was born in any of the
transmitted congenitally by breastfeeding, organ African countries on the list of those with increased
transplants, or blood transfusion.13 risk of HIV-1 group O infection (see Box 13–3).
In the United States and Canada, there have been • If the collecting facility has implemented testing for
seven documented cases of transfusion-associated HIV-1/2 that has been licensed for use in detecting
transmission of Chagas’ disease in the past 20 years, antibodies to HIV-1 group O, the questions concerning
all occurring in immunosuppressed recipients.14 There residence in or travel to specific African countries or
are 70 known species of Babesiai, and at least 5 are sexual contact with persons from those countries may
identified as infecting humans. Babesia microti utilizes be omitted. In addition, donors who were previously
the vector Ixodes scapularis to infect the human and deferred prior to the availability of the HIV-1 group O
transmit the parasite. The Babesia organism penetrates testing may be reentered provided it has been at least
the erythrocyte where the trophozoite multiplies; 1 year since the last potential exposure to HIV-1 group
upon lysis of the RBC, merozoites are released into O and the screening test results are non-reactive.15
the blood where they are free to infect other RBCs. • Have any of your relatives had Creutzfeldt-Jakob dis-
Transfusion-associated infection with Babesia carries ease? The FDA recommends prospective donors with a
an incubation period of 2 to 8 weeks; symptoms may family history of CJD be permanently deferred unless
include malaise, fatigue, anorexia, arthralgias, nausea, the diagnosis of CJD was confidently excluded.
vomiting, abdominal pain, and fever reaching temper-
atures of 40°C. The Physical Examination
• Received a dura mater (or brain covering) graft? Due
to an increased risk of exposure to CJD or vCJD, The donor center representative evaluates the prospective
prospective donors with history of a dura mater graft donor with regard to general appearance, weight, tempera-
require an indefinite deferral. ture, pulse, blood pressure, hemoglobin, and presence of
• Had any type of cancer, including leukemia? A history
of cancer, leukemia, or lymphoma is generally a cause for
indefinite deferral. Any donor presenting with a history BOX 13–3
of cancer should be reviewed by the blood bank medical
director. The exceptions include basal or squamous
African Countries at Increased Risk of HIV-1 Group O
cell cancer, carcinoma in situ of the cervix, and papillary Infection
thyroid carcinoma that has been surgically removed. Cameroon
• Had any problems with your heart or lungs? A history Benin
of cardiovascular, coronary, or rheumatic heart disease Central African Republic
is usually a cause for deferral; however, in the absence Chad
of disability or restrictions by the patient’s physician, Congo
the donor may be accepted on a case-by-case basis by Equatorial Guinea
the blood bank director. Active pulmonary tuberculosis Kenya
or other active pulmonary disease is cause for deferral. Gabon
• Had a bleeding condition or a blood disease? Donors Niger
indicating a history of a bleeding problem following Nigeria
surgery, invasive dental procedures, cuts, or abrasions Senegal
must be further evaluated by the blood bank director. Togo
Diseases of the blood such as hemophilia, von Zambia
Willebrand disease, sickle cell anemia, thalassemia,
Kaposi’s sarcoma, or polycythemia, or a history of Data from Guidance for industry recommendations for management of donors
at increased risk for human immunodeficiency virus type 1 (HIV-1) group O
receiving clotting factor concentrates are causes for infection. FDA, Rockville, MD, August 2009.
indefinite deferral.
2682_Ch13_289-330 22/05/12 11:48 AM Page 301
skin lesions. A blood bank physician should be available to should be greater than or equal to 11 g/dL and 33%,
evaluate any special considerations. respectively.1 The methods used for measuring hemoglobin
include copper sulfate or point-of-care instruments using
• General appearance: The donor center representative
spectrophotometric methodology. A hematocrit or packed
should observe the prospective donor for presence of ex-
cell volume can be determined manually by centrifugation.
cessive anxiety, drug or alcohol influence, or nervousness.
The blood is usually acquired via a finger stick.
If possible, this should be done in a gentle manner so as
• Skin lesions: Prior to donation, the donor’s arms should
to not deter the donor from donations in the future.
be inspected for skin lesions. Evidence of skin lesions
• Weight: Standards mandates a maximum of 10.5 mL of
(e.g., multiple puncture marks) is cause for indefinite de-
blood/kg of donor weight for whole blood collection in-
ferral. Skin disorders that are not cause for deferral include
clusive of pilot tubes for testing. If the donor weighs less
poison ivy and other rashes; these, however, should not
than 100 pounds, the amount of blood collected must be
be present in the area of the venipuncture site and may
proportionately reduced as well as that of the anticoagu-
need to be evaluated by a blood bank physician.
lant. The following formulas can be used to calculate the
adjusted volume of blood to be collected and anticoagu-
Informed Consent
lant to be used.
Volume to collect = (donor’s weight in kg/50) × 450 mL AABB Standards mandates that informed consent of allo-
Volume to collect/450 × 63 mL = reduced volume of geneic, autologous, and apheresis donors be obtained before
anticoagulant donation. The donor must be informed of the risks of the
63 mL – above calculated volume = amount of solution procedure and of the tests that are performed to reduce
to be removed the risk of infectious disease transmission to the recipient.
• Temperature: Standards mandates the donor temperature The donor must be able to ask questions concerning any
must be less than or equal to 37.5°C or 99.5°F.28 Donors element of the collection or testing process. If the donor is
are asked not to drink coffee or hot beverages while wait- a minor or is unable to comprehend the informed consent
ing to donate, as this may sometimes affect their temper- protocol, applicable state law provisions will intercede. An
ature. Oral temperatures that are lower than normal are example of an informed consent is shown in Figure 13–5.
not cause for deferral.
• Pulse: The donor’s pulse should be between 50 and Autologous Donors
100 bpm. Often, a donor who is athletic will have a pulse
less than 50 bpm, which is not cause for deferral. The An autologous donor is one who donates blood for his or
pulse should be counted for at least 15 seconds; any irreg- her own use; thus, such a donor is referred to as the donor-
ularities should be evaluated by a blood bank physician. patient. Most autologous blood is used to treat surgical blood
• Blood pressure: A potential donor’s systolic blood pressure loss in very specific situations where there is a reasonable
should be less than or equal to 180 mm Hg and the dias- opportunity to avoid homologous transfusions or when
tolic less than or equal to 100 mm Hg. Blood pressure compatible allogeneic blood is not available. The potential
readings above these levels should be evaluated by a blood advantage of using autologous blood over allogeneic blood
bank physician. includes a decreased risk of disease transmission, transfusion
• Hemoglobin: The donor’s hemoglobin level should be reactions, and alloimmunization. However, there is still a risk
greater than or equal to 12.5 g/dL and the hematocrit level of bacterial contamination, circulatory overload, cytokine-
greater than or equal to 38% for allogeneic donation. For mediated reactions, and misidentification of the product or
autologous donation, the hemoglobin and hematocrit level patient.16 The patients for whom autologous donation or
Donor Release:
I have read and I understand the information contained in the Responsibilities of a Blood Donor and Important Information About
Donating Blood Information sheet. I understand that if my behavior or physical condition puts me at risk to transmit AIDS or any other
disease, I should not donate blood. The medical history I have given is truthful and accurate to the best of my knowledge. I have not
participated in activities considered high risk for acquiring and transmitting AIDS.
I give my permission for all laboratory testing necessary to provide safe blood to the recipient including tests to detect exposure to
hepatitis, AIDS, and other transfusion-transmitted diseases. Some of these tests may be unlicensed and for research only. Some
positive test results are routinely reported to the State Health Department as required by law for notification of sexual partners. I realize
that LifeSouth is not a testing center and that by giving blood I am not guaranteed that disease testing will be performed.
Although donating blood is normally a pleasant experience, I realize that short-term side effects can occur, such as bruising, dizziness,
or fainting, and that, in rare cases, a donor may experience infection or nerve damage.
I voluntarily donate my blood to the LifeSouth Community Blood Centers to be used as the blood center deems advisable.
Figure 13–5. Sample informed consent. (LifeSouth Blood Center, Montgomery, AL, with permission.)
2682_Ch13_289-330 22/05/12 11:48 AM Page 302
transfusion holds the greatest advantage are those with very be limited to those needed to ensure the safety of the
rare blood types and those with multiple antibodies where donor, including cardiac history, bleeding disorders, major
compatible units in the general blood supply may be difficult illness, previous donor reactions, fainting problems, and
or impossible to find. so on. In addition, there should be questions to rule out
The disadvantages of autologous donation or transfusion the possibility of bacteremia in the donor or patient. This
include a higher cost due to added administrative processes would include information on current medications, antibi-
and special labeling requirements to ensure that units otics, recent infections, fever, recent minor procedures
get transfused to the proper patient. It should be noted that such as dental procedures, gastrointestinal problems, or
there is a high percentage of wasted units (30% to 50%), be- diarrhea.
cause patients end up not requiring any or all of the units Most autologous donor units are a standard 450 or
donated.16 In addition, the AABB Standards do not permit 500 mL ± 10%; however, if the donor weighs less than
“crossing over” of unused autologous units into the general 50 kg (110 lb), the total amount of blood collected must
inventory, except in exceptional circumstances. The decision be reduced proportionately. If the amount of blood to be
must be approved by the medical director on a case-by-case collected is determined to be 300 to 405 mL, the unit must
basis.17 be labeled as low volume. There is no requirement to
Although the use of autologous blood has decreased in reduce the volume of anticoagulant-preservative solution;
the past several years, it is still a viable and common alter- however, the plasma is not suitable for transfusion. If
native therapy for select patients. There are various methods the unit must be less than 300 mL, the anticoagulant-
and techniques for obtaining autologous blood, including: preservative solution must be adjusted, and approval by
the blood bank medical director is required. See the earlier
• Preoperative collection
section “The Physical Examination” for formulas to calcu-
• Acute normovolemic hemodilution
late the volume and anticoagulant adjustments.
• Intraoperative collection
Testing requirements for autologous donor units are
• Postoperative collection
somewhat less stringent than with allogeneic units. The col-
Preoperative Collection lecting facility must determine the ABO and Rh of the blood,
Preoperative collection occurs during the 5 to 6 weeks but antibody screening is optional. If the collection facility
immediately preceding a scheduled, elective surgical pro- and the transfusion facility are the same, then viral marker
cedure unless the red blood cells and plasma are scheduled testing is not required; however, if they are different, the col-
to be frozen. Procedures that typically might use preoper- lecting facility must test for HBsAg, anti-HBc, anti-HCV,
ative autologous blood include orthopedic procedures, HCV RNA, anti-HIV-1/2, HIV-1 RNA, anti-HTLV-I/II, WNV
vascular surgery, cardiac or thoracic surgery, and radical RNA, and STS on at least the first unit collected from the
prostatectomy. Although some women do participate in donor in every 30-day period. If any of the markers yield a
an autologous collection program during pregnancy for positive or reactive result, the patient’s physician and trans-
unforeseen complications, blood is seldom needed except fusing facility must be notified of the result.1
in cases in which the mother has multiple antibodies The transfusing facility must reconfirm the ABO and Rh
to high-frequency antigens or risk for placenta previa or of the unit, but a crossmatch is optional; however, an imme-
intrapartum hemorrhage.2 diate spin crossmatch would be a good safety check that the
The decision to use preoperative autologous blood re- selected unit is identified properly.
quires both an order from the patient’s physician (Fig. 13–6) Units collected for autologous use must be labeled
and an approval from the blood bank medical director. The appropriately. The label should include the patient’s full
maximal surgical blood order schedule (MSBOS) can provide name, medical record number or ID number, expiration
guidance for surgical procedures to estimate the number of date of the unit, and the name of the facility where the
units needed for transfusion. The last blood collection should donor-patient will be transfused. The label must also
occur no later than 72 hours (3 days) before the scheduled clearly state “For Autologous Use Only” (Fig. 13–7).
surgery to allow for volume replacement. Autologous units also generally have a distinct green label
Medical history and physical exam requirements for and tag (Fig. 13–8). This is done both to ensure the unit is
autologous donation are less stringent than those for linked correctly with the donor-patient and to make the
allogeneic donations. There is no minimum or maximum blood bank technologists aware that certain patients have
age requirement; however, the donor must be able to tol- autologous units on the shelf that must be transfused be-
erate the donation, and for younger donors, most centers fore allogeneic units; more specifically, the oldest units
limit the age to children whose veins can accommodate should be transfused first. Blood banks should have a
the phlebotomy needle and who can understand the system in place to ensure autologous units are selected
procedure. The minimum hemoglobin/hematocrit level is first.
11 g/dL and 33%, respectively. Blood pressure and pulse
are the same as with allogeneic donors unless otherwise Acute Normovolemic Hemodilution
defined by the blood bank medical director, and the Acute normovolemic hemodilution (ANH) results in the col-
donor’s temperature should not be elevated or indicate any lection of whole blood with the concurrent infusion of crys-
sign of possible infection. Medical history questions can talloid or colloid solutions, thus maintaining a normal blood
2682_Ch13_289-330 22/05/12 11:48 AM Page 303
*An appointment is recommended for this service. Please contact your local blood bank for information.
Branch Location:
Reset Form
If patient is also under treatment for, or has any, preexisting medical condition(s), please indicate below.
, MD Condition
, MD Condition
, MD Condition
LifeSouth Community Blood Centers will process and store this blood for subsequent replacement transfusion. The number of units drawn
and the interval between donations will be determined by the patient's hemoglobin and anticipated blood requirements, and will be at the
discretion of the blood center's Medical Director. Units will be held only until outdate (42 days from the date drawn) unless specific
arrangements are made with the blood center prior to that date. Blood should not be drawn from the donor-patient within 72 hours of the
time of anticipated surgery or transfusion. A cardiac release is required for all patients with a history of cardiac problems; units will not be
collected until the blood center has received written release from the patient's cardiologist.
Physicians will be notified in writing of abnormal test results as soon as possible. It is the responsibility of the ordering physician to notify
the patient.
#Units
• Packed Cells Signature, MD Date
• Whole Blood Physician (printed name)
• Fresh Frozen Plasma Phone
• Cryoprecipitated AHF
Figure 13–6. Autologous donation permission form. (LifeSouth Blood Center, Montgomery, AL, with permission.)
volume but decreasing the patient’s hematocrit. The ratio of dilution will reach 21% or less. It is recommended that
replacement is 3:1 for crystalloids and 1:1 for colloids.15 The the patient start with a hemoglobin of at least 12 g/dL. The
number of units collected depends of the patient’s ability to procedure is performed in surgery immediately prior to be-
tolerate the decrease in hemoglobin/hematocrit. A limited ginning the surgical procedure and is managed by anesthe-
hemodilution will reduce the hematocrit to 28%; severe siology. The blood is collected in standard blood bags
2682_Ch13_289-330 22/05/12 11:48 AM Page 304
See circular of information for urgency of the surgery or the patient cannot be scheduled
indications, contraindications,
cautions and methods of infusion for multiple preoperative donations. In addition, the risk of
AUTOLOGOUS DONOR misidentifying the patient and blood product is minimized,
This product may transmit infectious agents.
Caution: Federal law prohibits dispensing
and there are no labeling, testing, and storing costs.
without a prescription. The disadvantages include the high cost of the instrumen-
PROPERLY IDENTIFY INTENDED RECEPIENT
tation involved and training of the personnel to run the in-
DONOR NAME
strument. In addition, frequently the amount of blood that
is collected is not sufficient for the patient’s total needs, and
DONOR SIGNATURE allogeneic blood may still need to be given. The procedure
is counterindicated if there is potential for contamination of
Social Security # Date of Birth
the surgical site by bowel contents, amniotic fluid, urine,
clotting agents, and so on, or where there is a risk of bacterial
Figure 13–7. Autologous donation label. (LifeSouth Blood Center, Montgomery,
AL, with permission.)
contamination or activation of coagulation factors.
As with ANH, the blood generally does not leave the
OR; however, if some of the blood is to be stored postop-
containing anticoagulant or preservative and is stored in the eratively, it must be labeled with the patient’s full name,
room at room temperature. The blood is normally reinfused medical record number, date and time of collection, and
to the patient during or immediately following the surgery, with “For Autologous Use Only.” The blood may be stored
but within 8 hours of collection, thus maintaining the via- at room temperature for up to 6 hours or at 1°C to 6°C for
bility of both platelets and coagulation factors. up to 24 hours, as long as the latter temperature has begun
Blood units are reinfused in the reverse order of collec- within 4 hours from the end of collection. Hospitals
tion so that the last unit reinfused carries the highest hema- should establish their own policies and procedures for this
tocrit level. Because the blood generally does not leave type of collection.
the operating room (OR), the units generally are not tested,
labeled, or tracked, and the blood bank is generally not Postoperative Blood Salvage
involved. However, if the blood is not transfused during Postoperative blood salvage is collected from a drainage
surgery, it can be stored for up to 24 hours in a monitored tube placed at the surgical site. It is reinfused, with or with-
blood bank refrigerator if refrigerated within the first out processing, via a microaggregate filter to screen out any
8 hours. If the units leave the OR and are stored, they must debris. This blood is characterized as being dilute, partially
be appropriately tested and labeled as with predeposit hemolyzed, and defibrinated. It is recommended that no
autologous units. more than 1,400 mL be reinfused.2 Procedures that have
used postoperative blood collection include orthopedic
Intraoperative Collection (e.g., arthroplasty) and cardiac surgeries. Blood must be
Intraoperative autologous collection involves collecting reinfused within 6 hours of collection or it is to be dis-
shed blood from the surgical site; processing the blood carded. Hospitals should establish their own policies and
through an instrument that washes it with saline to remove procedures for this type of collection.
tissue debris, free hemoglobin, and plasma that may contain The advantage of this procedure is that it has a very low
activated coagulation factors; concentrating the residual red cost, and it can be used in conjunction with other autolo-
cells (to a hematocrit of 50% to 60%); and then reinfusing gous blood collection procedures to avoid the need for
those cells immediately. This process is repeated continually allogeneic blood transfusions. Because the blood does not
during the surgical procedure. This type of collection has leave the patient’s bedside, there is no risk of the blood
been used in cardiothoracic, major orthopedic, and cardiac being transfused to the wrong person. The disadvantage
surgeries, in addition to vascular surgeries, such as liver of post-op salvage is that generally it does not yield a large
transplantation. The advantage of using intraoperative au- volume of blood and by itself would not generally produce
tologous blood collection is that it may be used in cases enough blood for the patient’s needs. In addition, it carries
where preoperative donation is not possible due to the a significant risk of producing transfusion reactions due
to the presence of activated coagulation factors, fibrin
degradation products, and cytokines.
Although the blood bank is frequently not responsible
FOR AUTOLOGOUS USE ONLY
for managing ANH intraoperative recovery and post-op
Unit #
Patient salvage collection procedures, both the AABB and the
Hospital College of American Pathologists (CAP) have recommen-
SSN or Hospital # dations and guidelines that require the blood bank medical
Date needed director or supervisor to assist in the development and
Signature
LifeSouth Community Blood Centers, Gainesville, FL 32601
implementation of the programs. This includes assisting
with the development of procedures, maintaining the equip-
Figure 13–8. Autologous donation tag. (LifeSouth Blood Center, Montgomery, ment, monitoring quality control, and training personnel
AL, with permission.) and assessing competency.
2682_Ch13_289-330 22/05/12 11:48 AM Page 305
Directed Donation the donor and patient possible in cases where the patient
has become alloimmunized and refractory to random
A directed donation is a unit collected under the same require- donor platelets. Plateletpheresis donors may donate more
ments as those for allogeneic donors, except that the unit col- often. The interval between donations is at least 2 days,
lected is directed toward a specific patient. Often, when a not to exceed more than twice a week or more than
friend or family member needs blood, the donor center will 24 times a year.1
accommodate these directed donations so that the required Donors who have ingested aspirin, Feldene, or aspirin-
testing may be done as soon as possible; if the blood is com- containing medications should be deferred for 48 hours,
patible, it can be used by the patient. The tag for the directed because these medications interfere with platelet adhesion,
unit is a distinct color (e.g., yellow, salmon) to differentiate it and all of the platelets in a given therapeutic dose would
from autologous tags (Fig. 13–9). If the donor is a blood rela- be affected. In addition, donors on Plavix (clopidogrel) or
tive, the unit must be irradiated to prevent graft versus host Ticlid (ticlopidine) should be deferred for 14 days, which
disease so that viable T cells from the donor that enter the is the time required for the medication to clear the system
patient’s circulation do not mount an attack against patient’s and the platelet function to return to normal. If the donor
cells and tissue.18 A system should be in place to ensure has donated whole blood, or if 100 mL or more of red cells
directed units from blood relatives are irradiated. were not able to be returned to the donor on the previous
pheresis procedure, the donor must be deferred for 8 weeks
Apheresis Donation to allow time for the donor to replenish the lost red cell
mass. Certain exceptions can be made for compelling med-
Apheresis collection is an effective mechanism for collecting
ical reasons, provided a physician certifies that the donor
a specific blood component while returning the remaining
will not be harmed and the blood bank medical director
whole blood components back to the patient. Most apheresis
agrees.
instrumentation use an automated cell separator device
Although a platelet count is not required on the first
whose centrifugal force separates blood into components
donation, it is required if the interval between donations
based on differences in density. (Refer to Chapter 14,
is less than 4 weeks; in that case, the platelet count must
“Apheresis.”)
be above 150,000/µL. The total amount of plasma that can
Apheresis can be used to collect platelets, plasma, white
be removed along with the platelets is limited to 500 mL
cells (leukocytes), red cells, and stem cells. It is designed to
(600 mL for donors weighing more than 175 lb) or the
collect large volumes of the intended component and is the
amount of plasma stated in the directions circular for the
only effective method for collecting leukocytes and stem
automated instrument being used for the collection. Each
cells. The donor requirements for apheresis donation are
pheresis platelet unit is required to contain at least 3 × 1011
generally the same as for whole blood donation; however,
platelets. The automated instruments used today are very
there are some differences, depending on the component
efficient, and a single donor collection frequently yields
that is to be collected. As with whole blood donation, the
a high enough platelet count to allow the collection to
process is regulated by the FDA, and both the AABB and the
be split into two or three individual products. (Refer to
American Society for Apheresis (ASFA) provide comprehen-
Chapter 14.) Donor reactions to platelet pheresis collec-
sive guidelines and standards.
tions are most commonly a reaction to the citrate or antico-
Plateletpheresis agulant used in the procedure. Vasovagal and hypovolemic
reactions are very rare but have been reported.2
Today the majority (more than 75%) of platelet transfu-
Testing requirements are the same as for other allogeneic
sions are pheresis-derived platelets.2 A pheresis platelet
blood products, ABO group/Rh type, antibody screen, and
unit is equivalent to six to eight random donor platelets,
infectious disease markers. If the donor is donating repeat-
so a single product is a typical therapeutic dose for most
edly for a specific patient, repeat testing need only be done
adult patients. This significantly reduces the recipient’s
every 30 days. In addition, the platelet count on each prod-
donor exposure, makes routine leukoreduction of the
uct is determined and recorded but does not have to be
product practical, and allows compatibility matching of
recorded on the product label. If there are visible red cells in
the finished product, the amount of red cells must be deter-
mined. If the product contains more than 2 mL of red cells,
DIRECT DONATION a pilot sample must be attached to the product and used by
Unit # Component the transfusing facility to crossmatch the product with the
Patient intended recipient. FDA guidelines require that the donation
Patient Blood Type D.O.B records of regular platelet pheresis donors be reviewed by a
Hospital physician at least every 4 months.19
SSN or Hospital #
Date needed Plasmapheresis
LifeSouth Community Blood Centers, Gainesville, FL 32601
Plasma was the first product to be collected by apheresis
Figure 13–9. Directed donation tag. (LifeSouth Blood Center, Montgomery, AL, methods and was primarily used as a method for collecting
with permission.) “source plasma,” which is further manufactured into plasma
2682_Ch13_289-330 22/05/12 11:48 AM Page 306
derivatives. Today plasma apheresis is also used to collect Corticosteroids such as prednisone or dexamethasone
transfusable fresh frozen plasma. Plasmapheresis donors can also be used. These drugs are given to the donor prior
are classified as either “infrequent/occasional” or “serial,” to the collection procedure. They work by pulling the
depending on the frequency of donations. An infrequent granulocytes from the marginal pool into the general cir-
donor undergoes no more than one procedure in a 4-week culation, thus increasing the supply of cells available for
period. Serial donors may donate more frequently than collection. Careful scrutiny must be used to obtain an ac-
4 weeks but no more than every 48 hours and no more than curate health history on the donor to identify any medical
two donations in a 7-day period. condition that could be exaggerated by the presence of
The donor requirements for both infrequent and serial corticosteroids.
donors are the same as for allogeneic whole blood donors, Finally, the newest agents are growth factors. Now that
although serial donors have some additional requirements recombinant hematopoietic growth factors are available, they
to protect the donor from excessive red cell and plasma pro- are being used in leukapheresis procedures. The advantages
tein loss. The red cell loss must not exceed 25 mL/week or of these growth factors is that they can produce four to eight
200 mL in an 8-week period. As with any pheresis proce- times the volumes of cells in each collection compared with
dure, if the donor’s red cells cannot be returned, the donor other agents. In addition, these growth factors appear to be
must be deferred for 8 weeks before returning to a plasma quite well tolerated by the donor. Each collection facility
pheresis program. At the start of a serial pheresis program should develop policies outlining maximal doses of stimu-
and at 4-month intervals, the donor must be tested for total lating agents used in leukapheresis with appropriate con-
serum/plasma protein levels and quantitative immunoglob- traindications for donors.
ulin levels, and protein electrophoresis must be performed. Each leukapheresis product must be tested for ABO group
The levels must remain in the normal range, or the donor and Rh type as well as the HLA type of the leukocytes. Most
must be removed from the program until the levels return to leukocyte concentrates are contaminated with some red
normal. cells. As stated for platelet concentrates, if the amount of
If the plasmapheresis procedure is being performed man- contaminating red cells exceeds 2 mL, the product should
ually, there must be a mechanism in place to ensure positive be crossmatched with the recipient, and a pilot tube sample
donor and product identification, as well as safe return of must accompany the product.
the donor’s cells. There should be two separate forms of
identification so that both the donor and the phlebotomist Double RBC Pheresis
can verify the ID. Full names, signatures, unique numbers, In the 1990s, double units of red cells were collected using
or pictures may all be used. the apheresis equipment. The FDA finalized the guidance
recommendations for this procedure in 2001. Double red
Leukapheresis cell pheresis can be used to collect either allogeneic or
Apheresis is the only effective method for collecting leuko- autologous units. The donor must meet the requirements
cytes or, more specifically, granulocytes. The therapeutic ef- for whole blood donation and the recommendations estab-
fectiveness of granulocyte transfusions is still somewhat lished by the equipment manufacturer. The hemoglobin
controversial, but they have been shown to be effective in level must be determined by a quantitative method; copper
specific cases. A typical therapeutic dose is at least 1 × 1011 sulfate method is not acceptable. If the donor’s weight
granulocytes each day for 5 consecutive days. or hemoglobin level are at the minimum level, it is recom-
In order to collect a large enough volume of leukocytes mended that the donor be further evaluated by the blood
(more than 1 × 1011 granulocytes), the donor must be given bank physician to ensure the procedure is safe for the
certain drugs or sedimenting agents, and specific informed donor. If the procedure method used calls for saline infu-
consent must be obtained from the donor prior to adminis- sion to minimize volume depletion, then male donors
tering these drugs. AABB Standards states that any of these must weigh at least 130 pounds and be at least 5’1” tall.
drugs or agents used to facilitate leukapheresis will not be Female donors must weigh at least 150 pounds and be at
used on donors whose medical history suggests that such a least 5’5” tall. The hematocrit level for both sexes must be
drug will exacerbate previous disease.1 a minimum of 40%.2
One of the drugs typically given is hydroxyethyl starch Donors participating in double red cell pheresis programs
(HES), which is a common sedimenting agent. It enhances are deferred for 16 weeks following successful completion
the separation of the white cells from the red cells during of the donation procedures. They should not participate in
centrifugation, which increases the amount of leukocytes platelet or plasma pheresis during that period. If the proce-
collected and decreases the amount of red cell contamina- dure is discontinued prior to completion and the total red
tion in the final product. The disadvantage is that HES cell loss is less than 200 mL, the donor can donate again
is a colloid; it expands the donor’s blood volume and within 8 weeks provided all other donation criteria are
remains in the circulation for extended periods of time. As met. If the red cell loss is greater than 200 mL but less than
a result, caution must be taken to control the amount of 300 mL, the donor should be deferred for 8 weeks. If the
HES given to a donor and its accumulation in the donor’s total red cells lost is greater than 300 mL, the donor must be
circulation. deferred for the full 16 weeks.
2682_Ch13_289-330 22/05/12 11:48 AM Page 307
Once the donor has satisfied requirements of the screening The collection procedure for whole blood is outlined in
process and has been registered, whole blood collection can Box 13–5.
proceed. This procedure must be performed only by trained
personnel working under the direction of a qualified licensed Postdonation Instructions
physician. This section describes donor identification, asep-
tic technique, venipuncture, collection of pilot tubes and Most donor reactions will occur during or shortly after the
whole blood unit, postdonation instructions, and adverse donation. It is recommended that donors remain in an area
donor reactions. where they can be observed by the donation center staff and
be given instructions to follow for the next 24 hours. Most
Donor Identification blood centers have a designated postdonation area where
donors can sit and replenish their fluids. An example of post-
A numeric or alpha numeric system is used to link the donor donation instructions is shown in Figure 13–10.
to the donor record, pilot tubes, blood container, and all
components made from the original collection. Care must Donor Reactions
be taken to avoid duplicate numbers, voided numbers, or
other mistakes in the labeling system. These issues must be Most donors tolerate the withdrawal of a unit of blood without
investigated and kept on record. incident; however, in the event an adverse reaction does occur,
AABB Standards require that the trained phlebotomist the donor room staff must be well trained and able to react
must identify the donor record and ensure that the donor immediately to the donor’s needs. Donor reactions cover a
name and identification numbers match. The phlebotomist wide spectrum, from nervousness and hematomas at the phle-
should ask the donor to state or spell his or her name. At botomy site to convulsions and loss of consciousness. The
this time, the phlebotomist can attach all labels to blood donor staff should be trained in CPR. Reactions can generally
bags, donor record, and pilot tubes. be divided into three categories: mild, moderate, and severe.
Mild Reactions
Aseptic Technique
Reactions in this category encompass one or more of the fol-
For blood collection, most blood centers use an iodine com-
lowing: syncope or fainting, nausea or vomiting, hyperven-
pound such as PVP-iodine or polymer iodine complex. Using
tilation, twitching, and muscle spasm. Syncope may be
a tourniquet or blood pressure cuff, the venipuncture site is
idiopathic or may be brought about by the sight of blood.
identified, and the area is scrubbed at least 4 cm in all directions
The donor may show signs of sweating, dizziness, pallor, or
from the site for a minimum of 30 seconds. The area is then
convulsions. The following instructions apply for a donor
covered with a dry sterile gauze pad until the venipuncture is
who has fainted:
performed. Donors who are allergic or sensitive to iodine com-
pounds may use chlorhexidine gluconate and isopropyl alco- 1. Remove the tourniquet and withdraw needle
hol. All methods must be approved by the FDA (Box 13–4). 2. Place cold compresses on the donor’s forehead
BOX 13–4
BOX 13–5
3. Raise the donor’s legs above the level of the head exhibit a fall in systolic pressure to 60 mm Hg. The following
4. Loosen tight clothing and secure airway instructions apply:
5. Monitor vital signs
1. Check vital signs frequently
Donors who are extremely nervous may exhibit sudden 2. Administer 95% oxygen and 5% carbon dioxide
twitching or muscle spasms. If this happens, try to disengage
the hyperventilation sequence by conversing with the donor Severe Reactions
and having the donor breathe into a paper bag, if necessary.
A donor experiencing convulsions defines a severe reaction.
It is not advised to give oxygen to these donors.
Convulsions can be caused by cerebral ischemia, marked
If the donor starts to feel nauseated or vomits, the follow-
hyperventilation, or epilepsy. The former is associated with
ing instructions apply:
vasovagal syncope or reduced blood flow to the brain owing
1. Instruct the donor to breathe slowly to the shock symptoms, and hyperventilation is caused by
2. Apply cold compresses to the forehead marked depletion of carbon dioxide. The following should
3. Turn the donor’s head to one side and provide an appro- be followed by the donor room personnel:
priate receptacle
1. Call for help immediately; notify blood bank physician
4. The donor may be given water after vomiting has ceased
2. Try and restrain the donor to prevent injury to self or
others
Moderate Reactions 3. Ensure an adequate airway
A moderate reaction can include any of the reactions listed In the event of cardiac or respiratory difficulties, the
above in addition to loss of consciousness. The donor may donor room staff should perform CPR until medical help
have a decreased pulse rate, may hyperventilate, and may arrives.
2682_Ch13_289-330 22/05/12 11:48 AM Page 309
Donor Recommendations
Please read the following instructions and sign at the bottom.
• If you develop a headache and fever of 101oF or higher within 14 days following your donation or if you become diagnosed with
West Nile Virus infection.
• If you have any questions about your donation or if you remember something about your medical or personal history that may
affect your blood donation.
5. Drink more fluids (nonalcoholic) than usual in the next four hours, especially fruit juices.
6. Leave the bandage on for a few hours, and then you may remove it. If there is any bleeding from the puncture site, raise arm, and
apply direct pressure.
7. If you feel faint or dizzy, either lie down or sit down with head between the knees.
8. Do not perform strenuous activities or engage in critical work where safety requires your maximum abilities.
9. If any symptoms persist, either return to blood center or see your doctor.
Signatures
Figure 13–10. Sample postdonation instructions. (LifeSouth Blood Center, Montgomery, AL, with permission.)
2682_Ch13_289-330 22/05/12 11:48 AM Page 310
although available and approved by the FDA, is not and would be indefinitely deferred as a blood donor.
mandated for donor screening and has not been widely Donors who test repeat reactive for HCV screening tests
implemented in the United States at this time. If the initial must be deferred, and the components or products
HBsAg testing is reactive but the confirmatory testing prepared are discarded. If the confirmatory RIBA or NAT
is not (unconfirmed positive), all the current products testing are nonreactive, the donor may be considered
are discarded but the donor need not be permanently for reentry.
deferred, provided the antibody to hepatitis B core (anti-HBc)
testing is also nonreactive. The donor is deferred for Anti-HIV-1/2 and NAT
8 weeks and may be reinstated if the next HBsAg testing is
nonreactive. All donor units must be screened for the presence of the
If the donor unit is needed in an emergency that pre- human immunodeficiency virus (HIV-1/2) antibody using
cludes completion of viral marker testing, a notation an FDA-approved method. If the initial screening test is neg-
indicating testing is not yet completed must be conspicu- ative, the unit is suitable for transfusion; if it is positive, the
ously attached to the unit. If tests are subsequently found test must be repeated in duplicate. If any one of the dupli-
to be reactive or positive, the transfusion service must be cate tests is reactive, the unit must be discarded as well as
notified as soon as possible. any in-date components from prior donations. Screening
tests include EIA, ChLIA, and NAT. NAT testing is run using
Anti-HBc 16 to 24 donation samples per pool. Confirmation tests for
HIV include the Western blot (Wb) and the immunofluo-
Antibody to the core or interior protein on the hepatitis B rescence assay (IFA). Results are expressed as positive, neg-
virus has been implicated in hepatitis C disease. This test ative, or indeterminate.
was once part of surrogate testing, along with its counter-
part alanine transferase (ALT). In 1995, a National Insti- Anti-HTLV-I/II
tutes of Health consensus panel voted to discontinue the
ALT test for blood donors because of the increased sophis- The HTLV-I virus or human T-cell lymphotrophic virus
tication and sensitivity for anti-HCV testing;22 however, type I is the causative agent of adult T-cell leukemia and
testing for anti-HBc has remained a requirement of blood has been associated with a neurological disorder called
donors in the prevention of post-transfusion hepatitis B. HTLV-associated myelopathy. HTLV-II has been shown to
The methods employed are similar to those for HBsAg. have about 60% homology with type I and is prevalent
Presence of HBc antibody in the donor serum suggests among intravenous drug users in the United States.22
the possibility of HBV infection, either acute or chronic. Persons can contract both viruses from transfusion via in-
A positive result for anti-HBc along with a positive HBsAg fected lymphocytes. Screening for HTLV-I began in 1988;
would place the donor in a permanently deferred category. a combined HTLV-I/II was approved 10 years later in 1998.
A positive test for anti-HBc without an accompanying Screening test methodologies include both EIA and ChLIA.
positive test for HBsAg or HBC is not cause for donor In recipients of blood infected by HTLV-I and II, 20% to
deferral unless it occurs on more than one occasion or 60% will develop infection. Myelopathy can occur in a
on two consecutive tests. The products will not be used person infected with these viruses as a result of blood
for transfusion even if the donor is not deferred for a transfusion.23
positive result. As with the other viral markers, a donation that is repeat-
edly reactive may not be used for transfusion. It is recom-
Anti-HCV and NAT mended that if another kit is used by another manufacturer
and that test is also positive, the donor should be indefinitely
The hepatitis C virus (HCV) was identified in 198821 and deferred. Confirmatory tests include Western blot, RIPA, and
was initially referred to as non-A, non-B hepatitis. Screening NAT testing.
tests for anti-HCV involve EIA and ChLIA methods. In 1999,
NAT for HCV RNA was introduced and in 2002 the tests WNV RNA
were licensed by FDA and mandated for routine donor
screening in addition to the EIA or ChLIA methods. NAT is Testing for West Nile virus (WNV) began in 2003, and in
able to detect small amounts of viral nucleic acid in blood 2009 the FDA published their final guidelines for testing
before antibodies or viral proteins such as HCV core antigen whole blood and component donor samples. It is recom-
are detectable by current methods. Implementation of HCV mended that units be tested year-round using either a
NAT testing effectively reduced the window period for mini-pool (MP-NAT) or individual donor (ID-NAT)
detection of HCV by approximately 70%, from a mean of method.41 MP-NAT uses pools of 6 to 16 donor samples
82 days to 25 days. and is routinely used when risk of WNV infection in the
Confirmatory methods include the RIBA (recombinant geographical area is low. A switch to the ID-NAT format is
immunoblot assay) and HCV RNA. The test is reported as recommended when the risk is high. This generally coin-
positive, negative, or indeterminate. An individual who is cides with mosquito season during the summer months.
positive by RIBA is considered to have the HCV antibody Each blood collection facility must define the trigger point
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and have established procedures to switch from MP-NAT retested with each donation. This was reasoned because
to ID-NAT testing and when to switch back again. If a most donors who are infected in the United States have a
mini-pool is reactive, each sample in the pool should be chronic infection that was acquired when residing in a
retested individually. Those that remain nonreactive may country where the disease is endemic. Repeat reactive
be released for transfusion. Any individual units that test donors must be deferred indefinitely and the products quar-
reactive should be discarded, and the donor should be antined and destroyed. No approved confirmatory tests are
deferred from further donations for 120 days. In addition, available, so there are no mechanisms for reentry for de-
any currently in-date products from the same donor during ferred donors. The donor should be notified and counseled,
the previous 120 days should be retrieved and quarantined. and a “look-back” procedure should be performed, looking
The donor should be notified and counseled. Repeat test- back to all donations from that donor for the prior 10 years.
ing with the same or another individual NAT test and a test If the donor had been previously tested with a licensed test,
for WNV antibodies is recommended. “look back” 12 months from the last nonreactive test.
positive for clinically significant RBC antibodies, the neonate febrile transfusion reactions. Examples of BRMs include
must receive blood that does not contain the corresponding proinflammatory cytokines (interleukin-1, interleukin-6,
antigen or is compatible by the antiglobulin crossmatch. and tumor necrosis factor) and complement fragments
The anticoagulant most often used for neonate transfu- (C5a and C3a).26
sions is CPDA-1. A transfusion of 10 mL/kg in a unit with a There are three methods available in prestorage leukore-
hematocrit level of 80% should raise the hemoglobin by duction. In the first method, an in-line filter can be attached
3 g/dL.25 Some institutions, however, have begun using ad- to the whole blood unit and filtered via gravity; RBCs and
ditive solutions for neonatal transfusions when the volume plasma can then be prepared. In the second method, plasma
transfused is minimal. Concerns with additive solutions in- is initially removed from the whole blood unit, and then the
volve the constituents adenine and mannitol and their toxic packed cells are passed through an in-line reduction filter.
effects on the renal system. Most physicians advocating the In both these methods, random-donor platelets cannot be
use of additive solutions do so in the limited setting of small- prepared, as they would have been trapped in the filter. In
volume transfusions. Blood units with additive solutions are the third method, a sterile docking device can be used to at-
contraindicated for use in exchange transfusions. tach a leukocyte reduction filter to a unit of RBCs, which is
allowed to flow via gravity. Today, many institutions maintain
RBCs Irradiated 100% of their red cell inventory as prestored leukoreduced
products.
Patients who are immunocompromised or who are receiving In poststorage leukoreduction, leukocytes are removed in
a bone marrow or stem cell transplant, fetuses undergoing an the blood bank prior to issuing blood or at the bedside before
intrauterine transfusion, and recipients of blood from rela- transfusion. Whereas centrifugation can procure counts less
tives must receive irradiated blood. Irradiation inhibits the than 5 × 108, which can prevent most febrile hemolytic re-
proliferation of T cells and subsequent transfusion-associated actions to RBC concentrates, third-generation filters reduce
graft-versus-host disease. RBCs, platelets, and granulocyte leukocytes to levels of 5 × 106 or lower. Removing leukocytes
concentrates contain viable T lymphocytes that can become by centrifugation or filtration just before transfusion of blood
engrafted when transfused if the host’s immune system is should prevent reactions that are caused by leukocyte anti-
not capable of identifying or defending against the foreign bodies in patient’s plasma and leukocytes present in the
cells. transfused blood; however, it won’t prevent reactions caused
Both the FDA and AABB recommend a minimum dose of by BRMs that originate from the leukocytes present in the
gamma irradiation of 25 Gy to the central portion of the component during storage. Studies suggest that the age of
blood unit, with no less than 15 Gy delivered to any part of the RBC unit is a predictor of a febrile reaction and that the
the blood unit.2 Irradiation is generally performed using cytokine involvement may be cumulative.27
cesium-137 or cobalt-60. To confirm a product was irradi- RBCs that have been frozen, thawed, deglycerolized, and
ated, a radiochromic film label is affixed to the component washed also produce a leukoreduced product. Arnaud and
before it is placed into the metal canister of the irradiator. Meryman28 recently discovered that removing the buffy coat
Darkening of the film confirms irradiation requirements. at the time of collection lowered the level of leukocytes to
Each facility should have a protocol and procedure for acceptable limits (1.9 × 106) after freezing and deglyceroliza-
irradiating blood components, training of personnel using tion and could provide a possible economic alternative to
the irradiator, and issuing of irradiated components. The leukocyte filtration after freezing. This product has a shelf-
expiration date of irradiated RBCs is 28 days from the time life of 24 hours, because it is an open system and requires
of irradiation or the original outdate, whichever is sooner. special equipment to carry out the procedure. Leukoreduced
RBCs have been useful in trying to avoid the following reac-
RBCs Leukoreduced tions associated with products containing leukocytes: febrile
nonhemolytic transfusion reactions; transfusion-related
According to AABB Standards, leukoreduced red cells is a acute lung injury; and transmission of Epstein-Barr virus,
product in which the absolute WBC count in the unit is re- CMV, and human T-cell lymphotrophic virus.
duced to less than 5 × 106 and contains at least 85% of the
original RBC mass.1 There are two major categories of leuko- Frozen, Deglycerolized RBCs
reduced RBCs: prestorage and poststorage.
In prestorage leukoreduction, special filters procure Freezing RBCs with glycerol dates back to the 1950s.29
at least a 99.9% (a 2- to 4-log) removal of leukocytes by Frozen RBCs can be stored for up to 10 years for those pa-
employing multiple layers of polyester or cellulose acetate tients with rare phenotypes, for autologous use, and for the
nonwoven fibers that trap leukocytes and platelets but that military to maintain blood inventories around the world
allow RBCs to flow through. These filters provide a leuko- for U.S. military use. The resulting deglycerolized product is
cyte-reduced product with normal shelf-life and meet free of leukocytes, platelets, and plasma due to the washing
the requirement for 85% retention of original RBCs. The process. Since all donor plasma is deglycerolized when
impetus for prestorage leukoreduction involved biological removed, washed red cells can be used for patients with
response modifiers (BRMs) released from leukocytes dur- paroxysmal nocturnal hemoglobinuria and IgA deficiency
ing storage of the component that were found to promote with circulating anti-IgA.
2682_Ch13_289-330 22/05/12 11:48 AM Page 315
Cryoprotective agents can be categorized as penetrating would hemolyze upon suspension in hypertonic solutions;
and nonpenetrating. A penetrating agent involves small in this case, the cells would be washed in 12% NaCl and then
molecules that cross the cell membrane into the cytoplasm. 0.9% NaCl with 0.2% dextrose, omitting the 1.6% solution.
The osmotic force of the agent prevents water from migrat- Automated continuous-flow instruments can be utilized for
ing outward as extracellular ice is formed, preventing intra- washing. Once the RBCs have been deglycerolized, the unit
cellular dehydration. An example of a penetrating agent is is considered an open system with an expiration date of 24
glycerol. An example of a nonpenetrating agent is hydrox- hours and is stored at 1°C to 6°C.
yethyl starch (HES). This comprises large molecules that do
not enter the cell but instead form a shell around it, pre- Low Glycerol (20% Weight per Volume)
venting loss of water and subsequent dehydration. HES, as In this method, the cryoprotection of the glycerol is minimal,
well as dimethylsulfoxide, is used to freeze hematopoietic and a very rapid, more controlled freezing procedure is re-
progenitor cells. Two procedures used for freezing and quired. Liquid nitrogen (N2) is routinely used for this
deglycerolizing RBCs are the high-glycerol and low-glycerol method. The frozen units must be stored at about –120°C,
methods. The methods differ in the equipment used, the which is the temperature of liquid N2 vapor. Because of the
temperature of storage, and the rate of freezing. Most blood minimal amount of protection by the glycerol, temperature
centers practice the high-glycerol method, which is outlined fluctuations during storage can cause RBC destruction.
in Table 13–2. The quality-control procedures necessary for RBC freez-
ing include all of the standard procedures for monitoring
High Glycerol (40% Weight per Volume) refrigerators, freezers, water baths, dry thaw baths, and cen-
This method increases the cryoprotective power of the glyc- trifuges. They also include procedures to ensure good RBC
erol, thus allowing a slow, uncontrolled freezing process. The recovery (80%), good viability (70% survival at 24 hours
freezer is generally a mechanical freezer that provides storage post-transfusion), and adequate glycerol removal (less than
at −80°C. This particular procedure is probably the one most 1% residual intracellular glycerol; Box 13–7). Valeri and
widely used, because the equipment is fairly simple and the colleagues30 froze RBCs for up to 37 years by using the high-
products require less delicate handling. It does, however, re- glycerol method (40 w/v) and yielded an average RBC re-
quire a larger volume of wash solution for deglycerolization. covery of 75%, with 1% hemolysis. Although researchers are
RBCs are frozen within 6 days of collection when the pre- attempting to freeze cells for more than 10 years, the FDA
servative is CPD or CPDA-1 and up to 42 days when pre- has not yet been swayed to change the maximum storage
served in AS-1, AS-3, and AS-5. AABB Standards1 states that time. Studies have also shown that irradiated (25 Gy) red cells
RBCs must be placed in the freezer within 4 hours of open- that are subsequently frozen and deglycerolized yield similar
ing the system. It is advisable to freeze a sample of donor results in RBC recovery, post-transfusion survival, and resid-
serum in the event additional testing is required for donor ual hemoglobin as nonirradiated units.31 Cells should be
screening. frozen within 6 hours of collection unless they have been
The thawing process takes approximately 30 minutes and rejuvenated. Rejuvenation serves to increase the levels of
involves immersing units into a 37°C waterbath and washing 2,3-DPG and ATP in RBCs stored in citrate/phosphate/dextrose
the RBCs with solutions of decreasing osmolarity (e.g., 12% (CPD) or CPDA-1 using an FDA-approved solution. RBCs
NaCl, 1.6% NaCl, 0.9% NaCl, + 0.2% dextrose). An excep- can be rejuvenated up to 3 days after expiration and then
tion to this rule is a donor with sickle trait in which RBCs glycerolized and frozen.
Table 13–2 Key Steps in Freezing Red Cells Using High-Glycerol Concentration
PREPARATION GLYCEROLIZATION DEGLYCEROLIZATION
Weigh RBCs Place cells on a shaker and add 100 mL glycerol Thaw frozen cells at 37°C in water bath
Adjust to 260–400 g 0.9% NaCl Stop agitation and allow cells to equilibrate 5–30 min Deglycerolize cells using a continuous flow
washer
Prewarm RBCs and glycerol to 25°C
Set glycerol bottles in a water bath for Let partially glycerolized cells flow into freezing bag; Apply a deglycerolize label to transfer pack;
15 min at 25–37°C slowly add glycerol ABO, Rh, WB unit #s and expiration date
Label the freezing bag with name of Maintain glycerolized cells at 24–32°C until ready to Dilute unit with hypertonic 12% NaCl and let
facility, whole blood unit #s, ABO, freeze (not to exceed 4 hours) equilibrate for 5 min
Rh, date collected, date frozen,
cryoprotective agent, expiration,
and “red blood cells frozen”
Freeze at ≤ 65°C Wash with 1.6% NaCl until residual glycerol is
less than 1%; wash with 0.9% NaCl plus
0.2% dextrose; store at 1–6°C
2682_Ch13_289-330 22/05/12 11:48 AM Page 316
for neonates whose counts fall below 50,000/µL and who are
BOX 13–8 experiencing bleeding. Factors that may be associated with
Procedure for Preparing Random-Donor Platelets thrombocytopenia include immaturity of the coagulation
and Plasma from Whole Blood system, platelet dysfunction, increased platelet destruction,
dilution effect secondary to massive transfusion, or exchange
1. Maintain the whole blood at 20°C to 24°C before and during
platelet preparation.
transfusion and intraventricular hemorrhage. Either random
2. Set the centrifuge temperature at 22°C. The rpm and time
or apheresis platelets may be transfused and should increase
must be specifically calculated for each centrifuge. It will the platelet count by 50,000 to 100,000, given a dose of 5 to
generally be a short (2 to 3 minute), light (3,200 rpm) spin. 10 mL/kg.2
This spin should separate most of the RBCs but leave most of A procedure for the preparation of platelet aliquots for
the platelets suspended in the plasma. neonates is outlined in Box 13–9.
3. Platelet preparation should be done in a closed, multibag system.
4. Express off the platelet-rich plasma into one of the satellite bags. Platelets Leukoreduced
Enough plasma must remain on the RBCs to maintain a 70% to
80% hematocrit level. Platelets can be leukoreduced to help prevent febrile non-
5. Seal the tubing between the RBC and the plasma. Disconnect the hemolytic reactions. Random-donor platelets can be leuko-
RBC and store it at 4°C.
reduced by using a leukoreduction filter designed for
6. Recentrifuge the platelet-rich plasma at 22°C using a heavy spin
(approximately 3,600 rpm for 5 minutes). This will separate the platelets. Some apheresis equipment is designed to produce
platelets from the plasma. a leukoreduced product with or without an integrated filter.
7. Express the majority of the plasma into the second satellite bag, Random donor platelets must contain less than 8.3 × 105
leaving approximately 50 to 70 mL on the platelets. The volume leukocytes, and at least 95% of units sampled should meet
is important to maintain the pH above 6.2 during storage. this criterion.1 If random donor platelets have been pooled,
8. Seal the tubing between the bags and separate. Make segments a method must be used that results in a leukocyte count of
for both the platelets and the plasma for testing purposes. less than 5 × 106 in the final pooled product. Single-donor or
9. Allow the platelets to rest undisturbed for 1 to 2 hours at 20°C to apheresis platelets that have been leukoreduced must contain
24°C or until all platelet clumps have been resuspended in the
residual plasma. Be sure the platelet button is covered with the less than 5 × 106 leukocytes in at least 95% of units tested.
plasma. Gentle manipulation can be used if needed.
10. Weigh the plasma bag and determine the volume. Record the Single-Donor Plasma
volume on the bag.
11. Place the plasma in a protective container and freeze. The Frozen plasma from single donors may be made into fresh
plasma must be frozen in such a way that evidence of thawing frozen plasma (FFP), plasma frozen within 24 hours (PF24),
can be determined. Freezing some sort of indentation into the or plasma cryoprecipitate-reduced. Frozen plasma may be
bag, which is visible as long as the plasma remains frozen, is an
easy way to accomplish this. The freezing container is important
because the plastic bag becomes quite brittle when frozen at low
temperatures and can be cracked or broken easily. BOX 13–9
12. The plasma must be frozen solid within 8 hours or 24 hours, de- Preparation of Platelet Aliquots for Neonates
pending on which frozen plasma product is to be made. The rate
of freezing for the plasma will depend on the temperature of the 1. Select a unit to be aliquotted. Either AB or group-specific units
freezer and the amount of air circulation around the plasma. should be selected. CMV-negative or volume-reduced platelets may
13. Before freezing, be sure that any tubing segments and the trans- be requested by the physician. For volume-reduced platelets, when
fusion ports (ears) of the bag are tucked in or placed in such a infants cannot tolerate large intravenous infusions of plasma, stored
manner as to prevent or minimize possible breakage. platelets are centrifuged and the plasma is removed. The platelets
remain undisturbed at room temperature for 20 to 60 minutes
14. The label on the frozen plasma must include all of the standard
before being resuspended in residual plasma.
information. (See the following section on labeling).
2. If platelets are shipped from another facility, they should rotate
15. The plasma can be stored as FFP, single-donor plasma frozen
for at least 30 minutes at room temperature following volume
within 24 hours (PF24), or liquid recovered plasma. Be sure to
reduction.
record the plasma volume on the bag. Shelf-life is 12 months
when stored at –18°C or colder. 3. Remove cap from stopcock apparatus and attach to end of blood
set. Use a filter 170 to 260 µm.
16. Once the platelet concentrate has rested, the unit should be
placed on an agitator to maintain gentle agitation during 4. Remove second cap from stopcock and cap from syringe to be
storage. filled and place them on a piece of sterile gauze. Attach syringe
to stopcock.
17. Platelet concentrate shelf-life is 5 days from the date of collection.
If the system is opened, transfusion must occur within 6 hours. 5. Close the roller clamps at the spike end of the blood set above
The volume, expiration date, and time (if indicated) must be on the filter, and spike the platelet unit.
the label. 6. Open the roller clamp on the spike, and draw the platelets
18. All of the units for a single platelet dose (typically 6 to 8 units through the filter and into the syringe. Remove the syringe from
for an adult) can be pooled into a single bag before transfusion. the filter set, and force the excess air back out of the syringe.
Once pooled, the product must be transfused within 4 hours of 7. Label the syringe properly. Indicate volume of aliquot on platelet
pooling. The pooled unit must be given a unique pool number, product label.
which must be placed on the label. 8. The expiration is 4 hours from the time the unit was spiked.
2682_Ch13_289-330 22/05/12 11:48 AM Page 318
produced from whole blood or apheresis collections. FFP factor V, VII, VIII, and X.24 Thawed plasma is prepared
must be frozen within 8 hours of collection if the anticoag- from FFP and PF24 thawed at 30°C to 37°C and main-
ulant used was CPD, CD2D, or CPDA-1 and within 6 hours tained at 1°C to 6°C for up to 4 days after the initial
if the preservative was ACD. FFP is stored at −18°C or colder 24-hour post-thaw period has elapsed. Thawed plasma
for 1 year or at –65°C for 7 years, with FDA approval. FFP may be indicated in all of the same situations as FFP or
will contain the maximum levels of both stable and labile PF24, and like FFP and PF24, it should not be used to
clotting factors, about 1 international unit (IU) per mL. PF24 treat specific factor deficiencies where other products with
is frozen within 8 to 24 hours of collection and is stored higher factor levels are available, and it should not be used
at –18°C or colder. It contains all stable proteins found in purely as a volume expander. Many institutions now have
FFP, has normal levels of factor V, and has only slightly programs to routinely maintain a thawed plasma inventory
reduced levels of factor VIII. to facilitate emergency and trauma situations and to con-
Both PF24 and FFP are thawed at temperatures between vert all nontransfused FFP or PF24 into thawed plasma to
30°C and 37°C or in an FDA-approved microwave device. If prevent outdate.
a waterbath is used, the product must be placed in a protec- Liquid plasma is separated no later than 5 days after the
tive lining or overwrap so that the ports of the unit are not expiration date of whole blood, is stored at 1°C to 6°C, and
contaminated by contact with the water. Once thawing is can be transfused for up to 5 days after the whole blood’s
complete, the product may be stored at 1°C to 6°C for expiration date. Levels of coagulation factors are poorly
24 hours. If not transfused within the initial 24-hour period, characterized and depend upon storage conditions and
the thawed plasma may be stored for up to 5 days, but the cellular interactions over time. Indications include patients
product label must be changed to “thawed plasma.” A single undergoing massive transfusion with concurrent coagula-
unit of FFP or PF24, from whole blood collection, should tion deficiencies. Because very little blood is stored as whole
contain 150 to 250 mL of plasma, approximately 400 mg of blood, this product is not often produced or available.
fibrinogen, and 1 unit of activity per mL of each of the stable
clotting factors. FFP also contains the same level (1 unit/mL) Cryoprecipitated Antihemophilic Factor
of factors V and VIII. FFP or PF24 prepared from apheresis
collections may contain from 400 to 600 mL. Cryoprecipitate is the cold-precipitated concentration of
The use of FFP or PF24 is indicated in patients who are factor VIII, the antihemophilic factor (AHF). It is prepared
actively bleeding and have multiple clotting factor deficien- from FFP thawed slowly between 1°C and 6°C. The product
cies. Examples may include massive trauma, routine surgical is prepared from a single whole blood unit collected into
bleeding, liver disease, DIC, and when a specific disorder CPDA-1 or CPD and suspended in approximately 15 mL of
cannot be or has not yet been identified. They may also be plasma. The product contains most of the factor VIII and
used when a patient on warfarin must undergo surgery and part of the fibrinogen from the original plasma. It contains
there is not sufficient time for vitamin K to reverse the effect. at least 80 units of AHF activity and at least 150 mg of fib-
Either product may also be used for transfusion or therapeu- rinogen.25 Other significant factors found in cryoprecipitate
tic plasma exchange in patients with thrombotic thrombo- are factor XIII, von Willebrand factor, and fibronectin. Cry-
cytopenic purpura (TTP). FFP or PF24 are contraindicated oprecipitate has a shelf-life of 12 months in the frozen state
in patients with specific, known coagulation disorders that and must be transfused within 6 hours of thawing or within
can be better treated with specific factor concentrates or 4 hours of pooling. Like FFP and PF24, cryoprecipitate
vitamin K and should not be used as a volume expander should be thawed quickly at 37°C. Once cryoprecipitate is
when other, safer products are adequate and available. thawed, the FDA recommends storing at room temperature
Cryoprecipitate-reduced plasma is prepared from FFP (22°C to 24°C) until transfused. Cryoprecipitate is indi-
after thawing and centrifugation to prepare cryoprecipitate. cated in the treatment of factor XIII deficiency, as a source
In addition to removing factor VIII, the process also removes of fibrinogen for hypofibrinogenemia, and as a secondary
fibrinogen, factor XIII, von Willebrand factor (vWF), cryo- line of treatment for classic hemophilia (hemophilia A)
globulin, and fibronectin.24 The resulting cryo-poor plasma and von Willebrand disease. Cryoprecipitate should not be
must be refrozen within 24 hours and stored at –18°C or used to treat hemophilia A or von Willebrand disease if
colder for 1 year from the time of collection. This product virus-inactivated or recombinant factor preparations are
still contains albumin; factors II, V, VII, IX, X, XI; and available.24
ADAMTS13. This product is most often used for transfusion In recent years, cryoprecipitate has also been used to
or plasma exchange in patients with TTP but could also be make fibrin glue, a substance composed of cryoprecipitate
used as a source of those factors that remain. This product (fibrinogen) and topical thrombin. Generally, 1 to 2 units of
cannot be used as a substitute for FFP, PF24, or thawed cryoprecipitate are thawed and drawn into a syringe. Topical
plasma.24 thrombin with or without calcium is drawn into a second
syringe. The contents of the two syringes are simultaneously
Thawed Plasma and Liquid Plasma applied to the bleeding surface, where fibrinogen is con-
verted to fibrin.2 Fibrin glue is also available as commercially
Thawed plasma contains stable coagulation factors such prepared products that are viral-inactivated and licensed to
as fibrinogen and prothrombin but reduced amounts of control bleeding in cardiovascular surgeries.
2682_Ch13_289-330 22/05/12 11:48 AM Page 319
The whole blood donor requirements and preparation re- DNA technology; or monoclonal antibody purification.
quirements for cryoprecipitate are the same as those for Source plasma is defined as plasma collected by plasma-
platelets and FFP. The quality-control requirements mandate pheresis and intended for further manufacture into plasma
that the volume and AHF activity of the final product must derivatives. Recovered plasma is plasma recovered from
be tested on at least 4 units monthly. The volume should not whole blood donations. The plasma is frozen when sent to
exceed 25 mL, and 75% of all units tested must show a min- the manufacturer. The manufacturing process usually begins
imum of 80 IU of AHF activity. Records must be maintained with a separation of the cryoprecipitate from the plasma.
of all quality-assurance testing performed. The cryoprecipitate is then used to produce factor VIII
A procedure for producing cryoprecipitate is outlined in concentrate. The residual plasma is separated into various
Box 13–10. proteins by manipulating the pH, alcohol content, and
temperature and then is viral inactivated by any of several
Granulocyte Concentrates methods, including heat, solvent-detergent treatment, and
nanofiltration.2 Most derivative plasma is also further
See information on leukapheresis in Chapter 14. tested for hepatitis A and parvovirus.
Plasma Derivatives
Activated Factor VII (Factor VIIa)
The following products are different from blood compo-
Activated factor VII (rFVIIa) is produced by recombinant
nents because they are prepared by further manufacture of
DNA technology and has been approved for use in patients
pooled, human source, and recovered plasma; recombinant
with hemophilia A who have circulating antibodies or
inhibitors to factor VIII and in patients with congenital
factor VII deficiency. It has also been used in other situa-
BOX 13–10 tions such as trauma, massive transfusion, and liver trans-
plantation, where bleeding has proved difficult to control
Procedure for Production of Cryoprecipitate
and the patient’s life is threatened. In these other situa-
1. The venipuncture must be nontraumatic. tions, it has been most successful in controlling intracra-
2. The whole blood can be cooled before and during production, nial bleeding in patients with major head trauma and
because platelets are not usually produced along with cryoprecip- cerebral hematomas.2 In addition, the administration of
itate. The volume of plasma required to remain on the RBCs
and the platelet concentrate will reduce the amount of plasma rFVIIa has seen promising results in uncontrolled nonsur-
available for cryoprecipitate production, enough to significantly gical hemorrhages after implanting VADs (ventricular
reduce the final AHF activity in the precipitate. At least 200 mL assist devices) to treat end-stage cardiac failure.33 VADs
of plasma (205 g) should be used to ensure that the final product are frequently used in cardiac surgery procedures if wean-
will contain at least 80 AHF units. ing from cardiopulmonary bypass is refractory due to poor
3. The plasma must be frozen within 8 hours of collection and cardiac performance. The disadvantage to using rFVIIa is
within 1 hour from the time freezing was initiated.
that it has been associated with an increased risk of spon-
4. The second stage of cryoprecipitate preparation begins by
allowing the frozen plasma to thaw slowly in the refrigerator at taneous thrombosis and thromboemboli. In addition, the
1°C to 6°C. This takes 14 to 16 hours when plasma is thawed in product is very expensive and should used with caution
a standard blood bank refrigerator. If a circulating cryoprecipi- and restraint.
tate thaw bath (4°C water bath) is used, the thawing time is One theory for the mechanism of factor VIIa is that
reduced to about 4 hours. The endpoint is when the plasma rFVIIa binds to tissue factor that is released from injured
becomes slushy.
tissue and then activates factor IX and X. Factor X’s pres-
5. Centrifuge the plasma at 4°C for a “hard” spin.
ence on the surface of platelets leads to the generation of
6. Express the supernatant plasma into the attached satellite
bag. The cryoprecipitate will be a small white mass in the thrombin. Studies have shown that full thrombin genera-
original plasma bag. Leave only 10 to 20 mL of plasma on the tion occurred after the addition of up to 150 nm of recom-
precipitate. binant FVIIa.34
7. Separate and refreeze the cryoprecipitate immediately. Time
elapsed should be no more than 1 hour from the time the Factor VIII Concentrates (FVIII)
plasma reaches the slushy stage until the cryoprecipitate is re-
frozen. A delay in refreezing or exposure of the unit to elevated
temperatures during processing will significantly decrease the
Factor VIII concentrates are used to treat patients with
factor VIII activity level in the final product. The centrifuge tem- hemophilia A or classical hemophilia and have almost
perature must be at 4°C, and it is better if the centrifuge cups completely replaced cryoprecipitate as the product of
are well chilled. choice. Factor VIII concentrates may be prepared from
8. The final product should be placed in a protective container because large volumes of pooled plasma, but more commonly they
of the brittle nature of the plastic bag at freezer temperatures. Store are prepared by recombinant DNA technology. If prepared
at –18°C or colder up to 12 months from the date of whole blood
collection.
from pooled plasma, the plasma must be treated by pasteur-
9. If the supernatant plasma is refrozen at –18°C, it must be labeled
ization, solvent/detergent treatment, or monoclonal purifi-
as plasma cryoprecipitate reduced. cation to inactivate or eliminate viral contamination. In
pasteurization, stabilizers such as albumin, sucrose, or
2682_Ch13_289-330 22/05/12 11:48 AM Page 320
glycine are added to the factor VIII concentrate to prevent percentage of the first-generation product (20%).37 To date,
denaturation of the product. The product is heated to 60°C no transmission of hepatitis or HIV has been reported in
for 10 hours. The stabilizers are removed, and the product association with this product. The next-generation native
is lyophilized. This product is safe from HIV-1 and hepatitis recombinant FVIII has included a solvent/detergent step as
transmission. well as a purification step to help ensure a safe and effective
In solvent/detergent treatment, ethyl ether and tri(n-butyl) product.
phosphate and the detergent sodium cholate and Tween 80
are effective in disrupting the viral coat membrane, preventing Factor IX Concentrates
the transmission of lipid-envelope viruses like HIV and hep-
Factor IX concentrates are available in three forms: pro-
atitis B. The solvent and detergent are removed, and the final
thrombin complex concentrates, factor IX concentrates,
product is lyophilized. Combinations of TnBP (tri-n-butyl
and recombinant FIX. The first contains significant levels
phosphate) and polysorbate 20 during the manufacture of
of vitamin K–dependent factors: II, VII, IX, and X. The
FVIII/vWF concentrate Optivate® resulted in inactivation of
complex concentrate is prepared from large volumes of
most enveloped viruses over a wide range of conditions, in-
pooled plasma by absorbing the factors out using barium
cluding solvent/detergent concentration, protein concentra-
sulfate or aluminum hydroxide. The concentrate is then
tion, and temperature.
lyophilized and virally inactivated by methods previously
For monoclonal purification, immunoaffinity chro-
described (e.g., solvent/detergent). The prothrombin complex
matography is used to positively select out the vWF:FVIII
concentrates may contain activated vitamin K–dependent
complex from the plasma pool. Briefly, a murine mono-
factors.
clonal antibody directed at the vWF:FVIII complex is
Factor IX concentrate is developed by monoclonal
bound to a solid-phase matrix. On addition of pooled
antibody purification and is less thrombogenic than pro-
plasma, the complex will attach to the monoclonal anti-
thrombin complex concentrates. This product contains ap-
body. The product is in lyophilized form and is safe from
proximately 20% to 30% of FIX and is stored in the
viral transmission.36
refrigerator in lyophilized form. Prothrombin complex
Porcine Factor VIII concentrates should be used with caution in patients with
liver disease due to reports of DIC and thrombosis.25 This
This xenographic form of factor VIII is made from porcine is most likely due to failure of the liver to produce ade-
plasma and is beneficial for patients with hemophilia A quate amounts of antithrombin III and a decreased hepatic
who have developed inhibitors or antibodies to human clearance of activated factors.
factor VIII. Porcine factor VIII has been shown to provide Recombinant factor IX (rFIX) has been commercially
effective hemostatic control for patients with intermediate available in Europe and in the United States since 1997.
FVIII inhibitor levels. Other studies have shown that resid- It is produced in a Chinese hamster ovary cell line and not
ual porcine vWF in the preparation of the product induces thought to transmit human infectious disease. In 2001,
platelet activation, thus providing a mechanism for en- Roth and colleagues38 published a study evaluating this
hancing hemostasis apart from the action of circulating product in the treatment of patients with hemophilia B.
FVIII.35 Based on this study and other data accumulated by physi-
cians using rFIX for hemophilia B patients, the Committee
Recombinant Factor VIII
for Proprietary Medicinal Products (CPMP) considered
The gene for FVIII was sequenced nearly 16 years ago and the benefit–risk balance for rFIX for the treatment and
led to the production of recombinant human FVIII prophylaxis of bleeding in previously treated patients with
(rFVIII). The first generation rFVIII products are synthe- hemophilia B to be favorable. However, there were con-
sized by introducing human FVIII gene into BHK (baby cerns regarding some aspects of the study, as there was
hamster kidney) cells. The rFVIII is released into culture the possibility of inhibitors to rFIX and allergic reactions
medium and harvested, isolated, and purified using a com- to this product. As a result, a new clinical trial will be
bination of ion-exchange chromatography, gel filtration, conducted.
and immunoaffinity chromatography. The purification and
final formulation of rFVIII (BHK) uses human albumin Factor XIII Concentrates
as a stabilizer. The next-generation product is referred to
as rFVIII:FS; it is formulated using sucrose as a final Factor XIII deficiency is a severe autosomal-recessive
stabilizer instead of albumin and is available in three doses bleeding disorder associated with a characteristic pattern
(250, 500, and 1,000 IU). of neonatal hemorrhage and a lifelong bleeding diathesis.
When compared with the first-generation rFVIII, rFVIII-FS There are currently two plasma-derived virus-inactivated
demonstrated a predictable clinical hemostatic response factor XIII concentrates. One is available in Europe,
with an efficacy among previously treated patients and pre- South America, South Africa, Japan, and the United States
viously untreated patients with hemophilia A. In one study as an investigational new drug under the FDA. The sec-
with previously untreated patients, inhibitors to rFVIII-FS ond is available only on a “named patient” basis in
were present in 15% of patients, which was lower than the the UK.
2682_Ch13_289-330 22/05/12 11:48 AM Page 321
Immune Serum Globulin The FDA has approved two doses for treatment of ITP
and immunization against the D antigen: a 120-µg dose and
Immune serum globulin is a concentrate of plasma gamma a 300-µg dose.25,39 These are IV preparations that have been
globulins in an aqueous solution. It is prepared from heat-treated. An IM preparation is available as a 50-µg dose
pooled plasma by cold ethanol fractionation. Preparations and a 300-µg dose. The latter is considered a full dose pro-
are in the form of intravenous (IV) or intramuscular (IM) tective against 15 mL of D-positive RBCs.
solutions. The IV preparation generally contains more IgG In preventing immunization to the D antigen during
protein than the IM preparation, with a half-life of 18 to gestation, a number of scenarios with varying dosages
32 days. apply. During the first 12 weeks of pregnancy, a 50-µg dose
Immune globulin preparations are indicated for patients of RhIg is indicated for D-negative females for abortion or
with immunodeficiency diseases (i.e., severe combined im- miscarriage. After 12 weeks’ gestation, a full dose (300 µg)
munodeficiency and Wiskott-Aldrich syndrome) and for is indicated for abortion or miscarriage in D-negative
providing passive antibody prophylaxis against hepatitis women. The 120-µg dose is advised after 34 weeks’ gestation
and herpes. IVIg is also used in patients with idiopathic when amniocentesis is performed or in the event of obstetric
thrombocytopenic purpura, post-transfusion purpura, complication or following termination of pregnancy.
HIV-related thrombocytopenia, and neonatal alloimmune An antepartum dose (300 µg IM or IV) should be
thrombocytopenia. Individuals with a history of IgA defi- given to nonimmunized D-negative females at 28 weeks’
ciency or anaphylactic reactions should not receive im- gestation. Following delivery, a postpartum blood sample
mune globulin because of the presence of trace amounts is drawn from the mother. The sample undergoes a screen-
of IgA. ing test for fetomaternal hemorrhage (FMH) in which
Whereas there are no documented cases of HIV or hepa- D-positive RBCs from the newborn are detected. Addition-
titis B transmission, transmission of hepatitis C has been re- ally, the newborn’s Rh status is determined. If the newborn
ported with IVIg preparations. This has led to preparations is D-positive or if the Rh type cannot be determined on the
of immune globulin treated with solvent/detergent for viral newborn (i.e., positive DAT), the mother should receive a
inactivation. full dose of RhIg unless she has demonstrated previous ac-
tive immunization to the D antigen. If the screening test
Normal Serum Albumin (NSA) is negative for the presence of D-positive RBCs of fetal ori-
gin, the mother should receive a full dose of RhIg within
NSA is prepared from salvaged plasma, pooled and frac- 72 hours of delivery. If the screening test is positive, the
tionated by a cold alcohol process, then treated with heat FMH must be quantified using the Kleihauer-Betke test.
inactivation (60°C for 10 hours), which removes the risk RhIg is also used in the event Rh-positive platelet concen-
of hepatitis or HIV infection. It is composed of 96% albu- trates are transfused to an Rh-negative patient. A 300-µg
min and 4% globulin. It is available in 25% or 5% solu- dose IM (120-µg dose IV) is sufficient to protect against
tions. NSA is indicated in patients who are hypovolemic D-positive RBCs contained in up to 10 units of random
and hypoproteinemic and in clinical settings for shock and platelets or 1 pheresis product.
burn patients. The 25% preparation is contraindicated
in patients who are dehydrated, unless it is followed Synthetic Volume Expanders
with crystalloid infusions (e.g., normal saline) for volume
expansion. There are two categories of the synthetic volume expanders:
crystalloids and colloids. Ringer’s lactate and normal isotonic
Plasma Protein Fraction saline comprise the crystalloids; dextran and HES make up
the colloid solutions. Normal saline consists only of sodium
The preparation of plasma protein fraction (PPF) is similar and chloride ions; Ringer’s lactate consists of sodium, chlo-
to that of NSA, with fewer purification steps. PPF contains ride, potassium, calcium, and lactate ions. These solutions
83% albumin and 17% globulins. PPF is available in a 5% are useful in burn patients because of their ability to rapidly
preparation; its uses parallel that of NSA. PPF, however, is cross the capillary membrane and increase the plasma vol-
contraindicated for infusion during cardiopulmonary bypass ume. Colloids are used as volume expanders in hemorrhagic
procedures. Both PPF and NSA can be stored for 5 years at shock and burn patients. Dextran is prepared in a 6% and
2°C to 10°C and have not been reported to transmit HIV or 10% solution with a half-life of 6 hours. HES is available in
hepatitis. a 6% solution with an IV half-life of more than 24 hours.
Both colloids and crystalloids are free from viral transmis-
Rho(D) Immune Globulin sion. Table 13–3 provides a comparison of crystalloids and
colloids.
Rh immune globulin (RhIg) is a solution of concentrated
anti-Rho(D). It is prepared from pooled human plasma of Antithrombin III Concentrates
patients who have been hyperimmunized and contains
predominantly IgG anti-D. RhIg has two primary uses: Antithrombin III (AT-III) concentrates, or antithrombin
treatment of ITP and prevention of Rh HDN. (AT) as it is now called, is prepared from pooled human
2682_Ch13_289-330 22/05/12 11:48 AM Page 322
Hydroxyethyl starch
plasma and heat-treated to prevent viral transmission. This of Blood and Blood Components supporting Codabar in
product, pdAT, has been approved in the United States for the United States.
treating patients with hereditary AT deficiency in connec- As time progressed and complexities of the transfusion
tion with surgical or obstetrical procedures or for those medicine industry heightened, the ISBT working group de-
suffering from thromboembolism.40 AT-III is an inhibitor signed a totally new system based upon the bar code symbol-
of clotting factors IX, X, XI, XII, and thrombin. Patients ogy known as Code 128. This allowed for multidimensional
with plasma levels of AT-III less than 50% of normal are at bar codes and nonrepetitiveness of the DIN (donor identifi-
risk of thrombosis. cation number) in not less than 100 years. ISBT 128 is man-
A new recombinant AT (rhAT) concentrate produced aged by ICCBBA (International Council for Commonality in
using transgenic technology has been developed on a Blood Banking Automation). ICCBBA was established in
compassionate-use basis. It is produced by transgenic goats 1994 and is a not-for-profit organization whose mission is to
expressing recombinant human AT in their milk, under the support ISBT 128 and assist in its implementation from blood
control of the beta-casein promoter. It is purified from the establishments. Each blood center or transfusion service must
milk and concentrated. Initial studies using rhAT have indi- register with ICCBBA when using ISBT 128 coding labels.
cated effective support for AT-deficient patients who undergo The donor identification number (DIN) is comprised of
surgery and that it is a suitable alternative to pdAT. No anti- 14 characters that contain information relating to the coun-
bodies have been produced against rhAT in studies to date, try, the center of origin, the year of collection, a sequential
and adverse events, which are minimal, include sponta- number, and a check character. Each ISBT 128 donation
neously resolving skin hyperpigmentation at the site of drug number is unique on a worldwide basis. Each blood bank or
infusion. Table 13–4 provides a comprehensive list of blood transfusion service should have its own protocol for labeling
component characteristics. components. The original unit, its components, or any mod-
ifications thereof must be identified; serologic results of the
Labeling of Components unit must be reviewed and the appropriate labels attached
in such a way that is clear and readable to the naked eye. If a
Once the component has been made, it must be labeled in change must be made to the unit, such as a modification of
accordance with AABB Standards, FDA regulations, and the expiration date, the handwritten change must be legible.
ISBT (International Society of Blood Transfusion) Code 128. The unique identifier of the unit, the ABO and Rh type,
The latter system is an adaptation of the conventional expiration date, and component labels must be checked with
coding system known as Code 128. This code has been a second person. There must be a method in place linking
adapted for use in blood transfusion services throughout the respective donor to the unit. The donor must be classi-
the world by the ISBT—hence ISBT 128. The precursor to fied as autologous (Fig. 13–11) or volunteer (Fig. 13–12).
ISBT 128 was Codabar. In the 1970s, the Committee for The maximum number of unique identifiers that may be af-
Commonality in Blood Banking Automation appointed fixed to the unit is two; this may be in numeric or alphanu-
by the American Blood Commission (ABC) published a meric form. If the unit is shipped to a transfusion facility that
seven-volume report for labeling recommendations leading applies its own unique identifier for the unit, the original
to adoption of ABC Codabar to improve and simplify identifier of the collecting facility must not be removed.
the labeling of blood and its components. In 1985, the There must be a method in place for tracing the unit from
FDA published the Guideline for the Uniform Labeling its origin to its final disposition.
2682_Ch13_289-330 22/05/12 11:48 AM Page 323
Table 13–4 Blood Component Characteristics
STORAGE QUALITY INDICATIONS TRANSFUSION
COMPONENT SHELF-LIFE TEMPERATURE CONTROL VOLUME FOR USE CONTENT DOSAGE CRITERIA
Whole blood CPD-21 d CPDA-1 1–6°C Hct approx. 40% 450–500 mL Volume expansion, RBC ↑ hgb 1g/dL ABO, Rh
35 d ↑ O2 Plasma ↑ hct 3%
CP2D-21 d Platelets
ACD-21 d WBCs
Whole blood Original expira- 1–6°C 25 Gy to center of 450–500 mL Prevent GVHD RBC ↑ hgb 1 g/dL ABO, Rh
irradiated tion or 28 days canister volume expan- Plasma ↑ hct 3%
from irradiation sion ↑ O2 Platelets
RBCs CPD-21 d CPDA-1 1–6°C Hct ≤ 80% 250–300 mL ↑ O2 RBC ↑ hgb 1 g/dL ABO, Rh
35 d ↑ hct 3%
CP2D-21 d
ACD-21 d
AS-42 d
RBC aliquots CPDA-1 35 d 1–6°C Hct ≤ 80% varies ↑ O2 RBC 10 mL/kg ABO, Rh
(closed system) ↑ hgb 2 g/dL
RBC leukoreduced Closed system: 1–6°C < 5 × 106 250–300 mL Febrile rxn, ↑ O2 RBC ↑ hgb 1 g/dL ABO, Rh
same WBCs ≥ 85% Few platelets; ↑ hct 3%
Open system: RBC recovery residual plasma
24 hours
Washed RBCs 24 hours 1–6°C Hct 70–80% 180 mL IgA-negative RBC ↑ hgb 1 g/dL ABO, Rh
persons PNH WBC < 5 × 108 ↑ 3%
RBC deglycerolized 24 hr 1–6°C 80% RBC recovery 180 mL Rare phenotypes RBC ↑ hgb 1 g/dL ABO, Rh
< 1% glycerol ↑ O2 Saline ↑ hct 3%
< 300 mg hgb Dextrose < 1%
WBC, platelets
323
2682_Ch13_289-330 22/05/12 11:48 AM Page 324
324
PART III
Transfusion Practice
Table 13–4 Blood Component Characteristics—cont’d
STORAGE QUALITY INDICATIONS TRANSFUSION
COMPONENT SHELF-LIFE TEMPERATURE CONTROL VOLUME FOR USE CONTENT DOSAGE CRITERIA
Platelets, SD 5d 20–24°C ≥ 3 × 1011 200–400 mL Platelet Platelets ↑30k–60k/µL HLA compatible
pH ≥ 6.2 refractoriness
Platelets, irradiated 5d 20–24°C 25 Gy to center of Same Prevent GVHD Platelets Same Same
canister
Platelets, 5d 20–24°C < 5 × 106 SD pH≥ 6.2 Febrile rxns Platelets RD: ↑ 5–10k SD: HLA
leukoreduced < 8.3 × 105 RD SD: ↑ 30–60k
FFP 1 yr –18°C 8 hr CPD, CPDA-1, 200–250 mL Coagulation 1 U/mL clotting ↑Factor 20–30% ABO
7 yr –65°C CP2D 6 hr ACD deficiency factors 10–20 mL/kg
Liver disease
DIC
Massive trx
SDP 1–6°C liquid 5 days after WB Frozen 6 hr 150–250 mL Stabile clotting Same Same
expiration factors
Frozen: 5 yr
Cryoprecipitate Frozen: 1 yr –18°C FVIII:C 80 IU 10–25 mL Hemophilia A FVIII:C (80–120U) ↑Fibrinogen ABO
Thawed: 6 hr 20–24°C VWD FXIII VWF (40–70%) 5–10 mg/dL
Pooled: 4 hr deficiency FXIII (20–30%)
Fibrin sealant Fibrinogen
Hypofibrinogene- (150 mg/dL)
mia
FVIII concentrates Check vial 1–6°C 10–30 mL Hemophilia A FVIII 1U FVIII/kg body wt Reconstitute before
Trace other ↑ 2% infusion
clotting factors
FIX concentrates Check vial 1–6°C 20–30 mL Hemophilia B FIX 1U FIX/kg body wt Reconstitute before
Trace other ↑ 1.5% infusion
clotting factors
2682_Ch13_289-330 22/05/12 11:48 AM Page 325
Granulocytes 24 hr 20–24°C ≥ 1.0 × 1010 200–600 mL Neutropenia < 500 WBC 1–2 × 1010/ ABO, Rh, HLA
PMN/uL RBC infusion four
Plts plasma daily doses
Granulocytes, 24 hr 20–24°C ≥ 1.0 × 1010 200–600 mL Prevent GVHD Same Same Same
irradiated neutropenia
325
2682_Ch13_289-330 22/05/12 11:48 AM Page 326
Case 13-2
SUMMARY CHART
An allogeneic blood donor should weigh at least Postoperative salvage is an autologous donation in
110 lb (50 kg). which a drainage tube is placed in the surgical site
The pulse rate of a potential blood donor should be be- and postoperative bleeding is salvaged, cleaned, and
tween 50 and 100 beats per minute. reinfused.
The hemoglobin/hematocrit level of an allogeneic All whole blood units should be stored at 1°C to 6°C;
blood donor should be at least 12.5/38%. those units destined for platelet production should
A donor must be permanently deferred if he or she be stored at 20°C to 24°C until platelets have been
has had a confirmed positive test for HBsAg after the removed.
11th birthday. Donor units must be tested for the following viral
The deferral period for persons who have been treated markers: STS, anti-HIV-1/2, HIV-antigen, anti-HTLV
for malaria is 3 years following therapy. I/II, HBsAg, anti-HBc, and anti-HCV.
Persons who have had a blood transfusion are deferred RBCs must be prepared by a method that separates the
for 12 months owing to risk of exposure to hepatitis, RBCs from the plasma and results in a hematocrit level
HIV, or other viral diseases. of less than or equal to 80%.
A platelet pheresis donor should not have taken aspirin Irradiated RBCs must be given a radiation dose of at
for 3 days before donation because it decreases platelet least 25 Gy to the midplane of the canister, after which
function. the expiration date of the product changes to 28 days
from the time of irradiation or maintains the original
The interval between whole blood donations is
outdate, whichever comes first.
8 weeks or 56 days.
A person with a history of hemophilia A or B, von
Leukocyte-reduced RBCs are products in which the
absolute leukocyte count is less than 5 × 106.
Willebrand disease, or severe thrombocytopenia must
be permanently deferred from donating blood. Random-donor platelets must contain at least 5.5 ×
1010 platelets; single-donor platelets must contain at
Attenuated live viral vaccines such as smallpox,
least 3 × 1011 platelets; each carries a shelf-life of
measles, mumps, yellow fever, and influenza (live
5 days.
virus) carry a 2-week deferral.
Attenuated live viral vaccines such as German measles
FFP must be prepared within 8 hours of collection
for CPD, CPDA-1, and CP2D; it is stored at –18°C for
(rubella) and chickenpox (varicella zoster) carry a
12 months.
4-week deferral.
A blood donor who has a positive serologic test for
Cryoprecipitate is prepared from FFP and contains
at least 80 units of antihemophilic factor and
syphilis must be deferred for 12 months.
150 to 250 mg of fibrinogen; this product is indi-
Donors who have tested positive for the HIV antibody cated for hemophilia A, factor XIII deficiency, and
must be indefinitely deferred. hypofibrinogenemia.
Predeposit autologous donation refers to blood for the RhIg is a solution of concentrated anti-Rho(D), which
donor-patient that is drawn before an anticipated is manufactured from pooled hyperimmunized donor
transfusion (e.g., surgery) and stored until use. plasma. It is used to prevent Rho(D) immunization of
An autologous donor must have a hemoglobin of at an unsensitized Rh-negative mother after an abortion,
least 11 g/dL and a hematocrit level of at least 33%. miscarriage, amniocentesis, or delivery of an Rh-
Intraoperative autologous transfusion occurs when positive or Rh-unknown infant.
blood is collected during a surgical procedure and is One unit of random-donor platelets typically increases
usually reinfused immediately. the platelet count in a 70-kg adult by 5,000 to
Acute normovolemic hemodilution takes place in the 10,000/µL; 1 unit of apheresis platelets should increase
operating room when 1 to 3 units of whole blood are the platelet count in a 70-kg adult by 30,000 to
collected and the patient’s volume is replaced with col- 60,000/µL.
loid or crystalloid. The blood is reinfused during the
surgical procedure.
2682_Ch13_289-330 22/05/12 11:48 AM Page 328
15. Prothrombin complex concentrates are used to treat 6. Friedland, GH, et al: Lack of transmission of HTLV-III/LAV in-
which of the following? fection to household contacts of patients with AIDS or AIDS
related complex with oral candidiasis. N Engl J Med 314:344,
a. Factor IX deficiency 1986.
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of current and recent inmates of correctional institutions as
d. There is no difference in testing.
donors of whole blood, blood components, source leukocytes,
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than ______ and retain at least ______ of original 12. Food and Drug Administration. Guidance for Industry. Revised
RBCs. preventive measures to reduce the possible risk of transmission
a. 8 × 106/85% of Creutzfeldt-Jakob disease (CJD) and variant Creutzfeldt-
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Available at www.fda.gov/cber/guidelines.htm.
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c. 5 × 106
guidelines.htm.
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following treatment with porcine factor VIII (HYATE:C). Am J
Hematol 69:192, 2002.
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Chapter 14
Apheresis
Beth A. Hartwell, MD, MT(ASCP), SBB and Paul J. Eastvold, MD, MT(ASCP)
OBJECTIVES
1. Define apheresis and describe the physiology of the process.
2. Define leukapheresis, plateletpheresis, plasmapheresis, and erythrocytapheresis.
3. Compare and contrast the procedures of continuous flow centrifugation and intermittent flow centrifugation.
4. Describe the use of membrane technology for the collection of plasma.
5. List the components that can be collected using apheresis technology.
6. State the regulatory requirements for apheresis donations, including the frequency of donation.
7. Explain the rationale for the basic types of therapeutic apheresis.
8. List the indications for therapeutic apheresis, differentiating between conditions requiring plasma exchange and those requiring
cytapheresis.
9. List the different types of adsorbents and their clinical application.
10. Identify the factors that can be removed by plasmapheresis.
11. Describe the use of cytapheresis to collect hematopoietic progenitor (stem) cells.
12. Identify the possible adverse effects of apheresis.
platelets or red blood cells. This increases the ability to pro- Table 14–1 Types of Apheresis Procedures
duce the optimal components for patients and prevents and Their Application
wastage.
The use of apheresis on a therapeutic basis permits the COMPONENT
removal of disease-causing or unwanted cellular or plasma PROCEDURE REMOVED APPLICATION
constituents from a patient. Importantly, apheresis is also Donor Patient
used to harvest stem cells from the peripheral blood of
Plasmapheresis Plasma √ √
donors and patients, avoiding the need for extraction from
the bone marrow. Apheresis technology continues to evolve, Plateletpheresis Platelets √ √
and the procedure is now commonplace in blood donor
Leukapheresis White blood √* √**
centers and many hospitals and acute care settings.1 cells (WBC)
particular blood component for therapeutic purposes (ther- "Buffy Coat" Lymphocytes Basophils
White Blood Cells Granulocytes Neutrophils
apeutic apheresis).3 The process of removing plasma is Platelets Monocytes Eosinophils
termed plasmapheresis. In a similar manner, platelets
(plateletpheresis), red blood cells (erythrocytapheresis), or Red Blood Cells
leukocytes (leukapheresis) can be removed (or collected)
using apheresis technology. Table 14–1 outlines the types of
apheresis procedures and their applications. Figure 14–1. Sedimented blood sample.
2682_Ch14_331-351 22/05/12 11:48 AM Page 333
A B
Figure 14–6. (A) The COBE Spectra apheresis system. (B) The separation chamber uses a unique asymmetric design to minimize contamination from RBCs and WBCs.
(Courtesy CaridianBCT, Lakewood, CO.)
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2RBC 16 weeks
Table 14–3 Examples of Instruments Used for Donor Apheresis and Component(s) Collected
INSTRUMENT 2RBC PLASMA PLATELETS WBC COMBINATION
Haemonetics
• MCS+ LN8150 √ RBC/P
• MCS+ LN9000 √ PLT/P
• PCS-2 √
• Cymbal √
CaridianBCT
• COBE Spectra √ √ PLT/P
• Trima Accel √ √ √ RBC/PLT/P
Fenwal
• ALYX √ RBC/P
• Amicus √ RBC/PLT/P
• Autopheresis C √
Fresenius AS104 √ √ √
RBC = red blood cells; WBC = white blood cells (granulocytes); PLT = platelets; P = plasma
RBCs collected by apheresis are typically collected as a dou- Collection of plasma by apheresis is termed plasmapheresis.
ble unit (termed a 2RBC or double RBC procedure). Depend- In a plasmapheresis procedure, whole blood from the donor
ing on the instrument used, the plasma and platelets are is centrifuged, the plasma is diverted into a collection bag,
returned to the donor, or one or both of these components and the cellular components (RBCs, platelets, WBCs) are
may be collected as a concurrent apheresis product. A clini- returned to the donor. This allows a larger volume of plasma
cal advantage to the collection of apheresis RBCs is reduced to be collected from a donor, such that each apheresis unit
donor exposure for the recipient since the patient can (“jumbo” plasma) is the volume equivalent of at least two
potentially receive two units from the same individual. whole-blood-derived plasma units. In addition to direct
An FDA guidance issued in 2001 requires the collection patient use, collection of apheresis plasma can serve a variety
facility to follow specific donor selection criteria outlined of purposes. It can be used to augment the inventory of fresh
in each apheresis instrument manufacturer’s operator’s frozen plasma (FFP) of a particular ABO group, especially
manual.16 Donors must meet the appropriate collection cri- group AB. Plasma can be collected from donors with high
teria for a whole blood donation; however, many instruments titers of antibodies directed against specific infectious agents
have minimum gender-specific height and weight standards (hepatitis B, cytomegalovirus, varicella zoster) to prepare
as well. Since the volume of RBCs being collected during a immune globulin. These preparations are used to provide
2RBC procedure is greater than it would be for a whole blood prophylaxis against infectious organisms in exposed individ-
donation, the requirements for donor hematocrit are more uals. Finally, apheresis is used commercially to collect plasma
stringent. The hematocrit must be at least 40% regardless of for further manufacturing into such products as intravenous
gender, and the level (hemoglobin or hematocrit) must be immune globulin (IVIG), hepatitis immune globulin, and
determined by a quantitative method; the use of copper Rh-immune globulin.
sulfate is not acceptable.9,16 For donor purposes, collection is divided into frequent
If two units of RBCs are collected by apheresis, the donor and infrequent plasmapheresis. With infrequent plasma-
must wait 16 weeks before providing another donation that pheresis, donation occurs no more than once every 4 weeks,
includes RBCs. If one RBC and one plasma and/or platelet and the donor requirements are the same as for whole
unit are collected, the donor must wait 56 days before blood.17 With frequent, or serial, plasmapheresis, donation
donating another red cell product. These procedures may be occurs more frequently than once every 4 weeks. There must
performed on both allogeneic and autologous donors. The be at least 2 days between procedures and no more than two
RBC apheresis procedure is well tolerated by donors, and procedures in a 7-day period. In addition, these donors must
several collection facilities have noted a decreased incidence be evaluated periodically by a physician and must undergo
of donor reactions compared with collection of whole specific laboratory testing (total protein and serum protein
blood.9 Some of this may be attributed to the infusion of electrophoresis or measurement of immunoglobulin levels).9
saline during the procedure to replace lost volume. RBC loss must not be greater than 25 mL per week. FDA
2682_Ch14_331-351 22/05/12 11:49 AM Page 339
guidelines recommend 12 L (14.4 L for donors weighing circumstances (HLA-matched platelets for a specific patient)
more than 175 pounds) as the maximum allowable plasma but must be approved by the blood bank medical director.
volume donated per year.17 Finally, the total volume of plasma collected during any one
procedure cannot exceed 500 mL (or 600 mL if the donor
Platelets weighs 175 lb or more) or the volume of plasma cleared by
Platelets obtained by an apheresis procedure provide the the FDA for the instrument.9,20
equivalent of six to eight whole-blood-derived platelets Apheresis platelets produced on newer instruments are
(random-donor platelets). This significantly decreases the typically leukocyte-reduced and contain less than 5 x 106
donor exposure for a patient. In a plateletpheresis procedure, WBCs per unit, which meets the regulatory guidelines
platelets along with a portion of plasma are removed, and established by the FDA and the AABB.9,20
the remaining RBCs, WBCs, and majority of the plasma are Granulocytes
returned to the donor. The platelets are suspended in donor
plasma in a collection bag specifically designed for platelet The patient population that can benefit from granulocyte
storage. A routine plateletpheresis procedure typically takes transfusions is very limited.22,23 Patients who undergo
45 to 90 minutes. Like other donor apheresis procedures, aggressive chemotherapy may develop profound neutropenia
additional blood components may be collected concurrently, during the course of their treatment. The decrease in neu-
including a single RBC and/or plasma product. Furthermore, trophils places these patients at risk for acquiring bacterial
depending on the donor’s platelet count and the apheresis and fungal infections, which may become life-threatening.
instrument used, a high-yield product can be obtained that Granulocyte transfusions have been shown to be beneficial
is subsequently divided into two or three platelet products, in some severely neutropenic patients (neutrophil count less
each containing an acceptable number of platelets.18,19 than 500/µL) who meet the following criteria: documented
Donor selection criteria for the plateletpheresis donor are (clinically or by culture) infection for 24 to 48 hours that is
the same as for whole blood donation, with two additional unresponsive to standard antibiotic or antifungal therapy,
requirements.20 Prior to each plateletpheresis procedure, a bone marrow demonstrates myeloid hypoplasia, and there is
sample must be collected to determine the donor’s platelet a reasonable chance of bone marrow recovery (i.e., the neu-
count. The regulations concerning donor qualification can tropenia is reversible).24,25 Granulocyte transfusions have
be somewhat confusing. The platelet count must be at least also shown favorable results in the treatment of neutropenic
150,000/µL in order to provide an adequate platelet collec- neonates with sepsis.26,27
tion and for the donor to safely undergo the collection pro- Although granulocytes can be prepared from a whole
cedure. If the donor’s platelet count is less than 150,000/µL, blood donation,28 the yield is insufficient for treating a
he or she is deferred from platelet donation until a subse- pediatric or adult patient. Collection of granulocytes by
quent count is at least 150,000/µL. In some collection apheresis provides a higher yield product.29 Apheresis
centers, the platelet count can be measured immediately and collection requires close communication between the blood
used to qualify the donor. However, if the platelet count is center or apheresis center, the blood bank physician, and the
not immediately available, then the most recent platelet patient’s physician. Since granulocytes must be transfused
count can be used for qualification.21 If it is the initial as soon as possible after collection for optimal therapeutic
plateletpheresis collection for the donor, or if 4 weeks have effectiveness, typically there is insufficient time to perform
elapsed since the prior platelet donation, it is not necessary all infectious disease testing on the donor. Therefore,
that the platelet count be determined prior to beginning advance planning can allow one or more donors to be pre-
the procedure as long as it is evaluated after the platelet screened prior to the actual collection procedure.
collection. A minimum therapeutic dose is 1 × 1010 granulocytes per
Plateletpheresis collections should not be performed on day.24,25 The granulocyte yield is influenced by the donor’s
potential donors taking medications that interfere with neutrophil count and the collection process. In general, most
platelet function, as this would result in production of a collection facilities stimulate the donor with corticosteroids
suboptimal and therapeutically ineffective patient product. or a colony stimulating factor prior to the procedure and
Antiplatelet medications have differing deferral time periods: utilize a red cell sedimenting agent during the collection
48 hours for aspirin, aspirin-containing medications, and the process.30
anti-inflammatory drug Feldene, and 14 days for clopidogrel During centrifugation of whole blood, granulocytes are
(Plavix), ticlopidine (Ticlid), ticagrelor (Brilinta), and pra- found in the buffy coat between the RBC and plasma layers
sugrel (Effient).9 (see Fig. 14–2). Adding the red cell sedimenting agent,
The interval between plateletpheresis procedures must be hydroxyethyl starch (HES), allows better separation of
at least 2 days with no more than two procedures in a 7-day layers, resulting in an improved yield with reduced RBC con-
period. However, to protect the donor, if a double or triple tamination.31,32 Since HES is added directly to the apheresis
apheresis platelet is collected, 7 (rather than 2) days must circuit, some amount will enter the donor’s circulation dur-
elapse before the donor is again eligible to provide apheresis ing the collection procedure and ultimately be removed by
platelets. A donor may undergo no more than 24 platelet- the reticuloendothelial system. Immediate side effects of HES
pheresis procedures in a rolling 12-month period. Excep- are due to its colloid properties, including circulatory volume
tions to any of these time periods can be made in unusual expansion, with headaches and peripheral edema. Residual
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HES has been reported in donors up to 1 year after granulo- plasma when the pathological substance is found in the cir-
cyte apheresis; therefore, collection facilities are required to culation. Therapeutic apheresis has become an accepted
control the maximum cumulative dose of HES (or any other and standard therapy for many hematologic, neurological,
sedimenting agent) given to a donor during a specified time renal, metabolic, autoimmune, and rheumatic diseases,
interval.9 among others.33,34
A number of granulocytes are normally present in the
marginal, or noncirculating, pool. The administration of oral General Considerations
corticosteroids, such as prednisone or dexamethasone, can
mobilize these granulocytes and significantly increase the Therapeutic apheresis has placed blood banks and transfu-
number of circulating granulocytes. The use of steroids in sion services in the position of providing direct medical care
donors prior to granulocyte collection may exacerbate cer- for a patient. This situation has necessitated a change in the
tain medical conditions, such as diabetes, peptic ulcer, or perspective of the medical director and the technical staff.
hypertension, and should be used under the guidance of the Clearly defined policies must delineate the responsibility of
blood bank physician.26 the blood bank and attending physicians; typically the blood
The administration of granulocyte colony-stimulating bank physician provides TA as a consultative service. It is
factor (GCSF), a recombinant hematopoietic growth factor, imperative that the blood bank medical director be closely
to granulocyte donors has resulted in marked increases in involved with the attending physician in deciding whether
granulocyte yield. Although mild side effects, such as muscle there are clinical indications for the TA procedure requested.
and skeletal pain, have been reported with the use of these Issues such as who makes the decision about vascular access,
growth factors, they are usually well tolerated by donors.30 who orders laboratory tests to evaluate and monitor the
patient, and who chooses replacement fluids must be clearly
defined. The technical staff must be properly trained in the
Advanced Concepts care of very ill patients and be able to handle emergency sit-
uations. As with donor apheresis, written informed consent
Apheresis granulocytes contain a large number of viable must be properly obtained from the patient, outlining risks
lymphocytes. If transfused to a severely immunocompro- and benefits. Obviously, the number and type of risks will
mised patient, there is a significant risk for graft-versus-host vary depending on the type of TA procedure being performed
disease (GVHD). Therefore, the product should be irradi- and the anticipated duration of treatment. Proper documen-
ated prior to administration. Granulocyte function will not tation of all facets of the procedure is required.35
be affected. Even with the use of sedimenting agents, gran- Numerous studies performed during the last decade,
ulocyte preparations contain a significant number of RBCs many of them randomized, controlled clinical trials, have
(the component resembles a diluted RBC rather than a provided clinicians with sufficient data to more realistically
platelet concentrate). Compatibility testing is typically per- evaluate apheresis as a form of therapy and to define its role
formed, since the apheresis granulocyte product contains in the treatment of numerous disorders. The American
greater than 2 mL of RBCs.9 Leukocyte depletion filters Society for Apheresis (ASFA) has developed guidelines for
must not be used; a standard blood administration filter is therapeutic apheresis based on a systematic review of infor-
sufficient. mation from clinical trials, case studies, and anecdotal
reports and has designed a series of categories denoting the
likely effectiveness of apheresis in the treatment of various
Therapeutic Procedures clinical disorders.
The indication categories for therapeutic apheresis are as
The rationale of therapeutic apheresis (TA) is based on the
follows:
following:
• Category I. Apheresis is standard and acceptable, either
• A pathologenic substance exists in the blood that con-
as primary therapy or as a first-line adjunct to other initial
tributes to a disease process or its symptoms.
therapies. Efficacy is based on well-designed randomized
• The substance can be more effectively removed by aphere-
controlled clinical trials or a broad base of published
sis than by the body’s own homeostatic mechanisms.
experience.
Therefore, therapeutic apheresis, like donor apheresis, • Category II. Apheresis is generally accepted in a support-
involves the removal of a specific blood component, with ive role or as second-line therapy, rather than first-line
return of the remaining blood constituents to the patient. therapy.
However, with TA, since the component being removed is • Category III. Apheresis is not clearly indicated based
considered pathological (or contributing to the patient’s on insufficient evidence, conflicting results, or inability
underlying disease state), significantly larger volumes of to document a favorable risk-to-benefit ratio. Decision-
blood must be processed in order to remove as much of the making should be individualized.
offending agent as possible. The TA procedure is classified • Category IV. Apheresis has been demonstrated to lack
according to the blood component removed: a cytapheresis efficacy or be harmful, and should be discouraged in these
procedure may be used to selectively remove RBCs, WBCs, disorders. Clinical applications should be undertaken only
or platelets; a plasmapheresis procedure is used to remove under an approved research protocol.
2682_Ch14_331-351 22/05/12 11:49 AM Page 341
These guidelines are published and periodically updated procedure is longer than for donor apheresis. Vascular access
(Table 14–4).34,36 It should be noted that this text is not in- can be obtained via peripheral veins, central veins, or a com-
tended to provide detailed information on using TA to man- bination of both. If only one to two TA procedures are to be
age specific diseases. If needed, more thorough reviews performed, peripheral access can be used. In such instances,
should be consulted.30,34,35 two venous sites are necessary—one for removal and one for
return. Therefore, the patient must have adequate veins at
Vascular Access two sites capable of accommodating a 16- to 18-gauge nee-
Adequate vascular access is mandatory during TA, as larger dle. If several or frequent TA procedures are required, central
volumes of blood are processed and the duration of the venous access with a double-lumen catheter is desirable.
Photopheresis II
Plasma exchange II
Photopheresis IV
Hematologic Diseases
Immunoadsorption III
Neurological Disorders
Multiple sclerosis
Immunoadsorption III
Paraproteinemic polyneuropathy
• Category I. Apheresis is standard and acceptable, either as primary therapy or as a first-line adjunct to other initial therapies. Efficacy is based on
well-designed randomized controlled clinical trials or a broad base of published experience.
• Category II. Apheresis is generally accepted in a supportive role or as a second-line therapy, rather than first-line therapy.
• Category III. Apheresis is not clearly indicated based on insufficient evidence, conflicting results, or inability to document a favorable risk-to-benefit
ratio. Decision-making should be individualized.
• Category IV. Apheresis has been demonstrated to lack efficacy or be harmful, and should be discouraged in these disorders. Clinical applications
should be undertaken only under an approved research protocol.
symptoms.30 Thrombocytosis (at least 500,000/µL) can leukocyte-reduced, negative for hemoglobin S (donor does
occur in myeloproliferative disorders (essential thrombo- not have sickle cell trait), and partially phenotype-matched
cythemia, polycythemia vera, chronic myelogenous leukemia) for the Rh (C, c, E, e) and K1 antigens to avoid future
or as a reactive process in response to splenectomy, infection, alloimmunization.26,35 Some patients may be placed on a long-
chronic inflammation, or malignancy. Should the platelet term program of prophylactic red cell exchange to decrease
count reach levels above 1,000,000/µL, the patient is at risk the risk of complications associated with the disease.
for developing thrombotic or hemorrhagic complications. Other indications for red cell exchange are much less
The preferred method for lowering the platelet count is med- common. The procedure can be considered for treatment
ication;50 however, therapeutic apheresis may be indicated of overwhelming malaria or Babesia infections.54–56 Both
during an acute event to rapidly reduce the platelet count of these protozoa infect red blood cells, and a pronounced
until pharmacological therapy takes effect. During a platelet- parasitemia can occur. Red cell exchange has been shown
pheresis procedure, the platelet count will be decreased by to be beneficial for treating these patients when the para-
30% to 60%. More than one procedure may be required until site load is greater than 10%. A 1.5- to 2-volume red cell
the platelet count normalizes to desired levels (usually less exchange should significantly decrease the parasite
than 600,000/µL).34 There are no specific guidelines as to load.30,34 It is important to note that these patients are
the level the platelet count must be reduced to or a standard- often extremely ill and must be closely monitored during
ized procedure to reach a particular target platelet count. the procedure.
Finally, red cell exchange can be used to remove incom-
Leukapheresis patible RBCs from a patient’s circulation; for example, the
emergent transfusion of Rh-positive RBCs to an Rh-negative
Therapeutic leukapheresis has been used to treat patients female of child-bearing potential or an ABO-mismatched
with hyperleukocytosis, defined as a WBC or circulating transfusion.35
blast count of over 100,000/µL.34 These elevated levels place
the patient at risk for complications associated with Fluid Replacement
leukostasis, including organ dysfunction due to the forma-
tion of microthrombi in the pulmonary and cerebral microvas- In TA procedures, the extracorporeal circuit (tubing, collec-
culature.51 Leukostasis is more common in patients with tion chamber, blood warmer) is primed with normal saline,
acute myelogenous leukemia (AML) than with acute lym- providing the patient with an initial bolus of crystalloid. Dur-
phocytic leukemia (ALL).30 It is difficult to predict how ing therapeutic cytapheresis procedures, additional fluid
much blood volume should be processed to permit a suffi- replacement is often not necessary, since the majority of the
cient reduction in WBCs or blast cells to prevent leukostasis, patient’s plasma is being returned. Should hypovolemia be-
and WBC counts should be monitored during the procedure. come a concern, normal saline can be infused during the
A single procedure should reduce the WBC count by 30% to procedure.
60%; however, more than one procedure may be necessary In therapeutic plasmapheresis procedures, however, large
due to rapid mobilization of cells from the extravascular volumes of the patient’s plasma are retained. This fluid must
compartment. To achieve an adequate reduction in the WBC be replaced to maintain appropriate intravascular volume
count, up to 1 liter of fluid may be removed, necessitating and oncotic pressure. Several options are available, and the
the use of a replacement fluid. Use of a red cell sedimenting choice is determined by the disease being treated, the con-
agent, such as HES, may be of benefit during a leukapheresis dition of the patient, and the preference of the institution.
procedure.30,52 The most common replacement fluid for TPE is human
serum albumin (HSA) as a 5% solution.34,57 Although 5%
Erythrocytapheresis (Red Cell Exchange) HSA can be used to replace the entire volume removed, a
crystalloid such as normal saline may be used for up to one-
Erythrocytapheresis, or red cell exchange, removes a large third of the replacement volume. The availability of sufficient
number of RBCs from the patient and returns the patient’s HSA has been challenging in recent years, leading to the use
plasma and platelets along with compatible allogeneic donor of pentastarch as a portion of the replacement solution in
RBCs. The procedure is most commonly performed in addition to HSA.58,59
patients with sickle cell disease in order to decrease the num- Fresh frozen plasma (FFP) contains all the constituents
ber of hemoglobin S–containing RBCs, thereby treating of the removed plasma and thus would appear to be the
or preventing the complications (acute chest syndrome, optimal replacement fluid for TPE procedures. However, the
impending stroke, unrelenting painful crisis) associated with use of FFP is not without risk, including transmission of in-
the disease.30,34,53 fectious disease, additive effect contributing to citrate toxic-
The therapeutic goal is to decrease the level of hemoglo- ity, and sensitization to plasma proteins. Therefore, the use
bin S to less than 30%. This is usually accomplished with a of FFP is usually reserved for treatment of thrombotic
single red cell exchange procedure, requiring from six to thrombocytopenic purpura (TTP) and related disorders. For
ten RBC units, depending on the patient’s age and red patients with TTP who do not respond in a timely manner
cell volume. The donor RBCs selected for transfusion should to replacement with FFP, cryoprecipitate-reduced plasma is
be ABO- and Rh-compatible, relatively fresh (less than an alternative.35 FFP may also be used as replacement during
10 days is preferable to allow maximum in vivo survival), TPE on patients with a preexisting coagulopathy (severe
2682_Ch14_331-351 22/05/12 11:49 AM Page 345
Reservoir
Formed
Elements
treated, LDL-apheresis must be performed indefinitely, sometimes difficult to evaluate whether the deleterious effects
typically at 2- to 3-week intervals.26 were caused by the procedure or by the underlying disease.
Numerous adsorptive matrices have been developed with Some of the problems encountered are listed in Box 14–2.
varying clinical usefulness. These include charcoal for Some of the more common reactions and complications of
removal of bile acids, polymyxin B for removal of endotoxin, donor and therapeutic apheresis are discussed here.76–79
and cellulose acetate for removal of granulocytes or mono-
cytes, to mention a few. The majority of the adsorbent sys- Citrate Toxicity
tems are not available or in clinical use in the United States.30
Citrate toxicity is usually observed during cytapheresis
Photopheresis component collections when anticoagulated plasma is re-
turned at a rapid rate. It is also relatively common during
Photopheresis utilizes leukapheresis to collect the buffy therapeutic apheresis procedures. Citrate is the anticoagu-
coat layer from whole blood. These cells are treated with lant used in apheresis and is normally metabolized quickly
8-methoxypsoralen (8-MOP), exposed to ultraviolet A in the liver. If the amount of citrate infused exceeds the
(UVA) light, and then reinfused into the patient. The com-
bination of 8-MOP and UVA irradiation results in cross-
linking of leukocyte DNA, ultimately leading to apoptosis BOX 14–2
(cell death).30 Photopheresis has been shown to be effica-
cious and has been approved by the FDA for the treatment Adverse Effects of Apheresis
of cutaneous T-cell lymphoma.71 It has subsequently been • Citrate toxicity
used successfully to treat acute and chronic graft-versus-host • Vascular access complications (hematoma, sepsis, phlebitis,
disease,72 solid organ transplant rejection,73 and selected neuropathy)
immunologically mediated diseases.30,74,75 • Vasovagal reactions
• Hypovolemia
• Allergic reactions
Adverse Effects • Hemolysis
• Air embolus
Apheresis is accepted as a relatively safe procedure, but com- • Depletion of clotting factors
plications do occur, especially for donors at higher risks for • Circulatory and respiratory distress
presyncopal or syncopal reactions associated with component • Transfusion-transmitted diseases
collections due to donor factors such as younger age, female • Lymphocyte loss
sex, and small total blood volume.6,7,76 Adverse effects may be • Depletion of proteins and immunoglobulins
observed in therapeutic procedures as well.77 In this case, it is
2682_Ch14_331-351 22/05/12 11:49 AM Page 347
body’s ability to metabolize it, the level of ionized calcium Plasma Protein Interactions
will decrease, and the donor may feel numbness or tingling
around the mouth (paresthesias).35 This can be clinically The concentration of most plasma substances is reduced by
detected using the Chvostek’s sign or Trousseau’s sign. To 50% to 60% after one standard plasmapheresis treatment,
prevent this complication, calcium is infused intravenously with the rate of return to steady state concentrations varying
while the patient is undergoing the therapeutic plasma- among analytes. Plasma lipids, total protein, immunoglob-
pheresis. Intravenous calcium is not recommended on a ulins, and transferrin recover to steady state concentrations
routine basis. If unattended, the symptoms can lead to by 8 days postplasmapheresis, whereas ceruloplasmin con-
tetany and cardiac arrhythmia. Calcium supplementation by centrations take longer to reach prepheresis levels.
mouth may also be given. If FFP is used as replacement fluid
during a therapeutic plasma exchange, this phenomenon is Fatalities
more likely to occur because of the combined effects of the
anticoagulant in the FFP and the citrate used in the aphere- Rare fatalities occurring during therapeutic apheresis pro-
sis procedure itself. cedures have been reported. The majority of these have
been caused by circulatory (cardiac arrest or arrhythmia)
Vascular Access or respiratory complications (acute pulmonary edema or
adult respiratory distress syndrome). Of the cases reported,
Though plasmapheresis is helpful in certain medical condi- about half had received plasma as part or all of the replace-
tions, like any other therapy, there are potential risks and ment fluid. Because plasma has been associated with fatal-
complications that should be discussed with the patient be- ities, its use is recommended only in cases of TTP or
fore the procedure. Insertion of a rather large intravenous hemolytic uremic syndrome (HUS) in which there is a
catheter can lead to bleeding, lung puncture (depending on specific indication for its use. Plasma is also capable of
the site of catheter insertion), and, if the catheter is left in transmitting diseases such as hepatitis and the human
too long, infection. immunodeficiency viruses.
Vasovagal Reactions
CASE STUDY
A vasovagal episode (also called a vasovagal response,
vasovagal attack, and neurocardiogenic syncope) is a malaise Case 14–1
mediated by the vagus nerve. When it leads to syncope or
“fainting,” it is called vasovagal syncope, which is the most A 72-year-old man presented to the emergency depart-
common type of fainting. Another mechanism causing ment at approximately 10:00 a.m. after waking during the
hypotension during apheresis procedures is the vasovagal night with confusion and expressive dysphasia. These
reaction.76,78 In this reaction, hypovolemia results in a symptoms initially appeared to improve and then progres-
decrease in blood pressure. The compensatory response for sively worsened. Apart from a recent 3-week history of
this volume depletion is to increase sympathetic nervous left shoulder pain on abduction, his previous medical his-
system output with physiological compensation as previously tory was significant for excision of melanoma in situ and
described. During a vasovagal reaction, however, parasym- multiple basal cell carcinomas, mild gastroesophageal
pathetic output that normally counteracts sympathetic output reflux, and an appendectomy 52 years earlier. He was a
increases, resulting in a slowing of heart rate and decreased nonsmoker, did not drink alcohol, used no regular med-
vascular tone. This results in hypotension. Factors that have ications, and had no known drug allergies.
been associated with vasovagal reactions in whole blood
donors include younger age, low weight, first-time donation, Physical examination findings:
and inattentive collection staff.6,7 • Expressive dysphasia
• Cranial nerves intact
Miscellaneous Reactions • Vital signs: BP 150/90 mm Hg, heart rate (HR) 82 bpm,
respiratory rate (RR) 12, temperature 101.4°F.
Hypovolemia is observed more frequently with IFC instru- • All extremities moved equally, with normal strength.
ments. Careful monitoring of the volume in and out is • Multiple petechiae over both lower extremities and
necessary to prevent hypovolemia and hypervolemia. Allergic abdomen.
reactions are related to the replacement fluids. This type of • Computerized tomography (CT) of the brain showed
reaction is generally observed in patients in whom FFP is being no abnormalities.
administered (such as during a TPE procedure for TTP), but it • Carotid duplex ultrasound revealed no significant
has also been reported during infusion of albumin. Hemolysis carotid or vertebral artery disease.
is usually caused by a mechanical problem with the equipment, • Twelve-lead electrocardiograph (ECG) was normal.
such as a kink in the plastic tubing. Observing the return line • Chest x-ray showed normal heart size and no
is critical in avoiding this problem. Air embolism and clotting abnormalities.
factor deficiencies are not commonly observed.
2682_Ch14_331-351 22/05/12 11:49 AM Page 348
Laboratory findings during the first 48 hours: became anuric. He was then transferred to the intensive
• Anemia care unit (ICU).
• Thrombocytopenia
Negative direct antiglobulin test (DAT)
• Normal prothrombin time (PT) and partial thrombo-
plastin time (PTT) Peripheral smear: decreased platelets, rare nucleated RBC,
• Normal D-dimer and fibrinogen 2+ schistocytes.
• Elevated LDH Decreased ADAMTS 13
• Decreased haptoglobin 1. What are the top five possible diagnoses for this case?
2. What is the most likely diagnosis for this patient and
Hospital Course
why?
Overnight, the patient became increasingly agitated, re- 3. What are the main symptoms in cases of this type?
fused oral intake, and pulled out his intravenous 4. What is the main treatment for this disease?
catheter. His urine output decreased significantly and he
SUMMARY CHART
In an apheresis procedure, blood is withdrawn from a Membrane filtration technology uses membranes with
donor or patient and separated into its components. specific pore sizes, allowing plasma to pass through the
One or more of the components is retained, and the membrane while the cellular portion passes over it.
remaining constituents are recombined and returned The most common anticoagulant used in apheresis is
to the individual. acid citrate dextrose.
The process of removing plasma from the blood is Therapeutic apheresis is used to remove a pathological
termed plasmapheresis; removing platelets is termed substance, to supply an essential or missing substance
plateletpheresis or thrombocytopheresis; removing to alter the antigen–antibody ratio, or to remove
RBCs is termed erythrocytapheresis; removing leuko- immune complexes.
cytes is known as leukapheresis. The American Society for Apheresis (ASFA) has devel-
Apheresis equipment that uses intermittent flow oped categories to define the effectiveness of therapeu-
centrifugation (IFC) requires only one venipuncture, tic apheresis in treating a particular condition or
in which the blood is drawn and reinfused through disease. Therapeutic apheresis is most appropriate for
the same needle. Once the desired component is treating category I or II disorders.
separated, the remaining components are reinfused In therapeutic plasmapheresis procedures, the replace-
to the donor, and one cycle is complete. Apheresis ment fluids used to maintain appropriate intravascular
procedures performed on patients usually require volume and oncotic pressure include normal saline, FFP,
many cycles to reach an acceptable therapeutic cryo-reduced plasma, and 5% human serum albumin.
endpoint.
Complications of apheresis include vascular access
Continuous flow centrifugation (CFC) procedures issues, alteration of pharmacodynamics of medications,
withdraw, process, and return the blood to the individ- citrate toxicity, fluid imbalance, allergic reactions, equip-
ual simultaneously. Two venipuncture sites are neces- ment malfunction (hemolysis), and infection. Fatalities
sary. The process of phlebotomy, separation, and have occurred (primarily patient rather than donor).
reinfusion is uninterrupted.
25. Vamvakas, EC, and Pineda, AA: Determinants of the efficacy 50. Cortelazzo, S, et al. Hydroxyurea for patients with essential
of prophylactic granulocyte transfusions: A meta-analysis. thrombocythemia and a high risk of thrombosis. N Engl J Med,
J Clin Apheresis 12:74, 1997. 332:1132–1136, 1995.
26. Roback, JD (ed): Technical Manual, 16th ed. American Asso- 51. Procu, P, et al: Hyperleukocytic leukemias and leukostasis:
ciation of Blood Banks, Bethesda, MD, 2008. A review of pathophysiology, clinical presentation and man-
27. Sachs, UJ, et al: Safety and efficacy of therapeutic early onset agement. Leuk Lymphoma 39:1–18, 2000.
granulocyte transfusions in pediatric patients with neutropenia 52. Fenger-Eriksen, C, et al: Mechanisms of hydroxyethyl starch-
and severe infections. Transfusion 46(11):1909–1914, 2006. induced dilutional coagulopathy. J Thrombosis Hemostases
28. Kikuta, et al: Therapeutic transfusions of granulocytes 7:1099–1105, 2009.
collected by simple bag method for children with cancer and 53. National Heart, Lung, and Blood Institute. The management
neutropenic infections: Results of a single-centre pilot study. of sickle cell disease, 4th ed. NIH Publication No. 02-2117.
Vox Sang 91:70, 2006. Bethesda, MD: National Institutes of Health, 2002.
29. Leitner, G, et al: Preparation of granulocyte concentrates by 54. Spaete, J, et al: Red cell exchange transfusion for babesiosis in
apheresis. Vox Sanguinis 98:567–575, 2010. Rhode Island. J Clin Apheresis 24:97, 2009.
30. McLeod, BC (ed): Apheresis: Principles and Practice, 2nd ed. 55. Yarrish, RL, et al: Transfusion malaria: Treatment with
AABB Press, Bethesda, MD, 2003. exchange transfusion after delayed diagnosis. Arch Intern Med
31. Bryant, BJ, et al: Gravity sedimentation of granulocytapheresis 142:187, 1982.
concentrates with hydroxyethyl starch efficiently removes red 56. Cahill, KM, et al: Red cell exchange: Treatment of babesiosis
blood cells and retains neutrophils. Transfusion 50:1203, in a splenectomized patient. Transfusion 21:193, 1981.
2009. 57. Pusey, C, et al: Experience of using human albumin solution
32. Lee, JH, et al: A controlled study of the efficacy of hetastarch 4.5% in 1195 therapeutic plasma exchange procedures. Trans-
and pentastarch in granulocyte collections by centrifugal leuka- fusion Med 20(4):244–299, 2010.
pheresis. Blood 86:4662, 1995. 58. Goss, GA, and Weinstein, R: Pentastarch as partial replacement
33. McLeod, BC (ed): Clinical application of therapeutic hema- fluid for therapeutic plasma exchange: Effect on plasma
pheresis. J Clin Apheresis 14(4): 1999. proteins, adverse events during treatment, and serum ionized
34. Szczepiorkowski, Z (ed): Clinical applications of therapeutic calcium. J Clin Apheresis, 14:114–121, 1999.
apheresis: An evidence based approach, 5th ed. J Clin Aphere- 59. Agreda-Vásquez, GP, et al: Starch and albumin mixture as
sis 25:81, 2010. replacement fluid in therapeutic plasma exchange is safe and
35. Winters, JL (ed): Therapeutic Apheresis: A Physician’s Hand- effective. J Clin Apheresis 23:163, 2008.
book, 2nd ed. AABB, Bethesda, MD, 2008. 60. Itoh, T, et al: Predictive value of the original content of CD34+
36. Smith, JW, Weinstein, R, and Hillyer, KL: Therapeutic aphere- cells for enrichment of hematopoietic progenitor cells from
sis: A summary of current indication categories endorsed by bone marrow harvests by the apheresis procedure. J Clin
the AABB and the American Society for Apheresis. Transfusion Apheresis 21:176, 2006.
43:820, 2003. 61. Bishop, MR, et al: High-dose therapy and peripheral blood pro-
37. Kintzel, PE, Eastlund, T, and Calis, KA. Extracorporeal removal genitor cell transplantation: Effects of recombinant human
of antimicrobials during plasmapheresis. J Clin Apheresis granulocyte-macrophage colony-stimulating factor on the
18:67–70, 2003. autograft. Blood 83:610, 1994.
38. Boga, C, et al: Plasma exchange in critically ill patients with 62. Food and Drug Administration: Current good tissue practice
sickle cell disease. Transfus Apher Sci 37:17, 2007. for manufacturers of human cellular and tissue-based products:
39. Hastings, D, et al: Plasmapheresis therapy for rare but poten- Inspection and enforcement; proposed rule. 21 CFR 1271.
tially fatal reaction to rituximab. J Clin Apheresis 24:28, 2009. Federal Register 66:1507, 2001.
40. Gore, EM, Jones, BS, and Marques, MB: Is therapeutic plasma 63. Padley, D (ed): Standards for Cellular Therapy Product Serv-
exchange indicated for patients with gemcitabine-induced ices, 4th ed. AABB, Bethesda, MD, 2010.
hemolytic uremic syndrome? J Clin Apheresis 24:209, 2009. 64. FACT-JACIE: International Standards for Cellular Therapy
41. Sivakumaran, P, et al: Therapeutic plasma exchange for desen- Product Collection, Processing, and Administration, 4th ed.
sitization prior to transplantation in ABO-incompatible renal Online (factwebsite.org), 2008.
allografts. J Clin Apheresis 24:155, 2009. 65. Pineda, AA: Immunoaffinity apheresis columns: Clinical appli-
42. Wu, SG, Kuo, PH, and Yang, PC: Successful weaning after cations and therapeutic mechanisms of action. In Sacher, RA,
plasma exchange for polyneuropathy related to POEMS syn- et al: Cellular and Humoral Immunotherapy and Apheresis.
drome. J Clin Apheresis 24:170, 2009. American Association of Blood Banks, Arlington, VA, 1991.
43. Ezer, A, et al: Preoperative therapeutic plasma exchange in 66. Felson, DT, et al: The Prosorba column for treatment of refrac-
patients with thyrotoxicosis. J Clin Apheresis 24:111, 2009. tory rheumatoid arthritis: A randomized, double-blind, sham-
44. George, JN, et al: Lessons learned from the Oklahoma throm- controlled trial. Arthritis Rheum 42:2153, 1999.
botic thrombocytopenic purpura-hemolytic uremic syndrome 67. Mittelman, A, et al: Treatment of patients with HIV thrombo-
registry. J Clin Apheresis 23:129, 2008. cytopenia and hemolytic uremic syndrome with protein A
45. Scully, M: Thrombotic thrombocytopenic purpura and preg- (Prosorba® Column) immunoadsorption. Semin Hematol 26
nancy. ISBT Sci Series 2:226, 2007. (Suppl 1):15, 1989.
46. Lalmuanpuii, J, et al: Hypersensitivity to plasma exchange in a 68. Snyder, HW, Jr, et al: Successful treatment of cancer-chemotherapy–
patient with thrombotic thrombocytopenic purpura. J Clin associated thrombotic thrombocytopenic purpura/hemolytic
Apheresis 24:18, 2009. uremic syndrome (TTP/HUS) with protein A immunoadsorp-
47. Park, YA, et al: Is it quinine TTP/HUS or quinine TMA? tion. Blood 76(Suppl 1):4679, 1990.
ADAMTS 13 levels and implications for therapy. J Clin Aphere- 69. Doesch, AO, et al: Effects of protein A immunoadsorption in
sis 24:115, 2009. patients with advanced chronic dilated cardiomyopathy. J Clin
48. Patten, E: Pathophysiology of the immune system. In Kilins, J, Apheresis 24:141, 2009.
and Jones, JM (eds): Therapeutic Apheresis. American Associ- 70. Heustis, DW, and Morrison, FS: Adverse effects of immune
ation of Blood Banks, Arlington, VA, 1983. adsorption with staphylococcal protein A columns. Transfus
49. Klein, HG: Effect of plasma exchange on plasma constituents: Med Rev 10:62, 1996.
Choice of replacement solutions and kinetics of exchange. In 71. Edelson, R, et al: Treatment of cutaneous T-cell lymphoma by
MacPherson, JL, and Kaspirisin, DO (eds): Therapeutic Hema- extracorporeal photochemotherapy—preliminary results. N
pheresis, vol 2. CRC Press, Boca Raton, FL, 1985. Engl J Med 316:297, 1987.
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72. Rossetti F, et al: Extracorporeal photochemotherapy for the 76. Winters, J: Complications of donor apheresis. J Clin Apheresis
treatment of graft-vs-host disease. Bone Marrow Transplant 21:132, 2006.
18(Suppl 2):175, 1996. 77. Bramlage, CP, et al: Predictors of complications in therapeutic
73. Constanza-Nordin, MR, et al: Successful treatment of heart plasma exchange. J Clin Apheresis 24:225, 2009.
transplant rejection with photopheresis. Transplantation 53: 78. Yuan, S, et al: Moderate and severe adverse events associated
808, 1992. with apheresis donations: incidences and risk factors. Transfu-
74. Malawista, SE, Trock, DH, and Edelson, RL: Treatment of sion 50(2):478, 2010.
rheumatoid arthritis by extracorporeal photochemotherapy: 79. Shemin, D, Briggs, D, and Greenan, M: Complications of TPE.
A pilot study. Arthritis Rheum 34:646, 1991. J Clin Apheresis 22: 270, 2007.
75. Rook, AH, et al: Treatment of systemic sclerosis with extracor-
poreal photochemotherapy. Arch Dermatol 128:337, 1992.
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Chapter 15
Transfusion Therapy
Melanie S. Kennedy, MD
OBJECTIVES
1. Describe the blood products that are currently available for therapeutic use.
2. List the indications for each blood product, including the approximate volume of each product.
3. Select the appropriate blood product for patients with specific disorders.
4. State the expected incremental increase of a patient’s hematocrit level following transfusion of each unit of red blood cells
(RBCs) and platelet count following transfusion of each unit of platelets.
5. List the required procedures for preparing each blood component for transfusion.
6. Identify the groups of recipients who are at highest risk of infection from transfusion of cytomegalovirus-positive RBCs or
platelets.
7. Explain the role of irradiation in the prevention of transfusion-associated graft-versus-host disease (GVHD).
8. State the purpose of the surgical blood order schedule.
9. List the main advantages and disadvantages of autologous transfusion.
10. Identify the most important factors to consider when emergency transfusion is indicated.
11. Define massive transfusion.
12. Describe the various transfusion requirements of oncology patients.
13. Compare and contrast hemophilia A and von Willebrand’s disease.
14. State the respective blood products of choice for treating von Willebrand’s disease and hemophilia B.
15. Specify the steps involved in the proper administration of blood.
352
2682_Ch15_352-366 22/05/12 11:50 AM Page 353
Whole blood Symptomatic anemia with large-volume deficit Approx. Hct 40% 570 mL
Red blood cells: RBCs (adenine-saline Symptomatic anemia Approx. Hct 55% 330 mL
added), RBC pheresis
Red blood cells: deglycerolized, washed Severe allergic reactions Approx. Hct 75% 180 mL
Rare donors
Red blood cells: leukocyte-reduced Symptomatic anemia < 5 × 106 WBC 330 mL
Febrile reactions due to leukocyte antibodies
Reduce CMV transmission
Reduce HLA alloimmunization
Platelets: platelets pooled Bleeding due to thrombocytopenia or platelet ≥ 3 × 1011 platelets/unit 300 mL
function abnormality
Prevention of bleeding from marrow hypoplasia
Platelets pheresis see Platelets; platelets pooled (above) see Platelets 300 mL
Crossmatched or HLA-matched
Plasma Deficiency of labile and stable plasma All coagulation factors 250 mL
coagulation factors; TTP ADAMTS 13
Source: Circular of information for the use of human blood and blood components, American Association of Blood Banks, America’s Blood Centers, American Red Cross, Washington, DC, 2010.
2682_Ch15_352-366 22/05/12 11:50 AM Page 354
The transfusion of whole blood is limited to a few clinical of decompensation (need for increased oxygen-carrying
conditions. Whole blood should be used to replace the loss capacity). RBC transfusion is not to be used to enhance gen-
of both RBC mass and plasma volume.1,2 Thus, rapidly eral well-being, promote wound healing, prevent infection,
bleeding patients can receive whole blood, although RBCs expand blood volume when oxygen-carrying capacity is
and plasma are more commonly used and are equally effec- adequate, or prevent future anemia.
tive clinically. Each unit of transfused RBCs is expected to increase the
A definite contraindication to the use of whole blood is hemoglobin level 1 g/dL and the hematocrit level 3% in
severe chronic anemia. Patients with chronic anemia have a the typical 70-kg (154-lb) human, the same as whole
reduced amount of RBCs but have compensated by increas- blood. In pediatric patients, a dose of 10 to 15 mL/kg
ing their plasma volume to restore their total blood volume. will increase the hemoglobin about 2 to 3 g/dL or the
Thus, these patients do not need the plasma in the whole hematocrit 6% to 9%.
blood and, in fact, may adversely respond by developing pul- The increase in hemoglobin and hematocrit is evident
monary edema and heart failure because of volume overload. more quickly than with 1 unit of whole blood, because the
This is more likely to occur in patients with kidney failure adjustment in blood volume is less. As in the example for
or preexisting heart failure. whole blood, the RBC volume is increased to the same
For a 70-kg (155-lb) adult, each unit of whole blood amount, 1,200 mL, but the blood volume is increased only
should increase the hematocrit level 3% or hemoglobin 330 mL to 5,330 mL. The hematocrit level is increased
1 g/dL. After transfusion, the increase may not be apparent immediately to 22.5%.
until 48 to 72 hours when the patient’s blood volume adjusts RBCs prepared with additive solutions such as AS-1
to normal. For example, a patient with a 5,000-mL blood or AS-3 have greater volume than with citrate phosphate
volume and 20% hematocrit level has 1,000-mL RBCs. With dextrose (CPD) or citrate phosphate dextrose adenine
transfusion of 500-mL whole blood containing 200-mL (CPDA-1), 330 mL versus 250 to 275 mL (see Chapter 1),
RBCs, the blood volume will be 5,500 mL and will result but the additive solution units have less plasma. The RBC
in 21.8% hematocrit. When the patient’s blood volume mass is the same. Therefore, the hematocrit differs from 65%
readjusts to 5,000 mL, the hematocrit level will be 24% to 80% for CPDA-1 RBCs to 55% to 65% for additive solution
(1,200 mL divided by 5,000 mL). The increase is greater in RBCs (see Table 15–1).
a smaller person and less in a larger one.
Leukocyte-Reduced RBCs
Red Blood Cells
The average unit of RBCs contains approximately 2 × 109
RBCs are indicated for increasing the RBC mass in patients leukocytes. Donor leukocytes may cause febrile non-
who require increased oxygen-carrying capacity.1,2 These hemolytic transfusion reactions, transfusion-associated graft-
patients typically have pulse rates greater than 100 beats versus-host disease (TA-GVHD), and transfusion-related
per minute; have respiration rates greater than 30 breaths immune suppression. In addition, human leukocyte antigens
per minute; and may experience dizziness, weakness, (HLA) are responsible for HLA alloimmunization. Leuko-
angina (chest pain), and difficulty thinking. The decreased cytes may harbor cytomegalovirus (CMV).
RBC mass may be caused by decreased bone marrow To reduce HLA alloimmunization and CMV transmission,
production (leukemia or aplastic anemia), decreased RBC the leukocyte content must be reduced to less than 5 × 106,
survival (hemolytic anemia), or surgical or traumatic which can be achieved by using one of several leukocyte re-
bleeding. duction filters.5 With these filters, most RBC units are less
The human body compensates for anemia by increasing than 1 × 106; some are 1 × 104 leukocytes. In the United States,
plasma volume, heart rate, respiratory rate, and oxygen ex- the standard leukocyte content is less than 5 × 106; in Europe,
traction from the RBCs. Normally, only about 25% of the oxy- the standard is less than 1 × 106. Controversial is the effect of
gen is extracted, but with increased demand at the organ and leukocyte-reduced blood on length of hospital stay and post-
tissue level, up to 50% of the oxygen can be extracted. When surgical wound infection. However, febrile nonhemolytic
the demand exceeds 50% of the oxygen content, the compen- transfusion reactions, CMV transmission, and HLA alloimmu-
satory mechanisms fail, and the patient requires transfusion. nization are decreased by the use of leukocyte-reduced RBCs
No set hemoglobin levels indicate a need for transfusion. and platelets.6 The indications for transfusion of leukocyte-
The critical level is 6 g/dL or less. Consensus committees reduced RBCs and platelets are outlined in Table 15–2.
suggest trigger values of hemoglobin of less than 7 g/dL for most
patients and less than or equal to 8 g/dL for certain patients Washed RBCs and Frozen/Deglycerolized RBCs
with heart disease.3 Most patients can tolerate 7 g/dL, espe-
cially if on bed rest or at decreased levels of activity and given Patients who have severe allergic (anaphylactic) transfusion
supplemental oxygen.4 In fact, healthy individuals can tolerate reactions to ordinary units of RBCs may benefit from receiv-
hemoglobin levels as low as 5 g/dL with minimal effects. ing washed RBCs.1 The washing process removes plasma
Transfusion of RBCs is contraindicated in patients proteins, the cause of most allergic reactions. Washed RBCs
who are well compensated for the anemia. RBCs should are used for the rare patient who has had moderate to severe
not be used to treat nutritional anemia, such as iron defi- allergic transfusion reactions and has anti-IgA antibodies
ciency or pernicious anemia, unless the patient shows signs because of IgA deficiency.
2682_Ch15_352-366 22/05/12 11:50 AM Page 355
Table 15–2 Indications for Leukocyte- Bacterial testing is required for each platelet product.
Reduced RBCs and Platelets Plateletpheresis products and pooled platelets (from whole
blood) are cultured by the blood center. However, individual
ACCEPTED CONTROVERSIAL platelet products from whole blood are difficult to culture
Decrease febrile nonhemolytic Decrease hospital length of stay because of their small volume and thus are less commonly
transfusion reactions used for transfusion.
A plateletpheresis component is prepared from one donor
Decrease alloimmunization Decrease incidence of wound and must contain a minimum of 3 × 1011 platelets.5 One
to white blood cell antigens infections postsurgery
plateletpheresis should increase the adult patient’s platelet
Decrease transmission of Decrease incidence of cancer count to 20,000 to 60,000/µL. Each unit of platelets from
cytomegalovirus (CMV) recurrence postsurgery whole blood must contain at least 5.5 × 1010 platelets5 and
should increase the platelet count by 5,000 to 10,000/µL in
a 70-kg human. A pool of 5 units, then, will contain roughly
3 × 1011 platelets and should give a platelet count increase
Freezing RBCs allows the long-term storage of rare blood similar to plateletpheresis.
donor units, autologous units, and units for special pur-
poses, such as intrauterine transfusion. Because the process Refractory Patients
needed to deglycerolize the RBCs removes nearly all the
Advanced Concepts
plasma, these units, although more expensive, can be used
interchangeably with washed RBCs. However, the 24-hour Massive splenomegaly, high fever, sepsis, disseminated in-
outdate of washed or deglycerolized RBCs severely limits the travascular coagulation (DIC), and platelet or HLA anti-
use of these components. The expected hematocrit increase bodies can cause less-than-expected platelet count
for washed or deglycerolized RBCs is the same as that for increment and survival. The 10-minute to 1-hour post-
regular RBC units. transfusion platelet count increment is less affected by
splenomegaly, high fever, and DIC than by the presence of
platelet or HLA antibodies.1 If the 10-minute increment is
Platelets and Plateletpheresis
less than 50% of that expected on two occasions, the
Platelets are essential for the formation of the primary patient is considered refractory. Positive platelet crossmatch
hemostatic plug and maintenance of normal hemostasis. or positive HLA antibody screen is considered evidence of
Patients with severe thrombocytopenia (low platelet count) alloimmunization. Platelet crossmatching with available in-
or abnormal platelet function may have petechiae, ecchy- ventory can speed the provision of platelets for transfusion,
moses, and mucosal or spontaneous hemorrhage. The as HLA-typing the patient and recruiting HLA-compatible
thrombocytopenia may be caused by decreased platelet platelet donors can be time-consuming. HLA-matched
production (e.g., after chemotherapy for malignancy) or in- platelets should be irradiated.
creased destruction (e.g., disseminated intravascular coagu- A corrected count increment using a 10-minute to 1-hour
lation [DIC]). Massive transfusion, which is discussed later post-transfusion platelet count can provide valuable infor-
in this chapter, may also cause thrombocytopenia because of mation about patient response to a platelet component.1 The
the rapid consumption of platelets for hemostasis and the platelet count increment is corrected for differences in body
dilution of the platelets by resuscitation fluids and RBC size so that more reliable estimates of expected platelet in-
transfusion. crement can be determined. The expected corrected platelet
Platelet transfusions are indicated for patients who are increment is greater than 10,000/µL per m2. One formula for
bleeding because of thrombocytopenia or abnormally func- corrected count increment is:
tioning platelets (Box 15–1). In addition, platelets are indi- Absolute platelet increment/µL × body surface area (m2)
cated as prophylaxis for patients who have platelet counts
Number of platelets transfused (1011)
under 5,000 to 10,000/µL.7
in which the absolute platelet increment is the post-
transfusion platelet count minus the pretransfusion platelet
count, the body surface area is expressed as square meters,
BOX 15–1 and the number of platelets transfused is 3 for platelet-
pheresis or pooled platelets (the number of platelets in each
Indications for Platelet Transfusion unit expressed in 1011).
• Thrombocytopenia with bleeding or invasive procedure For example, a patient with a 10,000/µL platelet count
• Chemotherapy for malignancy (decreased production, less than has a body surface area of 1.3 m2. One unit of plateletphere-
5,000 to 10,000/µL) sis is given. The 1-hour post-transfusion platelet count is
• Disseminated intravascular coagulation (increased destruction, 50,000/µL. Put these into the formula:
less than 50,000/µL)
• Massive transfusion (platelet dilution, less than 50,000 to (50,000/µL – 10,000/µL) × 1.3
100,000/µL) 3
2682_Ch15_352-366 22/05/12 11:50 AM Page 356
The answer, 17,333, shows that the patient has a good The neutrophil count will increase to 1,000/µL or more in
increment (greater than 10,000/µL) and is not refractory response to infusion of granulocyte colony-stimulating factor
to platelets. An answer less than 5,000/µL indicates (GCSF)–mobilized granulocyte pheresis.
refractoriness. Plasma
In addition to HLA- and platelet-specific antigens, ABO
antigens are also expressed on the platelet membrane. Plasma includes fresh frozen plasma, plasma 24 (frozen within
Sometimes platelets are selected for transfusion without 24 hours), and thawed plasma. Plasma and plasma 24 contain
regard to ABO; however, group O recipients may have a all coagulation factors. After fresh frozen plasma and plasma
lower increment when given group A platelets than when 24 are thawed, they can become thawed plasma and stored
group-identical platelets are selected.7 Group A, B, and AB for 5 days at 4°C. The 5-day storage reduces outdating and
patients may also develop a positive direct antiglobulin test allows rapid response to urgent orders for bleeding patients.
owing to passive transfer of anti-A, anti-B, or anti-A,B when Thawed plasma after 5-day storage has less factor V and VIII
several ABO-incompatible platelet transfusions are given. but is still therapeutic.
Although platelet membranes do not express Rh anti- Plasma can be used to treat multiple coagulation deficien-
gens, platelet products contain small amounts of RBCs and cies occurring in patients with liver failure, DIC, vitamin K
thus can immunize patients to Rh antigens. Rh-immune deficiency, warfarin overdose, or massive transfusion.8 Some-
globulin can be given to girls and women of childbearing times, plasma is used to treat patients with single factor
potential to prevent Rh sensitization. Each 300-µg vial of deficiencies, such as factor XI deficiency.
Rh immune globulin is adequate for about 30 platelet- Vitamin K deficiency or warfarin overdose should be
phereses or 4 platelet pools of 5. treated with vitamin K orally, intravenously, or intramuscu-
larly if liver function is adequate and with an adequate
interval (4 to 24 hours) before a major or minor hemostatic
For the same reasons as RBCs, platelet components may
challenge such as surgery. Plasma is given if the patient is
also be leukocyte-reduced or washed. Washing platelet com-
actively bleeding or if time is not available for warfarin
ponents removes some platelets and plasma proteins and, if
reversal before surgery.
using an open method, requires a 4-hour expiration time.
Patients with liver disease or liver failure frequently develop
Therefore, platelet components should be washed only to
clinical coagulopathy due to impaired hepatic synthesis of
prevent severe allergic reactions or to remove alloantibodies
all coagulation factors and antithrombotic factors. Plasma
in cases of neonatal alloimmune thrombocytopenia.
is the product of choice for patients with multiple-factor
Granulocyte Pheresis deficiencies and hemorrhage or impending surgery. Usually
4 to 6 units of plasma will effectively control hemostasis.
Patients who have received intensive chemotherapy for Even so, plasma may not correct coagulation tests to normal
leukemia or bone marrow transplant or both may develop range because of dysfibrinogenemia. Mild hemostatic abnor-
severe neutropenia and serious bacterial or fungal infec- malities do not predict bleeding, so correction is not indicated
tion. Without neutrophils (granulocytes), the patient may for minor procedures, such as liver biopsy.9 In addition,
have difficulty controlling an infection, even with appro- plasma is not a concentrate so that volume overload may be
priate antibiotic treatment. Criteria have been developed a serious complication of transfusion.
to identify patients who are most likely to benefit from Congenital coagulation factor deficiencies are rarely treated
granulocyte transfusions: those with fever, neutrophil with plasma, because the dose requirement for surgical pro-
counts less than 500/µL, septicemia or bacterial infection cedures and serious bleeding is so great as to cause pulmonary
unresponsive to antibiotics, reversible bone marrow hy- edema as a result of volume overload, even in a young
poplasia, and a reasonable chance for survival.1 Prophy- individual with a healthy cardiovascular system. Factor
lactic use of granulocyte transfusions is of doubtful value concentrates (see the “Factor VIII” and “Factor IX” sections
for those patients who have neutropenia but no demon- below) offer more effective modes of therapy. Factor XI
strable infection. deficiency, however, is still treated by plasma infusion, requir-
Newborn infants may develop overwhelming infection ing 20% to 30% factor XI levels for adequate hemostasis. This
with neutropenia because of their limited bone marrow disease is milder than hemophilia A (factor VIII deficiency)
reserve for neutrophil production. In addition, neonatal neu- or hemophilia B (factor IX deficiency). Factor XI also has a
trophils have impaired function. Studies show granulocyte long half-life, so treatment is not needed on a daily basis.
transfusions to be beneficial for these patients. For an adult A coagulation factor unit is defined as the activity in 1 mL
or a child, the usual dose is one granulocyte pheresis product of pooled normal plasma, so 100% activity is 1 unit/mL or
daily for 4 or more days. For neonates, a portion of a granu- 100 units/dL. About 30% activity of each of the coagulation
locyte pheresis unit is usually given once or twice. factors is required for adequate hemostasis. Thus, less
Granulocyte components should be administered as soon than half of the plasma volume, or about 4 to 6 plasma units,
as possible and within 24 hours of collection.5 The granulo- is required to correct a coagulopathy such as in liver disease
cyte pheresis needs to be crossmatched because of the signif- or DIC. With continued hemorrhage, additional doses
icant content of RBCs. The patient must be monitored for are usually needed if the prothrombin time is more than
resolution of symptoms and clinical evidence of efficacy. 1.5 times normal or if the international normalized ratio is
2682_Ch15_352-366 22/05/12 11:50 AM Page 357
greater than 1.5. Because several key clotting factors, Cryoprecipitate was used to treat patients with von
such as factor VII, VIII, or IX, have half-lives less than Willebrand’s disease, a deficiency of vWF. Cryoprecipitate
24 hours, repeated transfusions are required to control post- is no longer considered the product of choice for factor
operative bleeding or to maintain hemostasis. For example, VIII deficiency or von Willebrand’s disease. Virus-safe
factor IX has a half-life of 18 to 24 hours, requiring daily factor VIII with assayed amounts of factor VIII and vWF
transfusions. is available.
Plasma is sometimes used as a replacement fluid during
plasma exchange (therapeutic plasmapheresis; see Chapter 14, Factor VIII
“Apheresis”). In cases of thrombotic thrombocytopenic
purpura, plasma provides a metalloprotease (ADAMTS13) Patients with hemophilia A or factor VIII deficiency have
and removes inhibitors, thus reversing the symptoms. spontaneous hemorrhages that are treated with recombinant
Plasma should not be used for blood volume expansion or or human plasma–derived factor VIII replacement.10 Plasma
protein replacement because safer products are available for derived factor VIII is prepared from plasma obtained from
these purposes—serum albumin, synthetic colloids, and bal- paid donors by plasmapheresis or from volunteer whole
anced salt solutions—none of which transmit disease or blood donors. Factor VIII is treated by different methods,
cause severe allergic reactions or transfusion-associated acute such as pasteurization, nanofiltration, and solvent detergent,
lung injury. to ensure sterility for HIV, hepatitis B virus, and hepatitis C
Plasma should be ABO-compatible with the recipient’s virus (HCV).11 The recombinant human product is virus-
RBCs, but the Rh type can be disregarded. safe. Recombinant human products and their indications are
listed in Table 15–3.
Cryoprecipitate Both the plasma derived and recombinant factor VIII are
stored at refrigerator temperatures and are reconstituted with
Cryoprecipitate is used primarily for fibrinogen replacement. saline at the time of infusion. This ease of handling allows
The AABB requires that at least 150 mg of fibrinogen be in self-therapy for individuals with hemophilia.
each unit of cryoprecipitate,5 although quality control levels The following example illustrates the calculation of a
are often over 250 mg. Generally, 5 cryoprecipitate units are factor VIII dose:
pooled, rinsing each bag with saline, at the blood center. A 70-kg hemophiliac patient with a hematocrit level
These pools are frozen and shipped to transfusion services of 30% has an initial factor VIII level of 4% (4 units/dL,
where the pools can be thawed and issued. Each pool con- 0.04 units/mL). How many units of factor VIII concentrate
tains 750 to 1,250 mg of fibrinogen. should be given to raise his factor VIII level to 50%?
Fibrinogen replacement may be required in patients with
liver failure, DIC, or massive transfusion and in rare patients {desired factor VIII (units/mL) – initial factor VIII
with congenital fibrinogen deficiency. A fibrinogen plasma (units/mL)} × plasma volume (mL) = units of factor VIII
level of about 100 mg/dL is recommended for adequate he- required.
mostasis with surgery or trauma. For example, a patient’s • Blood volume = weight (kg) × 70 mL/kg
fibrinogen must be increased from 30 mg/dL to 100 mg/dL, • 70 kg × 70 mL/kg = 4,900 mL
or an increment of 70 mg/dL (100 – 30 mg/dL): • Plasma volume = blood volume (mL) × (1.0 – Hct)
• To calculate the amount to be infused, first convert mil- • 4,900 mL × (1.0 – 0.30) = 3,430 mL
ligrams per deciliter to milligrams per milliliter by divid- • Solution: 3,430 mL × (0.50 – 0.04) = 1,578 units
ing by 100 (100 mL/dL). • The assayed value on the label can be divided into the
• Multiplying the answer 0.7 mg/mL by the plasma number of units required to obtain the number of vials to
volume, 3,000 mL, we thus require 2,100 mg (0.7 mg/mL × be infused.
3,000 mL).
• To calculate the number of pools needed, divide 2,100 mg
by 750 mg/pool, which equals 2.8 or almost 3 pools.
• Instead, plasma volume can be converted to deciliters by Table 15–3 Recombinant Human Products
dividing by 100 mL/dL or 70 mg/dL × 30 dL. PRODUCT INDICATIONS
Cryoprecipitate was used as a source for fibrin sealant, Factor VIII Hemophilia A, von Willebrand’s disease
which uses cryoprecipitate as the source of fibrinogen. FDA-
approved fibrin sealants, which have been treated to reduce Factor IX Hemophilia B
viral transmission, are preferred. Factor VIIa Inhibitors in hemophilia A or B
Cryoprecipitate was originally prepared as a source of fac- Factor VII deficiency
tor VIII. Each unit of cryoprecipitate must contain at least Acquired factor VII deficiency*
80 units of factor VIII. However, mild or moderate factor VIII • Liver disease
deficiency (hemophilia A) is now treated with desmopressin • Warfarin overdose
acetate (1-deamino-[8-D-arginine]-vasopressin [DDAVP]) or Massive hemorrhage*
factor VIII, or both, whereas severe factor VIII deficiency is
treated only with factor VIII. *Randomized controlled trials needed
2682_Ch15_352-366 22/05/12 11:50 AM Page 358
Only factor VIII products labeled as containing vWF The product is heat-treated and has proved to be virus-safe
should be used for patients with von Willebrand’s disease. over many years of use.
Albumin may be used to treat patients requiring volume
Factor IX replacement. Whether albumin or colloids other than crys-
talloid (i.e., saline or electrolyte) solutions are better for
Factor IX complex (prothrombin complex) is prepared treating hypovolemia with shock is controversial. In many
from pooled plasma using various methods of separation and plasmapheresis procedures, albumin is used routinely as the
viral inactivation. The prothrombin complex contains factors replacement fluid for the colloid that is removed during the
II, VII, IX, and X; however, the product is recommended procedures. Albumin can also be used in the treatment of
for factor IX–deficient patients (hemophilia B), patients burn patients to replace colloid pressure.
with factor VII or X deficiency (rare), and selected patients Albumin, with diuretics, can induce diuresis in patients
with factor VIII inhibitors or reversal of warfarin overdose.10 who have low total protein because of severe liver or
Activated coagulation factors present in prothrombin com- protein-losing disease. The 25% solution brings about five
plex may cause thrombosis, especially in patients with liver times its volume from extravascular water into the vascular
disease. Recombinant human factor IX (see Table 15–3) is space. Thus, patients receiving 25% albumin need to have
effective only in the management of factor IX deficiency. adequate extravascular water and compensatory mechanisms
The dose is calculated in the same manner as that for to deal with the expansion of the blood volume.
factor VIII concentrate, using the assayed value of factor IX
on the label, with the caveat that half the dose of factor Immune Globulin
IX rapidly diffuses into tissues, and half remains within the
intravascular space, so the initial dose must be doubled. Immune globulin prepared from pooled plasma is primarily
IgG. Although small amounts of IgM and IgA may be present
Antithrombin and Other Concentrates in some preparations, others are free of these contaminating
proteins. Products are available for intramuscular or intra-
Antithrombin is a protease inhibitor with activity toward venous administration. The intramuscular product must not
thrombin. Heparin accelerates the binding and inactivation be given intravenously because severe anaphylactic reactions
of thrombin by antithrombin. The hereditary deficiency of may occur. The intravenous product must be given slowly
antithrombin is associated with venous thromboses, whereas to lessen the risk of reaction.
the acquired deficiency is seen most frequently with DIC. Immune globulin is used for patients with congenital
Antithrombin concentrates are licensed for use in the hypogammaglobulinemia11 and for patients exposed to
United States for patients with hereditary deficiency of an- diseases such as hepatitis A or measles. For hypogamma-
tithrombin. The product is pasteurized to eliminate the risk globulinemia, monthly injections are usually given because
of HIV or HCV infections. Antithrombin has been shown to of the 22-day half-life of IgG. The recommended dose is
provide no significant clinical benefit in acquired deficiency. 0.7 mL/kg intramuscularly or 100 mg/kg intravenously. For
Thawed plasma is an alternative source of antithrombin. hepatitis A prophylaxis, 0.02 to 0.04 mL/kg intramuscularly
Protein C and protein S are vitamin K–dependent proteins is recommended.
synthesized in the liver. Protein S functions as a cofactor for The intravenous preparation of immune globulin is used
activated protein C, which, in turn, inactivates factors V and increasingly in the therapy of autoimmune diseases, such as
VIII, thus preventing thrombus formation. Deficiency immune thrombocytopenia11 and myasthenia gravis. Various
(hereditary or acquired) leads to a hypercoagulable state mechanisms of action have been postulated. Conceivably, the
(i.e., the tendency for thrombosis). Human plasma–derived infused immune globulin blocks the reticuloendothelial
protein C concentrates are approved for use in hereditary system or mononuclear phagocytic system.
deficiency states. Recombinant-human activated protein C Various hyperimmune globulins are available to prevent
has been used for DIC and sepsis. diseases such as hepatitis B, varicella zoster, rabies, mumps,
Recombinant human activated factor VII (rFVIIa; see and others. These are prepared from the plasma of donors
Table 15–3) has been used to control bleeding episodes in who have high antibody titers to the specific virus causing
hemophilia A and B patients with inhibitors.10,12 rFVIIa has the disease. The dose is recommended in the package insert.
been used in patients with a wide variety of bleeding disor- It should be noted that preparations such as hepatitis B
ders. However, large randomized controlled trials are needed hyperimmune globulin provide only passive immunity after
to define dose, indications, and adverse effects. Reports of an exposure. They do not confer permanent immunity and
use for liver disease, massive transfusion, and other bleeding so must be accompanied by active immunization.
disorders have been promising.13 Rh immune globulin (RhIG) was developed to protect the
Rh-negative female who is pregnant or who delivers an
Albumin Rh-positive infant (see Chapter 19, “Hemolytic Disease of
the Fetus and Newborn [HDFN]”). Much of the IgG in this
Albumin is prepared by chemical and physical fractionation preparation is directed against the D antigen within the Rh
of pooled plasma. Albumin is available as a 5% or a 25% system. Administration of this preparation allows attachment
solution, of which 96% of the protein content is albumin. of anti-D to any Rh-positive cells of the infant that have
2682_Ch15_352-366 22/05/12 11:51 AM Page 359
entered the maternal circulation. The antibody-bound cells components are indicated for recipients who are CMV-
are subsequently removed by the mother’s macrophages, pre- negative and at risk for severe sequelae of CMV infections.1
venting active immunization or sensitization. The risk is greatest for CMV-negative pregnant women
Rh immune globulin products, which can be administered (mainly for the benefit of the fetus), allogeneic CMV-negative
intravenously or intramuscularly, are approved for use in bone marrow and hematopoietic progenitor cell transplant
idiopathic thrombocytopenic purpura patients who are Rh- recipients, and premature infants weighing less than
positive.1 The proposed mechanism of action is blockage of 1,200 g.
the reticuloendothelial system by anti-D–coated RBCs, thereby
reducing the destruction of autoantibody-coated platelets. Irradiated Cellular Blood Components
For RBC transfusion accidents, the number of RhIG vials
is calculated by dividing the volume of Rh-positive packed Blood components are irradiated with gamma radiation to
RBCs transfused by 15 mL, the amount of RBCs covered by prevent graft-versus-host disease, which requires three con-
one vial. The number of vials can be large, so the entire dose ditions to occur:
is often divided and administered in several injections at sep- 1. Transfusion or transplantation of immunocompetent
arate sites and over 3 days. The intravenous preparation may T lymphocytes
also be used. Another approach is to perform an exchange 2. Histocompatibility differences between graft and recipi-
transfusion with Rh-negative blood and then calculate the ent (major or minor HLA or other histocompatibility
dose based on the number of Rh-positive RBCs remaining in antigens)
the circulation. For platelets pheresis, one vial is sufficient 3. Usually, an immunocompromised recipient15
for 30 or more products, because each unit contains fewer
than 0.5 mL RBCs. The dose for leukocyte concentrates can Common after allogeneic bone marrow or hematopoietic
be calculated by obtaining the hematocrit and volume of the progenitor cell transplantation, GVHD is a syndrome affect-
product from the supplier. ing mainly skin, liver, and gut (see Chapter 17, “Cellular
Immune globulins may cause anaphylactic reactions Therapy”).
(flushing, hypotension, dyspnea, nausea, vomiting, diarrhea, Transfusion-associated graft-versus-host disease (TA-
and back pain). Caution should be used in patients with GVHD), occurring less frequently, is caused by viable
known IgA deficiency and previous anaphylactic reactions T lymphocytes in cellular blood components (e.g., RBCs
to blood components. and platelets). The mortality rate is high;15 therefore, pre-
vention is key. Prevention centers on irradiating cellular
components before administration to significantly im-
Special Considerations for Transfusion
munocompromised individuals. Irradiation doses range
Certain categories of patients require the selection of blood from 2,500 to 5,000 cGy, with the higher doses being
products that are leukocyte-reduced, CMV-negative, or irra- more effective but more damaging to RBCs. Irradiation
diated. These special products are detailed here. decreases or eliminates the mitogenic (blastogenic) capac-
ity of the transfused T cells, rendering the donor T cells
Leukocyte-Reduced Cellular Blood Components immunoincompetent.
At risk for TA-GVHD are transfusion recipients with con-
Leukocyte-reduction filters are designed to remove more genital immunodeficiencies (severe combined immunodefi-
than 99.9% of leukocytes from RBCs and platelet products. ciency, DiGeorge syndrome, Wiskott-Aldrich syndrome),
The goal is fewer than 5 × 106 (1 × 106 in Europe) leukocytes Hodgkin’s lymphoma, bone marrow transplants (allogeneic
remaining in the RBC unit. Prestorage filtration in the labo- or autologous), intrauterine transfusion of fetuses, exchange
ratory or at the time of collection, rather than at the bedside, transfusion of neonates, donations from blood relatives, and
is more reliable for leukocyte reduction. HLA-matched platelets.1
Leukocyte-reduced RBCs and platelets can be used to pre- Immunocompetent recipients have experienced TA-
vent febrile nonhemolytic transfusion reactions, to prevent GVHD after receiving nonirradiated directed donations
or delay the development of HLA antibodies, and to reduce primarily from first-degree relatives. The related donor is
the risk of CMV transmission. Controversial effects of leuko- homozygous for one of the patient’s (host’s) HLA haplo-
cyte reduction include decreased mortality and length of types, so the patient is incapable of rejecting the donor’s
hospital stay.1 (graft’s) T lymphocytes, which then can act against the
HLA antigens encoded by the patient’s other haplotype. The
CMV-Negative Cellular Blood Components donor lymphocytes then reject the host. The level of
immunosuppression a recipient must have to develop
CMV is carried, in a latent or infectious form, in neutrophils TA-GVHD is unknown, although patients with the severe
and monocytes. Transfusion of these virus-infected cells in immunosuppressive conditions listed previously are most
a cellular product such as RBCs or platelets can transmit at risk. In addition, the dose of lymphocytes needed for
infection. CMV infection of the patients can be reduced by TA-GVHD to occur is unknown. For this reason, prevention
using leukocyte-reduction filters14 or by providing CMV depends on irradiation and not on reduction of lymphocytes
antibody–negative blood. CMV-negative or leukocyte-reduced by filtration.
2682_Ch15_352-366 22/05/12 11:51 AM Page 360
many units of blood to be performed without the need for Table 15–4 Example of a Massive
homologous blood (see Fig. 15–1). Several types of equip- Transfusion Protocol (MTP)
ment are available for collecting, washing, and filtering shed
blood before reinfusion. Washing of intraoperative or post- INITIAL TRANSFUSION STRATEGY
operative salvaged blood is recommended to remove the cel- Draw type and crossmatch 2 units group O RBCs
lular debris, fat, and other contaminants. Heparin or citrate uncrossmatched
solutions may be used for anticoagulation of the shed blood.
Continuing
For very-low-birth-weight infants, the blood should be reduced amount of all sizes of vWF multimers and is milder
selected to be CMV-seronegative or leukocyte-reduced to than type III, in which little or no vWF is produced. Type IIA
prevent CMV infection, which can be serious in premature is distinguished by a deficiency of high molecular weight
infants. Indeed, pregnant women should be given CMV- multimers, whereas type IIB is identified by abnormal high
negative or leukocyte-reduced cellular components if they molecular weight multimers that have an increased avidity
test negative for CMV. for binding to platelets. Type I patients have the ability to
Irradiation of the blood is recommended to prevent pos- make the full spectrum of vWF multimers but do not pro-
sible TA-GVHD when blood is used for intrauterine transfu- duce them in normal amounts. DDAVP, a synthetic vaso-
sion, for an exchange transfusion (see Chapter 19), or for pressin analog, can stimulate release of the vWF from the
transfusion of a premature (less than 1,200 g) neonate. vascular endothelium in type I patients. DDAVP, however, is
Transfusions in a full-term newborn infant do not require contraindicated in type IIB von Willebrand’s disease. Many
routine irradiation. factor VIII products are assayed for vWF and can be used for
Infants who are hypoxic or acidotic should receive blood type III von Willebrand’s disease or in type I or IIA disease
tested and negative for hemoglobin S. For more detail on in- when DDAVP treatment has failed.
trauterine and exchange transfusions, see Chapter 19. Hemophilia B is the congenital deficiency of factor IX.
Factor IX is activated by factors XIa and VIIa. The activated
Transfusion in Oncology factor IX (IXa), along with factor VIII, ionized calcium, and
phospholipid, activates factor X to Xa. Factor IX deficiency
The bone marrow of oncology patients may be suppressed should be treated with recombinant factor IX or prothrombin
because of chemotherapy, radiation therapy, or infiltration complex concentrates. Factor IX concentrates are made
and replacement of the bone marrow with malignant cells. virus-safe by various sterilization techniques.10
Repeated RBC and platelet transfusions may lead to the need All coagulation factors except vWF are made in the liver.
for rare RBC units or HLA-matched plateletpheresis compo- With severe liver failure, multiple coagulation factor defi-
nents because of incompatibility problems. Platelet transfu- ciencies occur. In addition, some of the coagulation factors
sion requirements may also necessitate a change from produced may be abnormal. The liver also produces many
Rh-negative to Rh-positive products. RhIG may be given to of the thrombolytic proteins, leading to imbalance between
a woman with childbearing potential to protect against im- the coagulation process and the control mechanism. Plasma,
munization. There is less than 0.5 mL of Rh-positive RBCs having normal amounts of all these proteins, can be used to
present in each Rh-positive plateletpheresis component. treat these patients.
A pool of platelets may contain as much as 4 mL of RBCs. Vitamin K aids in the carboxylation of factors II, VII, IX,
One 300-µg dose of RhIg can neutralize the effects of up to and X. With the absence of vitamin K or the use of drugs
15 mL of Rh-positive cells. Thus, one dose could be used for that interfere with vitamin K metabolism, such as warfarin,
30 plateletphereses or 4 platelet pools. the inactive coagulation proteins cannot be carboxylated to
In addition, some malignancies such as chronic lympho- active forms. Vitamin K administration rather than plasma
cytic leukemia and lymphoma are frequently complicated by transfusion is recommended to correct vitamin K deficiency
autoimmune hemolytic anemia, increased destruction of or warfarin overdose. Because several hours are required for
RBCs, and pretransfusion testing problems. vitamin K effectiveness, depending on route of administra-
Oncology patients with hematologic malignancies, such tion, signs of hemorrhage or impending surgery may require
as Hodgkin’s disease and lymphoma, are at increased risk of transfusion of plasma.
TA-GVHD because of the chemotherapy drugs used for treat- DIC is the uncontrolled activation and consumption of
ment. Therefore, these patients should receive irradiated coagulation proteins, causing small thrombi within the
cellular components. vascular system throughout the body. Treatment is aimed
at correcting the cause of the DIC: sepsis, disseminated ma-
Coagulation Factor Deficiencies lignancy, certain acute leukemias, obstetric complications,
or shock. In some cases, transfusion of plasma, platelets, or
Factor VIII is normally complexed with another plasma pro- cryoprecipitate may be required. Monitoring of the PT, PTT,
tein, vWF. Both proteins are necessary for normal hemosta- platelet count, fibrinogen, and hemoglobin and hematocrit
sis. Patients with hemophilia A, or classic hemophilia, have levels helps direct the choice of the next component to be
factor VIII deficiency. Hemophilia A patients have factor VIII used.
levels less than 50%, although clinical disease is generally Platelet functional disorders may be caused by drugs,
not apparent unless the factor VIII level is less than 10% uremia, or congenital abnormalities. Platelet transfusions
(normal is 80% to 120%). Individuals with a level less than in these patients should be reserved for treating hemor-
1% have severe and spontaneous bleeding, typically into rhage or the impending need for adequate hemostasis
muscles and joints. The vWF level is usually normal in (such as a surgical procedure) to decrease development of
patients with hemophilia A. platelet refractoriness. Drugs that interfere with platelet
Von Willebrand’s disease is defined by a deficiency of function, such as clopidogrel (Plavix®), are commonly
vWF. Type I von Willebrand’s disease is characterized by a used in cardiovascular disease and are irreversible for the
2682_Ch15_352-366 22/05/12 11:51 AM Page 363
life of the platelets. Therefore, discontinuing the drug for hospital armband. This prevents a specimen tube labeled
5 to 7 days before surgery will minimize hemorrhage and with one patient’s name being used for the collection of a
lessen the need for massive transfusion. In uremia, DDAVP specimen from another patient. The labels are applied to
may be beneficial as well as dialysis or RBC transfusions. the specimen tubes before leaving the bedside to avoid
DDAVP releases fresh, functional vWF from endothelial labeling the wrong tube. Electronic systems for patient and
cells. Dialysis removes by-products of protein metabolism label identification are available and should be adopted to
that degrade vWF and coat platelets, making both non- reduce clerical errors.
functional. RBC transfusion increases viscosity. Positive identification is carried out in the laboratory.
Clerical check is performed as results are generated and com-
General Blood Transfusion Practices pared to historical results. Another clerical check is done as
blood is issued from the blood bank. The final clerical check
Although blood administration and the hospital transfusion is performed at the patient bedside as the nurse compares
committee are not the responsibility of the transfusion serv- the patient armband with the blood bank tag attached to the
ice, nurses, physicians and administrators look to the trans- component to be transfused.
fusion specialists to guide policies and practices. The patient with difficult veins should have the intra-
venous infusion device in place before the blood is issued
Blood Administration from the transfusion service. All blood components must be
filtered (170-µm filter) because clots and cellular debris
Blood must be administered carefully for patient safety
develop during storage. Blood components are infused
(Fig. 15–2). The positive identification of the patient,
slowly for the first 10 to 15 minutes while the patient is
patient’s blood specimen, and blood unit for transfusion is
observed closely for signs of a transfusion reaction. The
essential. Careful identification procedures prevent a major
blood components should then be infused as quickly as
cause of transfusion-related deaths: ABO incompatibility.
tolerated or, at most, within 4 hours. The patient’s vital signs
Clerical errors represent the main cause of transfusion-
(pulse, respiration, blood pressure, and temperature) should
related deaths and acute hemolytic transfusion reactions.16
be monitored periodically during the transfusion to detect
The identification process begins with positive identifica-
signs of transfusion reaction (see Chapter 16, “Adverse
tion of the patient—that is, asking patients to state or spell
Effects of Blood Transfusion”). Signs and symptoms are fever
their name while you read their armband. The patient iden-
with back pain (acute hemolytic transfusion reaction), ana-
tification label is compared at the bedside to the patient’s
phylaxis, hives or pruritus (urticarial reaction), congestive
heart failure (volume overload), and fever alone (febrile non-
hemolytic transfusion reaction). A delayed hemolytic trans-
fusion reaction (jaundice, decreasing hematocrit level) may
be diagnosed 5 to 10 days after transfusion and thus is not
considered an immediate reaction.
In standard blood administration sets, the 170- to
Transfuse
< 4 hours 260-µm filter removes gross clots and cellular debris. A stan-
dard blood administration filter must be used for transfusion
of all blood components.
Use a filter Rapid transfusion, including exchange transfusion, re-
quires blood warming because the cold blood can cause
hypothermia in the patient, which increases the possibility
of cardiac arrhythmia and hemorrhage. A patient with
paroxysmal cold hemoglobinuria or with potent cold ag-
glutinins may also require blood warming. The blood
Identify patient
and blood unit
warmer should have automatic temperature control with
Have IV in place an alarm that will sound if the blood is warmed over 42°C.
Blood units must not be warmed by immersion in a water-
bath or by a domestic microwave oven because uneven
heating can cause damage to blood cells and denaturation
of blood proteins.
Monitor patient Only isotonic (0.9%) saline or 5% albumin should be
periodically
used to dilute blood components, because other intravenous
solutions may damage the RBCs and cause hemolysis (dex-
trose solutions such as D5W) or initiate coagulation in the
Figure 15–2. Blood administration and patient safety. (Reprinted with permis- infusion set (calcium-containing solutions such as lactated
sion from Kennedy, MS (ed): Blood Transfusion Therapy: An Audiovisual Program. Ringer’s solution). In addition, some drugs may cause
AABB, Bethesda, MD, 1985.) hemolysis if injected through the blood infusion set.
2682_Ch15_352-366 22/05/12 11:51 AM Page 364
After transfusion, the transfusion set may be flushed with serving as a guide for conducting audits.3,7,8,18 The results
normal saline. The empty blood bag can be discarded or of the audits can be used by the transfusion committee to
returned to the transfusion service, according to hospital recommend changes in practice by the hospital staff to im-
policy. A copy of the blood bag tag is placed in the patient’s prove patient care. The transfusion committee also reviews
chart. transfusion reactions to ensure that adverse reactions are
unavoidable. In addition, the transfusion committee
Hospital Transfusion Committee ensures that appropriate procedures (such as for blood
administration) are in place and are followed by hospital
The Joint Commission requires all blood transfusions to be personnel.
reviewed for appropriate use. A hospital transfusion com- The transfusion committee is most effective if the vari-
mittee, although not required, may serve as the peer review ous groups who order and administer blood, such as sur-
group for transfusions. Blood usage review may also be per- geons, anesthesiologists, oncologists, and nurses are
formed prospectively if criteria are approved by the trans- represented on the committee. The transfusion committee
fusion committee and the medical staff.17 The blood bank must have a mechanism for reporting activities and recom-
director may interact and correspond directly with the chair mendations to the medical staff and hospital administra-
of individual hospital departments concerning medical staff tion. Optimally, the transfusion committee ensures that the
blood component usage patterns. Appropriate criteria most appropriate, efficient, and safe use of the blood supply
for blood transfusion have been published (Table 15–5), is achieved.
Review of patients with transfusion reactions of hemolysis, severe allergic signs or anaphylaxis, circulatory overload, transfusion-related acute lung
injury, or infection
RBCs18
Indications Decreased oxygen-carrying capacity or acute loss of more than 20% blood volume
or hemoglobin less than 7 g/dL or hematocrit less than 21%
Exceptions Patients with hemoglobin less than 8 g/dL and acute coronary syndrome
Platelets7
Outcome Platelet count immediately before and 10 minutes to 1 hour post-transfusion except with antiplatelet drugs (e.g., Plavix®)
Plasma
Cryoprecipitate
Data from Simon, TL, et al: Practice parameter for the use of red blood cell transfusions. Arch Pathol Lab Med 122:130, 1998; Slichter, SJ: Evidence-based platelet transfusion guidelines. Hematology
127:172–178, 2007; and AuBuchon, JP, et al: Guidelines for blood utilization review, AABB, Bethesda, MD, 2001.
H/H = hemoglobin/hematocrit
PT = prothrombin time
PTT = partial thromboplastin time
INR = international normalized ratio
2682_Ch15_352-366 22/05/12 11:51 AM Page 365
A 55-year-old man is contemplating surgery for severe 1. What blood component(s) is (are) indicated? Why?
arthritis in the right hip. 2. Describe how the dose is calculated. What laboratory
result is desired?
1. What should be known to determine how many units 3. The patient receives chemotherapy, and 2 weeks
of RBCs should be crossmatched? later the hemoglobin is 6.8 g/dL and the hematocrit
2. How can it be determined if the patient can donate 20%. The patient complains of shortness of breath
blood (autologous predeposit) for use during surgery? when hurrying to the bus stop. The physician decides
3. The patient has a history of bleeding after a tonsillec- to order RBC transfusion. What dose of RBCs is
tomy at age 7 years. What tests should be done to indicated? How is this determined?
further study this potential problem?
4. If the patient is found to have von Willebrand’s Case 15-4
disease, which blood product might be necessary?
A 45-year-old man is admitted to the emergency depart-
Case 15-2 ment vomiting blood. His hemoglobin is 8 g/dL, platelet
count 80,000, PT/INR 18/2, fibrinogen 125 mg/dL. The
A 45-year-old woman complains of tiredness and weak- physician orders 4 units of RBCs, 4 units of plasma, and
ness. She appears pale. Laboratory results are as follows: 1 pool of platelets stat.
hemoglobin 6.2 g/dL, hematocrit 20%, MCV 75 fL, MCHC
28%. On further questioning, she reports excessive men- 1. Which blood group(s) should be selected for the RBC
strual bleeding, sometimes lasting for several weeks. transfusions?
2. For FFP and platelets, which blood type(s) should be
1. Does this patient need a transfusion? Justify your selected?
answer.
2. If the intern decides to give one unit of RBCs, what His type and screen is B positive with positive antibody
would be the resulting hemoglobin and hematocrit screen. The ED nurse calls and informs the blood bank
levels? that the massive transfusion protocol has been started.
3. Which components should be prepared?
Case 15-3
4. Which blood group and Rh type should be selected?
A 22-year-old woman presents with easy bruising and 5. What should be done about the positive antibody
fatigue. A complete blood count reveals hemoglobin screen?
SUMMARY CHART
Transfusion therapy is used primarily to treat two con- Plasma contains all coagulation factors and is indicated
ditions: inadequate oxygen-carrying capacity because for patients with multiple coagulation deficiencies that
of anemia or blood loss and insufficient coagulation occur in liver failure, DIC, vitamin K deficiency,
proteins or platelets to provide adequate hemostasis. warfarin overdose, and massive transfusion.
A unit of whole blood or RBCs in an adult should in- Cryoprecipitate contains at least 80 units of factor VIII
crease the hematocrit level 3% or hemoglobin level and 150 mg of fibrinogen, as well as vWF, and factor
1 g/dL. XIII.
RBCs are indicated for increasing the RBC mass in pa- Factor IX is used in the treatment of persons with
tients who require increased oxygen-carrying capacity. hemophilia B.
Platelet transfusions are indicated for patients who are Immunoglobulin (IG) is used in the treatment of con-
bleeding because of thrombocytopenia. In addition, genital hypogammaglobulinemia and patients exposed
platelets are indicated prophylactically for patients to hepatitis A or measles.
who have platelet counts under 5,000 to 10,000/µL. Massive transfusion is defined as the replacement of
Each dose of platelets should increase the platelet one or more blood volume(s) within 24 hours, or
count 20,000 to 40,000/µL in a 70-kg human. about 10 units of blood in an adult.
A plateletpheresis product is collected from one donor Emergency transfusion warrants group O RBCs when
and must contain a minimum of 3 × 1011 platelets. patient type is not yet known.
2682_Ch15_352-366 22/05/12 11:51 AM Page 366
Chapter 16
Adverse Effects of Blood Transfusion
Susan Ruediger, MLT, CSMLS, Ileana Lopez-Plaza, MD
OBJECTIVES
1. Define transfusion reaction.
2. Explain the risks of transfusions. Compare and contrast acute transfusion reactions and delayed transfusion reactions.
3. List the types of acute transfusion reactions and delayed transfusion reactions.
4. Differentiate the clinical signs and symptoms of the various types of transfusion reactions.
5. List laboratory findings associated with acute transfusion reactions.
6. Describe the pathophysiology, signs, symptoms, therapy, prevention, and clinical workup of transfusion reactions.
7. Describe the procedures to follow in the event of a suspected transfusion reaction.
8. Explain the importance of the patient’s history in relation to medications, transfusion history, and pregnancies.
9. List the logical steps and procedures to follow in a laboratory investigation of transfusion reactions.
10. Describe the appropriate reporting of transfusion reaction workups.
11. List accreditation agencies involved in determining policies regarding transfusion reactions.
12. State the regulatory record requirements and procedures to follow in reporting a fatal transfusion reaction.
367
2682_Ch16_367-390 22/05/12 11:54 AM Page 368
As with other transfusion service–related activities, the Iron overload Any patient with chronic RBC
transfusion
recognition and evaluation of transfusion reactions must fol-
low established regulations. Preventive measurements must Neonatal 1.3% overall
be performed when feasible. Transfusion reactions should
Pediatric 1.6% overall
be promptly recognized and evaluated. Any death that is
suspected to be related to a transfusion event must be Autologous 0.43%
reported to the federal government and other governmental
Plasma derivatives Varies among derivatives; severe
entities according to the state or jurisdiction.10 A summary
reactions are rare
of transfusion reaction–related regulations is provided in
Table 16–2.10–12 Therapeutic apheresis 1.6%
PLT = platelets; PPP = pre-pooled platelets; RBC = red blood cell; SDP = single donor platelet;
Recognition, Evaluation, TACO = transfusion-associated circulatory overload; TA-GVHD = transfusion associated graft
and Resolution versus host disease; TAS = transfusion associated sepsis; TRALI = transfusion-related acute
lung injury
The diagnosis and treatment of a transfusion reaction is a
multidisciplinary task. It starts with the patient at the bed-
side and is completed with the interpretation and recommen- nurses, physicians, intermediate-level practitioners, and
dations by the transfusion service physician. medical technologists involved in any steps in a transfusion.
The initial step occurs at the bedside and involves the Figure 16–1 describes the roles for each of the medical per-
patient, the transfusionist, and the physician responsible for sonnel involved in the recognition, evaluation, and resolu-
the patient at the time of transfusion. The intermediate step tion of a transfusion reaction.
and final steps involve the transfusion service technical staff
and the transfusion service physician. The execution of each Basic Immunohematology Testing
step is critical for the recognition, treatment, evaluation, and
recommendations for future transfusions. This underlines The initial transfusion reaction workup performed by the
the importance of education and competency assessment for technical staff consists of steps to rule out a hemolytic
2682_Ch16_367-390 22/05/12 11:54 AM Page 369
AABB = American Association of Blood Banks; CAP = College of American Pathologists; FDA = Food and Drug Administration
transfusion reaction.10 Figure 16–2 describes the basic identify its cause. If the workup rules out hemolysis, then
immunohematology workup performed for the evaluation other etiologies for the reaction are evaluated according to
of any acute transfusion reaction. If the workup suggests the patient’s signs and symptoms and the evolution of
the presence of hemolysis, then more specific testing the transfusion reaction. The latter are discussed further
is conducted to confirm the presence of hemolysis and in the sections for specific types of transfusion reactions.
• Generate a final
transfusion report, including
interpretation of the
transfusion reaction and
recommendations for future
transfusions
Figure 16–1. Roles in recognition, evaluation, and resolution of a transfusion reaction. IV = intravenous; TRALI = transfusion-related acute lung injury
2682_Ch16_367-390 22/05/12 11:54 AM Page 370
Basic Testing
Post-Transfusion Reaction Sample
No No Hemolysis No
Discrepancy Negative* Positive Discrepancy
Discrepancy Hemolysis present Discrepancy
Notify TS Go to
Notify TS Physician Second Draw: physician secondary Discrepancy revealed
testing if
If another patient is involved Hemolysis present on Go to requested If another patient is involved
in discrepancy, take second draw secondary by TS in discrepancy, take
necessary step to prevent testing if physician necessary steps to prevent
another adverse event. Notify TS physician requested another adverse event
by TS
Go to secondary testing if Go to secondary testing if physician Notify TS physician
requested by TS physician requested by TS physician
Go to secondary testing if
requested by TS physician
Figure 16–2. Basic immunohematology workup for the evaluation of a transfusion reaction. The purpose of basic immunohematology testing during the evaluation of
an acute transfusion reaction is to identify the presence of hemolysis. TS = transfusion service
Figure 16–3 describes the secondary testing performed incompatible blood, as little as 10 mL, can cause rapid he-
according to the presenting signs and symptoms and the molysis.2 The severity of symptoms is closely related to the
evolution of the transfusion reaction as requested by amount and rate of incompatible blood transfused. The
the transfusion service physician. Table 16–3 summarizes patient’s underlying clinical condition can obscure recogni-
the medical personnel and their tasks in transfusion reac- tion of the reaction. The interaction of preformed antibodies
tion recognition, evaluation, and resolution.13 in the recipient with the donor red cell antigens is the basis
for the pathophysiology. The most severe reactions are asso-
Acute Transfusion Reactions ciated with ABO incompatibility.1
Figure 16–5 shows the pathophysiology of the transfu-
Acute transfusion reaction is defined as a reaction in which sion reaction. Previously formed IgM or IgG antibodies in
signs and symptoms present within 24 hours of a transfu- the recipient recognize the corresponding donor red cell
sion. Most of the acute reactions can be grouped according antigens, immune complexes are formed, the complement
to the common etiology that gives similar presenting signs cascade is activated, vasoactive amines and inflammatory
and symptoms, as illustrated in Figure 16–4.14 mediators are released into the plasma, and the coagula-
tion cascade is activated.2 The activated lytic arm (mem-
Acute Hemolytic Transfusion Reaction brane attack complex) of the complement cascade causes
hemoglobinemia (Fig. 16–6) and hemoglobinuria, the
Acute hemolytic transfusion reaction (AHTR) consists of hallmarks of intravascular hemolysis (Fig. 16–7). The
acute hemolysis with accompanying presenting symptoms secreted vasoactive amines and inflammatory mediators
within 24 hours of transfusion. Etiology may be of immune are responsible for most of the systemic symptoms. The
or nonimmune origin. activation of the coagulation cascade causes diffuse
In the immune mediated acute hemolytic transfusion intravascular coagulopathy (DIC) and the consequent
reaction, accompanying signs and symptoms include abdom- bleeding. A combination of the vascular collapse and the
inal, chest, flank, or back pain; pain at infusion site; feeling DIC-initiated end organ microthrombi contribute to renal
of impending doom; hemoglobinemia; hemoglobinuria; failure. Without the completion of the complement cas-
hypotension; renal failure; shock; and diffuse intravascular cade, the red cell undergoes extravascular hemolysis. This
coagulopathy. Red or dark urine or diffuse oozing may be is typical of non-ABO antibodies and delayed hemolytic
the only sign in the anesthetized patient. A small volume of transfusion reactions.2
2682_Ch16_367-390 22/05/12 11:54 AM Page 371
Secondary Testing
Post-Transfusion and Pre-Transfusion Reaction Sample
Figure 16–3. Secondary testing for the evaluation of an acute transfusion reaction. A basic immunohematology test indicative of hemolysis will require additional
testing in order to investigate the cause of hemolysis. The transfusion service physician may request additional testing as part of the evaluation of suspected nonhemolytic
transfusion reactions. ALTR = allergic transfusion reaction; BNP = B-type natriuretic peptide; C3d = complement fragment 3d; DAT = direct antiglobulin test; HLA = human
leukocyte antigens; HNA = human neutrophil antigens; IgG = immunoglobulin G; LDH = lactic dehydrogenase; TACO = transfusion-associated circulatory overload;
TAS = transfusion-associated sepsis; TRALI = transfusion-related acute lung injury; WBC = white blood cells
Patient’s physician Evaluate the patient, make recommendations for immediate intervention, and request transfusion reaction
workup
TS technologist Perform basic testing to identify hemolysis and perform secondary additional testing to identify the etiology
of hemolysis
TS physician Order supplemental testing to identify etiologies of hemolysis or other testing to assist in the diagnosis
of nonhemolytic transfusion reactions
Quality management (transfusion Track and trend transfusion reaction occurrences; perform root cause analysis; recommend corrective
service, transfusion committee, and preventive measures
Biovigilance)
TS = transfusion service
2682_Ch16_367-390 22/05/12 11:54 AM Page 372
Figure 16–4. Acute transfusion reactions. Acute transfusion reactions are divided according to one of three key presenting symptoms: fever, allergic, or pulmonary.
AHTR = acute hemolytic transfusion reaction; FNHTR = febrile nonhemolytic transfusion reaction; TACO = transfusion-associated circulatory overload; TAS = transfusion-
associated sepsis; TRALI = transfusion-related acute lung injury
Less frequently, ABO-incompatibility-related acute and the patient’s physician notified. The intravenous access is
hemolytic transfusion reaction may be observed with the maintained and used for supportive therapy. Any additional
transfusion of plasma containing sufficient quantities of iso- therapeutic intervention at this time should be appropriate
hemagglutinins (by volume or antibody titers) against the to the patient’s clinical course with emphasis on maintaining
recipient ABO antigens.15 This syndrome has been observed adequate blood pressure and urine output. As soon as possi-
with the transfusion of platelets, mainly group O single ble, the patient’s nurse should notify the transfusion service,
donor platelets transfused to a non-O recipient.16 Recent complete the transfusion reaction documentation, draw the
studies have shown that whole blood–derived pooled post-transfusion reaction samples, and collect a urine sample,
platelets may contain similar levels of isohemagglutinins.17 if possible. Once the transfusion service receives the docu-
Nonimmune hemolysis most frequently presents as ments, samples, and blood component bag, the technologist
asymptomatic hemoglobinuria, although it has been occa- should begin the basic reaction evaluation (see Fig. 16–2) and
sionally associated with renal dysfunction, and rare related notify the transfusion service physician. If a discrepancy is
death has been reported.18 Its pathophysiology is independ- identified during clerical verification, the transfusion service
ent of the presence of antibodies. It can be caused by chem- technologist should take any steps necessary to determine
ical damage or mechanical damage such as improper whether another patient is involved in order to prevent
shipping or storage temperatures; incomplete deglyceroliza- another adverse event.
tion of frozen red blood cells; needles used for transfusion If the postreaction blood sample appears hemolyzed upon
with an inappropriately small bore size; rapid pressure inspection, the technologist should request a second sample
infuser or roller pumps; improper use of blood warmers; to verify that the hemolysis is not secondary to a difficult
concurrent infusion of unapproved fluids via the transfusion blood draw. Perform a direct antiglobulin test (DAT) on the
line; and bacterial contamination. postreaction sample. A negative DAT does not exclude
When an immune AHTR is suspected, the transfusion must immune hemolysis. If an immune hemolysis is suspected, all
be discontinued immediately, a clerical verification performed, prereaction testing is to be repeated using the postreaction
Immune Complexes
Bleeding
Vascular Renal
Bronchospasm
collapse failure
Figure 16–5. Overview of the pathophysiology of an acute immune hemolytic transfusion reaction. In an acute immune hemolytic anemia due to ABO incompatibility,
the full complement cascade is activated by the immune complexes formed by the interaction between the incompatible IgM isohemagglutinin and the ABO antigen. The
activation of the membrane attack complex damages the red cells. The release of inflammatory mediators, and the activation of the coagulation cascade cause the signs and
symptoms associated with the reaction. IgM = immunoglobulin M
2682_Ch16_367-390 22/05/12 11:54 AM Page 373
INTRAVASCULAR HEMOLYSIS
RBC
Globin
HEPATOCYTE
Methemalbumin
Fe Bilirubin
Hemosiderin
Fecal urobilinogen
Hemoglobin
Methemoglobin
Consultation with appropriate medical specialists early Skin flora and, less frequently, gut flora associated with
in the course is also recommended. In acute nonimmune transient bacteremia in an asymptomatic donor are consid-
hemolytic transfusion reactions in which asymptomatic ered the sources of bacterial contamination. Bacterial endo-
hemoglobinuria is the only sign observed, adequate hydra- toxins generated during storage may contribute to
tion is recommended. TAS-related morbidity and mortality. Table 16–4 outlines
Improper patient identification at the time of sample col- bacterial organisms commonly implicated in TAS. Not sur-
lection or transfusion is the most common cause of an acute prisingly, bacterially contaminated platelets are considered
immune hemolytic transfusion reaction.1 Therefore, it is the most frequent component causing TAS, due to room tem-
imperative that the patient be properly identified at the bed- perature storage requirements. The contamination rate in
side. To increase overall transfusion safety, implementation association with red blood cell transfusions is related to the
of new technology such as handheld bar-code scanners and inhibition of the growth and viability of most bacteria at the
radio-frequency chips for patient identification, and a similar component storage required temperatures (1°C to 6°C) and
system for refrigerators in pharmacy for the release of med- thus the lower risk for TAS.
ication may further reduce misidentification due to human Laboratory workup for the diagnosis of TAS consists of
error.2 ruling out hemolysis and performing a gram stain and cul-
The incidence of hemolytic reactions associated with the ture of the implicated component, as well as getting a blood
transfusion of platelets containing incompatible isoagglu- culture from the patient. The sample from the implicated
tinins has been reduced by 83% overall.15 This has been done container must be obtained from within the container and
by limiting the volume of incompatible plasma transfused not a segment attached to the component. Blood cultures
with platelet transfusions or by measuring the isohemagglu- from the patient should be drawn from a venous site different
tinin titers of group O platelets16,17 to decide if such platelets from the transfusion site. The returned component should
may be used for transfusion to a non-O recipient. The use of be inspected for color changes, presence of bubbles, hemol-
platelet additive solutions as a substitute for plasma to store ysis in a red blood cell component, absence of swirling, or
platelets may further limit the risk of a hemolytic transfusion presence of clumping in a platelet component. A positive
reaction associated with the transfusion of ABO-incompatible blood culture from the patient without confirmation of the
platelets.19 same organism in the transfused component is not sufficient
for the diagnosis. Isolation of the same organism in both the
Transfusion-Associated Sepsis implicated blood bag and the patient’s blood is key for the
diagnosis of TAS. It is important to remember that a colo-
Transfusion-associated sepsis (TAS) is an acute nonimmune nized or infected central venous catheter used for transfusion
transfusion reaction presenting with body temperatures usu- may be the source of the bacteria causing the signs and
ally 2°C or more above normal and rigors that can be accom- symptoms observed during transfusion.
panied by hypotension. The symptoms usually present Treatment consists of discontinuing the transfusion imme-
shortly after the transfusion begins. TAS occurs when a diately upon suspecting a TAS, providing supportive treat-
bacteria-contaminated blood component is transfused. ment, and possibly initiating antibiotic therapy. Antibiotic
Abruptness of presentation may be similar to AHTR; milder therapy can be initially started with a broad-spectrum cover-
cases may mimic a febrile nonhemolytic transfusion reaction age and then adjusted to more specific coverage based on the
(FNHTR). The number of organisms transfused may influ- organism identified and its antimicrobial susceptibility.
ence the clinical presentation and outcome. Mortality risks TAS prevention is directed toward decreasing the risk
include contamination by a gram-negative rod, patient’s age, of bacterial contamination at the time of collection and
volume transfused, and platelet storage time.13 detecting contamination prior to transfusion. Interventions
to prevent the transfusion of a contaminated component a hypothermic patient to normal body temperature should
are focused only on platelet components. Box 16–1 lists not be considered to be a FNHTR.13
interventions currently in use to decrease the risk of TAS The etiology of FNHTR has been attributed to two differ-
associated with platelet transfusions. The interventions ent white cell–related mechanisms. The first one is immune
used to detect bacterial growth prior to storage are more mediated and is due to the presence of preformed antibodies13
sensitive but delay the distribution of platelets for clinical reacting against white cells in the blood component for which
use. The interventions used to detect bacterial contamina- antigen–antibody specificity is shared, which causes the
tion at the time of transfusion are more rapid but less sen- release of endogenous pyrogens. The second mechanism is
sitive. A donor who has given blood more than 20 times closely related to platelet storage changes, which involve the
may be at increased risk due to scarring on the antecubital production and release of biologically active cytokines21,22 by
fossa, which makes skin preparation less effective.13 the white cells present in the component during storage.
A 50% reduction in reported TAS and related fatalities has FNHTR is considered a diagnosis by exclusion. It tends to be
been observed after the introduction of mandatory testing self-limited, so treatment may not be required, as fever will
for the detection of bacterial growth in single donor platelets resolve within 2 to 3 hours. However, the presence of rigors
before distribution, with one institution showing a 69.7% increases the metabolic rate and many patients express great
reduction after implementing an automated microbial detec- discomfort. Rigors will not respond to antipyretic therapy, but
tion system.3 A similar standard now applies to pre-pooled treatment with meperidine may quickly resolve them. How-
and leukoreduced whole blood–derived platelets. Neverthe- ever, because of its respiratory-depressant effect, meperidine
less, because of the existing risk of contamination at the time must be used with extreme caution.
of collection, single donor platelets carry a lower risk of con- There are several approaches to prevent the FNHTR. The
tamination when compared to pre-pooled and leukoreduced use of prestorage leukocyte reduction has shown to reduce the
whole blood–derived platelets, due to the single venipunc- number of white blood cells present in packed red blood cells
ture used in the collection of the former and the multiple and platelet concentrates before their release of cytokines, thus
venipunctures required with the latter.20 making this intervention the most effective in preventing and
reducing the incidence of FNHTR.23
Febrile Nonhemolytic Transfusion Reaction
Allergic Transfusion Reactions
Febrile nonhemolytic transfusion reaction (FNHTR) is an
acute complication of transfusion presenting with at least a Allergic transfusion reactions (ALTR) are acute, immune
1°C increase in body temperature that can be accompanied complications of transfusion presenting with a variety of
by chills, nausea or vomiting, tachycardia, increase in blood symptoms that can vary according to the reaction’s degree
pressure, and tachypnea. Occasionally, shaking chills is the of severity. ALTR occurs as a response of recipient antibod-
only initial presenting symptom, followed by an increase in ies to an allergen present in the blood component. ALTR
body temperature up to 30 minutes after discontinuing the can range from minor urticarial effects to fulminant anaphy-
transfusion.13 An asymptomatic rise in body temperature in lactic shock and death. The more common, milder reactions
consist of weals, hives, erythema, or pruritus. Severe reac-
tions (anaphylactoid or anaphylactic) are rare and can
present with bronchoconstriction (wheezes), angioedema
BOX 16–1 (periorbital edema, tongue swelling), gastrointestinal symp-
toms (diarrhea), and cardiovascular instability (hypoten-
Interventions to Prevent Transfusion-Associated sion, cardiac arrhythmia, loss of consciousness, shock,
Sepsis cardiac arrest).
At Collection Symptoms associated with milder reactions can present
• Donor history any time during or after the transfusion. However, symptoms
• Proper phlebotomy technique associated with more severe reactions will generally appear
• Single arm collection technique shortly after the transfusion has been started and minimal
• Diversion pouch volume has been transfused. The pathophysiology of ALTR
is due to the activation of mast cells in the recipient triggered
Prior to Storage
most frequently by an allergen present in the plasma of the
• Prestorage leukoreduction blood component. Patient preformed IgE antibodies interact
• Pathogen inactivation with the donor-derived allergen. The binding of the allergen
• Automated culture to the IgE bound to the mast cell results in the release of his-
At Time of Transfusion tamine and other granule contents (type I hypersensitivity
• Visual inspection reaction).24 ALTR resulting from non-IgE-mediated release
• Biochemical markers of mast cell mediators are termed anaphylactoid.2
• Microscopy/stains The triggering factor for most severe ALTR is almost
• Immunoassays never identified, but the most classic example of such a
reaction is the IgA-deficiency-related anaphylactic reaction.
2682_Ch16_367-390 22/05/12 11:54 AM Page 376
The classic presentation in this population is the develop- of cases)32 react with recipient leukocytes, causing aggregates
ment of anaphylaxis shortly after starting a transfusion in a that occlude the pulmonary circulation. Implicated leukocyte
patient who has never been previously transfused. Absolute antibodies include HLAI (4% to 23%), HLAII (34% to 47%),
IgA deficiency (IgA levels less than 0.05 mg/dL) is found in both (18% to 21%), and HNA (8% to 28%). Antibodies against
1:700 individuals of European descent, with only 40% or less class II human leukocyte antigens (HLA), anti-HLA-A2 (anti-
forming class-specific anti-IgA antibodies.25 A similar type body against a class I HLA), and HNA-3a (antibody against
of reaction, associated with haptoglobin deficiency, has been human neutrophil antigens) are frequently associated with
described in the Japanese population.26 severe (ventilatory support required) and fatal cases.32
Mild ALTR involving isolated symptoms or signs of Blood components implicated are usually donated by
urticaria or hives are the only type of transfusion reactions in multiparous females and are mostly associated with large
which discontinuation and restart of the transfusion is allowed volumes of plasma; however, blood components with less
if administration of antihistamines improves symptoms. A than 20 mL of plasma and the presence of anti-HNA-3a
transfusion reaction report to the transfusion service should have also been associated with immune TRALI cases.32 In
still be initiated; however, laboratory workup to rule out rare instances (6%), the antibodies will be present in the
hemolysis is not required.2 In the event that symptoms do not recipient, reacting with the transfused donor leukocytes.32
subside with the administration of antihistamines or when Immune TRALI may occur in patients with no underlying
other symptoms accompany the hives or urticaria, the trans- lung injury or even in healthy individuals.
fusion must not be restarted. In general, milder reactions are The other pathway (nonimmune TRALI) consists of a
treated with antihistamines. More severe reactions may require two-hit event. The risk of developing nonimmune TRALI
treatment with corticosteroids. depends on the patient’s predisposition to this disorder. The
In the presence of anaphylactic symptoms, prompt inter- first hit (i.e., lung trauma or an infectious or inflammatory
vention to maintain oxygenation and stabilize blood pressure disease in the patient) may result in priming of the patient’s
must be initiated, including the administration of subcuta- neutrophils. Therefore, a proinflammatory priming event of
neous epinephrine.2 In patients with proven absolute IgA the patient’s endothelium, which primes the patient’s neu-
deficiency, anti-IgA testing should be performed. Patients trophils, is a basic requirement for nonimmune TRALI. The
with known anti-IgA antibodies or a reliable history of ana- second hit (i.e., transfused biologically active substances
phylactic transfusions must receive blood components and accumulated during storage or antileukocyte antibodies)
intravenous immunoglobulins deficient in IgA. These causes the activation of the primed neutrophils. Both the
patients may receive washed red blood cells and platelets. immune and nonimmune TRALI lead to a final common
However, plasma and cryoprecipitate must be obtained from pathway that causes damage to the endothelium, leading to
IgA deficient donors.2 Anti-IgA antibodies are naturally pulmonary capillary permeability and resulting in noncar-
occurring antibodies, so their presence cannot be predicted. diogenic pulmonary edema.
Therefore, it is recommended that similar precautions be TRALI is a clinical diagnosis based on clinical (Box 16–2)
taken for all patients proven deficient and who are unknown and radiological parameters (Figs. 16–9 and 16–10). There is
and known to have anti-IgA antibodies. a growing appreciation that milder forms of respiratory distress
deferral of all female apheresis PLT donors; a loss of 22.5% marker of congestive heart failure, may be used to aid in the
from deferral of parous female apheresis PLT donors; and a diagnosis of TACO. A post-transfusion to pretransfusion
loss of 5.4% from deferral of parous female apheresis PLT BNP ratio of 1.5, with a post-transfusion level equal or
donors with a positive screening test result for antibodies to greater than 100 picograms per milliliter as a cutoff point,
HLAs, as reported in this study.34 Thus far, implementation provides a sensitivity of 81% and a specificity of 89% for
of the different preventive interventions has been successful diagnosis of TACO.35 TACO is also associated with increased
in decreasing the risk of immune TRALI and the mortality morbidity and mortality.33
associated with large plasma volume blood components. Treatment consists of putting the patient in a sitting
Judicious application of the best preventable option, taking position, administering supplemental oxygenation, and start-
into consideration the effect on female donor availability, will ing diuresis. Patients with diminished cardiac reserves or
facilitate such implementation. with chronic anemia and those who are very young or very
old are considered at risk of developing TACO, even with a
Transfusion-Associated Circulatory Overload small transfusion volume. Other patients at risk include
those who receive rapid or massive transfusion of blood. For
Transfusion-associated circulatory overload (TACO) is an patients at risk of developing TACO, the transfusion rate
acute, nonimmune complication of transfusion presenting with should be slowed when feasible. Dividing the blood compo-
respiratory distress and hypoxemia that can be accompanied nent into smaller aliquots to be transfused over a longer
by cough, headache, chest tightness, hypertension, jugular vein period of time should be considered.
distention, elevated central venous pressure, and elevated pul-
monary wedge pressure during or after transfusion. TACO Adverse Events Associated with Massive
occurs when the patient’s cardiovascular system’s ability to Transfusions
handle additional workload is exceeded, manifesting as con-
gestive heart failure. The chest radiography is characterized by Massive transfusion is defined as the replacement of one total
the presence of pulmonary edema, cardiomegaly, and distended blood volume in 24 hours or the replacement of 50% of the
pulmonary artery (see Figs. 16–10 and 16–11). blood volume in 3 hours.36 Massive blood replacement may
The laboratory assay of brain natriuretic peptide (BNP), be accompanied by metabolic and coagulation abnormalities
consisting of the measurement of a peptide secreted from the related to transfusion volume and infusion rate. Patient-
ventricles in response to increased filling pressures and a related comorbid factors such as resuscitation maneuvers,
underlying disease, severity and duration of hypotensive
shock, and hypothermia may further contribute to the meta-
bolic complications and coagulation abnormalities.
Hypothermia can incite metabolic and hemostatic derange-
ments. Studies have shown that the transfusion of one unit of
red blood cells with a temperature of 4°C can drop the core
body temperature 0.25°C in an average-sized patient.36 During
massive transfusion, the rate of citrate delivery may exceed
the liver’s capacity for its clearance. Metabolic alkalosis
can develop secondarily to the accumulation of bicarbonate,
the metabolic by-product of citrate. Citrate toxicity will result
in hypocalcemia and cause symptoms such as tingling, shiv-
ering, light-headedness, tetany, and hyperventilation. Rarely,
hypomagnesemia-associated myocardial depression can be ob-
served with severe citrate toxicity. These complications are
mostly observed in patients with liver failure. Citrate-induced
hypocalcemia is rarely associated with clinical consequences
in other massively transfused patients.
Packed red blood cells leak potassium into the plasma or
additive solution of the blood component during storage.
Rapid infusion of a large volume of packed red blood cells
may put patient populations such as neonates and patients
with cardiac, hepatic, or renal dysfunction at risk of devel-
oping hyperkalemia.36 The transient hyperkalemia related to
massive transfusion appear to be related to the patient’s acid-
base balance, ionized calcium levels, and rate of infusion
Figure 16–11. Transfusion-associated circulatory overload (TACO) chest
of the packed red blood cells. Extreme hyperkalemia or
radiograph showing pulmonary edema, obtained 2 hours after initial presentation
of respiratory distress and significant increase in systemic blood pressure during the hypokalemia can compromise the myocardial function.
transfusion of packed red blood cells. (Courtesy of Spizarny, David, MD, Department Coagulation abnormalities can develop secondarily to
of Radiology, Henry Ford Health System.) hemodilution or diffuse intravascular coagulopathy. Use
2682_Ch16_367-390 22/05/12 11:54 AM Page 379
of large volumes of asanguineous fluids and packed red Transfusion-related adverse events during massive trans-
blood cells during resuscitation in hemorrhagic shock fusion can be avoided through careful patient monitoring
will create platelet and coagulation factor deficiencies. for the development of sign and symptoms, changes in lab-
Thrombocytopenia with platelet counts below 50,000/mm3, oratory values, and the appropriate use of medications and
hypofibrinogenemia, and coagulation factor levels below blood components. The infusion of prewarmed resuscitation
25% generally occur after the replacement of two blood fluids and refrigerated blood components will help prevent
volumes.37 Tissue injury may disrupt the procoagulant-an- exacerbation of hypothermia. Hyperkalemia can be reversed
ticoagulant balance, leading to diffuse intravascular coag- by slowing the transfusion rate and by maintaining the acid-
ulopathy and putting the massively transfused patient base balance in the patient. Treatment strategies for the cor-
at risk of bleeding secondary to a combination of platelet rection of bleeding should be guided by baseline and serial
and coagulation factor consumption and secondary assessment of laboratory parameters. Table 16–5 summa-
fibrinolysis. rizes the types of acute transfusion reactions.
Allergic Mild Erythema Clinical diagnosis Temporary discontinue For repeated reactions,
Pruritus DAT not required transfusion consider premedication
Treat with antihistamines with antihistamines
If symptoms improve
restart transfusion
Allergic Severe Angioedema DAT negative Discontinue transfusion For IgA absolute deficient
Wheezing IgA deficiency workup Maintain vascular access patients provide IgA
Hypotension when indicated Treat with subcutaneous deficient blood
Anaphylaxis epinephrine components
Maintain blood pressure
Provide respiratory
support
TRALI Severe hypoxemia CXR: bilateral infiltrates Discontinue transfusion Use male only plasma
No evidence of left atrial Donor test for HLA/HNA Maintain vascular access Exclude or screen female
hypertension antibodies Supplemental oxygen platelet donors
Recipient test for Mechanical ventilation
HLA/HNA antigens
TACO Severe hypoxemia CXR: pulmonary edema, Upright posture Slower transfusion rate
↑ Blood pressure cardiomegaly, distended Supplemental oxygen Transfuse in smaller
Jugular vein distension pulmonary artery Diuresis volumes
↑ Central venous pressure BNP
AIHTR = acute immune hemolytic transfusion reaction; ANIHTR = acute nonimmune hemolytic transfusion reaction; DAT = direct antiglobulin test; DIC = diffuse intravascular coagulopathy;
FNHTR = febrile nonhemolytic transfusion reaction; LDH = lactic dehydrogenase; PRBC = packed red blood cell; TAS = transfusion-associated sepsis
2682_Ch16_367-390 22/05/12 11:54 AM Page 380
Other markers
DAT
of hemolysis
Repeat immunohematology on
previous specimen—if available
Figure 16–14. Immunohematology and other testing in delayed serologic/hemolytic transfusion reactions. The evaluation of a suspected delayed serologic/hemolytic
delayed transfusion reaction may consist of one or more of the basic and secondary tests performed during the evaluation of an acute immune hemolytic transfusion
reaction. DAT = direct antiglobulin test; LDH = lactic dehydrogenase
of the amount of transfused red cells that may have a post-transfusion of a nonirradiated cellular blood component.
shortened survival. Unlike the hematopoietic progenitor transplant, TA-GVHD
Baseline and follow-up samples to assess ongoing hemol- leads to profound marrow aplasia with a mortality rate greater
ysis in the patient should also be performed (hemoglobin; than 90%.2 Death occurs 1 to 3 weeks after first symptoms
total and direct bilirubin; LDH, haptoglobin). DSHTR in gen- appear. Three conditions must exist for TA-GVHD to develop
eral are mild and may require only careful monitoring of the in a recipient: HLA antigen difference between donor and
patient.18 Treatment, if necessary, should be based on the recipient, presence of donor immunocompetent cells in the
patient symptomatology. When antigen typing suggests a blood component, and a recipient incapable of rejecting the
large burden of antigen-positive cells and other laboratory donor immunocompetent cells.40 The number of lymphocytes
results are consistent with ongoing hemolysis, a red cell in a bag is determined by the age of the blood component and
exchange should be considered. The prompt identification the irradiation status. Fresher blood components contain more
and accurate record-keeping of clinically significant red cell viable T lymphocytes.
alloantibodies are required blood bank practices. Whenever At-risk immunodeficient patient populations include
a transfusion is being considered with any patient new to an infants and patients with cancer or compromised immune
institution, it is important to inquire about his or her trans- systems. In patients with an intact immune system, the
fusion history. The use of partially or fully phenotypically TA-GVHD can occur when the patient is transfused with a
matched red cell units for some patient populations such as cellular blood component from a donor homozygous for an
those with sickle cell disease1 may decrease the risk of HLA haplotype that is shared with the heterozygous recipi-
alloimmunization and thus the risk of DSHTR. If a sickle cell ent (Fig. 16–15). In such settings, the recipient’s immune
HTR syndrome is suspected, withholding further transfu- system does not recognize the homozygous HLA haplotype
sions may be required.39 of the transfused donor lymphocytes as foreign and there-
fore will not eliminate them. In contrast, the transfused lym-
Transfusion-Associated Graft-Versus-Host Disease phocytes are able to recognize the recipient nonshared
haplotype as foreign and will mount an immune attack on
Transfusion-associated graft-versus-host disease (TA-GVHD) the recipient.
is defined as a delayed immune transfusion reaction due to an At-risk immunocompetent patient populations include
immunologic attack by viable donor lymphocytes contained individuals receiving cellular blood components from
in the transfused blood component against the transfusion blood relatives and patients receiving HLA matched or
recipient. The presenting reaction with a maculopapular rash crossmatched platelets or granulocytes. The degree of pop-
(often pruritic, typically starting centrally and extending to ulation genetic diversity influences the TA-GVHD risk
the extremities), fever, watery diarrhea (accompanied by of a transfusion from a random donor. For example,
bloody stools and abdominal pain), elevated liver function in Japan the TA-GVHD risk from a random donor transfu-
tests, and pancytopenia occurs between 3 and 30 days sion is as high as 1 in 874 transfusions because of the
2682_Ch16_367-390 22/05/12 11:54 AM Page 382
BOX 16–3
A1 B8 A1 B8
Indications for the Transfusion of Irradiated Cellular
Donor Recipient Blood Components
A1 B8 A2 B44 Immunocompromised State (Indicated by Patient
Condition)
• Intrauterine transfusions
Figure 16–15. Transfusion-associated graft-versus-host disease (TA-GVHD) in • Low birth weight infants
an immunocompetent patient. The triggering event involves the transfusion of a • Neonates receiving a whole blood exchange
cellular blood component from a donor (graft) with a homozygous HLA haplotype
to a recipient (host) with a heterozygous HLA type of which a haplotype is shared
• Neonates undergoing extracorporeal membrane oxygenation
with the donor. In this figure, the HLA haplotype shared is A1,B8. There is no foreign • Congenital immunodeficiencies
haplotype to be recognized by the recipient. However, the recognition of the foreign • Hematopoietic progenitor cell transplantation
HLA haplotype (A2, B44) by the donor triggers an “unopposed” immune attack • Solid organ transplantation
against the recipient. • Acute leukemia
• Hodgkin’s disease
• Patients with B-cell malignancies
population HLA genetic homogeneity, as compared with • Patients receiving fludarabine
France where more HLA genetic heterogeneity exists and
Immunocompetent State (Indicated by Origin
the TA-GVHD risk from a random donor transfusion is 1 of Blood Component to Be Transfused)
in 16,835 transfusions.41
• Directed donations from blood relatives
In cases of TA-GVHD, diagnostic testing includes:
• HLA-matched platelets or granulocytes
• Skin biopsy for superficial perivascular lymphocyte infil- • Crossmatched platelets or granulocytes
trate, necrotic keratinocytes, and bullae formation. • Granulocyte components
• Bone marrow examination for hypocellular or aplastic
marrow, with only the presence of macrophages. HLA = human leukocyte antigen
• Liver biopsy for degeneration and eosinophilic necrosis of
small bile ducts, intense periportal inflammation, and lym-
phocytic infiltration.
• Molecular studies from the patient’s buccal swabs However, in Europe newer techniques for lymphocyte inac-
or skin fibroblasts, or otherwise from those of first- tivation such as photochemical treatment of platelets and
degree relatives to distinguish between the recipient and plasma have been established and used as an alternative to
donor cells. gamma irradiation.43
and glycoprotein (GP) IV.41 The diagnosis is confirmed by associated organ damage.47 Table 16–6 summarizes the cat-
demonstrating the presence of antibodies in the patient’s egories of delayed transfusion reactions.
plasma against human platelet antigens and the absence of
the implicated antigen specificity in the patient’s platelets. “Silent” Transfusion-Related Adverse
PTP is a self-limiting syndrome with the platelet count Events
usually returning to normal within 2 weeks. However, up to
a 13% mortality related to intracranial hemorrhage has been There are some incidents or errors associated with transfusions
reported.41 The standard of treatment consists of the infusion that do not necessarily present with transfusion-associated
of intravenous immunoglobulin, 0.5 to 1 grams per kilogram symptoms. Therefore, the authors assign the term silent to
per day over a period of 2 to 10 days, with an average these adverse events. However, any of these have the potential
response time of 4 days after initiating therapy.41 Patients to cause an adverse event if gone unnoticed. Such events
with a documented history of PTP should receive, if possible, include transfusion sample collection errors, red blood cell
antigen-negative blood products for subsequent transfusions. alloimmunization, platelet alloimmunization, and transfusion
over- or underdosing.
Iron Overload
Sample Collection Errors
Iron overload is a delayed, nonimmune complication of
transfusion, presenting with multiorgan (i.e., liver, heart, Transfusion sample collection errors consist of those samples
endocrine organs) damage secondary to excessive iron that have the wrong blood in the tube (major, risk 1:1,200 to
accumulation. Each unit of red blood cells contains approx- 1:2,800) or those samples that are provided with missing
imately 250 mg of iron. After 10 to 15 red cell transfusions, required information (minor, risk 1:71 to 1:165).48 Surveys
excess iron is present in the liver, heart, and endocrine have shown an associated risk between the number of minor
organs.4 Chronic red cell transfusion recipients have the collection errors occurring at an institution and the risk for
greatest risk for developing iron overload, with a cumulative the occurrence of a major collection error.49 Major collection
50 to 100 red blood cell unit transfusions causing greater errors are often associated with a properly labeled tube con-
morbidity than the underlying anemia. Therefore, prevent- taining all the required information but drawn from the wrong
ing the accumulation of iron stores by chelation is extremely patient. In the absence of a previous historic blood type in the
important for this patient population. The chelating agents institution for that individual, a great risk occurs for a mis-
bind to iron in the tissues, helping its removal through the transfusion of incompatible blood. Thus, it is important to
urine and feces. Currently, there are three chelating agents track and trend the major and minor collection error rates
available: parenteral deferoxamine, oral deferiprone, and to provide the appropriate intervention or corrective or pre-
oral deferasirox.2 In certain chronic red cell transfusion ventive action. Current preventive efforts for collection errors
patient populations, red cell exchange transfusion may be include improved patient identification mechanisms such as
used as an alternative to transfusion therapy and may help improved computer technology and barrier systems to prevent
delay transfusion-related iron accumulation, preventing transfusion without precise recipient identification.
AHG = antihuman globulin; DAT = direct antiglobulin test; DHTR = delayed hemolytic transfusion reaction; DSHTR = delayed serologic/hemolytic transfusion reaction; HPA = human platelet
antigen; PRBC = packed red blood cells; PTP = post-transfusion purpura; TA-GVHD = transfusion-associated graft-versus-host disease
2682_Ch16_367-390 22/05/12 11:54 AM Page 384
Red Blood Cell Alloimmunization populations and transfusion circumstances that warrant
some additional discussion: infusion of plasma derivatives,
In chronically transfused patients, the risk for them devel- therapeutic apheresis procedures using blood components
oping antibodies against the red cell antigens (RBC alloim- as fluid replacement, neonatal transfusions, and transfusion
munization) increases by 2% to 8%; in the sickle cell patient of autologous blood components.
population, this is as high as 40%.4 Factors influencing
the rate of alloimmunization are antigenic differences, dose, Infusion of Plasma Derivatives
frequency of transfusion, recipient immune status, and
immunogenicity of the donor HLA antigens.4 The presence Today, nonviral transfusion reactions are the most common
of red cell antibodies not only makes allocation of compati- risks seen in association with the transfusion of plasma
ble, antigen-negative units difficult but it also may increase derivatives such as albumin and intravenous immunoglob-
the risk of DSHTR/DHTR. ulin (IVIg), and human-derived coagulation factor concen-
trates.8 These adverse events can be acute or delayed. Most
Platelet Alloimmunization are transient and do not appear to have clinical sequelae.
Nevertheless, on rare occasions, these plasma derivatives
The presence of antibodies against class I HLA antigens is have the potential for life-threatening adverse reactions.
the most common immune cause of platelet transfusion Albumin infusion has been associated with hypotensive
refractoriness.4 These antibodies form after exposure to cor- reactions when used as the sole replacement fluid during
responding antigen expressed on contaminating WBCs in plasmapheresis in patients taking ACE inhibitors as treat-
transfused blood components and can interfere with ade- ment for hypertension. IVIg infusion has been associated
quate platelet count increments to platelet transfusions with numerous adverse effects such as renal dysfunction,
(platelet transfusion refractoriness). The Trial to Reduce related to the use of sucrose as a stabilizing agent; dose-
Alloimmunization to Platelets (TRAP) study50 showed that related aseptic meningitis; positive direct antiglobulin test
HLA alloimmunization developed in 45% of the recipients with or without hemolysis; anaphylaxis; TRALI; and throm-
of nonleukoreduced platelets. The rate of HLA alloimmu- boembolic events. Infusion of human-derived coagulation
nization decreased to 17% when an intervention to decrease factor concentrates may be associated with allergic reactions
the number of WBCs present by leukoreduction was used. and, depending on the coagulation protein being replaced,
Antibodies against the human platelet antigen system are a with thrombosis.
less common cause of platelet transfusion refractoriness (rate
of 2% to 10% in multiply transfused patients).4 The leuko- Therapeutic Apheresis
reduction of cellular platelets does not seem to decrease the
latter risk. Another adverse effect of the development of During therapeutic apheresis, transfusion reactions can
antibodies against the HPA is post-transfusion purpura. occur, or inherent symptoms to the procedure may be aggra-
vated when the replacement fluids used consist of blood
Transfusion Overdosing and Underdosing components.9 Adverse effects most frequently reported
include perioral or acral paresthesias, seen more frequently
For many years, transfusion practices have been providing when citrated plasma rather than albumin solution is used
blood components in standardized dosing that do not nec- as the replacement fluid. Anaphylactoid reactions have been
essarily take into account the component contents, patient reported with the use of albumin as the replacement fluid in
blood volume, or the baseline laboratory parameters initiat- patients receiving ACE inhibitors. There are also reported
ing the request for transfusion. For pediatric transfusions, cases of bacterial contamination of albumin solutions.
the volume to be transfused is mostly based on the patient’s
weight. However, in adults, for the transfusion of RBCs, Neonatal Transfusions
2 units have been the standard of practice despite evidence
that, in many scenarios, a 1-unit transfusion may suffice.4 Recognizing transfusion adverse events in neonates may be
Similarly a 2-unit transfusion practice has been adopted difficult in the presence of other concomitant clinical factors.
when transfusing plasma despite recommendations of cal- The more frequently acute immune-mediated transfusion
culating the dose in a way similar to pediatric transfusion reactions observed in adults and older children are rarely
practice, which is based on body weight.51 This persistent seen in neonates due to the latter’s underdeveloped immune
practice can lead to overtransfusion, as demonstrated with system at birth.52 More importantly, neonates may be more
red cell transfusions,4 or to undertransfusion of plasma, as vulnerable to metabolic complications as a result of their
has been observed during the correction of coagulopathy.51 brittle physiological balance.52 Some symptoms may be
associated with the composition of the blood component
Transfusion-Related Adverse Events anticoagulant or preservative solutions, or the temporary dis-
in Special Patient Scenarios continuation of fluid replacement during transfusion.
Neonates may be at risk of developing hyperglycemia or
A transfusion-related adverse event study is usually focused hypoglycemia, hypocalcemia, or hyperkalemia in association
on recipients (mainly adults and older children) of allogeneic with transfusion. These metabolic symptoms often present
blood components. However, there are special patient with similar symptoms that may include jitteriness, tremors,
2682_Ch16_367-390 22/05/12 11:54 AM Page 385
convulsions, hypotonia, lethargy, apnea, and cyanosis. The Blood Typing: B-positive
treatment for the transfusion-associated metabolic changes Antibody Screen: Negative
will depend on their expected duration and the infant’s Immediate Spin Crossmatching: Compatible
underlying condition. Careful planning for replacement bal-
ance between transfusion and other fluids will minimize Upon medical evaluation, the patient was diagnosed
such adverse events. with acute pyelonephritis and anemia. He was promptly
Development of antibodies is extremely rare in infants due started on antibiotics. Shortly after admission, the patient
to the inability of the neonatal lymphocytes to recognize for- was transfused with 2 units of packed red blood cells. The
eign antigens.52 On the rare occasions when an acute immune- patient’s clinical course was significant for persistent
mediated transfusion reaction occurs, the reaction is the result hypotension despite hydration, requiring the addition of
of passively transfused antibodies. Rarely, infants with necro- blood pressure support medication. Due to persistent ane-
tizing enterocolitis or sepsis may develop intravascular hemol- mia, he received five additional units of packed red blood
ysis due the exposure of cryptogenic antigens in the autologous cells. On day 3 after admission, a new sample was sent to
red cells that react with antibodies present in the donor plasma the transfusion service with a request for two additional
that share a similar specificity (T-antigen activation, polyag- units of packed red blood cells.
glutination syndrome).52 Some neonatal patients are also at Transfusion Service Evaluation:
risk of developing graft-versus-host disease. Finally, the infant Day 3 Sample 1
is at risk of developing TAS or mistransfusion in a similar fash- Blood type: O-positive
ion to the adult or older children population. Antibody Screen: Neg
Hemolysis: Yes
Autologous Blood Transfusions
The day 3 sample revealed a blood type discrepancy.
There are several methods used to collect autologous blood: The patient’s medical staff was immediately notified and a
preoperative autologous donation (PAD; planned collection day 3 second sample was requested. The laboratory tech-
and storage of patient’s own blood until the time of surgery), nologist examined both pre- and postsamples for clerical
intraoperative hemodilution (IOH; collection of a patient’s errors. Upon initial review, no name discrepancy or other
own blood at the beginning of the surgery that is then identifying information was identified among the different
returned to the patient at the end of surgery), and perioper- blood bank samples and transfusion records. Both day
ative blood recovery (POBR; the recovery of a patient’s blood 3 samples were noticed to have “orange” appearing plasma
from the surgical field or postoperative drainage that is when compared to the admission sample. Repeat testing
washed and returned to the patient). was performed on the pre- and post-transfusion samples
Autologous transfusions are perceived to be risk-free by with the following results.
many, including patients and their physicians. However, with
the exception of transfusion-transmitted diseases and a slight Day 3 Sample 1 Day 3 Sample 2 Admission Sample:
difference in incidence, all other adverse events associated Blood type: Blood type: O-positive Blood type: B-positive
with allogeneic transfusions (FNHTR, TAS, TACO, mistrans- O-positive
fusion, AHTR) may also occur in association with the trans- Antibody Screen: Antibody Screen: Neg Antibody Screen: Neg
fusion of autologous blood. These adverse events may be Neg
associated with increased morbidity and even death. The Hemolysis: Yes Hemolysis: Yes Hemolysis: No
most efficient way to avoid adverse events associated with ABO discrepancy and hemolysis were identified.
the transfusion of autologous blood is to follow the same Transfusion service records indicated that the patient
rules and regulations that apply to the use of allogeneic had been transfused with seven B-positive packed red
blood, as well as the manufacturer’s instructions when uti- blood cells. No discrepancies were identified.
lizing perioperative blood recovery. Additional testing was performed with the following
results:
Day 3 Sample 2
CASE STUDIES
Direct AHG: Positive (PS 3+, IgG 3+, and C3d 1+)
Case Study 16-1 Eluate: ABS cell I: Neg
ABS cell II: Neg
A 47-year-old male with a history of intravenous drug abuse A cells: Neg
presented to the emergency department with a 1-week B cells: 3+
history of back pain, fever, shaking chills, and red urine. Transfusion Service Physician Follow-up
Blood Test Results (Initial) The transfusion service physician promptly notified the
Hgb: 7 g/L (reference range: 14 to 17.4 g/dL) patient’s physician regarding the ABO incompatibility and
Total Bilirubin: 1 mg/dL (reference range: 0.2 to 1.2 mg/dL) the likelihood of an acute hemolytic transfusion reaction.
Urinalysis: Blood: large; microscopy: many WBCs and granular The patient was evaluated for ongoing signs and symp-
casts toms of acute hemolysis and was found to be unchanged
Continued
2682_Ch16_367-390 22/05/12 11:54 AM Page 386
from baseline with the exception of a temporary decrease labeled, and sent to the transfusion service with a copy of
in urine output that was treated with hydration. the transfusion record listing the reaction. The transfused
Additional laboratory assays were performed and results autologous salvaged units had been discarded and there-
are shown below: fore were not available for evaluation.
LDH 813 (reference range: 100 to 190 IU/L) Transfusion Service Evaluation
Total Bilirubin 3.7 (reference range: 0.2 to 1.2 mg/dL) The laboratory technologist examined both pre- and
Direct Bilirubin 0.3 (reference range: 0.1 to 0.4 mg/dL) postreaction samples for clerical errors. The historic pa-
Haptoglobin Undetectable (reference range: 30 to 200 mg/dL) tient records were checked and verified. No defects were
Urinalysis Blood 3 +; many WBC, RBC, granular and red revealed. Additional testing was performed with the fol-
cell casts lowing results:
During a retrospective review of the patient’s clinical Postreaction sample:
course, an increase in body temperature, a decrease in Blood type: O-positive; no discrepancies identified
blood pressure, an increase in heart rate and respiratory Antibody Screen: Neg
rate, and shaking chills and back pain were associated DAT: Neg
with each of the transfusion episodes. These were thought Hemolysis: No hemolysis
to be related to the patient’s underlying condition and AHG Crossmatch: Both units, previously electronic crossmatched,
therefore not recognized as transfusion-related signs and compatible
symptoms. A more detailed examination of the initial Repeat ABO confirmatory testing on allogenic unit segments:
sample sent to the transfusion service the day of admis- O-positive (both units)
sion revealed that the tube’s original label had a different Transfusion Service Physician Follow-up
name and medical record number than the “typenex
The transfusion service physician notified the anesthesi-
label” placed on top. Transfusion records of the second
ologist that the transfusion reaction showed no discrep-
name were reviewed, showing that a type and antibody
ancies or evidence of hemolysis based on the evaluation
screen had been performed but no crossmatches were
of the 2 units of allogenic blood issued by the transfusion
requested. The emergency department initiated an inves-
service and transfused during surgery. The anesthesiolo-
tigation to find the cause of the major collection error,
gist contacted the hospital’s cell saver charge personnel
revealing that the patients involved in the sample collec-
to determine if the cause of the hemoglobinuria could
tion error were in the same room when samples were
have been caused by the improper use of the cell saver. It
drawn and that the samples were labeled at the nurses’
was revealed that the proper washing solution (0.9%
station where addressographs are kept.
NaCl) was used and there was no malfunction of the cell
Upon follow-up, the patient was found to be improv-
saver. However, the surgeon had requested the use of a
ing and was discharged home 10 days after reaction.
more potent suction because he stated that the one that
Interpretation came with the cell saver kit was too slow. It was deter-
This case represents an acute immune hemolytic trans- mined that the hemoglobinuria was caused by the use of
fusion reaction due to a major collection error with fail- an inappropriate suction during blood salvage, causing
ure to recognize transfusion reaction–related signs and hemolysis of the harvested blood that was then transfused
symptoms. to the patient. The patient’s urine cleared overnight and
the postoperative course was unremarkable.
1. Define acute transfusion reactions.
2. What does the orange-appearing plasma or hemoglo- Interpretation
binemia indicate? This case represents a nonimmune acute hemolytic trans-
3. The severity of symptoms with acute hemolytic reac- fusion reaction due to failure to follow the manufacturer’s
tions are most likely related to what major factor? guidelines for the cell saver’s use. Communication with
4. What are the key clerical checks to be performed by other health professionals served as the key to resolving
the laboratory technologist? this incident.
The transfusion was stopped two-thirds of the way culture were performed on the unit of blood implicated
through. The nursing personnel performed a clerical with the reaction, and two sets of blood cultures were
check by verifying the patient identification band to the drawn from the patient.
transfusion record and the unit crossmatch tag to confirm
Gram stain-unit of blood: Gram-positive cocci in clusters
that the blood component was given to the correct
patient. The patient’s physician was notified. The nurse, Transfusion Service Physician Follow-up:
who had monitored the patient during past transfusions, The transfusion service physician was immediately noti-
expressed her concern to the physician regarding the fied of the gram-stain results. The results were relayed to
severity of the reaction when compared to previous reac- the patient’s physician, who started the patient on broad-
tions observed. The physician requested a transfusion spectrum antibiotic treatment until identification and sen-
reaction workup. A properly labeled postreaction sample sitivity of the cultures were complete. Shortly after, the
was drawn and sent along with the unit of blood, attached blood center was notified of the transfusion service physi-
tubing, and solutions to the transfusion service for a cian’s preliminary findings, and all available donor prod-
transfusion reaction workup. The patient was treated with ucts were discarded. The following day, culture results
Tylenol and discharged with instructions to continue revealed positive growth in both the patient and the unit
monitoring her temperature. of blood, and the organism was identified as Staphylococ-
Transfusion Service Evaluation cus aureus. A follow-up written notification prepared by
The laboratory technologist examined both pre- and the transfusion service physician was sent to the blood
postreaction samples, the blood bag, and the tubing for center for donor evaluation. Further investigation by the
clerical errors. The attached solution was verified to be blood center revealed that the donor arm was not cleaned
0.9% normal saline, and historic patient records were per standard operating procedures, which led to contam-
checked and verified. No defects were revealed. Repeat ination of the unit. The blood center phlebotomist was
testing was performed on the postreaction sample with retrained on proper cleansing techniques.
the following results: Interpretation
Postreaction Sample: This case represents a transfusion-associated sepsis due
Blood type: O-negative to a failure to follow standard operating procedures for
DAT: Neg the arm preparation prior to donation, most likely result-
Hemolysis: No hemolysis ing in contamination of the unit from the skin flora of the
donor.
Three hours later, the patient returned to the emer-
gency department with an increase in temperature and a 1. What laboratory workup is performed to diagnose
decrease in blood pressure. The transfusion service physi- transfusion-associated sepsis?
cian was consulted, and further testing of the unit of 2. What symptoms usually present with transfusion-
blood was ordered. Immediately, a gram stain and blood associated sepsis?
SUMMARY CHART
Transfusion reactions are classified according to postreaction sample from the acute transfusion reac-
symptom time interval. Less than 24 hours: acute tion evaluation or test will need to be followed with
transfusion reactions; greater than 24 hours: delayed comparison to the pretransfusion testing results. If
transfusion reactions. Both further classify into immune necessary, additional testing in duplicate (pre- and
or nonimmune. postreaction samples) could include repeat basic
The transfusion reaction workup is designed to rule in immunohematology testing, eluate, and antigen typing
or rule out hemolysis. to identify the cause of the immune hemolysis.
Acute transfusion reaction evaluation or testing Non-immune hemolysis occurs when the RBC suffers
includes clerical check, examination for visual hemol- mechanical or chemical damage and is manifested as
ysis, DAT, and patient ABO group confirmation. an asymptomatic hemoglobinuria
Acute transfusion reactions include acute hemolytic Transfusion-associated sepsis occurs when bacteria are
reactions, transfusion-associated sepsis, febrile non- introduced to the patient via a contaminated blood
hemolytic reactions, allergic reactions, TRALI, and TACO. product, manifested by an increase in body tempera-
Immune hemolysis occurs when previously formed ture or more than 2°C, rigors, and hypotension. When
IgM (ABO) or IgG (non-ABO) antibodies in the recip- this condition is suspected, additional testing includes
ient recognize the corresponding donor RBC antigen gram staining and cultures of the blood component
and result in complement-mediated intravascular and the patient. Isolation of the same organism is key
hemolysis. Evidence of immune hemolysis in the for the diagnosis.
Continued
2682_Ch16_367-390 22/05/12 11:54 AM Page 388
SUMMARY CHART—cont’d
Febrile nonhemolytic reactions occur when the recip- graft-versus-host disease, post-transfusion purpura,
ient is exposed to the donor cytokines present in the and iron overload.
WBC or plasma and is manifested by an increase in Delayed serologic/hemolytic reactions occurs second-
body temperature of more than 1°C with or without ary to an anamnestic or primary immune response
chills. Workup must exclude hemolytic (transfusion directed to red cell antibodies that may (delayed
reaction workup testing) and septic reactions (symp- hemolytic transfusion reaction) or may not (delayed
toms and patient evaluation). serologic transfusion reaction) be associated with clin-
Allergic reactions can be mild (hives or itching) or se- ical evidence of shortened red cell survival of the trans-
vere (anaphylaxis) and are mainly caused by the release fused RBCs. Besides standard basic immunohematology
of histamine from the interaction between the allergen testing (ABO/Rh, antibody screen and when indicated
present in the donor plasma and the recipient preformed antibody identification), additional testing will include
IgE antibodies. A classic example of a severe allergic re- DAT and, when indicated, an eluate and antigen typing
action is the one seen due to the presence of anti-IgA of the units recently transfused.
antibodies in a patient with absolute IgA deficiency. Transfusion-associated graft-versus-host disease occurs
TRALI occurs most frequently when donor leukocyte when the transfusion-derived donor lymphocytes
antibodies react with the WBCs in the recipient’s lung attack and destroy the recipient immune system, caus-
vasculature, damaging the endothelium and causing ing pancytopenia and death. It is prevented by the
noncardiogenic pulmonary edema. High plasma volume gamma irradiation of blood components for transfu-
blood components from parous female donors are the sion to patient populations at risk.
most commonly associated blood components. There- Post-transfusion purpura is an acquired profound throm-
fore, focus is on prevention achieved by using only male bocytopenia that occurs when a patient with preformed
plasma components or plasma components collected platelet antibodies is transfused with a blood component
from WBC antibody–negative female donors. containing the platelet antigen and sharing specificity
TACO occurs when the patient’s cardiovascular system is with the preformed antibodies, leading to the destruction
unable to handle the transfused volume, resulting in con- of the donor and recipient platelets. Anti-HPA-1a is the
gestive heart failure. This is an underreported complica- most common implicated antibody specificity.
tion of transfusion, often responsive to patient diuresis. Iron overload occurs due to long-term accumulation
Delayed transfusion reactions include delayed of iron in the body tissues from multiple RBC transfu-
serologic/hemolytic reactions, transfusion-associated sions. This causes organ damage.
7. Which transfusion reaction presents with fever, macu- 15. Which of the following is characteristic of iron overload?
lopapular rash, watery diarrhea, abnormal liver function, a. Delayed, nonimmune complication
and pancytopenia? b. Chelating agents are used
a. Transfusion-associated sepsis c. Multiorgan damage may occur
b. Transfusion-related acute lung injury d. All of the above
c. Transfusion-associated graft-versus-host disease
d. Transfusion-associated allergic reaction References
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reduces but does not eliminate the risk of septic transfusion
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by: 2009.
4. Hendrickson, JE, and Hillyer, CD: Noninfectious serious
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c. Improper use of a blood warmer. hemovigilance reports of transfusion-related acute lung injury
d. All of the above in the United Kingdom and the impact of preferential use of
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a. Signs or symptoms presenting during or after 46:1899–1908, 2006.
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b. Immune or nonimmune. blood transfusion: Evaluation and incidence at a large academic
c. Infectious or noninfectious. hospital. Transfusion 38:296–300, 1998.
d. All of the above 8. Callum, J, and Nahirniak, S: Adverse effects of human-derived
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11. With febrile nonhemolytic transfusion reactions: tions, 3rd ed. AABB Press, Bethesda, MD, 2007, pp 501–523.
9. McLeod, BC, Sniecinski, I, Ciaravella, D, et al: Frequency of
a. They are self-limited. immediate adverse effects associated with therapeutic aphere-
b. Fever resolves within 2 to 3 hours. sis. Transfusion 39:282–289, 1999.
c. Treatment is required. 10. Price, TH (ed): Standard for Blood Banks and Transfusion Serv-
d. A and B are correct ice, 26th ed. AABB, Bethesda, MD, 2009.
e. All of the above 11. College of American Pathologists Laboratory Accreditation
Program checklists. Northfield, IL: College of American Pathol-
12. Absolute IgA deficiency is a classic example of a severe ogists, 2009.
allergic reaction. Results indicating an absolute IgA 12. Code of Federal Regulation. Title 21 CFR Part 606-660.
Washington, D.C.: U.S. Government Printing Office, 2008.
deficiency: 13. Davenport, RD: Management of transfusion reactions. In
a. < 0.05 mg/dL Mintz, PD (ed): Transfusion Therapy Clinical Principles
b. < 0.50 mg/dL and Practice, 2nd ed. AABB Press, Bethesda, MD, 2005,
c. < 0.50 gm/dL pp 515–539.
14. Ramirez-Arcos, S, Goldman, M, and Blajchman, MA: Bacterial
d. < 5 mg/dL contamination. In Popovsky, MA (ed): Transfusion Reactions,
13. How are mild allergic transfusion reactions with isolated 3rd ed. AABB Press, Bethesda, MD, 2007, pp 163–206.
15. Fung, MK, Downes, KA, and Shulman, IA: Transfusion of
symptoms or hives and urticaria treated? platelets containing ABO incompatible plasma: A survey of
a. Transfusion is stopped and transfusion reaction 3156 North American Laboratories. Arch Pathol Lab Med
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b. Transfusion is stopped and antihistamines adminis- 16. Josephson, CD, Mullis, NC, Van Demark, C, et al: Significant
number of apheresis-derived group O platelet units have a
trated; when symptoms improve, transfusion is “high titer” anti-A/A,B: Implications for transfusion policy.
restarted. Transfusion 44:805–808, 2004.
c. Stop transfusion and prepare washed red cells. 17. Cooling, LL, Downs, TA, Butch, SH, et al: Anti-A and anti-B
d. Continue transfusion with a slower infusion rate. titers in pooled group O platelets are comparable to apheresis
platelets. Transfusion 48:1206–1213, 2008.
14. TRALI presents with the following symptoms: 18. Davenport, RD: Hemolytic transfusion reactions. In Simon, TL,
a. Respiratory distress Dzik, WH, Snyder, EL, Stowell, CP, and Strauss, RG (eds):
Rossi’s Principles of Transfusion Medicine, 3rd ed. Lippincott,
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versus single donor (apheresis) platelets in the United States: and replacement of major blood loss with plasma-poor red-cell
A systematic review of disparate risks. Transfusion 49: concentrates. Anesth Analg 81:360–365, 1995.
2743–2758, 2009. 38. Ness, PM, Shirey, RS, Thoman, SK, et al: The differentiation of
21. Muylle, L, Joos, M, Wouters, E, et al: Increased tumor necrosis delayed serologic and delayed hemolytic transfusion reactions:
factor alpha (TNFα), interleukin 1, and interleukin 6 (IL-6) Incidence, long-term serologic findings, and clinical signifi-
levels in the plasma of stored platelet concentrates: Relation- cance. Transfusion 30:688–693, 1990.
ship between TNFα and IL-6 levels and febrile transfusion 39. Petz, LD, Cahoun, L, Shulman, IA, et al: The sickle cell he-
reactions. Transfusion 33:195–199, 1993. molytic transfusion reaction syndrome. Transfusion 37:382–392,
22. Heddle, NM, Klama, L, Singer, J, et al: The Role of the plasma 1997.
from platelet concentrates in transfusion reactions. N Engl J 40. Billingham, R: The biology of graft-versus-host reactions. In
Med 331:625–628, 1994. The Harvey Lecture series, 1966–1967. Academic Press,
23. King, KE, Shirey, RS, Thoman, SK, et al: Universal leukoreduc- Orlando, FL, 1968, pp 62:21–78.
tion decreases the incidence of febrile nonhemolytic transfu- 41. McFarland, JG: Posttransfusion purpura. In Popovsky, MA
sion reactions to RBCs. Transfusion 44:25–29, 2004. (ed): Transfusion Reactions, 3rd ed. AABB Press, Bethesda, MD,
24. Hennino, A, Berard, F, Guillot, I, et al: Pathophysiology of 2007, pp 275–299.
Urticaria. Clin Rev Allergy Immunol 30:3–11, 2006. 42. Alyea, EP, and Anderson, KC: Transfusion-associated graft-
25. Sadler, G, Malllory, D, Malmaut, D, et al: IgA anaphylactic versus-host disease. In Popovsky, MA (ed): Transfusion Reac-
transfusion reactions. Transfusion Med Rev 9:1–8, 1995. tions, 3rd ed. AABB Press, Bethesda, MD, 2007, pp 229–249.
26. Koda, Y, Watanabe, Y, Soejima, M, et al: Simple PCR detection 43. Dwyre, DM, and Holland, PV: Transfusion-associated graft-
of haptoglobin gene deletion in anhaptoglobinemic patients versus-host disease. Vox Sanguinis 95:85–93, 2008.
with antihaptoglobin antibody that causes anaphylactic trans- 44. Shulman, NR, Aster, RH, Leitner, A, et al: Immunoreactivity
fusion reactions. Blood 95:1138–1143, 2000. involving platelets. V. Post-transfusion purpura due to a com-
27. Popovsky, MA: Transfusion associated circulatory overload: plement fixing antibody against a genetically controlled platelet
The plot thickens. Transfusion 49:2–4, 2009. antigen. A proposed mechanism for thrombocytopenia and its
28. Goldman, M, Webert, KE, Arnold, DM, et al: Proceedings of a relevance in autoimmunity. J Clin Invest 40:1597–1620, 1961.
consensus conference: Towards an understanding of TRALI. 45. Kickler, TS, Ness, PM, Herman, JH, et al: Studies on the patho-
Transfus Med Rev 19:2–31, 2005. physiology of post-transfusion purpura. Blood 68:347–350,
29. Gajic, O, Rana, R, Winters, JL, et al: Transfusion-related acute 1986.
lung injury in the critically Ill: Prospective nested case-control 46. Strickler, RB, Lewis, BH, Corash, L, et al: Post-transfusion pur-
study. Am J Respir Crit Care Med 176:886–891, 2007. pura associated with an autoantibody directed against a previ-
30. Curtis, BR, and McFarland, JG: Mechanisms of transfused- ously undefined platelet antigen. Blood 69:1458–1463, 1987.
related acute lung injury (TRALI): Anti-leukocyte antibodies. 47. Kim, HC, Dugan, NP, Silber, JH, et al: Erythrocytapheresis ther-
Crit Care Med 34 (suppl.):S118–123, 2006. apy to reduce iron overload in chronically transfused patients
31. Bux, J, and Scahs, UJH: The pathogenesis of transfusion-related with sickle cell disease. Blood 83:1136–1142, 1994.
acute lung injury (TRALI). Br J Haematol 136:788–799, 2007. 48. Dzik, WH, Murphy, MF, Andreu, G, et al: An international
32. Bux, J: Antibody-mediated (immune) transfusion-related acute study of the performance of sample collection from patients.
lung injury. Vox Sanguinis 100:122–128, 2011. Vox Sanguinis 85:40–47, 2003.
33. Gajic, O, Grooper, MA, and Hubmayr, RD: Pulmonary edema 49. Lamadue, JA, Boyd, JS, and Ness, PM: Adherence to a strict
after transfusion: How to differentiate transfusion-associated specimen-labeling policy decreases the incidence of erroneous
circulatory overload from transfusion-related acute lung injury. blood grouping of blood bank specimens. Transfusion
Crit Care Med 34:S109–113, 2006. 37:1169–1172, 1997.
34. Rios, JA, Schlumph, KS, Kakaiya, RM, et al: Blood donations from 50. The Trial to Reduce Alloimmunization to Platelets Study
previously transfused or pregnant donors: A multicenter study Group: Leukocyte reduction and ultraviolet B irradiation of
to determine the frequency of alloexposure. NHLBI Retrovirus platelets to prevent alloimmunization and refractorines to
Epidemiology Donor Study-II. Transfusion 51: 1197–1206. platelet transfusions. N Engl J Med 337:1861–1869, 1997.
35. Zhou, L, Giacherio, D, Cooling, L, et al: Use of B-natriuretic 51. Dzik, WH: Component therapy before bedside procedures.
peptide as a diagnostic marker in the differential diagnosis of In Mintz, PD (ed): Transfusion Therapy Clinical Principles
transfusion-associated circulatory overload. Transfusion and Practice, 2nd ed. AABB Press, Bethesda, MD, 2005,
45:1056–1063, 2005. pp 1–26.
36. Uhl, L: Complications of massive transfusion. In Popovsky, MA 52. Pisciotto, PT, and Luban, NLC: Complications of neonatal
(ed): Transfusion Reactions, 3rd ed. AABB Press, Bethesda, MD, transfusion. In Popovsky, MA (ed): Transfusion Reactions,
435–457, 2007. 3rd ed. AABB Press, Bethesda, MD, 2007, pp 459–499.
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Chapter 17
Cellular Therapy
Melanie S. Kennedy, MD, and Scott Scrape, MD
Introduction Umbilical Cord Blood Processing Transfusion Therapy for HPC Transplantation
Purpose and Goals of HPC Transplantation Cell Enrichment HLA Alloimmunization
Categories of Transplantation Cell Purging and Reduction CMV Transmission
Donor/Patient Selection Product Storage and Shipping Transfusion-Associated Graft-Versus-
Autologous Donors/Patients Shipping Fresh Products Host Disease
Unrelated Donors and National Long-Term Frozen Storage Transfusion in the ABO-Mismatched
Marrow Donor Program Shipping Frozen Products Transplant
Related Donors Thawing and Preparing Products for Case Study
Umbilical Cord Blood Transplant Case Study 17-1
HPC Collection Transplanting the Recipient Summary Chart
Bone Marrow Collection (HPC-M) Infusion of HPCs Review Questions
Leukapheresis (HPC-A) ABO Incompatibility References
Umbilical Cord Blood (HPC-C) Adverse Recipient Reactions
Detection and Measurement of HPCs Engraftment
Indications for HPC Transplantation Graft Rejection
Processing HPC Products Graft-Versus-Host Disease
Initial Testing Relapse of Disease and Donor
Initial Processing Lymphocyte Infusion
Preparation for Freezing HPCs Outcomes
OBJECTIVES
1. List and define the categories of hematopoietic progenitor cell (HPC) donors.
2. List five assessments of an HPC donor.
3. Name a registry for HPC unrelated donors.
4. State the chance of an HLA match between siblings.
5. Describe the differences in health screening of cord blood donors compared with other HPC donors.
6. Compare and contrast the collection of HPC-M and HPC-A.
7. Name the antigen that defines HPCs.
8. Identify three diseases treated with HPC transplantation.
9. Explain the reason few HPC transplants are performed for nonmalignant diseases.
10. List five tests performed on all HPC products.
11. Explain when cultures are necessary during processing of HPCs.
12. State the temperature for frozen storage of HPCs.
13. Identify the cryoprotectant generally used for HPCs.
14. List two methods of determining HPC viability.
15. Describe the freezing process of HPCs.
Continued
391
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OBJECTIVES—cont’d
16. Contrast myeloablative with nonmyeloablative recipient conditioning.
17. Compare major, minor, and bidirectional ABO incompatibility between HPC donor and recipient.
18. Define engraftment for neutrophils, platelets, and all cell lines.
19. Define graft-versus-host disease (GVHD).
20. Define acute versus chronic GVHD.
21. State the purpose of donor lymphocyte infusion.
22. List three important conditions to prevent when transfusing blood products.
23. Select the correct ABO type(s) when transfusing RBCs and plasma to a B recipient with an O donor.
Introduction
BOX 17–1
Cellular therapy encompasses a wide variety of cells for Categories of HPCs for Transplantation
transplantation. This chapter will focus on the cells used
Autologous
for blood and bone marrow transplantation. The bone
• From self
marrow contains the hematopoietic stem cells (HSC) from
Allogeneic
which all the cells of the blood are formed. These stem cells
• From one human to another
differentiate into hematopoietic progenitor cells (HPC),
• Related or unrelated
which are committed to specific cell lines. The myeloid
Syngeneic
line differentiates into erythrocytes, platelets, neutrophils,
• Identical twin or triplet
basophils, eosinophils, monocytes, and macrophages. The
lymphoid line forms dendritic cells and lymphocytes,
including T cells, B cells, and NK cells. Therefore, a
bone marrow transplant repopulates not only myeloid malignancies or metastatic solid tumors. These individuals
cells (erythrocytes, platelets, and granulocytes), but also are generally in disease remission, so the blood or bone
lymphocytes and dendritic cells. marrow is less likely to be contaminated with malignant
The population of hematopoietic stem cells in the bone cells.
marrow is small, about 0.01%. These cells are not a uniform Allogeneic HPC transplantation is used for patients who
population but are in varying states of development. As they cannot donate their own cells because of multiple cycles of
mature, the daughter cells commit to specific cell lines and chemotherapy, irradiation of bone marrow, disease in mar-
are known as hematopoietic progenitor cells (HPC). row (e.g., aplastic anemia), or genetic disease. There is a one
in four chance of an HLA match with siblings for a related
Purpose and Goals of HPC transplant, so many patients must receive HPCs from an un-
Transplantation related, HLA-matched, or mismatched donor (Box 17–2).
These donors are often registered with the National Marrow
HPCs are collected and transplanted for three main reasons: Donor Program (NMDP) or are part of other registries.
(1) to serve as bone marrow replacement following total- Syngeneic HPC transplantation is performed infre-
body irradiation or chemotherapy given to treat primary quently because of the relative rarity of identical twins or
marrow and nonmarrow disorders, (2) to provide a graft- triplets. Identical twins or triplets do not have minor his-
versus-leukemia (or tumor) reaction, or (3) to replenish tocompatibility differences, which means graft rejection,
diseased or destroyed bone marrow. graft-versus-host disease, or graft versus leukemia does not
Categories of Transplantation
HPC transplants fall into three general categories: autolo- BOX 17–2
gous, allogeneic, and syngeneic (Box 17–1). Autologous
means the source of HPCs is the patient, who donates the HLA Matching for Related Transplantation
cells for transplantation. Allogeneic includes both related Sibling—HLA
and unrelated donors. Syngeneic is when the donor and re- • 25% identical
cipient are identical twins. The type of transplantation used • 50% one haplotype
is dependent on, among other things, the type of disease • 25% mismatch
being treated and the availability of the donor. Parents—HLA
Autologous bone marrow or peripheral blood HPCs are • 100% one haplotype
selected for most patients, especially those with marrow
2682_Ch17_391-402 22/05/12 11:56 AM Page 393
occur. In contrast, HLA-matched siblings have minor in- The donor selects the most convenient date for the donation.
compatibilities, which can cause graft-versus-host disease. After HPCs are collected, the fresh product is shipped to the
recipient’s institution. The recipient may be anywhere in the
Donor/Patient Selection world that has an HPC transplant program.
Autologous donors are usually patients who have a hemato- The NMDP also coordinates umbilical cord blood transplants,
logic malignancy. The patient should be in clinical remission locating the appropriate cord blood bank that has the best
to decrease the risk of collecting malignant cells with the matched cord blood. Cord blood is most appropriate for chil-
HPCs. Pulmonary function tests and cardiac stress tests are dren needing transplantation because of their smaller size.
performed, as are the tests listed above. Infectious disease Cord blood is immunologically naïve, so there is less risk of
testing is important for the autologous donor because of pos- graft-versus-host disease than with adult HPCs. In addition,
sible reactivation after the transplant. Also, if positive, the HLA mismatches are better tolerated by the recipient.
HPC product is stored to prevent cross-contamination of Before delivery, the mother must consent to the collection
other products. of umbilical cord blood. The mother is screened for health
issues, infectious disease risks, and family history for genetic
Unrelated Donors and National Marrow Donor diseases. The cord blood is collected at or after delivery, with
Program no risk to the mother or infant.
Allogeneic unrelated donors are selected from a registry, such HPC Collection
as the National Marrow Donor Program (NMDP), which is
the largest registry in the world (Box 17–3). These donors HPCs intended for transplantation are collected in one of
previously agreed to join the registry and have basic infor- three ways: bone marrow collection, known as HPC-marrow
mation, such as ABO, Rh, and HLA type recorded. The (HPC-M); peripheral blood progenitor cell (leukapheresis)
NMDP’s goal is to match 90% of requests, although the per- collection, called HPC-apheresis (HPC-A); and umbilical
centage is less for ethnic and racial minorities. The NMDP cord blood collection, referred to as HPC-cord (HPC-C).
has targeted these underrepresented groups for recruiting
drives to increase the HLA diversity of the registry. Bone Marrow Collection (HPC-M)
The recipient’s institution initiates the search for the best
matched donor. After the search, the selected donor is con- Bone marrow is harvested similar to a diagnostic bone mar-
tacted for initial screening and more testing, including high- row aspiration, except with a stronger and longer needle.
resolution (DNA) HLA typing (see Chapter 21, “The HLA The procedure must be done using general or epidural
System”). After the donor’s workup, the recipient’s institu- anesthesia. The posterior iliac crest is the most frequently
tion accepts the match and selects dates for the transplant. used. Multiple aspirations are performed, moving the nee-
dle within the skin puncture to various points within the
marrow and making additional bone marrow punctures.
The marrow aspirates are transferred within a sterile sys-
BOX 17–3 tem. The usual volume goal for marrow is 10 to 15 mL/kg,
National Marrow Donor Program (NMDP) using the weight of the donor or recipient, whoever is
smaller, with 1,500 mL or more collected.
• Contains the largest registry of recruited donors
Because of the volume collected, some donors may need
• Coordinates searches for HLA-matched adult donors and cord
blood
RBC transfusion. Many donors donate their own blood days
• Recruits donors to provide match for 90% of recipients
to weeks before the HPC-M collection to be transfused dur-
• Emphasizes recruitment of racial and ethnic minorities for HLA
ing or after the collection.
diversity The risks of HPC-M collection include anesthesia, pain
• Provides for collection and transport of the HPC product (acute and chronic), bruising, allogeneic blood transfusion,
and, in rare cases, nerve damage.
2682_Ch17_391-402 22/05/12 11:56 AM Page 394
Leukapheresis (HPC-A) the rate of cord blood transplant is increasing as the availabil-
ity increases.2
HPCs are more frequently harvested from peripheral blood Cord blood is collected after the baby’s delivery and
by leukapheresis (see Chapter 14, “Apheresis”). Because few clamping of the cord and either before or after delivery of
HPCs are in circulating blood (0.05%), numerous days the placenta (Box 17–5). Most collections are performed
would be required to collect sufficient quantities for a suc- soon after delivery of the placenta either in the sterile deliv-
cessful transplant. However, if the HPCs are “mobilized” ery room or a separate sterile room. The HPC-C is collected
from the marrow to the peripheral blood with cytokines, the by gravity into a FDA-approved collection bag containing
number of leukapheresis procedures can usually be reduced CPD anticoagulant, using a needle or cannula in the cord
to one or two. The leukapheresis procedure generally blood vessel. Only 100 to 150 ml are usually collected.3 The
processes 3 to 4 blood volumes to collect a product of 150 collection is of no risk to the mother or infant.
to 500 mL. The higher volumes are the result of the difficulty
in stabilizing the white cell layer during the collection. Detection and Measurement of HPCs
Autologous donors (patients) also may be given
chemotherapy to take advantage of the rebound of the bone HPCs can be identified by, among other things, the pres-
marrow and the increase in circulating HPCs (Box 17–4). ence of the surface CD34 antigen and the absence of
The donors receive the cytokine analog, granulocyte lineage-specific antigens. The CD34 antigen is a cell sur-
colony-stimulating factor (G-CSF) filgrastim to stimulate face transmembrane protein expressed on hematopoietic
the release of more HPCs into the peripheral blood. Less progenitor cells and vascular endothelium throughout the
commonly, granulocyte-macrophage colony-stimulating body. The CD34 antigen is used routinely to determine
factor (GM-CSF) is used. The usual dose of either cytokine when a patient or donor should be collected by leukaphere-
analog is 10 µg per kilogram of body weight per day sis. Cellular therapy labs measure CD34+ cells to determine
(10 µg/kg/day). the quantity of HPCs harvested by bone marrow, apheresis,
Allogeneic donors, mobilized by G-CSF, are collected on or cord blood collection. The HPCs in a sample can be
day 5 and, if needed, day 6. These donors do not receive quantified by flow cytometry using antibody to the CD34
chemotherapy. Autologous donors may be slower to mobi- antigen. HPCs also express HLA, but not ABO blood group
lize and need more time to achieve a high enough HPC antigens.
level to be collected. A few may require plerixafor, an an- The CD34 antigen is measured in autologous and allo-
tagonist to the chemokine receptor 4 on CD34+ cells, to geneic donors who are to be collected by leukapheresis. The
aid in mobilization. donor should have a minimum of 10 CD34+ cells/µL in the
As a side effect of these cytokines, the donor may have peripheral blood for collection to begin. The collection goal
bone pain from the marrow expansion. Other side effects for an adult is 2 to 6 × 106 CD34 positive cells per kilogram
may include headache, myalgia (muscle pain), nausea of recipient body weight (2–6 × 106/kg). Generally, the yield
and vomiting, fatigue, and insomnia. These signs and of CD34+ cells is less for autologous than for allogeneic
symptoms quickly resolve when the collection meets donors, so more days of collection are required.
the goal and the G-CSF (and plerixafor) is discontinued.
Commonly, the platelet count decreases with cytokine stim- Indications for Transplantation
ulation and HPC collection. A few donors need platelet
transfusions. Rare side effects are chronic thrombocytope- The most common indication for HPC transplantation is to
nia and splenic bleeding. treat malignant diseases, such as acute leukemia, chronic
myelogenous leukemia, multiple myeloma, Hodgkin’s dis-
Umbilical Cord Blood (HPC-C) ease and non-Hodgkin’s lymphoma, and solid childhood tu-
mors (Box 17–6). HPC transplantation can extend survival
Umbilical cord blood is rich in HPCs and is another donor and cure disease.4,5 However, the risks must be weighed
source for transplantation. Although umbilical cord blood is against the chance of recurrence of the malignancy and ex-
used less frequently than HPC-A products for transplantation, pected survival.
BOX 17–4
The cellular therapy laboratory receives the HPC products Autologous HPCs are frozen while the donor or recipient is
from the collection facilities for further processing, storage, prepared for the transplant. The volume of the collected
and transportation. The lab is essential to assure the quality product is reduced to limit the aliquots for freezing, and
and quantity of the product before storage, transportation, and RBCs are removed from bone marrow products, because the
dispensing. Careful labeling and preventing mix-ups during freezing and thawing process causes the cells to lyse and re-
processing and storage are fundamental. Processing must lease nephrotoxic hemoglobin and RBC stroma. HPC-A
meet the regulations of the FDA1 and should meet the products have a smaller RBC volume, with hematocrit of 3%
requirements of the AABB Standards for Cellular Therapy to 10% in a volume from 150 to 500 ml. The larger products
Product Services.10 need to be volume reduced before freezing. Contaminating
granulocytes, which are higher in HPC-A products, can
Initial Testing cause febrile reactions with chills in the recipient because of
lysing during thawing.
Upon receipt, the product is examined for proper labeling, After processing, the products are transferred to freezing
clumps of platelets, hemolysis, and integrity of the collection bags in small aliquots of about 45 to 70 ml. The cryoprotec-
container. A sample from the product is tested for CBC, tive chemicals—dimethyl sulfoxide (DMSO) or DMSO and
platelets, WBC differential, CD34+ cells (HPCs), and viabil- hydroxyethyl starch (HES)—are slowly added to a final con-
ity. Sampling is performed in a laminar flow hood, with the centration of 5% to 10% DMSO.13 DMSO and HES cross the
use of a sterile connecting device or integral sampling tubes cell membrane and protect the cells from ice damage and cell
attached to the collection set. dehydration during the freezing and thawing process. The
Cell counts are usually performed on automated instru- amount of DMSO needs to be limited because of its toxicity.
ments, with the differential confirmed manually to confirm For that reason, many labs concentrate the buffy coat as
mononuclear cells, which contain the HPCs. CD34+ cells are much as possible and thus reduce the DMSO required.
2682_Ch17_391-402 22/05/12 11:56 AM Page 396
Umbilical Cord Blood Processing specific for the malignant cell antigen can be used to bind
and then remove (purge) the malignant cells.
Umbilical cord blood requires special processing. Both the T, B, and NK cells can be reduced by using antibodies
mother’s blood sample and the cord blood are tested for ABO, to specific antigens that define these cells. The risk of
Rh, and, for the mother’s sample, antibodies (Box 17–7). The graft-versus-host disease is reduced, but the rate of relapse
product is tested for the total nucleated cell (TNC) count and is increased.
the CD34 content. Testing for hemoglobinopathies is per-
formed. Serologic and DNA testing for HLA A, B, and DRB1 Product Storage and Shipping
is performed on both mother and cord to assure no mix-ups
occurred. It is wise to store samples of viable cells and DNA Factors involved with product storage and shipping include
of both the cord and the mother. shipping fresh products, freezing for long-term frozen stor-
The maternal sample is tested for HIV-1,-2; HBV; HCV; age, shipping frozen products, and thawing and preparing
and HTLV-I,-II. Also, HBsAg, anti-HBc, and syphilis status is products for transplant.
determined. The cord blood is cultured for cytomegalovirus
(CMV). In some cases, WNV by nucleic acid tests (NAT) is Shipping Fresh Products
performed.
Several ways of processing the cord blood product exist; Fresh products can be shipped by air for long distances at
one method is described. The cord blood is diluted with dex- room temperature or at 4°C. An insulated container with
tran and albumin or hetastarch, which improves the viability cooling packs at appropriate temperature should be used to
of the HPCs. The cells are allowed to sediment, and the buffy ship the product. Complete paperwork must accompany the
coat and plasma are transferred to another bag. Centrifuga- product for security checks and customs. A courier hand-
tion is used to separate and then remove plasma. The final carrying the product, as practiced by the NMDP, can avoid
volume is about 20 ml. Then 5 mL of 50% DMSO is slowly irradiation of the product by airport security x-ray, which
added for a final content of 10%. At the end of processing, a would damage the HPCs.
sample from the product is tested for viability and cell count.
Bacterial and fungal cultures are performed. Long-Term Frozen Storage
To prevent mix-ups, one national cord blood bank uses a
The HPCs are best preserved in liquid nitrogen below –196°C
master bar code label that can be scanned to generate addi-
or in the vapor phase (just above the liquid nitrogen). A
tional labels for transfer bags and the final aliquots.
disadvantage of the vapor phase is that as the level of
liquid nitrogen lowers, the temperature at the top of
Cell Enrichment the vapor phase is warmer and further increases when
CD34+ cells can be separated from the other cells by a device the freezer is opened. However, the cells need steady
using antibodies and magnetic beads with attached antibod- temperature, as warming damages them. A problem with
ies. Mouse anti-CD34 is incubated with the product, mag- the liquid phase is that it can harbor viruses and can cross-
netic beads with sheep antimouse are added, and the cells contaminate the products. Careful processing so that
are separated by magnetic force, giving an enriched product the bags are clean externally and the canisters are cleaned
of CD34+ cells. Another method of enrichment is fluorescent after breakage lessens the chance of cross-contamination.
cell sorting. Some cellular therapy labs use an overwrap to quarantine
products.
Cell Purging and Reduction The freezing rate must be controlled, as either too slow
or too fast freezing can damage the cells and reduce viability.
Unfortunately, leukemic cells also are mobilized from the The best rate is 1°C to 3°C per minute. Automatic controlled
marrow by cytokine therapy and are collected by HPC-A. rate freezers are available for this purpose.
Bone marrow can also contain leukemic precursors. The Mechanical –80°C freezers are also used for short-term
cellular laboratory can use a process called cell purging storage of weeks to 2 years. This method does not require
to remove the malignant cells from the HPCs. Antibodies controlled-rate freezing.
Freezers must have alarms that are monitored 24 hours
a day, 7 days a week to prevent loss of products. Backup
BOX 17–7 freezers must be readily available in case a freezer fails.
Cord Blood Testing Shipping Frozen Products
• ABO and Rh
• CD34 cells and TNC Frozen HPC products can be shipped short distances in liq-
• Hemoglobinopathies uid nitrogen; however, the shipping container must be kept
• HLA A, B, DRB1 upright. The canister has a large mouth opening to accom-
• Infectious diseases modate the bags, leading to dangerous situations for the
• Antibody screen—mother shipping company and loss of the HPC product. Available
are “dry shippers,” which are packed with absorbent material
2682_Ch17_391-402 22/05/12 11:56 AM Page 397
for the liquid nitrogen. Dry shippers are much safer and can Table 17–1 ABO Incompatibility
be shipped longer distances.
RECIPIENT DONOR POSSIBLE OUTCOME
Thawing and Preparing Products for Transplant Major O A Hemolysis of donor RBCs
Delayed RBC engraftment
Thawing and preparing the HPCs for transplant includes
testing the product for TNC count, CD34 count, and viabil- Minor A O Hemolysis of recipient RBCs
Passenger lymphocyte
ity and culturing for contamination. Each product bag must syndrome
be thawed quickly at 37°C and infused through the recipi-
ent’s IV within 1 to 2 hours. Significant loss of HPCs has
been found when the product is kept at room temperature
for a short time. Some transplant units thaw each bag at the leukocyte-reduction filter must never be used because many
bedside to minimize delay in starting the infusion. of the HPCs would be removed.
The thawed products can be washed, especially if DMSO
toxicity is a problem for the recipient. Based on recipient ABO Incompatibility
body weight, no more than 10 ml/kg of a product having
ABO-incompatible products require special care. Major in-
10% DMSO should be given during one infusion.
compatibility occurs when the recipient has ABO antibody
against the donor; for example, the recipient is group O and
Transplanting the Recipient
the donor is A (Table 17–1). The recipient’s plasma contains
The conditioning of the recipient includes gamma irradia- anti-A, which can hemolyze the donor RBCs in the product.
tion, high-dose chemotherapy, or both to ablate (eliminate) Because HPC-A and HPC-C products have only a few milli-
the recipient’s marrow and immune system. In this way, the liters of RBCs, a serious hemolytic reaction is less likely.
recipient can accept the HPC transplant. Most conditioning However, bone marrow contains a much higher portion of
regimens completely ablate the bone marrow and immune RBCs, requiring the cellular therapy laboratory to remove
system. Once this conditioning has occurred, the recipient most of the RBCs and thus prevent a serious acute hemolytic
must receive a transplant or he or she will die. The transplant reaction. The recipient may also have delayed RBC engraft-
is thus “rescue.” ment if group O RBCs containing anti-A are selected for
More recently, some recipients have received nonmye- transfusion.14
loablative conditioning, which allows part of their bone mar- Minor ABO incompatibility is less likely to cause adverse
row and immune system to survive. The theory is to reaction. In Table 17–1, the first recipient is A and the donor
establish a stable chimerism (two cell populations) so that is O. The donor may have high-titered anti-A or anti-A,B,
donor cells recognize malignant cells as foreign and destroy which may cause hemolysis of the recipient’s group A cells.
them. Potential recipients who are not suitable for myeloab- In addition, the donor’s T lymphocytes are primed to make
lative conditioning may be candidates for nonmyeloablative anti-A and may cause ongoing hemolysis of the recipient’s
transplantation. These recipients include those who have se- RBCs and may attack other cells bearing the A antigen. This
rious comorbidity or are elderly. is called passenger lymphocyte syndrome.
Bidirectional mismatches have both major and minor ABO
Infusion of HPCs incompatibility (Table 17–2). Thus, both the recipient and the
graft may have adverse effects following the transplant.
Fresh products (allogeneic) are infused slowly to minimize More than one donor cord blood may be selected for
volume overload and adverse reactions. Leukapheresis prod- transplant to an adult or teenager. The two or three cords are
ucts can be infused over an hour or two, and bone marrow, selected for HLA match, so as many as three different ABO
because of the larger volume, can be infused over 2 to 4 hours. types may be infused. However, cord blood products are RBC
The tradition was not to use a filter, although a regular blood and plasma reduced, so ABO reactions are less frequent than
transfusion filter has been recommended more recently. A when bone marrow is used.
Table 17–2 Major, Minor, and Bidirectional ABO Incompatibility for Donors and Recipients
RECIPIENT
ABO Type (Antibody Produced) A (anti-B) B (anti-A) AB O (anti-A, anti-B)
Donor
Adverse Recipient Reactions Acute GVHD is defined as occurring within the first
100 days and chronic GVHD after 100 days.18 About 25%
The recipient may receive premedication to lessen or avoid to 50% of allogeneic recipients develop chronic GVHD.19
reactions, similar to the process for blood transfusion.15 The most affected body sites are the skin, gastrointestinal
DMSO has an odor like the sulfur in garlic or rotten eggs, so tract, liver, lung, and eyes (Table 17–3). Immunosuppres-
the recipient may have this odor. The most common adverse sive drugs are required to treat GVHD, which increases the
reactions are flushing and nausea. In addition, vomiting and risk of infection.
changes in heart rate and blood pressure may occur.
Contamination with granulocytes may lead to fever and Relapse of Disease and Donor Lymphocyte
chills because of the release of cytoplasmic granules and their Infusion
content by freezing and thawing.16 Red blood cells are not
preserved by DMSO, so the free hemoglobin and red blood Leukemia and lymphoma can reoccur because of inadequate
cell stroma may lead to renal damage. conditioning or contamination of the autologous graft. The
Other transplant-related complications include liver or incidence of relapse is lower in recipients with chronic
lung toxicity caused by myeloablative chemotherapy, which, GVHD. With allogeneic transplants, the donor can be asked
in the autologous recipient, may mimic graft-versus-host to donate another leukapheresis with the goal to collect
disease. T cells to attack the malignancy. The process is called donor
leukocyte infusion (DLI). For DLI collection, the donor is not
Engraftment stimulated with cytokines, such as G-CSF or plerixafor.
The purpose of DLI is to induce graft versus leukemia,
After the HPC products are infused, engraftment should which can aid in controlling the leukemia or lymphoma re-
occur. Early engraftment is generally defined as the interval lapse. DLI works best for CML and less well for other hema-
from transplant to the absolute neutrophil count greater tologic malignancies.
than 500/µL. This occurs in 9 to 30 days. Platelet engraft-
ment is defined as platelet count greater than 20,000/µL Outcomes
without transfusion and requires 15 or more days. RBC
engraftment and immune reconstitution occurs 90 days or Following myeloablative therapy, autologous HPC trans-
longer. The standard measurement of success is engraft- plantation has proved to provide an improved quality of
ment of all three cell lines at 100 days. The rate of engraft- life and increased disease-free survival for patients with
ment varies by the source of the HPCs, the number and multiple myeloma, acute myelogenous leukemia (AML),
viability of CD34+ cells, cellular processing, the condition- non-Hodgkin’s lymphoma, and Hodgkin’s disease. Several
ing regimen of the recipient, and other variables.17 For ex- cancer centers have also proven the benefits of two se-
ample, engraftment of neutrophils and platelets occur quential autologous transplants for patients with multiple
earlier with higher CD34 counts in the HPC product. Cord myeloma.
blood and leukapheresis products generally engraft earlier Allogeneic transplants have had less success, mainly be-
than bone marrow products. However, because of the cause of acute GVHD and infections caused by immunosup-
smaller CD34+ cell dose in cord blood, engraftment can be pressive therapy.
delayed or, in about 20%, never occur. Cord blood trans-
plantation is most successful in children weighing less than Transfusion Therapy for HPC
45 kg. Transplanting two or three HPC-C products may be Transplantation
used successfully in children weighing more than 45 kg and
in adults. The longer the engraftment takes, the higher Once a patient is recognized as an HPC transplant candidate,
the risk of severe viral or fungal infection. Major ABO the blood bank personnel should be vigilant to provide blood
mismatch can also delay RBC engraftment. products which prevent alloimmunization to HLA antigens,
CMV infection, or graft-versus-host disease. This can be ac-
Graft Rejection complished with cellular products that have been leukocyte
reduced, gamma-irradiated, and, in special circumstances, cause a type of microchimerism, surviving for months to
CMV seronegative. years in the recipient’s circulation.25 Although TA-GVHD
is uncommon, the mortality rate is over 90%. The clinical
HLA Alloimmunization symptoms may include fever, maculopapular rash, bloody
diarrhea, or pancytopenia and may begin 1 to 4 weeks after
Leukocyte-reduced cellular products help to minimize the transfusion.
incidence of alloimmunization to HLA antigens. HLA al- Gamma irradiation for all cellular blood components is
loimmunization can interfere with platelet transfusion recommended in the pre-HPC transplantation period and
support in the critical pre- and post-transplantation peri- when a patient is considered a potential HPC transplant
ods. Platelet engraftment may be delayed weeks, leading candidate. Irradiation is required, beginning at condition-
to increased risk of hemorrhage if the patient is refractory ing. The recommended dose of radiation is 2,500 cGy to
to platelet transfusion. the center of a freestanding irradiation canister, with a
In the multi-institutional Trial to Reduce Alloimmuniza- minimum of 1,500 cGy at any other area of the canister.
tion to Platelets (TRAP) study, 20 a significant number of pa- Usually, the patient receives irradiated blood products for
tients (17% to 20%) developed HLA antibodies, even though life, but no studies are available to support this practice.
all of their RBC and platelet components had been leukocyte
reduced. However, leukocyte reduction decreases the inci-
Transfusion in ABO-Mismatched Transplant
dence of HLA antibodies and is believed to benefit patients
on platelet therapy. ABO-mismatched transplants present interesting transfu-
Some studies have shown that minimizing the HLA sion problems. RBCs and platelets or plasma will have
alloimmunization may reduce the incidence of comple- separate preferred ABO blood groups in most cases
ment fixing HLA antibodies, which may result in a positive (Table 17–4). The table is based on the selection of the
serological HLA crossmatch with the donor before HPC ABO blood groups that are compatible with both the re-
transplantation. 21 These recipients are at increased risk of cipient and the donor.
graft rejection with an HLA-mismatched related donor and In the first line of Table 17–4, both the donor and the re-
perhaps with an unrelated HLA-matched donor. cipient can receive group O RBCs, so group O is selected for
RBCs. For plasma and platelets, anti-A must be avoided, so
CMV Transmission A or AB products are selected. If group A or AB platelets
are unavailable, the next choice would be group O. In the
Leukocyte-reduced products benefit HPC transplantation
second line of the table, the recipient is A and the donor is
candidates and recipients by decreasing the risk of transfusion-
B (bidirectional). Group O is the only choice for RBCs. To
transmitted CMV infections. CMV-seropositive rates among
avoid anti-A- or anti-B-containing plasma or platelets, AB is
whole blood donors in different areas of the United States
preferred, with B as the second choice and A the third choice.
range from 40% to 80%.22,23 However, very few CMV-
An important point is that group O plasma or platelets
seropositive donors have active CMV infections.
should be avoided except in unusual circumstances, such as
Allogeneic recipients are immunosuppressed because of
when HLA-matched platelets are required and the preferred
drugs used to treat acute and chronic GVHD. Thus, these re-
ABO group is not available.
cipients are at risk for acquiring a life-threatening CMV in-
As the HPC cells engraft, the recipient will slowly become
fection from a CMV-infective blood component.24 The use
the donor’s ABO group. In the first line of the table, the A
of CMV-seronegative or leukocyte-reduced cellular compo-
RBCs of the recipient will become fewer over several weeks,
nents decreases the risk of CMV transmission.
eventually becoming undetectable. During that time, small
The supporting data for leukocyte-reduced or CMV
proportions of donor group O cells will be detected as
seronegative products were obtained from studies of neona-
mixed-field reactions. Eventually, the recipient will type as
tal and hematologic malignancy patients and anecdotally
group O, generally at the same time as RBC engraftment is
applied to HPC transplantation recipients. Although no for-
achieved.
mal recommendations exist for how long CMV-seronegative
In the second line of the table, the group A recipient has
patients should use CMV-seronegative blood, a prudent
anti-B, so the group B donor RBCs may be hemolyzed during
course may be to continue use until immunosuppression is
the infusion. In addition, the engraftment of RBCs may be
withdrawn.
delayed because of the circulating anti-B. Only group O
For CMV-seropositive recipients, the most common cause
RBCs or AB plasma and platelets should be transfused. Even-
of CMV disease is reactivation of latent CMV from prior ex-
tually, the donor’s B cells will engraft in the recipient, with
posure. Therefore, CMV seronegative blood products are not
the loss of the recipient’s anti-B. Generally, the donor’s lym-
recommended for seropositive recipients.
phocytes will not make anti-A because of the A antigens on
Transfusion-Associated Graft-Versus-Host Disease the recipient’s endothelial tissue.
In all ABO-incompatible transplants, transfusing the
Transfusion-associated graft versus disease (TA-GVHD) blood groups of choice for RBCs, plasma, and platelets is
is caused by donor T lymphocytes engrafting in a suscep- extremely important to prevent hemolysis and delayed RBC
tible immunosuppressed host. Donor lymphocytes can engraftment.
2682_Ch17_391-402 22/05/12 11:56 AM Page 400
A O O A, AB A, AB O
A B O AB AB B, A
A AB A AB AB A, B
B O O B, AB B, AB O
B A O AB AB A, B
B AB B AB AB B, A
AB O O AB AB A, B
AB A A AB AB A, B
AB B B AB AB B, A
O A O A, AB A, AB B, O
O B O B, AB B, AB A, O
O AB O AB AB A, B
SUMMARY CHART
The categories of HPC donors are autologous (self), Freezing of HPCs requires the slow addition of DMSO
allogeneic (another person, related or unrelated), and and freezing in a controlled-rate freezer at 1°C to
syngeneic (twin or triplet). 3°C/min.
HPC donors are assessed by history and physical, lab Myeloablative conditioning completely destroys the
tests, EKG, chest x-ray, and donor health history. bone marrow and the immune system. Nonmyeloab-
The National Marrow Donor Program (NMDP) re- lative conditioning partially destroys bone marrow and
cruits donors, stores data, and searches for donors re- the immune system.
quested by recipient institutions. Major ABO incompatibility in HPC transplantation is
A recipient has a 25% chance of an HLA match with a when the donor’s RBCs are incompatible with the re-
sibling. cipient’s antibody.
Umbilical cord blood donation requires the consent, Minor ABO incompatibility is when the donor’s plasma
history, and lab testing of the mother. is incompatible with the recipient’s RBCs.
HPC-M is the collection of bone marrow by aspiration. Engraftment is defined as PMNs greater than 500/µL
The donor undergoes general or epidural anesthesia (about 10 days) and platelets greater than 20,000/ µL
and may need RBC transfusion. (about 15 days), and recipient is no longer RBC trans-
HPC-A is the collection of HPCs from the peripheral fusion dependent (90 to 100 days).
blood by leukapheresis. The HPCs are mobilized by Graft-versus-host disease is defined as donor T cells
cytokine. attacking the recipient’s cells and tissues.
Transplantation is used to treat leukemia, lymphoma, Acute GVHD occurs during the first 100 days after
and multiple myeloma. transplant; chronic occurs after 100 days.
Allogeneic transplantation is not routinely used for Donor lymphocyte infusion is used to treat relapse of
nonmalignant diseases because of the serious risks. chronic myelogenous leukemia.
HPC products are tested for CBC, WBC differential, In HPC transplant recipients, leukocyte-reduced and
CD34+ cells, and viability. irradiated cellular products are selected to reduce the
Bacterial and fungal cultures are required after thawing. risk of HLA alloimmunization, CMV transmission, and
GVHD. CMV seronegative cellular blood products may
HPCs are stored at 20°C to 25°C or 4°C and –196°C.
also be selected.
DMSO is used as a cryoprotectant.
The blood type selected for blood transfusion must be
Viability can be determined by dye exclusion or by compatible with both the donor and the recipient.
flow cytometry.
7. The cellular marker used to quantify the collection of 6. Burt, RK, Loh, Y, Pearce, W, et al: Clinical applications of
HPCs is: blood-derived and marrow-derived stem cells for nonmalignant
diseases. JAMA 299:925–936, 2008.
a. CD4. 7. Bitter, RE, Schofer, C, Weipoltshammer, K, et al: Recruitment
b. CD33. of bone marrow derived cells by skeletal and cardiac muscle in
c. CD34. adult dystrophic mdx mice. Anat Embryol 199:391–396, 1999.
d. CD59. 8. Petersen, BE, Bowen, WC, Patrene, KD, et al: Bone marrow as
a potential source of hepatic oval cells. Science 284:1168–1170,
8. The recommended dose of gamma radiation administered 1999.
to a blood product to reduce the risk of graft-versus-host 9. Cogle, CR, Yachnis, AT, Laywell, ED, et al: Bone marrow trans-
differentiation in brain after transplantation: A retrospective
disease is: study. Lancet 363:1432–1437, 2004.
a. 1,500 cGy to any point within the canister. 10. Padley, D (ed): Standards for Cellular Therapy Product Serv-
b. 1,500 cGy to the midplane of the canister. ices, 4th ed. AABB, Bethesda, 2009.
c. 2,500 cGy to the midplane of the canister. 11. Klein, MA, Kadidlo, McCollough, J, et al: Microbial contamina-
tion of hematopoietic stem cell products: Incidence and clinical
d. 2,500 cGy to any point within the canister.
sequelae. Biol Blood Marrow Transplant 12:1143–1149, 2006.
9. During HPC processing, cultures must be performed: 12. Larghero, J, Rea, D, Esperou, H, et al: ABO-mismatched mar-
row processing for transplantation: Results of 114 procedures
a. At initial testing. and analysis of immediate adverse events and hematopoietic
b. Before freezing. recovery. Transfusion 46:309–402, 2006.
c. After thawing. 13. Berz, D, McCormack, EM, Winer, ES, et al: Cryopreservation
d. After infusion. of hematopoietic stem cells. Am J Hematol 82:463–472, 2007.
14. Worel, N, Greinix, HT, Schneider, B, et al: Regeneration of ery-
10. HPC products are required to be tested for: thropoiesis after related and unrelated donor BMT or peripheral
blood HPC transplantation: A major ABO mismatch means
a. Hepatitis C. problems. Transfusion 40:543–560, 2000.
b. Epstein-Barr virus. 15. Sauer-Heilborn, A, Kadidlo, D, and McCullough, J: Patient care
c. Variant CJD. during infusion of hematopoietic progenitor cells. Transfusion
d. Herpes simplex virus. 44:507–516, 2004.
16. Camels, B, Lemarie, C, Esterni, B, et al: Occurrence and sever-
11. Which of the following terms describe an HPC trans- ity of adverse events after autologous hematopoietic cell infu-
plant where donor and recipient are the same person? sion are related to the amount of granulocytes in the apheresis
product. Transfusion 47:1268–1273, 2007.
a. Allogeneic
17. Bittencourt, H, Rocha, V, Chevret, S, et al: Association of CD34
b. Autologous cell dose with hematopoietic recovery, infections, and other
c. Syngeneic outcomes after HLA-identical sibling bone marrow transplan-
d. Hematopoietic tation. Blood 99:2726–2733, 2002.
18. Goker, H, Haznedaroglu, IC, and Chao, NJ: Acute graft-
12. Which of the following terms describe an HPC trans- versus-host disease: Pathobiology and management. Exp
plant where donor and recipient are identical twins? Hematol 29:259–277, 2001.
19. Lee, SJ, Vogelsang, G, and Flowers, MED: Chronic graft-versus-
a. Allogeneic
host disease. Biol Blood Marrow Transplant 9:215–233, 2003.
b. Autologous 20. Slichter, SJ: Leukocyte reduction and ultraviolet B irradiation
c. Syngeneic of platelets to prevent alloimmunization and refractoriness to
d. Hematopoietic platelet transfusions: The trial to reduce alloimmunization to
platelets study group. N Engl J Med 337:1861–1869, 1997.
21. Kaminski, ER, Hows, JM, Goldman, JM, and Batchelor, JR:
References Pretransfused patients with severe aplastic anaemia exhibit
1. Code of Federal Regulations, CFR—Title 21 Part 1271. Human high numbers of cytotoxic T lymphocyte precursors probably
cells, tissues, and cellular and tissue-based products. Washing- directed at non-HLA antigens. Br J Haematol 76:401–405,
ton, DC, U.S. Government Printing Office, 2010. 1990.
2. Grewal, SS, Barker JN, Davies SM, et al: Unrelated donor 22. Goodrich, JM, Bowden, RA, Fisher, L, Keller C, Schoch, G,
hematopoietic cell transplantation: Marrow or umbilical cord and Meyers, JD: Ganciclovir prophylaxis to prevent cy-
blood? Blood 101:4233–4244, 2003. tomegalovirus disease after allogeneic bone marrow transplant.
3. Wagner, JE, Barker, JN, Defor, TE, et al: Transplantation of un- Ann Intern Med. 118:173–178, 1993.
related donor umbilical cord blood in 102 patients with malig- 23. Winston, DJ, Ho, WG, Bartoni, K, et al: Ganciclovir prophylaxis
nant and nonmalignant diseases: Influence of CD34 cell dose of cytomegalovirus infection and disease in allogeneic bone
and HLA disparity on treatment related mortality and survival. marrow transplant recipients: Results of a placebo-controlled,
Blood 100:1611–1618, 2002. double-blind trial. Ann Intern Med 118:179–184, 1993.
4. Attal, M, Harrousseau, JL, Facon, T, et al: Single versus double 24. Broeckh, M, Nichols, WG, Papanicolaou, G, et al: Cytomegalovirus
autologous stem cell transplantation for multiple myeloma. in hematopoietic stem cell transplants: Current status, known
N Engl J Med 349:2495–2502, 2003. challenges, and future strategies. Biol Blood Marrow Transplant
5. Lenhoff, S, Hjorth, M, Holmberg, E, et al: Impact on survival of 9:543–558, 2003.
high dose therapy with autologous stem cell support in patients 25. Lee, TH, Paglieroni, T, Ohto, H, et al: Survival of donor leuko-
younger than 60 years with newly diagnosed multiple myeloma: cyte subpopulations in immunocompetent transfusion recipi-
A population based study. Nordic Myeloma Study Group. Blood ents: Frequent long-term microchimerism in severe trauma
95:7–11, 2000. patients. Blood 93:3127–3139, 1999.
2682_Ch18_403-426 22/05/12 11:58 AM Page 403
Chapter 18
Transfusion-Transmitted Diseases
Elizabeth F. Williams, MHS, CLS(NCA), MT(ASCP)SBB; Patsy C. Jarreau, MHS,
CLS(NCA), MT(ASCP); Michele B. Zitzmann, MHS, CLS(NCA), MT(ASCP);
and Christine Pitocco, MS, MT(ASCP)BB
OBJECTIVES
1. Describe the pathology, epidemiology, laboratory testing, and prophylaxis/treatment of the following diseases: hepatitis A
through G, HIV 1 and 2, human T-cell lymphotropic viruses I and II, and West Nile virus (WNV).
2. Explain the implications of the following diseases for blood transfusions: Epstein-Barr virus, cytomegalovirus (CMV),
parvovirus B19, herpesvirus 6 and 8, general bacterial contamination, syphilis, Babesia microti, Trypanosoma cruzi, malaria
(Plasmodium species), and Creutzfeldt-Jakob disease and variant Creutzfeldt-Jakob disease.
3. Describe procedures for look-back and recipient follow-up.
4. Describe pathogen inactivation for plasma and cellular components.
403
2682_Ch18_403-426 22/05/12 11:58 AM Page 404
Table 18–2 Disease Burden From Hepatitis A, B, and C in the United States
HEPATITIS A HEPATITIS B HEPATITIS C
2007 2000 2007 2000 2007 2000
Number of acute cases reported 2979 13,397 4519 8036 849 No data
Estimated number of acute clinical cases 45,000 57,000 22,000 22,000 4,000 5,700
Estimated number of new infections 25,000 143,000 43,000 81,000 25,000 35,000
Number of persons with chronic infections No chronic infection 1.25 million 2.7 million
children. By 1998, the levels had dropped to historic lows. (see Table 18–2).10 The clinical picture of HBV infection is
Health professionals now routinely vaccinate children, trav- highly variable. Most people with acute illness will recover
elers to certain countries, and other persons who may be with no liver damage, 15% to 25% of chronically infected
at risk.9 The vaccine is produced from inactivated HAV. It persons develop liver disease, and an estimated 3,000 per-
is believed that the risk is low for pregnant women and that sons in the United States die from HBV-related illness per
no special precautions should be taken for immunocom- year.10 The individual may be completely asymptomatic or
promised persons.7 Other prevention methods include may present with typical signs of disease, including jaundice,
improvement in water purification, good hygiene, and im- dark urine, hepatomegaly, anorexia, malaise, fever, nausea,
proved sanitation. abdominal pain, and vomiting. For children younger than
Immune globulin can be used pre-exposure to protect 5 years, fewer than 10% show signs of jaundice and clinical
those traveling to high HAV–endemic areas or postexpo- illness. However, infections acquired at birth or between
sure to prevent infection in those exposed within a family, ages 1 to 5 years result in a chronic infection 90% and 30%
after an outbreak at a day-care center, or from a common of the time, respectively. For those older than 5 years, up to
source of exposure such as a restaurant. Immune globulin 50% will have clinical illness, but only 2% to 10% will
should be used postexposure within 2 weeks for maximum develop a chronic infection. Mortality rates in acute HBV are
protection.8 approximately 0.55% to 1% as compared with a 15% to 25%
death rate following chronic HBV infection.9
Hepatitis B
Epidemiology and Transmission
Hepatitis B (HBV) is a partially double-stranded circular HBV is transmitted through exposure to bodily fluids con-
DNA virus of the Hepadnaviridae family.5 taining the virus from an infected individual. Concentrations
of the virus are at high levels in blood, serum, and wound
Clinical Manifestations and Pathology
exudates; at moderate levels in semen, vaginal fluids, and
Acute viral hepatitis has declined 82%, from 8.5 cases per saliva; and at low levels in urine, feces, sweat, tears, and
100,000 in 1990 to 1.5 cases per 100,000 population in 2007 breast milk. Transmission may be sexual, parenteral, or peri-
natal.4 Percutaneous transmission may occur through needle
Markers in HAV Infection stick (drug use, occupational hazard, acupuncture, tattooing,
or body piercing), hemodialysis, human bite, transfusion of
Icterus unscreened blood or blood products, or sharing razors. Per-
Enzymes
mucosal transmission can occur through sexual intercourse
or vertically from mother to infant (transplacental or through
HAV breast milk).4 According to the Surveillance for Viral Hepa-
(Stool) titis Report of 2007, higher rates of hepatitis B continue
Anti-HAV
IgG among adults, especially among males between the ages of
Exposure Anti-HAV 30 to 44 years.10
IgM
5 20 30 Laboratory Diagnosis
Days Years HBV consists of several proteins or antigens to which the
Figure 18–1. Markers in acute HAV infection. The typical pattern of HAV infection
body can make antibodies (Fig. 18–2). A surface antigen
includes early shedding of virus in the stool, appearance of IgM anti-HAV, and protein, HBsAg, is on the outer envelope of the virus. It can
immunity on recovery. also be found floating free in the plasma. Antibodies can be
2682_Ch18_403-426 22/05/12 11:58 AM Page 406
Dane Core Coat Hepatitis B immune globulin injections (HBIG) and the
particle particle protein vaccine given soon after exposure or within 12 hours
(HBcAg) (HBsAg) of birth, if the mother is infected, may prevent infection.
(HBeAg)
Three other treatments licensed by the FDA are interferon
Detergent
(IFN)-α-2b,11 lamivudine, and adefovir dipivoxil.12 Once a
+ patient has been diagnosed, family members should be
Treatment
28nm
200 x 22nm
tested. If uninfected, they should be vaccinated. If infected,
42nm
they should be treated.
SDS
Hepatitis C
+ Hepatitis C (HCV) is a member of the Flaviviridae virus
family and is caused by a virus with an RNA genome.4
DNA Polymerase
activity Hepatitis C is transmitted parenterally, although the sexual
and fecal-oral routes as modes of transmission have been
Figure 18–2. Diagram of the intact Dane particle (HB virion) as seen by electron
microscopy. Detergent treatment disrupts the particle into a core particle and outer coat documented.4 In 2007, a total of 849 confirmed cases of
protein, releasing DNA (double- and single-stranded) and DNA polymerase activity. HCV were reported with an overall national rate of 0.3 cases
per 100,000 population.10 Blood transfusions were a major
source of infection before 1992, before routine screening
was implemented. Testing has reduced the incidence of
produced to two proteins within the core: hepatitis B core
transmission.7
antigen (HBcAg) and hepatitis Be antigen (HBeAg). Viral
replication levels of these markers along with the host’s
Clinical Manifestations and Pathology
production of IgM or IgG antibodies are all used to make
an initial diagnosis and follow the course of infection The incubation period of HCV is 2 to 26 weeks. The average
(Table 18–3).6 incubation period is 7 to 8 weeks, followed by seroconver-
HBV DNA is the first marker to appear and can be sion occurring in 8 to 9 weeks.7 Of all HCV cases, 60%
detected by polymerase chain reaction (PCR) testing before to 70% are asymptomatic, with an additional 10% to 20%
HBsAg reaches detectable levels.3 There have been only having nonspecific symptoms such as anorexia, malaise,
incremental benefits of HBV minipool NAT over a sensitive fatigue, or abdominal pain. Most symptomatic cases are very
HBsAg assay.3 HBsAg is detectable 2 to 12 weeks postexpo- mild. Of HCV-infected individuals, 75% to 85% become
sure during the acute stage and becomes undetectable in chronic carriers, 60% to 70% develop chronic liver disease,
12 to 20 weeks after development of anti-HBsAg (Fig. 18–3). 5% to 20% develop cirrhosis over a period of 20 to 30 years,
If the patient develops chronic HBV, the level of HBsAg 1% to 5% will die from cirrhosis or liver cancer, and an
remains high. HBsAg can be used to monitor the stages of estimated 12,000 persons in the United States die from
HBV from the acute, active infection to recovery or a chronic HCV-related illness per year.9
infection. It is also used to screen donor blood.7
HBeAg appears after the HBsAg and, in recovering patients, Epidemiology and Transmission
disappears before HBsAg. In chronic patients, it remains Because most HCV cases are asymptomatic, the worldwide
elevated. HBeAg is present during the time of active replication incidence is unknown (see Table 18–2). It is estimated
of the virus and is considered a marker of high infectivity.7 that there are over 4 million people in the United States and
HBcAg is present in the serum but is undetectable. How- 170 million chronic carriers worldwide.7 The risk of post-
ever, IgM anti-HBc is the first antibody to appear, and it per- transfusion HCV has declined dramatically since the intro-
sists for about 6 months. Appearance of this antibody duction of testing.
indicates current or recent acute infection.7 HCV can be transmitted percutaneously through needle
ALT testing is no longer required in the United States. stick, hemodialysis, human bite, transplant, or transfusion,
However, it continues to be performed because the European or by acupuncture, tattooing, or body piercing. It can also be
Union requires it for recovered plasma.3 transmitted permucosally through sexual intercourse, contact
with infected toothbrush or razor, or perinatally. Hepatitis C
Prophylaxis and Treatment is transmitted mainly by exposure to contaminated blood,
The donor questionnaire is used to identify individuals with IV drug use being the main source of infection.7
at risk for HBV infection. For those who are not eliminated
by that process, testing for HBsAg and anti-HBc can be Laboratory Diagnosis
performed. Diagnosis of HCV is difficult. Not only are symptoms so mild
An HBV vaccine was licensed in 1981 and introduced in in acute cases as to make separation of acute HCV from
1982. Hepatitis vaccination programs will eliminate domestic chronic HCV difficult, but also separating HCV from other
HBV transmission, and the increased vaccination of adults forms of liver disease is not easy. Diagnosis depends on bio-
with risk factors will help accelerate the progress toward chemical changes suggestive of HCV, detection of HCV RNA
elimination.10 or anti-HCV in serum, or a known exposure to the virus.
2682_Ch18_403-426 22/05/12 11:58 AM Page 407
Table 18–3 Molecular and Serologic Tests in the Diagnosis of Viral Hepatitis
VIRUS TEST REACTIVITY INTERPRETATION
Anti-HBc
HBV DNA HBsAg Total IgM Anti-HBs HBeAg Anti-HBe
+ + + + – + – Acute infection
+ – – – – – – Window period
– – + + + Recovered infection
Anti-HCV Recombinant
Antigens
Screening
HCV RNA EIA 5-1-1 c100-3 c33c c22-3
– + – – – – False-positive
Total IgM
+ + + Acute HAV
– + – Recovered HAV/vaccinated
Total IgM
+ + + Acute HEV
+ + – Recovered HEV
Taken from AABB Technical Manual, 15th ed., pp. 670–671. Bethesda, Maryland, 2005 with permission.
HBsAg = hepatitis B surface antigen; anti-HBc = antibody to hepatitis B core antigen; anti-HBs = antibody to HBsAg; HBeAg = hepatitis Be antigen; anti-HDV = antibody to hepatitis D virus; anti-HAV =
antibody to hepatitis A virus; anti-HCV = antibody to hepatitis C virus; anti-HEV = antibody to hepatitis E virus.
*Those with HBeAg are more infectious and likely to transmit vertically.
†Anti-5-1-1 and anti-c100-3 generally appear later than anti-c22-3 and anti-c33c during seroconversion and may disappear spontaneously, during immunosuppression or after successful antiviral therapy.
2682_Ch18_403-426 22/05/12 11:58 AM Page 408
HBsAg Anti-HBs disease, with a higher risk of fulminant hepatitis (2% to 20%)
HBeAg Anti-HBc IgM but a lesser risk of developing chronic hepatitis.
Anti-HBc IgG
Anti-HBe Those at highest risk of infection are IV drug users. This
infection can also be transmitted sexually. It is believed that
20 million people are infected worldwide, but the number
LFT of new infections appears to be declining, most likely due to
SYMP the implementation of the Hep B vaccine.4
HDV is detected by testing for IgM or IgG anti-HDV or
HDAg and HDV RNA in the serum. Tests for HDV are not
required for blood donations. If a donor has HBV, the unit
will not be used for transfusion. As HDV cannot exist
without HBV, testing for HBV will eliminate any infections
with HDV.
Hepatitis E
Weeks after exposure
Hepatitis E (HEV) is a member of the Caliciviridae family of
Figure 18–3. Markers in HBV infection.
nonenveloped RNA viruses. It is rare in the United States,
and there are no recorded cases of transfusion transmission.5
Today, anti-HCV testing via EIA or ChLIA methodology As in HAV, HEV is spread through the fecal-oral route, usu-
is performed on all donor units.3 Currently licensed con- ally through contaminated drinking water in developing
firmatory tests such as recombinant blot immunoassay countries. A carrier state does not develop after the acute,
(RIRA) or HCV RNA are performed on all positive tests3 usually self-limiting, illness.7
(see Table 18–3).
Recombinant immunoblot assays (RIBA), licensed by the Clinical Manifestations and Pathology
FDA, can be used to confirm anti-HCV tests. Seventy to 90% Symptoms are the same as for any hepatitis. Generally, these
of all RIBA-positive tests are also positive for HCV by NAT cases are short-lived but can be prolonged. HEV causes an
methods. Those units that are positive for HCV RNA acute, self-limiting hepatitis that may last from 1 to 4 weeks
approach 100% infectivity. The repeatedly reactive EIA tests in most people.14
that are RIBA-negative or indeterminate (approximately 37%
of EIA repeatedly reactive donors) are rarely infectious.3 Epidemiology and Transmission
HEV usually occurs in developing countries and is respon-
Prophylaxis and Treatment sible for acute, sporadic cases of infection that can be short-
Currently there is no HCV vaccine. Prevention consists of lived or prolonged. The fecal-oral route is the most common
worldwide screening of blood and blood products; destruc- form of transmission. Most cases in the United States are
tion or sterilization of needles and surgical or dental instru- found in travelers returning from an endemic country.14 HEV
ments; universal precautions; and education about the does not progress to a chronic state. Rare cases have been
risks. reported among persons with no history of travel.14
Patients with chronic HCV infection are usually treated
with pegylated interferon and ribavirin.4 Optimal therapy is Laboratory Diagnosis
now considered to be pegylated IFN and ribavirin combina- Both IgM and IgG antibody to HEV (anti-HEV) may occur
tion for chronic HCV.13 In a small study by Gerlach and col- following HEV infection. The titer of IgM anti-HEV declines
leagues,13 50% of patients with acute HCV spontaneously rapidly during early convalescence; IgG anti-HEV persists
and permanently cleared the virus within the first 3 to and appears to provide at least short-term protection against
4 months. Patients who were still viremic at 3 months and disease (see Table 18–3). Antibodies are usually identified
were treated at that point had an 80% clearance rate. How- using highly sensitive enzyme immunoassays that are recom-
ever, most cases were not symptomatic and therefore were binant and synthetic HEV antigens.7
not noticed and treated until the patient was in the chronic
phase. Treatment with INF-α and ribavirin in these chronic Prophylaxis and Treatment
cases achieved only a 30% to 54% sustained viral clearance.13 Water supplies must be cleaned and sewage disposal handled
properly to prevent the spread of HEV. Currently, no com-
Hepatitis D mercially available vaccines exist for the prevention of
hepatitis E.
Hepatitis D (HDV) is a defective, single-stranded RNA virus
that is found only in patients with HBV infection. It requires Hepatitis G Virus
HBsAg in order to synthesize an envelope protein and repli-
cate. It was previously called the delta antigen. If HBV and GB virus C (GBV-C) and hepatitis G virus (HGV) are two
HDV are contracted concurrently, this coinfection, as com- genotypes of the same enveloped RNA virus that belongs to
pared with HBV alone, appears to cause a more severe acute the Flaviviridae family. Approximately 1% to 2% of U.S.
2682_Ch18_403-426 22/05/12 11:58 AM Page 409
donors have tested positive for HGV, making this virus more HIV-1 HIV-2
common than HCV.15 However, recent reports do not impli-
cate GBV-C/HGV as a cause of hepatitis.
gp120 gp41 gp125 gp36
Clinical Manifestations and Pathology p24 RNA p26 RNA
Although acute, chronic, and fulminant hepatic failure cases
have been associated with GBV-C/HGV, there are other stud- RT RT
ies that do not implicate GBV-C/HGV. One article showing a
Figure 18–4. Schematic representation of human immunodeficiency virus
strong association with fulminant hepatitis dealt with a cer- genomes, HIV-1, and HIV-2. RT = reverse transcriptase.
tain mutated strain of GBV-C/HGV.16 In another article by
Halasz,17 33 GBV-C/HGV individuals were identified who
had no coinfection with other known hepatitis viruses. No
evidence of liver disease, clinical or biochemical, was found. causes a slowly progressing immune disorder. The causative
In fact, there is some evidence that patients with HIV who viruses, HIV-1 and HIV-2, are similar in structure, varying
have a coinfection with HGV have a slower progression to primarily in the envelope proteins (Fig. 18–4 and Table 18–4).
AIDS.15 Overall data do not support GBV-C/HGV as a major Almost all cases in the United States result from infection
cause of liver failure. with the HIV-1 virus. HIV-2 is prevalent in West Africa but
very rarely diagnosed in the United States; when it is diag-
Epidemiology and Transmission nosed in the States, it is usually linked to an association
HGV is transmitted by the blood-borne route. Parental with West Africa.
transmission through contaminated blood and the pres-
ence of the virus in bile, stool, and saliva suggest trans- Profile
mission through the fecal-oral and respiratory routes.7
Clinical Manifestations and Pathology
It has been found in 20% to 24% of intravenous drug users
and in higher rates among people with HIV.18 Vertical Primary infection with HIV may be asymptomatic or may
or perinatal transmission from mother to child has been result in a mild, chronic lymphadenopathy with symptoms
documented.19 similar to those seen in infectious mononucleosis. Symptoms
Most adult infections appear to be transient, with viral may occur within 6 to 12 weeks of infection and persist for
clearance followed by antibody to the viral envelope (E2) a few days to 2 weeks. HIV enters the cell by the binding of
production. Vertical or perinatal infections and other infec- the virus glycoprotein 120 with cell surface receptors. Cells
tions established early in life can last for years but do not possessing these receptors include CD4+ lymphocytes,
cause liver disease.17,19 macrophages, and other antigen-presenting cells. The disease
may have a long, clinical latency period with the absence of
Laboratory Diagnosis clinical symptoms. During this period, antibody concentra-
Reverse transcription polymerase chain reaction (RT-PCR) tion and viral load reach equilibrium. As the viral load in-
for GBV-C/HGV-RNA is used to diagnose a current, ongoing creases and the CD4 count decreases, the patient progresses
infection. Anti-E2 along with a negative PCR for GBV- toward clinical AIDS. When the CD4 count is less than
C/HGV-RNA indicates a past infection and recovery. Individ- 200/µL, the patient is classified as having clinical AIDS. The
uals who never develop the GBV-C/HGV E2 antibody are still resultant immunodeficiency allows the onset of opportunis-
infected.20 These assays are first generation, and the evalua- tic infections such as Pneumocystis carinii pneumonia,
tion has not been completed on the sensitivity and specificity Kaposi’s sarcoma, fungal infections, and a host of others.
of these assays.17 About 50% of patients do not progress to clinical AIDS for
10 years or more.22
Prophylaxis and Treatment
Interferon-α treatment has been used with conflicting
results. In most cases, the level of the GBV-C/HGV-RNA
returned to normal levels once therapy was discontinued. Table 18–4 Components of the HIV Virus
Only a small percentage of cases with low pretreatment viral BANDS OBSERVED
loads had a predictable sustained response.21 Gene HIV-1 HIV-2 Protein
HIV Types 1 and 2 Gag p18, p24, p15 p16, p26, p55 Core
HIV-1 and HIV-2 are well recognized as the etiologic agents Pol p31 Endonuclease
of AIDS. AIDS was first diagnosed in 1981, but the causative p51, p65 p68 Reverse transcriptase
agent was not identified until 1984.
HIV is a retrovirus that is spherical in shape, with an Env gp41 gp36 Transmembrane protein
approximate diameter of 100 nm. It consists of an envelope gp120, gp160 gp140, gp125 Envelope unit
of glycoproteins, core proteins, and an inner core of viral
RNA and reverse transcriptase. Infection with the virus gp = glycoprotein (number indicates molecular weight); p = protein
2682_Ch18_403-426 22/05/12 11:58 AM Page 410
EIA Screen
Treatment with highly active antiretroviral therapy has paraparesis (HAM/TSP). A few case reports in the literature
lengthened life and improved quality of life for those infected suggest that HTLV-II may impact the development of neuro-
with HIV. Use of this therapy has resulted in the most stable logical diseases, including HAM, but subsequent studies
HIV morbidity and mortality rates since 1998.22 have failed to support this with convincing evidence.28
Epidemiological data suggest blood donors infected with
Human T-Cell Lymphotropic Viruses HTLV-I or HTLV-II have an excess of infectious syndromes,
Types I/II (HTLV-I/II) such as pneumonia, bronchitis, and urinary infections.29
HTLV-I is associated with uveitis and infective dermatitis of
HTLV-I and HTLV-II are RNA retroviruses. HTLV-I causes a children, Sjögren’s syndrome, polymyositis, and facial nerve
T-cell proliferation with persistent infection.26 Once the RNA palsy.7
has been transcribed into DNA, it is integrated randomly into
the host cell’s genome.27 Once integrated into the DNA, the Epidemiology and Transmission
provirus can either complete its replication cycle or remain HTLV-I is transmitted vertically (breastfeeding), sexually
latent for many years. (transmission from male to female more common), and
parenterally (blood transfusion or IV drug abuse).7 Because
Profile recipients of RBCs, platelets, and whole blood, but not fresh
frozen plasma, have seroconverted, it is believed that trans-
Clinical Manifestations and Pathology mission requires introduction of infected living white blood
HTLV-I was the first retrovirus to be associated with a human cells (WBCs).26,27 This theory is supported by the fact that
disease. That association was with adult T-cell lymphoma/ units stored for at least 7 days before transfusion are less
leukemia (ATL), a highly aggressive, mature T-cell non- likely to transmit the virus.
Hodgkin’s lymphoma with a leukemic phase.3 ATL does not There appears to be a strong correlation between disease
respond well to chemotherapy, and mean survival time with development and host factors such as cytotoxic T lympho-
acute ATL is less than 1 year. Immunodeficiency similar to cytes and HLA types. Susceptibility to ATL seems to corre-
that of patients with AIDS develops, making the ATL patient late with polymorphisms of the tumor necrosis factor α
susceptible to other hematologic malignancies.27 HTLV-I is (TNF-α) that result in an increased production of TNF-α.27
also associated with the progressive neurological disorder In individuals with HAM/TSP, both cellular and humoral
known as HTLV-I-associated myelopathy or tropical spastic immune responses are increased as compared with those of
2682_Ch18_403-426 22/05/12 11:58 AM Page 412
asymptomatic carriers and seronegative controls.28 However, disease.26 The best prophylaxis is to prevent exposure. How-
ATL seems to occur in persons who were infected as infants, ever, as the majority of infected individuals are asympto-
with a latent period of approximately 67 years, whereas matic, it is difficult to prevent spread to an uninfected
HAM/TSP is generally seen in individuals who are infected individual vertically or sexually. Infected mothers should not
in childhood or as an adult, with a variable latency as short breastfeed.
as weeks to months. There is a 40% to 60% probability of
seroconversion within 51 days following an infected blood West Nile Virus
transfusion.26 For both diseases, the host’s ability to keep the
proviral load low correlates with asymptomatic carriers.27,28 West Nile Virus (WNV) is a member of the Flavivirus fam-
Worldwide, it is estimated that 10 to 20 million people are ily and is a human, avian, and equine neuropathogen. It is
infected with HTLV-I and HTLV-II.27,28 It is endemic in parts a single-stranded RNA lipid-enveloped virion31 that is
of southern Japan, central and West Africa, the Caribbean, common in Africa, West Asia, and the Middle East. WNV
the Middle East, Melanesia, Papua New Guinea, the Solomon is a member of the Japanese encephalitis virus antigenic
Islands, and in Australian aborigines. In the United States, complex that includes St. Louis encephalitis virus preva-
HTLV-I and HTLV-II are seen primarily in IV drug users. lent in the Americas, Japanese encephalitis virus prevalent
HTLV-I is also seen in immigrants from endemic areas, in East Asia, and Murray Valley encephalitis virus and
whereas HTLV-II is seen in some Native American popula- Kunjin virus prevalent in Australia.32 WNV was first doc-
tions.26,28 Indications from the high numbers of carriers and umented in the Western Hemisphere when 149 cases were
low numbers of individuals diagnosed with actual disease are reported in New York in 1999. In the United States in
that most carriers are asymptomatic their entire lives.26,27 2009, a total of 720 human cases of WNV-associated ill-
The risk of transmitting HTLV through infected blood ness with 32 fatalities were reported in 44 states and the
is estimated to be between 10% to 30%.3 Leukocyte reduc- District of Columbia.33
tion and serologic testing greatly reduces the risk of HTLV
transmission.3 Profile
First Donation or
Previous Donation Previous Donation
Negative Repeat Reactive
donor notified
indefinite deferral neg, or Ind pos
blood components, organ transplants, pregnancy, and breast immunohistochemistry can be used by testing brain tissue
milk. During the epidemic outbreak of 2002, 23 persons with virus-specific monoclonal antibodies.37
were reported to have been infected through transfusion.2 Clinically, the serologic tests for IgM antibodies to WNV
using ELISA can be used for testing symptomatic patients.
Laboratory Diagnosis Because the virus is in the bloodstream before either symp-
Viremia usually lasts approximately 6 days and peaks around toms or antibodies develop, blood screening tests for WNV
the onset of symptoms. Once clinical symptoms occur, the that identify the virus itself were needed. In June 2003, two
IgM WNV-specific antibody titer increases, and the virus commercial WNV-screening NATs were distributed, and im-
concentration in the blood stream decreases. Until July 2003, plementation of donor blood testing began “under phase III
diagnosis depended on the clinical findings and specific lab- investigational new drug (IND) FDA approval.”2 As of July
oratory tests. IgM antibody-capture enzyme-linked im- 14, 2003, all civilian blood donations were being screened
munosorbent assay (ELISA) was the method used to detect by NAT. Units are initially screened individually or in pools
IgM antibody to the WNV in serum and cerebrospinal fluid of 6 or 16, depending on the kit manufacturer. Individual
(CSF). In the 1999 and 2000 outbreak in New York, 95% of samples are tested only if the pool is positive with NAT.2 The
all infected patients for whom CSF was tested had a demon- implementation of NAT testing for West Nile virus resulted
strable IgM antibody. However, because all Flaviviruses are in the detection of 183 confirmed cases, with an additional
antigenically similar, cross-reactivity has been observed in 47 cases of infected units being detected by testing targeted
testing persons who have been vaccinated for a Flavivirus, individual units.38
such as yellow fever or Japanese encephalitis, or who have If an individual donor tested NAT-positive, all current
been recently infected with another Flavivirus, such as blood components were discarded, and any unused compo-
St. Louis encephalitis or dengue fever. The plaque reduction nents donated within the last 14 to 28 days by that individual
neutralization test is the most specific test for arthropod- were retrieved. Additional NAT tests were performed by
borne Flaviviruses and helps to distinguish false-positive IgM another laboratory for confirmation, using a different ampli-
antibody-capture ELISA from cross-reactivity.36 In fatal cases, fication technique or different primers. The original sample
2682_Ch18_403-426 22/05/12 11:58 AM Page 414
collected from the donor was assayed for WNV-specific IgM affected infant, many of which will have clinically apparent
antibody. The donor was questioned again about “recent disease. Intrauterine transfusions with CMV-positive com-
travel history, other exposure history, and review of symp- ponents is also a high risk to the fetus.41
toms compatible with WNV illness before or after illness.”39 Individuals at moderate risk are recipients of solid organ
Donors were classified as viremic if the initial donor sample transplants, persons with HIV, and individuals who may
was NAT-positive by pool and by individual testing, and the require an allogeneic marrow transplant in the future. When
individual sample was repeatedly reactive using alternate the individual becomes immunosuppressed, a reactivation
NAT protocols.2 With the implementation of NAT testing, of a latent infection is possible, resulting in a clinically
approximately 2.5 million units were screened for WNV apparent infection.41
from June to mid-September 2003. Only 1,285 (0.50%) were Low-birth-weight neonates and autologous marrow recip-
WNV-positive by NAT. Of the positive units, 601 (0.02%) ients are considered to be at low risk. Preterm, multitrans-
were considered viremic, and results are pending for an fused neonates weighing less than 1,200 grams are currently
additional 209.2 considered to be at a lower risk than once considered, as
WNV has become a major area of focus for transfusion a result of better transfusion techniques and management
safety, especially since there have been large outbreaks in the of their condition. However, leukocyte-reduced or CMV-
United States when comparing WNV with other transmissi- negative units reduce the risk of CMV infection in these low
ble diseases tested by NAT testing. There is a similar interval birth-weight neonates.3 The neonate may be exposed at the
in which it is detected, but when comparing testing by time of delivery, through breastfeeding or contact with
minipool method, WNV has a much shorter duration of seropositive individuals. Approximately 1% of all newborns
viremia.38 are infected with CMV, but most are asymptomatic at birth.42
The fetus that is exposed to the mother’s reactivation of the
Prophylaxis and Treatment virus during pregnancy rather than a primary exposure
Individuals should avoid mosquitoes and wear mosquito re- rarely has any damage.42 The autologous marrow recipient
pellant and appropriate clothing if they are going to be in a is not as immunosuppressed as the allogeneic marrow
mosquito-infested area. Once infected, there is no licensed recipient, and therefore CMV infection does not present a
treatment, only supportive therapy. Research is ongoing for problem.41 CMV infection from transfusion is between 1%
the use of ribavirin, interferon-α,31 and West Nile immune to 3%.3
globulin to treat WNV. Having survived the illness, a person
is immune for life. Epidemiology and Transmission
Transmission occurs from person to person through contact
with infected body fluids, which may include urine, semen,
Other Viruses
saliva, blood, cervical secretions, and breast milk. CMV is
Cytomegalovirus the most frequently transmitted virus from mother to fetus.42
Seronegative individuals who have received organ trans-
Cytomegalovirus (CMV) is a member of the herpesvirus plants or hematopoietic progenitor cells from a seronegative
group40 and is found in all geographic locations and donor and AIDS patients who are not currently infected with
socioeconomic groups, with a higher prevalence in develop- CMV are also at risk.3 The rate of transmission of CMV to
ing countries. In areas with lower socioeconomic conditions, bone marrow recipients or to neonates has been documented
the prevalence approaches 100%. Of adults in the United at 13% to 38%.43
States, 50% to 85% have been exposed to CMV by the age of
40 years. Laboratory Diagnosis
Antibodies formed to CMV last a lifetime and can be de-
Clinical Manifestations and Pathology tected by ELISA. Other laboratory tests include fluorescence
When exposure occurs after birth to an individual with assays, indirect hemagglutination, and latex agglutination.
a competent immune system, there are generally few If the patient is symptomatic, active infection can be detected
symptoms. Rarely, mononucleosis-like symptoms with fever by viral culture of urine, throat swabs, and tissue samples.40
and mild hepatitis occur. Once an individual is exposed, CMV DNA tests are currently being investigated for use
CMV can remain latent in the tissues and leukocytes for in detecting blood donors who have been exposed but have
years, with reactivation occurring from a severe immune not yet seroconverted. Tests for anti-CMV would be negative
system impairment.40 for these individuals when, in fact, their blood might be
Those at the highest risk of a CMV infection are fetuses capable of transmitting the disease. In one study by Roback
and individuals receiving allogeneic marrow transplants. and colleagues, previously described CMV PCR assays were
CMV-seronegative recipients transplanted with CMV- compared.43 The performances of the seven assays varied
seronegative allogeneic marrow are at risk if they receive greatly in sensitivity, specificity, and reproducibility.43
untested and non-WBC-reduced blood components. The
risk of seroconversion and serious disease is 20% to 50%. Prophylaxis and Treatment
CMV-seronegative women who become infected in the first Currently, there is no treatment for CMV for a healthy indi-
two trimesters have a 35% to 55% chance of delivering an vidual; vaccines are still in the research and development
2682_Ch18_403-426 22/05/12 11:58 AM Page 415
stage. Infants are being evaluated using antiviral drug ther- erythroid progenitor cells.49 The cytotoxicity of erythroid
apy, and ganciclovir is being used for patients with depressed precursors can lead to serious illness in individuals with
immunity. chronic hemolytic anemia, such as sickle cell disease and
Blood and blood components are not universally screened thalassemia, who may have a transient aplastic crisis. Severe
for CMV because of the generally benign course of this dis- RBC aplasia or chronic anemia may manifest in patients with
ease and the high percentage of virus carriers. To prevent chronic or acquired immunodeficiency or malignancies or
CMV transmission, leukocyte-reduced blood or blood from in organ transplant recipients. Hydrops fetalis and fetal death
seronegative donors may be used. Leukoreduction using can occur when the virus is transmitted during pregnancy.
high-efficiency filters such that the final level of leukocytes The viremic stage occurs shortly after infection. A donor
is less than or equal to 5 ⫻ 106 leukocytes per component would be asymptomatic but capable of transmitting the virus
appears to work well with high-risk neonates (weighing less during this period. This is a concern for donor centers
than 1,200 grams) and transplant recipients.3 In an AABB because the rate of seroconversion is high after exposure. In
bulletin, prestorage leukoreduction was encouraged rather one study, B19 DNA was found in approximately 1 out of
than bedside leukoreduction. 800 donations. B19 is very resistant to heat and detergent
and has been found through PCR to be present in plasma
Epstein-Barr Virus components. Because of the lack of inactivation in the man-
ufacturing process, B19 has been implicated in several stud-
Epstein-Barr virus EBV is a ubiquitous member of the her-
ies, with transmission through factor concentrates and, in
pesvirus family. As many as 95% of the adult population in
one study, through an antithrombin III concentrate.50
the United States have been exposed to the virus by the age
There has been ongoing regulatory concern about the
of 40 years and maintain an asymptomatic latent infection
safety of plasma derivatives that has led some manufacturers
in B lymphocytes for life. Infections occurring in infants or
and regulatory authorities to require B19 DNA qualification
young children are usually asymptomatic. In adolescence
testing of plasma and release testing of manufactured lots.51
and young adulthood, EBV causes infectious mononucleosis
The FDA recommends manufacturers of plasma-derived
in 30% to 50% of patients.44
products to engage in practices that will reduce the time
Although transfusion transmission is rarely an individ-
between product collection and process testing in order for
ual’s first exposure to the virus and reactivation usually
the collection establishments to be notified of positive test
occurs only in immunocompromised individuals, there are
results within the in-date period of any blood components
a few cases in the literature of transfusion-associated EBV.
that are intended for transfusion.52
EBV is not detected by current practices and could cause
In a study by Weimer and colleagues,50 plasmas were
severe consequences in immunocompromised patients, par-
tested for B19 DNA levels by PCR, and those with high titers
ticularly organ transplant patients.45
were eliminated from the pool used in the manufacture of
EBV has been called the “kissing disease” because the
antithrombin III (ATIII). After the manufacturing process,
virus usually replicates in the cells of the oropharynx, pos-
PCR was used to compare ATIII concentrates made from a
sibly in infected B cells. The virus is shed in the saliva and is
pool in which high titers were excluded and ATIII concen-
most frequently associated with infectious mononucleosis.
trates that were made from a pool that had not been tested
EBV was first discovered in 1964 in Burkitt’s lymphoma
for B19 DNA. None of the concentrates manufactured with
cells.46 Since then it has been associated with many illnesses
high-titer plasma eliminated from the pool were PCR-
besides infectious mononucleosis and cancers such as
positive, whereas 66% of the concentrates that were not
nasopharyngeal carcinoma, non-Hodgkin’s lymphoma, oral
tested prior to manufacturing were PCR-positive. This indi-
hairy leukoplakia in AIDS patients, T-cell lymphomas, and
cates the effectiveness of eliminating high-titer plasmas in
Hodgkin’s disease.7
reducing the B19 DNA to undetectable levels.
Parvovirus B19 Disease transmission of parvovirus B19 is rare. Very high
levels of parvovirus B19 (up to 1012 IU/ml) in plasma of
Human B19 parvovirus (B19) is a small, single-stranded acutely infected asymptomatic donors may pose a greater
DNA nonenveloped virus.3 It causes a common childhood risk for plasma derivatives. This is due to the pooling of
illness called “fifth disease” and is usually transmitted larger plasma units by manufacturers when processing these
through respiratory secretions. Fifth disease presents with products.52 There have been no confirmed reports that
a mild rash described as “slapped cheek” when occurring immunoglobulin and albumin products have transmitted
on the face and a lacy red rash when occurring on the parvovirus B19 infection.52
trunk and limbs. Approximately 50% of adults have been
exposed as children or adolescents and have protective Human Herpesvirus 6 and Human Herpesvirus 8
antibodies. Primary infection in an adult is usually asymp-
tomatic, but a rash or joint pain and swelling may occur Human herpesvirus 6 (HHV-6) is a very common virus that
transiently.47,48 causes a lifelong infection. Seroprevalence approaches 100%
As in all viral infections, the virus must enter the cell in some populations. The virus replicates in the salivary
through a specific cell receptor. B19 parvovirus enters the gland and then remains latent in lymphocytes, monocytes,
red blood cell (RBC) via the P antigen and replicates in the and perhaps other tissues.
2682_Ch18_403-426 22/05/12 11:58 AM Page 416
In childhood, HHV-6 causes roseola infantum, also hours after the transfusion, and the connection may be
known as exanthem subitum or sixth disease. Symptoms are unrecognized. This may be because many patients who
those of a mild, acute febrile disease. In immunocompetent receive platelets are already immunosuppressed because of
adults, it is very rare to find infection or reaction from sites their condition or treatment, and the sepsis may be attrib-
other than the salivary gland where secretions from saliva uted to the immunosuppression.3
are a known source of transmission. Side effects are not
common but can include lymphadenopathy and fulminant Epidemiology and Transmission
hepatitis. Bacterial contamination usually originates with the donor,
HHV-6 has been associated with a number of diseases either through skin contamination at the phlebotomy site or
other than roseola infantum, such as multiple sclerosis and an asymptomatic bacteremia. It may also occur through
lymphoproliferative and neoplastic disorders. There is no contamination during processing.3 Contamination rates in
evidence to support a TTD association; with the high level the United States have been estimated to be 0.2% for RBCs
of seropositivity in the population, blood components are and as high as 10% for platelets. It is estimated that a febrile
not being tested for HHV-6.3 transfusion reaction caused by contaminated blood occurs
HHV-8 is another human herpesvirus. Unlike HHV-6, it once for every 10 to 20,000 units and that a death occurs
is not common in the population but has been seen in once for every 6 million units.56
Africa.53 Only 3% of donors are seropositive in the United According to the CDC,57 Yersinia enterocolitica is the
States.54 It is associated with several diseases that generally most common isolate found in RBC units, followed by the
affect the immunosuppressed patient. These include Kaposi’s Pseudomonas species. Together, these two account for more
sarcoma (KS), primary effusion lymphoma, and multicentric than 80% of all bacterial infections transmitted by RBCs. In
Castleman’s disease. Spread is generally through sexual con- a study by Kunishima,58 Propionibacterium acnes, a common
tact. However, in KS patients, 30% to 50% have circulating isolate of human skin, was the most common bacterial con-
lymphocytes harboring HHV-8, which lends support to taminant in RBCs. It is a slow-growing anaerobic bacteria
the premise that exposure to HHV-8 could be transfusion- that can go unrecognized if tested in aerobic conditions or
associated. There has been no evidence to support this to by using short-term bacterial cultural methods. Although
date.54 In post-transplant patients who develop KS, it P. acnes has been implicated in only a few cases of transfu-
appears to be due to reactivation. The transmission of sion-related sepsis, studies are needed to confirm long-term
HHV-8 has been associated with organ transplants and in- safety, as it has been associated with sarcoidosis.
jection drug use.53 Staphylococcus epidermidis, and Bacillus cereus (both
gram-positive) are the organisms most frequently recovered
Bacterial Contamination from donated blood and contamination of platelets.55
Overview
Laboratory Diagnosis
As the infection risk for other diseases has decreased due Before the unit of RBCs or platelets is issued, the unit should
to better donor testing, bacterial contamination has come be inspected for discoloration (dark purple or black), which
to the forefront and has become a great concern as a strongly indicates contamination. The unit may have no
transfusion-transmitted disease.55 Although the incidence of visible evidence of contamination at the time of issue. How-
transfusion-associated bacterial sepsis is low, the morbidity ever, clots in the unit and hemolysis may also indicate con-
and mortality rates are high. Common sources of bacterial tamination. Because the bacteria in the unit consume the
contamination include donor skin and blood. Less common oxygen, the cells may lyse, resulting in discoloration in the
sources are the environment and disposables.3 Platelets have unit as compared with the segments that remain normal in
been the most frequent source of septic transfusion reactions, color.3
due to the fact that room temperature storage promotes To detect bacterial contamination, both the donor blood
bacterial growth.3 In 2004, implementation of bacterial component and the recipient’s blood should be tested. It is
contamination screening methods has lowered the rate of better to test the component itself and not the segments, as
contamination. they may be negative.3 The FDA has cleared two culture
methods for quality-control monitoring of bacterial con-
Clinical Manifestations and Pathology tamination in platelets. Both methods can be used for
The most common signs and symptoms of transfusion- leukocyte-reduced apheresis platelets, whereas only one
associated sepsis are rigors, fever, and tachycardia.56 Other can be used for leukocyte-reduced whole blood–derived
symptoms may include shock, low back pain, disseminated platelets.
intravascular coagulation (DIC), and an increase or decrease Bacterial screening of platelets was implemented in the
in systolic blood pressure.3 The mortality rate from sepsis United States from 2003 to 2004. This has reduced the risk
and toxemia due to bacterially contaminated RBC units is of transfusing contaminated platelets to patients. Between
greater than 60%.3 Although the number of contaminated 2004 and 2006, the American Red Cross documented a
platelet units is much greater, the mortality rate is not as high residual risk of clinically relevant septic shock reactions of
as it is for RBCs. However, platelet contamination is consid- 1 in 74,807, and a fatality rate of at least 1 in 498,711, with
ered to be underreported. Sepsis due to platelets can occur platelets that were distributed (to outside facilities) after
2682_Ch18_403-426 22/05/12 11:58 AM Page 417
routine bacteria detection by culture techniques.59 This Between 2005 and 2006, the number of reported cases of
shows a 50% reduction in reported reactions and fatalities in primary and secondary syphilis increased 11.8%.62
a 10-month period after bacteria screening was implemented. The standard serologic tests for syphilis (STS) usually do
The FDA has allowed individual blood collection facilities not detect a donor in the spirochetemia phase who has not
to apply for extension of platelet storage from 5 to 7 days. yet seroconverted. Spirochetemia is short, and seroconver-
This extension is based on the track record and successful sion usually occurs after this phase.3 However, the STS is still
implementation of a bacterial-detection system.3 required for blood donors despite the fact that in 1978 a fed-
eral advisory panel recommended that this requirement be
Prophylaxis and Treatment eliminated. The FDA withheld the proposed rule to drop STS
The 26th edition of the AABB Standards states that “the from donor testing. The 26th edition of the AABB Standards
blood bank or transfusion service shall have methods to continues to require the STS.1
detect or inactivate bacterial contamination in all platelet Polymerase chain reaction followed by Southern blotting
components.”1 Use of apheresis platelets, careful phle- and a labeled probe have been used to confirm the presence
botomy technique, and phlebotomy diversion are listed by of treponemal antigen. The test is capable of detecting as few
the AABB as methods to limit bacterial contamination of as one treponeme in CSF.61 Nontreponemal EIAs, fluorescent
platelets. treponemal antibody absorption (FTA-ABS), treponema
Use of apheresis platelets rather than pooled whole pallidum immobilization (TPI), and treponema pallidum
blood–derived platelets from multiple donors reduces the in- hemagglutination (TPHA) are the methodologies utilized.
cidence of contamination occurring during phlebotomy. Blood donations that are reactive may not be used unless a
However, apheresis platelets cannot meet all platelet trans- confirmatory test such as FTA is determined to be nonreac-
fusion needs, and whole blood–derived platelets are still tive.3 Donors that have confirmed positive results can be
needed. Therefore, proper arm preparation for phlebotomy reinstated for donation after 12 months with documentation
is of paramount importance. Standard 5.6.2 in the 26th edi- of treatment.3
tion of Standards states that the use of green soap will no
longer be allowed.1 Improved bacterial disinfection has been Tick-Borne Bacterial Agents
correlated with the use of an iodine-based scrub. In donors
Lyme disease, Rocky Mountain spotted fever (RMSF), and
allergic to iodine, chlorhexidine or double isopropyl alcohol
ehrlichiosis are all bacterial diseases spread by a tick bite.
skin disinfectant may be used.55
Lyme disease is caused by the spirochete Borrelia burgdorferi
Phlebotomy diversion consists of collecting the first 20 to
and RMSF (Rickettsia rickettsii) and ehrlichiosis (Ehrlichia
30 mL of blood in a separate container to be used for testing.
species) are caused by bacteria that are obligate intracellular
This reduces the quantity of skin contaminants entering the
pathogens.
unit during phlebotomy and appears to be very effective in
reducing Staphylococcus species contamination.60 Several
Transfusion-Associated Parasites
blood bag manufacturers have developed systems with a
diversion pouch. At least three parasites have been associated with transfusion-
Leukodepletion of units can be helpful in removing associated infections: Babesia microti, Trypanosoma cruzi,
phagocytized bacteria along with the leukocytes. Some and malaria (Plasmodium species). Several additional parasites
advocate this for RBCs to reduce Yersinia contamination. have been identified in association with transfusion-associated
Other methods under consideration listed in the AABB disease. These include Leishmania species, other Trypanosoma
Technical Manual include endotoxin assays, detection of species, Toxoplasma gondii, and the microfilarial parasites.
by-products of bacterial metabolism, NAT, and pathogen Most of these infections occur on rare occasions and typically
inactivation methods.3 involve patients who are severely immunocompromised. The
Water baths used in a blood bank can have high bacterial risk for acquiring a blood transfusion containing these
counts unless disinfected frequently. An overwrap is recom- parasites may be underreported in endemic areas but has
mended for any components placed in the water bath, with always been very low in the United States. However, in
inspection of the outlet ports before use. October 2003, the AABB put forth a recommendation to
If transfusion-associated sepsis is suspected, treatment blood collection facilities that all individuals who had been
should begin immediately without waiting for laboratory in Iraq should be deferred for 1 year from the last date of
confirmation. Treatment should include IV antibiotics and departure. This was done after cases of leishmaniasis were
necessary therapy for whatever symptoms are present, such reported in personnel stationed in Iraq.
as shock, renal failure, and DIC.3
Babesia Microti
Syphilis
Babesiosis, a zoonotic disease, is usually transmitted by the
Treponema pallidum, the causative agent of syphilis, is a spiro- bite of an infected deer tick. Infection is caused by the proto-
chete. It is usually spread through sexual contact but can be zoan parasite, Babesia, which infects the RBCs. Most human
transmitted through blood transfusions. In 2006 in the cases of Babesia infection that occur in the United States are
United States, 36,000 cases of syphilis was reported.61 caused by the Babesia microti parasite.63 There have been
2682_Ch18_403-426 22/05/12 11:58 AM Page 418
reported cases of simultaneous transmission of B. microti and of atovaquone and azithromycin can be as effective in
Borrelia burgdorferi, the causative agent of Lyme disease, be- patients without a life-threatening illness.69 Apheresis has
cause the tick vectors are the same for the two organisms.64 also been successful in patients who fail to respond to
Other reported species are B. duncani, formally called WA1- antibiotic therapy.70,71
type Babesia, CA1-type Babesia, and B. divergens–like agents There is no test currently available to screen for asymp-
such as the MO1-type Babesia.65 Babesia infection may also tomatic carriers of Babesia. Many blood banks have added
be acquired by blood transfusion and solid organ transplant. questions to their donor questionnaire that address topics
Estimates between 70 and 100 cases of transfusion-transmitted such as living in an endemic area and previous Babesia in-
Babesia (TTB) have occurred over the last 30 years in the fection. Some blood banks have chosen to defer individuals
United States, with at least 12 fatalities in transfusion recip- who reside in areas that are heavily tick-infested in the sum-
ients diagnosed with babesiosis.66,67 mer months.72 This practice may have little value, as donors
may remain asymptomatic for months after exposure to the
Clinical Manifestations and Pathology organism. Donors with a history of babesiosis should be
Most cases of babesiosis are asymptomatic. Symptomatic deferred from donating blood for an indefinite period of
patients usually develop a malaria-type illness characterized time.3 Because B. microti can be transmitted by blood
by fever, chills, lethargy, and hemolytic anemia. The risk for donated from asymptomatic donors, effective measures for
developing severe complications, which include renal failure, preventing transmission are needed. The AABB Transfusion
DIC, and respiratory distress syndrome, increases for elderly, Transmitted Disease Committee (TTD) has described
asplenic, or immunocompromised patients. Reported incu- Babesiosis as a red category agent.52 These agents are given
bation periods for symptomatic patients range from 1 to a low to high scientific or epidemiological risk regarding
8 weeks after transfusion; therefore it is important that physi- blood safety with a potential for severe outcomes.51
cians consider babesiosis when diagnosing a febrile illness
following a transfusion.3 Trypanosoma Cruzi
Epidemiology and Transmission Trypanosoma cruzi is a flagellate protozoan that is the etio-
Areas of the United States, such as the northeast, mid- logic agent of Chagas’ disease (American trypanosomiasis).
Atlantic, upper Midwestern states are said to have endemic It is estimated that 300,000 people are infected within the
transmission.65 The incidence is higher during the spring United States.73 The disease is naturally acquired by the bite
and summer months, which corresponds to the increase in of a reduviid bug, thus making it a zoonotic infection. Insect
tick activity and outdoor recreation of humans. Persons in- transmission is the most common mode of infection but the
fected with Babesia may not have clinical signs of illness for organism has also been transmitted by blood transfusion and
an extended time. Infected persons who donate blood during organ transplants.
the asymptomatic period pose the greatest risk to the blood
supply, as they probably have infectious organisms circulat- Clinical Manifestations and Pathology
ing in their bloodstreams. Units of packed RBCs (liquid The acute phase of Chagas’ disease is initiated when the
stored and frozen deglycerolized) and platelet units, which organism enters the host. The reduviid bug bite produces a
contain RBCs, have been associated with transmission.66,68 localized nodule, referred to as a chagoma. The chagoma is
B. microti can survive in refrigerated, uncoagulated blood for usually painful and may take up to 3 months to heal. Clinical
21 to 35 days.3 There have been 63 transfusion-transmitted symptoms may be mild or absent; therefore, many cases are
babesiosis cases in the United States between 2004 and not diagnosed until the chronic phase of the disease. Symp-
2008.65 toms include anemia, weakness, chills, intermittent fever,
edema, lymphadenopathy, myocarditis, and gastrointestinal
Laboratory Diagnosis symptoms. Death may occur within a few weeks or months
Prompt diagnosis is essential, as Babesia responds well to after initial infection.
antibiotic therapy but can be fatal in certain risk groups Following the acute phase, the disease may enter a latent
if not properly treated. There is no specific test to diagnose phase, which can last up to 40 years.73 During this phase,
an infection with B. microti. Thick and thin blood smears the patient is usually asymptomatic but has parasites circu-
stained with Giemsa or Wright stain can be examined lating in the bloodstream. Transfusion-associated Chagas’
for intraerythrocytic organisms. A single negative smear disease is most likely to occur during this phase.
does not rule out an infection. Serologic studies such Chagas’ disease usually progresses to the chronic phase
as immunofluorescence assays can be used to detect circu- years or decades after the acute phase.73 In the chronic
lating antibody.65 Currently there is no screening test for phase, the organism begins to cause damage to cardiac tissue,
blood donors. thus causing cardiomyopathy. Since Chagas’ disease is not
very common in the United States, it can be easily misdiag-
Prophylaxis and Treatment nosed. One study revealed that 72% of Chagas’ disease
Babesiosis can be effectively treated with antibiotic therapy. patients in the United States had been treated for other car-
There is no specific drug of choice, but quinine and clin- diomyopathies for as long as 9 years before Chagas’ disease
damycin are very effective. In addition, the combination was diagnosed.73
2682_Ch18_403-426 22/05/12 11:58 AM Page 419
Epidemiology and Transmission test, called the Abbott Prism Chagas, is highly sensitive and
Chagas’ disease is endemic in Central and South America specific for the detection of antibodies to T. cruzi.74
and some areas of Mexico.3,73 Infected individuals pose a risk The AABB TTD committee has given T. cruzi an orange
of infecting recipients of their donated blood. There have category rating.51 An orange category agent is considered a
been four reported cases of T. cruzi infection acquired by low scientific or epidemiological risk regarding blood
blood transfusion in North America.3,73 All cases occurred saftey.51 T. cruzi was assigned a moderate rating by the TTD
in immunocompromised patients. This number could be committee based on public and regulatory attention to
higher if we consider there may be other cases not detected introducing blood donor screening.51
in immunocompetent patients receiving blood. T. cruzi can In the United States, medication for Chagas’ disease may
survive in platelets stored at room temperature, RBC units only be obtained by contacting the CDC.
at 4°C, and during cryopreservation and thawing.73 In addi-
tion to blood transfusions, Chagas’ disease can be transmit- Malaria (Plasmodium Species)
ted congenitally or transplacentally or through solid organ
Malaria, another intraerythrocytic protozoan infection, may
transplantation.73
be caused by several species of the genus Plasmodium
Screening for Chagas’ disease was implemented in Janu-
(P. malaria, P. falciparum, P. vivax, and P. ovale). Natural trans-
ary 2007, shortly after licensing of the screening test in the
mission occurs through the bite of a female Anopheles mos-
United States confirmed 1 in 121,000 positive blood donors
quito, but infection may also occur following transfusion of
after 3 months of screening.3 By January of 2008, there were
infected blood. Malaria is very rare in the United States, but
1,112 repeat antibody-positive blood donors identified.3
it is the most common parasitic complication of transfusion.3
Laboratory Diagnosis There are approximately 1,500 cases of malaria diagnosed in
the United States each year.75 Between 1963 and 2009, there
Acute Chagas’ disease is diagnosed by detecting the organism
have been 96 reported cases of transfusion-transmitted
in the patient’s blood. Blood smears stained with Giemsa or
malaria in the United States.75
Wright stain may be examined for the characteristic C- or
U-shaped trypomastigote (Fig. 18–8). Anticoagulated blood Clinical Manifestations and Pathology
or the buffy coat may also be evaluated for motile organisms.
Symptoms include fever, chills, headache, anemia, hemoly-
Chronic Chagas’ disease is diagnosed serologically. Such
sis, and splenomegaly. There may be variations in symptoms
testing includes complement fixation, immunofluorescence,
among the different species of Plasmodium. Malaria often
and ELISA. False-positive reactions are common; therefore
mimics other diseases, and its diagnosis is often delayed due
it is recommended that patient specimens be analyzed using
to lack of suspicion in nonendemic areas.
more than one assay. Trypomastigotes are rare or absent in
the peripheral blood during the chronic phase, so examina- Epidemiology and Transmission
tion of blood smears is not useful.
Malaria is endemic in tropical and subtropical areas and in
Prophylaxis and Treatment West Africa. The World Health Organization (WHO) esti-
mated that in 2008, malaria caused 190 to 311 million clin-
National screening of the blood supply was initiated in 2007.
ical episodes and 708,000 to 1,003,000 deaths.75 Many
Since that time, more than 1,000 donors with T. cruzi infec-
people associate malaria with a history of traveling to an
tion have been identified.74 The FDA approved a second test
endemic area. However, other transmission modes are pos-
to screen blood, tissue, and organ donors in April 2010. The
sible, including blood transfusions and congenital infection.
Transfusion-associated malaria is acquired by receiving
blood products from an asymptomatic carrier. Plasmodium
can survive in blood components stored at room temperature
or 4°C for at least a week, and deglycerolized RBCs can
transmit disease.3
Laboratory Diagnosis
Examination of thick and thin blood smears is performed to
diagnose infection with malaria. Although each species of
Plasmodium varies morphologically, diagnosis can be quite
difficult. Depending on the species of Plasmodium and the
stage of the parasite’s life cycle, timing is crucial when eval-
uating the blood smear. A single negative smear does not rule
out a diagnosis of malaria.
persons who have traveled to an endemic area are deferred There is no epidemiological evidence linking classic CJD
for 1 year, and those who have had malaria or who have im- to TTD. However, in vCJD cases, prion particles have been
migrated from or lived in an endemic area are deferred for found in lymphoreticular tissues, including the tonsils,
3 years.1,3 spleen, and lymph nodes. As blood is intimately involved
Chloroquine is generally effective for chemoprophylaxis with the lymphoreticular system, concerns arose regarding
and treatment of all four species of Plasmodium, except the ability of vCJD individuals to transmit the prion to
P. vivax acquired in Indonesia or Papua New Guinea, which recipients of blood or blood products.77
is best treated with atovaquone-proguanil, with mefloquine Currently, there is no reliable diagnostic test that can
or quinine plus tetracycline or doxycycline as alternatives.76 detect asymptomatic individuals. Therefore, deferral of
Therapy has become more complicated due to the increase donors with connection to the United Kingdom and parts of
in resistance of P. falciparum and, more recently, P. vivax to Europe is used to prevent transmission.3
chloroquine. It is important for the physician to carefully
evaluate the species of Plasmodium causing the illness, the Pathogen Inactivation
estimated parasitemia, and the patient’s travel history. This
information is necessary to prescribe appropriate therapy The safety of the blood supply in the United States has im-
and decrease the chance of resistance by the organism. proved greatly over the years, with improved screening of
Some individuals have a natural immunity to certain species donors and testing of the blood product. However, pathogen
of malaria, caused by a genetic alteration in their RBCs. These inactivation methods have been developed to account for
include persons who have sickle cell anemia or trait, G6PD residual risks associated with serologic window periods,
deficiency, or RBCs that lack the Duffy blood group antigen. virus variants, and laboratory errors and for organisms for
which testing is not performed routinely.79 The possibility
Prion Disease of newly emerging pathogens also exists as evidenced by
WNV that can be transmitted by blood.
Creutzfeldt-Jakob Disease
Plasma Derivatives
Creutzfeldt-Jakob disease (CJD) is one of the transmissible
spongiform encephalopathies (TSE). These are rare diseases Heat inactivation, the first pathogen inactivation interven-
characterized by fatal neurodegeneration that results in tion, has been used since 1948 to treat albumin.3 Even before
spongelike lesions in the brain. Although a definitive diag- the introduction of third-generation testing for HBsAg, heat
nosis can be made only at autopsy, neurological signs and inactivation prevented the transmission of HBV. The trans-
symptoms and disease progression are used to make a pre- mission of viruses or bacteria has been prevented due to
liminary diagnosis. Animals, such as sheep, goats, cattle, albumin’s pasteurization method (60°C for 10 hours).3
cats, minks, deer, and elk, and humans can be affected by In 1973, third-generation assays for HBsAg were licensed.
TSE. In humans, sporadic CJD is the most common form, Only one case of HBV transmission by immune globulin was
representing 85% to 90% of all cases, generally occurring in ever documented before then. Intramuscular immune glob-
late middle age (average age 60 years). An inherited form ulin has never transmitted HIV or HCV. All immunoglobulin
due to a gene mutation accounts for another 5% to 10% of plasma pools were screened for HBsAg, and only those that
cases, and iatrogenic CJD acquired through contaminated were negative were used. Viral inactivation included cold-
neurosurgical equipment, cornea or dura mater transplants, ethanol (Cohn-Oncley) fractionation and anion-exchange
or human-derived pituitary growth hormones accounts for chromatography (for one IV immunoglobulin). However, in
less than 5% of cases. The sporadic, inherited, and iatrogenic 1994, the FDA required viral clearance processing or proof
CJD are considered the classic CJD.76,77 In 1996, a variant of absence of HCV by NAT testing because of outbreaks of
form of CJD (vCJD) affecting younger individuals was noted, HCV from anion-exchange chromatography in Ireland and
and epidemiological evidence linked vCJD to bovine spongi- Germany that did not use a viral clearance procedure. NAT
form encephalopathy, possibly from eating contaminated is now used in the processing of all source plasmas.3
beef. Of the 129 cases reported from 1996 to 2002, most Coagulation factors had a high rate of viral transmission
were in the United Kingdom.76 until the early 1980s. Chronic hepatitis was the biggest prob-
The causative agent of all TSEs is believed to be a “prion,” lem until HIV emerged. More than 50% of all hemophiliacs
which is described as a self-replicating protein. It does not receiving concentrates became infected with HIV. Since
contain nucleic acid but is formed when the confirmation of 1987, these clotting factors have become very safe with im-
the normal cell surface glycoprotein, the prion protein, is plementation of a variety of virus inactivation steps, and
changed to an abnormal form. This abnormal form accumu- there have been no cases of HIV transmission. Today, all
lates in the brain and makes the brain tissue highly infectious. manufacturers use methods that either remove the virus or
It is resistant to inactivation by heat, radiation, and formalin.78 inactivate it. The lipid-enveloped viruses—HIV, HBV, HCV,
The median duration of illness for vCJD is 13 to HTLV, EBV, CMV, HHV-6 and HHV-8—are all inactivated by
14 months.77 However, the incubation period in humans use of organic solvents and detergents. This process is not
varies from 4 to 20 years and may eventually prove to be effective with non-lipid-enveloped viruses such as HAV and
longer in some cases. parvovirus B-19.3,80
2682_Ch18_403-426 22/05/12 11:58 AM Page 421
The current risk of enveloped virus transmission is very donors of any abnormality with the predonation evaluation,
low because of the combination of treatments such as heat laboratory testing, or recipient follow-up. A report should
treatment, solvent and detergent treatment, and nanofiltra- be submitted to the collecting agency when the recipient of
tion.81 These methods are often used in combination during a blood component develops a TTD.1
the manufacturing process. With the exception of one case Current donations that test positive for HBV, HCV, HIV, or
of HCV transmission in IV immune globulin in 1994, there HTLV cannot be used for transfusion.1 All prior donations
have been no cases of HBV, HCV, or HIV since 1985 by any from these donors become suspect. The timeline and stan-
U.S. licensed plasma derivative.80 dards using the look-back procedure to identify recipients of
a component from the implicated donation or other donations
Cellular Components by the same donor differ depending on the disease. Any prior
components still in date must be quarantined, and the dispo-
Pathogen inactivation using psoralen activated by ultraviolet sition depends on results of licensed supplemental tests.3
light has been tested with platelet concentrates. It has been If on recipient follow-up it is noted that a patient devel-
shown to inactivate cell-associated viruses, cell-free viruses, oped HBV, HCV, HIV, or HTLV after receiving a single unit
and selected prokaryotic organisms. Whether this process from one donor, that donor is permanently deferred. If the
will work against intracellular bacterial organisms has not recipient received donations from several donors, all donors
been established. There are three licensed platelet pathogen do not have to be excluded. These implicated donors may
reduction systems that are currently in use in the United be called in for retesting. If a donor has been implicated in
States and Canada: the Cerus Corporation INTERCEPT more than one case of TTD, this donor should be retested
Blood System, CaridianBCT Biotechnologies Mirasol PRT, and possibly permanently deferred.3 Once a donor has been
and the MacoPharma’s Theraflex UV.51 These companies also implicated in a TTD, other recipients of a component from
have processes for pathogen reduction in plasma.51 the suspected donor should be contacted. The donor must
CaridianBCT Biotechnologies is currently using a photo- be placed on the appropriate donor deferral list if subsequent
chemical process for red cell pathogen reduction. This sys- tests are positive.3 Donors who have been permanently
tem incorporates riboflavin and UV light.51 A process for deferred due to positive test results must be notified of
RBCs that uses a chemical cross-linker specific for nucleic the fact. Notification and a thorough explanation of the
acid is being designed by Cerus Corporation.51 positive tests results and their implications must be given to
Limitations of pathogen reduction systems include agents the donor.3 Follow-up testing should be performed by the
with intrinsic resistance, such as prions, some bacterial donor’s own physician.3
spores, and nonenveloped viruses.51 Some viruses may not Autologous donations positive for HBV, HCV, HIV, HTLV,
be inactivated if they are of high titer, including B19V and or syphilis can be used. If they are not transfused at the col-
HBV.51 lecting facility, the collecting facility must notify the transfu-
sion service. Testing must be repeated every 30 days on at least
Quarantine and Recipient Tracing the first unit to be shipped. Information about abnormalities
(Look-Back) must be transmitted to the patient and the patient’s physician.1
Any fatalities due to a TTD must be reported to the direc-
All blood banks and transfusion services are required to have tor of the Center for Biologics Evaluation and Research within
a process to detect, report, and evaluate any complication of 1 working day, followed by a written report within 7 days.81
transfusion, including recipient development of HBV, HCV, Table 18–5 summarizes the laboratory tests for transfusion-
HIV, or HTLV. There must be an established method to notify transmitted diseases.
Hepatitis B Virus (HBV) B surface 1971 ChLIA Antigen neutralization 1 in 200,000 and 1 in
antigen (HBsAq) 500,0001,2
Hepatitis B core 1986 ChLIA Ultrasensitive HBV 1 in 200,000 and 1 in
antibody (HBc) DNA detection 500,0001,2
by PCR
Confirmatory 2009 NAT All TMA-reactive
testing donations confirmed
by PCR
Continued
2682_Ch18_403-426 22/05/12 11:58 AM Page 422
Syphilis (Treponema Antibody testing— 1950s *NAT EIA, as well as a test 0* (No cases of trans-
pallidum) qualitative for regain (a pro- fusion-transmitted
screening test tein-like substance syphilis have been
detects presence that is present during recorded for more
of antibodies to acute infection and than 30 years).
Treponema for several months
pallidum following resolution
of infection).
West Nile Virus (WNV) WNV RNA 2003 *NAT (same 9 documented cases
detection type of assay (since the introduc-
as used for tion of blood donor
HBV, HIV-1, screening).
and HCV).
ChLIA = Chemiluminescent immunoassay; *NAT = Nucleic Acid Testing (Polymerase chain reaction (PCR) for anti-HBc; Transcription mediated amplification (TMA) for HBV DNA); ELISA = Enzyme-
Linked, Immunosorbent Assay Test System; RIBA = Recombinant Immunoblot Assay; EIA = Enzyme Immunoassay; IFA = Indirect Immunofluorescence Assay; RIPA = Radioimmunoprecipitation
Assay.
References: Cited in American Red Cross, Infectious Disease Testing, on: http://www.redcrossblood.org/learn-about-blood/what-happens-donated-blood/blood-testing
1. Stramer S. Current risks of transfusion-transmitted agents—A Review. Arch Pathol Lab Med. 2007; 131: 702-707.
2. Zou S, et al. Current Incidence and residual risk of hepatitis B infection among blood donors in the United States. Transfusion 2009; 49:1609-1620.
3. Dodd RY, et al. Current prevalence and incidence of infectious disease markers and estimated window-period risk in the American Red Cross blood donor population. Transfusion 2002; 42: 975-97.
4. Young C, Losikoff P, Chawla A, Glasser L, Forman E. Transfusion-acquired Trypanosoma cruzi infection. Transfusion 2007;47:540-4. in Perkins HA and Busch MP, Transfusion-associated infections:
50 years of relentless challenges and remarkable progress. Transfusion 2010; 8-9. doi: 10.1111/j.1537-2995.2010.02851.x
2682_Ch18_403-426 22/05/12 11:58 AM Page 423
SUMMARY CHART
The first and most important step in ensuring that Transfusion-associated CMV infection is a concern for
transfused blood will not transmit a pathogenic virus seronegative allogeneic organ transplant recipients and
is careful selection of the donor. fetuses. Reactivation of a latent infection can occur when
HAV is usually spread by the fecal-oral route in com- an individual becomes severely immunocompromised.
munities where hygiene is compromised. The risk of CMV infection for low-birth-weight neonates
On infection with HBV, the first serologic marker to is not as great as it was in the past due to better transfu-
appear is HBsAg, followed by HBeAg and IgM anti-HBc sion techniques and management of their conditions.
within the first few weeks of exposure. The WB confirmation test detects the presence of anti-
HBIG is an immune globulin prepared from persons HIV and determines with which viral proteins the
with a high titer of anti-HBs and is used to provide pas- antibodies react.
sive immunity to health-care workers and others who The window period for HIV can be shortened by using
are exposed to patients with HBV infection. the polymerase chain reaction, which detects HIV in-
A combined vaccine for HAV and HBV is available to fection before tests for antigen or antibody are positive.
provide immunity. Bacterial contamination is the most frequent cause of
HDV infection is common among drug addicts and can transfusion-transmitted infection.
occur simultaneously with HBV infection; diagnosis Because routine screening for parasitic infections is not
depends on finding anti-HDV or HDV RNA in the currently available, many blood banks have added
serum. questions to their donor questionnaire that address
Of all HCV infections, 60% to 70% are asymptomatic. topics associated with risk for parasitic infection.
With the implementation of NAT testing for HCV, the Pathogen inactivation methods are under development
window period has been reduced to 10 to 30 days. to remove the residual risk of transfusion-associated
HCV is the leading cause of liver transplants in the disease due to the window period, virus variants,
United States. laboratory mistakes, and new, emerging diseases.
Diagnosis of HIV-1 and HIV-2 infection is dependent Look-back is a process mandated by the FDA that
on the presence of antibodies to both envelope and directs collection facilities to notify donors who test
core proteins; HIV-positive persons with fewer than positive for viral markers, to notify prior recipients of
200 CD4+ T cells per µL are considered to have AIDS the possibility of infection, and to quarantine or dis-
in the absence of symptoms. card implicated components currently in inventory.
7. Currently, steps taken to reduce transfusion-transmitted 15. Individuals exposed to EBV maintain an asymptomatic
CMV include: latent infection in:
a. Plaque reduction neutralization test. a. B cells.
b. NAT testing. b. T cells.
c. Leukoreduction. c. All lymphocytes.
d. Minipool screening. d. Monocytes.
8. HBV remains infectious on environmental surfaces for 1: 16. Fifth disease is caused by:
a. Day. a. CMV.
b. Week. b. EBV.
c. Month. c. Parvovirus B19.
d. Year. d. HTLV-II.
9. HBV is transmitted most frequently: 17. Transient aplastic crisis can occur with:
a. By needle sharing among IV drug users. a. Parvovirus B19.
b. Through blood transfusions. b. WNV.
c. By unknown methods c. CMV.
d. By sexual activity d. EBV.
10. Which of the following is the most common cause of 18. Reasons why syphilis is so rare in the United States
chronic hepatitis, cirrhosis, and hepatocellular carci- blood supply include all of the following except:
noma in the United States? a. 4°C storage conditions.
a. HAV b. Donor questionnaire.
b. HBV c. Short spirochetemia.
c. HCV d. NAT testing.
d. HDV
19. Nucleic acid amplification testing for HIV was instituted
11. The first retrovirus to be associated with human disease in donor testing protocols to:
was: a. Identify donors with late-stage HIV who lack antibodies.
a. HCV b. Confirm the presence of anti-HIV in asymptomatic
b. HIV HIV-infected donors.
c. HTLV-I c. Reduce the window period by detecting the virus
d. WNV earlier than other available tests.
d. Detect antibodies to specific HIV viral proteins,
12. All of the following statements are true concerning
including anti-p24, anti-gp41, and anti-gp120.
WNV except:
a. 1 in 150 infections results in severe neurologic 20. Screening for HIV is performed using the following
disease. technique:
b. Severe disease occurs most frequently in the over-50 a. Radio immunoassay
age group. b. WB
c. Deaths occur more often in those over 65 years who c. Immunofluorescent antibody assay
present with encephalitis. d. NAT
d. Fatalities occur in approximately 38% of infected
21. The first form of pathogen inactivation was:
individuals.
a. Chemical.
13. The primary host for WNV is: b. Heat.
a. Birds. c. Cold-ethanol fractionation.
b. Horses. d. Anion-exchange chromatography.
c. Humans.
22. What is the most common parasitic complication of
d. Bats.
transfusion?
14. Tests for WNV include all of the following except: a. Babesia microti
a. ELISA. b. Trypanosoma cruzi
b. NAT. c. Plasmodium species
c. Plaque reduction neutralization test. d. Toxoplasma gondii
d. Immunofluorescent antibody assay.
2682_Ch18_403-426 22/05/12 11:58 AM Page 425
23. Which organism has a characteristic C- or U-shape on 17. Halasz, R, et al: GB virus C/hepatitis G virus. Scand J Infect
stained blood smears? Dis 33:572, 2001.
18. Nunnari, G, et al: Slower progression of HIV-1 infection in
a. Trypanosoma cruzi persons with GB virus C coinfection correlates with an intact
b. Plasmodium vivax T-helper 1 cytokine profile. Ann Intern Med 139:26, 2003.
c. Plasmodium falciparum 19. Zanetti, AR, et al: Multicenter trial on mother-infant transmis-
d. Babesia microti sion of GBV-virus. J Med Virol 54:107, 1998.
20. Sathar, MA, et al: GB virus C/hepatitis G virus (GBV-C/HGV):
24. Which transfusion-associated parasite may have asymp- Still looking for a disease. Int J Exper Pathol 81:305, 2000.
tomatic carriers? 21. Jarvis, LM, et al: The effect of treatment with alpha-interferon
on hepatitis G/GBV-C viraemia. The CONSTRUCT Group.
a. Babesia microti Scand J Gastroenterol 33:195, 1999.
b. Trypanosoma cruzi 22. Centers for Disease Control and Prevention: HIV/AIDS
c. Plasmodium species Prevalence Estimates, United States, 2006. Available at
d. All of the above www.cdc.gov/mmwr/preview/mmurhtm/mm5739a2.htm.
Accessed August 11, 2010.
25. Which disease is naturally caused by the bite of a deer tick? 23. Centers for Disease Control and Prevention: HIV and AIDs
in the United States. Available at www.cdc.gov/hiv/resources/
a. Chagas’ disease factsheets/us.htm. Accessed August 11, 2010.
b. Babesiosis 24. American Red Cross: Blood Testing. Available at www.
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25. Centers for Disease Control: Advancing HIV prevention:
New strategies for a changing epidemic—United States, 2005.
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5. Lemon, SM: Type A viral hepatitis: Epidemiology, diagnosis, 30. American Association of Blood Banks: Dual enzyme immuno
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15. Alter, H, et al: The incidence of transfusion associated hepatitis 39. Centers for Disease Control and Prevention: Dispatch:
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41. Laupacis, A, et al: Conference report: Prevention of posttrans- 62. Centers for Disease Control: Sexually Transmitted Diseases—
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42. Ely, JW, Yankowitz, J, and Bowdler, NC: Evaluation of pregnant 63. Centers for Disease Control: Babesiosis Fact Sheet. Available
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43. Roback, JD, et al: Multicenter evaluation of PCR methods for de- 64. Machon, CR, et al: Textbook of Diagnostic Microbiology.
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1964. 67. Stramer, SL, Hollinger, FB, Katz, LM, et al: Emerging infectious
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49. Brown, KE, et al: Resistance to parvovirus B19 infection due 70. Jocoby, GA, et al: Treatment of transfusion-transmitted babesio-
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54. Pellett, PE, et al: Multicenter comparison of serologic assays Fact Sheet No. 180. Revised November 2002. Available
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US donors. Transfusion 43:1260, 2003. Accessed August 26, 2010.
55. Brecher, ME, and Hay, SN: Bacterial contamination of blood 77. Centers for Disease Control and Prevention: Bovine spongi-
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56. Kuehnert, MJ, et al: Transfusion-transmitted bacterial infection and answers regarding Creutzfeldt-Jakob disease infection-
in the United States, 1998 through 2000. Transfusion 41;1493, control practices. Available at www. cdc.gov/ncidod/dvrd/vcjd/
2001. indexhtm. Accessed August 26, 2010.
57. Centers for Disease Control: Red blood cell transfusions con- 78. McKnight, C: Clinical implications of bovine spongiform
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1996, and initiation of a national study to detect bacteria- 79. Purmal, A, et al: Process for the preparation of pathogen-
associated transfusion reactions. MMWR 46:553, 1997. inactivated RBC concentrates by using PEN110 chemistry:
58. Kunishima, S, et al: Presence of Propionibacterium acnes in Preclinical studies. Transfusion 42:139, 2002.
blood components. Transfusion 41:1126, 2001. 80. Tabor, E: The epidemiology of virus transmission by plasma
59. Eder, A, Kennedy, J, Dy, B, et al: Bacterial screening of apheresis derivatives: Clinical studies verifying the lack of transmission
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Red Cross Experience (2004–2006). Transfusion 47:1134–1142, 39:1160, 1999.
2007. 81. Food and Drug Administration: Transfusion/Donation
60. de Korte, D, et al: Diversion of first blood volume results in a Fatalities. Available at www.fda.gov/BiologicsBloodVaccines/
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tions. Vox Sang 83:13, 2002. default.htm. Accessed September 3, 2010.
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Chapter 19
Hemolytic Disease of the Fetus
and Newborn (HDFN)
Melanie S. Kennedy, MD
OBJECTIVES
1. State the definition and characteristics of hemolytic disease of the fetus and newborn (HDFN).
2. Describe the role of the technologist in the diagnosis and clinical management of HDFN.
3. Compare and contrast ABO versus Rh HDFN in terms of:
• Pathogenesis
• Incidence
• Blood types of mother and baby
• Severity of disease
• Laboratory data: anemia, direct antiglobulin test (DAT), and bilirubin
• Prevention and treatment
4. Define Rh-immune globulin and describe its function.
5. Identify the indications and contraindications for administration of Rh-immune globulin.
6. List the tests used for detecting fetomaternal hemorrhage.
7. Outline the protocol for testing maternal and cord blood in cases of suspected HDFN.
8. Given maternal and infant ABO blood group phenotypes, state the possible ABO donor blood group(s) that would be selected
for an exchange transfusion. Be specific as to donor blood groups for both the plasma and the red blood cells (RBCs).
9. State the blood components and the maximum age of the donor unit preferred for intrauterine or exchange transfusions.
10. State the source of cells and plasma (serum) to crossmatch an RBC unit for a newborn.
427
2682_Ch19_427-438 22/05/12 11:58 AM Page 428
Etiology
Immune
Although the clinical findings in the fetus and newborn were + System
noted as early as the 17th century, it was not until 1939 that
Levine and Stetson reported a transfusion reaction from trans- +
fusing a husband’s blood to a postpartum woman. They pos-
tulated that the mother had been immunized to the father’s
antigen through fetomaternal hemorrhage. The antigen was
later identified as Rh(D).
HDFN is caused by the destruction of the RBCs of a fetus
by antibodies produced by the mother. Only antibodies of
the immunoglobulin G (IgG) class are actively transported
across the placenta; other immunoglobulin classes, such as
IgA and IgM, are not. Most IgG antibodies are directed HDFN
against bacterial, fungal, and viral antigens, so the transfer
of IgG from the mother to the fetus is beneficial. However,
in HDFN, the antibodies are directed against those antigens
on the fetal RBCs that were inherited from the father.
Rh HDFN
In Rh(D) HDFN, the Rh-positive firstborn infant of an Rh-
negative mother usually is unaffected because the mother
has not yet been immunized. During gestation and partic- Figure 19–1. Pathogenesis of hemolytic disease of the fetus and newborn
ularly at delivery, when the placenta separates from the caused by D incompatibility between the fetus and mother. The pathogenesis of
uterus, variable numbers of fetal RBCs enter the maternal other blood group incompatibilities (except ABO) follows the same pattern.
2682_Ch19_427-438 22/05/12 11:58 AM Page 429
villus sampling and trauma to the abdomen can increase Table 19–1 Antibodies Identified in
the risk of fetomaternal hemorrhage. At delivery, the inci- Prenatal Specimens as a
dence is more than 50%. In the majority of cases, the vol-
Cause of HDFN
ume of fetomaternal hemorrhage is small; however, as little
as 1 mL of fetal RBCs can immunize the mother.3 COMMON RARE NEVER
The number of antigens on the fetal RBCs corresponds to Anti-D Anti-Fya Anti-Lea
heterozygous RBCs, because all fetal antigens incompatible
with the mother have been inherited from the father, who Anti-D + C Anti-s Anti-Leb
can give only one set of genes to the fetus. Anti-D + E Anti-M Anti-I
Of all the RBC antigens, D is the most antigenic. For this Hemolysis occurs when maternal IgG attaches to specific
reason, only Rh-negative blood is transfused to Rh-negative antigens of the fetal RBCs (see Fig. 19–1). The antibody-
females of childbearing potential. Other antigens in the Rh coated cells are removed from the circulation by the
system, such as C, E, and c, are also potent immunogens macrophages of the spleen. The rate of RBC destruction de-
(although less than D; Table 19–1). These other Rh antibod- pends on antibody titer and specificity and on the number
ies have been associated with moderate to severe cases of of antigenic sites on the fetal RBCs. Destruction of fetal RBCs
HDFN. Anti-E and anti-c have caused severe HDFN that and the resulting anemia stimulate the fetal bone marrow to
required intervention and treatment. produce RBCs at an accelerated rate, even to the point that
Of the non–Rh system antibodies, anti-Kell is consid- immature RBCs (erythroblasts) are released into the circula-
ered the most clinically significant in its ability to cause tion. The term erythroblastosis fetalis was used to describe
HDFN. Kell antigens are present on immature erythroid this finding. When the bone marrow fails to produce enough
cells in the bone marrow, so severe anemia occurs not only RBCs to keep up with the rate of RBC destruction, erythro-
by destruction of circulating RBCs but also by precursors.5 poiesis outside the bone marrow is increased in the
Almost any IgG RBC antibody is capable of causing HDFN, hematopoietic tissues of the spleen and liver. These organs
although the disease caused by these antibodies is usually become enlarged (hepatosplenomegaly), resulting in portal
mild to moderate in severity. Nevertheless, all pregnant hypertension and hepatocellular damage.
women with IgG RBC antibodies should be followed Severe anemia and hypoproteinemia (caused by decreased
closely for HDFN. Vengelen-Tyler lists and discusses hepatic production of plasma proteins) lead to the develop-
64 different RBC antibody specificities reported to cause ment of high-output cardiac failure with generalized edema,
HDFN.6 effusions, and ascites, a condition known as hydrops fetalis.
2682_Ch19_427-438 22/05/12 11:58 AM Page 430
In severe cases, hydrops fetalis can develop by 18 to 20 weeks’ Pregnant female with antibody
gestation. In the past, hydrops fetalis was almost uniformly
fatal; today, most fetuses with this condition can be treated Pregnancy history
successfully. IgG antibody specificity
Father’s antigen status
The process of RBC destruction continues even after such
an infant is delivered alive—in fact, it continues as long as
maternal antibody persists in the newborn infant’s circula- Potential of HDFN
tion. The rate of RBC destruction after birth decreases be-
cause no additional maternal antibody is entering the infant’s No Yes
circulation through the placenta. However, IgG is distributed
both extravascularly and intravascularly and has a half-life Routine prenatal care Maternal antibody titer
of 25 days, so antibody binding and hemolysis of RBCs con-
tinue for several days to weeks after delivery.
ⱖ16 ⬍16
spin and room temperature incubation phases are omitted, Fetal DNA Testing
and anti-IgG rather than polyspecific antiglobulin reagent is If the mother has anti-D and the father is likely to be het-
used. These steps reduce detection of IgM antibodies, which erozygous for the D antigen, amniocentesis or chorionic vil-
cannot cross the placenta. Other antibody screening meth- lous sampling can be performed as early as 10 to 12 weeks’
ods, such as solid phase or gel column, may be used. gestation to determine whether the fetus has the gene for the
If the antibody screen is nonreactive, repeat testing is rec- D antigen. During the second trimester, maternal plasma can
ommended before RhIG therapy in Rh-negative prenatal pa- be tested for fetal DNA to determine genotype.9 Testing can
tients and in the third trimester if the patient has been be performed for the genes coding c, e, C, E, K, Fya, Fyb, Jka,
transfused or has a history of unexpected antibodies.8 Jkb, M and others.
Antibody Identification Antibody Titers
If the antibody screen is reactive, the antibody identity must The relative concentration of all antibodies capable of cross-
be determined. Follow-up testing will depend on the anti- ing the placenta and causing HDFN is determined by anti-
body specificity. Cold reactive IgM antibodies such as anti-I, body titration. The patient serum or plasma is serially
anti-IH, anti-Lea, anti-Leb, and anti-P1 can be ignored. Lewis diluted and tested against appropriate RBCs to determine the
system antibodies are rather common in pregnant women highest dilution at which a reaction occurs.9 The method
but have not been reported to cause HDFN. must include the indirect antiglobulin phase using anti-IgG
Antibodies such as anti-M and anti-N can be IgM or IgG or reagent. The result is expressed as either the reciprocal of
a combination of both. Both anti-M and anti-N can cause mild the titration endpoint or as a titer score.
to moderate HDFN, although rarely. To establish the im- The titration must be performed exactly the same
munoglobulin class, the serum can be treated with a sulfhydryl way each time the patient’s serum is tested. The recom-
reagent, such as dithiothreitol or 2-mercaptoethanol, and then mended method is the saline antiglobulin tube test, with
retested with appropriate controls. The J-chain of IgM antibod- 60-minute incubation at 37°C and the use of anti-IgG
ies will be destroyed by this treatment; IgG antibodies will reagent.8 The RBCs used for each titration should have the
remain reactive. same genotype (preferably homozygous for the antigen of
Many Rh-negative pregnant women have weakly reactive interest), approximately the same storage time, and the
anti-D, particularly during the third trimester. Most of these same concentration. The first serum or plasma specimen
women have received RhIG, either after an event with in- should be frozen and run in parallel with later specimens.
creased risk of fetomaternal hemorrhage or at 28 weeks’ ges- Only a difference of greater than 2 dilutions, or a score
tation (antenatal). The passively administered anti-D will be change of more than 10, is considered a significant change
weakly reactive in testing and will remain demonstrable for in titer.
2 months or longer. This must be distinguished from active The method chosen is critical for the appropriate clinical
immunization. A titer higher than 4 almost always indicates correlation. Methods using enhancing media or gel column
active immunization; with a titer under 4, active immuniza- result in higher titers, as shown by comparative proficiency
tion cannot be ruled out, but it is less likely. testing. Therefore, the critical titer level for these other meth-
If the antibody specificity is determined to be clinically ods must be determined by reviewing the outcome of several
significant and the antibody is IgG, further testing is re- pregnancies complicated by HDFN.
quired. Other than anti-D, the most common and most sig- For the recommended method, 16 is considered the criti-
nificant antibodies are anti-K, anti-E, anti-c, anti-C, and cal titer. If the initial titer is 16 or higher, a second titer should
anti-Fya (see Table 19–1). be done at about 18 to 20 weeks’ gestation. A titer repro-
ducibly and repeatedly at 32 or above is an indication for
Paternal Phenotype and Genotype color Doppler imaging to assess middle cerebral artery peak
A specimen of the father’s blood should be obtained and systolic velocity (MCA-PSV) after 16 weeks’ gestation.10
tested for the presence and zygosity of the corresponding When the titer is less than 32, it should be repeated at
antigen. If the mother has anti-D and the father is D-positive, 4-week intervals, beginning at 16 to 20 weeks’ gestation
a complete Rh phenotype can help determine his chance of and then every 2 to 4 weeks during the third trimester.
being homozygous or heterozygous for the D antigen. A Antibody titer alone cannot predict severity of HDFN.8 In
more sensitive and precise genotype can be determined by some sensitized women, the antibody titer can remain mod-
DNA methods. The information is helpful in determining erately high throughout pregnancy while the fetus is becom-
further testing of the mother and in counseling her about ing more severely affected. Similarly, a previously sensitized
potential treatment plans and complications of HDFN. woman can have consistently high antibody titer whether
In cases of antibody specificity other than D, testing the pregnant or not, and whether the fetus is Rh-positive or Rh-
father can save a great deal of time, expense, and worry if negative. In others, the titer can rise rapidly, which portends
he is shown to lack the corresponding antigen. For exam- increasing severity of HDFN. However, antibody titers con-
ple, only 9% of the random population is positive for the sistently below the laboratory’s critical titer throughout the
Kell antigen. The mother must be counseled in private as pregnancy reliably predict an unaffected or mild-to-
to the paternity of the fetus to ensure accurate paternal moderately affected fetus, with the exception of anti-K with
phenotyping. a K-positive fetus.
2682_Ch19_427-438 22/05/12 11:58 AM Page 432
Titration studies at time of delivery are not recommended, • Cordocentesis blood sample has hemoglobin level less
because they provide no clinically useful information. than 10 g/dL.
• Amniotic fluid ∆OD 450 nm results are high.
Color Doppler Middle Cerebral Artery Peak
Intrauterine transfusion is performed by accessing the
Systolic Velocity
fetal umbilical vein (cordocentesis) and injecting donor
At about 16 to 20 weeks’ gestation, further diagnosis and RBCs directly into the vein.12 The goal of intrauterine trans-
treatment are begun. Patients with a history of a severely af- fusion is to maintain fetal hemoglobin above 10 g/dL. Once
fected fetus or early fetal death may require earlier interven- intrauterine transfusion is initiated, the procedure is repeated
tion. The measurement of the fetal middle cerebral artery every 2 to 4 weeks until delivery. The initial intrauterine
peak systolic velocity (MCA-PSV) with color Doppler ultra- transfusion is rarely performed after 36 weeks’ gestation. In-
sonography can reliably predict anemia in the fetus.10 Color trauterine transfusion apparently suppresses the fetal bone
Doppler indicates the direction of blood flow, using red for marrow RBC production. During the first weeks after birth,
arterial flow and blue for venous. The middle cerebral artery the infant may require additional RBC transfusion.
is used because of its easy accessibility. The measurement is Cordocentesis, intrauterine transfusion, and amniocente-
based on the reduced blood viscosity at low hematocrit and sis have several risks, including infection, premature labor,
the resulting faster velocity. The peak systolic (arterial) ve- and trauma to the placenta, which may cause increased an-
locity is plotted on a standardized graph to determine the tibody titers because of antigenic challenge to the mother
critical point for cordocentesis. MCA-PSV is noninvasive and through fetomaternal hemorrhage.
poses no adverse effects for the fetus.
Phototherapy
Cordocentesis
After delivery, the neonate can develop hyperbilirubinemia of
Advanced sonography allows clinicians to obtain a sample unconjugated bilirubin. Phototherapy at 460 to 490 nm is used
of fetal blood through a procedure called cordocentesis. to change the unconjugated bilirubin to isomers, which are less
Using high-resolution ultrasound with color Doppler en- lipophilic and less toxic to the brain.13 Relatively high doses
hancement of blood flow, the umbilical vein is visualized are given by using two banks of lights to surround the infant’s
at the level of the cord insertion into the placenta. A spinal body. In infants with mild-to-moderate hemolysis or history of
needle is inserted into the umbilical vein, and a sample of intrauterine transfusion, phototherapy is generally sufficient.
the fetal blood is obtained. The fetal blood sample can then
be tested for hemoglobin, hematocrit, bilirubin, blood type, Intravenous Immune Globulin
direct antiglobulin test (DAT), and antigen phenotype and
Intravenous immune globulin (IVIG) is increasingly used to
genotype.
treat hyperbilirubinemia of the newborn caused by HDFN.
The IVIG competes with the mother’s antibodies for the FC
Amniocentesis
receptors on the macrophages in the infant’s spleen, reducing
For management of HDFN, amniocentesis is uncommonly the amount of hemolysis.
used, because MCA-PSV is noninvasive and gives the same
information.11 The concentration of bilirubin pigment in Exchange Transfusion
the amniotic fluid estimates the extent of fetal hemolysis.
Exchange transfusion is the use of whole blood or equiva-
The amniotic fluid is tested by a spectrophotometric scan
lent to replace the neonate’s circulating blood. Exchange
at steadily increasing wavelengths, so the change in the
transfusion is rarely required because of advances in pho-
optical density (∆OD) at 450 nm (the absorbance of biliru-
totherapy and the use of IVIG. In addition, less than
bin) can be calculated. The measurement is plotted on
one-half of the cases of HDFN are caused by anti-D, so the
a graph according to gestational age. An increasing or
majority is generally less severe. Exchange transfusions are
unchanging ∆OD 450 nm as pregnancy proceeds predicts
used primarily to remove high levels of unconjugated biliru-
worsening of the fetal hemolytic disease and the need for
bin and thus prevent kernicterus. Premature newborns are
frequent monitoring and intervention if indicated. High
more likely than full-term infants to require exchange trans-
values indicate severe and often life-threatening hemolysis
fusions for elevated bilirubin because their livers are less able
(fetal hemoglobin less than 8 g/dL) and require urgent
to conjugate bilirubin. Other advantages of exchange trans-
intervention.
fusion include the removal of part of the circulating maternal
antibody, removal of sensitized RBCs, and replacement of in-
Intrauterine Transfusion
compatible RBCs with compatible RBCs; all of these help in-
Intervention in the form of intrauterine transfusion terrupt the bilirubin production caused by hemolysis.
becomes necessary when one or more of the following
conditions exists: Serologic Testing of the Newborn Infant
• MCA-PSV indicates anemia. Serologic testing of the cord blood is used to confirm HDFN
• Fetal hydrops is noted on ultrasound examination. and prepare for possible transfusion.
2682_Ch19_427-438 22/05/12 11:58 AM Page 433
ABO Grouping fetuses and neonates whose blood types are unknown or are
ABO antigens are not fully developed in newborn infants, so Rh-negative.
newborns may show weaker reactions than older children For intrauterine transfusion, the hematocrit level of the
and adults. In addition, infants do not have their own isoag- RBCs should be greater than 70% because of the small vol-
glutinins but may have those of the mother, so reverse group- ume transfused and the need to correct severe anemia.
ing cannot be used to confirm the ABO group. For the rare exchange transfusions, one practice is to pre-
pare RBCs from whole blood units and then replace the
Rh Typing plasma with group AB plasma to reduce the amount of blood
Rarely, the infant’s RBCs can be heavily antibody-bound with group antibodies transfused. This procedure is not necessary
maternal anti-D, causing a false-negative Rh type, or what if both the neonate and the mother are the same ABO group.
has been called blocked Rh. An eluate from these RBCs will Blood transfused to the fetus and premature infant should
reveal anti-D, and typing of the eluted RBCs will show reac- also be irradiated to prevent graft-versus-host disease (see
tion with anti-D. Chapter 15, “Transfusion Therapy”). Blood for exchange
transfusion should be irradiated and should not contain
Direct Antiglobulin Test hemoglobin S, because the decreased oxygen tension that
The most important serologic test for diagnosing HDFN is may occur early in the neonatal period may cause hemoglo-
the DAT with anti-IgG reagent. A positive test result indi- bin S trait blood to sickle. Traditionally, blood units less than
cates that the antibody is coating the infant’s RBCs; however, 7 days from collection from the donor are selected. Special
the strength of the reaction does not correlate well with the circumstances, such as the need for units of the mother’s
severity of the HDFN. A positive test result may be found in blood when high-incidence antibodies are involved, have
infants without clinical or other laboratory evidence of he- shown that older blood units can be safe and effective for the
molysis (e.g., mother received RhIG). newborn when infused slowly.
Elution RhIG
The routine preparation of an eluate of all infants with a pos-
itive DAT result is unnecessary. Elution in cases of known Active immunization induced by RBC antigen can be pre-
HDFN and postnatal ABO incompatibility is not needed, be- vented by the concurrent administration of the correspon-
cause eluate results do not change therapy. The preparation ding RBC antibody. This principle has been used to prevent
of an eluate may be helpful when the cause of HDFN is in immunization to D antigen by the use of high-titered RhIG.
question. As noted earlier, the resolution of a case of blocked During pregnancy and delivery, fetal and maternal blood
Rh requires an eluate. are mixed. If the mother is Rh-negative and the fetus is Rh-
positive, the mother has up to a 16% chance of being stim-
Newborn Transfusions ulated to form anti-D.2 As little as 1 mL of fetal RBCs can
elicit a response. Before delivery, the risk of sensitization is
The newborn may receive small aliquot transfusions, which 1.5% to 1.9% in susceptible women, indicating that a signif-
can be used to correct anemia when the bilirubin is icant amount of fetal RBCs can enter the maternal circulation
successfully treated with phototherapy or IVIG. The sup- during pregnancy.2 However, the greatest risk of immuniza-
pression of erythropoiesis by small aliquot or exchange tion to Rh is at delivery.
transfusions may cause anemia to occur after the immediate
neonatal period. Mechanism of Action
Although full-term newborn infants normally have rather
high hemoglobin levels (14 to 20 g/dL), a level below 10 g/dL The administered RhIG attaches to the fetal Rh-positive
may require transfusion, especially if the neonate has hy- RBCs in the maternal circulation. The antibody-coated RBCs
poxia needing oxygen supplementation. A hemoglobin level are removed by the macrophages in the maternal spleen. The
lower than 7 g/dL is considered severe anemia. A cord blood amount of antibody necessary to prevent alloimmunization
sample closely correlates with the levels during gestation. has been determined experimentally and is known to be less
than that required to saturate all D-antigen sites. The mech-
Selection of Blood for Intrauterine anism of action of RhIG is uncertain. Evidence indicates it
and Neonatal Transfusion interferes with B-cell priming to make anti-D, although other
modes of action may occur.14
Most centers treating HDFN use group O RBCs for intrauter-
ine and neonatal transfusions. The RBCs must be antigen- Indications
negative for the mother’s respective antibodies. Donors are
usually cytomegalovirus (CMV)-negative as well. Physicians This section describes the clinical indications for administer-
in these centers are transfusing neonates for other indica- ing RhIG to the mother during pregnancy and after delivery.
tions, such as RBC replacement for blood samples taken
for laboratory tests. This allows a small inventory of group O Antenatal
CMV-negative donor units to be set aside for intrauterine Because of the known risk of Rh immunization during preg-
and neonatal transfusions. Rh-negative units are selected for nancy, RhIG should be given early in the third trimester, or
2682_Ch19_427-438 22/05/12 11:58 AM Page 434
The RhIG must be injected according to the product Table 19–3 Comparison of ABO Versus Rh
label. The IV product can also be given intramuscularly. HDFN
The intramuscular form must be given intramuscularly
only. IV injections of intramuscular preparations can cause CHARACTERISTIC ABO Rh
severe anaphylactic reactions because of the anticomple- First pregnancy Yes Rare
mentary activity of these products. RhIG also contains IgA
and may be contraindicated in patients with anti-IgA and Disease predicted by titers No Yes
IgA deficiency who have had anaphylactic reactions to Antibody IgG Yes (anti-A,B) Yes (anti-D, etc.)
blood products.
Bilirubin at birth Normal range Elevated
Other Considerations Anemia at birth No Yes
RhIG is of no benefit once a person has been actively Phototherapy Yes Yes
immunized and has formed anti-D. However, care must
Exchange transfusion Rare Uncommon
be taken to distinguish women who have been passively
immunized by antenatal administration of RhIG from Intrauterine transfusion None Sometimes
those who have been actively immunized by exposure to
Rh-positive RBCs.
Care must also be taken that fetal Rh-positive RBCs in the mothers with potent anti-A,B. Most occur in group A in-
maternal circulation are not interpreted as maternal, because fants in white populations. In the black population, how-
then the mother would be erroneously assumed to be weak ever, group B infants are more often affected, and the
D-positive. The difference is distinguished by a quantitative overall incidence of ABO HDFN is several times greater
test such as the Kleihauer-Betke or by flow cytometry using than in other groups.
antibody to hemoglobin F. The mother’s history of prior transfusions or pregnancies
RhIG is not indicated for the mother if the infant is seems unrelated to the occurrence and severity of the dis-
found to be D-negative. The blood type of fetuses in abor- ease. Thus, ABO HDFN can occur in the first pregnancy and
tions, stillbirths, and ectopic pregnancies usually cannot in any, but not necessarily all, subsequent pregnancies. How-
be determined; therefore, RhIG should be administered in ever, tetanus toxoid administration and helminth parasite in-
these circumstances. RhIG must not be given to the new- fection during pregnancy have been linked to the production
born infant. of high-titered IgG ABO antibodies and severe HDFN.
There is no risk of transmission of the viral diseases hep- Even high-titered IgG antibodies that are transported
atitis A and B and HIV with the administration of RhIG. across the placenta seem incapable of causing significant
RBC destruction in an ABO-incompatible fetus. These in-
ABO HDFN fants are delivered with mild anemia or normal hemoglo-
ABO incompatibility between the mother and newborn in- bin levels. The mild course of ABO HDFN is related more
fant can cause HDFN. Maternal ABO antibodies that are IgG to the poor development of ABO antigens on fetal RBCs
can cross the placenta and attach to the ABO-incompatible than the characteristics of the maternal antibody. ABO
antigens of the fetal RBCs. However, destruction of fetal antigens are not fully developed until after the first year of
RBCs leading to severe anemia is extremely rare. More com- life. Group A infant RBCs are serologically more similar to
monly, the disease is manifested by the onset of hyperbiliru- A2 adult cells, with group A2 infant RBCs much weaker.
binemia and jaundice within 12 to 48 hours of birth. The The weakened A antigen on fetal and neonatal RBCs is
increasing levels of bilirubin can be treated with photother- more readily demonstrable with human than with mono-
apy. Severe cases requiring exchange transfusion are ex- clonal anti-A reagents. As expected, group A2 infants are
tremely rare. A comparison of ABO versus Rh HDFN is less likely to have ABO HDFN.
shown in Table 19–3. The laboratory findings in ABO HDFN differ from those
As the incidence of HDFN caused by Rh(D) has declined, shown in Table 19–3 for Rh disease. Microspherocytes and
ABO incompatibility has become the most common cause of increased RBC fragility in the infant are characteristic of ABO
HDFN. Statistically, mother and infant are ABO-incompatible HDFN, but not of Rh HDFN. The severity of the disease is
in one in every five pregnancies. independent of the presence of a positive DAT result or
demonstrable anti-A, anti-B, or anti-A,B in the eluate of the
Factors Affecting Incidence and Severity infant’s RBCs.
The bilirubin peak is later, at 1 to 3 days. Phototherapy is
ABO antibodies are present in the plasma of all individuals usually sufficient for slowly rising bilirubin levels. With rap-
whose RBCs lack the corresponding antigen. These anti- idly increasing bilirubin levels, IVIG or exchange transfusion
bodies, the result of environmental stimulus, occur more with group O RBCs may be required. The serious conse-
frequently as high-titered IgG antibodies in group O indi- quences of Rh and other blood groups causing HDFN, such
viduals than in group A or B individuals. Hence, ABO as stillbirth, hydrops fetalis, and kernicterus, are extremely
HDFN is nearly always limited to A or B infants of group O rare in ABO HDFN.
2682_Ch19_427-438 22/05/12 11:58 AM Page 436
Prenatal Screening
Case 19-2
Many investigators have tried to use the immunoglobulin
class and titer of maternal ABO antibodies to predict ABO Mother: gravida 4 para 2
HDFN. These tests are laborious and at best demonstrate the History:
presence of IgG maternal antibody, but they do not correlate Firstborn child unaffected
well with the extent of fetal RBC destruction. Consequently, Second-born child mildly affected, positive DAT at birth
detection of ABO HDFN is best done after birth. and required no treatment
Third child stillborn
Postnatal Diagnosis Mother had anti-D during second and third pregnancies
Current pregnancy:
No single serologic test is diagnostic for ABO HDFN. When
ABO/Rh(D) typing: O-positive
a newborn develops jaundice within 12 to 48 hours after
Antibody screen: Positive
birth, various causes of jaundice need to be investigated, and
Antibody identification: Anti-D
ABO HDFN is only one. The DAT on the cord or neonatal
Antibody titer:
RBCs is the most important diagnostic test. In all cases of
Saline: negative
ABO HDFN requiring transfusion therapy, the DAT result
AHG: 128 (1:128)
has been positive.16 On the other hand, the DAT result can
Gestational age: 8 weeks
be positive even in the absence of signs and symptoms of
clinical anemia in the newborn infant. However, these in- Same father as the three previous pregnancies (he is
fants may have compensated anemia, or the RBCs are not homozygous for the D antigen)
destroyed by the reticuloendothelial system.
1. What is the most likely Rh(D) type of the fetus?
Collecting cord blood samples on all delivered infants
2. When should the titer be repeated?
is highly recommended. The sample should be collected
3. Is there any intervention that should be done
by venipuncture to avoid contamination with maternal
now? Why?
blood and Wharton’s jelly (the material surrounding the
4. When should MCA-PSV be performed?
blood vessels) and should be anticoagulated for storage. If
the neonatal infant develops jaundice, ABO, Rh, and DAT Case 19-3
results can be assessed. The DAT result is neither strongly
nor consistently positive, although 90% of the cases com- Mother: gravida 3 para 2
plicated by jaundice are positive.16 When the DAT result History:
is negative but the infant is jaundiced, other causes of Firstborn child unaffected, mother’s antibody screen
jaundice should be investigated. In the rare cases in which negative
ABO incompatibility can be the only cause of neonatal Second-born child mildly affected, ABO/Rh(D) typing
jaundice, but the DAT result is negative, the eluate of the A-positive, positive DAT, jaundice
cord RBCs always reveals ABO antibodies. The eluate can Mother O-positive, antibody screen negative
also be helpful when the mother’s blood specimen is not Current pregnancy:
available. ABO/Rh(D) typing: O-positive
Antibody screen: Positive
Antibody identification: Anti-Lea
CASE STUDIES Gestational age: 32 weeks
the infant’s hemoglobin is reported as 4.3 g/dL and biliru- The results indicate the cord blood specimen is all
bin as 3.9 mg/dL. Typing results of this specimen are as adult blood and the heel-stick specimen is nearly all adult
follows: blood.
Anti-A Anti-B Anti-D DAT 2. How could that happen?
0 0 0 +/–
Further testing is done on the heel-stick specimen:
1. What is the infant’s blood type? Why is the infant so
Anti-A Anti-B RBC Eluate
anemic? What further testing is indicated?
4°C0 + Anti-D
Further testing shows the following:
These results indicate that the infant is B-positive and
Cord Blood Heel-stick has HDFN caused by anti-D.
Anti-I 4+ 3+
Kleihauer-Betke 0/1,000 23/1,000
SUMMARY CHART
HDFN is the destruction of the RBCs of the fetus and Although anti-D is the most antigenic of the Rh anti-
neonate by IgG antibodies produced by the mother. bodies, anti-Kell is considered the most clinically sig-
Only antibodies of the IgG class are actively trans- nificant of the non-Rh-system antibodies in the ability
ported across the placenta. to cause HDFN.
In Rh HDFN, the Rh-positive firstborn infant of Prenatal serologic tests for obstetric patients include
an Rh-negative mother is unaffected because the an ABO, Rh, and antibody screen during the first
mother has not yet been immunized; in subsequent trimester of pregnancy.
pregnancies, fetal cells carrying the Rh antigen A cord blood workup includes tests for ABO and Rh
immunize the Rh-negative mother and stimulate pro- as well as DAT; the most important serologic test for
duction of anti-D. diagnosis of HDFN is the DAT with anti-IgG reagent.
In ABO HDFN, the firstborn infant may be affected RhIG administered to the mother within 72 hours fol-
as well as subsequent pregnancies in which the lowing delivery is used to prevent active immunization
mother is group O and the newborn is group A, B, by the Rh(D) antigen on fetal cells; RhIG attaches to
or AB; the IgG antibody, anti-A,B in the mother’s cir- fetal Rh-positive RBCs in maternal circulation, blocking
culation, crosses the placenta and attaches to the immunization and subsequent production of anti-D.
ABO-incompatible antigens of the fetal RBCs. A Kleihauer-Betke test or flow cytometry is used to
Erythroblastosis fetalis describes the presence of imma- quantitate the number of fetal Rh-positive cells in
ture RBCs or erythroblasts in the fetal circulation be- the mother’s circulation as a result of a fetomaternal
cause the splenic removal of the IgG-coated RBCs causes hemorrhage.
anemia; the term commonly used now is HDFN.
Chapter 20
Autoimmune Hemolytic Anemias
Denise M. Harmening, PhD, MT(ASCP), CLS(NCA); Karen Rodberg,
MBA, MT(ASCP)SBB; and Ralph E. B. Green, B. App. Sci, FAIMS, MACE
OBJECTIVES
1. Define autoantibody and compare the types of immune hemolytic anemias with respect to thermal amplitude, method of red
blood cell (RBC) destruction, and the type of immunoglobulin that characteristically coats the RBCs.
2. Characterize autoantibodies that react at temperatures below 37°C and identify the common specificities of benign cold
autoagglutinins.
3. Describe problems encountered in the laboratory testing of specimens containing cold autoagglutinins.
4. Explain the techniques used to investigate serologic findings and detect underlying clinically significant alloantibodies in the
presence of cold autoantibodies.
5. Describe pathological cold autoagglutinins, including laboratory testing and treatment.
6. Differentiate between idiopathic warm autoimmune hemolytic anemia (WAIHA) and drug-induced immune hemolytic
anemia.
7. Describe the clinical and laboratory findings in WAIHA, including indicators of RBC hemolysis, difficulties in serologic testing,
and selection of blood for transfusion.
8. Explain procedures used to investigate serologic findings and detect underlying clinically significant alloantibodies in the
presence of warm autoantibodies.
9. Compare the mechanisms for drug-induced hemolysis and give examples of medications causing each type.
10. Describe the Donath-Landsteiner test.
439
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Definition Characterization
Antibodies that are directed against the individual’s own In individuals who have not been recently transfused, the
RBCs are termed autoantibodies or autoagglutinins. Most presence of a positive DAT, a positive autocontrol, or serum
autoantibodies react with high-incidence RBC antigens. autoantibody does not necessarily confer the diagnosis of
They typically agglutinate, sensitize, or lyse RBCs of most autoimmune hemolytic anemia (AIHA). In themselves, these
random donors as well as their own. RBC survival may be findings merely indicate the presence of autoantibody. It has
shortened by this circulating humoral antibody. Some indi- been reported that approximately 0.1% of normal blood
viduals will produce an autoantibody that readily attaches donors and up to 15% of hospitalized patients have positive
to their own RBCs but does not cause RBC destruction. DATs with no evidence of hemolytic anemia.7 Before the
Approximately 1 in 1,000 healthy blood donors will have a presence of an AIHA is established, it is important to
positive direct antiglobulin test (DAT) but will have a nor- verify the presence of immune-mediated RBC destruction.
mal hematocrit and be asymptomatic. In these individuals, Evidence of increased RBC destruction is not always accom-
the presence of autoantibody alone does not necessarily panied by decreased hemoglobin and hematocrit levels.
cause decreased RBC survival.1 These may become evident only when the individual is
2682_Ch20_439-474 22/05/12 12:00 PM Page 441
no longer able to compensate for increased RBC destruction. in the detection of IgG-coated RBCs. Therefore, with the
In many instances, even before decreases in hemoglobin and increase in the use of methods other than conventional
hematocrit are obvious, there will be evidence of compensa- test tube technique, it is possible that a greater number of
tion for increased RBC destruction, including increases in individuals may be found to be DAT-positive, with or with-
reticulocyte count, unconjugated (indirect) bilirubin levels, out detectable evidence of RBC destruction. As mentioned
and lactate dehydrogenase (LDH) levels, and a decrease in above, the incidence of positive DATs in normal blood donor
haptoglobin. populations has been reported to be as high as 1 in 1,000 in
The individual who has immune RBC destruction may the U.S. population,1 whereas the incidence in hospitalized
experience either compensated or uncompensated anemia. patients ranges from 0.3 to 1%, using anti-IgG antiglobulin
In compensated anemia, the rate of RBC production will reagent10,11 and as high as 15% using polyspecific antiglobu-
nearly equal the rate of RBC destruction. These individuals lin reagent.12 Many of the latter group have only complement
will demonstrate an increased reticulocyte count but may bound to the RBCs. The differences between individuals who
have only a mild decrease in hemoglobin and hematocrit are affected (i.e., those who have AIHA) and those who are
levels, depending on the rate of RBC production. Those unaffected by autoantibodies are not clearly understood.
individuals with uncompensated anemia demonstrate a Among the possibly significant factors are:
rate of RBC destruction that exceeds the rate of RBC pro-
• Thermal amplitude of antibody reactivity13
duction. Hemolytic anemia is often demonstrated in a
• IgG subclass of the antibody14
blood smear by macrocytosis (evidence of a young cell
• Amount of antibody bound to the RBCs14
population) or spherocytosis (evidence of cell membrane
• Ability of the antibody to fix complement in vivo15
damage). The reticulocyte count of these individuals is
• Activity of the individual’s macrophages7
generally greater than 3%,8 and the unconjugated bilirubin
• Quantitative or qualitative change in band 3 and proteins
levels and LDH levels are also increased due to accelerated
4.1 and 4.2 in the RBC membrane structure16
RBC destruction; however, haptoglobin levels are markedly
decreased due to its role of clearing hemoglobin from the The opposite situation also occurs; in some patients with
plasma. With intravascular RBC destruction, hemoglobine- hemolytic anemia, autoantibodies cannot be demonstrated
mia and hemoglobinuria may occur. by routine techniques. Some patients have more IgG than
Because there are other causes of hemolysis (e.g., hered- normal on their RBCs but less than the amount detectable
itary spherocytosis, hemoglobinopathies, and RBC enzyme by the routine antiglobulin test.17 In other cases, the
defects), AIHA must be confirmed by additional serologic patient’s RBCs are sensitized with anti-IgA or anti-IgM, not
testing. Diagnostic tests in a symptomatic patient include: routinely demonstrable using commercial antiglobulin
reagents.18–20 Because polyspecific antihuman globulin
1. Direct antiglobulin test (DAT) using polyspecific and
(AHG) reagents are required by the FDA to contain anti-IgG
monospecific antiglobulin reagents (refer to Chapter 5,
and anti-C3d,21,22 antibodies to other immunoglobulins
“The Antiglobulin Test”)
(e.g., IgA and IgM) are not consistently present in the
2. Characterization of the autoantibody in the serum or elu-
reagent, and cells sensitized with IgA or IgM may not give
ate using standard antibody detection and identification
a positive direct antiglobulin test.
procedures (refer to Chapter 9)
Autoantibodies can be characterized by their optimal tem-
Based on these results and the clinical evaluation of the perature of reactivity. About 70% of the reported cases of
patient, AIHA may be diagnosed and classified as cold reac- AIHA are those that react best at warm temperatures (30°C
tive, warm reactive, or drug-induced. The expected labora- to 37°C), whereas cold reactive (4°C to 30°C) autoagglu-
tory findings for each type are discussed in the following tinins account for about 18%. Drug-induced autoagglutinins
sections. In their book Immune Hemolytic Anemias, Petz and are present in about 12% of the reported cases of AIHA.
Garratty devote several chapters to the differential diagnosis Characterization of autoantibodies is important because
of the hemolytic anemias and another chapter to drug- treatment of the patient and resolution of the serologic prob-
induced immune hemolytic anemia. The reader is referred lems will differ according to the optimal temperature of
to their text for a complete discussion.7 reactivity and the immunoglobulin responsible; treatment
As previously stated, some individuals may have autoan- of the patient will depend on the nature of the autoantibody.
tibodies in their sera and on their RBCs but display no evi- The remainder of this chapter details the clinical and labo-
dence of decreased RBC survival. All normal RBCs have a ratory aspects of cold reactive, warm reactive, and drug-
small amount of IgG and complement on their surfaces. induced autoantibodies.
Studies have shown that there may be 5 to 90 molecules of
IgG/RBC and 5 to 500 molecules of complement/RBC in the Cold Reactive Autoantibodies
average healthy individual.7 Because conventional tube test-
ing will normally detect 100 to 500 molecules of IgG/RBC Cold reactive autoantibodies are frequently encountered
and 400 to 1,100 molecules of complement/RBC, these in serologic testing. Most of the time, the antibodies
individuals are not routinely identified as DAT-positive.9 detected are not clinically significant but nevertheless can
It should be noted that column agglutination (gel) or present challenges for the blood banker. Occasionally, cold
solid phase methodologies may have an increased sensitivity autoantibodies are clinically significant and cause immune
2682_Ch20_439-474 22/05/12 12:00 PM Page 442
hemolytic anemia. The antibody specificity may be of of normal people do not typically interfere with testing, they
interest to the technologist or may be a clue to the disease are one of the more common causes of serologic problems;
process, but the important distinction between clinically therefore, one should be able to recognize these circum-
significant and benign cold autoantibodies is their thermal stances and employ methods to resolve problems associated
range. Any antibody that reacts at or near body tempera- with these antibodies. If a cold agglutinin is strong enough
ture (30°C to 37°C), regardless of specificity, is potentially to interfere with ABO typing, it may also interfere with indi-
clinically significant; this includes “cold” autoantibodies. rect antiglobulin testing even if the antibody detection
method does not intentionally include an RT incubation
Benign Cold Autoantibodies step. In these cases, troubleshooting a positive antibody
screening should include RT testing to prove the existence
When testing is performed at 4°C, the most commonly of a cold reacting antibody.
encountered autoantibody is a benign cold agglutinin that
may be found in the serum of most normal, healthy individ- ABO Typing
uals. Normally these antibodies present no serologic problem If an individual’s RBCs are heavily coated with cold agglutinins,
because routine antibody detection tests are not performed they may directly agglutinate, causing false-positive reactions
at this temperature. The typical cold agglutinin has a rela- with routine ABO reagents. In most cases, valid results can be
tively low titer (less than 64 at 4°C). Occasionally, these obtained by using patient’s cells that have been washed once
benign cold autoantibodies may agglutinate cells at room or twice with normal saline warmed to 37°C. The cold autoan-
temperature (20°C to 24°C); however, even in this situation, tibody is eluted from the cells during warm washing. For ex-
strongest reactivity is found at 4°C. Table 20–1 compares the ample, group O cells coated with cold autoantibody might
characteristics of benign (normal) cold autoantibodies with show the following reactions before and after warm washing:
those of pathological cold autoantibodies, which are dis-
cussed later in this chapter. Most cold agglutinins react ANTI-A ANTI-B
strongly with enzyme-treated cells; therefore, cold agglu-
tinins are quite likely to be detected when ficin-treated cells Serum-suspended RBCs 1+ 1+
are tested. Cold autoantibodies are IgM immunoglobulins Warm-washed, saline-suspended RBCs 0 0
and therefore can activate complement in vitro. In serum
testing, reactivity may be seen in the antiglobulin phase
if polyspecific antihuman serum (i.e., containing anticom- If more potent autoagglutinins are present, it may be nec-
plement) is used in antiglobulin testing. Table 20–2 contrasts essary to incubate the patient’s whole blood sample at 37°C
titers and thermal amplitudes typical of benign versus patho- prior to warm washing.23 In some cases, it may be inconven-
logic cold autoagglutinins. ient to adhere closely to a protocol of maintaining the sample
at 37°C from the point of collection; similar success has been
Laboratory Tests Affected by Cold Autoagglutinins found with warming the sample in a waterbath or heat block
Cold agglutinins sometimes interfere with routine serum and to 37°C for 15 to 30 minutes and then warm washing the
cell testing performed at room temperature (RT). The extent RBCs. The warm incubation elutes the cold agglutinin from
to which they cause problems depends on whether antibody the patient’s RBCs, and the warm washes remove it to pre-
detection tests are performed at room temperature and vent it from reattaching in vitro. In the rare situation in
how strongly the antibody reacts at this temperature which washing with warm saline is not effective, thiol
(i.e., the concentration and thermal amplitude of the anti- reagents (e.g., dithiothreitol) can be used to disperse the
body). Although the cold autoantibodies found in the serum autoagglutination.24
Table 20–1 Characteristics of Normal (Benign) Cold and Pathological Cold Autoantibodies
CHARACTERISTIC NORMAL (BENIGN) PATHOLOGICAL
Titer at 4C ≤ 64 ≥ 1,000
Table 20–2 Benign Cold Agglutinin Compared With Pathological Cold Agglutinin: Titers
and Thermal Amplitudes*
Benign Cold Agglutinin
Tube # 1 2 3 4 5 6 7 8 9 10 11 12 Titer
37°C 0 0 0 0 0 0 0 0 0 0 0 0 0
30°C 0 0 0 0 0 0 0 0 0 0 0 0 0
20°C 3+ 2+ 1+ 0 0 0 0 0 0 0 0 0 4
4°C 4+ 4+ 3+ 2+ 1+ 0 0 0 0 0 0 0 16
Tube # 1 2 3 4 5 6 7 8 9 10 11 12 Titer
Temp ↓
37°C 1+ ± 0 0 0 0 0 0 0 0 0 0 1
30°C 2+ 1+ 0 0 0 0 0 0 0 0 0 0 2
20°C 4+ 4+ 3+ 3+ 2+ 1+ 0 0 0 0 0 0 32
See Procedure 20-1 on the textbook’s companion and the tests with the A1, B, O and autologous cells are
website. repeated with autoadsorbed serum.
Because serum ABO grouping is performed at room tem- See Procedure 20-2 on the textbook’s companion
perature, cold autoagglutinins frequently cause discrepancies website.
in the serum ABO (reverse) typing also. In the following
example, the cell typing results suggest the cells are group Although it may be possible to resolve this discrepancy
AB. In a group AB individual, one does not expect the with prewarmed testing, one must remember that not all
serum to agglutinate either the A1 or B cells, as seen in ABO isoagglutinins are reactive at 37°C, and erroneous test
this example. Although a number of explanations for this results may be obtained; therefore, autoadsorption proce-
discrepancy exist, a cold agglutinin is the most likely dures are preferred.
cause. Group O and autologous cells should also be tested
in the investigation of a serologic ABO discrepancy. If a AUTOLOGOUS
cold autoagglutinin is present, the autologous and group A1 RBCs B RBCS O RBCs RBCs
O cells (the negative controls) will most likely be positive
as well: Autoadsorbed 0 0 0 0
Serum
ANTI-A ANTI-B
RBCs 4+ 4+ Rh(D) Typing
AUTOLOGOUS As in ABO cell grouping, false-positive reactions can be seen
A1 RBCs B RBCs O RBCs RBCs with Rh reagents when RBCs coated with cold autoagglutinins
Serum (or 1+ 1+ 2+ 1+
are tested. This was previously a common problem encoun-
Plasma) tered in typing with polyclonal high-protein anti-D reagent
where the anti-D and the Rh control were both positive, ren-
dering the test invalid. Today, the Rh reagents in common use
Such a discrepancy is easily resolved if the cold reactive are monoclonal or a blend of monoclonal and low-protein
autoantibody is removed by an autoadsorption technique, anti-D reagents, which normally yield valid results. When a
2682_Ch20_439-474 22/05/12 12:00 PM Page 444
monoclonal reagent is used, many manufacturers consider a antibody screening is no longer performed at this tempera-
negative reaction with any of the ABO reagents as a control ture. Antibodies reactive only at room temperature are usually
for the D typing; however, an Rh control reagent is available considered to be of no clinical significance. Benign cold
from some manufacturers. As stated above, if a discrepancy autoagglutinins do not react at 37°C, but they may interfere
exists in the ABO typing, washing the cells with warm saline with testing at the antiglobulin phase if polyspecific antihu-
usually gives acceptable results. This also holds true when man reagent is used, inasmuch as they may bind to cells at
testing with low-protein anti-D. Thiol reagents may be re- lower temperatures when the serum and cells are mixed
quired when washing with warm saline is ineffective. together initially or during centrifugation following the 37°C
Cold reactive IgM autoagglutinins can activate the comple- incubation, and complement may be activated. Although the
ment cascade in vitro, causing complement components to antibody elutes from the cell surface during the incubation
be bound to the RBC surface, which can lead to false-positive or washing phases, the complement remains bound. Polyspe-
reactions in the weak D (antiglobulin) test if cells from a clot- cific antihuman serum will agglutinate the cells coated with
ted sample and polyspecific antihuman serum are used. In this complement. When enzyme-treated cells are used, reactions
instance, the Rh control test will also be positive. The use of in all phases may be stronger. Often, omitting room temper-
monospecific anti-IgG for weak D testing or RBCs collected ature incubation and the use of anti-IgG for the indirect
into ethylenediaminetetraacetic acid (EDTA) can eliminate the antiglobulin test (IAT) are all that is needed to avoid detecting
problem of complement-binding cold agglutinins in D typing. cold agglutinins at the IAT.
The following examples illustrate these results: Because most clinically significant antibodies capable of
causing accelerated RBC destruction are detected by the
antiglobulin test, reactions in this phase must be investi-
ANTI-D Rh CONTROL
gated. Reactivity caused by cold autoagglutinins can mask
Immediate spin phase 0 0 the presence of clinically significant alloantibodies. Although
Indirect antiglobulin phase 1+ 1+ the use of anti-IgG antiglobulin reagents will eliminate most
(poly AHG) problems with cold autoagglutinin reactivity in the IAT
phase, it may be necessary to remove the cold reacting au-
Indirect antiglobulin phase 0 0 toantibody by cold adsorption procedures to thoroughly in-
(anti-IgG)
vestigate the reactivity observed in antiglobulin testing.
RBCs collected in EDTA 0 0 Rabbit erythrocyte stroma is known to be rich in I antigen
and is commercially available and easy to use. There have
been some reports that clinically significant antibodies have
Similar problems can be encountered with other antisera been adsorbed using rabbit erythrocyte stroma, so caution is
used in RBC phenotyping (e.g., anti-K, anti-S, anti-Fya) that advised.25
require the use of an indirect antiglobulin test. The use of Other techniques useful in differentiating between cold
anti-IgG antiglobulin reagent or a sample collected in EDTA autoantibodies and alloantibodies are prewarm technique or
is recommended when cold autoagglutinins are present. For- testing with cold autoadsorbed serum.23 Cold autoadsorp-
tunately, most samples collected for the blood bank today tion technique is preferred because the prewarm test has
are collected in EDTA. become quite controversial. There have been reports of
clinically significant alloantibodies being unintentionally
Direct Antiglobulin Test “prewarmed away,” either due to poor technique or because
When a properly collected specimen is used (EDTA- the antibodies were partially IgM, yet this technique persists
anticoagulated RBCs), the DAT of a patient with benign cold in practice and does have value in investigating an identified
autoagglutinins is negative; however, one frequently obtains antibody’s thermal amplitude.
a positive result using polyspecific antihuman serum if a The principle of the prewarm test is that by first warming
clotted specimen is used, because complement can be acti- the cell suspension and serum prior to mixing, avoiding
vated in vitro. This in vitro sensitization is usually weak— room temperature centrifugation after 37°C incubation, and
less than or equal to 1+. If monospecific reagents are used, washing with saline warmed to 37°C, any reaction between
these cells are reactive with anti-C3d but not with anti-IgG. the cold autoagglutinin and RBC antigens can be prevented,
A negative control of 6% albumin or saline, used in parallel thus avoiding complement activation. While cold reacting
with the antiglobulin reagents, is recommended for direct antibodies should not react by this method, alloantibodies
antiglobulin testing, particularly when there has been diffi- that are reactive at 37°C (i.e., potentially clinically signifi-
culty obtaining valid ABO or Rh typing. cant) would still bind to the cells and be detectable at the
antiglobulin phase.
Antibody Detection and Identification
See Procedure 20-3 on the textbook’s companion
The frequency with which cold autoagglutinins interfere with website.
detection and identification of RBC alloantibodies depends
to a large extent on the routine procedures used in patient An example of the results of testing a serum that contains
testing. As shown in Table 20–2, cold agglutinins react best a cold autoagglutinin and anti-Fya using routine antiglobulin
at 4°C but are generally not detected because routine testing with polyspecific antiglobulin and testing with the
2682_Ch20_439-474 22/05/12 12:00 PM Page 445
Table 20–3 Typical Reactivity Observed With Patient Serum Containing a Clinically Significant
Alloantibody (anti-Fya) and Cold Autoagglutinin
STANDARD ANTIGLOBULIN PREWARMED ANTIGLOBULIN
TESTING WITH TESTING WITH STANDARD ANTIGLOBULIN
RBCs TESTED POLYSPECIFIC AHG POLYSPECIFIC AHG TESTING WITH ANTI-IgG
Fy(a+b-) 2+ 2+ 2+
Fy(a+b+) 2+ 2+ 2+
Fy(a–b+) 1+W 0 0
prewarmed technique is shown in Table 20–3. Reactions are adsorption should not be performed if a patient has been
present in routine antiglobulin tests with both Fy(a+) and recently transfused, because donor RBCs will be present in
Fy(a–) cells. In a prewarmed test, only the reactions expected the patient’s circulation. Alloantibodies and autoantibodies
of the anti-Fya are evident. The weak reactions of the cold will be adsorbed if an “auto” adsorption is performed on a
autoagglutinin are eliminated by prewarm technique. The recently transfused patient. In this situation, it is best to
prewarm technique is simple and successful in most cases; use allogeneic adsorption or cold adsorption using rabbit
however, if the cold autoantibody is very potent, it may be erythrocyte stroma.
difficult to maintain the cells and serum at 37°C to avoid the
antigen-antibody interaction and complement activation. See Procedure 20-4 on the textbook’s companion
Although prewarm technique may appear to be very help- website.
ful in resolving problems caused by cold autoagglutinins, If anti-IgG antiglobulin reagent is used, the problems
this technique should not be used indiscriminately. Cases caused by most cold agglutinins can be avoided. The use of
have been reported in which clinically significant alloanti- anti-IgG reagents is an attractive alternative when prewarm-
bodies have been missed after prewarming.26 Prewarming ing is not effective and there is not enough time to adsorb
should be used only when the reactions obtained indicate the serum.
the likely presence of a cold autoagglutinin (i.e., positive
autocontrol and reactivity noted below 37°C). Because there Compatibility Testing
is no immediate spin reading or room temperature incuba- The difficulties encountered in antibody detection and
tion in the prewarmed procedure, IgM immunoglobulin identification tests are also found in compatibility tests,
components of a newly forming alloantibody may not be because the most commonly encountered cold autoantibody
detected in this testing. Therefore, this testing must not be (autoanti-I) is directed against an antigen that is found on
performed with patients transfused within the previous the RBCs of most random donors and on most reagent RBCs.
3 months or patients without an accurate transfusion history. Compatibility tests, like antibody identification tests, can
A cold autoagglutinin is not apt to be the answer if weak be performed using prewarmed or autoadsorbed serum or
reactions are present only in the antiglobulin phase with allogeneic adsorbed serum or using anti-IgG antiglobulin
anti-IgG (i.e., there is no evidence of reactivity at immediate reagent.
spin or room temperature). Two other common cold agglutinins, anti-IH and anti-H,
When strong cold autoantibodies are present or if one distinguish between group O reagent RBCs and group A, B,
wishes to identify a room temperature–reactive alloanti- or AB donor cells. Because these antibodies react with the
body, autologous adsorption must be performed to remove H antigen that is only weakly expressed on the RBCs of
the autoantibody. Cold autologous adsorption, described group A, B, or AB individuals, they are not always obvious
in Procedure 20-2 on the companion website, may be per- as autoantibodies when encountered. As discussed in the fol-
formed if the patient has not been transfused within lowing section on specificity, anti-IH and anti-H react best
3 months. In autoadsorption procedures, an aliquot of with group O cells; they react least with group A1 and A1B
patient cells is incubated with an equal volume of the cells, perhaps only at 4°C. Anti-IH and anti-H are found most
patient’s serum at 4°C. Autoantibody is adsorbed onto the often in the serum of group A1 and A1B persons; therefore,
cells, and alloantibody remains in the serum. It may be nec- the units selected for compatibility testing (group A or AB)
essary to repeat the adsorption several times if the autoan- are those that give the weakest, if any, reactivity. On the other
tibody is of high titer. In order to enhance the adsorption hand, group O cells (e.g., antibody screening cells) give the
process, the patient’s RBCs may be treated with enzymes strongest reactions.
before adsorption to increase the amount of autoantibody
removed by the adsorption; however, enzyme pretreatment Specificity of Cold Autoagglutinins
should not be performed without confirming that the serum The specificity of the cold autoantibody is often associated
antibody is reactive with enzyme-treated cells. Autologous with the patient’s diagnosis. It is important to be familiar
2682_Ch20_439-474 22/05/12 12:00 PM Page 446
Table 20–4 Typical Reactivity Observed at 4°C With Serum Containing Autoanti-I with Iadult
and icord Cells
SERUM SERUM DILUTION Iadult CELLS Icord CELLS
1:2 1+ 0
1:4 1+W 0
1:8 0 0
1:2 4+ 2+
1:4 4+ 1+
1:8 3+ 0
with the properties and characteristics of these types of with both adult and cord cells, but the preference for the
autoantibodies. adult cells is still obvious. Alloanti-I is frequently present in
the serum of i adults.27
Anti-I, Anti-i. Most cold reactive autoantibodies have anti-I Anti-i is a relatively uncommon autoantibody. As shown
specificity. The I antigen is fully expressed on the RBCs of in Table 20–5, this antibody reacts in a manner antithetical
virtually all adults, whereas it is only weakly expressed on to anti-I. Cord cells and i adult cells have the strongest
cord RBCs. At birth, an infant’s RBCs express the i antigen. expression of the i antigen; adult I cells have the least.
As an infant matures, the antigen expressed is converted
from the i antigen to the I antigen; the amount of I antigen Anti-H, Anti-IH. Cold agglutinins found in the sera of group A1
increases until the adult levels are reached at about 2 years and A1B individuals (and occasionally group B) may have
of age.23 Very rarely do adult RBCs lack the I antigen; if they anti-H specificity. This antibody distinguishes between cells
do, they are termed i adults and may produce alloanti-I, of various ABO groups. Group O and A2 cells react best be-
which is a potentially clinically significant alloantibody cause they have the largest amounts of H antigen. Group A1
because it is typically an IgG antibody that reacts at 37°C. and A1B cells have the least H antigen, so they react weakly.
The reactivities of several examples of anti-I are given in Because the A1 and A1B individual’s own cells demonstrate
Table 20–4. As shown, anti-I specificity may be apparent a very weak expression of the H antigen, anti-H and anti-IH
when a serum is tested with adult and cord RBCs. Benign are actually autoantibodies that may react only with autolo-
cold autoantibodies, for example, react with adult RBCs but gous RBCs at 4°C, but that can be easily managed by the
not with cord RBCs. Pathological cold autoantibodies react selection of type-specific RBCs. The pattern of reactivity seen
Group A1 Iadult 4+ 0 to 1+ 0 to 1+ 0 to 1+ 4+
Group A2 Iadult 4+ 0 to 1+ 2+ 2+ 4+
Bombay Oh Iadult 4+ 0 to 1+ 0 0 to 2+ 4+
Group O iadult 0 to 1+ 4+ 4+ 0 to 1+ 4+
Group O icord 0 to 1+ 4+ 4+ 0 to 1+ 4+
Group A1 iadult 0 to 1+ 4+ 0 to 1+ 0 4+
Group A2 iadult 0 to 1+ 4+ 2+ 0 to 2+ 4+
Bombay Oh iadult 0 to 1+ 4+ 0 0 4+
*Anti-Pr is a less commonly encountered cold autoagglutinin that frequently mimics autoanti-I.
2682_Ch20_439-474 22/05/12 12:00 PM Page 447
with anti-H is shown in Table 20–5. See Chapter 6, “The 30°C. The antibody is usually an IgM immunoglobulin
ABO Blood Group System,” for a discussion of the ABO that quite efficiently activates complement.
system and H substance.
It is very important not to confuse cold reactive anti-H Clinical Picture. CHD occurs predominantly in older individu-
with the anti-H found in the serum of Oh (Bombay) individ- als, with a peak incidence in those over 50 years of age.
uals who lack the H antigen. Cold reactive anti-H may be Antibody specificity in this disorder is almost always anti-I,
found in A1 or A1B individuals as an autoantibody even less commonly anti-i, and rarely anti-Pr. It is rarely severe
though the cells of the antibody maker (A1 or A1B) may give and is usually seasonal because the winter months often pre-
considerably weaker reactions. The anti-H in the Oh person cipitate the signs and symptoms of this chronic hemolytic
is a potent alloantibody, which reacts at 4°C to 37°C with all anemia. Acrocyanosis of the hands, feet, ears, and nose is fre-
cells except the rare Oh cell, and is capable of causing rapid quently the patient’s main complaint along with a sense of
intravascular RBC destruction. numbness in the extremities. Changes take place when the
Anti-IH, another of the usually harmless cold autoagglu- person is exposed to the cold, because the cold autoantibody
tinins, is also found more commonly in the serum of A1 and agglutinates the patient’s RBCs as they pass through the skin
A1B individuals. This antibody agglutinates only RBCs that capillaries, resulting in localized blood stasis. During cold
have both the I and the H antigens. As with anti-H, group O winter weather, the temperature of an individual’s blood falls
and group A2 cells react best. The difference between these to as low as 28°C in the extremities, activating his or her cold
two antibodies is that group O icord cells and group O iadult autoantibody. The antibody then agglutinates the RBCs and
cells react as strongly as group O Iadult cells with anti-H but fixes complement as the cells flow through the capillaries of
not with anti-IH (see Table 20–5). the skin, causing signs of acrocyanosis. These patients may
also experience hemoglobinuria, because the complement
fixation may result in intravascular hemolysis. Figure 20–1
Other Cold Reactive Autoagglutinins
illustrates the relationship between ambient temperatures
A number of other less commonly encountered cold autoag- and LDH concentration (a reflection of the severity of
glutinins have been described, such as anti-Pr, anti-Gd, and hemolysis) over a period of 18 months.31 However, this
anti-Sdx (anti-Rx).28 Cold autoantibodies with the specificity intravascular hemolytic episode is not associated with fever,
of anti-M have also been described.29 Most researchers agree chills, or acute renal insufficiency, any one of which is char-
that specificity of cold reactive autoantibodies is primarily acteristic of patients with paroxysmal cold hemoglobinuria
of academic interest and usually not clinically important; (PCH) or severe WAIHA.
however, development of autoantibodies with specificities Patients usually display weakness, pallor, and weight loss,
for integral components of the RBC membrane, such as the which are characteristic symptoms of a chronic anemia. CHD
glycophorins or band 3, may be precursors for developing usually remains quite stable; however, if it does progress in
other autoimmune disorders such as systemic lupus erythe- severity, it is insidious in intensity. Physical findings such as
matosus or rheumatoid arthritis.30 hepatosplenomegaly are infrequent because of the mecha-
nism of hemolysis. Other clinical features of CHD include
jaundice and Raynaud’s disease (symptoms of cold intoler-
Pathological Cold Autoagglutinins ance, such as pain and bluish tinge in the fingertips and toes
Differentiating the serologic characteristics of a benign cold as a result of vasospasm). Patients with severe CHD usually
autoagglutinin from a pathological cold agglutinin may aid live more comfortably in warmer climates.
the clinician in making a diagnosis. This is very important Laboratory Findings. Laboratory findings in CHD include retic-
in terms of the treatment that is required for pathological ulocytosis and a positive DAT due to complement only. It is
cold autoantibodies as opposed to no treatment required for suggested that a simple serum screening procedure be per-
benign cold autoantibodies. formed initially to test the ability of the patient’s serum
to agglutinate autologous saline-suspended RBCs at 18°C
Cold Hemagglutinin Disease (Idiopathic Cold AIHA) to 20°C. If this test result is positive, further steps may be
Most cold autoagglutinins do not cause RBC destruction, taken to determine the titer and thermal amplitude of the
but in some patients they can cause hemolytic anemia that patient’s cold autoantibody, which typically reacts as patho-
varies in severity from mild to life-threatening intravascu- logical cold autoagglutinins as described in Table 20–2. If
lar lysis. Cold reactive immune hemolytic anemia may be it is negative, the diagnosis of CHD is unlikely. The periph-
a chronic, idiopathic (no identifiable cause) condition or eral smear of a patient with CHD may show agglutinated
an acute, transient disorder often associated with an infec- RBCs, polychromasia, mild to moderate anisocytosis,
tious disease such as Mycoplasma pneumoniae pneumonia and poikilocytosis (Fig. 20–2). Autoagglutination of antico-
or infectious mononucleosis. Cold agglutinin syndrome, agulated whole blood samples is characteristic of CHD
also called cold hemagglutinin disease (CHD) or idio- and occurs quickly as the blood cools to room temperature,
pathic cold AIHA, represents approximately 18% of the causing the binding of cold autoantibodies to the patient’s
cases of AIHA. A moderate chronic hemolytic anemia is RBCs in vitro. As a result of this autoagglutination, it is
produced by a cold autoantibody that optimally reacts extremely difficult to perform automated blood counts
at 4°C but also reacts at temperatures between 25°C and and preparation of blood smears with these patients’ samples.
2682_Ch20_439-474 22/05/12 12:00 PM Page 448
500 25
450
20
250 5
200 0
150
⫺5
100
⫺10
50
0 ⫺15
ly
st
er
ry
ch
ril
ay
ne
ly
st
er
r
be
be
be
be
be
be
ar
Ju
Ju
gu
Ap
gu
ua
ob
ob
ar
Ju
nu
em
em
em
em
em
em
Au
Au
br
M
ct
ct
Ja
O
O
pt
ov
ec
Fe
pt
ov
ec
Se
Se
N
D
Month
Figure 20–1. Seasonal hemolysis in cold agglutinin disease. As the ambient temperature decreases, the amount of hemolysis (reflected in serum lactate dehydrogenase
levels) increases. During warmer months, LDH levels return to normal. (Adapted with permission from Lyckholm, LJ, and Edmond, MB: Seasonal hemolysis due to cold-
agglutinin syndrome. N Engl J Med 334:437, 1996.)
Leukocyte and platelet counts are usually normal. Box 20–1 The issue of transfusion in CHD patients is most rele-
lists the clinical criteria for diagnosis of CHD. vant in those undergoing surgical procedures that use
hypothermia (lowering of the body temperature to 22°C
Selection of Blood for Transfusion. Most patients with CHD do to 30°C), such as cardiac procedures. For patients with
not require transfusion; however, when they do, it is some- CHD, blood for transfusion can be warmed by an approved
times challenging to perform the pretransfusion testing. As blood warmer, or the operative procedures can be per-
previously described, potent cold autoantibodies interfere formed without subjecting the patient to hypothermia.32
with most routine tests. Perhaps the most difficult problem Patients with benign cold autoagglutinins do not require
is detecting and identifying alloantibodies. Procedures to these special arrangements.
manage these problems were described earlier in this chapter,
but cold autoadsorption of the serum should be the method Cold Autoantibodies Related to Infection (Secondary Cold
of choice, if possible. It is important to provide RBCs com- AIHA)
patible with any clinically significant alloantibodies. Most CHD can also occur as a transient disorder secondary to
patients tolerate transfusion of blood that is incompatible infection. Episodes of cold AIHA often occur after upper
with the autoantibody. Units of iadult RBCs are extremely respiratory infections. Approximately 50% of patients suf-
rare and should be reserved for the rare iadult patient with fering from pneumonia caused by M. pneumoniae have cold
alloanti-I.
BOX 20–1
agglutinin titer levels higher than 64. In the second or third children who have had viral illnesses such as measles,
week of the patient’s illness, CHD may occur in association mumps, chickenpox, infectious mononucleosis, and the
with the infection, and a rapid onset of hemolysis is ill-defined flu syndrome. Originally, PCH was described in
observed. Pallor and jaundice are characteristically present. association with syphilis, with an autoantibody formed in
Acrocyanosis and hemoglobinuria are uncommon and not response to Treponema pallidum infection; however, with the
consistently present. Usually, resolution of the episode effective treatment of syphilis with antibiotics, PCH is no
occurs within 2 to 3 weeks because the hemolysis is self- longer commonly reported in relation to syphilis.
limiting, resolving when the infection subsides. The offend- In PCH, RBC destruction is caused by a cold autoanti-
ing cold autoantibody is an IgM immunoglobulin with a body referred to as a biphasic autohemolysin, which binds
characteristic anti-I specificity. Very high titers of cold to the patient’s RBCs at lower temperatures and fixes
autoagglutinins are seen almost exclusively in patients with complement. Hemolysis occurs when the sensitized RBCs
M. pneumoniae. It has been theorized that the cold agglu- circulate and are exposed to 37°C and the sensitized cells
tinin produced in this infection is an immunologic response undergo complement-mediated intravascular lysis. In
to the mycoplasma antigens and that this antibody cross- contrast to the other cold reactive autoagglutinins, the
reacts with the I antigen on RBCs. antibody in PCH is an IgG immunoglobulin with biphasic
The antibodies produced in primary CHD and in this dis- activity. The classic antibody produced in PCH is called
order secondary to M. pneumoniae both have anti-I specificity, the Donath-Landsteiner antibody and is an autoantibody
and the RBCs are sensitized with complement components. with anti-P specificity. Other specificities have been
If the complement cascade does not proceed to C9 (cell death reported, including anti-i33 and anti-Pr-like.34 These anti-
by lysis), the macrophages of the reticuloendothelial system bodies are not identifiable using regular serologic tech-
can still clear the sensitized RBCs through their receptors for niques. The antibody specificity is only demonstrated
C3b fragments, thereby causing hemolysis. in the Donath-Landsteiner test, which is the test used to
Infectious mononucleosis also may be associated with confirm the diagnosis of PCH.
a hemolytic anemia resulting from a cold autoantibody. Al- The Donath-Landsteiner test involves the collection of a
though infrequent, it has been well documented that a high- fresh blood sample from the patient. The sample is main-
titered IgM anti-i with a wide thermal range plays a major tained at 37°C after collection and the serum is then sepa-
role in hemolytic anemia associated with this viral infection. rated. Three sets of three test tubes, labeled A1–A2–A3,
Acute illness with a sore throat and a high fever, followed B1–B2–B3, and C1–C2–C3, containing the patient’s serum
by weakness, anemia, and jaundice, are characteristic fea- are incubated at various temperatures with group O RBCs
tures of infectious mononucleosis. Lymphadenopathy and that express the P antigen (i.e., RBCs of common pheno-
hepatosplenomegaly are common findings. A larger percent- type). In this test, tubes 1 and 2 of each set contain 10 drops
age of patients with infectious mononucleosis has been of patient’s serum, and tubes 2 and 3 of each set contain
reported to develop anti-i, but only a small number of these 10 drops of fresh normal serum as a complement source.
patients develops the antibody of sufficient titer and thermal One volume of 50% suspension of washed P+ RBCs is added
amplitude to induce in vivo hemolysis. to each tube, and all tubes are mixed. After mixing, the three
A tubes are then placed in a melting ice bath for 30 minutes
Treatment for CHD. Therapy for CHD is generally unneces-
and subsequently for 1 hour at 37°C (biphasic incubation).
sary. Most patients require no treatment and are instructed
The three B tubes are placed in a melted ice bath for
to avoid the cold, keep warm, or move to a milder climate.
90 minutes. The three C tubes are kept at 37°C for 90 minutes.
Patients with moderate anemia are given the same instruc-
After the appropriate time has passed, the tubes are cen-
tions and are urged to tolerate the symptoms rather than
trifuged, and supernatant fluids are examined for hemolysis.
to use drugs on a therapeutic trial basis. There is some ad-
Table 20–6 summarizes the reactions of a positive Donath-
vantage to the use of plasma exchange in more severe
Landsteiner test.
cases, inasmuch as IgM antibodies have a predominantly
As the name PCH implies, paroxysmal or intermittent
intravascular distribution; however, response to plasma
episodes of hemoglobinuria occur upon exposure to cold.
exchange is still variable in this patient population, and
These acute attacks are characterized by sudden onset of
repeated plasma exchanges are often required on a fre-
fever, shaking chills, malaise, abdominal cramps, and back
quent basis to maintain low plasma levels of autoaggluti-
pain. All the signs of intravascular hemolysis are evident,
nating antibodies.
along with hemoglobinemia, hemoglobinuria, and biliru-
Corticosteroids also have been used but generally have a
binemia, depending on the severity and frequency of the
poor response. In some patients whose RBCs are strongly
attack (Fig. 20–3). This results in a severe and rapidly pro-
coated with C3, successful results have been reported with
gressive anemia with hemoglobin levels frequently as low as
corticosteroids. Some favorable responses also have been re-
4 or 5 g/dL. Polychromasia, nucleated RBCs, and poikilocy-
ported with the alkylating drug chlorambucil. Splenectomy
tosis are demonstrated in the peripheral smear, findings that
is generally considered ineffective.
are consistent with hemolytic anemia. These signs and symp-
Paroxysmal Cold Hemoglobinuria. Paroxysmal cold hemoglobin- toms, as well as hemoglobinuria, may resolve in a few hours
uria (PCH) is the least common type of AIHA, with an in- or persist for days. Splenomegaly, hyperbilirubinemia, and
cidence between 1% and 2%. It is most often seen in renal insufficiency may also develop.
2682_Ch20_439-474 22/05/12 12:00 PM Page 450
+ = with hemolysis
0 = without hemolysis
* Tubes with normal serum are used as control
Note: The patient’s blood should be clotted at 37°C and the serum separated at this temperature to avoid the loss of the autoantibody by cold autoadsorption prior to testing. Fresh normal serum
should be included in the reaction medium as a source of complement, as PCH patients may have low levels of serum complement. Omit the patient serum-only tubes (A1, B1, C1) if a limited
amount of blood is available (e.g., a young child).
Hemosiderinuria
plained. WAIHA may be idiopathic with no underlying dis-
ease process or may be secondary to a pathological disorder.
Box 20–2 lists the disorders most commonly associated with
WAIHA.
Signs and symptoms appear when a significant anemia
Time (d) has developed. Pallor, weakness, dizziness, dyspnea, jaun-
Figure 20–3. Indicator of acute intravascular hemolysis. Within a few hours of dice, and unexplained fever are occasionally presenting
an acute hemolytic event, free hemoglobin is cleared from plasma, and the serum complaints. Hemolysis is usually acute at onset and may sta-
haptoglobin falls to undetectable levels. Hemoglobinuria ceases soon after this. If
bilize or may continue to accelerate at a variable rate. The
no further hemolysis occurs, the serum haptoglobin level recovers and methemal-
bumin disappears within several days. The urinary hemosiderin can provide more peripheral blood smear usually exhibits polychromasia and
lasting evidence of the hemolytic event. (From Hillman, RS, and Finch, CA: Red Cell macrocytosis, reflecting reticulocytosis, or even the presence
Manual, 7th ed. FA Davis, Philadelphia, 1996, p 112, with permission.) of nucleated RBCs, which is evidence of a hyperactive bone
2682_Ch20_439-474 22/05/12 12:00 PM Page 451
Table 20–7 Comparison of Paroxysmal Cold Hemoglobinuria (PCH) and Cold Hemagglutinin
Disease (CHD)
FACTORS PCH CHD
Harmening DM: Clinical Hematology and Fundamentals of Hemostasis, 4th ed. FA Davis, Philadelphia, 2002, p 212, with permission.
marrow (Fig. 20–4). Spherocytosis and occasionally RBC time of intense hemolysis is associated with a high mortality
fragmentation, indicating extravascular hemolysis, may also rate. Products of hemolysis, such as bilirubin (particularly
be demonstrated in a blood smear from a patient with the unconjugated indirect fraction) and urinary urobilino-
immune hemolytic anemia. An uncommon manifestation of gen, are increased. In severe cases, depleted serum hapto-
WAIHA is reticulocytopenia. This may be associated with a globin, hemoglobinemia, hemoglobinuria, and increases
hypoplastic marrow that is secondary to an underlying dis- in LDH may be laboratory markers that help confirm the
ease state. Because antigenic determinants on erythrocyte diagnosis.
precursors can also react with the patient’s RBC autoanti-
bodies, reticulocytes may be destroyed as they are released RBC Hemolysis
from the bone marrow. Therefore, reticulocytopenia at a
Most patients with WAIHA have both IgG and complement
on their RBCs (67%). A minority have either IgG only (20%)
or complement only (13%). Fewer still are patients with
BOX 20–2 WAIHA with IgA (2% to 3%) or IgM (less than 2%) coating
Diseases Frequently Associated With WAIHA their RBCs.7 The IgG subclass of coating antibody has been
studied in hopes of finding a correlation with severity of
Hemolytic Anemia
• Reticuloendothelial neoplasms, such as chronic lymphocytic
leukemia, Hodgkin’s disease, non-Hodgkin’s lymphomas, myelofi-
brosis, and myelodysplastic syndromes
• Collagen disease, such as systemic lupus erythematosus and
rheumatoid arthritis
• Infectious diseases, such as viral syndromes in childhood and
adults
• Immunologic diseases, such as hypogammaglobulinemia, dysglob-
ulinemia, and other immune-deficiency syndromes
• Gastrointestinal diseases, such as ulcerative colitis
• Carcinoma (nonovarian)
• Pregnancy
• Chronic renal failure
Adapted from Sokol, RJ, Booker, DJ, and Stamps, R: The pathology of autoim-
mune haemolytic anaemia. J Clin Path 45:1047–1052, 1992, with permission
Figure 20–4. Autoimmune hemolytic anemia (peripheral blood).
2682_Ch20_439-474 22/05/12 12:00 PM Page 452
hemolysis, with IgG1 predominating (87%). Unexpectedly, Serologic Characteristics and Laboratory Tests
approximately the same percentages of patients with a posi- Affected
tive DAT but no evidence of hemolysis and normal blood
donors with a positive DAT had IgG1 on their RBCs. Nance Because warm reactive autoantibodies are typically IgG im-
and Garratty reported that, in general, the strength of the munoglobulins, they react best by the indirect antiglobulin
DAT correlated with the presence of multiple IgG subclasses technique. As a rule, they do not directly agglutinate saline-
on the RBCs, which in turn correlated with the severity of suspended RBCs after 37°C incubation. The antibodies may
the hemolysis.38 activate complement and are usually enhanced by enzyme
In general, IgG3 antibodies are the most destructive to techniques. Most of these autoantibodies react with a high-
RBCs, followed by IgG1. IgG2 antibodies are less destructive, incidence RBC antigen, often with a general specificity
and IgG4 shows little or no RBC destruction. The subclasses, within the Rh blood group system, but there are reports of
or isotypes, of IgG are distinguished by the number of disul- autoantibodies associated with most of the other blood
fide bonds present in the hinge region of the molecule, group systems. Identification of autoantibodies is discussed
accounting for their different electrophoretic mobility and later in this chapter.
biological properties. Refer to Chapter 3 for a complete dis- Warm autoantibodies can interfere with most routine
cussion of the properties of the IgG subclasses. All IgG sub- blood bank tests, and they may present more of a serologic
classes, except IgG4, possess the ability to bind complement dilemma than cold autoagglutinins. While most cold autoan-
via the classic pathway of activation, with IgG3 being tibodies can be avoided if room temperature incubation is
more efficient than IgG1, which in turn is more efficient omitted, if testing is performed at 37°C, or if anti-IgG
than IgG2. antiglobulin is used, with WAIHAs, both clinically signifi-
Immune RBC destruction resulting from sensitization cant alloantibodies and the autoantibodies themselves react
with IgG antibody is primarily extravascular, taking place in best at the indirect antiglobulin phase; therefore, more com-
the fixed reticuloendothelial system (RES) cells of the liver plicated and time-consuming procedures for resolving the
and spleen. The spleen is 100 times more efficient than the problems may have to be used.
liver in removing IgG-sensitized RBCs. Macrophages are
equipped with two important biological receptors on their ABO Typing
membranes: Since most warm autoantibodies are not direct agglutinins,
ABO grouping is usually not affected. Even though the
1. Receptors for the FC fragments of IgG1 and IgG3
patient’s cells may be heavily coated with antibody, the
immunoglobulins
antibodies typically do not cause spontaneous agglutination
2. Receptors for the C3b fragment of complement
of RBCs at room temperature when reagent anti-A and
Sensitized RBCs are phagocytized by interaction with RES anti-B are added. Similarly, warm autoantibodies in the
mononuclear phagocytes, depending on which protein coats serum usually do not directly agglutinate saline-suspended
the erythrocytes. If only IgG coats the RBCs, gradual phago- A1 and B cells.
cytosis of the erythrocytes occurs. If both IgG and C3b coat
the RBCs, there is a rapid phagocytosis, because the C3b frag- Rh(D) Typing
ment augments the action of IgG, enhancing sequestration False-positive Rh typing can occur when the patient’s cells
and phagocytosis of the coated erythrocytes. If only C3b coats are coated with immunoglobulins. The high-protein Rh
the RBCs, transient immune adherence occurs. It has been antisera, previously the mainstay of Rh typing, demonstrated
estimated that more than 100,000 molecules of the comple- numerous testing problems. Potentiators were added to
ment fragment would be required to induce phagocytosis; many high-protein Rh typing reagents that caused direct
therefore, the activity of the macrophages and the severity of agglutination of RBCs strongly coated with IgG. For this rea-
hemolysis via phagocytosis of sensitized RBCs depend on son, a negative control consisting of patient cells and the
various factors, summarized in Box 20–3. matching Rh diluent had to be tested in parallel with the
D typing. The results of the D antigen typing were valid only
when the control test result was negative.
The current reagents available for D typing are predom-
BOX 20–3 inantly monoclonal antisera containing no more than
Factors Affecting Activity of Macrophages 7% protein additive. Monoclonal antisera have a low inci-
dence of false-positive test results, but false positive results
• Subclass of IgG, especially IgG1 and IgG3
still may occur if the RBCs are heavily coated with IgG.39
• Presence of complement (C3b) fragments
Some manufacturers offer an Rh control reagent designed
• Quantity of immunoglobulin or complement
to be tested in parallel with the anti-D serum. Other man-
• Number and activity of helper T cells (CD4)
ufacturers recommend that a negative test with an anti-
• Number and activity of suppressor T cells (CD8)
serum of a similar protein concentration (e.g., ABO antisera)
Adapted from Pittiglio, D, and Sacher, RA: Clinical Hematology and Fundamentals
is sufficient to detect a false-positive reaction. If a patient
of Hemostasis. FA Davis, Philadelphia, 1987, p 153, with permission. types group AB Rh positive (i.e., all tubes in the ABO cell
typing and the D typing are positive), a separate Rh control
2682_Ch20_439-474 22/05/12 12:00 PM Page 453
(commercial matched diluent or 6% albumin) must be determining its specificity. The main goal of additional
tested alongside to ensure that the ABO/Rh typing is valid. testing should be to detect and identify all clinically
If the diluent or 6% albumin control is positive, it may be significant alloantibodies that might be masked by the
necessary to pretreat the cells using EDTA/glycine acid autoantibody. If the patient has had a previous transfusion
(EGA) or choloroquine diphosphate (CDP) to remove or has been pregnant, there is an inherent risk of previous
coating IgG immunoglobulins from the RBCs to obtain a alloimmunization.
valid ABO/Rh test.40
Evaluation of Autoantibody
See Procedures 20-5 or 20-10 on the textbook’s
companion website. A positive DAT can result from RBC alloantibodies coating
recently transfused donor cells or drug-induced antibodies
If the DAT of the EGA-treated or CDP-treated RBCs is and RBC autoantibodies. IgG immunoglobulins will most
negative, it is then possible to use these cells for weak D test- likely be present on the cells in each case. It is important to
ing; however, an Rh control serum or 6% albumin should differentiate the causes of a positive DAT because selection
again be tested in parallel to detect false-positive reactivity. of RBCs for transfusion and treatment protocols differ. To
Note: Even if some IgG remains on the RBCs after EGA or make the distinction between these causes, one must have
CDP treatment, they are usually suitable for testing with the patient’s medical history, including an accurate history
directly agglutinating monoclonal reagents, including anti-D, of previous transfusions and pregnancies, diagnoses, and
anti-C, anti-E, anti-c, anti-e, anti-K, anti-Jka, anti-Jkb and medications.
others, as long as the negative control is valid. If a patient has had a recent transfusion, the possibility
Another technique to detect a weak D type is the rosette that alloantibodies and not autoantibodies are coating the
test (see Chapter 19), which is commonly used in the detec- circulating transfused donor cells must be considered. In
tion of fetal-maternal hemorrhage. This screening test, used most cases, by examining the DAT microscopically for
to detect fetal D+ cells in the circulation of the D– mother, mixed-field agglutination (which indicates a mixed-cell
will also detect D+ cells in any cell population. If the patient’s population of DAT+ and DAT– RBCs) and by determining
cells are D+, the rosette test will be strongly positive. Because the specificity of the antibody in the eluate, it is possible to
the rosette test does not incorporate an antiglobulin phase, establish alloantibodies as the cause (see Chapter 16). Be-
a patient with a positive DAT can be accurately typed for the cause warm autoantibodies are frequently associated with
D antigen by this method. It is not absolutely necessary to certain diseases, such as systemic lupus erythematosus, and
determine the correct weak D typing of a patient with with medications, such as Aldomet, the patient’s diagnosis
WAIHA, because D– (Rh negative) RBCs can be transfused and drug history are informative tools in helping establish
if necessary. autoantibodies as the cause of the positive DAT. The speci-
ficity of the antibody compared to the patient’s RBC pheno-
DAT type is also helpful in differentiating between autoantibody
A positive DAT is expected in association with warm autoan- and alloantibody.
tibodies. As autoantibody is produced, it adsorbs onto the To identify the specificity of a warm reactive autoantibody,
antigen of that defined specificity present on the patient’s an eluate prepared from the patient’s RBCs must be tested,
own RBCs. The RBCs may then be coated with IgG alone in addition to the patient’s serum, with a panel of reagent
(20%), IgG and complement (67%), or complement alone RBCs.
(13%).7 In rare cases, the DAT may be negative or cells may
See Procedure 20-6 on the textbook’s companion
be coated only with IgA or IgM.18–20
website for instructions in preparing a digitonin
Antibody Detection and Identification acid eluate. Note: A commercial kit is also available
for the preparation of acid eluates.
The serum of a patient with warm autoantibodies may con-
tain only autoantibody or, if the patient has been previously If the patient has a well-documented history showing
transfused or pregnant, a mixture of autoantibody and that he or she has not been transfused within the past
alloantibody. When smaller amounts of autoantibody have 3 months, and there is no evidence of a mixed-cell popula-
been produced, the autoantibody may be detected only on tion in any phenotyping results, it is reasonably safe to
the patient’s cells, adsorbed in vivo, with no free autoanti- assume that antibody activity in the eluate is autoantibody.
body detectable in the serum. If the amount of antibody pro- But if the patient has a history of recent transfusion or preg-
duced exceeds the number of RBC antigen sites available, nancy, the serum may contain alloantibody in addition to
when the antigen-antibody equilibrium is reached, serum autoantibody.
antibody will be detected in the indirect antiglobulin phase The specificity of an autoantibody may be different in
of testing. When warm autoantibodies are present in the the serum and in the eluate. Warm autoantibodies in the
serum or on the patient’s cells, the extent of further testing serum may show a relative anti-e specificity, reacting weakest
could be based on the patient’s history, but reliable transfu- with e– RBCs, while the eluate may show pan-agglutination
sion histories are notoriously difficult to obtain. It must of all RBCs tested. This apparent difference in antibody
be confirmed that the antibody coating the patient’s cells specificity may be qualitative or quantitative. The concen-
is an autoantibody, and limited effort should be expended tration of antibody removed from the cells in the elution
2682_Ch20_439-474 22/05/12 12:00 PM Page 454
process may be greater than the quantity of antibody in the There are numerous reports of autoantibodies with speci-
serum. Table 20–8 illustrates an example of such reactivity. ficities other than Rh, many of which appear to be directed
It should also be noted that elution procedures vary in their against RBC antigens of high incidence or a null phenotype.
ability to remove coating immunoglobulins (i.e., a heat Among the other specificities are autoanti-U, autoanti-Wrb,
or freeze/thaw eluate generally reacts less strongly than autoanti-Ena, autoanti-Kpb, autoanti-Vel, and autoanti-Ge.42–44
an acid eluate or one of the chemical eluates, such as The reader is referred to Petz and Garratty7 for a detailed dis-
dichloromethane or ether). cussion of autoantibody specificity. Apparent specificities
A majority of the IgG antibodies detected in eluates pre- such as these can cause confusion, especially when the
pared from the WAIHA patient’s RBCs or in the patient’s patient has had a recent transfusion. Determining the
serum have a complex Rh-like specificity, similar to those patient’s phenotype is one of the most valuable tools used to
shown in Table 20–9. Occasionally, an autoantibody may classify the antibody as “auto” or “allo.” As previously dis-
have what is termed “simple” anti-e specificity that reacts cussed, monoclonal antisera and commercially available IgG
with all red cells except R2R2 (D+C–E+c+e–) cells. Much removal agents have made this challenge easier. In recently
more frequently, reactivity is observed with all RBCs of transfused patients, molecular genotyping of the patient’s
normal Rh phenotype. In order to identify the specificity white blood cells may be helpful in predicting the patient’s
of a complex Rh-like autoantibody, one must have an exten- phenotype. Most researchers agree that it is not necessary to
sive library of rare cells, which includes Rhnull and D-- cells. do extensive studies to identify the specificity of the autoan-
Testing of these cells should allow one to categorize the tibody, but testing of at least one example of RBCs that is
antibody as anti-nl (normal), which reacts with all cells of phenotypically similar to those of the patient is important to
common/normal Rh phenotypes but not those that are par- help distinguish between autoantibody (the phenotypically
tially deleted or Rhnull; anti-pdl (partially deleted), which re- similar RBCs will be reactive) and multiple alloantibodies
acts with all cells except Rhnull; or anti-dl (deleted), which (the phenotypically similar RBCs will be nonreactive).
reacts with all cells.41 This information is of historic value Specificity of the autoantibody, if obvious, may influence
because of early work done by Weiner, Vos, and Race. For- selection of blood for transfusion. Some practitioners prefer
tunately, this level of antibody identification is of academic to transfuse RBCs that are compatible with the autoantibody
interest, since few laboratories have access to these rare if simple specificity can be assigned, such as anti-e, and if
RBCs for testing purposes, let alone have them available for there are no preexisting circumstances that would prevent
transfusion. this transfusion (e.g., one would not select Rh-positive
Table 20–8 Warm Autoantibody With Relative Anti-e Specificity Noted in Serum Testing, but
Broad Reactivity Noted in Eluate
SERUM ELUATE
LAST
Cell D C E c e Cell RT LISS 37° C IAT Eluate IAT WASH IAT
R1 R1 - 44 1 + + 0 0 + 1 0 0 3+ 4+ 0✓
R1 R1 - 39 2 + + 0 0 + 2 0 0 3+ 4+ 0✓
R2 R2 - 23 3 + 0 + + 0 3 0 0 ± 4+ 0✓
r r - 33 4 0 0 0 + + 4 0 0 3+ 4+ 0✓
r r - 26 5 0 0 0 + + 5 0 0 3+ 4+ 0✓
r’ r - 4 6 0 + 0 + + 6 0 0 3+ 4+ 0✓
r” r - 17 7 0 0 + + + 7 0 0 3+ 4+ 0✓
R0 r – 13 8 + 0 0 + + 8 0 0 3+ 4+ 0✓
R1 r – 14 9 + + 0 + + 9 0 0 3+ 4+ 0✓
R1 R2 - 8 10 + + + + + 10 0 0 2+ 4+ 0✓
Auto Control 11 11 0 0 3+ 4+ 3+
(untt’d RBCs)
Auto Control 12 12 0 0 3+ 4+ 0✓
(EGA-tt’d)
0✓ = negative IAT reading with check cells added and reactive, as expected; untt’d = untreated; EGA-tt’d = EDTA glycine acid treated.
2682_Ch20_439-474 22/05/12 12:00 PM Page 455
Table 20–9 Typical Serologic Reactions of Warm Autoantibodies With RBCs of Selected Rh
Phenotypes
Rh PHENOTYPE AUTOANTI-e ANTI-nl ANTI-pdl ANTI-dl
R1R1 (normal) + + + +
R2R2 (normal) 0 + + +
rr (normal) + + + +
D– – (partially deleted) 0 0 + +
e– RBCs for an Rh-negative patient with autoanti-e). Select- procedure. This phenotyping information can be used to
ing donor units that are compatible with the patient’s au- select RBCs that are phenotypically similar to those of the
toantibody, however, cannot guarantee normal RBC survival. patient by matching the common RBC antigens: Rh, Kell,
Unfortunately, the transfused cells will probably be destroyed Kidd, Duffy, S, and s. Allogeneic adsorptions performed
as rapidly as the patient’s own cells, regardless of phenotype. using this method to select phenotypically similar donor
or reagent RBCs will remove autoantibody, but it will
Detection and Identification of Alloantibodies also adsorb an alloantibody to any high incidence RBC
antigen, if present.
All researchers agree that detection and identification of all
alloantibodies is of primary concern when one must trans-
See Procedure 20-11 on the textbook’s companion
fuse a patient with WAIHA, especially when the patient has
website.
had a previous transfusion or pregnancy. When autoantibody
is found in the serum, it will typically mask any alloantibod-
4. If it is not possible to determine the patient’s RBC phe-
ies present. In this situation, one can use several techniques:
notyping, differential allogeneic adsorptions may be per-
1. If the autoantibody demonstrates a simple specificity, formed by selecting three donor units that are known to
such as anti-e, test a panel of cells negative for the corre- lack common RBC antigens and are of complimentary Rh
sponding antigen (e– in this case) and positive for all antigen combinations. Typically, these cells include each
common clinically significant RBC antigens (Rh, Kell, of the following Rh types: R1R1 (D+C+E–c–e+), R2R2
Duffy, Kidd, S, and s) to exclude or identify the presence (D+C–E+c+e–), and rr (D–C–E–c+e+). Allogeneic ad-
of underlying alloantibodies. sorptions performed using this method will remove
2. If the patient has not had a recent transfusion (within the autoantibody but will also adsorb an alloantibody to any
past 3 months), determine the patient’s RBC phenotype high-incidence RBC antigen, if present. Ficin or ZZAP
using either monoclonal antisera, which does not require treatment of the allogeneic adsorbing cells is recom-
antiglobulin testing, or using the patient’s RBCs treated mended because it increases antibody uptake but also al-
with an agent known to remove coating IgG im- ters the phenotype of the adsorbing cells—for example,
munoglobulins. The cells may be treated with chloro- ficin will render the RBCs M–N–S– Fy(a–b–) and ZZAP
quine diphosphate (CDP) solution or EGA prior to will render them K–k–s–. Keep in mind the cell treatment
phenotyping. (The EGA procedure will denature all Kell used when interpreting the pattern of results in the
system antigens, among others.) If there is a sufficient adsorbed sera.
quantity of patient RBCs available, prepare autologous
In typical warm adsorption procedures, the patient’s
cells to be used for autoadsorption procedures. Pretreat
serum and the prepared adsorbing cells are incubated at
the cells to remove coating autoantibody, using either of
37°C, allowing the autoantibody to bind to antigen sites on
the methods described above or a chemical such as ficin
the prepared cells, leaving unadsorbed alloantibody in the
or ZZAP (a combination of ficin and DTT).
serum. The number of adsorptions needed to remove all au-
toantibody depends on the amount of autoantibody present
See Procedure 20-7B on the textbook’s companion
in the patient’s serum and the test procedure used. Usually
website. This treatment will free antigen sites for
the strength of reactivity in the indirect antiglobulin phase
adsorption of autoantibody.
of the antibody screen or panel is a good indicator of how
3. If autoadsorption studies are not possible because the pa- many adsorptions will be necessary. If there is strong reac-
tient was recently transfused, and there is evidence of tivity (3+ to 4+) of autoantibody in the serum and gel or
reticulocyte production, it may be possible to determine polyethylene glycol (PEG) enhancement is used in testing,
the patient’s phenotype using a reticulocyte harvesting then more than one adsorption will be necessary. Testing the
2682_Ch20_439-474 22/05/12 12:00 PM Page 456
adsorbed serum with the DAT– (e.g., EGA or CDP-treated) It is impossible to detect all clinically significant alloanti-
autologous RBCs or allogeneic adsorbing cells used is a use- bodies using allogeneic adsorptions, like those directed
ful way of determining if autoantibody has been adsorbed against high-incidence antigens. For example, an anti-k
and the adsorbed serum is ready to test with additional would be adsorbed onto virtually all random donor cells,
selected cells. Table 20–10 shows an example of a patient because the k antigen is present on the cells of more than
with a warm autoantibody demonstrable by LISS indirect 99% of the population (including the R1R1, R2R2, and rr ad-
antiglobulin test (IAT) and alloanti-E detected in the autoad- sorption cells) unless ZZAP treatment of the adsorbing cells
sorbed serum. is used. An antibody such as this would be differentiated
from the autoantibody only if a cell negative for the high-
See Procedure 20-7 on the textbook’s companion incidence antigen happened to be present among the cells
website. used for adsorption. Nevertheless, allogeneic (differential)
When performing adsorption procedures, the following adsorption technique is valuable when there is no alternative.
circumstances must be considered: Table 20–12 shows an example of a warm autoantibody
with underlying anti-K and anti-Jka. The allogeneic adsorp-
• Autoadsorption procedures are never recommended in tions were performed using phenotyped donor RBCs that are
patients who have been transfused within the previous of phenotypes complimentary to each other as a “set.” Note
3 months. In vitro studies have determined that as few as that the phenotype of the donor RBCs is unknown for P1, M,
10% of antigen-positive RBCs are sufficient to adsorb the N, Lea, and Leb. Because antibodies to these antigens would
alloantibody.45 This small amount of antigen-positive cells not be expected to be clinically significant unless they were
may not be obvious in phenotyping. reactive at 37°C, they can be ignored. If interpretation of the
• If the patient is severely anemic, it may not be possible to reactivity pattern in the adsorbed sera suggested one of these
obtain sufficient autologous cells for multiple adsorptions. antibodies was present, the adsorbing cells could be pheno-
• Whenever an adsorption is performed, whether with au- typed at that point for the antigen of interest.
tologous or allogeneic cells, the serum is diluted to some When alloantibodies are detected in the serum of a patient
extent. Some saline remains in “packed” RBCs. A weakly with WAIHA, it is desirable to test the patient’s untransfused
reactive alloantibody could be diluted and missed if mul- RBCs for the absence of the corresponding antigen, if not al-
tiple adsorptions are performed. ready done. If no pretransfusion sample is available, underly-
• If allogeneic adsorptions are performed, it is possible to ing antibodies must be assumed to be alloantibody in nature.
adsorb an alloantibody to a high-incidence RBC antigen
that is present on the allogeneic cells that is not present
on the patient’s own cells. Selection of Blood for Transfusion
When the patient has had a recent transfusion or is Many patients with WAIHA never require transfusion; they
severely anemic, cells of selected phenotypes for adsorption can be managed with medical treatment. Occasionally, how-
can be used. If the patient’s phenotype is unknown and ever, the anemia is so severe that transfusion cannot be
cannot be determined, adsorptions can be performed using avoided. In addition, patients who have a nonhemolytic
a trio of selected cells (R1R1, R2R2, and rr). These cells WAIHA pose problems when blood is needed for a surgical
should also lack one or more antigens for the more com- procedure. In these cases, after thorough serologic investi-
monly encountered clinically significant alloantibodies gation, the primary concern is to ensure compatibility with
(i.e., anti-D, anti-C, anti-E, anti-c, anti-e, anti-S, anti-s, any alloantibodies in the patient’s serum. Compatibility with
anti-K, anti-Fya, anti-Fyb, anti-Jka, anti-Jkb). As shown in the autoantibody is controversial. If the autoantibody shows
Table 20–11, if the patient’s serum is adsorbed with cells a simple specificity, such as anti-e, local practice may be to
from donors of selected phenotypes and then the adsorbed select donor units that are negative for the corresponding
sera are tested, alloantibodies to the common antigens can antigen, when practical.
be detected. As previously mentioned, this may be difficult if the
Alternatively, if a pretransfusion phenotype is available donor RBCs selected precipitated alloantibody formation
for the patient, phenotypically matched RBCs can be used that would bring bigger challenges. For example, because
for adsorption. (See discussion of antigen typing the patient virtually all Rh-negative (D–) cells are e+, an Rh-negative
with a positive DAT in this chapter.) With the exception of (D–) patient with autoanti-e specificity should not receive a
possible adsorption of an antibody to a high-incidence anti- transfusion of Rh-positive (D+) RBCs that lack the e anti-
gen, adsorption with donor RBCs of similar phenotype to gen.46 Singh and colleagues report hemoglobin increments
the patient’s is a useful alternative to autologous adsorption. in patients with AIHA comparable to the expected increment
The patient should not form alloantibodies to RBC antigens even when e+ units were transfused to patients with an ap-
that he or she possesses; therefore, it is advised to focus parent autoanti-e.47 Finding compatible units for patients
mainly on the antigens the patient lacks. For example, if the with a broad specificity warm autoagglutinin is virtually im-
patient’s RBCs phenotype is E–c–S–K–Fy(a–)Jk(b–) and possible. Even if the units are nonreactive with the adsorbed
donor RBCs are available that are E–c–K–Jk(b–), ficin treat- serum, these units will be “least incompatible” when cross-
ment of the donor RBCs would render them also S– and matched with unadsorbed serum. Also, the selection of in
Fy(a–), a phenotype-similar to the patient’s. vitro least incompatible RBCs may not translate to in vivo
2682_Ch20_439-474 22/05/12 12:00 PM Page 457
Table 20–10 Warm Autoantibody With Underlying Alloanti-E
FICIN AUTO-ADS
Cell D C E c e P1 M N S s Lea Leb K k Fya Fyb Jka Jkb OTHER CELL RT LISS 37°C IAT SERUM IAT
R1 R1 - 44 1 + + 0 0 + + + + 0 + 0 + 0 + + 0 + + Cw+ 1 0 0 3+ 0✓
R1 R1 - 39 2 + + 0 0 + + + + 0 + 0 + 0 + + + + 0 Co(b+) 2 0 0 3+ 0✓
R2 R2 - 23 3 + 0 + + 0 + 0 + 0 + 0 + 0 + 0 + + 0 3 0 2+ 4+ 3+
r r - 33 4 0 0 0 + + 0 + 0 + + + 0 0 + 0 + 0 + 4 0 0 3+ 0✓
r r - 26 5 0 0 0 + + + + + + + 0 + + + + + + 0 5 0 0 3+ 0✓
r’ r - 4 6 0 + 0 + + 0 + 0 + + 0 0 + + 0 + 0 + 6 0 0 3+ 0✓
r” r - 17 7 0 0 + + + + + + + 0 0 + 0 + + 0 0 + 7 0 1+ 4+ 2+
R0 r – 13 8 + 0 0 + + + + + + 0 0 0 0 + 0 0 + + V+ VS+ 8 0 0 3+ 0✓
R1 r – 14 9 + + 0 + + + 0 + + + + 0 0 + + 0 + + 9 0 0 3+ 0✓
R1 R2 - 8 10 + + + + + 0 + 0 + + 0 + 0 + 0 + + + Kp(a+) 10 0 1+ 4+ 2+
Auto Control 12 12 0 0 3+ 0✓
(EGA-tt’d)
Anti-E is also detectable at 37°C as a directly agglutinating antibody, which is not uncommon with many examples of Rh antibodies. Adsorption x3 with ficin-treated autologous RBCs removed the warm autoantibody and left alloanti-E detectable in the autoadsorbed
serum. EGA-treated autologous RBCs react with the unadsorbed serum but are nonreactive with the autoadsorbed serum. ADS = Adsorbed.
0✓ denotes negative IAT reading with check cells added and reactive as expected.
457
2682_Ch20_439-474 22/05/12 12:00 PM Page 458
Table 20–11 Differential Adsorption Technique for Detecting Alloantibodies in the Serum of a
Patient With Warm-Reacting Autoantibodies
EXAMPLE OF PHENOTYPE ANTIBODY REMOVED ANTIBODY REMAINING
RBC OF ADSORBING RBCs BY ADSORPTION IN ADSORBED SERUM
most compatible RBCs. The transfused donor cells are likely Its mechanism for effectiveness is not understood and
to be destroyed as rapidly as the patient’s own RBCs. It is appears to be most effective when used in conjunction with
much more important that donor units be selected that are corticosteroid therapy.7 The use of intravenous immune
compatible with any alloantibodies identified. globulin in patients unresponsive to corticosteroid treatment
is controversial and appears to have limited efficacy.50
Treatment of WAIHA
Splenectomy
Therapy is generally aimed at first treating the underlying dis- If steroid therapy fails, or if a patient requires large doses of
ease, if one is present. General measures to support cardiovas- steroids to control hemolysis, splenectomy is usually recom-
cular function are important for patients who are severely mended. The decision to perform a splenectomy requires
anemic. Transfusion should be avoided, if possible, because it clinical evaluation and judgment. There are three reasons for
may only accelerate the hemolysis; however, transfusion performing a splenectomy:
should not be avoided if the anemia is life-threatening. The
1. Failure of steroid therapy
volume of red blood cells transfused should be conservative,
2. Need for continuous high-dose steroid maintenance
aiming for a relief in symptoms, not restoring a normal hema-
3. Complications of steroid therapy
tocrit. In many cases, even small amounts of transfused RBCs
(even 1/2 to 1 unit of packed RBCs) are sufficient to relieve the Splenectomy results in decreased production of antibody and
symptoms of the anemia.48 Forms of treatment other than removes a potent site of RBC damage and destruction. Patients
transfusion are described in the following section. who had a good initial response to steroids respond better with
splenectomy than do those who failed initial steroid therapy.
Corticosteroid Administration and Use It has been reported that as many as 60% of patients with
of IV Immunoglobulin WAIHA benefit from splenectomy if steroid dosages greater
One form of therapy involves the use of corticosteroids, such than 15 mg per day are also used to maintain remission.
as prednisone. Initially, high doses (100 to 200 mg) of
prednisone are maintained until the patient’s hematocrit Immunosuppressive Drugs
level stabilizes. Patients who have not had transfusions seem The use of immunosuppressive drugs is usually the last
to respond to steroid therapy more rapidly than those who approach for managing WAIHA. The field of organ trans-
are transfused. plantation has seen advances in development of immunosup-
Several mechanisms have been proposed for the action of pressive drugs, some of which have been used effectively in
prednisone,49 including: the treatment of WAIHA that has failed other forms of ther-
apy. Azathioprine (Imuran) and cyclophosphamide are ex-
• Reduction of antibody synthesis
amples of immunosuppressive drugs that interfere with
• Altered antibody activity
antibody synthesis by destroying dividing cells. Detrimental
• Alteration of macrophage receptors for IgG and C3, which
side effects of these drugs are infection, infertility, risk of
reduces the clearance of antibody-coated RBCs
birth defects, and development of malignancies. Cyclosporin
The dosage of prednisone should be reduced when the has also been used, but the risk of renal damage is high. One
hematocrit begins to rise and the reticulocyte count drops. of the most promising drugs is Rituximab, a monoclonal an-
The steroids are withdrawn slowly over 2 to 4 months. tibody that targets antibody-producing B lymphocytes, but
A beneficial response to the administration of prednisone it also has adverse effects, especially when used long-term.
is demonstrated in 50% to 65 % of all cases of WAIHA. An
androgenic steroid, danazol, has also been beneficial in Mixed-Type Autoantibodies
prednisone-resistant cases.49
Intravenous immunoglobulin (IVIG) has also been used Individuals demonstrating antibody activity that appears to
in patients who do not respond to prednisone therapy. have both “warm” and “cold” components are considered to
2682_Ch20_439-474 22/05/12 12:00 PM Page 459
Table 20–12 Warm Autoantibody With Underlying Alloanti-K and -Jka
UNADSORBED R1R1 R2R2 rr
SERUM ALLO- ALLO- ALLO-
ADS ADS ADS
{
SERUM SERUM SERUM
LISS
CELL D C E c e P1 M N S s Lea Leb K k Fya Fyb Jka Jkb CELL RT 37°C IAT IAT IAT IAT
R1 R1 - 10 + + 0 0 + ? ? ? 0 + ? ? 0 + + 0 + + Adsorbing 0✓
cells
R2 R2 - 18 + 0 + + 0 ? ? ? + + ? ? 0 + + + + 0 chosen by 0✓
phenotype
r r - 14 0 0 0 + + ? ? ? + 0 ? ? + + 0 + 0 + 0✓
R1 R1 - 16 1 + + 0 0 + + + 0 + 0 + 0 0 + 0 + + 0 Set of 1 0 0 3+ 0✓ 0✓ 3+
3 antibody
R2 R2 - 25 2 + 0 + + 0 + 0 + + + 0 + + + + + 0 + detection 2 0 0 3+ 3+ 3+ 0✓
cells
rr-9 3 0 0 0 + + 0 + + 0 + 0 + 0 + + 0 + + 3 0 0 3+ 0✓ 0✓ 2+
r r - 17 4 0 0 0 + + + + + + 0 0 + 0 + + 0 0 + Selected 4 0✓ 0✓ 0✓
RBCs to
R0 r – 13 5 + 0 0 + + + + + 0 + 0 0 + + 0 0 0 + confirm and 5 3+ 3+ 0✓
R1 R2 - 12 7 + + + + + 0 + 0 + + 0 + 0 + 0 + 0 + alloabys 7 0✓ 0✓ 0✓
Auto Control 8 8 0 0 3+ NT NT NT
(untt’d RBCs)
Auto Control 9 9 0 0 3+ 0✓ 0✓ 0✓
(EGA-tt’d)
Each column in the adsorbed sera columns must be interpreted separately for purposes of antibody identification, keeping in mind the phenotype of the adsorbing cells after ficin treatment; for example, the R1R1, R2R2, and rr adsorbing cells would all become
Fy(a–b–). This means that if anti-Fya or –Fyb were present in the adsorbed sera, they would be present in all three aliquots.
Differential adsorption x3 with ficin-treated allogeneic RBCs removed the warm autoantibody and left alloanti-K and alloanti-Jka detectable in the alloadsorbed serum. EGA-treated autologous RBCs react with the unadsorbed serum but are nonreactive with the
autoadsorbed serum.
? = phenotype of adsorbing cell is unknown
0✓ = negative IAT reading with check cells added and reactive as expected
NT = cell not tested
459
2682_Ch20_439-474 22/05/12 12:00 PM Page 460
have a mixed-type AIHA. Serologic testing will show autoan- DAT Negative Autoimmune Hemolytic Anemia
tibody components typical of both warm and cold AIHA.
The mere presence of a warm and cold autoantibody in a Although the vast majority of patients presenting with signs
patient does not define “mixed-type” AIHA. Although the and symptoms of autoimmune hemolytic anemia will have
cold autoagglutinin in these individuals will demonstrate a positive DAT and other serologic evidence to support their
strongest reactivity at 4°C, it is usually reactive at 30°C or diagnoses, there is a minority of affected patients who appear
above. It is the thermal amplitude of the cold autoantibody to have a negative DAT when tested with commercial
that is key to its pathogenicity. The cold agglutinin is an IgM reagents (polyspecific AHG, anti-IgG, and anti-C3). Some-
hemagglutinin capable of binding complement. The warm times, the amount of IgG coating the RBCs is lower than the
component is an IgG antibody; therefore, the patient will detectable limit of commercial IgG reagents. However, if the
typically demonstrate a positive DAT with both IgG and hematologist has a laboratory value to confirm his diagnosis,
complement on the RBCs. This type of AIHA is rare. he may order a “super Coombs’” test. Few laboratories have
Patients with mixed-type AIHA often present with ex- the reagents and experience to perform this test, more accu-
tremely acute hemolysis and frequently require transfusion. rately called a “DAT hemolytic anemia workup,” but the lab-
Because there are two separate autoantibodies present, ad- oratories that do this sort of testing have found the following
sorption procedures must include both cold and warm ad- tests to be most productive: cold saline/cold LISS wash prior
sorptions to completely remove autoagglutinins. In routine to DAT testing (to detect low-affinity IgG that is lost if wash-
adsorption procedures, the patient’s serum or plasma may ing is done at RT), direct polybrene DAT, DAT testing with
first be adsorbed at 4°C with one or more aliquots of selected anti-IgA and anti-IgM (noncommercial reagents standard-
cells and then adsorbed at 37°C with additional aliquots of ized for RBC agglutination), and DAT by solid phase or
selected cells, using the procedures previously described; column agglutination method. Researchers have also used
however, the procedures may be performed on the same flow cytometry, enzyme-linked antiglobulin assays, mono-
aliquot of adsorbing cells if the order is reversed. Warm ad- cyte monolayer assays (MMAs), and concentrated eluates.
sorption followed by cold adsorption in the same tube saves Even with these esoteric methods, immunoglobulins are
time and adsorbing cells. The warm antibody will remain only detected on the patient’s RBCs about half of the time.7
bound at 4°C while the cold antibody is secondarily ad- Table 20–13 shows a summary contrasting the main charac-
sorbed. It has been found that corticosteroid treatment is teristics of warm and cold autoantibodies.
most often effective in treating individuals with mixed-type
AIHA.51 Drug-Induced Immune Hemolytic
Anemia
IgM Warm Autoantibodies
Therapeutic drugs may have unintended consequences, in-
An unusual type of WAIHA is one associated with IgM cluding immune destruction of RBCs, white blood cells
warm autoantibodies. The serologic characteristics are often (WBCs), and platelets, although the incidence is rare. He-
unimpressive when compared to the clinical havoc these an- molytic anemia, leukopenia, and thrombocytopenia can
tibodies wreak. Usually the patient’s RBCs are strongly occur separately, but in some patients more than one cell line
coated with complement and frequently cause difficulty in is affected. The cells may be coated with antibody, antibody
obtaining valid ABO/Rh typing and DAT because the cells and complement, or complement alone. The discussion in
spontaneously agglutinate. This spontaneous agglutination this section is limited to RBC problems, but many of the
is not prevented by warm washing because the antibody is same principles also apply to platelets and leukocytes.
a warm agglutinin. Treatment of the RBCs with 0.01M DTT Drug-mediated problems may come to the attention of
is necessary to remove enough IgM from the RBCs to allow the blood bank technologist in one of two ways:
for valid ABO/Rh and DAT results. IgM can sometimes be
detected on the RBCs, if suitable anti-IgM is available, but 1. A request for diagnostic testing on a patient with
flow cytometry is the most reliable technique in detecting suspected hemolytic anemia
the IgM.In antibody detection tests, there is usually no re- 2. Unexpected results in routine testing; for example, a
activity of the serum at room temperature, a weak agglutinin positive autologous control in the antiglobulin phase of
at 37°C, and very weak or no reactivity at the antiglobulin antibody screening or compatibility testing, or a positive
phase. If enzyme-treated RBCs are tested, many examples DAT
react strongly or are lysed by these antibodies. If antibody Drugs should be suspected as a possible explanation for
specificity is done, these antibodies may show a specificity immune hemolysis or a positive DAT when there is no other
for antigens on glycophorins (e.g., anti-Ena, anti-Pr, anti-Ge, reason for the serologic and hematologic findings and if the
anti-Wrb).51 If the IgM warm autoantibodies detected are patient has a recent history of taking high doses of antibiotics
associated with a severe clinical anemia (hgb less than or other drugs associated with drug-induced immune he-
5 g/dL), the prognosis is poor. Some patients have been molytic anemia (DIIHA). Drug-induced positive DATs and
treated with plasma exchange, IVIG, steroids, or transfu- hemolytic anemia are relatively rare (estimated at 1 in
sion, but treatment is not always successful and the disease 1 million), so other potential causes should be considered
may be fatal.7 and investigated first.52
2682_Ch20_439-474 22/05/12 12:00 PM Page 461
Site of hemolysis Usually extravascular (no cell lysis) Extravascular/intravascular (cell lysis)
Adapted from Harmening, DM: Clinical Hematology and Fundamentals of Hemostasis, 4th ed. FA Davis, Philadelphia, 2002, p 213, with permission.
Table 20–14 Test Results Observed in the Presence of Anti-Drug Antibody Reactive by the
Drug Adsorption Mechanism
PATIENT SERUM DRUG-COATED UNCOATED
TEST OR ELUATE RBCs RBCs RESULTS
Patient’s serum test X X — If agglutination is present and controls are all negative,
indicates the presence of antidrug antibody. If
negative, no antidrug present.
Drug-Dependent or Immune Complex (“Innocent when the antibody is of the IgG class, because the drug-
Bystander”) Mechanism antidrug complex is thought to elute from the cells during
the washing procedure before the antiglobulin test.58,65
Although the occurrence is rare, many drugs have been Other routine blood bank tests are negative in all phases;
implicated in causing immune-mediated problems by the so- the antibody is directed against a drug or the drug-RBC
called immune complex mechanism. This mechanism was membrane “neoantigen,” not a true RBC antigen. There-
first described in the early 1960s, and it was thought that fore, the antibody screening procedures and compatibility
drugs operating through this mechanism combine with tests are negative, unless an alloantibody is also present.
plasma proteins to form immunogens.63 The antibody pro- An eluate tested with reagent RBCs will also be nonreac-
duced (often IgM, but IgG antibodies may also be present) tive. A summary of typical serologic results is given in
recognizes determinants on the drug. If the patient ingests the Table 20–15.
same drug (or a drug bearing the same haptenic group) after To confirm that a positive DAT is caused by a drug-
immunization, the formation of a drug-antidrug complex may antidrug reaction through the immune-complex mecha-
occur. The complement cascade may be activated because of nism, it must be demonstrated that the antibody in the
this antigen-antibody interaction. RBCs are thought to be in- patient’s serum reacts only if the drug is added to the test
volved in this process only as “innocent bystanders.”64 The system. The patient’s serum is incubated with a solution
soluble drug-antidrug complex nonspecifically adsorbs loosely of the drug in question and ABO-compatible (and antigen-
to the RBC surface. Complement, when activated, sensitizes negative if there is alloantibody present) RBCs. Comple-
the cell and may proceed to lysis (Fig. 20–7). ment activation is the usual indicator of an antigen-antibody
More recently, the concept of “neoantigen” formation reaction; therefore, one should observe for hemolysis after
has been proposed. In this model, the drug interacts in a incubation and use reagents containing anti-C3 for the
noncovalent manner with a specific membrane component antiglobulin test.
and forms a new antigen (“neoantigen”) determinant con-
sisting of both drug and membrane components. This A general procedure for demonstrating antibodies
explanation is the basis of the unifying hypothesis dis- reacting by the immune complex mechanism, sug-
cussed above. gested by Garratty,57 is given in Procedure 20-8 on
Because complement activation is involved in the immune the companion website.
complex, clinically affected patients frequently present with
For the test results to be interpreted correctly, adequate
acute intravascular hemolysis. Up to half of the patients
controls must be performed. The patient’s serum must not
affected also have renal failure. When other causes for
react with the cells when saline or the diluent used to dis-
hemoglobinemia and hemoglobinuria have been excluded, a
solve the drug is substituted for the drug solution, and the
drug-antidrug reaction should be considered. When obtain-
drug solution must not hemolyze the suspension of cells
ing the medication history, it is important to realize that this
nonspecifically. Examples of typical reactions with the pa-
group of patients needs to take only small doses of the drug
tient’s serum and control are given in Table 20–16. An eluate
to be affected. The patient recovers rapidly once the drug is
from the patient’s cells is usually nonreactive even if the drug
withdrawn.
and a source of complement are added. Very little antibody,
If polyspecific antihuman serum is used in DAT testing,
if any, remains on the cells after washing.
the patient’s DAT will usually be positive. If monospecific
In most blood banks, confirmatory testing is done only
reagents are used, the DAT may be positive due to comple-
when the patient has hematologic complications and not
ment alone. Tests with anti-IgG are usually negative, even
when he or she simply has a history of taking the drug and
a positive DAT. Some of the drugs known to cause immune
Complement complex–mediated problems are in frequent use, and there
are large numbers of patients with positive DATs but no ev-
idence of hemolysis. A full workup may only be of aca-
Complex
formation demic interest and is not required before release of RBCs
for transfusion. Treatment involves discontinuing the use
of the drug. Although hemolysis by this mechanism is rare,
Drug
the onset is usually sudden and may be characterized by
intravascular hemolysis and renal failure. Therefore, imme-
diate cessation of the drug is essential. Steroid treatment
Cell
may also be given.
Immune complex C3 and occasionally IgG and IgM Cells, only if serum was incubated with the drug prior to testing.
Routine testing with reagent RBCs is negative.
Drug adsorption IgG—rarely C3 may be present Cells only if they are coated with the specific drug prior to testing.
Routine testing with reagent RBCs is negative.
Drug-independent IgG All “normal” RBCs. Routine testing with reagent RBCs is positive
Membrane modification IgG, IgM, IgA, C3 No cells tested. The mechanism is nonimmunologic protein adsorption.
proteins (e.g., IgG, IgM, IgA, and complement) can bind to because it has been so well studied. The antibodies produced
the membrane (Fig. 20–8).66 Consequently, RBCs from ap- are serologically indistinguishable from those seen in patients
proximately 3% of patients receiving Keflin may exhibit a with WAIHA. Presence of the drug is not required to obtain
positive DAT with polyspecific and monospecific reagents. positive reactions. Positive DATs are encountered in approx-
The uptake of immunoglobulins or complement components imately 10% to 20% of patients receiving Aldomet as an an-
is not the result of a specific antigen-antibody reaction, so tihypertensive; however, very few (0.5% to 1%) of this group
this mechanism is nonimmunologic. Because antibodies with of patients subsequently develop significant immune he-
blood group specificities are not involved, tests with the molytic anemia,7,69 and this occurs only after patients have
patient’s serum and eluate are negative (see Table 20–15). been taking Aldomet for approximately 6 months. If the drug
Numerous cases of cephalosporin-associated hemolytic is stopped, the hemolytic anemia gradually resolves. Other
anemia have been reported, but, as stated earlier, RBC drugs that also cause autoantibody production are L-dopa,
destruction seems to have been mediated through the drug mefenamic acid (Ponstel), procainamide, and diclofenac.69,70
adsorption or immune complex mechanism rather than the The second- and third-generation cephalosporins, fludara-
membrane modification mechanism. There is no treatment bine, and cladribine, have also been associated with autoan-
approach because hemolytic anemia associated with the tibody formation.
ingestion of these drugs has not been described in relation Several mechanisms by which Aldomet causes the produc-
to membrane modification. tion of autoantibodies have been proposed.4,62,68 Kirtland,
Horwitz, and Mohler5 propose that methyldopa alters the
Autoantibody Formation function of T-suppressor cells and suggest that this upset in
the immune system would allow production of antibody
Unlike the drugs acting through the previously described against self. Worlledge and associates68 suggest that the drug
mechanisms that induce production of an alloantibody alters RBC membrane components, similar to the theory of
against a determinant on a drug or on a combination of the “neoantigen” formation.
drug and RBC membrane, alpha-methyldopa (Aldomet) in- The serologic features of this type of drug-induced problem
duces the production of an autoantibody that recognizes RBC are very different from those of the other drug-related immune
antigens.67,68 Although Aldomet is used rarely to treat hyper- hemolytic anemias. As shown in Table 20–17, the antibody in
tension in current practice, a review of its history is important the eluate does react with normal RBCs in the absence of the
Table 20–16 Test Results Observed in the Presence of Anti-Drug Antibody Reactive by the
Immune Complex Mechanism
TEST PATIENT FRESH SERUM* DRUG RBCs RESULTS
Immune complex Quinidine IgM or IgG Positive—often C3 Often negative Small doses of drugs may cause
Phenacetin only, but IgG may acute intravascular hemolysis
be present with hemoglobinemia/
hemoglobulinuria; renal
failure common.
Drug adsorption Penicillins IgG Strongly positive Often negative 3%–4% of patient on large doses
Streptomycin (10,000,000 U) of penicillin,
Cephalosporin which is one of the most
common causes of immune
hemolysis, usually extravascular.
Methyldopa induced Methyldopa IgG Strongly positive Positive—warm 0.8% develop a hemolytic anemia
(Aldomet) autoantibody iden- that mimics a warm AIHA; 15%
tical to that found of patients receiving methyldopa
in WAIHA develop a positive DAT.
Adapted from Harmening, DM: Clinical Hematology and Fundamentals of Hemostasis, 4th ed. FA Davis, Philadelphia, 2002, p 215, with permission.
2682_Ch20_439-474 22/05/12 12:00 PM Page 466
Immunoglobulin Polyclonal IgG— Polyclonal IgM in infec- Polyclonal IgG Polyclonal IgG
characteristics occasionally IgM and tion monoclonal
IgA may be present kappa chain IgM in
cold agglutinin disease
Thermal reactivity 20°–37°C (optimum 4°C–32°C, rarely to 4°C–20°C biphasic 20°C–37°C (optimum 37°C)
37°C) 37°C (optimum 4°C) hemolysin
Titer of free antibody Low to moderate High (>1,000 at 4°C) Moderate to low (< 64) Depends on the mechanism of drug,
(< 32) may be antibody, and RBC interaction
detectable only with
enzyme-treated cells
Reactivity of eluate Usually pan-reactive Nonreactive Nonreactive Pan-reactive with methyldopa type;
with antibody nonreactive in all other cases
screening cells
Most common Anti-Rh precursor; Anti-I, anti-i, anti-Pr Anti-P Anti-e-like, methyldopa antidrug
specificity anti-common Rh;
anti-LW; anti-U
Site of RBC destruction Predominantly spleen Predominantly liver, Intravascular Intravascular and spleen
with some liver rarely intravascular
involvement
Adapted from Harmening DM: Clinical Hematology and Fundamentals of Hemostasis, 4th ed. FA Davis, Philadelphia, 2002, p 217, with permission.
After evaluating this information, one can decide whether testing, drug-coated cells or solutions of the drug can be
drugs are a possible cause of the problem and which of prepared for confirmatory tests. Testing procedures for the
the mechanisms may be involved. It is wise to also note investigation of drug-induced AHIA require a library of
that many drugs cause an immunologic reaction by both control sera and cells and expertise in this testing. Without
drug-dependent and drug-independent mechanisms. appropriate controls, it is possible to misinterpret test
When other causes (e.g., transfusion reaction) have been results and report either false-positive or false-negative
excluded and the clinical situation warrants additional results.
CASE STUDIES
Case 20-1
A 71-year-old man from northern Minnesota is admitted to the hospital with complaints of severe fatigue, shortness of
breath, “pounding heart,” and numbness in his fingers. He first noticed the symptoms when setting up his lake shelter for
ice fishing as he does every year. Although he was mostly annoyed, his wife was concerned and made him go to the doctor.
An automated CBC done at the doctor’s office showed a hemoglobin value of 7.8 g/dL and hematocrit of 23.5%. His doctor
has ordered a bone marrow biopsy, a type and crossmatch for 3 units of packed red blood cells, and a “direct Coombs’”
test. A sample of his blood is drawn and sent to the blood bank for the crossmatch and DAT.
The type and screen results are:
Note: The technologist also commented that the EDTA tube appeared “clotted” when it was first received in the
blood bank.
Case 20-2
A 52-year-old female high school band teacher nearly fainted one afternoon while demonstrating some trombone-playing
techniques to one of her students. She went to her physician complaining of shortness of breath, mild depression, irri-
tability, and a loss of appetite (which was uncharacteristic). Her physician noted signs of anemia and had some hematology
tests done. Most notable was her hemoglobin of 5.2 g/dL, hematocrit of 14.8%, and reticulocyte count of 10.5%. He sent
blood samples to the hospital blood bank for type and crossmatch for 4 units of red blood cells for outpatient transfusion
ASAP. The patient was not thrilled at the prospect of a transfusion since she’d had a bad experience one time previously
when transfused for multiple fractures incurred in a motorcycle accident in her wilder days.
2682_Ch20_439-474 22/05/12 12:00 PM Page 468
The blood bank tested her samples and got the following results:
The patient’s serum was warm autoadsorbed x3 with ficin-treated aliquots of her RBCs. An antibody identification panel
with the autoadsorbed serum gave the following results:
Case 20-3
A 32-year-old female is admitted to the hospital in active labor at term with her first baby. After 8 hours of labor with not
much progress, there are signs of fetal distress and she is taken for an emergency Caesarian section. The baby is soon de-
livered and is healthy. After 3 days of care, mother and baby are discharged. A week later, the mother returns to the emer-
gency room of the hospital with complaints of extreme weakness, mild jaundice, and some back pain. An automated CBC
reveals a hemoglobin of 6.9 g/dL and hematocrit of 21.3%. The ER physician orders 3 units for transfusion STAT. A sample
is drawn and sent to the blood bank.
The type and screen results are:
Note: The technologist performing the testing commented that the EDTA plasma and serum were dark red, but
the phlebotomist denied having difficulty drawing the samples.
Anticefotetan was identified in the patient’s serum and eluate. The patient must be warned that she should never receive
cefotetan again. Renal failure and fatality were seen in 19% of such cases in one study.7
SUMMARY CHART
Immune hemolytic anemia is defined as shortened The classic antibody produced in PCH is called the
RBC survival mediated through the immune response, Donath-Landsteiner antibody, and it has the specificity
specifically by humoral antibody. of autoanti-P. This biphasic antibody binds to patient
In alloimmune hemolytic anemia, patients produce al- RBCs at low temperatures and fixes complement.
loantibodies to foreign RBC antigens introduced into Hemolysis occurs when coated cells are warmed to 37°C
their circulation, most often through transfusion or and complement-mediated intravascular lysis occurs.
pregnancy. In WAIHA, most patients (67%) have both IgG and
Alloimmune hemolytic anemia is self-limiting; when complement on their RBCs, but 20% have only IgG
the foreign RBCs are cleared from circulation, RBC and 13% have only complement.
destruction stops. Warm reactive autoantibodies are generally enhanced
In autoimmune hemolytic anemia (AIHA), patients by enzyme techniques and often have a broad speci-
produce antibodies against their own RBC antigens. ficity within the Rh blood group system.
In AIHA, the autoantibody is directed against the
When transfusing a patient with WAIHA, the primary
concern is detection and identification of all alloanti-
patient’s own RBCs; therefore, there is a consistent
bodies that are masked by the warm autoantibody.
source of antibody and antigen present for continuous
RBC destruction. In the immune complex drug mechanism, the soluble
drug-antidrug complex nonspecifically adsorbs loosely
In drug-induced immune hemolytic anemia, patients
to the RBC surface, yielding a positive DAT with anti-C3
produce antibody to a particular drug or drug com-
and sometimes with anti-IgG.
plex, with subsequent damage to RBCs.
AIHA may be classified as cold reactive (18%), warm
In the drug-adsorption (hapten) mechanism, drugs
such as penicillin or cefotetan bind firmly to proteins
reactive (70%), or drug-induced (12%); diagnostic
of the RBC membrane. The DAT will show reactivity
tests include the DAT and characterization of the
with anti-IgG and often with anti-C3.
autoantibody in the serum or eluate.
In AIHA, serum antibody will not be detected until
In the membrane modification drug mechanism, drugs
such as the cephalosporins modify the RBCs so that
the amount of antibody produced exceeds the num-
plasma proteins can bind to the membrane nonim-
ber of RBC antigen sites available on the patient’s
munologically. The DAT will often demonstrate reac-
own RBCs.
tivity with both anti-IgG and anti-C3.
The common antibody specificity in both benign and
pathological cold autoagglutinins is anti-I.
In the autoantibody drug mechanism, the drug (e.g.,
alpha-methyldopa/Aldomet) induces production of an
In CHD, the DAT will be positive because of comple- autoantibody that recognizes RBC antigens. Both the
ment coating of the RBCs; an antibody titer greater autoantibody and eluate are reactive with normal
than 1,000 may cause visible agglutination of antico- RBCs. The DAT is reactive with anti-IgG.
agulated blood at room temperature.
2682_Ch20_439-474 22/05/12 12:00 PM Page 471
15. A patient who is taking Aldomet has a positive DAT. An 13. Garratty, G, Petz, LD, and Hoops, JK: The correlation of cold
eluate prepared from his RBCs would be expected to: agglutinin titrations in saline and albumin with haemolytic
anaemia. Br J Haematol 35:587, 1977.
a. React only with Aldomet-coated cells 14. Engelfriet, CP, et al: Autoimmune hemolytic anemias: Serolog-
b. Be neutralized by a suspension of Aldomet ical studies with pure anti-immunoglobulin reagents. Clin Exp
c. React with all normal cells Immunol 3:605, 1968.
d. React only with Rhnull cells 15. Rosse, WF: Quantitative immunology of immune hemolytic
anemia: The relationship of cell-bound antibody to hemolysis
16. One method that can be used to separate a patient’s and the effect of treatment. J Clin Invest 50:734, 1971.
RBCs from recently transfused donor RBCs is: 16. De Angelis, V, et al: Abnormalities of membrane protein com-
position in patients with autoimmune haemolytic anaemia.
a. Chloroquine diphosphate treatment of the RBCs Br J Hematol 95:273, 1996.
b. Reticulocyte harvesting 17. Gilliland, BC, Baxter, E, and Evans, RS: Red cell antibodies in
c. EGA treatment acquired hemolytic anemia with negative antiglobulin serum
d. Donath-Landsteiner testing tests. N Engl J Med 285:252, 1971.
18. Garratty, G: Autoimmune hemolytic anemia. In Garratty, G
17. Monoclonal antisera is valuable in phenotyping RBCs (ed): Immunobiology of Transfusion Medicine. Marcel Dekker,
with positive DATs because: New York, 1994, p 493.
19. Stratton, F, et al: Acquired hemolytic anemia associated with
a. Both polyspecific and monospecific antihuman serum IgA anti-e. Transfusion 12:197, 1972.
can be used in antiglobulin testing 20. Sturgeon, P, et al: Autoimmune hemolytic anemia associated
b. Anti-C3 serum can be used in antiglobulin testing exclusively with IgA of Rh specificity. Transfusion 19:324,
c. It usually does not require antiglobulin testing 1979.
21. Hoppe, PA: The role of the Bureau of Biologics in assuring
d. It does not require enzyme treatment of the cells prior
reagent reliability. In Considerations in the Selection of
to antiglobulin testing Reagents. American Association of Blood Banks, Washington,
DC, 1979, p 1.
18. Autoadsorption procedures to remove either warm or 22. Garratty, G, and Petz, LD: An evaluation of commercial
cold autoantibodies should not be used with a recently antiglobulin sera with particular reference to their anticomple-
transfused patient. Recently means: ment properties. Transfusion 11:79, 1971.
a. 3 days 23. Roback, JD (ed): Technical Manual, 16th ed. American
Association of Blood Banks, Bethesda, MD, 2008.
b. 3 weeks
24. Reid, ME: Autoagglutination dispersal utilizing sulfhydryl
c. 6 weeks compounds. Transfusion 18:353, 1978.
d. 3 months 25. Storry, JR, Olsson, MI, and Moulds, JJ: Rabbit red blood cell
stroma bind immunoglobulin M antibodies regardless of blood
group specificity (letter). Transfusion 46:1260, 2006.
26. Judd, WJ: Controversies in transfusion medicine: Prewarmed
References tests: Con Transfusion 35:271, 1995.
27. Chaplin, H, et al: Clinically significant allo-anti-I in an
1. Allan, J, and Garratty, G: Positive direct antiglobulin tests in I-negative patient with massive hemorrhage. Transfusion 26:57,
normal blood donors (abstract). Proceedings of the Interna- 1986.
tional Society of Blood Transfusion, Montreal, 1980. 28. Marsh, WL: Aspects of cold-reactive autoantibodies. In Bell,
2. Izui, S: Autoimmune hemolytic anemia. Curr Opin Immunol CA (ed): A Seminar on Laboratory Management of Hemolysis.
6:926, 1994. American Association of Blood Banks, Washington, DC, 1979,
3. Barthold, DR, Kysela, S, and Steinberg, AD: Decline in suppres- p 79.
sor T cell function with age in female NZB mice. J Immunol 29. Combs, MR, et al: An auto-anti M causing hemolysis in vitro.
112:9, 1974. Transfusion 31:756, 1991.
4. Banacerraf, B, and Unanue, ER: Textbook of Immunology. 30. Garratty, G: Target antigens for red-cell-bound autoantibodies.
Williams & Wilkins, Baltimore, 1979. In Nance, ST (ed): Clinical and Basic Science Aspects of
5. Kirtland, HH, Horwitz, DA, and Mohler, DN: Inhibition of Immunohematology. American Association of Blood Banks,
suppressor T cell function by methyldopa: A proposed cause Arlington, VA, 1991, p 33.
of autoimmune hemolytic anemia. N Engl J Med 302:825, 31. Lyckholm, LJ, and Edmond, MB: Seasonal hemolysis due to
1980. cold-agglutinin syndrome. N Engl J Med 334:437, 1996.
6. van Loghem, JJ: Concepts on the origin of autoimmune 32. Aoki, A, et al: Cardiac operation without hypothermia for
diseases: The possible role of viral infection in the etiology of the patient with cold agglutinin. Chest 104:1627, 1993.
idiopathic autoimmune diseases. Semin Hematol 9:17, 1965. 33. Shirey, RS, et al: An anti-i biphasic hemolysin in chronic parox-
7. Petz, LD, and Garratty, G: Immune Hemolytic Anemias, 2nd ed. ysmal cold hemoglobinuria. Transfusion 26:62, 1986.
Churchill Livingstone, Philadelphia, 2004. 34. Judd, WJ, et al: Donath-Landsteiner hemolytic anemia due to
8. Hillman, RS, and Finch, GA: Red Cell Manual, 7th ed. FA an anti-Pr-like biphasic hemolysin. Transfusion 26:423, 1986.
Davis, Philadelphia, 1996. 35. Reid, M: Association of red blood cell membrane abnormalities
9. Roback, JD (ed): Technical Manual, 16th ed. American with blood group phenotype. In Garratty, G (ed): Immunobi-
Association of Blood Banks, Bethesda, MD, 2008. ology of Transfusion Medicine. Marcel Dekker, New York,
10. Okuno, T, Germino, F, and Newman, B: Clinical significance 1994, p 257.
of autologous control (abstract). American Society Clinical 36. Salloum, E, and Lundberg, WB: Hemolytic anemia with
Pathologists 16, 1984. positive direct antiglobulin test secondary to spontaneous
11. Lau, P, Haesler, WE, and Wurzel, HA: Positive direct antiglob- cytomegalovirus infection in healthy adults. Acta Hematol
ulin reaction in a patient population. Am J Clin Pathol 65:368, 92:39, 1994.
1976. 37. Benraad, CEM, Scheerder, HAJM, and Overbeeke, MAM:
12. Judd, WJ, et al: The evaluation of a positive direct antiglobulin Autoimmune haemolytic anaemia during pregnancy. Eur J
test in pretransfusion testing. Transfusion 20:17, 1980. Obstet Gynecol Reprod Biol 55:209, 1994.
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38. Nance, S, and Garratty, G: Subclass of IgG on red cells of 53. Snapper, I, Marks, D, Schwarta, L, et al: Hemolytic anemia
donors and patients with positive direct antiglobulin tests secondary to mesantoin. Ann Intern Med 39:619, 1953.
(abstract). Transfusion 23:413, 1983. 54. Harris, JW: Studies on the mechanism of a drug-induced
39. Rodberg, K, Tsuneta, R, and Garratty, G: Discrepant Rh pheno- hemolytic anemia. J Lab Clin Med 47:760, 1956.
typing results when testing IgG-sensitized RBCs with mono- 55. Kerr, RO, et al: Two mechanisms of erythrocyte destruction in
clonal Rh reagents (abstract). Transfusion 35(Suppl):67S, 1995. penicillin-induced hemolytic anemia. N Engl J Med 298:1322,
40. Edwards, JM, Moulds, JJ, and Judd, WJ: Chloroquine dissoci- 1972.
ation of antigen-antibody complexes: A new technique for 56. Ries, CA, et al: Penicillin-induced immune hemolytic anemia.
typing red blood cells with a positive direct antiglobulin test. JAMA 233:432, 1975.
Transfusion 22:59, 1982. 57. Garratty, G: Laboratory Investigation of Drug-Induced Immune
41. Issitt, P, and Anstee, D: Applied Blood Group Serology, 4th ed. Hemolytic Anemia and/or Positive Direct Antiglobulin Tests.
Montgomery Scientific Publications, Durham, NC, 1998, p 1021. American Association of Blood Banks, Washington, DC, 1979.
42. Win, N, et al: Autoimmune haemolytic anaemia in infancy with 58. Garratty, G: Drug-induced immune hemolytic anemia. In
anti-Kpb specificity. Vox Sang 71:187, 1994. Garratty, G (ed): Immunobiology of Transfusion Medicine.
43. Becton, DL, and Kinney, TR: An infant girl with severe autoim- Marcel Dekker, New York, 1994, p 523.
mune hemolytic anemia: Apparent anti-Vel specificity. Vox 59. Eckrich, RJ, Fox, S, and Mallory, D: Cefotetan-induced immune
Sang 51:108, 1986. hemolytic anemia due to the drug-adsorption mechanism.
44. Reynolds, MV, Vengelen-Tyler, V, and Morel, PA: Autoimmune Immunohematology 10:51, 1994.
hemolytic anemia associated with auto anti-Ge. Vox Sang 60. Bernini, JC, et al: Fatal hemolysis induced by ceftriaxone in a
41:61, 1981. child with sickle cell anemia. J Pediatr 126:813, 1995.
45. Laine, EP, Leger, RM, Arndt, PA, et al: In vitro studies of the 61. Lascari, AD, and Amyot, K: Fatal hemolysis caused by
impact of transfusion on the detection of alloantibodies after ceftriaxone. J Pediatr 126:816, 1995.
autoadsorption. Transfusion 40:1384–1387, 2000. 62. Garratty, G: Drug-induced immune hemolytic anemia. Hema-
46. Wilkinson, SL: Serological approaches to transfusion of tology Am Soc Hematol Educ Program 73–79:2009.
patients with allo- or autoantibodies. In Nance, ST (ed): Im- 63. Shulman, NR: Mechanism of blood cell destruction in individ-
mune Destruction of Red Blood Cells. American Association uals sensitized to foreign antigens. Trans Assoc Am Physicians
of Blood Banks, Arlington, VA, 1989, p 227. 76:72, 1963.
47. Singh, M, Thompson, H, and Pallas, C: Response to transfusions 64. Dameshek, W: Autoimmunity: Theoretical aspects. Ann N Y
in autoimmune hemolytic anemia. Abstracts of South Central Acad Sci 124:6, 1965.
Association of Blood Banks Annual Meeting, Tucson, AZ, 1997. 65. Petz, LD, and Mueller-Eckhardt, C: Drug-induced immune
48. Jeffries, LC: Transfusion therapy in autoimmune hemolytic hemolytic anemia. Transfusion 32:02, 1992.
anemia. Hematol Oncol Clin North Am 8:1087, 1994. 66. Spath, P, Garratty, G, and Petz, LD: Studies on the immune
49. Anderson, DR, and Kelton, JG: Mechanisms in intravascular response to penicillin and cephalothin in humans: Immuno-
and extravascular cell destruction. In Nance, ST (ed): Immune hematologic reactions to cephalothin administration. J Immunol
Destruction of Red Blood Cells. American Association of Blood 107:860, 1971.
Banks, Arlington, VA, 1989, p 39. 67. Carstairs, KC, et al: Incidence of a positive direct Coombs’ test
50. Flores, G, et al: Efficacy of intravenous immunoglobulin in in patients on alpha-methyldopa. Lancet 2:33, 1966.
the treatment of autoimmune hemolytic anemia: Results in 68. Worlledge, SM, Carstairs, KC, and Dacie, JV: Autoimmune he-
73 patients. Am J Hematol 44:237, 1993. molytic anemia associated with methyldopa therapy. Lancet
51. Garratty, G, Arendt, P, and Domen, R: Severe autoimmune 2:135, 1966.
hemolytic anemia associated with IgM warm autoantibodies 69. Petz, LD: Drug-induced hemolytic anemia. Transfus Med Rev
directed against determinants on or associated with gly- 7:242, 1993.
cophorin A. Vox Sang 72:124–130, 1997. 70. Lopez, A, et al: Autoimmune hemolytic anemia induced by
52. Arndt, PA, and Garratty, G: The changing spectrum of drug- diclofenac. Ann Pharmacother 29:787, 1995.
induced immune hemolytic anemia. Semin Hematol 42: 71. Harmening, DM: Clinical Hematology and Fundamentals
137–144, 2005. of Hemostasis, 5th ed. FA Davis, Philadelphia, 2009, pp 252–265.
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Leukocyte Antigens
and Relationship Testing Part IV
Chapter 21
The HLA System
Donna L. Phelan, BA, CHS(ABHI), MT(HEW) and Gerald P. Morris, MD, PhD
OBJECTIVES
1. Define the abbreviations HLA, MLR, SSO, SSP, SBT, TRALI, and MHC.
2. Describe the three regions of the HLA complex located on the short arm of chromosome 6.
3. List the characteristics of HLA genes.
4. List the three exceptions to the practice of naming all serologic specificities on the basis of correlation with an identified
sequence that eliminates the need for a provisional w designation.
5. Describe the current nomenclature for HLA genes.
6. Define the term haplotype.
7. Describe the difference between HLA phenotype and HLA genotype.
8. List the characteristics, importance, and clinical relevance of HLA class I and class II gene products.
9. Describe the characteristics of HLA antibodies.
10. Define linkage disequilibrium, a characteristic of HLA antigens.
11. Describe the techniques used for HLA antigen and allele detection.
12. Describe the techniques for HLA antibody detection.
13. Describe crossmatch technology to evaluate tissue compatibility.
14. Describe the role of HLA typing in paternity testing, disease association, platelet transfusion, TRALI, and transplantation.
15. Explain the role of virtual crossmatches with single antigen antibody testing.
475
2682_Ch21_475-494 22/05/12 12:01 PM Page 476
medical sciences and familiarity with general immunology Figure 21–1. The HLA complex on the short arm of chromosome 6.
concepts.
The emphasis of the chapter is on the principles and con-
cepts of HLA structure and function, HLA procedures, and alleles. DR molecules use DRA but can use alleles coded by
the clinical application of HLA testing to immunogenetics DRB1 (the classic DR specificities), DRB3 (DR52 molecules),
(e.g., paternity and disease association), transfusion prac- DRB4 (DR53), and DRB5 (DR51).
tices, and transplantation. Although HLA testing is a tech- The class III region encodes structurally and functionally
nologically complex area, this chapter provides a general diverse molecules, including C2, C4, Bf (the complement
overview of the topic. factors), 21-hydroxylase, and tumor necrosis factor. In addi-
tion, two other genes, glyoxalase-1 (GLO) and phosphoglu-
Historical Perspective comutase-3 (PGM-3) are linked with the HLA complex.
One very important characteristic of HLA genes is that
Evidence for human leukocyte blood groups was first they are highly polymorphic, and several alleles exist at
advanced in 1954 by Jean Dausset,1 with the observation each locus. The antigenic specificities, defined by serologic
that patients whose sera contained leukoagglutinins had reactivity, are designated by numbers following the locus
received a larger number of blood transfusions than other symbol (e.g., HLA-A1, HLA-A2, HLA-B5, HLA-B7, and so
patients. He determined that these agglutinins were not on). Table 21–1 lists the current specificities of the HLA
autoantibodies as had been thought previously but rather system recognized by the WHO Nomenclature Committee
were alloantibodies produced by the infusion of cells bear- for Factors of the HLA System.3 Several HLA alleles demon-
ing alloantigens not present in the recipient. Dausset2 strate serologic cross reactivity due to structural similarity.
determined that patients with alloantibodies segregated For example, HLA-A9, HLA-A23, and HLA-A24 all react
into two groups, based upon their reactivity; antibodies with antibodies specific for HLA-A9, but HLA-A23 and
from group 1 specifically recognized antigens on group 2. HLA-A24 can be “split” from A9 by unique reactivity with
Through this method, Dausset identified HLA-A2, the first antibodies specific for each. Two exceptions to HLA
known HLA molecule. nomenclature are the Bw4 and Bw6 epitopes, where the “w”
is present to distinguish them as significant serologic epi-
Nomenclature topes found on multiple HLA-A and HLA-B alleles, rather
than independent HLA specificities (Table 21–2).
The HLA genetic region is a series of closely linked genes
Continued investigation into HLA using molecular tech-
that determine major histocompatibility factors—that is,
niques of DNA sequence analysis revealed many HLA allelic
surface antigens or receptors responsible for the recogni-
variants that were not detectable by traditional serologic
tion and elimination of foreign tissues. The region is
also referred to as the major histocompatibility complex
(MHC). The HLA complex contains an estimated 35 to
Class II
40 genes physically grouped into three regions located
on the short arm of chromosome 6: class I, class II, and
class III regions (Fig. 21–1). DP DN DO DQ DR
The class I region encodes genes for the classic transplan-
B2A2B1A1 A B B2A2B1A1 B1B2B3/4/5 A
tation molecules HLA-A, HLA-B, and HLA-C. It also encodes
for additional nonclassic genes, including HLA-E, HLA-F,
and HLA-G. The class II region encodes genes for the mole- DPW1 DQ1 DR1
DR2 DR52
cules HLA-DR, HLA-DP, and HLA-DQ composed of both α DPW2
DPW3
DQ2
DQ3 DR3
DR53
and β chains. DP molecules are the product of DPA1 and etc. etc. etc.
DR51
DPB1 alleles (Fig. 21–2); DPB2 and DPA2 are pseudogenes, Figure 21–2. The loci that code for the major categories of HLA class II gene
genes with mutations that prevent gene activation or tran- products. (From Rodey, GE: HLA Beyond Tears. De Novo, Atlanta, GA, 1991, p 12,
scription. DQ molecules are the product of DQA1 and DQB1 with permission.)
2682_Ch21_475-494 22/05/12 12:01 PM Page 477
B46
B47
B48
B49 (21)
B50 (21)
B51 (5)
B5102
B5103
B52 (5)
Continued
2682_Ch21_475-494 22/05/12 12:01 PM Page 478
B54 (22)
B55 (22)
B56 (22)
B57 (17)
B58 (17)
B59
B60 (40)
B61 (40)
B62 (15)
B63 (15)
B64 (14)
B65 (14)
B67
B70
B71 (70)
B72 (70)
B73
B75 (15)
B76 (15)
B77 (15)
B78
B81
B82
Bw4
Bw6
Data from Marsh, SGE, et al: Nomenclature for factors of the HLA system. Tissue Antigens 75:291, 2010.
group reactivity (i.e., A2 reactivity is described as being inherited together. The entire set of A, B, C, DR, DQ,
HLA-A*02). and DP genes located on one chromosome is called a
5. Following the allele family, separated by a colon (:), is haplotype. Genetic crossovers and recombination in the
a two digit numeral that defines the specific allele. For HLA region are uncommon (less than 1%), and thus a
example, the serologically defined HLA-B27 specificity is complete set of alleles located on a chromosome is usually
actually made up of 43 distinct allelic variations. These inherited by children as a unit (haplotype). Figure 21–3
alleles are defined as HLA-B *27:01 through *27:36. It is illustrates the segregation of HLA haplotypes in a family.
mandatory to include the leading zeros that are part of The two haplotypes of the father are labeled a and b, and
the defined alleles. the mother’s haplotypes are c and d. Each offspring inherits
6. The following two-digit series, separated by a colon, con- two haplotypes, one from each parent. Thus, only four pos-
vey other, less clinically relevant but scientifically impor- sible haplotypes—ac, ad, bc, and bd—can be found in the
tant information. Alleles that differ only by synonymous offspring. It can be calculated that 25% of the offspring will
nucleotide substitutions within the coding sequence have identical HLA haplotypes, 50% will share one HLA
are distinguished by the use of the fifth and sixth digits haplotype, and 25% will share no HLA haplotypes. An im-
(i.e., noncoding silent substitutions). portant corollary is that a parent and child can share only
7. Lastly, alleles that only differ by sequence polymorphisms one haplotype, making an identical match between the two
in the introns are distinguished by the use of the seventh unlikely. It should also be apparent that uncles, grandpar-
and eighth digits. See Table 21–3 for examples of all ents, and cousins are very unlikely to have identical haplo-
loci with the new nomenclature changes.3 types with any given child. These are important factors
when looking for a well-matched organ or blood donor.
Antigens and Antibodies The HLA phenotype, then, represents the surface markers
or antigens detected in histocompatibility testing of a single
It is necessary to evaluate the HLA antigen composition in individual. The HLA genotype represents the association of
prospective donor-recipient pairs before organ transplantation the alleles on the two chromosomes as determined by family
and in candidates for platelet therapy refractory to random studies, and the term haplotype refers to the allelic makeup
donor platelets. Even more important is the evaluation and of a single chromosome, illustrated in Figure 21–4.
identification of HLA antibodies in the serum of recipients
before transplantation and transfusion. Evidence clearly indi- Crossing Over
cates that presensitization to HLA antigens may cause rapid
rejection of transplanted tissue or poor platelet survival follow- An event that infrequently complicates HLA-typing interpre-
ing transfusion.4 HLA-antigen testing is also used in disease tation and haplotype determination is crossing over, or
correlation, relationship testing, and anthropologic studies.
Each person has two alleles for each locus. Both alleles
of a locus are expressed co-dominantly—that is, there is Father Mother
equal expression of both alleles. The presence of one allele A1 Cw2 B8 DR3 A3 Cw3 B7 DR2
does not suppress the expression of the other. If there are a c
two different alleles on one locus, the person is heterozy- b d
gous. If both alleles on that locus are the same, the person A2 Cw3 B18 DR4 A11 Cw5 B37 DR6
is homozygous.
A1 Cw2 B8 DR3
Inheritance Child 1
a
c
The physical linkage of the HLA genes on chromosome 6 A3 Cw3 B7 DR2
results in all of the genes on a single chromosome typically
A1 Cw2 B8 DR3
a
Child 2
d
A11 Cw5 B37 DR6
Table 21–3 HLA Nomenclature
A2 Cw3 B18 DR4
EXISTING NOMENCLATURE NEW NOMENCLATURE
b
Child 3
A*01010101 A*01:01:01:01 c
A3 Cw3 B7 DR2
B*150102 B*15:01:02
Structure
recombination. During meiosis, exchange of material between
the paired chromosomes can occur. During chromosomal Class I (HLA-A, HLA-B, and HLA-C) molecules consist of
replication, replicated chromosomes often overlay each other, a heavy chain with a molecular weight of 45,000 daltons
forming x-shaped chiasmata (Fig. 21–5). When the chromo- associated non-covalently with β2-microglobulin, a non-
somes are pulled apart during meiotic division, breaks can polymorphic protein of 12,000 dalton molecular weight
occur at the crossover site, resulting in complementary ex- found in serum and urine. The heavy chain folds into three
change of genetic material. The farther apart two loci are on a domains and is inserted through the cell membrane via a
given chromosome, the more likely it is that genetic exchanges hydrophobic sequence.5
will take place. For example, recombination between HLA-A Class II (HLA-DR, HLA-DQ, and HLA-DP) molecules
and HLA-DP occurs commonly, whereas recombination consist of two similar-sized chains of a molecular weight of
between HLA-DQ and HLA-DR is a rare event. Crossing over 33,000 (α) and 28,000 (β) daltons associated noncovalently
has the effect of rearranging the genes on the chromosome to throughout their extracellular portions. In these molecules,
produce new haplotypes in the general population. both chains are inserted through the membrane via hy-
drophobic regions. The extracellular portions of these chains
Linkage Disequilibrium fold into two domains.6
Class I molecules are present on all nucleated cells, den-
An important characteristic of HLA antigens is the existence dritic cells, and platelets, whereas class II molecules have a
of linkage disequilibrium between the alleles of the loci. Link- much more restricted distribution. They are found only on
age disequilibrium is when HLA genes occur more frequently B lymphocytes, activated T lymphocytes, macrophages,
in the same haplotype than would be expected by chance monocytes, and endothelial cells. Although class I and class II
alone. In the Rh system, the blood groups C, D, and e are molecules have some obvious structural differences, they
found together more often than would be expected based on are thought to be very similar in overall three-dimensional
their individual gene frequencies. This is commonly found configuration (Fig. 21–6). The folding of class I and class II
within the HLA system.4 In a random mating population at molecules forms a pocket that is used to present short linear
Hardy-Weinberg equilibrium, the occurrence of two alleles peptides to T cells for immune recognition.7,8
from closely linked genes will be the product of their indi- Early structural models of class I molecules indicated
vidual gene frequencies. If the observed value of the joint that the α-1 and α-2 domains consisted of stretches of
frequency is significantly different from the expected fre- amino acids that were arranged into helical structures
quency (the product of the individual allele frequencies), the rather than sheets typical of globular proteins. The crys-
alleles are said to be in linkage disequilibrium. tallography studies of Bjorkman and colleagues5 elucidated
For example, if HLA-A1 and HLA-B8 gene frequencies are the three-dimensional structure of the class I, HLA-A2
0.16 and 0.1, respectively, in a population, the expected oc- molecule. The α1 and α2 domains form a platform overlaid
currence of an HLA haplotype bearing both A1 and B8 by two helical structures to form the peptide-binding
should be 0.16 × 0.1 × 100, or 1.6%. In certain white popu- site (Fig. 21–7). This groove holds processed peptides
lations, however, the actual occurrence of this haplotype is for presentation to T cells. Class I and class II molecules
as high as 8%, far in excess of the expected frequency. The are also alike in that most of the polymorphism is
HLA alleles frequently associated through disequilibrium are expressed in the portion of the molecule farthest from
listed in Table 21–4. Disequilibrium between the B and DR the cell membrane involved in presenting peptide antigens
loci alleles may account for problems in correlating B locus to T cells.9
2682_Ch21_475-494 22/05/12 12:01 PM Page 481
These may be “public” (binding to epitopes shared by more HLA-A Locus Cregs
than one HLA gene product) or cross-reactive (binding to
1
structurally similar HLA epitopes).4 36
The monoclonal HLA antibody (MoAb) is produced by 23
fusing HLA antibody-producing B cells with plasmacytoma
lines.10 Plasmacytoma cells arise from the malignant trans- 3
24
formation of plasma cells or differentiated B cells. These
9
cells continue to secrete antibody after transformation. 11
MoAbs detect a broader range of epitopes because they are
2
derived through xenoimmunization—immunization across
different species. Monoclonal antibodies are still used for 66
serologic typing, especially class II; however, they are more 28 68
currently used as capture antibodies in newer-generation 25 10
immunoassays. 69
26
43
Cross Reactivity
34
Cross reactivity is a phenomenon in which an antiserum di- 74
rected against one HLA antigenic determinant reacts with 29
33
other HLA antigenic determinants as well. Cross-reactive
antigens share important structural elements with one an- 30
31 32
other but retain unique, specific elements. HLA serologists
very frequent frequent rare
recognized very quickly that many of the HLA alloantibodies
were serologically cross-reactive with HLA specificities.11–13 Figure 21–8. Cross reactions within the HLA-A locus. (From Bender, K: The HLA
Dausset and colleagues14 suggested that these antibodies System. Biotest Diagnostics, 1991, p 21, with permission.)
might detect public specificities shared by multiple HLA
gene products. The broadly reactive antibodies used in van
Rood and von Leeuwen’s original computer-derived HLA HLA Antigen Detection
clusters also defined many of the current major cross-reactive
groups (CREGs). The agglutination methods initially used to define HLA anti-
The majority of cross-reactive alloantibodies detect HLA gens have been succeeded by a precise microlymphocytotox-
specificities of allelic molecules coded by the same locus. On icity test.24 Cytotoxicity techniques require only 1 to 2 µL
the basis of these reactions, most specificities can be grouped of serum and are sensitive and reproducible. For this pur-
into major CREGs (Figs. 21–8 and 21–9).15,16 For example, the pose, acid-citrated dextrose or phenol-free heparinized blood
HLA-A locus antigens A2, A23, A24, and A28 share a common
determinant and therefore make up the A2 CREG, or A2C. HLA-B Locus Cregs
HLA-A28 also shares a common determinant with A26, A33,
and A34, defining the A28 CREG. There are also at least three 51 52
46 53
interlocus cross reactions detected by alloantisera and MoAbs
5 35
that occur between the HLA-A and HLA-B loci: HLA-A23, 37 62
HLA-A24, HLA-A25, and HLA-A32 with HLA-Bw4;14,17–19 38 70 63
HLA-A11 with HLA-Bw6;20 and HLA-A2 with HLA-B17.21–23 39 16 75
Recently, cross-reactive groups have been defined molecularly 15
67 76
using amino acid residues. CREGs are used predominantly in 77
17
the definition of antibodies present in recipient antisera and in 14
the definition of antigens to avoid in potential organ donors 18
8
when performing virtual crossmatches.
59 49
21
Techniques of Histocompatibility 22
50
Testing
12
27
The principles used in histocompatibility testing are concep- 13
tually similar to those used for red blood cell (RBC) testing. 73
7 40
HLA antigens are identified using sera with known anti-HLA 42 41
48 47
antibodies and DNA-based molecular typing, recipient
serum is screened for the presence of HLA antibodies using very frequent frequent rare
immunoassays, and compatibility is directly determined by Figure 21–9. Cross-reactions within the HLA-B locus. (From Bender, K: The HLA
crossmatching of donor cells and recipient sera. System. Biotest Diagnostics, 1991, p 22, with permission.)
2682_Ch21_475-494 22/05/12 12:01 PM Page 483
laboratories with low throughput, where oligonucleotide SBT has become the preferred method of high-resolution
probes are immobilized on membranes and hybridized with typing for hematopoietic stem cell transplantation.
amplified DNA.
All known HLA alleles can be identified with one or a HLA Antibody Detection Techniques
combination of allele-specific oligonucleotide probes. Differ-
ent strategies are used, depending on the extent of resolution Preformed antibodies to the tissue of the donor and recipient
required. Figure 21–12 illustrates a basic strategy for may cause significant complications in transplantation or
oligonucleotide typing. Although SSO is a powerfully reliable transfusion. Recipient lymphocytotoxic HLA antibodies to
and accurate technology, it has been superseded by the more donor antigens have been associated with accelerated graft
rapid technique of PCR-SSP. rejection and with poor response to platelet transfusion.
Antibody in donor plasma to recipient leukocytes has been
Sequence-Specific Primers associated with severe pulmonary infiltrates and respiratory
In the sequence-specific primers (SSP) technique, oligonu- distress following transfusion, known as transfusion-related
cleotide primers are designed to obtain amplification of spe- acute lung injury (TRALI). Thus, the clinical management
cific alleles or groups of alleles. The typing method is based of patients, pre- and post-transplant, includes screening
on the principle that a completely matched primer will be for and determining the specificity of anti-HLA class I and
more efficiently used in the PCR reaction than a primer with II antibodies that may be present.
one or more mismatches. The specificity of the typing system Crossmatching involves serologic and cellular procedures.
is part of the PCR reaction. Assignment of alleles is based on Serologic crossmatching is performed by cytotoxicity and
the presence or absence of amplified product normally de- flow cytometric techniques.24 Enzyme-linked immunosor-
tected by agarose gel electrophoresis and transillumination. bent crossmatch assays (ELISA) are in development. Detec-
tion and identification techniques of HLA antibodies are
Sequence-Based Typing similar to those for RBC antibodies. The unknown serum is
Direct nucleotide sequencing of HLA genes is utilized for tested against a panel of cells or soluble antigen of known
high-resolution typing and is required in the definition HLA phenotype. Targets from a large panel of donors must
of a new allele. Most commonly, sequence-based typing be selected if antibodies to all HLA specificities are to be de-
(SBT) is performed by terminal-end incorporation of flu- tected. A panel of at least 30 carefully selected targets is re-
orescently labeled nucleotides during PCR reactions quired for initial screening in the determination of panel
amplifying the most polymorphic regions of the HLA reactive antibody (PRA), and a panel of at least 60 cells is re-
genes: exons 2, 3, and 4 of class I genes, and exon 2 of the quired for accurate antibody identification.
class II β chain genes. The fluorescently tagged DNA frag-
ments are sorted by capillary electrophoresis and inter- Microlymphocytotoxicity
preted by computer software to provide a DNA sequence. The microlymphocytotoxicity method depends on the pur-
Generated DNA sequences are then compared to the pose of the screen. When seeking alloantisera as typing
known DNA sequences of HLA alleles to find a match, reagents, it is essential to screen using the method for the typ-
identifying the HLA allele. Detection is not based on the ing procedure, the standard CDC being the most common.
use of sequence-specific oligonucleotide probe, and prior When screening recipient serum samples, a more sensitive
knowledge of the nucleotide sequences is not required.30,31 technique—Amos-modified, extended incubation, or antihu-
Therefore, previously undefined alleles can be detected. man globulin—should be employed. The Amos-modified
technique introduces a wash step after the initial serum-cell
incubation. The wash step removes anticomplementary activ-
ity: aggregated immunoglobulin in the serum that can activate
complement, making it unavailable for binding on the cell
membranes. Standard CDC tests rarely detect 100% of the
Isolate DNA PCR Amplify
antigen-binding specificities of cross-reactive antibodies.32,33
Sensitivity of the CDC test can be greatly enhanced by
Cells DNA Millions of Copies adding an anti-human globulin reagent following serum and
cell incubation.32 Addition of goat anti-human kappa chain
increases the likelihood of complement binding and subse-
quent cell injury, especially in circumstances in which the
amount of HLA-antibody binding is below the threshold of
Hybridize with
DNA probes
detection in the standard technique. The anti-human globulin
test functions like a complement-independent technique with
respect to HLA alloantibodies. It is also similar to the addition
of Coombs in testing for antibodies to red blood cell antigens.
If antibodies to class II molecules (DR and DQ) are to be iden-
Detection of probes DNA bound to membranes tified, then separated or labeled B-lymphocyte suspensions
Figure 21–12. Strategy for allele-specific oligonucleotide typing. of known phenotype must be used. Serum can be screened
2682_Ch21_475-494 22/05/12 12:01 PM Page 485
using freshly prepared lymphocytes or lymphocytes frozen in ELISA, flow screening can distinguish between IgG and IgM
bulk or in trays. Lymphocytes frozen in trays have the advan- antibodies with the use of either anti-IgG or anti-IgM sec-
tage of rapid preparation, enabling serum to be screened in ondary antibodies. Flow screening can also detect noncom-
just a few hours. plement-fixing antibodies, because binding of the antibodies
The lymphocytotoxicity assay has several disadvantages. rather than complement fixation is measured. Flow cytomet-
First, viable T and B lymphocytes are essential for an accurate ric antibody screens utilize T and B lymphocytes as targets
assessment of the presence of antibody. Many laboratories or, in a newer technique, employ purified HLA antigens
routinely use frozen cells to maintain a consistent, represen- coated onto microparticles 2 to 4 µm in diameter.
tative panel of antigens. The process of freezing renders lym- Flow cytometry may be used to screen for the presence
phocytes more fragile and susceptible to cell lysis, resulting of HLA antibodies and determine antibody specificity in the
in false positives. A second problem is the necessity to main- same manner as the ELISA assays by utilizing either pooled
tain a reliable and consistent antigen panel that reflects the or specific HLA antigen-coated microparticles as targets.
ethnic composition of the patient population; HLA antigen Lymphocytes as targets have two problems:
frequencies are known to vary among different races. The
1. Difficulty in distinguishing between HLA-specific and
requirement for activating complement in the cytotoxicity
non-HLA antigens on lymphocytes
assay results in a third disadvantage: the inability to detect
2. Inability to distinguish class I or II antibody specificity,
non-complement-fixing antibodies. Differentiation between
as B lymphocytes express both class I and II markers
HLA-specific and non-HLA-specific antibodies and between
IgG and IgM antibodies is not possible with the standard The use of microparticles/beads coated with purified class I
cytotoxicity assays. Three techniques have been introduced or II antigens circumvents these problems. HLA antibodies
as alternatives to the lymphocytotoxicity assay for the detection present in patient sera react specifically with the beads. After
of anti-HLA antibodies. They employ ELISA, flow cytometry, incubation of serum with beads, followed by staining with a
and Luminex technologies, and overcome many of the pitfalls fluorescently labeled antihuman IgG antibody, the anti-HLA
of the standard cytotoxicity assays. IgG-positive serum shows a fluorescent channel shift as com-
pared with the negative serum. Percent PRA is represented
Enzyme-Linked Immunosorbent Crossmatch Assays by the percentage of pooled beads that react positively with
The enzyme-linked immunosorbent crossmatch assays the serum.
(ELISA) test uses purified HLA antigens instead of lym-
phocytes or cells as targets for antibodies that may be pres- Multiplex Immunoassay (Luminex)
ent in the patient’s sera. The increased specificity of the The highly polymorphic nature of HLA requires testing
assay, due to the use of purified HLA antigens, offers the a large number of cell types to accurately screen for alloan-
advantage of recognizing false-positive non-HLA reactions tibodies by either serologic, lymphocytotoxicity cross-
and distinguishing class I and II specificities. In addition, matching or by ELISA with cell lysate techniques. The
this method also differentiates between IgG and IgM anti- development of the multiplex flow assay using recombi-
body isotypes by the use of appropriate secondary detec- nant individual HLA proteins, the Luminex single antigen,
tion antibody. ELISA can be used as a screening assay for improved upon ELISA, increasing assay throughput,
the detection of anti-HLA antibodies and as a method to sensitivity, and simplified determination of anti-HLA
determine antibody specificity. The screening assay utilizes specificity.34–38 The Luminex assay involves testing patient
a pool of purified HLA antigens. Results are interpreted as sera against a panel of recombinant soluble HLA antigens,
either positive or negative. The specificity determination each bound to specific microbeads. Each microbead is
assay utilizes a panel of purified antigens, rather than a labeled with a distinct set of fluorophores, enabling direct
pool, permitting the evaluation of PRA and HLA antibody evaluation of antibody binding specificity. Anti-HLA anti-
specificity. body binding is detected by labeling with PE-conjugated
HLA class I and II antigens are purified from either trans- anti-Ig and measured on a specialized flow cytometer. Posi-
formed cell lines or from platelets with known HLA pheno- tive reactions are described as the mean fluorescence inten-
types. The affinity-purified HLA antigens, either pooled or sity (MFI) of positive beads.
specific, are bound directly to wells of microtiter plates. The The increased sensitivity of the Luminex assay is clini-
specific binding of antibody from the test serum sample with cally relevant, as detection of donor-specific antibody
any of the antigens is detected by subsequent incubation (DSA) by immunoassay is a strong predictor of antibody-
with alkaline phosphatase-conjugated antibody that recog- mediated rejection, particularly in patients with negative
nizes human IgG. A quantitative measure of the reaction is or equivocal serologic crossmatch results.39–41 However,
obtained by spectrophotometry after the appropriate enzyme several studies have demonstrated that not all DSA de-
substrate is added. tected by Luminex are correlated with positive crossmatch
and that these antibodies may not preclude transplanta-
Flow Cytometry tion.42–44 There is currently much interest in defining DSA
The flow cytometric antibody screen detects antibody bind- concentrations that preclude tissue compatibility, though
ing directly. Therefore, complement activation is not neces- no clear cutoffs between clinically relevant and irrelevant
sary as in the case of the lymphocytotoxicity assay. As with antibodies have been agreed upon.
2682_Ch21_475-494 22/05/12 12:01 PM Page 486
alloantibody profile, has led to improved organ allocation, population studied. To date, over 500 diseases have been
directing shared organs to centers with the most likely com- studied.53 A list of the most significant HLA and disease
patible matches.48–50 associations is given in Table 21–5.
Virtual crossmatching is not infallible, however, as it is crit- The exact cause for the association of HLA to disease is
ically limited by the information used to determine compati- unclear. There is no question that genetic factors coded
bility: Accurate evaluation of immunologic compatibility within or near the HLA complex confer susceptibility for
requires full knowledge for donor HLA type and recent alloan- a variety of diseases. This susceptibility may somehow be
tibody profile of the recipient. Knowledge of the donor organ related to altered immunologic responsiveness in many
HLA type is often limited to HLA-A, HLA-B, and HLA-DR anti- cases. It is also probable that the diseases in question may
gens, while multiplex alloantibody testing is most commonly be a result of both multiple gene interactions and environ-
limited to anti-HLA antibodies, ignoring the contribution of mental factors. In some cases, it has been documented to
allotypic antibodies against MICA and other alloantigens. This result from presentation of unique pathogenic epitopes to
reinforces the necessity of serologic or flow crossmatch with T cells. Although the study of HLA and disease associations
donor cells being performed prior to any solid organ transplan- is very important in understanding disease susceptibility
tation to minimize the risk of hyperacute rejection. and manifestation, HLA alone is not clinically useful as a
diagnostic tool.
Clinical Significance of the HLA System
Platelet Transfusion
The identification of HLA antigens was driven by their
Hematopoietic stem cell transplantation and more aggressive
potential clinical application to transplantation. The HLA
use of chemotherapy in the treatment of malignancies have
system is still of primary clinical importance in transplanta-
led to a dramatic increase in platelet transfusions in the past
tion, but it has recently become of great interest to individ-
decade. Human leukocyte class I antigens are expressed
uals in the field of human genetics and to investigators
variably on platelets.54–58 Alloimmunization to the HLA
of disease associations. HLAs are associated with disease
results in refractoriness to random donor platelet transfu-
susceptibility to a greater extent than any other known
sions. Refractoriness is manifested by the failure to achieve
genetic marker in humans, and the HLA system has the high-
a rise in the circulating platelet count 1 hour after infusion
est exclusion probability of any single system in resolving
of adequate numbers of platelets. The refractory state is often
cases of disputed paternity.51
associated with lymphocytotoxic HLA antibodies.
Paternity Considering the highly polymorphic nature of the HLA
system, it is impossible to obtain sufficient numbers of HLA-
Relationship testing involves analyzing genetic markers typed donors to provide HLA-matched platelets for all alloim-
from the mother, child, and alleged father to determine munized patients. Duquesnoy and associates60 demonstrated
whether the tested man could be the biological father of a that platelet transfusions from donors mismatched only for
child. To accomplish this, the laboratory uses several tech- cross-reactive antigens can effectively provide hemostasis for
niques to accurately identify the polymorphic genetic mark- refractory patients. For example, an HLA-A1,B7; HLA-
ers in a paternity trio. Combinations of various genetic A11,B22 recipient might benefit from the platelets of an A1,B7;
markers are used, including RBC markers and enzymes, A3,B27 donor because A3 and A11 and B27 and B22 are cross-
serum proteins, HLA typing, and DNA testing.51 Previously, reactive. Table 21–6 lists the major cross-reactive groups and
the most common combination of testing included RBC the private specificities associated with each. As a result of
markers together with HLA typing. Owing to increased these observations, the donor pool necessary to sustain an
costs of HLA typing reagents and the significantly high HLA-matched platelet program can be reduced from 8,000 to
power of exclusion when testing multiple DNA loci, DNA 10,000 to manageable numbers of 2,000 to 5,000.
methodologies are used increasingly more than HLA typing Good platelet survival and HLA matching are not
(see Chapter 22, “Parentage Testing”). absolute. For example, poor transfusion results are some-
times obtained despite a perfect HLA match. Poor recovery
Disease Association may be a result of sensitization to non-HLA antigens, such
as platelet-specific antigens. In contrast, excellent trans-
It has been determined that HLA antigens are associated with fusion results are at other times obtained in the presence
disease susceptibility to a greater extent than any other of complete HLA mismatch. Good recovery may be a
known genetic marker.52 Many genetic markers have been function of:
suspected to be associated with disease, the most extensively
• A restricted pattern of alloimmunization—private versus
studied being blood groups, enzymes, and serum proteins.
public antibodies
Relative risk indicates how many times more frequently the
• Variable expression of HLA antigens on the platelet
disease occurs in individuals positive for the marker than
surface
in individuals negative for the marker. In contrast, the asso-
ciation between HLA-B27 and ankylosing spondylitis has Leukocytes are more immunogenic than platelets, and re-
a relative risk ranging from 60 to 100, depending on the fractoriness is probably initiated by HLA antigens on the
2682_Ch21_475-494 22/05/12 12:01 PM Page 488
B8 9.8
A26 4.8
B38 4.6
DQA1*0301/DQB1*0302 2
B17 5.3
B13 4.1
B7 2.9
B8 3.3
DRB1*0401/DQB1*0302 9.5
Myasthenia gravis B8 3.4
DRB1*03 2.5
*Relative risk of the disease in the white population. Frequencies for other races can be found in Tiwari, JL, and Terasaki, Pl: HLA and Disease. Springer-Verlag, New York, 1995, p 33.
contaminating leukocytes. Evidence for this is based on a might be useful for those patients who are unable to produce
study by Brand and others,60 in which they demonstrated a a response to HLA-matched platelets.
decreased rate of alloimmunization to random donor
platelets when contaminating leukocytes were removed Transfusion-Related Acute Lung Injury
before transfusion. Herzig and colleagues61 were also able
to improve the transfusion response to HLA-matched Transfusion-related acute lung injury (TRALI) is the clinical
platelets by removing the leukocytes. This type of approach syndrome of new acute lung injury (ALI) that develops with
2682_Ch21_475-494 22/05/12 12:01 PM Page 489
Table 21–6 Major CREGs is important for confirmation, particularly to evaluate the
potential risks of transfusing blood products from donors
MAJOR CREG ASSOCIATED SEROLOGIC ALLELES associated with TRALI. Demonstration of anti-HLA or anti-
A1 CREG A1, 36, 3, 9 (23, 34), 10 (25, 26, 34, 66), 11, 19 HNA antibodies will likely not affect treatment decisions
(29, 30, 31, 32, 33), 28 (68, 69), 43, 74, 80 regarding a patient with symptoms of TRALI, but they are
A2 CREG A2, 9 (23, 24), 28 (68, 69), B17
important to determine whether the donor associated with
the suspected causative blood product should be removed
B5 CREG B5 (51, 52), 15 (62, 63, 75, 76, 77), 17 (57, 58), from the donor pool.
18, 35, 12 (49, 50), 53, 70 (71, 72), 78, 37
B7 CREG B7, 42, 22 (54, 55, 56), 27, 40 (60, 61), 13, 41, Transplantation
46, 47, 48, 81, 37, 73
Clinical transplant immunology is a difficult field. Unlike
B8 CREG B8, 14 (64, 65), 16 (38, 39), 18, 59, 67 animal experimentation, in which studies are performed
B12 CREG B12 (44, 45), 21 (49, 50), 13, 40 (60, 61), 41, under controlled conditions in selected inbred strains,
48, 82 transplant immunology deals with actual patients with
different medical histories and backgrounds. Individuals
4c CREG A9 (23, 24), A25, A32, Bw4 working in transplant immunology must determine how
6c CREG Bw6, Cw1, Cw3, Cw7 best to select recipients and donors, when to increase or
decrease immunosuppressive treatment, and how to pre-
condition potential recipients so that their immune systems
will accept a graft. Decisions are based on the relative mer-
a clear temporal relationship to transfusion, in patients with its of laboratory findings viewed against complex medical
or without alternate risk factors for ALI.62 TRALI manifests histories. Differences of opinion exist from center to center
as fever, hypoxemia, and pulmonary edema occurring about the significance of immunologic test results and their
acutely (within 6 hours) following transfusion of blood com- considerations in clinical treatment protocols.
ponents. The phenomenon of respiratory distress following
transfusion has been observed for 60 years, though TRALI Hematopoietic Stem Cell Transplantation
has only been recognized as a distinct clinical entity since Hematopoietic stem cell transplantation (HSCT) is the term
1985.63 TRALI is the most severe adverse outcome from used for the transplantation of multipotent hematopoietic
transfusion, with estimates of prevalence ranging from 0.02% stem cells (HSC). Under normal conditions, HSC in the bone
to 0.16% of patients receiving transfusions.63,64 Treatment is marrow continuously generates red blood cells, leukocytes,
supportive, with supplemental oxygen and respiratory sup- and platelets. However, under certain conditions, including
port if necessary. Most cases resolve within 96 hours, though hematologic malignancies, congenital and acquired aplasias,
a mortality rate of 5% to 10% is reported.65 and after intense radiation and chemotherapy, the bone
The exact pathophysiology of TRALI is unclear, though marrow’s hematopoietic capability is compromised. In these
TRALI is strongly associated with the presence of allotypic instances it is beneficial to transplant HSC, either collected
antibodies present in donor blood products. Antibodies from the patient prior to bone marrow ablation (autologous
against HLA class I and class II molecules have been found HSCT) or from a healthy donor (allogeneic HSCT) to recon-
in 50% to 89% of products associated with TRALI.63,66,67 stitute normal hematopoietic function. Allogeneic HSCT
Additionally, antibodies to HNA-1a, HNA-1b, HNA-2a, enables reconstitution with fully functioning, healthy HSC,
and HNA-3a, alloantigens found specifically on neu- replacing those compromised by malignancy or metabolic
trophils, have been described in as many as 72% of blood defects, with demonstrated benefit in immune-mediated
products associated with TRALI.63 The current model of antitumor function (graft versus leukemia, GVL). However,
TRALI suggests that these allotypic antibodies activate it has adverse consequences, most notably the development
neutrophils in the lung, which release cytokine and of graft-versus-host disease (GVHD). Both GVHD and GVL
chemokine mediators that cause pulmonary edema.63 In are directly related to the degree of alloantigenic mismatch.
rare cases, TRALI has been described to be mediated by This has led to the development of several donor-recipient
allotypic antibodies in the recipient that react with donor matching strategies, each with distinct trade-offs between
granulocytes. However, no causative antibody can be GVHD and GVL.
found in over 15% of reported cases, demonstrating the Because of the continuous difficulty of finding well-
limitations of current testing.68 matched related (approximately 33% of patients) and
Suspected cases of TRALI are typically evaluated by ana- unrelated (less than 50%) donors for HSCT, umbilical cord
lyzing the donor blood product for anti-HLA and anti-HNA blood transplantation (UCBT) has increased over the
antibodies. This can be performed by immunoassay or by past 15 years. UCBT is an accepted alternative to HSCT,
serologic techniques. However, the diagnosis of TRALI is pri- because the functional and phenotypic immaturity of UCB
marily upon clinical symptoms correlated with a recent lymphocytes or the reduced T-cell dose contribute to UCB
transfusion event. While laboratory evaluation is not re- reduced alloreactivity. Therefore, limited HLA mismatches
quired to make a diagnosis of TRALI or begin treatment, it are better tolerated, and there is a decreased incidence
2682_Ch21_475-494 22/05/12 12:01 PM Page 490
of GVHD. The most limiting factor of UCBT is cell dosage and colleagues73 found that matching based on public
in that quite frequently there are insufficient hematopoi- cross-reactive antigens can provide the same association
etic stem or progenitor cells in one UCB unit. UCB dose is with graft outcome as private antigens. Matching for
of paramount importance in engraftment and survival after public antigens also promotes and increases the transplan-
unrelated UCBT. The single most important factor for the tation of minority patients. When matching for highly
potential increase of HSCT is expanding the donor pool sensitized recipients, by either private or public antigens,
to include both related and unrelated individuals. To meet identification of HLA serum antibodies is important.
the needs of patients who do not have a matched, related Oldfather and others74 observed that crossmatch results
donor, the U.S. Congress authorized a federal contract in can be predicted in highly sensitized recipients based on
1986 to establish a national marrow donor registry. careful analysis of serum HLA-antibody specificities. Thus,
In response to a request for proposal, the National single antigen antibody testing plays an enormous role in
Marrow Donor Program (NMDP) was formed with the detection of antibodies and, ultimately, the prediction
cosponsorship of the American Association of Blood of crossmatch results.
Banks, American Red Cross, and the Council of Commu- The third strategy is based on the induction of tolerance
nity Blood Centers. The goals were to recruit a large num- to donor-specific antigens. The evidence that transfusion
ber of informed HLA-typed volunteers to be listed as of blood products before transplantation promotes graft
potential marrow donors, to combine all available donor acceptance suggests that tolerance induction may be
HLA data into a centralized registry, and to establish a feasible. In 1973, Opelz and coworkers75 reported that
national coordinating center for facilitating donor searches blood transfusion might promote renal allograft survival
and communication between donor and transplant cen- in patients receiving kidneys from crossmatch-negative
ters. The NMDP currently operates the Be The Match donors. In their retrospective study, graft survival rates at
registry of volunteer donors and umbilical cord blood units 1 year were significantly improved in patients who had
in the United States. It is the world’s largest hematopoietic received more than 10 transfusions (66%), compared with
cell registry, listing more than 8 million individuals and patients who had received 1 to 10 units (43%) or no trans-
160,000 cord blood units. As of January 2010, the NMDP had fusions (29%). Salvatierra and colleagues76 applied this
facilitated more than 38,000 unrelated transplants worldwide. observation to the potential benefits of donor-specific
blood transfusions and transplants between living related
Kidney Transplantation individuals. The effects of blood transfusion on the success
Kidney transplantation is used to treat end-stage renal of renal transplantation are complex and paradoxical.
disease. Transplantation is preferred over dialysis in treat- Transfusion of blood usually leads to HLA alloimmuniza-
ing patients with chronic renal failure because it is more tion, and when this leads to HLA antibody production, it
cost-effective, and it usually returns patients to a state is difficult to find compatible donors. Yet graft survival
of relatively normal health. The best graft survival rates rates are improved when pretransplant blood is given to
occur when kidneys are obtained from HLA-identical, the recipient with no resulting sensitization. However,
ABO-compatible siblings, but such donors are available for with new drug therapies, transfusion protocols have not
relatively few patients.69,70 Three general strategies are been utilized due to the risk of recipient sensitization to
used by transplantation surgeons and immunologists to potential organ donors.
minimize graft rejection: Two modalities—plasmapheresis and intravenous
immune globulin (IVIG)—are used in the treatment of
1. Immunosuppressive agents
rejection post-transplant and in the pretransplant desen-
2. Reduction of graft “foreignness”
sitization of the patient.77 Plasmapheresis has been demon-
3. Induction of tolerance
strated to remove HLA-specific antibody in many different
Immunosuppressive agents such as azathioprine, pred- clinical settings.78,79 IVIG has been used to modulate
nisone, thymoglobulin, cyclosporine, and tacrolimus are immune responses and suppress alloantibody. Several
used to diminish the destructive immunologic responses groups have had success using IVIG to decrease levels of
to the graft. These agents are nonselective and carry risks anti-HLA antibody and to lower panel-reactive antibody
of serious side effects, especially life-threatening infec- among highly sensitized patients awaiting transplanta-
tion.72 Extensive efforts are used to minimize graft “for- tion.80,81 Most recently, rituximab has been added to
eignness” through matching of donor and recipient antirejection and desensitization treatments to destroy
antigens. Antigen disparities that most influence graft antibody producing B cells.
rejection include the ABO blood group antigens and the
HLA antigens. Although it is still not clear what combina- Heart Transplantation
tions of HLA gene products promote optimal graft survival Heart transplantation is used to treat cardiomyopathies
rates, it is evident that 0 and 1 antigen mismatches result and end-stage ischemic heart disease. Because of the
in increased graft survival.72 In highly sensitized recipi- organ’s extremely short total ischemic time (3 hours for
ents, it is necessary to match for HLA-A and HLA-B hearts, compared with 72 hours for kidneys), HLA match-
because of the presence of class I HLA antibodies. Sanfilippo ing and prospective crossmatching is not feasible. Total
2682_Ch21_475-494 22/05/12 12:01 PM Page 491
ischemic time is the amount of time there is no blood the HLA matching between donor and recipient may
flow through the organ. The single most important HLA play an important role in live-donor lung transplantation
pretransplant test is the HLA-antibody screen. Recipients (2% to 3%) in an attempt to improve post-transplant con-
with no preformed HLA antibodies receive transplants ditions and graft survival rates.85 Virtual crossmatching also
without crossmatching. Those with preformed HLA anti- plays an important role in those sensitized patients awaiting
bodies require either pretransplant crossmatches to deter- lung transplantation.
mine recipient-donor compatibility or donor HLA phenotype
to perform virtual crossmatching. Pancreas and Islet Cell Transplantation
The primary indication for pancreas transplantation is di-
Liver Transplantation abetes. The majority of pancreas transplants performed are
Orthotopic liver transplantation has become an established simultaneous pancreas and kidney transplants (81%), with
and successful therapeutic modality for patients with pancreas following kidney (12%) and pancreas alone (5%).
end-stage liver disease. Immunologic factors in recipient/ HLA matching, as reported by one of the largest pancreas
donor matching for liver transplantation and recipient transplant centers, has a major effect on graft survival,86
presensitization have largely been ignored in the past particularly in the pancreas after kidney and pancreas
because of the liver’s unique abilities to act as a sink for alone transplants. Because of increased risks of myocardial
anti-HLA antibodies and to regenerate itself if destroyed complications with pancreas transplantation, islet cell
by antibody. The consequences of HLA presensitization transplantation has been actively pursued. Although islet
and ABO incompatibility were recently underlined in cell transplantation is technically simple, difficulty has
three reports.82–84 In the first, a retrospective analysis of been encountered in achieving sustained engraftment in
preformed HLA antibodies demonstrated 1-year graft humans due to insufficient cell numbers. To overcome
survival rate of 40% in the presensitized individuals as this, sufficient islet mass is attained by transplanting islets
compared with 83% in the nonsensitized individuals. In from two donor pancreases when cell numbers are low. To
the second, survival of patients with emergency ABO- date, the effect of HLA matching has not been studied, but
incompatible transplants was 30% compared with 76% in data are being stored for future analyses.
patients with emergency ABO-compatible grafts and 80%
in patients with elective ABO-compatible grafts. United Network for Organ Sharing
In an effort to provide solid organs (a rare commodity)
Lung Transplantation equitably, the United Network for Organ Sharing (UNOS)
An overall review of the indications for lung transplantation was established in 1986. This organization received the
during the past several years reveals that emphysema and federal contract to operate the national Organ Procure-
cystic fibrosis account for the majority of double-lung trans- ment and Transplantation Network (OPTN) and to de-
plants. The major indications for single-lung transplantation velop an equitable scientific and medically sound organ
include pulmonary fibrosis (33%) and emphysema (41%). allocation system. The OPTN is charged with developing
In addition, single-lung transplants for primary hyperten- policies that maximize use of organs donated for trans-
sion are being performed instead of heart-lung transplanta- plantation, ensuring quality of care for transplant patients,
tion. As with hearts, short cold ischemic times for lungs and addressing medical and ethical issues related to organ
preclude prospective histocompatibility testing. However, transplantation in the United States.
SUMMARY CHART
The HLA genetic region is a series of closely linked The HLA genotype represents the association of the
genes located on the short arm of chromosome 6 that alleles on the two C6 chromosomes as determined
determine major histocompatibility factors—that is, by family studies, and the term haplotype refers to the
surface antigens or receptors that are responsible for allelic makeup of a single C6 chromosome.
recognizing and eliminating foreign tissues. The majority of HLA alloantibodies are IgG and can be
The HLA class I region encodes genes for the classic grouped into private antibodies (binding to an epitope
transplantation molecules HLA-A, HLA-B, and HLA-C; unique to one HLA gene product), public antibodies
the class II region encodes genes for the molecules (binding to epitopes shared by more than one HLA
HLA-DR, HLA-DP, and HLA-DQ; the class III region gene product), or cross-reactive (binding to struc-
encodes genes for C2, C4, Bf (complement factors), turally similar HLA epitopes) antibodies.
21-hydroxylase, and tumor necrosis factor.
Continued
2682_Ch21_475-494 22/05/12 12:01 PM Page 492
SUMMARY CHART—cont’d
Techniques of histocompatibility testing include anti- The general strategies employed by transplantation
gen and allele typing; HLA antibody detection and immunologists include use of immunosuppressive
identification, in which recipient serum is tested drugs, reduction of graft “foreignness,” and induction
against a panel of cells or a panel of single antigens; of tolerance.
and crossmatching, in which specific donor cells and Platelet refractoriness is manifested by the failure to
recipient sera are tested for compatibility. The ability achieve a rise in circulating platelet count 1 hour after
to perform “virtual” crossmatches due to the specific infusion of adequate numbers of platelets.
and sensitive single antigen screening assay has refined TRALI is the most severe adverse outcome from trans-
pretransplant histocompatibility testing and improved fusion manifested by fever, hypoxemia, and pulmonary
organ allocation algorithms. This has led to improved edema.
clinical outcomes.
13. Svejgaard, A, and Kissmeyer-Nielsen, F: Crossreactive human 37. Ishida, H, et al: Evaluation of flow cytometric panel reactive
HL-A iso-antibodies. Nature 219:868, 1968. antibody in renal transplant recipients—examination of 238
14. Legrand, L, and Dausset, J: The complexity of the HLA gene cases of renal transplantation. Transpl Int 18:163, 2005.
product: Possible evidence for a “public” determinant common 38. Patel, AM, et al: Renal transplantation in patients with pre-
to the first and second HLA series. Transplantation 19:177, transplant donor-specific antibodies and negative flow cytom-
1975. etry crossmatches. Am J Transplant 7:2371, 2007.
15. Rodey, G, et al: ASHI HLA class I public epitope workshop: 39. Amico, P, et al: Incidence and prediction of early antibody-
Phase I report. Transpl Proc 19:872, 1987. mediated rejection due to non-human leukocyte antigen-
16. Rodey, GE, et al: Public epitopes and the antigenic structure of antibodies. Transplantation 85:1557, 2008.
HLA molecules. Crit Rev Immunol 7:229, 1987. 40. Amico, P, et al: Clinical relevance of pretransplant donor-
17. Scalamogne, M, et al: Crossreactivity between the first and sec- specific HLA antibodies detected by single-antigen flow-beads.
ond segregant series of the HLA system. Tissue Antigens 7:125, Transplantation 87:1681, 2009.
1976. 41. Vlad, G, et al: Relevance of different antibody detection
18. Kostyu, DD, Cresswell, P, and Amos, DB: A public HLA antigen methods for the prediction of antibody-mediated rejection
associated with HLA-A9, Aw32, and Bw4. Immunogenetics and deceased-donor kidney allograft survival. Hum Immunol
10:433, 1980. 70:589, 2009.
19. Muller, C, et al: Monoclonal antibody (Tu 48) defining alloanti- 42. Reinsmoen, N, et al: Acceptable donor-specific antibody levels
genic class I determinants specific for HLA-Bw4 and HLA- allowing for successful deceased and living donor kidney trans-
Aw23, -Aw24 as well as -Aw32. Hum Immunol 5:269, 1982. plantation after desensitization therapy. Transplantation 86:820,
20. Belvedere, M, Mattiuz, PL, and Curtoni, ES: An antibody cross- 2008.
reacting with LA and four antigens of the HLA system. Im- 43. Phelan, D, et al: Living donor renal transplantation in the
munogenetics 1:538, 1975. presence of donor-specific human leukocyte antigen anti-
21. McMichael, AJ, et al: A monoclonal antibody that recognizes body detected by solid phase assay. Hum Immunol 70:584,
an antigenic determinant shared by HLA-A2 and B17. Hum Im- 2009.
munol 1:121, 1980. 44. Morris, G, et al: Virtual crossmatch by identification of donor-
22. Ahern, AT, et al: HLA-A2 and HLA-B17 antigens share an al- specific anti-human leukocyte antigen antibodies by solid
loantigenic determinant. Hum Immunol 5:139, 1982. phase immunoassay. A 30-month analysis in living donor
23. Claas, F, et al: Alloantibodies to an antigenic determinant kidney transplantation. Hum Immunol 3:268, 2010.
shared by HLA-A2 and B17. Tissue Antigens 19:388, 1982. 45. Lucas, ZG, et al: Early renal transplant failure associated with
24. Troup, CM, and Walford, RL: Cytotoxicity test for the typing subliminal sensitization. Transplantation 10:522, 1970.
of human lymphocytes. Am J Clin Pathol 51:529, 1969. 46. Patel, R, and Briggs, WA: Limitation of the lymphocyte cyto-
25. Eisen, SA, Wedner, HJ, and Parker, CW: Isolation of pure toxicity crossmatch test in recipients of kidney transplants
human peripheral blood T-lymphocytes using nylon wool having preformed anti-leukocyte antibodies. N Engl J Med
columns. Immunol Commun 1:571, 1972. 284:1016, 1971.
26. Lowry, R, et al: Improved B cell typing for HLA-DR using nylon 47. Bray, RA: Flow cytometry in the transplant laboratory. Ann NY
wool column enriched B lymphocyte preparations. Tissue A Acad Sci 677:138, 1993.
ntigens 14:325, 1979. 48. Bray, RA, et al: Transplanting the highly sensitized patient: The
27. Vartdal, F, et al: HLA class I and II typing using cells positively Emory algorithm. AM J Transplant 6:2307, 2006.
selected from blood by immunomagnetic isolation: A fast and 49. Vaidya, S, et al: Prediction of crossmatch outcome of highly
reliable technique. Tissue Antigens 28:301, 1986. sensitized patients by single and/or multiple bead luminex
28. van Rood, JJ, van Leeuwen, A, and Ploem, JS: Simultaneous assay. Transplantation 82:1524, 2006.
detection of two cell populations by two-color fluorescence 50. Bielmann, D, et al: Pretransplant risk assessment in renal allo-
and application to the recognition of B cell determinants. graft recipients using virtual crossmatching. Am J Transplant
Nature 262:795, 1976. 7:626, 2007.
29. Tiercy, JM, Jannet, M, and Mach, B: A new approach for the 51. Polesky, HF: Impact of molecular (DNA) testing on determi-
analysis of HLA class II polymorphism: HLA oligo typing. nation of parentage. Arch Pathol Lab Med. 123:1060, 1999.
Blood Rev 4:9, 1990. 52. Tiwari, JL, and Terasaki, PI: HLA and Disease. Springer-Verlag,
30. Zemmour, J, and Parham, P: HLA class I nucleotide sequences. New York, 1985.
Hum Immunol 31:195, 1991. 53. Mourant, AE, Kopec, AC, and Domaniewska-Solczak, K: Blood
31. Marsh, SGE, and Bodmer, J: HLA class II nucleotide sequences. Groups and Diseases. Oxford University Press, New York,
Hum Immunol 31:207, 1991. 1978.
32. Fuller, TC, et al: Antigenic specificity of antibody reactive in 54. Colombani, J: Blood platelets in HL-A serology. Transpl Proc
the antiglobulin-augmented lymphocytotoxicity test. Trans- 3:1078, 1971.
plantation 34:24, 1982. 55. Svejgaard, A, Kissemeyer-Nielson, F, and Thorsby, E: HL-A typ-
33. Fuller, TC, and Rodey, GE: Specificity of alloantibodies against ing of platelets. In Terasaki, PI (ed): Histocompatibility Testing
antigens of the HLA complex. In Theoretical Aspects of HLA: 1970. Munksgaard, Copenhagen, 1970, p 160.
A Technical Workshop. American Association of Blood Banks, 56. Leibert, M, and Aster, RH: Expression of HLA-B12 on platelets,
Arlington, VA, 1982, p 51. on lymphocytes, and in serum: A quantitative study. Tissue
34. Pei, R, et al: Simultaneous HLA class I and class II antibodies Antigens 9:199, 1977.
screening with flow cytometry. Hum Immunol 59:313, 1998. 57. Aster, RH, Szatkowski, N, and Liebert, M: Expression of HLA-
35. Pei, R, et al: Single human leukocyte antigen flow cytometry B12, HLA-B8, W4, and W6 on platelets. Transpl Proc 9:1965,
beads for accurate identification of human leukocyte antibody 1977.
specificities. Transplantation 75:43, 2003. 58. Duquesnoy, RJ, Testin, J, and Aster, RH: Variable expression of
36. El-Awar, N, Lee, J, and Terasaki, PI: HLA antibody identifica- W4 and W6 on platelets: Possible relevance to platelet trans-
tion with single antigen beads compared to conventional meth- fusion therapy of alloimmunized thrombocytopenic patients.
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59. Duquesnoy, RJ, Filip, DJ, and Rody, GE: Successful transfusion 74. Oldfather, JW, et al: Prediction of crossmatch outcome in
of platelet “mismatched” for HLA antigens to alloimmunized highly sensitized dialysis patients based on the identification
thrombocytopenic patients. Am J Hematol 2:219, 1977. of serum HLA antibodies. Transplantation 42:267, 1986.
60. Brand, A, van Leeuwen, A, and Eernisse, JG: Platelet immunol- 75. Opelz, G, et al: Effect of blood transfusions on subsequent kid-
ogy with special regard to platelet transfusion therapy. Excerpta ney transplants. Transpl Proc 5:253, 1973.
Medica International Congress 415:639, 1978. 76. Salvatierra, O, Jr, et al: Deliberate donor-specific transfusions
61. Herzig, RH, Herzig, GP, and Biell, MI: Correction of poor prior to living related renal transplantation: A new approach.
platelet transfusion responses with leukocyte-poor HLA- Ann Surg 192:543, 1980.
matched platelet concentrates. Blood 46:743, 1975. 77. Montgomery, RA, et al: Plasmapheresis and intravenous immune
62. Toy, P, et al: Transfusion-related acute lung injury: Definition globulin provides effective rescue therapy for refractory humoral
and review. Crit Care Med 33:721, 2005. rejection and allows kidneys to be successfully transplanted into
63. Popovski, MA, and Moore, SB: Diagnostic and pathogenic cross-match positive patients. Transplantation 70:887, 2000.
considerations in transfusion-related acute lung injury. Trans- 78. Ross, CN, et al: Renal transplantation following immunoad-
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64. Sillimann, CC, et al: Transfusion-related acute lung injury: 785, 1993.
Epidemiology and a prospective analysis of etiologic factors. 79. Hodge, EE, et al: Pretransplant removal of anti-HLA antibodies
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65. Popovski, MA, et al: Transfusion-related acute lung injury: A based therapy after heart-kidney transplant. Transpl Proc
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32:589, 1992. 80. Kickler, T, et al: A randomized, placebo-controlled trial of
66. Kopko, PM, et al: HLA class II antibodies in transfusion-related intravenous gamma globulin in alloimmunized thrombocy-
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67. Eder, AF, et al: Transfusion-related acute lung injury surveil- 81. Tyan, DB, et al: Intravenous immunoglobulin suppression of
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68. Engelfriet, CP, and Reesink, HW: Transfusion-related acute 82. Karuppan, S, Ericzon, BG, and Moller, E: Relevance of a posi-
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69. Siegler, HF, et al: Comparisons of mixed leukocyte skin graft 83. Gugenheim, J, Samuel, D, and Reynes, M: Liver transplantation
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70. Hamburger, J, et al: The value of present methods used for the donor-specific soluble HLA in the circulation of liver transplant
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71. Alexander, JW: Impact of transplantation on microbiology and 85. Shaw, LR, Miller, JD, and Slutsky, AS: Ethics of lung transplan-
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public and private specificities. Transplantation 38:483, 1984.
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Chapter 22
Relationship Testing
Robert W. Allen, PhD, and Chantal Ricaud Harrison, MD
Introduction Terminology and the Interpretation of Results Accreditation and Quality Issues
History Exclusion Criteria for Classical Systems Case Studies
Goals of Testing Exclusion Criteria for DNA Systems Case 22-1
Criteria for Selection of Genetic Systems Inclusionary Calculations Case 22-2
Types of Genetic Systems and Technology Used Paternity Index Summary Chart
RBC Antigens Probability of Paternity Review Questions
RBC Enzymes and Serum Proteins Probability of Exclusion References
Immunoglobulin Allotypes Nonclassic Situations Bibliography
HLA Advantages and Disadvantages of Genetic
DNA Polymorphisms: RFLP Systems
DNA Polymorphisms: PCR Social and Legal Issues
OBJECTIVES
1. State the goals of parentage testing.
2. List the criteria used to select a genetic system for parentage testing.
3. Briefly describe the testing technologies used for the different types of genetic systems.
4. Outline the advantages and disadvantages of the different types of genetic systems.
5. Define and give examples of false direct and indirect exclusions.
6. List at least two causes of false exclusions produced using serologic testing and one cause of false exclusion with DNA typing.
7. Define paternity index, probability of paternity, and power of exclusion.
8. List organizations involved and resources available in quality improvement for parentage testing laboratories.
495
2682_Ch22_495-508 22/05/12 12:04 PM Page 496
it can also be applied to determining maternity and other original genes. This group of genetic systems is often
kinships. termed the classic systems.
The molecular genetics technology developed over the
History next decade laid the foundation for a revolution in parentage
testing, heralded by the description by Jeffreys4 in 1985 of a
The processes currently used to resolve cases of questioned new type of polymorphism in human DNA: hypervariable
parentage can be traced back to the statistical analysis minisatellites often referred to as variable number tandem
published by Bernstein1 in 1924 demonstrating that the ABO repeats (VNTR). New genetic systems, tested by technolo-
blood group frequency distribution in Austria was most com- gies that directly assess the variability of DNA, have now
patible with a three-allele theory. ABO blood group test become the norm. These are called DNA polymorphisms.
results were first used to establish nonpaternity in Vienna in Over the past decade, there has been a rapid evolution of
1926. As other blood groups were described and their inher- the DNA polymorphism testing technologies used for
itance established, they were used with the ABO system for relationship testing and forensic analysis. Early on, VNTR
paternity studies. In 1955, Smithies2 described the charge systems were tested by treating the extracted DNA with re-
polymorphism of haptoglobin, which could be revealed striction enzymes followed by Southern blotting and
through gel electrophoresis; this opened the door to a new hybridization to VNTR probe(s). This technology, referred
type of genetic system: enzymes and proteins. to as restriction fragment length polymorphism (RFLP)
The next step occurred in 1972, when the extreme poly- testing, dominated the field for several years. Figure 22–1
morphism of the human leukocyte antigen (HLA) system is a schematic representation of RFLP analysis from
was demonstrated at the Evian workshop organized by genomic DNA.
Dausset.3 All these genetic systems were expressed on dif- However, RFLP testing ceded precedence to polymerase
ferent elements of the blood (red blood cells [RBCs], pro- chain reaction (PCR) technology. Originally, PCR was often
teins, and leukocytes), but they had one important aspect used to detect polymorphisms caused by point mutations
in common: The detection of the polymorphisms were all or sequence-specific polymorphisms but soon was used to
dependent on a complex biochemical expression of the analyze VNTR-type loci in which the repeated unit was small
Six blood groups have been routinely used in paternity test- FGA 4q28
ing: ABO, Rh, MNSs, Kell, Duffy, and Kidd. These systems HUMTHO1 11pl5–15.5
have been in use for a very long time, and their potential
pitfalls in interpretation are well known from the vast, world- TPOX 2p23–2pter
wide experience accumulated. Phenotype determination is VWA 12p12–pter
based on standard RBC agglutination techniques with regulated
2682_Ch22_495-508 22/05/12 12:04 PM Page 498
reagents and, with some extra built-in quality control steps, chapter, it comprises multiple linked loci expressed as class I
is indistinguishable from testing performed daily in most and class II antigens. In relationship testing with classic
blood banks or transfusion services. This is why, prior to the methodology (non-DNA), only antigens expressed by the A
introduction of DNA testing methods, the majority of labo- and the B loci are part of the testing performed.
ratories performing relationship testing were associated with The usual method for revealing the HLA phenotype is
a blood bank or a transfusion service. In performing serologic termed microlymphocytotoxicity. HLA phenotyping depends
testing, duplicate testing of each specimen was required, and on the evaluation of the reactions of live lymphocytes with
it was also recommended that testing be done by two indi- a panel of cytotoxic antibodies of known specificity in the
viduals using two different sources of antisera. Such test presence of complement. Testing is performed in 60 or
redundancy ensured the accuracy of test results traceable to 72 microwell trays preloaded with reagent antisera. A suffi-
interpretive errors and antiserum reagent variability. There may cient number of antisera should be used so that all HLA-A
be only one or two laboratories that still conduct antigen and HLA-B specificities recognized by the 1980 HLA Nomen-
testing for cases of questioned parentage. The College of clature Committee of the World Health Organization can be
American Pathology, which administers the only proficiency reliably identified. Additional antigens should be tested if
test for parentage, no longer reports participant responses appropriate antisera can be obtained. The larger the number
(because no laboratory submits them) for serologic testing; of antigens that can be defined, the more powerful the
therefore it is difficult to know the number of laboratories system becomes—that is, the better it can exclude falsely
still using this technology as part of their testing. accused men. Because of the large number of existing anti-
gens, no single tray can identify all relevant antigens. Some
RBC Enzymes and Serum Proteins antigens occur more frequently in specific racial groups, and
specially designed trays are available for African Americans
Testing of RBC enzymes and serum proteins is also rarely, if
and Asians. Such trays should be selected when appropriate.
ever, performed. Testing methodology consists of separating
Antisera are of human origin (alloantisera) or are mono-
the different allelic proteins based upon net charge using
clonal (created in the laboratory), and monospecific sera are
electrophoresis, followed by staining. Subtyping of the PGM1
generally not available for a large number of antigens. It is
and Gc alleles may be done using isoelectric focusing, which
required that each antigen be defined by at least two different
allows separation of molecules with only slightly differing net
operationally monospecific sera, by one monospecific and
charge, thereby increasing the extent of polymorphism of the
two multispecific sera, or by three multispecific sera. Phe-
system. When isoelectric focusing is used, it is customary to
notypes must be verified by reading two independent trays
refer to the system with a subscript letter i (e.g., PGM1i or Gci).
or tray sets. Each tray or tray set must be read independently.
Phenotypes are identified by the number or respective
In interpreting phenotypes, a certain amount of expertise
position of the bands detected and comparison with control
is needed, requiring knowledge of cross reactivity between
specimens expressing at least two known allotypes that are
antigens and splits of broader reactivity (e.g., A9 splitting into
tested in parallel with the unknown specimens. Interpreta-
A23 and A24) and familiarity with the unexpected reactivity
tion of the band patterns must be performed independently
of the antisera used (false-negative or extra reactions).
by two observers. Rare variants exist in most systems, and
Because of the variability of HLA antisera, it is recommended
the laboratories should maintain a file of variants to permit
that all individuals in a parentage case be tested in the same
identification of rare phenotypes when they are encountered.
laboratory, ideally with the same reagents.
Immunoglobulin Allotypes
DNA Polymorphisms: RFLP
Three immunoglobulin chains demonstrate a polymorphism
RFLP refers to polymorphisms in DNA that can be revealed
that has been applied to paternity testing. The gamma heavy
by restriction enzyme digestion because of nucleotide
chain expresses the Gm polymorphism, the kappa light chain
sequence differences that either create or destroy restriction
expresses Km (also termed Inv), and the alpha heavy chain
sites; this alters their linear arrangement on a chromosome
expresses Am. Determination of the phenotype is done by
and, hence, the size of DNA fragments produced by the
hemagglutination inhibition using indicator RBCs coated
accompanying digestion. Restriction enzymes recognize
with antibodies of known phenotype and reagent anti-Gm,
specific DNA sequences of four to six bases and cut the
anti-Km, or anti-Am. Reagents are not widely available,
double-stranded DNA at that site. These enzymes have been
genetic interpretation is complex, and phenotypic interpre-
isolated from bacteria and are named after the bacteria from
tation is often not possible in infants younger than 6 months
which they were isolated (e.g., Eco RI from Escherichia coli
of age because of interference with maternal immunoglobu-
RY13; Hae III from Haemophilus aegyptius).
lins. For these reasons, few laboratories in the world now
After DNA is incubated with the restriction enzyme (diges-
use these genetic systems. They are described here solely for
tion step), it is electrophoresed in an agarose gel to separate
the sake of completion and historical perspective.
the DNA fragments according to length. DNA present in the
HLA gel is then denatured and transferred by blotting (Southern
blotting) to a membrane that is then subsequently hybridized
The HLA complex represents the most polymorphic genetic with a labeled probe to reveal the DNA fragments correspon-
system in the human genome. As discussed in the previous ding to the locus tested. Phenotypes are defined as the length
2682_Ch22_495-508 22/05/12 12:04 PM Page 499
of the DNA fragments in kilobases (kb), estimated through polymorphisms, it is VNTR-type polymorphisms that were
comparison to a sizing ladder consisting of DNA fragments of the target of RFLP analysis by parentage laboratories because
known size. Some variability in migration may occur within of their high discriminatory power characteristics. As shown
a gel, depending on the position of the electrophoretic lane in Figure 22–2, the fragment size of VNTR alleles depends on
(band shifting), and it is recommended that mixtures of DNA the number of repeats they contain and the restriction enzyme
of each alleged parent with each child be electrophoresed utilized. This is an important concept: The phenotype of the
together in a single lane to maximize resolution and to normal- same individual at the same locus will differ between two lab-
ize potential electrophoretic anomalies. Ultimately, the goal of oratories if each uses a different restriction enzyme. This is
the process is to confirm or refute sharing of alleles between why, when reporting RFLP results, it is mandatory to identify
alleged parent and child. Figure 22–2 illustrates this process. not only the locus and probe tested but also the restriction en-
Although RFLP analysis is a suitable technique to reveal zyme used. In the United States, the most common restriction
insertion or deletion or single nucleotide substitution–type enzymes used were Hae III and Pst I, whereas in Europe, Hinf
I was more popular. Table 22–2 illustrates the different band
sizes obtained on the same individuals at the same locus when
using different restriction enzymes.
An important limitation of RFLP analysis of VNTR poly-
morphisms is that allele identification is based upon an
estimate of the size of a restriction fragment that generally
consists of thousands of nucleotides. As in any quantitative
measurement, there is variability, which is referred to as
sigma (σ). One variable is the ability to distinguish two
alleles that are similar in size (alleles differing by only a few
repeats). Resolution may vary greatly among laboratories,
depending on the methodology utilized, especially the
technical conditions used during electrophoresis. The limit
of resolution is referred to as delta (δ).
Because of these two sources of variability, allele frequency
distribution of VNTR alleles using RFLP analysis is continu-
ous as opposed to the discrete distribution of allele frequencies
as in the classic genetic systems. For example, in most labo-
ratories, a DNA fragment measured at 1.60 kb cannot be
considered different from a fragment measured at 1.57 kb or
1.63 kb. Through the use of validation, each laboratory must
identify the limit of resolution for which two closely spaced
alleles cannot be distinguished from one another. This range
of “matching sizes” is called a bin. The allele frequency used
in the statistical calculations is actually the frequency of all
the alleles falling into the bin for the allele size measured.
Since the bin size varies with the technical characteristics
of the laboratories, the final statistical result (i.e., likelihood
ratio, probability of parentage, or probability of exclusion)
may differ between two laboratories for the same locus
with the same restriction enzyme, even if their band sizes are
identical.
These VNTR loci are very polymorphic and thus are very a thousandfold; this made it possible to use buccal swabs as
powerful for excluding falsely accused men and in providing a source of DNA instead of blood samples, which were often
compelling inclusionary evidence in true biological relation- difficult to collect from newborns. The reduced quantity
ships. However, their mutation rate can be up to 2 per 1,000, of DNA needed also allowed for direct DNA testing from
which is small but not negligible.5 In the past 5 years, labo- prenatal samples of amniotic fluid or chorionic villi (CVS).
ratories have stopped using RFLP to test VNTR genetic A second advantage of STR analysis is the increased res-
systems, moving instead to technologies that involve PCR olution of alleles associated with polyacrylamide gel elec-
amplification of VNTR-type polymorphisms. trophoresis (PAGE) and capillary electrophoresis (CE),
which can reliably distinguish two DNA fragments differing
DNA Polymorphisms: PCR by a single base pair. With the enhanced resolution of the
electrophoresis systems came the ability to designate STR
The transition of relationship testing laboratories from serol- alleles as discrete alleles, naming each based upon the
ogy and HLA to RFLP analysis was short lived, largely be- number of repeats of the tetranucleotide or pentanucleotide
cause of the characterization of VNTR loci consisting of contained within the PCR amplicon. Eliminating the use of
tetranucleotide repeats that could be amplified using PCR. bins in the matching process and in determining allele fre-
Figure 22–3 shows PCR amplification of STR loci using flu- quencies also greatly improved the acceptance of STR typing
orescent detection. The figure shows STR amplification in court. Third, the technology allows simultaneous testing
products separated in one of four capillaries in the array that of several loci by multiplex amplification and very fast allele
are labeled with either red or green fluorescent dye (depend- identification by capillary electrophoresis. This is conducive
ing on the particular STR locus). Electropherograms are to automation and a faster turnaround time.
displayed as fluorescent bands (right panel) or as a his- The panels of STR loci currently in use consist mostly of
togram, the latter of which is the more common method for repeats with core sequences of four bases (tetranucleotide
STR profile presentation. The banding results are correlated repeats), although a few loci based on core sequences of five
with the histogram by capillary number. The lane or his- bases (pentanucleotides) have recently been introduced. A
togram panel shown in capillary #2 represents an allelic group of 12 loci dominates the field in conjunction with a
ladder containing all common alleles in the population for 13th locus, amelogenin, that identifies the sex of the sample
the different STR loci amplified and is used as a reference to donor and nothing else. This group has been selected by the
identify the alleles in unknowns. FBI as the core testing panel for a nationwide databank of crime
DNA typing using STR loci and PCR amplification elimi- scene DNA evidence material and reference samples from con-
nated many of the shortcomings associated with RFLP analy- victed felons. Box 22–2 lists the STR loci included in the FBI
sis. The amount of DNA required for testing dropped about Combined Offender DNA Index System (CODIS) database.
Figure 22–3. PCR amplification of STR loci using fluorescent detection. Genomic DNA recovered from a biological sample is added to a multiplex PCR reaction containing
fluorescently labeled primers for multiple STR loci. Following amplification, PCR products are separated by size using polyacrylamide gel or, in this case, capillary electrophoresis.
The gel or capillary is then irradiated with a laser, causing the STR alleles to emit light at several wavelengths depending on the particular fluorescent dye coupled to the
different primers. In this figure, STR alleles are displayed in one of two formats—horizontal bands in a gel track much like traditional RFLP profiles or in the newer histogram
format that is the norm among STR typing laboratories that use capillary electrophoresis platforms to separate STR alleles. In addition, whereas current STR multiplex kits contain
PCR primers targeting as many as 16 different loci and labeled with 4 different fluorescent dyes, the allele pattern shown in Figure 22–4 has been simplified to show only the
STR loci amplified from primers labeled with green or red fluorescent dyes. Size of the PCR amplicons and the color emitted serve to identify the particular genetic marker
visualized and the particular allele present in a sample.
2682_Ch22_495-508 22/05/12 12:04 PM Page 501
homozygosity in a true biological parent/child pair. Such Although the mechanism underlying alterations in repeat
alleles exist at a low but significant frequency in most of the number resulting from mutation in VNTR loci is not certain,
classical systems. current evidence does not support unequal recombination
between sister chromosomes during meiosis as the mecha-
Exclusion Criteria for DNA Systems nism. Rather, it appears the replicative complex responsible
for DNA replication “slips and jumps” as it moves down a
One consequence of the technology shift to DNA-related chromosome. If the complex “slips” and remains in one spot
methods was the discovery of the rather high mutation rate on the chromosome while replicating, a new repeat may be
characteristic of the VNTR systems.5,8 Such mutation rates added to the daughter strand. If the complex “jumps” over
were sufficiently high to cause a laboratory to be wary of sin- a repeat due to a loopout of the DNA template or for some
gle DNA mismatches between alleged parent and child as other reason, a repeat may be lost from the daughter chro-
compelling evidence of exclusion. Mutations associated with mosome. The net effect of either occurrence will be the
VNTR loci are generally the result of DNA replication errors production of a gamete harboring a genetic marker distinct
that occur during meiosis and alter the number of repeats in from the parent. If this gamete is involved in conception, the
the tandem array. Changes in the number of repeats is usu- offspring will differ from the parent. When an inconsistency
ally small (i.e., 1% to 3% of allele size for VNTR loci detected in the inheritance pattern is detected in a DNA system, the
with RFLP methods and single repeat changes for STR loci), term exclusion is not used; instead, the terms mismatch or
and repeats can be added to or subtracted from daughter inconsistency are used to indicate that the alleles between the
alleles with approximate equal frequency.5,8 There is a con- child and the alleged father do not match. The term exclusion
firmed difference in mutation rate in the male versus female is reserved for the final interpretation of the entire set of
lineages, with males exhibiting a 10- to 20-fold higher rate genetic systems tested. A minimum of two mismatches is
than females for some loci, possibly due to an increased required before an opinion of nonpaternity (or nonmater-
number of cell divisions that occur during spermatogenesis nity) is rendered. Interestingly, because of the large number
(Table 22–3). of STR loci tested routinely, some laboratories require three
mismatches to conclusively exclude an alleged parent, espe-
cially if all three mismatches involve single repeat changes.
Table 22–3 Summary of Mutation Rates
for STR Loci Inclusionary Calculations
PATERNAL MATERNAL Three types of statistical evaluations can be calculated when
LOCUS LINEAGE LINEAGE the alleged father is not excluded: the parentage index (PI),
CSF1PO 0.002021 0.000283 the probability of paternity (PP), and the probability of
exclusion (PE). The three calculations producing the values
D13S317 0.001743 0.000436 defined by these terms are based on the assumption that the
D16S539 0.001127 0.000481 “random man” with whom the tested man is compared is
biologically unrelated to him or to the mother. The standard
D18S51 0.00253 0.000748 calculations are not valid if a brother, father, uncle, or other
D19S453 0.000745 0.000596 close biological relative of the tested man is a possible bio-
logical father of the child.
D21S11 0.001709 0.001295 Another important aspect of the calculations is the need
D2S1338 0.001526 0.000245 to have accurate allele frequencies for each genetic system
tested, estimated from databases drawn from an adequate
D3S1358 0.001691 0.000211 sample population of the appropriate ethnic group. When
D5S818 0.001742 0.0003 referring to a sample size for a database, the term adequate
will depend largely upon the degree of polymorphism exhib-
D7S820 0.001348 0.000073 ited by the genetic marker in question. In general, the larger
D8S1179 0.002031 0.000333 the number of alleles exhibited by the marker, the larger the
population sampling needed to create a valid allele frequency
FGA 0.003713 0.000522 database. In contrast, databases created for STR loci may
PENTA D 0.000259 < 0.000253 consist of only 200 to 300 individuals because there are far
fewer alleles possible.
PENTA E 0.00026 < 0.000253
parent. The denominator mathematically states the proba- allele in the child) is 16. Alleged father #1 (AF1) is heterozy-
bility of producing the child through a mating of the known gous for this allele; thus, his chance of passing it on to
parent with a randomly selected, unrelated individual in the an offspring is 50% (i.e., 0.5). Alleged father #2 (AF2) is
same ethnic population. The PI is calculated one genetic homozygous for allele 16 and thus will pass it to 100% of his
system at a time and is sometimes referred to as the system offspring. The population frequency for allele 16 is 0.232.
index (SI). If all the genetic systems tested are independent Therefore, the system index calculation for AF1 takes the
from one another, the overall final PI is the product of all following form:
the system indices calculated. When the PI is greater than
SI = [(0.5M passing 18)(0.5AF passing 16)]/[(0.5M
100, the evidence for paternity is considered very strong.
passing 18)(0.232RM passing 16)]
Figure 22–4 shows STR typing in a paternity case involving
SI = 0.5/0.232 = 2.16.
two alleged fathers, and Table 22–4 outlines the testing
results for the case. For the D3S1358 system, the mother and Therefore, based upon this test result alone, AF1 is
child share the 18 allele, thus the obligatory gene (paternal 2.16 times more likely to be the father of the child than is a
Figure 22–4. STR typing in a paternity case involving two alleged fathers. DNA was extracted from buccal swabs from two alleged fathers (#1 and #2), a child, and a
mother and subjected to STR analysis using the Identifiler STR typing kit (Applied Biosystems, Foster City, CA). Products were separated and analyzed using capillary elec-
trophoresis on an ABI310 Genetic Analyzer (Applied Biosystems, Foster City, CA). Shown in the figure are the results from those loci labeled with the green fluorescent dye
known as JOE consisting of 5 of the 15 STR loci co-amplified with the Identifiler kit. Shown under each peak in the histogram are labels containing the allele identification
(labeled “al”), the size of the amplicon (labeled “sz”), and the intensity of the fluorescence (labeled “ht”). The results of the analysis are shown in Table 22–4.
2682_Ch22_495-508 22/05/12 12:04 PM Page 504
Table 22–4 System Indices Produced in a Paternity Case with Two Alleged Fathers
SYSTEM MOTHER CHILD ALLEGED FATHER #1 PI (AF#1) ALLEGED FATHER #2 PI (AF#2)
D3S1358 15, 18 16, 18 16, 17 2.1598 16 4.32
PI = Parentage Index
who would not be excluded; this number is called random in inclusion cases give extremely consistent results between
men not excluded (RMNE). The probability of exclusion is laboratories.
1 – RMNE. If p is the sum of all frequencies of the possible However, they do have some serious drawbacks when
paternal alleles, then RMNE = p (2 – p) for each genetic compared with the newer technologies that test DNA
system. The overall RMNE for all the genetic systems tested polymorphisms. In particular, each genetic system, except
is the product of the RMNE values for each system, and the for the HLA-A and HLA-B antigens, has a relatively low
overall probability of exclusion is 1 – overall RMNE. In the average power of exclusion. The highest power of exclu-
case shown in Table 22–4, the overall RMNE is: sion for a classical system is achieved by PGM1i at 0.32,
followed by MNSs and Gci at 0.31 and Rh 0.27, respec-
0.410 × 0.560 × 0.197 × 0.316 = 0.0075,
tively. Virtually all other systems have a power of exclusion
and the combined probability of exclusion is 0.9925. The below 0.20. In contrast, the HLA-A and HLA-B antigens,
probability of exclusion represents the proportion of analyzed as a haplotype, have a power of exclusion of
untested men who would have been excluded by the extent 0.87. By testing all the classic systems, a high power of
of testing performed (99.25% in this case) and can some- exclusion can be achieved, but it is almost impractical to
times be more easily understood by a layperson, such as go beyond 0.99. In addition, to get the full benefit of these
a jury member or a judge. The PP is a more accurate way systems, one must be competent with a multiplicity of
to quantify the situation, because it includes all the genetic techniques.
information available, whereas the probability of exclusion Fairly rigid restrictions exist on the type, amount, and
does not take into account the phenotype of the alleged quality of samples needed for testing. Because of the small
father. number of alleles existing in each system, often no more than
two or three, serologic test batteries lack efficacy in nonclas-
Nonclassic Situations sic cases. Finally, because serologic markers represent the
products of genes, mutations in those genes altering expres-
The majority of parentage testing cases consist of the classic
sion can complicate the interpretation of parentage test
trio of mother, child, and alleged father. However, nonclassic
results, producing false direct and false indirect exclusions
cases are more and more frequently encountered. This is
of alleged parents who are true parents of a child.
largely because the newer DNA technologies are better able
The DNA polymorphisms by RFLP are extremely power-
to give reliable answers in these situations. It is not unusual
ful genetic systems with average powers of exclusion almost
to attempt to establish the presence or absence of a biological
always above 0.50 and often reaching above 0.90 per genetic
relationship between an alleged parent and a child when the
system. Thus, testing only four different loci by DNA-RFLP
known parent is unavailable for testing.9 Calculations in
ensures very high levels of PP for nonexcluded men. The
such situations follow the typical procedure with the excep-
sample required is any tissue or body fluid from which suf-
tion that a “random known parent” is substituted into the
ficient DNA can be extracted. Peripheral blood, buccal
PI equation in place of the known transmission frequency
smears, amniotic fluid, chorionic villi, and tissue biopsies
for the allele shared between the known parent and the child,
can be analyzed. Once extracted, DNA can be kept frozen
which is generally 0.5 to 1 (depending on the zygosity).
for an indefinite period. However, the technique is time-
When a “random known parent” must be included in the
consuming and labor-intensive, and turnaround times are
calculation, both alleles in a heterozygous child could have
long. The main drawback, however, may be the lack of
come from the known parent and their respective transmis-
precise correlation in the final PI value between laboratories
sion probabilities are set to equal the population frequency
testing the same loci. This is because, as previously dis-
for each.9
cussed, the phenotypes consist of measured band sizes, with
Occasionally, the alleged parent is deceased, and a recon-
the unavoidable difficulties caused by a built-in incertitude
struction of his or her possible genetic makeup is attempted
in measurement. This can be very confusing when results
by testing all available and known parents, siblings, and
from a case tested in several different laboratories are com-
other undisputed children of the alleged father. Other bio-
pared, and the results may give a false impression of unreli-
logical relationships, such as siblings, may be in question in
ability to someone who is unfamiliar with the limitations of
adoption, immigration, or inheritance cases.
the methodology.
Advantages and Disadvantages The DNA polymorphisms analyzed using PCR avoid
of Genetic Systems this difficulty because they return to a discrete allele dis-
tribution. When identifying alleles of an STR genetic
The classic systems have the advantage of the vast experience system based on fragment length polymorphisms, one can
accumulated over the decades during which they have been actually identify the number of repeats and not simply a
in place in many countries. Phenotypes are well recognized length of DNA subject to an imprecision in measurement.
and consistently reproducible from laboratory to laboratory. This technique has the least restriction in the amount and
Potential pitfalls have been thoroughly studied, gene frequency type of sample needed but the highest requirement for
distributions are available for almost every population in environmental controls to prevent sample contamination
the world, and the mutation rate has been shown to be because it is based on high amplification of a very small
extremely low (less than one in a million). Statistical analyses amount of DNA.
2682_Ch22_495-508 22/05/12 12:04 PM Page 506
A great advantage of DNA-PCR technology is the ability the documented evidence every step in the handling of
to amplify as many as 15 or more loci in a single amplification the case.
reaction. Current instrument technology allows the routine
resolution of 15 STR loci with analysis within 4 to 5 hours. Accreditation and Quality Issues
Individually, these systems are not as powerful as individual
DNA-RFLP systems, and so two to three times the number Approximately 30 years ago, the American Association of
of STR markers need to be analyzed to achieve the same Blood Banks (AABB) took the lead in promoting the establish-
discriminatory power as DNA-RFLP systems. Both types ment of standardization and quality in the field of paternity
of DNA polymorphisms have in common a significantly testing and created a standing committee on parentage testing.
higher mutation rate than the classic systems have. As This committee offered educational opportunities such as
previously discussed, this has resulted in abandoning the workshops and publications. With the participation of the
concept of direct and indirect exclusion that has been so American Medical Association, the American Bar Association,
effective in classic systems. A mismatch observed in a DNA and the Office of Child Support Enforcement, the Committee
polymorphism system should lead to the calculation of a on Parentage Testing organized an international conference in
system index, which takes into account the rate of mutation 1982 in Airlie, Virginia, where a group of international experts
for that system. A single mismatch should never lead to an established a consensus approach for the interpretation of
interpretation of nonpaternity but rather to the testing of inclusionary evidence. An accreditation program for parentage
additional systems. testing laboratories was implemented, and the first edition of
Standards for Parentage Testing Laboratories was published in
Social and Legal Issues 1990. A complementary Accreditation Requirements Manual
followed in 1991. The College of American Pathologists, with
Currently, more than 400,000 cases of relationship testing joint sponsorship by the AABB, initiated a proficiency testing
are performed every year in the United States (www.aabb.org/ program for parentage testing laboratories in 1993.
sa/facilities/Documents/rtannrpt08.pdf). The majority of Continuing involvement by professional organizations in
these cases are done to establish paternity for children, with the promotion of quality improvement and in the provision
the aim of obtaining child support. Child support enforce- of opportunities for continuing education in the field of pa-
ment agencies in all states actively pursue the identification ternity testing is essential to maintain a high level of accuracy
of the biological father of children who are eligible for their and consistency in ascertaining biological relationships (see
Aid to Families with Dependent Children for the purpose Chapter 23, “Quality Management”).
of seeking child support. Contracts with child support
agencies account for most of the activity of many relation-
ship testing laboratories. The majority of cases outside CASE STUDIES
the child support enforcement agencies also have a legal
implication, such as child support, custody in divorce Case 22-1
cases, inheritance rights, immigration, and more recently,
the identification of human remains in natural disasters or An African American trio consisting of a mother, child,
in cases of murder. Occasionally, cases also have criminal and alleged father is tested to establish paternity. The
implications relating to incest or statutory rape. STR testing following results are obtained:
is also widely used in other areas of medicine, including Genetic System Mother Child Alleged Father
monitoring engraftment in patients who received a bone ABO O B A
marrow transplant, assessing the purity of DNA presumably D3S1358 15,18 16,18 16,17
extracted from pregnant mothers that may actually repre- THO1 7,9 7,9 6,7
sent a mixture of maternal and fetal DNA, and the identifi- D13S317 11,12 12 8,11
cation of pathological specimens thought to perhaps D16S539 9,12 9 9,12
be mislabeled or contaminated with someone else’s tissue D2S1338 18,20 17,20 17,18
during processing.
Regardless of the application for the technology, every 1. Which of the genetic systems tested excludes the AF?
step in the collecting, storing, processing, and testing of the a. THO1 and D16S539
samples must be carefully documented as to time of occur- b. ABO and D13S317
rence and person performing the task. An unbroken chain c. ABO and THO1
of custody must be maintained. The individuals tested must d. No system excludes the AF
be carefully verified, and procedures must be in place to 2. Given the test results in question 1, if this was the only
ensure that unauthorized persons do not have access to the testing performed, would you exclude the AF based
samples or test results. Without careful attention to these upon currently accepted standards for parentage testing?
aspects, the results may not be valid in court. If called to a. Yes
testify on the validity and significance of the testing results, b. No
the laboratory director must be able to re-create from
2682_Ch22_495-508 22/05/12 12:04 PM Page 507
SUMMARY CHART
Relationship (parentage testing) refers to the testing of STR present on the Y chromosome can verify only
genetic markers that are inherited to determine the male lineage.
presence or absence of a biological relationship. Mitochondrial DNA can verify only maternal lineage.
The ultimate goal of relationship (parentage testing) is The term mismatch is used when the bands between
to confirm a specific biological relationship with the the child and the alleged father do not match; a mini-
individual in question, usually a father or a mother or mum of two mismatches is required before an opinion
sometimes a sibling. of nonpaternity (or nonmaternity) is rendered.
The blood group systems used most often in paternity A direct exclusion occurs when a marker is detected
testing are ABO, Rh, MNSs, Kell, Duffy, and Kidd. in the child and is absent in the mother and the alleged
The HLA complex represents the most polymorphic father or when the alleged father’s phenotype demon-
genetic system in the human genome; only antigens strates two markers and the child has neither one
expressed by the A and B loci are considered for of them.
relationship testing with non-DNA-based typing. An indirect exclusion occurs when a single marker is
RFLP refers to polymorphisms of the DNA that can be detected in the child and a different single marker is
detected by restriction enzymes; polymorphisms detected detected in the alleged father.
in relationship testing relate to the presence of VNTR. False direct exclusions can occur as the result of muta-
In PCR, the DNA fragment of interest is amplified in tions that are significant enough to alter the final prod-
amount thousands of times, and the resulting product uct, lack of precursor substance, suppressor activity at
is normally identified directly through fluorescence re- a locus unlinked to the one tested, or chimeric state of
sulting from fluorescently labeled primers incorpo- one of the tested individuals.
rated in the PCR reaction. False indirect exclusions occur secondary to the pres-
STRs have repeats that are 4 to 5 bp long. ence of silent alleles (e.g., Fy).
2682_Ch22_495-508 22/05/12 12:04 PM Page 508
Chapter 23
Quality Management
Lucia M. Berte, MA, MT(ASCP)SBB, DLM, CQA(ASQ)CMQ/OE
OBJECTIVES
1. Explain the differences between compliance and quality management.
2. List the three building blocks of quality.
3. Describe the framework of a quality system for a blood bank and a medical laboratory.
4. List 12 quality management system (QMS) essentials and the blood bank operations to which they are applied.
5. Describe a process using a flowchart.
6. Explain the role of validation in introducing a new process.
7. Name at least five blood bank process controls.
8. Describe the differences between a form and a record.
9. Explain the importance of document control for procedures.
10. State the differences between remedial and corrective action.
11. Describe the role of auditing in a QMS.
12. Identify at least six sources of input that can be used to improve processes.
13. Define the activities in a problem-solving process.
14. Describe the four types of quality costs.
15. Explain how to transition the QMS of a blood bank into that for a whole laboratory.
509
2682_Ch23_509-525 22/05/12 12:04 PM Page 510
Quality Assurance
BOX 23–1
QA is a set of planned actions that ensure that systems and Common Blood Bank QC Activities and QA Indicators
elements that influence the quality of the product or service
are working as expected, individually and collectively.8 QA QC Activities QA Indicators
looks beyond the performance of a test method or piece of Collection Equipment • Number of donor forms with
equipment; it addresses how well an entire process, which • Microhematocrit instrument incomplete or incorrect
is a sequence of activities, is functioning. This is particularly information
• Hemoglobin instrument
important in those processes that cross functional or depart- • Number and types of
• Apheresis equipment unusable units and blood
mental lines. For example, a blood center could monitor the • Blood-weighing scales components
number of times and reasons why a set of collected whole • Number of blood typing
Blood Components
blood units transported from the collection site to the discrepancies in donors and
component processing site did not arrive in time or was not • Red blood cell hematocrit patients
in an acceptable condition to make blood components. In • Cryoprecipitated • Number of and reasons for
antihemophilic factor invalid tests
the transfusion service, it is important to monitor the source,
• Platelet counts in platelet units • Number of and reasons for
the number of times, and the reason why specimens
• Residual leukocyte counts in labeling check failures
collected for compatibility testing do not meet predetermined leukocyte-reduced components
acceptance criteria. Box 23–1 lists common QC and QA • Number and source of
• Bacterial contamination of improper and incomplete
activities and indicators practiced by most blood banks.9 platelet units requests for blood
Reagents components
Quality Management Systems • Number and location of
• Copper sulfate patients without proper
A quality guideline published by the FDA10 set the meaning, • Reagent red blood cells identification at time of
emphasis, and organization of quality activities for blood • Reagent antisera specimen collection or
banks. Accrediting agencies such as the Joint Commission • Test kits for infectious disease transfusion
and AABB have established their quality requirements to be testing • Number of, source of, and
reasons for unacceptable
more comprehensive and more coordinated than either QC Laboratory Equipment specimens
or QA. A QMS provides a framework for applying quality • Heating instruments • Number of times wrong
principles and practices uniformly across all blood bank • Water baths component or ABO was
operations, starting with donor selection and proceeding • Thawing devices for blood selected for crossmatch
through transfusion outcomes. components or use
In its Standards for Blood Banks and Transfusion Services,8 • pH meters • Number and type of
the AABB defined quality system essentials (QSEs) for • Cell counters transfusion complications
blood collection and transfusion service facilities and the • Centrifuges, refrigerated and • Number of and reasons for
serologic turnaround time failures
blood bank operations to which they are applied. Box 23–2
lists the QSEs and blood bank operations on which the AABB • Cell washers
assesses blood banks in its accreditation program. • Blood irradiators
The next sections of this chapter describe the blood • Refrigerators
bank’s role and responsibilities in fulfilling QMS essentials, • Freezers
which extend far beyond historic QC and QA practices. • Platelet incubators
Figure 23–2 demonstrates that the QMS essentials are the • Blood warmers
building blocks that support blood bank operations in the • Shipping containers
path of workflow for both donor centers and transfusion
services.
Quality Management System requirements are met. The facility determines the qualifi-
Essentials cations needed for each job and hires qualified personnel
who are trained and who maintain competence in their
The QMS essentials are in a logical order that can be assigned duties.
explained as follows for any new or changed product or Before products and services can be provided to cus-
service. The blood center or transfusion service organization tomers, equipment and materials need to be purchased and
develops its mission, vision, values, goals, and objectives maintained in inventory. Equipment needs to be managed
around the products and services it plans to offer. The facility according to manufacturer, regulatory, and accreditation
should have a customer focus by determining the needs and requirements. Before any work can commence, the facility
expectations of its customers and designing its processes needs to design, document, and validate work processes
and procedures to meet those needs and all regulatory and with appropriate management to ensure their correct perform-
accreditation requirements. The blood center or transfusion ance. Documents provide instructions for how the work
service should have the physical facilities to support the generates records of work performance. Patient and donor
products and services offered and ensure that safety information is managed in a way to ensure that requirements
2682_Ch23_509-525 22/05/12 12:04 PM Page 512
for confidentiality and information integrity are met. The Customer Focus
process of capturing information on nonconformances is
managed to identify recurring process problems that can Although both blood centers and transfusion services toil on
affect patient safety, laboratory credibility, and the operating behalf of patients, the real customers of these facilities is the
budget. Internal and external assessments measure and mon- entity or person who receives and must be satisfied with a
itor the facility’s performance to identify opportunities for product or service.8 For blood centers, the customers are the
improvement. The facility should constantly strive for donors, who want a safe and satisfying donation experience,
continual improvement. and the transfusion services served by the blood center,
which want properly tested and labeled blood components
Organization of the appropriate types on demand. For transfusion services,
customers are the physicians, who want the blood transfu-
The type and size of the organization determine the config- sions they order to occur in a timely manner, and the nurses,
uration of the blood bank’s QMS. In hospitals, there is who want the correctly issued blood components in a timely
usually an organization-wide quality function or department manner for administration to patients. For all types of
that prioritizes and coordinates quality projects, approves facilities, internal customers are the employees.
Component Collections, Preparation, Testing, Labeling, Distribution, Compatibility Testing*, Issue, Administration
Process Management
Sample arrives
Sample is processed
Automated
MANUAL TESTING AUTOMATED TESTING testing
process
Or
ABO discrepancy is
resolved
and / or No
D discrepancy is
resolved Is the Yes
antibody screen and/or
positive?
Antibody Send
No identification out
process process
Are
red cell units
needed now? Yes
Crossmatch
No process
Sample and request are archived
Figure 23–4. An example of a flowchart for the pretransfusion testing process. (Adapted from Berte, LM (ed): Transfusion Service Manual of SOPs, Training Guides and
Competence Assessment Tools, 2nd ed. American Association of Blood Banks, Bethesda, MD, 2007.)
process control in serologic testing is the use of green- The blood bank should have a written policy document
colored antiglobulin serum to ensure that the antiglobulin stating that the blood bank maintains a process and relevant
serum was indeed added at the antiglobulin phase of testing. procedures for correcting erroneous entries or results on a
Another common process control is the addition of IgG- paper record or in the computer. A second document (such as
coated reagent red blood cells after the antiglobulin phase a process flowchart) should describe the sequence of activities
reading to ensure that the antiglobulin serum was working. in identifying the need for a correction, obtaining any necessary
Computer process controls include automatic comparison approvals for the changed information, making the correction
of current blood type interpretation with the previous in the computer or on paper (or both), and notifying all
computer records on the same donor or patient to prevent appropriate parties of the correction. Procedure documents
ABO errors and warning signals when ABO-incompatible instruct staff members how to record the need for a correc-
units are issued for transfusion. tion, how to obtain any necessary approvals for making the
change, how to properly record a change to an entry on a
Documents and Records paper record, how to properly record a change to an entry in a
computer record, and how to notify appropriate parties of
Documents are approved information contained in a the change and document the notification. The process and
written or electronic format. Documents define the QMS procedures should be written in a way that ensures that all
for external inspectors and internal staff. Examples of regulatory and accreditation requirements are met. Recording
documents include written policies, process flowcharts, the actions taken throughout the process provides a tracking
procedures and instructions, forms, manufacturers’ pack- record to provide evidence that the requirements were met.
age inserts, computer software and instrument operator The blood bank needs a system to control identification,
manuals, and copies of regulations and standards. approval, revision, and archiving of its policies, process
Records capture the results or outcomes of performing descriptions, procedures, and related forms. The document
procedures and testing on written forms or electronic control system includes instructions for creating documents
media, such as manual worksheets, instrument printouts, in approved formats, assigning document identification and
tags, or labels. Both documents and records must be con- version designation, approving new and revised documents,
trolled to provide evidence that regulations and standards preparing a master document list, and maintaining document
are being met. history files. This “change control” process usually requires
the completion and routing of a form that contains information
Document Control about the reason for a new document or a change to an
A structured document control system links a facility’s existing one. Other important change information includes
policies, processes, and procedures and ensures that only when the change was requested; who wants the change; what
the latest approved copies of documents are available for other documents, if any, are affected by this change; and
use. A typical document control system is structured as a dates for approval, training, and in-use.
pyramid, as shown in Figure 23–5. The following example A master copy of the new, approved version is added to
best illustrates how a blood bank should link its policies, the master document history file, which contains copies of
processes, and procedures. all previous versions of that same document. The hard copies
within this master file capture the original signatures and
provide a paper backup when electronic files cannot be
Quality Management System Documentation
accessed. The master document list is updated with the new
version number. When a revised version of a document
is ready for release, the distribution process needs to be
controlled to ensure that copies of the obsolete document
(e.g., procedures in a working manual) are replaced with the
Policy new version and the obsolete copies destroyed. Employees
(What will should not keep and refer to copies of procedures and forms
be done)
stashed in lockers, drawers, and personal files. Use of
unapproved, outdated documents could lead to errors or
omissions that could cause harm to patients.
Process
(How it happens) Records Management
Forms are specially designed documents—either paper or
electronic—on which are recorded the results or outcomes
of performing a given procedure. Forms are subject to
Procedure the document control activities described in the previous
(How to do it) paragraph. The document identification system should
link the form to its respective procedure. Instructions for
completion of forms, when needed, can be conveniently
Figure 23–5. A simple structure for organizing QMS documents. placed on the front or back side of the form. When a form
2682_Ch23_509-525 22/05/12 12:04 PM Page 517
(either paper or electronic) is filled out, it becomes a record. Table 23–1 Common Classifications
Regulations and accreditation requirements mandate the of Various Types of
review of records by authorized personnel and specify the
Nonconformances
type of records and length of time they are stored for possible
future reference. State, local, and facility requirements for NONCONFORMANCE
record retention periods may also apply. TYPE DEFINITION
Explain the error / complaint / incident / nonconformance. Attach copies of any relevant records, as needed.
Describe what you did to take care of the immediate problem (remedial action):
Will remedial action be effective in eliminating future nonconformances of this same type?
Circle one: Yes No
DATABASE ADMINISTRATOR
Entered into database by: ______________________________________ Date: _____________________
used to identify these contributing factors and to determine in the investigation. The completed report is returned to
the best way to remove them through implementing corrective the quality officer, who reviews it for completeness and
action. appropriateness of remedial and corrective action. If an
Most corrective action involves making changes in the identified error or accident needs to be reported to the FDA,
process. All employees performing that process must then the corresponding process is initiated.
be informed of the changes and retrained when necessary. In a good nonconformance management program, the
Sometimes the corrective action involves retraining only nonconformances are also mapped to the specific involved
specific individuals who may not have been adequately processes in the blood bank’s workflow path. This infor-
trained initially or who have been taking unapproved mation is trended to determine which processes have the
deviations from the established processes or procedures. most problems. Identifying problematic processes provides
The nonconformance reporting process needs to be significant support for defending when a process needs to
clearly defined so that information is tracked and acted upon be changed. Facility staff must make a conscious decision
and feedback is provided. The person responsible for the not simply to respond with remedial actions but also to use
quality function in the blood bank (usually called the QA nonconformance information for removing the underlying
officer or quality manager) reviews all nonconformance root causes of the problem and to make improvements that
reports, assigns an accession number, and forwards the report truly contribute to the safety and efficacy of transfusion
form to the sections or departments that will be involved medicine.
2682_Ch23_509-525 22/05/12 12:04 PM Page 519
Area/Function Assessed
Identifying Opportunities for Improvement in the previous four activities, there should be little new
Opportunities for improvement for both blood collection information learned of which the facility is not already
facilities and transfusion services can be identified from aware)
several main sources: • Reports from other departments in the hospital’s
organization-wide quality committee function, such as
• The nonconformance trending process pointing to nursing or emergency department problems in dealing
operational areas that are not functioning as well as with the blood bank
intended
• Customer feedback such as complaints, solicited feedback, Using Teams
or suggestions from external customers the facility serves The hospital blood bank or laboratory’s quality committee,
and from internal customers (employees) or the blood center’s quality council, should set priorities for
• Information derived from monitoring quality indicators the problems that need the most immediate attention. Many
of operations, particularly when it is compared with that organizations have successfully used teams to solve problems
of peer groups in other institutions or to design process improvements. Names such as process
• Internal audit feedback, whereby objective evidence improvement teams, quality action teams, continuous improve-
collected by the auditor should support the facility’s ment teams, and corrective action teams have all been used to
understanding of why corrective action is needed and refer to groups of people representing different parts of a
should be taken given process who have been brought together to identify
• Feedback from periodic external compliance inspections and implement ways to remove the causes of the problem
(however, if the blood bank is already seriously involved and thereby improve the process. Teams need good team
2682_Ch23_509-525 22/05/12 12:04 PM Page 521
Plan-Do-Check-Act Process
Step1: Plan
A mission-consistent, customer-oriented action plan
• Identify opportunities for improvement from data sources
• Prioritize improvement activities
• Develop an action plan for the selected activity, either
- initiating a new process, or
- improving an existing process
• Identify
- customer needs
- participants
- timeframes
- outcome measurements
- success criteria
Step 2: Do
Put the plan into action
• Implement the action plan
- do a pilot project first
- broaden only after success
• Collect performance data
Step 3: Check
Has the planned and implemented change created intended improvement?
• Analyze collected data
• Compare performance data to established success targets and original performance data to
determine if improvement was achieved
• Identify any unexpected peripheral benefits
• Identify unanticipated problems in other areas
Step 4: Act
Decide what to do next
• Determine if customer needs were met
• Take action based on the results:
• Success:
- revise the processes for further improvements (optional), and
- assess again to determine if improvement is maintained, and
- if a pilot project, standardize to the bigger group
• Lack of success—re-do the action plan and repeat
Figure 23–8. Plan-Do-Check-Act Process. A common quality improvement process.
2682_Ch23_509-525 22/05/12 12:04 PM Page 522
Plan
• Determine product/service
specifications
• Provide products/services to
meet customer needs
• Prepare SOPs
• Define training requirements
• Describe process change
system
• Verify processes are reliable
and consistent
• Establish QC standards
Improve
• Train staff in use of
problem-solving methods
and tools
• Provide time to address Implement
problems • Appraise conformance to
• Provide information as standards
needed • Act on difference
• Monitor impact of process
improvements
• Recognize success
Assess
Perform on-going audits to
ensure:
• Design requirements are
met
• Process requirements are
met
• Finished product/services
meet specifications
• Finished product/services
meet customer needs
• All production activities are
controlled
A formalized problem-resolution process is just one piece of Figure 23–10. All the quality system essentials (QSEs)
continual improvement. Figure 23–9 illustrates the whole supporting the path of workflow remain the same because
cycle that encourages continual improvement. these quality elements are universal. In fact, at the point of
compatibility testing in the blood bank path of workflow, the
A Quality Management System for laboratory’s (and transfusion service’s) path of workflow is
Medical Laboratories entered for all transfusion service testing.
A review of the International Organization for Standard-
A laboratory-wide QMS can be derived by simply replacing ization quality standards demonstrates that blood bank
the blood bank path of workflow (see Fig. 23–2) with and laboratory QSEs are included in the international
the medical laboratory’s path of workflow, as shown in standards.15,16 Therefore, all the discussion in the section on
Test Order, Sample Collection, Transport, Receipt and Process, Testing, Reporting,Sample Archiving
QSEs applies equally to hospital laboratories. It is not only unacceptable for compatibility testing that need recollec-
possible but also highly desirable to expand the blood bank’s tion, retesting when controls don’t give the proper results,
QMS building efforts so that the entire laboratory benefits instrument failures, and downtime. Internal failures are
from improved organization, coordination, and effectiveness those caught and corrected before they adversely affect
of its many processes.17 customers or patients. However, operating funds have been
expended and wasted because the activity was not per-
The Cost of Quality formed correctly the first time. Additional operating funds
are then expended to correct the failure, thus eroding the
Although it may sound like much work and expense to operating budget.
implement a QMS in a blood bank, transfusion service, or
medical laboratory, the costs involved are significantly lower External Failure Costs
than the expenses the facility experiences when there are
major and minor nonconformances that need correction. External failure costs are “bad” quality costs that are expensive
Expenses are multiplied when the same nonconformance and hard to measure because they include both actual
recurs and needs to be “fixed” again. A basic understanding and intangible costs incurred to correct a nonconformance
of the four different types of quality costs is vital to that has reached the customer. Examples of external failure
comprehending the value of quality management to the costs include customer complaint resolution, misdiagnoses,
customers and patients served by the facility.18 recalls, and lawsuits. Consider the time, effort, and expense
involved in investigating nonconformances related to issuing
Prevention Costs an erroneous report or blood component. The cost to correct
these and other “never” events greatly erodes the facility’s
The cost of implementing processes and controls to prevent operating budget and definitely erodes customer confidence
the occurrence of nonconformances is considered a “good” in the facility’s quality and credibility.
quality cost. In the blood bank, transfusion service, and Reports have shown that some businesses waste up
medical laboratory, prevention costs include validation to 40% of their operating budgets correcting internal and
activities, preventive maintenance, work process training, external failures! Whereas there are no published data for
and quality management activities such as improvement blood banks, transfusion services, and medical laboratories,
teams and quality system training. A small amount of money there is little reason to believe that the expenditures for
spent in these activities greatly reduces the chance of process internal and external are significantly less. The important
problems in the path of workflow that could compromise issue is for a facility to identify its prevention, appraisal, and
the quality of the services provided or patient safety. Every internal and external failure costs and to use prevention and
budget should contain funds for prevention activities. appraisal activities to reduce failure costs wherever possible.
The old adage “An ounce of prevention is worth a pound of
Appraisal Costs cure” was never more appropriate!
The cost of implementing processes and controls to appraise
or evaluate the blood bank, transfusion service, or medical Importance of a Facility Quality
laboratory’s performance is also considered a “good” quality Management System
cost. There are internal and external appraisal costs. Internal
evaluation costs include those for calibration materials and Today, working in a QMS environment is needed to achieve
reagents, equipment calibration, quality control materials the standards of excellence necessary for surviving the
and reagents, any interim inspections of blood products or changes facing the nation’s health-care industry and providing
records, and the internal audit program. External evaluation the level of patient safety that our donors and patients both
costs include those for proficiency testing and periodic expect and deserve. Purchasers of health-care services want
licensure or accreditation inspections. The budget usually evidence that health-care providers such as hospitals and
always contains funds for these activities because they are blood centers are involved in organization-wide quality
mandated by regulatory and accreditation agencies. Appraisal improvement programs that increase the safety of donors
helps ensure that problems are caught and corrected so as to and patients. Only those organizations demonstrating
minimize any negative impact on customers and patients. measurable quality improvements are approved for agree-
ments for products and services. The cultural change needed
Internal Failure Costs to create a QMS takes time, and organizations that have not
started must begin immediately to keep pace. Consumers of
Internal failure costs are one of two costs considered to blood center, hospital, and transfusion services accept no less
be “bad” quality costs (the other is external failure costs). than total quality. Organizations that provide less will not
Examples include discarded donated blood units, samples survive.
2682_Ch23_509-525 22/05/12 12:04 PM Page 524
SUMMARY CHART
Blood bank compliance with federal regulations and cGMP requires that facilities design their processes
accreditation requirements is mandated by the FDA, and procedures to ensure that blood components are
the Joint Commission, the AABB, and the CAP. manufactured consistently to meet the quality standards
Compliance inspections measure the state of the facility’s appropriate for their intended use.
program with respect to the applicable requirements at Process validation challenges all activities in a new
a single point in time and are usually conducted every 1 process before implementation to provide a high degree
to 2 years. of assurance that the process will work as intended.
Quality control procedures in blood banking may Routine QC procedures, review of records, and capture
include daily testing of the reactivity of blood typing of nonconformances when the process did not perform
reagents, positive and negative controls in infectious as expected are routine process control measures that
disease testing, calibration of serologic centrifuges, and monitor whether a process is functioning as needed.
temperature monitoring of refrigerators, freezers, and Nonconformance management is a name for processes
thawing devices. that detect, report, evaluate, and correct events in
Quality assurance is a set of planned actions to provide blood bank operations that do not meet the facility’s
confidence that processes and activities that influence or other requirements.
the quality of the product or service are working as An internal audit reviews a specific facility process and
expected individually and collectively. determines—by examining documents and records,
A QMS provides a framework for uniformly applying interviews, and observations—whether the facility is
quality principles and practices across all blood bank meeting applicable requirements and its own policies,
operations, starting with donor selection and proceeding processes, and procedures.
through transfusion outcomes. A process improvement team is a group of people
Process control is a set of activities that ensures a given who represent different activities in a given process
process will keep operating in a state that is continuously and who have been brought together to identify and
able to meet process goals without compromising the implement ways to solve process problems.
process itself.
9. The difference between the blood bank and laboratory 9. American Association of Blood Banks: Technical Manual, 16th
QMSs is that: ed. Bethesda, MD, 2008.
10. Food and Drug Administration, Center for Biologics Evalua-
a. The laboratory has a different path of workflow. tion and Research: Guideline on Quality Assurance in Blood
b. The blood bank does not include computer systems. Establishments (Docket No. 91N-0450). Food and Drug
c. The QSEs are different. Administration, Rockville, MD, 1995.
d. The blood bank excludes testing. 11. Department of Health and Human Services: Code of Federal
Regulations, Title 45, Parts 160 and 164. U.S. Government
10. The QSEs for the blood bank QMS can be used for the Printing Office, Washington DC, revised annually.
laboratory because: 12. Centers for Medicare and Medicaid Services: Code of Federal
Regulations, Title 42, Parts 430 to end. U.S. Government
a. The paths of workflow are identical. Printing Office, Washington, DC, revised annually.
b. Both the laboratory and blood bank experience 13. Scholtes, PR, et al: The Team Handbook, 3rd ed. Goal-QPC,
accreditation inspections. Salem, MA, 2003.
c. The QSEs are universal. 14. McCloskey, LA, and Collet, DN: TQM: A Primer Guide to Total
Quality Management. GOAL/QPC, Methuen, MA, 1993.
d. The QSEs are required by international standards.
15. International Organization for Standardization: ISO 9001:2008
Quality management systems—Requirements. International
References Organization for Standardization, Geneva, 2008.
16. International Organization for Standardization: ISO 15189:2007
1. Food and Drug Administration, Center for Biologics Evaluation
Medical laboratories—Particular requirements for quality and
and Research: Guideline on General Principles of Process Vali-
competence. International Organization for Standardization,
dation. Food and Drug Administration, Rockville, MD, 2011.
Geneva, 2007.
2. Food and Drug Administration, Department of Health and
17. Clinical and Laboratory Standards Institute: A Quality Man-
Human Services: Code of Federal Regulations, Title 21, Parts
agement System Model for Laboratory Services; Approved
200–299. U.S. Government Printing Office, Washington, DC,
Guideline, GP26, 4th ed. Wayne, PA, 2011.
revised annually.
18. Campanella, J (ed): Principles of Quality Costs: Principles,
3. Food and Drug Administration, Department of Health and
Implementation, and Use, 3rd ed. American Society for Quality
Human Services: Code of Federal Regulations, Title 21, Parts
Press, Milwaukee, WI, 1999.
600–799. U.S. Government Printing Office, Washington, DC,
revised annually.
4. The Joint Commission: Comprehensive Accreditation Manual Bibliography
for Hospitals. Joint Commission Resources, Oakbrook Terrace,
Berte, LM (ed): Transfusion Service Manual of SOPs, Training
IL, 2010.
Guides and Competence Assessment Tools, 2nd ed. American
5. The Joint Commission: Comprehensive Accreditation Manual
Association of Blood Banks, Bethesda, MD, 2007.
for Laboratories and Point-of-care Testing. Joint Commission
Clinical and Laboratory Standards Institute: Training and Competence
Resources, Oakbrook Terrace, IL, 2010.
Assessment, 3rd ed. Approved guideline GP21-A3. Clinical and
6. College of American Pathologists: Inspection Checklists for
Laboratory Standards Institute, Wayne, PA, 2009.
Laboratory Accreditation. College of American Pathologists,
Laboratory Documents: Development and Control. Approved
Northfield, IL, 2010.
guideline GP2A-5. CLSI, Wayne, PA, 2006.
7. American Association of Blood Banks: Standards for Blood Banks
Tague, NR: The Quality Toolbox, 2nd ed. ASQC Press, Milwaukee,
and Transfusion Services, 27th ed. American Association of
2005.
Blood Banks, Bethesda, MD, 2011.
8. International Organization for Standardization: ISO 9000:2005
Quality management systems—Fundamentals and vocabulary.
International Organization for Standardization, Geneva, 2005.
2682_Ch24_526-539 22/05/12 12:05 PM Page 526
Chapter 24
Utilization Management
Julie L. Cruz, MD, and Steven F. Gregurek, MD
OBJECTIVES
1. Distinguish the differences between blood utilization review and blood utilization management.
2. Describe the purpose and goals of a blood utilization management program.
3. Explain the principles and limitations of the Lean approach within the context of blood utilization management.
4. Describe how to perform a value stream assessment within the blood bank environment.
5. Develop a strategy for creating standardization and metrics used in blood utilization management.
6. Explain how adoption of transfusion guidelines can improve the value stream and help to develop utilization review criteria.
7. Explain how to assess the current and future state of blood utilization within a hospital system.
8. Identify the differences among the prospective, concurrent, and retrospective blood utilization reviews.
9. List intervention strategies and describe how they may be employed with the various blood utilization review models.
10. Explain the importance of a well-planned policy deployment and the need for continuous improvement within the context of
blood utilization management.
526
2682_Ch24_526-539 22/05/12 12:05 PM Page 527
application of Lean strategies in manufacturing. Lean man- than a combative “blood bank versus the hospital” mentality.
ufacturing (aka “the Toyota Production System”) is a pro- All must share a vision that existing processes must be
duction practice focusing on eliminating waste to generate changed in order to improve service to patients and to lower
efficiencies and ultimately deliver exactly what the customer costs. It is critical that the change leaders reach every stake-
wants precisely when it is needed. In fact, hospital systems holder in the process to repetitively communicate the impor-
and medical laboratories are increasingly training personnel tance of focusing on patients while also addressing the
and modifying their systems to adopt Lean strategies, and it concerns and needs of employees who will inevitably be
has been noted that transfusion services already apply some required to change work habits. The physician groups with
Lean concepts such as standardization.3 This chapter out- the greatest blood use should be selectively targeted for
lines one approach to designing a blood utilization manage- policy agreement and educational activities. Armed with a
ment program utilizing Lean tools and concepts. Each thorough understanding of appropriate indications, risks,
individual institution should develop a blood utilization and the blood utilization process, nurses can be instrumental
management strategy best suited to the unique challenges in achieving success and compliance. Once the program is
within its own system.4 launched, they can help ensure that the blood order indica-
tion and dose are appropriate, that the necessary interval
Approval and Support laboratory data are obtained, and that essential clinical in-
formation is relayed.9 Continued approval and support from
Blood utilization programs cannot exist in isolation within these areas of hospital culture leadership are necessary to
the hospital. Guidelines for transfusion and phone calls to drive acceptance and provide the motivation and awareness
clinicians regarding appropriateness of transfusion orders to achieve the long-term goals of the blood utilization program.
have the potential to lead to animosity and accusations of
intrusiveness by clinicians accustomed to ordering auton- Lean Approach to Blood Utilization
omy. These physicians may perceive a loss of control over Management
their medical decisions.5 Hospital leadership must support
the inception of the blood utilization management program When selecting a strategy for blood utilization management,
and continue to empower the program once a credible plan it is important to consider a systems-based approach that is
is designed and approved. It is extremely important to obtain compatible with the unique challenges of the safe and effec-
this commitment from hospital leadership before proceeding tive administration of blood components. The authors find
with a program of even low complexity. Lean to be a particularly useful blood utilization manage-
Blood utilization programs may have multiple dedicated ment strategy, although other quality management systems
personnel for auditing, educational activities, and reporting may also be employed. Tools for identifying and eliminating
and can require significant resources. However, most improper waste while improving quality and ensuring customer satis-
ordering is related to overtransfusion. If properly executed, faction are key components of any utilization strategy.
blood utilization management can produce significant indi-
rect and direct cost savings.6 These potential savings should Overview
be summarized and presented to the administrative staff and
should not be limited to projected savings related simply to Simply put, Lean is a systematic approach to identifying ac-
decreased purchasing of blood components. Assessment of tions within a process that create value, then optimizing the
the potential cost savings by preventing severe transfusion alignment of these actions to deliver to customers exactly
reactions associated with significant morbidity and mortality what is desired, in the quantity desired, precisely when it is
such as TRALI (transfusion-related acute lung injury), required. It is based upon an approach first developed by the
circulatory overload, bacterial contamination, and hemolytic Toyota Corporation following World War II to better manage
transfusion reactions should also be discussed.7 Although operations, including customer relations, supply chain man-
demonstrable cost savings are often a priority for adminis- agement, development, and production.10 Central to accom-
trative support of the program, it is important to remember plishing this goal is the elimination of waste (also described
that other key benefits, including enhanced safety and by the Japanese term muda).
greater stewardship of a limited resource, can be achieved. Waste is any action that does not create value as defined
These things often cannot be easily quantified in dollars but by the customer. DOWNTIME is a useful acronym utilized
should be considered just as critical in terms of rationale for in Lean for identifying types of waste: Defects, Overproduction,
development and continued support of the program.8 Waiting, Nonvalue Added Processing, Transportation, Inven-
Like hospital administration, senior medical and nursing tory Excess, Motion, Employee/People waste. Table 24–1
leadership must also be actively engaged, educated, and delineates types of waste identified in Lean and gives exam-
recruited to devise and support a successful program. In ples found in transfusion medicine.10 Although some trans-
particular, consensus regarding transfusion guidelines and fusion services may consider the ordering clinician to be the
utilization program objectives should be achieved between customer, the patient is the true customer, and value is
the blood bank and physician leadership. Education, commit- ultimately determined by the therapeutic benefit (or harm)
ment, and compromise are necessary to create a cooperative the patient receives from blood component transfusion. The
environment and promote system-wide acceptance rather system strives for “just in time” delivery as determined by
2682_Ch24_526-539 22/05/12 12:05 PM Page 528
Table 24–1 Types of Waste and Corollaries blood utilization program, as some actions fitting the strict
to Transfusion Medicine Lean definition of waste are necessary to enhance safety.
Infectious disease testing is an example. However, signifi-
TRANSFUSION MEDICINE cant improvement opportunities can still be identified.
TYPES OF WASTE COROLLARY Blood utilization management attempts to optimize value
Inventory waste • Available units diverted from (safety and therapeutic benefit) while minimizing waste.
• Misallocation of patients with greater need Here, the discussion and applications of Lean concepts
resources • Unused issued components are by no means representative of Lean in its entirety but
improperly returned merely illustrate the possibilities for application in blood
Overproduction • Not trusting of timely delivery if utilization management. Those interested in applying Lean
“just in case” ordered as needed (pull not met) concepts are referred to the literature, and a number
• FFP thawed for the OR that expires of professional organizations and consultancies offer
unused resources.12
• Unused washed units allowed to
expire on floor
The traditional Lean model focuses on the elimination
of waste (muda) to drive production toward value (as
People • Personnel diverted to perform defined by the customer). However, in transfusion medi-
unnecessary tasks cine, there is an additional facet to be considered—peril.
Motion • Inappropriate “STAT” order All transfusions (even optimally selected, fully tested,
appropriately stored and matched components produced
Transport • Delivered components that were not under good manufacturing practices and properly admin-
transfused
istered by skilled personnel) pose an inherent baseline risk
Processing waste • Failure to discontinue standing of adverse events. For example, despite intense mitigation
• Lack of order for HLA-matched platelets strategies that have reduced the risk of TRALI, residual
communication • CMV seronegative transfused when risk remains.13
• Customer need leukoreduced appropriate
not clearly defined
Improper blood utilization and management practices
decrease value and drive the result toward peril and waste.
Adapted from Womack, JP, and Jones DT: Lean Thinking: Banish Waste and Create Wealth in Thus, there are not only financial, labor, and resource losses
Your Corporation. Free Press, New York, 2003. but also human losses in terms of the patient’s health and
well-being. For example, the transfusion of a blood component
to a healthy person creates no value but increases waste and
customer need (also known as pull). Laboratorians are most peril. The Cruz-Gregurek model, shown in Figure 24–1,
familiar with this concept as turnaround time. demonstrates this concept. Optimal value is the penultimate
Lean utilization is not a one-time strategy that is deployed goal and is accomplished by developing standardized meth-
and delivers a fixed benefit. Rather, it relies upon a monitoring ods targeted to reduce waste and peril. As noted in the figure,
of metrics carefully chosen to reflect value and a process of some inappropriate practices create waste and peril. These
continuous improvement (kaizen). It is designed to move practices represent high-yield opportunities for intervention.
the system ever closer to pure value (perfection) with minimal In addition to financial and safety benefits, such interven-
waste.11 Perfect value with zero waste is unattainable in a tions are expected to reduce variability in practice and
WASTE PERIL
2682_Ch24_526-539 22/05/12 12:05 PM Page 529
provide better stewardship through optimization of a limited ordered) occurs out of the blood bank’s control. As the figure
resource. demonstrates, all the processes are considered under the
penumbra of the utilization program. This maximizes oppor-
Value Stream Assessment tunity for the identification and elimination of waste, which
can be generated along the stream at any point.
Application of the Lean paradigm begins with iteration of Considered another way, traditional methods to mitigate
the value stream and assessment of the current state. The peril focus on interventions designed to increase the safety of
value stream mapping team identifies all subprocesses nec- the individual blood component, such as donor deferral ques-
essary to bring the finished product to the customer (in our tions, infectious disease testing, the crossmatch, and the
case, the patient). Value stream mapping (VSM) is a strategy second label check. Quality assurance processes ensure that
development tool to aid in identifying opportunities safe, pure, and potent components are manufactured in the
throughout the process for waste elimination. After identi- blood center and released for transfusion by the blood bank.
fying problems with the current state, the strategy develop- The transfusion service mantra is “the right blood, right patient,
ment team creates an improved “future state” that will be right time.”9 The “right patient” in this context refers to iden-
translated into a “Lean transformation plan” and subsequent tity (e.g., John A. Smith in bed A vs. John J. Smith in bed B).
improvement (kaizen) projects. When considering the value In contrast, the utilization program ensures the patient actu-
stream, it becomes apparent that “improvements” in one area ally requires each transfusion. Figure 24–3 demonstrates the
can cause downstream impacts with the potential to increase paradigm shift, illustrating that greater risk reduction can be
waste over the total system.14 For this reason, it is critical achieved by considering both individual component safety
that executive management be involved in the VSM process, measures and utilization optimization strategies.
or at least endorse the VSM team’s future state value stream. To receive benefits from transfusion, the patient must
As applied to transfusion medicine, the traditional, or have a deficiency or pathological lesion that can be remedied
“current state,” map of the value stream reveals two disjointed by functioning stored components. This process can be
processes, as shown in Figure 24–2. One process is under tight perfected by ensuring optimal timing and administering the
quality control within the purview of the transfusion service, optimal dose for correction without exposing the patient to
but the portion of the value stream involving the patient, the increased transfusion risk. In this case, “the right patient”
clinician, and the nursing staff is disconnected with respect to refers to his or her suitability to receive the requested trans-
the assessment of the patient and the decision to transfuse. fusion, taking into account both individual clinical factors
Here, the blood bank serves merely as a dispensary, and what and established guidelines. In this context, blood utilization
happens after the component is issued (and before it is management is the complement to the crossmatch. The
Traditionally within control of the blood bank Traditionally outside control of the blood bank
• Strong control of unit-based risk mitigation (layers of safety) • Patient-based risk mitigation
• Blood supplier qualification • Decision to transfuse
• Donor qualification • Risk/benefit assessment
• Units tested for infectious diseases • Choice of component(s)
• Produced under good manufacturing processes • Desired component modification
• Transfusion service • Patient identification (phlebotomy and transfusion)
• Specimen labeling requirements • Dose
• Type and screen • Timing of administration
• Crossmatch • conditions under which unit is maintained from release
• Standard procedures to end of transfusion or return
• Strict quality assurance
Risk of Risk of
Transfusion-Related Transfusion-Related
Adverse Event Adverse Event
Component-
Component- Patient
Centered
Centered Selection
Risk Mitigation
Risk Mitigation
Figure 24–3. Traditional transfusion service risk mitigation strategies are focused on increasing the safety of the individual component, yet inherent residual risk remains.
Blood utilization management confers additional safety by incorporating consideration of patient-based factors to ensure transfusion is appropriate and optimally selected,
hence a greater safety benefit is achieved.
crossmatch assesses the safety of a particular component for For instance, yet unrecognized blood-borne pathogens and
the patient, while utilization management ensures that the noninfectious complications whose mechanisms are not yet
patient is appropriate for the transfusion. well understood. This is well understood from the immuno-
Although cost savings are often emphasized, the safety of hematological model of red blood cell antigen-antibody inter-
blood transfusion is the single most important aspect of action. Unfortunately, the sickest and most physiologically
effective blood utilization. According to Dzik, “the ‘decision vulnerable patients are also the most frequently transfused.16
to transfuse’ is perhaps the most critical step in patient trans- Examples of such individuals include cardiac, gastrointestinal,
fusion safety.”15 Despite the layers of safety designed to critical care, massive trauma, and oncology patients.
reduce risk associated with blood components, inherent risk
remains, thus zero risk is equivalent to no transfusion.7 Standardization and Metrics
Appropriate blood utilization and management also rec-
ognizes the risk of undertransfusion (i.e., failure to transfuse Having introduced the concept of utilization and discussed its
when benefits outweigh risk) and inappropriate or subopti- importance in transfusion medicine, we can now turn to the
mal component selection. From this perspective, the safest basic elements important in any utilization program. Metrics
transfusion is the one that is given appropriately. Although must be chosen to allow an assessment of the current state and
many transfusion risks are preventable, rare errors account scope of the problem. Evidence-based transfusion guidelines
for a significant number of fatal transfusion-related events. must then be written and deployed. Finally, review criteria must
Fortunately, rigorous attention to procedure and good man- be established to monitor physician compliance with guidelines.
ufacturing practice has decreased the incidence of these un-
toward events. However, it is important to recognize that Metrics
despite our best efforts, total elimination of all transfusion
risk (peril) is impossible. Assessment of the current state is an important initial step
Certainly there are many interventions the blood supplier in Lean utilization. A baseline status must be established by
or transfusion service may implement to improve transfusion selecting metrics that will both adequately reflect the system
safety (e.g., manufacturing plasma exclusively from male as is and serve as indicators of success (or failure) in execution
donors to prevent HLA antibody-associated TRALI, bacterial of the future state. Those chosen should be meaningful
detection in platelet components), but residual risk remains. within the system, readily evaluable, and widely disseminated.
2682_Ch24_526-539 22/05/12 12:05 PM Page 531
This will allow for individuals, cross-functional kaizen transfusion practices. It is critical to obtain input from clin-
teams, and departments at all levels of the system to contin- ical staff and support of guidelines to improve compliance
uously monitor their own subprocess performance and by ensuring they are broad and flexible enough to meet the
progress toward improvement targets and to benchmark majority of clinical situations. If guidelines are too restrictive
themselves against other components of the value stream. or ignore significant clinical literature consensus opinions
Many of these may already be trended and tracked by or guidelines in favor of more stringent recommendations
the blood bank or quality assurance personnel, such as from the transfusion medicine literature, compliance will be
crossmatch-to-transfusion ratio and number of mislabeled poor. Published evidence-based guidelines and consensus
or unlabeled specimens. However, instead of automatically opinions are widely available and should be referenced.18–24
including existing metrics, one should consider their potential
impact across the entire value stream. Consider not only Current State and Scope of Problem
what is measured and reported, but also how. For example,
the number of mislabeled or unlabeled specimens in a The next step is to assess the scope of inappropriate transfu-
particular time period is often tracked and reported to the sion. A retrospective and representative sample of records
transfusion committee and to individual nursing units. Yet from transfused patients should be reviewed to identify
might this information have greater impact if reported existing issues and patterns of misuse. The review should
not just as the number of “defects” or opportunities for compare the indications used for actual transfusions against
sentinel events, but also communicated as extension of the transfusion guidelines. Specific areas and physician prac-
turnaround time? tice groups should be ranked by total utilization and appro-
Waste begets waste within a system, and in this example priateness. Next, specific goals should be determined by
the interval between the blood order and the availability of the members of the planning committee who are considering
the component for transfusion is significantly prolonged the available resources and the desired monetary and safety
while a subsequent properly labeled specimen is obtained outcomes.
and delivered to the blood bank. Such delays may contribute Program complexity should be determined, including
to the perception that blood bank turnaround time is too type, frequency, and scope of audit. Special attention should
long and may be partially responsible for the overproduction be given to each type of blood component. Will all compo-
issues previously noted in Table 24–1. Would selecting met- nents be reviewed equally? Will there be disproportionate
rics that both include the absolute numbers of such speci- representation of more frequently ordered components?
mens and quantify the delay in the system caused by them Consideration must also be given to which patient populations
(in minutes or hours) increase the ability to drive behavior? will be audited, such as pediatric, neonatal, high-complexity
Would it better demonstrate that as waste is removed, flow surgery, hematology-oncology, and so on. Particular popula-
improves, thus decreasing strain on blood bank resources tions may require tailored auditing and intervention methods.
and resulting in more timely provision of appropriate units? To ensure the program moves forward expeditiously, a
These types of questions must be considered, and ultimately timetable should be created. Hospital leadership will often
the metrics chosen will reflect the individual institution’s request this, along with early projections of the expected
goals, priorities, and beginning state. costs and benefits of the program.
After the determination of metrics and the assessment of
Transfusion Guidelines the current state, it is important to ask the following ques-
tions regarding the future desired state: Is the current state
Once metrics are chosen, facility guidelines must be drafted, adequate without any intervention? How will the program
approved, and disseminated. The involvement of a multidis- increase safety or eliminate waste? Are there enough resources
ciplinary team is critical. A well-written transfusion guideline available for successful implementation and continuance of
will contain the indications for transfusion and thresholds the program? Will our program become too restrictive and
used in screening and auditing of transfusion records.17 If potentially cause undertransfusion or prohibit compliance?
the existing guidelines are not current, then current evidence- The answers should be used to alter the design as indicated.
based medicine should be reviewed and the guidelines
amended to reflect best practices. Evidence-based guidelines Review Criteria
should be carefully scrutinized and included only if they
fit within the scope of the hospital’s practice and patient After the transfusion guidelines are updated and the scope
population. In cases where the evidence is limited, adoption of the program is defined, criteria should be generated for
of expert consensus panels should be considered in conjunc- use in the audit process. If the transfusion service is small
tion with local practices. and sufficient personnel are available, 100% manual review
New guidelines should not be adopted until input is of records could be considered. However, as the number of
obtained from physician and nurse champions. Physician transfusions increases, this method can quickly exhaust
groups with high utilization should also be offered the available resources. Screening criteria can be selected to flag
opportunity to review and comment upon the proposed cases with a high probability of inappropriate use and unflag
guidelines. Emphasis should be placed on the hazards those with low probability. This maximizes the efficiency of
of transfusion, therapeutic indications, and conservative the review process. Algorithms can be developed that include
2682_Ch24_526-539 22/05/12 12:05 PM Page 532
laboratory data and information from blood order sets. process is particularly effective when a transfusion indication
Screening criteria that are too specific will fail to identify must be linked to a decision support function when com-
many inappropriate transfusions, while overly sensitive puterized physician order entry is available. This process has
criteria will result in detailed review of many appropriate led to significant cost savings.28
transfusions, making the program inefficient. In most cases,
screening thresholds using fixed lab values or excluding Retrospective Review
certain conditions or indications are used.18
Retrospective blood utilization review can be performed
Blood Utilization Review after the transfusion event has taken place and can occur
when convenient without interrupting other staff duties.
As previously discussed, blood utilization review attempts Detailed chart reviews can be performed by nursing or physi-
to optimize transfusion utilization by minimizing inappro- cian staff to determine appropriateness. Large numbers of
priate transfusion. Most programs emphasize overutilization transfusions can be reviewed and compared to previous data
of blood products; only a few institutions monitor for un- if medical records are readily available. Hence, this method
derutilization of blood. The rare studies that have addressed is often used to generate trending reports on a large scale,
underutilization have failed to show significant practice including utilization within the hospital, particular physician
deviations. However, with continued efforts to lower trans- practice group, or diagnosis and billing code.
fusion thresholds and reduce overutilization, the risk may In retrospective reviews, the feedback communication
increase.25,26 This may be especially true if educational efforts usually consists of letters, focused educational efforts, or
to improve utilization are strongly focused on potential both. The goal of either method is to educate clinicians and
transfusion-associated adverse events. raise their awareness of current guidelines, thereby improving
Blood utilization review is comprised of three broad future transfusion decisions.29 The major disadvantage of
categories of audits: retrospective, concurrent, and prospec- this methodology is that transfusion events occur in the past
tive. These strategies have direct corollaries from managed and the inappropriate transfusion is not prevented, merely
care.2 Two subtypes of prospective review—targeted and identified later. Another drawback is feedback and clinician
discontinuous—are particularly useful when there are education occur remotely from the event, decreasing the
resource constraints. likelihood that the clinician will remember the specifics
The feature common to all methods is the requirement of the transfusion decision. Of note, a longitudinal study of
for comparison of transfusion records from individual the impact of retrospective review alone in the sister field
patients to criteria developed from standardized guidelines. of drug utilization review failed to demonstrate significant
Each transfused component is assessed for medical necessity, improvement.30
adherence to indication guidelines, component dosing, and
timing of administration. It is then categorized as either Concurrent Review
appropriate or inappropriate. Results of the audit are often
submitted for further review and possible intervention strate- Managed care audit models define concurrent review as a
gies. Some methods are better suited for particular end goals. type of retrospective utilization review that occurs shortly
Reviews can be manual, semiautomated, or automated. after delivery of service, usually during the hospital stay.31
Manual reviews are often time-consuming, involve detailed Concurrent review is a type of retrospective review that
medical record review of the entire selected audit population, occurs shortly after the transfusion while case details are still
and require the involvement of clinical health-care profes- fresh in the mind of the ordering practitioner. It provides the
sionals such as nurses or physicians. Semiautomated reviews blood bank with the opportunity to obtain further informa-
include computerized screening criteria that flag particular tion to facilitate review.21 In blood utilization models, it
records for manual review. A screening process may add allows review of the relevant guidelines with the ordering
efficiency to the process by removing records for review that physician to encourage future compliance. This type of
are below a predetermined screening threshold and by flag- review process is more labor-intensive than retrospective
ging the remaining records for review.27 It is important to reviews because of the additional requirement to contact the
note that the screening threshold is different from the review ordering physician. Also like the retrospective audit, inter-
criteria threshold. Screening thresholds must be carefully ventions occur after transfusion and focus on preventing
selected to prevent missing inappropriate transfusions, yet future occurrence.
not be so strict as to compromise efficiency and speed. They
are usually delineated in terms of a specific algorithm. Prospective Review
Automated review is carried out completely by algorithm
and can be very quick and expeditious. Such reviews lack Prospective blood utilization review is the most complex
the detail of a manual system but can be an excellent method method and requires the interaction of many of the key play-
to display trending. Most common utilization benchmarks ers discussed below. This type of audit methodology uses
in the blood bank are examples of automated review. Many manual or semiautomated methods and requires real-time
times the screening process or automated review can be per- evaluation of component requests. If prospective review is
formed entirely electronically.15 The automated screening performed, the blood bank staff will be intimately involved
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in the audit process. Blood bank personnel are responsible identifiable. However, there is evidence that this strategy fails
for correctly screening transfusion orders in a timely manner to provide long-term improvements, likely because it is a
that does not delay preparation of the component. The “point” intervention rather than one that concentrates on
clinician’s order is reviewed in real time in light of available overall transfusion practice.33
patient laboratory data and clinical information. It is com-
pared to evidence-based criteria and the facility’s established Discontinuous Prospective Review
transfusion guidelines. The component is issued as ordered Discontinuous prospective review is a process that can be
if transfusion is indicated, within guidelines, and the com- employed by facilities with limited transfusion medicine
ponent optimally selected. If the order appears to deviate resources or by facilities with only occasional deviations
from facility-approved transfusion guidelines, or if the from transfusion utilization benchmarks. It is a method to
requested component is suboptimal, blood bank personnel conserve resources when intervention that is more aggressive
can communicate with the clinician to obtain more informa- is not expected to yield a significantly better response. Typ-
tion or offer guidance and education. Mechanisms must be ically, it is combined with retrospective or concurrent review
in place for notifying the on-call physician evaluator. Possi- strategies and is selectively triggered when significant devi-
ble outcomes include approval of the order after additional ations are identified. It may be limited to a particular product
information is provided, consultation and agreement upon type, DRG (diagnosis related group—a categorization system
a more appropriate component, or a more informed decision utilized for medical billing, based on diagnosis) clinical serv-
resulting in cancellation of the transfusion order. ice, or individual clinician. In fact, a multi-institutional study
If the prospective review process is used, the corresponding revealed that less than 30 diagnostic categories accounted
blood bank standard operating procedure should address for more than 75% of the cost of transfusion.34 Once
four issues: improvement has been demonstrated and a steady state
achieved, intense review may be discontinued until signifi-
1. The type of screening method must be incorporated into
cant deviations are again identified. The risk in employing
the procedure, including required computer data entry or
this strategy is that during periods of “relaxation,” appropriate
algorithm. Validation of the screening algorithm should
monitoring does not occur, and the review process is not
be specifically addressed to prevent errors.
reactivated in a timely manner.
2. There must be specific instructions for handling flagged
blood orders detailing the circumstances under which
Intervention Strategies
products are issued, denied, or the physician consulted.
3. There must be a process that describes what action to Each particular hospital and situation may require different
take in unusual circumstances or if the physician evalu- interventions or perhaps a combination of strategies to
ator cannot be reached. mitigate inappropriate practices.35 Interventions may be
4. An efficient method of recording details of the event, categorized as educational, punitive, or confrontational.
including the pertinent discussion, disposition of the com- They may be employed individually or collectively, depend-
ponent order, and the name of the approving physician ing on the culture of the institution. Tinmouth and
evaluator, is needed. colleagues conducted a comparative study and reported
This process should not cause unnecessary delay in issu- that “no particular intervention or combination of inter-
ing components for patients in urgent need.15 However, the ventions appeared more effective in reducing utilization”
majority of transfusions are not performed urgently, and of blood components.36
there is ample time for consultation to make the best thera- Educational strategies may require a hospital-wide focus
peutic choice for the patient. A well-written standard oper- if inappropriate utilization occurs in multiple departments
ating procedure with adequate training time is essential to or in groupings that are not easily categorized. Lectures,
ensure a robust, consistent process. Obstacles to performing grand rounds, and job aids are all useful resources. If greater
prospective review are the considerable time and resources inappropriate usage is found among specific physician
required and the potential for conflict between the transfusion practice groups or in patients with a particular disease,
service and clinician.29 classification-focused educational interventions are useful.
Small group lectures or discussions (“lunch and learn”) and
Targeted Prospective Review phone conversations are excellent vehicles to relay current
Targeted prospective review is a subtype of prospective re- practice guidelines.
view used during drug utilization review to pinpoint a high- Punitive strategies involve reporting aberrant ordering
priority, expensive, or potentially hazardous medication.32 practices to certain peer groups or hospital administrative
When this concept is applied to the blood bank, targeted groups relevant to the ordering physician. These groups
prospective review involves ongoing prospective monitoring must agree with the mission of the blood utilization program
of one or a few specifically selected products or indications. and endorse the metrics and guidelines or such reports will
These should be chosen from areas where intervention and be ignored. Transfusion utilization may be included in
behavior modification are expected to have high-yield annual “physician report cards.” Practitioners may then be re-
results. This process is most useful when resources are limited quired to complete annual training that emphasizes evidence-
and specific inappropriate utilization “targets” are readily based practice and the facility guidelines criteria.
2682_Ch24_526-539 22/05/12 12:05 PM Page 534
Confrontational methods involve preventing or obstruct- from physician practice groups that frequently order com-
ing certain physician orders. With institutional approval, ponents or have expected utilization issues. Groups with
system-wide constraints can be placed on transfusion orders. poor utilization should be notified and educated before any
Restrictions prevent the release of specific blood components stricter policies are enforced. Mechanisms must be included
for indications that are blatantly ineffective or better reme- for bypassing any potential for delay in a truly emergent
died by other lower risk or more cost-effective strategies. An situation such as massive hemorrhage.
example would be restricting all “fresh” blood to certain age
populations. Gatekeeping is another confrontational method Blood Order Sets
that requires approval of any transfusion order that does not
meet predefined criteria. Decision support logic added to In Lean manufacturing, visual control is important as a
blood order sets is an example of a gatekeeping role that has means of standardization.10 A utilization management equiv-
been shown to reduce inappropriate blood utilization.37 This alent is the use of blood order sets. This is a standardized
intervention can also be used to deny release of components method to clarify indications and enforce guidelines during
for transfusion order requests when indications are clearly physician order entry. This process may be paper or elec-
inappropriate. If product release is denied or delayed, it must tronic. Such tools should be simple, intuitive to use, and not
follow specific direction from the transfusion medicine cluttered or overwhelming. They must also be readily avail-
physician or must follow strict adherence to explicit instruc- able, whether electronic or paper-based. It is important to
tions for denial within the standard procedures. keep in mind that blood order sets are not foolproof methods
Although the preceding strategies focus on correcting for preventing inappropriate orders. Order sets that are too
aberrant ordering behavior, positive feedback should occur complex, difficult to access, or too restrictive will result in
as well. The development of physician champions has been the development of “workarounds” to bypass the screening
successful in improving quality in other areas of health process. Those who had employed this tactic reported they
care.38 Physicians who practice consistently within estab- were directed to do so by a more superior physician. Such
lished guidelines should be recognized and, where possible, “solutions” on the part of clinical staff impede flow in the
asked to share their practice philosophy with peers. These blood bank, increase waste, and inhibit the ability to provide
individuals may make excellent “utilization ambassadors,” “just in time” service according to customer pull.28,37
assisting in shifting the institutional culture toward the Generally, any blood utilization program will use a
desired goal. As practicing physicians, they will also have an screening tool such as the blood order set. Care should be
additional level of credibility with other clinicians who are taken to address issues such as massive and emergent trans-
absent in communications with laboratory personnel or fusion. It may be necessary to address special populations
transfusion service medical directors. such as neonates and pediatric patients with separate
specifically tailored order sets. Blood should not be denied
Behavioral Influences simply because a specific clinical indication was not
included among the choices on the blood order form. These
There are three types of behavioral influences that can rare instances require consultation with the blood bank
impact program success and should be considered in the physician before issuing or denying product and may pro-
planning process: direct, local, and external. Direct influences vide opportunities for continuous improvement of the form
are the communications and actions between the physician through updated versions.
or nursing staff involved in ordering a blood product and the
blood bank staff involved in manipulation and dispensation Education
of the product. Local influences include directing forces
particular to the institution or locality, including habitual Numerous studies suggest that educational efforts alone may
ordering practices, administrative expectations, blood bank impart a substantial improvement to blood utilization.17,29,39
resources, and physician group demands. External influences Physician training in transfusion medicine is variable and
include standard of care issues and obstacles outside the often limited to behavior learned “on the floor.” Pressure
immediate control of the hospital, such as conflicts between from specialists or more senior medical staff may influence
current evidence-based studies or consensus agreements. transfusion-related decisions made by medical residents.
Blood supplier resources are also an external influence. With Educational activities can be focused on a single practitioner
a common goal to improve safe and effective blood utiliza- or group. A direct phone call may be made to a physician
tion practices, all three of these must be addressed to ensure who appears to order inappropriately. Another example is a
quality and uniformity of care while avoiding unnecessary small group discussion with a physician group about current
conflict and confusion. guidelines for plasma transfusion. Formal lectures at grand
If planned interventions include component denial or rounds are also excellent methods for relaying transfusion
administrative actions, then it is of great importance to have guidelines to the community. Resident education is an ideal
transfusion committee endorsement before implementing way to instill proper transfusion practice. If educational
such actions. Consideration should be given to methods efforts are burdensome on available resources and time,
of creating official hospital policy and seeking administrative consideration should be given to participation in hospital
support. In addition, consensus and support should be obtained grand rounds or sponsorship of invited expert speakers.
2682_Ch24_526-539 22/05/12 12:05 PM Page 535
Brief prepackaged computer-based training modules can be and others with a thorough understanding of blood use,
included annually to reinforce guidelines. including knowing appropriate indications and risk. The
Sufficient lead time must be allocated for training and team must be skilled in communication and negotiation
competency of technical staff. Staffing responsibilities and since multiple interdepartmental actions are required. Effec-
limitations should be addressed. Even a small retrospective tive negotiation in the planning stage ensures a smooth
program should develop consistent methods for auditing, implementation and favors acceptance, and it provides a
recording, and reporting of data, thereby creating trans- buffer to administrative problems that may arise later. At a
parency and improving training efficiency. Complex programs minimum, planning must include the medical director and
involving real-time prospective audits require a team approach supervisor of the blood bank. Even with limited-scope
with readily available call schedules, written expectations for utilization programs, the medical director is responsible for
physician evaluators, and consistent documentation of out- maintaining guidelines and approving blood bank proce-
comes. Transfusion service personnel should help validate dures. The medical director is also ultimately responsible for
the implementation or adjustment of the program. The blood the safety and efficacy of transfused blood components. This
bank staff can be crucial in identifying and reporting any individual has the authority to appoint appropriate surro-
unexpected delays in issuing or preparing product. gates to these duties, including screening procedures, deny-
ing product in prospective programs, and providing feedback
Policy Deployment regarding inappropriate transfusion in retrospective audit.
A thorough understanding of current evidence and guide-
Just as a utilization management program needs careful assess-
lines is necessary for key players and should be promoted
ment and thoughtful development of policies and guidelines,
throughout the hospital system. As complexity of the utiliza-
initiating such a program requires planning. Enacting sweeping
tion program increases, additional input and advice should be
changes at the launch of the program may overwhelm partici-
sought from such sources as the transfusion committee, blood
pants, leading to poor compliance and unforeseen risks.
utilization champions, and transfusion safety officers. Blood
Employing the total quality management principle of hoshin
utilization champions are the physicians, practitioners, and
kanri, the program should prioritize the elimination of waste
nurses who order and administer blood components. They are
as outlined in the future state VSM, via policy deployment and
useful in bridging the gap between the blood bank and clini-
control. Hoshin kanri is a strategy of examining all of the de-
cians and can be recruited through newsletters, e-mails, or
sired goals and ordering projects based on balancing priority
directly at hospital lectures and presentations. Current publi-
with ease of accomplishment to ensure progress. Change is
cations and local and national organizations provide resources
thereby enacted in smaller pieces rather than through broad
for implementation of blood management and utilization pro-
instantaneous reform. For example, the program may first take
grams and can be very useful for planning teams unfamiliar
actions to address inappropriate red blood cell use. Once
with this process. Dedicated blood management personnel are
improvement is noted, measures to address plasma use can be
essential to the success of the utilization program, and the
instituted, and so on. An institution may first address the
duties and assignments should be described in detail in a
largest amount of waste, the greatest opportunity for expense
standard operating procedure. The utilization program should
reduction, the area of greatest peril, or any other area according
be conducted with the same level of rigor as other functions
to institutional priorities. Stepwise or phased deployment also
within the blood bank. Each facility will have staffing needs
provides an opportunity to incorporate lessons learned into the
determined based on its particular goals.
design of subsequent phases.40
The skill and training required of an evaluator is based
Personnel upon the complexity of the blood utilization program. The
initial screening is usually accomplished by blood bank staff,
Once the scope and nature of the blood management pro- sometimes in conjunction with automated screening algo-
gram is agreed upon, attention focuses on allocation of rithms. With a retrospective review at a small institution, the
resources and identification of personnel for planning, transfusion safety officer (TSO) may be able to accomplish
approval, and implementation. Although the planning stage retrospective auditing. However, a large prospective review
involves many steps, a well-designed program begets a with potential denial of product will require control by a
smooth implementation. Since blood management programs dedicated on-call physician or designee. Prospective audits
involve the interaction of multiple departments throughout may require the interaction of several physician evaluators.
the institution, the planning team must develop specific Review benchmarks should be developed and the goals of
policies, guidelines, and procedures that fall within the con- the utilization program should be clearly and carefully defined.
straints and expectations of the entire hospital system. Plan-
ners will lay the foundation for a successful utilization Transfusion Safety Officer
program, and once the program is implemented, they may
continue in other leadership roles. Continued involvement The transfusion safety officer (TSO) is a unique career role
allows them to serve as a resource for continuous improve- that developed in Canadian and European blood banks.
ment and to provide motivation and inspiration. TSOs are typically medical laboratory scientists or nurses
As the planning committee is established, team leadership (BSN). Essentially, they coordinate and participate in most
should include personnel well versed in blood bank operations functions within the blood utilization program, including
2682_Ch24_526-539 22/05/12 12:05 PM Page 536
but not limited to audits of transfusion records, quality transfusions are appropriate and have a therapeutic effect.35 The
assurance, and educational functions. This role requires program should not be structured in such a way as to limit the
excellent communication skills, clinical knowledge, and ability capabilities of the primary clinician, especially in emergencies.
to manipulate database software. This allows the TSO to per- Figure 24–5 illustrates one model of process flow for a
form audits, educate physicians and nurses, and assist with utilization management program. Most important is recog-
the development of other transfusion safety programs.41 nizing that the program design requires a cyclic model to
Coordinating auditing functions and educational activities ensure continuous improvement and maintain currency.
and ensuring that resources and personnel are correctly tasked Members of the utilization team may be responsible for spe-
is essential during implementation. In addition to prospec- cific phases of the cycle or may follow a particular compo-
tive or retrospective auditing, meticulous records should be nent or issue through the entire cycle. Regular updates and
kept for trending purposes. The coordinator must have suf- communications among team members are essential for
ficient knowledge of blood banking if no TSO is appointed. monitoring progress and ensuring that learning experiences
are shared. Important key players are listed in Table 24–2.
Information Technology
Continuous Improvement
For most programs, the inclusion of a team member from
information technology will be important. Data mining Continuous improvement (also known as kaizen) through
and analysis will be critical in assessing the current state Lean or another quality process is required for long-term suc-
and designing reports for continuous monitoring. A great cess of a blood utilization management program.12 Within the
deal of information can be gleaned and processed by well- strategy established in the future state map, improvement
constructed queries to the Library Information System (LIS), teams should be deployed at all organizational levels. For
electronic medical record, billing system, and other data- example, blood bank technologists, nursing staff, and ordering
bases. Often these systems do not readily interface, and practitioners should be included in the strategy. One study has
in-house design of well-tailored validated programs may demonstrated that without such measures, improvements in
provide the best solution. However, before any such design utilization tend to decrease after as little as 3 years.33 It follows
is undertaken, there must be agreement upon and clear that the long-term success of the program should be considered
communication of the precise data and metrics that will be a safety and cost-saving investment, which is dependent on the
desired. Redesign and rework of such programs is resource adequate allocation of resources. While teams may be empow-
intensive. Additional waste is generated if the ultimate result ered to make recommendations for improvements, implemen-
does not serve the purpose for which it was intended. tation of process changes must be endorsed by management to
ensure safety and total value stream optimization. Monitoring
Design Considerations and continuous improvement may include most of the same
players involved in the planning stages. The medical director
At first glance, the apparent increased complexity of the blood must be aware of trending patterns and anticipate changes to
management model versus simple component dispensation the screening criteria. A significant reduction in usage may
(order filling) may seem prohibitive. However, delays can be require changes in staffing and ordering components from
prevented by well-designed, properly implemented standard vendors. An increase in inappropriate utilization or a failure to
operating procedures. Intervention strategy styles should be improve may require reallocation of resources and more intense
paired with an appropriate audit type, since not all are comple- auditing procedures. Additional personnel resources for chart
mentary to each other. Figure 24–4 shows a schematic of review and clinician interaction may be required.
the interactions between blood utilization review and interven- The transfusion committee is an ideal forum for the review
tion strategies and the potential impact on the ordering clini- and assessment of the program’s success. Because of its
cian. It is important to remember that even in facilities where multidisciplinary nature, feedback can be shared between
there are significant improvement opportunities, many of the the blood bank and clinicians, preventing the development
Figure 24–5. Sample utilization management process flow: The cyclic nature is
critical to continuous improvement and program currency. CASE STUDIES
Case Study 24-1
Table 24–2 Key Players in a Utilization You are the blood bank supervisor at a medium-sized
Management Program hospital. Administration has been impressed by reported
ROLE EXAMPLES cost-reduction realized in other facilities with active blood
utilization and management programs. You have been
Planners • Medical director of blood directed to begin such a program in your facility.
bank
• Transfusion committee 1. What will you tell administration about cost reduction
• Supervisor of blood bank as the goal for a blood utilization and management
• Transfusion safety officer
• Delegated clinical leadership program?
• Quality assurance 2. What will your first steps be?
• Information technology 3. Who will be included on the blood utilization man-
agement team?
Approvers • Executive staff for physicians
Policy approval, resource • Physician groups with high 4. How will you establish appropriate utilization criteria?
provision, program blood utilization 5. What type(s) of review might you consider?
support, and awareness • Nursing leadership
• Hospital administration Case Study 24-2
Implementers • Coordinator (transfusion safety You are the transfusion safety officer of a large, growing
Day-to-day operations officer) tertiary-care hospital facility. Your facility’s blood utilization
• Evaluators (auditors, controller,
screeners) management program consists of monthly retrospective
• Educators (consultant, analysis of blood usage by diagnosis and procedure codes.
lecturer) Your results are reported at the quarterly transfusion
• Physician ambassadors committee, where review determines whether placement
• Nursing ambassadors of a letter in the physician’s file is warranted. You notice
• Suppliers (blood bank staff
following utilization an increasing trend in both plasma usage and plasma
procedure) wastage. You must report your findings and analysis at
the next transfusion committee meeting.
Reviewers • Medical director of blood
Reassessment and bank 1. How will you assess the source of the problem?
refinement of existing • Transfusion committee 2. How will you assess the decrease in value as a result
program • Supervisor of blood bank of the problem?
• Transfusion safety officer
• Hospital leadership and 3. What strategies should be used to develop possible
administration (for policy improvements?
changes) 4. How will the corrective action be monitored?
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SUMMARY CHART
Cost savings and risk reduction can be realized from a Meaningful metrics are chosen to reflect the progress
well-designed blood utilization management program. of the system toward facility-defined goals. Trending
The program should be multidisciplinary, tailored to and tracking these metrics allows monitoring of program
the needs of the institution, rely upon evidence-based effectiveness and indicates opportunities for continuous
criteria, and employ mechanisms for continuous mon- improvement.
itoring and improvement. Lean or another quality Blood utilization review consists of retrospective, con-
process can be utilized to organize the system and current, and prospective methods. Based on current
provide consistency and value. and future needs, a facility may choose a single method
Blood utilization management is a process designed to or may use them in combination.
ensure that blood transfusions are administered in ac- When review processes identify deviations, interventions
cordance with evidence-based guidelines and prede- are triggered. Like review processes, there are multiple
termined criteria. Effective program design and good interventions, and selected types are determined based on
compliance ensure that patients receive transfusions institutional culture and needs. However, not all review
only when necessary and medically appropriate. processes and interventions are mutually compatible.
Concepts derived from Lean manufacturing or from Key personnel in the blood utilization management in-
other quality systems may be employed in the design clude the transfusion service medical director, blood
and organization of an effective blood utilization man- bank supervisory personnel, and representatives from
agement program. nursing and the facility’s practicing physicians. Some
Standardization is achieved through thoughtfully institutions may employ a transfusion safety officer. A
prepared transfusion guidelines and carefully selected representative from information technology may also
evidence-based criteria. Blood order sets may also prove invaluable to the team.
be employed to promote uniformity and complete Support and approval by hospital administration is
documentation. crucial to the survival and success of the program.
9. Metrics should be: 18. Goodnough, LT: Transfusion triggers. Surgery 142:S67, 2007.
19. Blajchman, MA, et al: New strategies for the optimal use of
a. Chosen to indicate progress during the process of
platelet transfusions. Hematology Am Soc Hematol Educ
continuous improvement. Program 198, 2008.
b. Tracked and disseminated only to members of the 20. Wong, MP: Guidelines for frozen plasma transfusion. BC Med J
blood utilization management team. 49:311, 2007.
c. The same for all institutions. 21. Spiess, BD: Red cell transfusions and guidelines: A work in
progress. Hematol Oncol Clin North Am 21:185, 2007.
d. Selected only by the transfusion service leadership.
22. Slichter, SJ: Evidence-based platelet transfusion guidelines.
10. Perfect value with zero risk and no peril is attainable Hematology Am Soc Hematol Educ Program 172, 2007.
23. American Society of Anesthesiologists Task Force on Periop-
for any facility willing to invest in a blood utilization erative Blood Transfusion and Adjuvant Therapies: Practice
management program. guidelines for perioperative blood transfusion and adjuvant
a. True therapies: An updated report by the American Society of Anes-
b. False thesiologists Task Force on Perioperative Blood Transfusion
and Adjuvant Therapies. Anesthesiology 105:198, 2006.
24. Hill, SR, et al: Transfusion thresholds and other strategies
References for guiding allogeneic red blood cell transfusion. Cochrane
1. Restuccia, JD: The evolution of hospital utilization review Database Syst Rev 2002:CD002042, 2002.
methods in the United States. Int J Qual Health Care 7:253, 25. Saxena, S, et al: Monitoring for underutilization of RBC
1995. components and platelets. Transfusion 41:587, 2001.
2. Assistant Secretary for Planning and Evaluation: Managed Care 26. Vincent, JL, and Piagnerelli, M: Transfusion in the intensive
Terminology. http://aspe.hhs.gov/Progsys/forum/mcobib.htm, care unit. Crit Care Med 34:S96, 2006.
2010. Cited February 4, 2010. 27. So-Osman, C, et al: Triggers and appropriateness of red blood
3. Kim, CS, Spahlinger, DA, and Billi, JE: Creating value in health cell transfusions in the postpartum patient—a retrospective
care: The case for Lean thinking. J Clin Outcomes Manage audit. Vox Sang 98:65, 2010.
16:6, 2009. 28. Fernandez-Perez, ER, Winters, JL, and Gajic, O: The addition
4. Goodnough, LT, and Shander, A: Blood management. Arch of decision support into computerized physician order entry
Pathol Lab Med 131:695, 2007. reduces red blood cell transfusion resource utilization in the
5. Shea, AM, et al: Use and perceptions of clinical practice intensive care unit. Am J Hematol 82:631, 2007.
guidelines by internal medicine physicians. Am J Med Qual 29. Tinmouth, A: Reducing the amount of blood transfused by
22:170, 2007. changing clinicians’ transfusion practices. Transfusion 47:132S,
6. Laird, J, and Soutar, R: Effective transfusion audit can improve 2007.
and alter clinical practice: Something that is often questioned. 30. Hennessy, S, et al: Retrospective drug utilization review, pre-
Transfus Med 18:141, 2008. scribing errors, and clinical outcomes. JAMA 290:1494, 2003.
7. Hendrickson, JE, and Hillyer, CD: Noninfectious serious 31. Murray, ME, and Henriques, JB: An exploratory cost analysis
hazards of transfusion. Anesth Analg 108:759, 2009. of performing hospital-based concurrent utilization review.
8. Shulman, IA, and Saxena, S: The transfusion services Am J Manag Care 9:512, 2003.
committee—responsibilities and response to adverse trans- 32. Navarro, R: Managed Care Pharmacy Practice, 2nd ed. Jones
fusion events. Hematology Am Soc Hematol Educ Program and Bartlett, Sudbury, 2009.
483, 2005. 33. Tobin, SN, Campbell, DA, and Boyce, NW: Durability of
9. Gray, A, et al: Promoting safe transfusion practice: Right blood, response to a targeted intervention to modify clinician trans-
right patient, right time. Br J Nurs 17:812, 2008. fusion practices in a major teaching hospital. Med J Aust
10. Womack, JP, and Jones, DT: Lean Thinking: Banish Waste and 174:445, 2001.
Create Wealth in Your Corporation. Free Press, New York, 34. Beguin, C, et al: Concentration of transfusion resources on
2003. a few pathologies and a few patients: Analysis of the compre-
11. Melanson, SE, et al: Applying Lean/Toyota production system hensive in-hospital patient database. Transfusion 47:217, 2007.
principles to improve phlebotomy patient satisfaction and 35. Damiani, G, et al: Appropriateness of fresh-frozen plasma usage
workflow. Am J Clin Pathol 132:914, 2009. in hospital settings: A meta-analysis of the impact of organiza-
12. Womack, JP, and Jones, DT: Lean Solutions: How Companies tional interventions. Transfusion (epub) August 31, 2009.
and Customers Can Create Value and Wealth Together. Free 36. Tinmouth, A, et al: Reducing the amount of blood transfused:
Press, New York, 2005. A systematic review of behavioral interventions to change
13. Chapman, CE, et al: Ten years of hemovigilance reports of physicians’ transfusion practices. Arch Intern Med 165:845,
transfusion-related acute lung injury in the United Kingdom 2005.
and the impact of preferential use of male donor plasma. Trans- 37. Scheurer, D, et al: Effectiveness of computerized physician
fusion 49:440, 2009. order entry with decision support to reduce inappropriate
14. Dickson, EW, et al: Application of lean manufacturing tech- blood transfusions. J Clin Outcomes Manage 17:10, 2010.
niques in the emergency department. J Emerg Med 37:177, 38. Holland, R, et al: Creating champions for health care quality
2009. and safety. Am J Med Qual (epub) December 4, 2009.
15. Dzik, S: Use of a computer-assisted system for blood utilization 39. Brandt, MM, et al: Transfusion insurgency: Practice change
review. Transfusion 47:142S, 2007. through education and evidence-based recommendations.
16. Cobain, TJ, et al: A survey of the demographics of blood use. Am J Surg 197:279, 2009.
Transfus Med 17:1, 2007. 40. Hutchins, DC: Hoshin Kanri: The Strategic Approach to
17. Garrioch, M, et al: Reducing red cell transfusion by audit, Continuous Improvement. Gower, Burlington, VT, 2008.
education and a new guideline in a large teaching hospital. 41. Dzik, WH: Emily Cooley Lecture 2002: Transfusion safety in
Transfus Med 14:25, 2004. the hospital. Transfusion 43:1190, 2003.
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Chapter 25
Transfusion Safety and Federal Regulatory
Requirements
Judy Ellen Ciaraldi, BS, MT(ASCP)SBB, CQA(ASQ), and Alan E. Williams, PhD
OBJECTIVES
1. Describe why laws governing the regulation of biological products were enacted.
2. Define a biological product manufacturer.
3. Describe the regulatory process.
4. List the requirements for and responsibilities of being registered with the FDA.
5. List the requirements for and responsibilities of FDA licensure.
6. Describe the FDA’s inspectional authority of biological product manufacturers.
7. Distinguish between licensed and unlicensed manufacturers and between interstate and intrastate commerce.
8. Describe the role of the FDA current good manufacturing practice (CGMP) in biological product manufacturing.
9. List the possible enforcement actions.
10. List the FDA’s five layers of safety for protecting the blood supply.
540
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Introduction
BOX 25–1
The Department of Health and Human Services (DHHS) is Commonly Used Abbreviations
the United States government’s principal health agency for
BLA Biologics License Application
protecting the health of all Americans. The Centers for Disease
BPDR Biological Product Deviation Report
Control and Prevention (CDC), the Centers for Medicare
DHHS Department of Health and Human Services
and Medicaid Services (CMS), the National Institutes of
CBER Center for Biologics Evaluation and Research
Health (NIH), and the Food and Drug Administration (FDA)
CFR Code of Federal Regulations
are the primary agencies of the DHHS with responsibilities
CGMP Current Good Manufacturing Practice(s)
directly related to blood. DHHS is part of the executive
CMS Center for Medicare and Medicaid Services
branch of the U.S. government.
CSO Consumer Safety Officer
In addition to regulating other products such as food,
FDA Food and Drug Administration
veterinary medicine, and tobacco, the FDA promulgates
FD&C Act Federal Food, Drug, and Cosmetic Act
and enforces regulations to ensure the safety and efficacy
FR Federal Register
of biological products, drugs, and devices, which include
FY 2008 Fiscal Year 2008 (October 1, 2007 to September 31,
human blood and blood components, blood collection
2008)
supplies and instruments, blood establishment computer
OBRR Office of Blood Research and Review
systems, blood bank reagents, and infectious disease tests.
OCBQ Office of Compliance and Biologics Quality
Regulations for biological products promulgated under the
ORA Office of Regulatory Affairs
Public Health Service (PHS) Act and the Federal Food,
PHS Act Public Health Service Act
Drug, and Cosmetic (FD&C) Act are found in Parts
210-211, 600-680, and 800-820 of Title 21, Code of Fed-
eral Regulations (CFR).1 These regulations set forth the
regulatory requirements under the statutes, including ad-
herence to current good manufacturing practice (CGMP) labeling requirements, and authorizing entry of federal offi-
requirements. In addition, the regulations address licens- cials into manufacturing facilities to conduct inspections.
ing of biological products and establishment registration, The Biologics Control Act was incorporated into the
as well as product-specific standards for blood and blood Public Health Service (PHS) Act in 1944 as Section 351.
components. The new PHS Act required a biologics license to be in effect
This chapter describes the history of biologics regulations, before a biological product enters into interstate com-
the FDA’s regulatory process and requirements, the FDA’s merce.4 A biologics license application can be approved
inspection and enforcement activities, and the CGMP only after the manufacturer demonstrates that both the
requirements for blood and blood components. The appro- product and the manufacturing facility meet standards to
priate citations for specific regulations are provided next to ensure the continued safety, purity, and potency of the
the topic. Common abbreviations used here and throughout products.
the chapter are listed in Box 25–1. The 1902 Act did not include all biological products;
specifically, it did not address the regulation of blood and
History of Biologics Regulation blood components. In 1970, the definition of a biological
product within the PHS Act was expanded to include blood
Public Health Service Act and blood components and derivatives.
The regulation of biological products in the United States Federal Food, Drug, and Cosmetic Act
began when Congress passed the Biologics Control Act of
1902 (also known as the Virus-Toxin Law).2 The Federal Food, Drug, and Cosmetic (FD&C) Act was
In 1901, there was a serious epidemic of diphtheria that passed in 1938 to further define the government’s regula-
resulted in a great demand for the diphtheria antitoxin. tory authority. This act requires manufacturers to demon-
At that time, there were no requirements for safety testing. strate that a drug is safe and effective before marketing
A horse from which serum was obtained to produce the it. In addition, it authorizes facility inspections of drug
diphtheria antitoxin had contracted tetanus, which re- manufacturers and requires drug manufacturers to register.
sulted in tetanus infection in persons who received the The FD&C Act prohibits the introduction or delivery for
diphtheria antitoxin. The Biologics Control Act of 1902 introduction into interstate commerce any food, drug,
was passed following the deaths of 13 children who device, or cosmetic that is adulterated or misbranded.5
received injections of diphtheria antitoxin contaminated A drug or device is misbranded if its labeling is false or
with tetanus.3 misleading. A drug is adulterated if the methods used in
The Biologics Control Act required biological products to its manufacture do not conform with CGMP to ensure the
be manufactured in a manner that ensured the safety and pu- drug is safe, and has the identity and strength, and meets
rity of the product. The Act included provisions for licensing the quality and purity characteristics which it is repre-
products, suspending or revoking the license for violations, sented to possess.
2682_Ch25_540-555 22/05/12 12:06 PM Page 542
The FD&C Act has been amended many times to incor- The Office of Regulatory Affairs (ORA) is responsible for
porate new provisions. For example, in 1976 the FD&C performing FDA postmarket inspections of blood and blood
Act was amended to strengthen the FDA’s authority to component manufacturing facilities and other surveillance
regulate medical devices. In 1992, the Prescription Drug activities.
User Fee Act (PDUFA) was passed to expand the FDA’s The FDA has identified five overlapping layers of
capability to hire reviewers using industry application and safety that work together to prevent an unsuitable blood
supplement submission processing fees in exchange for component from being transfused to a patient.7 Blood and
FDA’s agreement to complete drug reviews within speci- blood component manufacturers must ensure they have
fied time frames. Device user fees were first enacted processes in place to perform and monitor each of these
in 2002. In 1997, the Food and Drug Administration procedures:
Modernization Act (FDAMA) was enacted to implement
1. Donor screening: Donors are informed about potential
many changes, including those to bring the regulation of
risks and are required to answer questions about factors
biological products in closer harmony with the regulations
that may affect the safety of their blood. For example,
for drugs. Some of the provisions in FDAMA include
donors with a history of intravenous drug abuse are rou-
eliminating the need for establishment licenses, streamlin-
tinely deferred.
ing the approval process for manufacturing changes, and
2. Blood testing: After donation, each unit of donated blood
incorporating risk-based regulation of medical devices. In
undergoes a series of tests for infectious diseases.
2007, the FDA Amendments Act (FDAAA) renewed the
3. Donor lists: Blood establishments must keep a current list
Prescription Drug and Device User Fee programs, increased
of deferred donors and use it to make sure they do not
the public transparency of current regulatory actions, and
collect blood from anyone on this list.
provided the FDA with numerous new authorities to assess
4. Quarantine: Donated blood must be quarantined until it
and act on safety issues that may only be recognized after
is tested and shown to be free of infectious agents.
a drug is marketed.
5. Problems and deficiencies: Blood centers must investi-
Food and Drug Administration gate manufacturing problems, correct all deficiencies,
and notify the FDA when product deviations occur in
Originally, the Laboratory of Hygiene of the Marine Hospital distributed products.
Service was charged with enforcing the Biologics Control
Act. In 1930, this laboratory was renamed the National In- Regulatory Process
stitutes of Health (NIH). In 1937, the Division of Biologics
Control (later renamed as the Bureau of Biologics) was Manufacturers, Manufacturing, and Biological
formed within the NIH to regulate biological products. The Products
FDA began in 1927 as the Food, Drug, and Insecticide
Administration, a law enforcement agency within the Biological product manufacturers are defined as any legal
U.S. Department of Agriculture. The name was changed to person or entity engaged in the manufacture of biological
FDA in 1930. In 1968, the FDA was transferred to the Public products subject to licensure under the PHS Act. The
Health Services within what was then known as the Depart- term “manufacturer” also includes any legal person or en-
ment of Health, Education, and Welfare. The authority for tity who is an applicant for a license where the applicant
biological products was transferred from the NIH to the FDA assumes responsibility for compliance with applicable
in 1972. In May 1980, education functions were removed product and establishment standards (21 CFR 600.3(t)).
from the department and it became known as the Depart- Manufacturing for blood and blood products means the
ment of Health and Human Services.2,6 collection, preparation, processing, or compatibility test-
Since 1972, the FDA has overseen the safety, purity, and ing by chemical, physical, biological or other procedures
potency of the U.S. blood supply by ensuring that blood and of any blood product that meets the definition of a drug
blood component manufacturers conduct their operations as defined in section 201(k) of the act. It includes the
and manufacture their products in accordance with appli- manipulation, sampling, testing, or control procedures
cable laws and regulations, including CGMP. Biological applied to the final product or to any part of the process
products are regulated in two FDA centers: the Center for and to the packaging, labeling, repackaging, or otherwise
Drugs Evaluation and Research (CDER), which regulates changing the container, wrapper, or labeling of any blood
therapeutic biological products, and the Center for Biologics product package in furtherance of the distribution of the
Evaluation and Research (CBER), which regulates biological blood product from the original place of manufacture to
and related products, including blood, vaccines, allergenics, the person who makes final delivery or sale to the ultimate
tissues, cellular and gene therapies, and related devices. The consumer (21 CFR 607.3(d)).
Office of Blood Research and Review (OBRR) and the Office Biological products are defined as “any virus, therapeutic
of Compliance and Biologics Quality (OCBQ) in CBER are serum, toxin, antitoxin, vaccine, blood, blood component or
responsible for overseeing the regulation and enforcement derivative, allergenic product, protein (except any chemi-
of biologics regulations for blood and blood components. cally synthesized polypeptide), or analogous product or
2682_Ch25_540-555 22/05/12 12:06 PM Page 543
arsphenamine or derivative of arsphenamine (or other triva- amendments to the Acts to further define the agency’s reg-
lent organic arsenic compound), applicable to the preven- ulatory authority.
tion, treatment, or cure of diseases or condition of human
beings” (42 USC 262(i)). Biological products, including Regulations
blood and blood components, are unique in that they may
be considered as biological products under the PHS Act and Agencies promulgate regulations to establish a specific
as drugs or devices (depending on their mode of action) standard or requirement of conduct set by the federal
under the FD&C Act. This means that both the CGMP reg- government under the statutory authority granted by
ulations for finished pharmaceuticals (21 CFR 210 and 211) Congress (21 CFR 10.90). Regulations are an interpreta-
and the CGMP regulations for blood and blood components tion of the statutes and are legally binding on both the
(21 CFR 606) apply. Table 25–1 provides a comparison of manufacturing industry and the government agencies
biological products and drugs. charged with enforcing the laws. The regulations define
There are a variety of tools used by the FDA to commu- specific minimum standards that manufacturers must
nicate and enforce the regulatory requirements, agency rec- meet. They often do not provide specific procedures on
ommendations, and product standards. These include how to meet the standards. There may be many acceptable
regulations and guidance documents. ways to achieve a required objective, and a requirement
that is too specific may become outdated.
Statutes and Laws Proposed and final regulations are published in the
Federal Register (FR), an official publication of the federal
Statutes are the acts passed by Congress and signed into government, under the authority of the Administrative
law by the President to establish the FDA and to grant Procedure Act, which usually requires notice and an
the agency authority to fulfill its mission. Statutes such as opportunity for the public to comment on regulations.
the PHS Act and the FD&C Act require biological product, FDA regulations are codified in Title 21 of the CFR. The
drug, and device manufacturers to demonstrate to the regulations applicable to blood and blood components
FDA that their products are safe and effective (for drug are found in subchapter F, Parts 600 to 680, and subchap-
products) or safe, pure, and potent (for biological prod- ter C, Parts 210 to 211, of Title 21 of the CFR. Major
ucts) before they are marketed in interstate commerce in regulations pertaining to blood and blood components are
the United States. They also require that all biological listed in Box 25–2.
product, drug, and device manufacturers follow the appli-
cable CGMP regulations and label their products appro- Guidance Documents
priately. The FD&C and PHS Acts grant the FDA the
authority to oversee and enforce the requirements Guidance documents describe the agency’s interpretation
described in the Acts, including inspecting the manufac- of or policy on a regulatory issue. The recommendations in
turing facilities and imposing penalties for violations when the FDA’s guidance documents are not legally binding on
appropriate. Over the years, there have been several the manufacturing industry or other government agencies.
Registration and Licensure product standards, and are routinely inspected by FDA inves-
tigators. Registered blood establishments that do not hold an
Registration approved BLA may only distribute their products in intrastate
commerce.
The FD&C Act requires manufacturers to register their estab- The regulations exempt some blood establishments from
lishments with the FDA and list the products they manufac- registration (21 CFR 607.65). The exemption applies to:
ture. Regulations for blood establishment registration are found
in the CFR (21 CFR 607). The following blood establishments • Transfusion services that neither collect nor process blood
are required to register with the FDA (21 CFR 607.20): components and that only perform compatibility testing
(crossmatching) and transfusion, pool Platelets and Cry-
• Collection centers oprecipitated AHF units immediately before transfusion,
• Community blood banks or issue bedside leukocyte-reduction filters with blood
• Component preparation facilities components and are either CLIA certified or have met
• Hospital blood banks equivalent requirements as determined by CMS
• Plasmapheresis centers • Carriers that transport blood products
• Product testing laboratories • Brokers who do not take possession, manipulate, or rela-
• Storage and distribution centers bel the product
• Brokers who take possession and manipulate and/or
relabel the product Table 25–2 summarizes the registration of blood establish-
ments. Additional information about registration can
Blood establishments must register within 5 days after be- be found on the CBER website at www.fda.gov/Biologics
ginning their manufacturing operations or within 5 days BloodVaccines/GuidanceComplianceRegulatoryInformation/
after the submission of a Biologics License Application EstablishmentRegistration/BloodEstablishmentRegistration/
(BLA) (21 CFR 607.21). Blood establishments may register default.htm.
by one of two methods: by completing a hard copy of the
registration form (Form FDA-2830) and submitting it to Licensure
the FDA or by completing an electronic registration form
found on the CBER website (www.fda.gov/BiologicsBlood The PHS Act requires a biologics license to be in effect if a man-
Vaccines/GuidanceComplianceRegulatoryInformation/ ufacturer wants to distribute biological products in interstate
EstablishmentRegistration/BloodEstablishmentRegistration/ commerce. A biologics license will be issued only after the
default.htm). manufacturer demonstrates that the product is safe, pure, and
The manufacturer must list all products and manufactur- potent and that the facility where the product is manufactured
ing processes performed at the establishment on the registra- meets applicable requirements to ensure the biological product
tion form and must complete a form for each manufacturing continues to be safe, pure, and potent (21 CFR 601.2(d)).
facility. Registered establishments are required to update their Unlike most licensed biological products, which typically
registration each year (21 CFR 607.21). In addition, all reg- have one Biologics License Application (BLA) approval per
istered establishments are responsible for complying with the product, blood establishments manufacture multiple licensed
applicable FDA regulations, including CGMP and applicable products under one license and one BLA approval.
Hospital blood banks Transfusion services that do not collect blood; only perform crossmatching
and transfusion, pool Platelets and Cryoprecipitated AHF units immediately be-
fore transfusion, and issue bedside leukocyte-reduction filters, and are either
CLIA certified or have met equivalent requirements as determined by CMS
Plasmapheresis centers
Brokers (take possession and manipulate and/or relabel product) Brokers (do not take possession, manipulate, or relabel product)
2682_Ch25_540-555 22/05/12 12:06 PM Page 546
The review of a BLA involves two steps: evaluating the not to distribute because of deficiencies in the submission.
submitted application documents (e.g., standard operating The 30-day wait is waived for some moderate changes,
procedures [SOPs], forms, labels, product quality control, which are reported as a Changes Being Effected Supple-
etc.) and conducting a pre-license inspection of the man- ment (CBE) (21 CFR 601.12(c)(5)). For submissions re-
ufacturing facility (21 CFR 601.2). The applicant submits porting these moderate changes, the product is distributed
the necessary application forms and information about before the FDA has approved the change; therefore imple-
their operations to CBER (21 CFR 601.2).8 Consumer mentation of the change and distribution of the product
safety officers (CSOs) in OBRR evaluate the application made using the change are performed at the manufac-
and supporting documents. The CSOs will inform the turer’s own risk. The FDA will review the submission, and
applicant if additional information is needed to complete if deficiencies are identified, will notify the manufacturer
the evaluation of the documents. As part of the application to submit the necessary information. In some cases, the
review, CSOs and ORA investigators will conduct a pre- FDA may require the manufacturer to stop distribution
license inspection of the manufacturing facility. The FDA until any identified deficiencies have been corrected.
investigators will observe the manufacturing processes Minor changes are reported to the FDA in an annual
to determine if the product is prepared according to the report (21 CFR 601.12(d)). Minor changes do not need
regulations, CGMP, product standards, and the manufac- to be approved by the FDA before the product made using
turing description provided in the application. the minor change is distributed into interstate commerce.
The applicant should respond to any inspectional obser- The FDA will review the annual report to determine if the
vations noted during the inspection. After the applicant has changes have been reported in the proper category. The FDA
addressed all deficiencies noted during the evaluation of the will notify the manufacturer if the annual report contains
submitted application documents and the facility inspection, changes that should be submitted as a supplement for
the CSO will make a decision regarding licensure. Once the FDA approval and may require the manufacturer to stop
manufacturer is licensed, the license number must appear distribution until the supplement is reviewed and approved.
on the label of the licensed products in order for the products
to be distributed into interstate commerce. Licensed manu- Unregistered Transfusion Services
facturers are responsible for complying with all applicable
The regulations exempt some blood establishments from reg-
FDA regulations, including CGMP and applicable product
istering with the FDA because they do not perform the man-
standards, and are inspected by FDA investigators at least
ufacturing steps requiring registration and are either CLIA
once every 2 years (21 CFR 600.21).
certified or have met equivalent requirements as determined
Licensed manufacturers are required to inform the FDA
by CMS (21 CFR 607.65). The majority of unregistered
about any intended change in manufacturing from previ-
blood establishments operate as transfusion services that
ously approved procedures (21 CFR 601.12). Originally,
only perform compatibility testing (crossmatching) and
when a manufacturer submitted a change to the FDA, the
transfusion, pool Platelets and Cryoprecipitated AHF units
manufacturer had to wait for CBER to approve the change
immediately before transfusion, and issue bedside leukocyte-
before the products could be distributed into interstate com-
reduction filters. Even though they are not registered or
merce. In 1997, CBER issued a new regulation to streamline
licensed, these manufacturers must still comply with the ap-
some FDA activities and reduce the reporting burden by the
plicable FDA regulations, including CGMP and applicable
manufacturing industry.9 The regulations in the CFR were
product standards. There are certain instances when the FDA
revised to be consistent with similar requirements in
will inspect these blood establishments; these are typically
FDAMA. Title 21 CFR 601.12 is the regulation that addresses
“for-cause” inspections, such as investigating a transfusion-
how licensed applicants report their manufacturing changes
related fatality.
to the FDA. Manufacturing changes are now divided into the
In 1983, the FDA entered into an agreement with the
following three categories as determined by the potential of
Health Care Financing Administration (HCFA), now called
the change to adversely affect the safety, purity, and potency
the Centers for Medicare and Medicaid Services (CMS).11
of the product. CBER has published guidance to help appli-
The Memorandum of Understanding (MOU) between the
cants determine the appropriate reporting category:10
FDA and CMS consolidated within CMS the responsibility
• Major changes (based on associated risk) are reported as for inspecting and surveying unregistered transfusion serv-
a Prior Approval Supplement (PAS) (21 CFR 601.12(b)). ices in order to minimize duplication of effort and to reduce
These submissions must be approved before the applicant the burden on the affected blood establishments. Transfusion
can distribute the product made using the change into services that engage in the compatibility testing and trans-
interstate commerce. fusion of blood and blood components, but that neither rou-
• Moderate changes (based on associated risk) are reported tinely collect nor process blood components, are now
as a Changes Being Effected in 30 Days Supplement covered by CMS. CMS, state survey agencies (including
(CBE30) (21 CFR 601.12(c)). These submissions must be those in CLIA-exempt states), and accreditation organiza-
sent to the FDA for review and approval. The applicant tions (such as CAP and AABB) conduct routine biennial sur-
may distribute the product made using the change in in- veys of transfusion services on behalf of the FDA. The FDA
terstate commerce 30 days after the FDA has received the does not routinely survey transfusion services, although the
submission, unless the FDA has informed the applicant FDA may inspect any transfusion service at any time.
2682_Ch25_540-555 22/05/12 12:06 PM Page 547
Table 25–3 summarizes the regulatory oversight of blood described in the ORA Investigations Operations Manual
and blood component manufacturing facilities. (IOM) and in written compliance programs and compliance
policy guides. The compliance programs provide instruc-
Overview of the FDA’s Inspection tions on how to conduct the inspection and specify the
Process process for recommending appropriate regulatory actions.12
The compliance policy guides inform investigators and com-
Inspection Authorities pliance officers about FDA policies and interpretations on
specific regulatory issues.13 These documents can be found
The statutory provisions granting the FDA the authority on the ORA website at www.fda.gov/AboutFDA/Centers
to enter biological product manufacturing facilities for the Offices/ORA/default.htm.
purposes of conducting inspections are found in both the Each FDA inspection is conducted using a risk-based
PHS and FD&C Acts. The FDA conducts pre-license and systems approach by covering some or all of the following
pre-approval inspections, routine CGMP inspections, and manufacturing systems in a blood establishment operation
for-cause inspections. to ensure compliance with applicable regulations and
FDA investigators responsible for conducting inspections standards:12
receive intensive training in the technical and regulatory • Quality Assurance System: various planned activities that
aspects of the products and manufacturing facilities they in- provide confidence that all procedures and processes that
spect. The investigators for blood-related manufacturing are influence product manufacture and overall quality are
specialized into two teams: (1) Team Biologics, which is re- monitored to ensure they are working as expected.
sponsible for inspecting plasma derivative, reagent, vaccine, • Donor (Suitability) Eligibility System: the system that pro-
and allergenics manufacturers, and (2) the Biologics Cadre, tects donor safety, determines a donor’s suitability for
who inspect blood, plasma, and tissue establishments. This blood collection (including donor deferral from either his-
reorganization was designed to facilitate inspections with im- tory screening or testing), notifies donors of unsuitability
proved consistency and appropriate citations. for donation and evaluates donors for reentry.
• Product Testing System: the system that tests for commu-
Inspection Procedures nicable diseases, performs blood grouping and typing, and
crossmatches blood for transfusion by direct testing or
As part of the biologics license application process, CBER
electronically.
and ORA investigators conduct prelicense and preapproval
• Quarantine/Inventory Management System: the system per-
inspections of manufacturing facilities that have submitted
taining to product storage, distribution, and retrieval; quar-
a BLA or a supplement to an approved BLA. Following
antine; and distribution (release for use or destruction).
approval of the BLA, ORA investigators who are trained as
• Production and Processing System: process controls in the
members of the Biologics Cadre will inspect the manufac-
manufacture of specific blood and blood components, and
turing facility at least once every 2 years (21 CFR 600.21).
equipment quality control, calibration, and maintenance.
The Biologics Cadre also inspects unlicensed, registered
blood establishments that only distribute blood and blood FDA investigators discuss their observations with the man-
components in intrastate commerce. All FDA inspections ufacturer as the inspection progresses. At the end of the
are conducted according to the policies and procedures inspection, investigators list significant observations on
Table 25–3 Summary of the Regulatory Oversight of Blood and Blood Component
Manufacturing Facilities
MANUFACTURERS THAT MANUFACTURERS THAT MANUFACTURERS THAT DO NOT
ENGAGE IN INTERSTATE AND ENGAGE IN INTRASTATE PERFORM MANUFACTURING STEPS
INTRASTATE COMMERCE COMMERCE ONLY THAT REQUIRE REGISTRATION
Requirements for Must be registered Must be registered Exempt from registration
registration
Requirements for Must hold an approved biologics Do not need to be licensed Do not need to be licensed
licensure license application
Applicable CGMP 21 CFR 210, 211, 606 21 CFR 210, 211, 606 21 CFR 210, 211, 606
regulations include:
Inspections Conducted by FDA: CBER Conducted by FDA: ORA Covered under CMS (CMS, state survey
(pre-license and pre-approval) investigators agencies, accredited organizations); FDA
and ORA investigators may conduct “for- cause” inspections
the Form FDA-483, Inspectional Observations. Investiga- must take appropriate corrective actions and may not
tors present the Form FDA-483 to the manufacturer and engage in interstate commerce of the product while the
listen to the proposed corrective actions. Investigators may license is suspended.
also discuss less serious observations with the manufac- A manufacturer’s license can be revoked for several
turer to point out areas that could potentially cause reasons: the FDA’s inability to gain access to the facility to
significant problems. Both significant and less serious conduct a meaningful inspection, the discovery of significant
observations, as well as any discussions with the manufac- CGMP deficiencies, or a determination that the product
turer, are included in the Establishment Inspection Report is not safe or effective for its intended use or is misbranded
(EIR) prepared by the FDA investigators.14 Most inspec- (21 CFR 601.5). The FDA may also revoke a license when it
tions result in voluntary compliance by the manufacturer observes that the deficiencies have been ongoing and have
to take corrective action, but inspections with numerous not been corrected after numerous inspections and prior
significant observations may trigger advisory, administra- notice. The FDA may revoke a license or call for a voluntary
tive, or legal actions, such as Warning Letters, license revocation if the manufacturer no longer makes the product
suspension, injunction, and prosecutions. Each year, the and therefore the FDA cannot conduct a meaningful inspec-
FDA conducts an average of 1,200 inspections of blood tion or evaluation. Revocation will prohibit the manufacturer
and blood component manufacturers. from distributing the product into interstate commerce.
When a biological product manufacturer violates any of the Seizure is an action taken to condemn violative products
laws or regulations the FDA enforces, the manufacturer is and remove them from distribution. The FDA and the U.S.
often given an opportunity for voluntary correction before Marshals Service take possession of the product typically
the FDA pursues enforcement actions. There are three types under a court order. The court usually gives the owner of the
of FDA actions: advisory, administrative, and judicial .15 seized product 30 days to decide on a course of action. If the
owner does not communicate proposed actions, the FDA will
Advisory Actions: Warning Letter dispose of the product. The owner may contest the charges
and litigate in court to have the seized products returned. The
Advisory actions alert manufacturers that they have violated owner is required to provide a monetary deposit (e.g., a bond)
FDA regulations and provide an opportunity for corrective to ensure the court’s orders will be carried out.
action before further compliance action is taken. Warning An injunction is a civil process initiated to stop or prevent
Letters are one example of an advisory action. The FDA will violation of the law, such as to halt the flow of violative prod-
issue a Warning Letter to a biological product manufacturer ucts in interstate commerce and to give the manufacturer an
for violations of regulatory significance. A Warning Letter is opportunity to correct the conditions that caused the viola-
a written communication from the FDA to the manufacturer tion to occur. The FDA may seek to obtain an injunction
notifying them that their product, practice, or other activity when a health hazard has been identified or the manufac-
is in violation of the law. The Warning Letter serves as a prior turer has a history of violations and the evidence suggests
notice that the FDA may decide to take further action, and that serious violations will continue.
it offers the manufacturer an opportunity to correct the An injunction may be used whether or not the manufac-
deficiencies listed in the letter. The FDA will conduct a turer holds a biologics license. If there is no license to sus-
follow-up inspection of the manufacturing facility to deter- pend or revoke, an injunction may be the only way for the
mine if corrective actions have been implemented. Warning FDA to halt the flow of violative products in interstate com-
Letters may have significant financial and legal ramifications merce and to correct the conditions that caused the violation
for a company. These letters are available on the FDA’s to occur. If a manufacturer violates the terms of the injunc-
website at www.fda.gov/BiologicsBloodVaccines/Guidance tion, a court may impose fines or even hold the company or
ComplianceRegulatoryInformation/ucm135850.htm. specific corporate officers in contempt and the FDA may take
further enforcement actions. As of 2009, only one blood and
Administrative Actions: Suspension, Revocation blood component manufacturer was under injunction.
A prosecution is a criminal action directed against a man-
Administrative actions of license suspension or license ufacturer or responsible individual(s) or both. The FDA may
revocation apply only to licensed manufacturers. These consider referring a matter to the Department of Justice for
actions are more formal than advisory actions, such as prosecution when fraud, health hazards, or continuing signif-
issuing Warning Letters, and the agency usually must fol- icant violations are encountered. The FD&C Act allows indi-
low certain procedures, such as providing notice and an viduals or corporations for whom prosecution is being
opportunity for a hearing. In addition, an affected com- considered to be offered an opportunity to explain to the
pany can seek a judicial review of the FDA’s administrative agency why the prosecution is not appropriate. Prosecution
decisions. The FDA may suspend a manufacturer’s license will proceed without a hearing if the violations are fraudulent
if a danger to health exists (21 CFR 601.6). The FDA can or the responsible individuals are likely to flee. Table 25–4
administer this action immediately, and the manufacturer summarizes the enforcement actions.
2682_Ch25_540-555 22/05/12 12:06 PM Page 549
Injunction • Civil process to stop or prevent violation of the law (e.g., to stop producing a product; to
comply with particular regulations)
• Manufacturer can contest injunction in court
• Court may impose penalties
• Applies to both licensed and unlicensed manufacturers
Prosecution • FDA will refer cases to Department of Justice for possible prosecution
• Applies to both licensed and unlicensed manufacturers
• FDA will seek to prosecute for criminal conduct, including fraud or practices that lead to
unsafe products or a pattern of violations
Current Good Manufacturing Practice deviating from established standards. During inspections,
Regulations the FDA observed that there were often no controls in
place to monitor the whole manufacturing process. The
Under the FD&C Act, a drug is adulterated if the methods CGMP regulations were published to provide direction for
used in, or the facilities or controls used for, its manufacture, this control and include the elements of quality assurance,
processing, packing, or holding do not conform to or are not quality control, and process validation. The CGMP regu-
operated or administered in conformity with current good lations for blood and blood components were published in
manufacturing practice. Drugs must meet the requirements 1975.17 Blood and blood component manufacturers must
of the FD&C Act as to safety and must have the identity and follow both the CGMP regulations in 21 CFR 210-211 and
strength and meet the quality and purity characteristics the more specific CGMP regulations for blood and blood
which it purports or is represented to possess. components. The more specific requirements are in
CGMP regulations for finished pharmaceuticals are 21 CFR Part 606 supplement and do not supersede the
found in 21 CFR 210 and 211, and CGMP regulations spe- more general CGMP requirements set forth in 21 CFR
cific for blood and blood components are found in 21 CFR 210 and 211.
Part 606. The CGMP regulations describe the minimum
current practice and require all manufacturing facilities to Quality Control Unit
implement and follow these requirements. The FD&C Act
The CGMP regulations require each manufacturer to desig-
similarly provides that a medical device is adulterated if
nate a quality control unit that has the responsibility and
the methods used in, or the facilities or controls used for,
authority to approve or reject all components, containers,
its manufacture, packaging, storage, or installation are not
closures, labeling, and drug products. Although the regula-
in conformity with device CGMP regulations, which the
tions identify this unit as a “quality control unit” (21 CFR
FDA has issued in the Quality System Regulations found
211.22), many manufacturers identify it as a quality assur-
in 21 CFR 820.
ance unit.
In 1963, the FDA published the first drug CGMP regu-
Under 21 CFR 211.22, the quality control unit has the
lations after observing that unsuitable products were being
responsibility and authority, at a minimum, to:
released to the public.16 The FDA believed that this was
due to an increase in number and complexity of the tests • Approve and reject components, containers (e.g., collec-
being performed and untrained or undertrained personnel tion bags), supplies, and drug products
2682_Ch25_540-555 22/05/12 12:06 PM Page 550
• Review production records for accuracy and completeness Alternative Procedures (“Variances”)
• Investigate errors and deviations
• Review and approve standard operating procedures The regulations allow for blood and blood component man-
ufacturers to perform procedures that vary from what is
The responsibilities and procedures applicable to the quality required in some sections of the CFR with the approval of
control unit must be in writing. The CGMP regulations do not CBER’s Director (21 CFR 640.120). Specifically, manufac-
prescribe how each manufacturer should develop its quality turers may request from CBER a variance to a regulation in
control unit or program but provide minimum requirements subchapter F of the CFR (Parts 600 to 680). This provision
that must be adhered to when developing the program. This is provides flexibility to accommodate changes in technology
to allow flexibility so that each manufacturer can develop a and unanticipated circumstances that may warrant a depar-
program that will work best in its own manufacturing environ- ture from regulations. 20
ment. The FDA has published a guidance document to assist The procedures addressed by 21 CFR 640.120 are some-
blood and blood component manufacturers in developing a times called “variances” and fall into two categories: an alter-
quality assurance program.18 The FDA generally recommends native procedure that is incorporated into blood establishment
that the quality control functions be separate from the manu- SOPs and an exception that is a singular event related to a
facturing operations because both have their own goals that specific emergency situation. Manufacturers wishing to im-
may sometimes conflict. plement a procedure that varies from the regulations in these
specific sections of the CFR must submit a written request to
Quality Reviews CBER. A request for exception or alternate procedure from a
licensed blood establishment must be submitted as a PAS
The CGMP regulations require manufacturers to retain man-
supplement and contain sufficient information to show that
ufacturing and blood collection records, such as donor selec-
the safety, purity, or potency of the product manufactured at a
tion records (21 CFR 211.180 and 606.160). At least annually,
variance from the regulations will not be adversely affected.
the data from written records and quality standards of each
Manufacturers cannot implement an alternate procedure
product must be reviewed to determine the need for changes
or exception until they have received approval from CBER.
in product specifications or manufacturing or control proce-
Examples of alternative procedures and exceptions that
dures (21 CFR 211.180(e)). The FDA’s Guideline for Quality
the FDA has approved are posted on the CBER website at
Assurance in Blood Establishments18 identifies the systems in
www.fda.gov/BiologicsBloodVaccines/BloodBloodProducts/
a blood and blood component manufacturing operation and
RegulationoftheBloodSupply/ExceptionsandAlternative
the critical control points that should be reviewed. The quality
Procedures/default.htm.
control unit or other assigned individuals are responsible for
the quality activities, including the annual reviews. Contract Manufacturing
The review should identify trends that could lead to the
release of an unsuitable product. The quality control unit Under the current regulations, one manufacturer may employ
must investigate any unexplained discrepancy or the failure the services of a contract manufacturer to perform some or
of a lot or unit to meet any of its specifications (21 CFR all of the product manufacturing steps.21 The contractor is
606.100(c)). The FDA expects the outcome of such investi- not under the direct control of the original biological product
gations will be the development and implementation of cor- manufacturer, but the original manufacturer should ensure
rective and preventive actions. Implementing corrective and that the manufacturing steps performed by the contractor
preventive actions could result in changes to product speci- conform with FDA regulations. Contract manufacturers must
fications or manufacturing and control procedures. The be registered with the FDA because they are performing
quality control unit must monitor these changes to ensure a manufacturing step on a regulated product (21 CFR
they adequately address the error and will result in the man- 607.20(a)). In addition, the contractor must follow CGMP
ufacture of a quality product. The review, investigation, and and all applicable regulatory product standards. Contractors
corrective actions must be documented. The quality control should notify the original manufacturer of any changes in
unit should have the authority to stop production if it deter- their operations, because these changes could affect the safety,
mines the quality of the product is being adversely affected. purity, or potency of the product. The original manufacturer’s
The quality control unit should share the results of the re- quality control unit is responsible for approving or rejecting
view with the management of the manufacturing operations drug products manufactured, processed, packed, or held
in order to affect the necessary changes to correct and pre- under contract by another company (21 CFR 211.22).
vent ongoing problems. Records of the reviews may be re- Examples of contract manufacturing include an outside
quested and reviewed during an FDA inspection.19 testing laboratory performing infectious disease testing
on donors or products and a blood center irradiating blood
Additional Requirements of Biological components for a hospital transfusion service.
Product Manufacturing
Short Supply Arrangements
All biological product manufacturers must comply with all
applicable biologics regulations in the CFR. This section lists A short supply arrangement is an example of a cooperative
some of the additional elements of biologics regulation. manufacturing arrangement. Short supply was introduced in
2682_Ch25_540-555 22/05/12 12:06 PM Page 551
1948, and the provisions governing short supply are found Table 25–5 Biological Product Deviations
in the CFR (21 CFR 601.22).21 The short supply regulations Reported to the CBER in FY
allow a licensed biological product manufacturer to arrange
200823
for the partial manufacture of a biological product at an
unlicensed facility under controlled conditions. Specifically, MANUFACTURING SYSTEM % OF TOTAL (44,125)
it allows an unlicensed product, such as recovered plasma, Postdonation information 68.3
to be shipped in interstate commerce and used to manufac-
ture a licensed final product. Short supply arrangements are Quality control and distribution 11.2
most typically established when a blood or blood component Labeling 5.2
manufacturer wants to market recovered plasma. The short
supply arrangement must be in writing and is between the Donor screening 5.2
blood establishment and the licensed final product manu- Miscellaneous 4.3
facturer. The written arrangement specifies the necessary
manufacturing procedures and labeling. Routine testing (ABO, Rh, antibody 2.5
screen)
Brokers frequently act as intermediaries between suppli-
ers and licensed final product manufacturers. Selling recov- Blood collection 1.8
ered plasma to a broker does not relieve the supplier of the
Component preparation 1.1
responsibility of obtaining a short supply arrangement. In
addition, brokers who take physical possession of plasma Donor deferral 0.3
must register with the FDA.
Infectious disease testing 0.1
Recalls are accompanied by a health hazard evaluation of Table 25–6 Transfusion Related Fatalities
the recalled product by FDA medical staff and scientists to Reported to the CBER in FY
determine if the product caused or could cause harm to a re-
200826
cipient (21 CFR 7.41). The recall will be classified as a class
I, II, or III recall based on the potential for a product to cause COMPLICATION % OF TOTAL (46)
serious health problems as outlined below: Transfusion-Related Acute Lung Injury (TRALI) 34.8
• Class I: Situation in which there is a reasonable probability Hemolytic transfusion reaction (ABO) 21.7
that the use of the product will cause serious adverse
health consequences or death Hemolytic transfusion reaction (non-ABO) 15.2
• Class II: Situation in which use of the product may cause Microbial infection 15.2
temporary or medically reversible adverse health conse-
quences or the probability of serious adverse health con- Transfusion Associated Circulatory Overload 6.5
(TACO)
sequences is remote
• Class III: Situation in which use of the product is not likely Anaphylaxis 6.5
to cause adverse health consequences
In FY 2008, CBER classified 2,070 recalls.24 Most of the re-
calls involved blood and blood components that were either www.fda.gov/BiologicsBloodVaccines/SafetyAvailability/Reporta
not likely to cause an adverse reaction or would at most cause Problem/TransfusionDonationFatalities/ucm113649.htm.
only temporary health problems. Additional information about
biological product recalls can be found on the CBER web- Medical Devices
site at www.fda.gov/BiologicsBloodVaccines/SafetyAvailability/
Recalls/default.htm. CBER regulates medical devices used in the collection, pro-
cessing, testing, manufacturing, and administration of blood,
Fatality Reporting blood components, human cells, tissues, and cellular- and
tissue-based products.27 Certain products meeting the defi-
Blood and blood component manufacturers should ini- nition of a medical device and used in blood collection or
tially notify CBER of any transfusion-related fatalities or processing are subject to license under Section 351 of the
donation-related deaths. The regulations require the blood PHS Act. Other medical devices used in blood collection or
and blood component manufacturer to formally notify processing are regulated under the device authorities in Sec-
CBER within 7 days after the fatality is confirmed (21 CFR tion 510(k) of the FD&C Act. Licensed biological products
606.170(b)).25 The 7-day written report should describe that meet the definition of a device are reviewed in the same
all information related to the fatality. Collection facilities manner as other licensed biological products. Examples of
are required to report donation-related deaths, and the such licensed biological products are blood grouping
facility that performed the compatibility tests must report reagents and infectious disease tests used for donor screen-
transfusion-related fatalities. Fatalities are reported to ing. Examples of devices used in blood collection or process-
CBER to: ing regulated by CBER under the FD&C Act are blood
irradiators, blood warmers, blood establishment computer
• Ensure that fatalities are thoroughly investigated
systems, blood filters, and apheresis collection instruments.
• Determine if appropriate corrective actions have been
Manufacturers of licensed biological products that are de-
taken to prevent a recurrence
vices must register with the FDA and list their products
• Determine if there are trends that may warrant FDA
under 21 CFR 607, and manufacturers of cleared biological
action
devices must register with the FDA and list their products
FDA investigators may visit the reporting facility to follow under 21 CFR 807. Both must follow device CGMP, known
up on the fatality reports. In FY 2008, CBER received as the Quality Systems Regulations (21 CFR 820). Additional
72 transfusion recipient fatality reports (46 were assessed information about biological devices can be found on the
to be transfusion-related fatalities) and 10 postdonation CBER website at www.fda.gov/BiologicsBloodVaccines/
fatality reports.26 Transfusion-Related Acute Lung Injury DevelopmentApprovalProcess/510kProcess/default.htm.
(TRALI) was the most common cause of transfusion- Additional information about the FDA and regulatory issues
related fatalities (Table 25-6). Additional information on related to blood and blood components can be found at
reporting fatalities can be found on the CBER website at www.fda.gov/BiologicsBloodVaccines/default.htm.
2682_Ch25_540-555 22/05/12 12:06 PM Page 553
SUMMARY CHART
The FDA promulgates and enforces regulations to en- The blood establishments required to register with the
sure the safety and efficacy of biological products, FDA under the FD&C Act include collection centers,
drugs, and devices, which include blood and blood community blood banks, component preparation
components, blood collection supplies and instru- facilities, hospital blood banks, plasmapheresis centers,
ments, blood grouping reagents, and donor screening product testing laboratories, storage and distribution
tests for infectious diseases. centers, and brokers who take possession and manip-
A drug or device is misbranded if its labeling is false ulate and/or relabel the product.
or misleading. The PHS Act requires an approved biologics license to
One reason a drug is considered adulterated is if the be in effect before biological products are distributed
methods used to manufacture it do not conform with in interstate commerce.
CGMP to ensure the drug is safe and has the identity The FDA may suspend or revoke a biologics license if
and strength and meets the quality and purity charac- there are continued violations that result in harm to a
teristics which it is represented to possess. blood or plasma donor or blood or blood component
The ORA is responsible for performing routine FDA recipient.
inspections and other surveillance activities. CGMP is the minimum current practice for methods to
The FDA’s five overlapping layers of safety of blood be used in and the facilities or controls to be used for
and blood components include determining donor the manufacture, processing, packing, or holding of a
eligibility, testing the donation for certain infectious drug to ensure that the drug meets the requirements of
diseases, checking donor deferral registries, quarantin- the FD&C Act as to safety and has the identity and
ing unsuitable products, and investigating and correct- strength and meets the quality and purity characteris-
ing manufacturing problems. tics that it purports or is represented to possess.
Biological products are defined as any virus, therapeutic CBER must approve alternative procedures or excep-
serum, toxin, antitoxin, vaccine, blood, blood compo- tions from the regulations before the product made at
nent or derivative, allergenic product, protein (except a variance from the regulations is distributed.
any chemically synthesized polypeptide), or analogous Deviations in biological product manufacturing that
product or arsphenamine or derivative of arsphenamine affect the safety, purity, or potency of distributed prod-
(or other trivalent organic arsenic compound) applica- ucts must be reported to the FDA if the affected prod-
ble to the prevention, treatment, or cure of diseases or uct was distributed.
conditions of human beings. A product recall is the removal or correction of a mar-
Statutes are the laws and acts passed by Congress keted product that the FDA considers to be in violation
and signed by the President to establish the FDA and of the laws.
to grant the agency authority to fulfill its mission. Following a confirmed fatality involving blood collec-
The Acts enforced by the FDA that are most relevant tion or transfusion, the manufacturer must notify
to blood regulation are the FD&C Act and the CBER as soon as possible and provide a written report
PHS Act. within 7 days of the incident.
5. Which of the following government organizations in- 2. Commemorating 100 Years of Biologics Regulation. Science
spect blood and blood component manufacturers? and the regulation of biological products from a rich history
to a challenging future. Center for Biologics Evaluation
a. CBER and Research, FDA, March 2002. Available at www.fda.
b. ORA gov/AboutFDA/WhatWeDo/History/ProductRegulation/
c. CMS 100YearsofBiologicsRegulation/default.htm.
d. All of the above 3. The Coroner’s Verdict in the St. Louis Tetanus Cases. 1901
New York Medical Journal 74; Special Article: Fatal results
6. Which of the following is true about CGMP? from diphtheria antitoxin. Minor comments: Tetanus from
antidiphtheria serum. JAMA, 1901; 37:1255, 1260.
a. CGMP is the minimum current practice for methods
4. Public Health Service Act. Regulation of Biological Products.
and facilities used to manufacture a drug to ensure Title 42 United States Code, Chapter 6A, Part F, Section 262
that it is safe, pure, and potent. (42USC262). Available at www.fda.gov/RegulatoryInformation/
b. The FDA will approve a Biologics License Application Legislation/ucm148717.htm.
if the manufacturer does not have a quality control 5. Federal Food, Drug and Cosmetic Act. Drugs and Devices.
Chapter V, Subchapter A. Available at www.fda.gov/Regulatory
plan. Information/Legislation/FederalFoodDrugandCosmeticAct
c. The quality control unit must perform all the quality FDCAct/FDCActChapterVDrugsandDevices/default.htm.
functions. 6. History of the FDA. Available at www.fda.gov/AboutFDA/
d. Blood and blood components do not have to be in WhatWeDo/History/default.htm.
compliance with the drug CGMP regulations. 7. Keeping blood transfusion safe: FDA’s multi-layered protections
for donated blood. Available at www.fda.gov/BiologicsBlood
7. A donor calls the blood bank and informs them that Vaccines/SafetyAvailability/BloodSafety/ucm095522.htm.
within a year prior to his donation, he had intimate 8. FDA Guidance for Industry: For the Submission of Chemistry,
Manufacturing and Controls and Establishment Description
contact with a person diagnosed with HIV. Which of the Information for Human Blood and Blood Components
following action is NOT required by the FDA? Intended for Transfusion or For Further Manufacture and for
a. Identify and quarantine all blood and blood com- the Completion of the Form FDA 356h “Application to Market
ponents produced from the blood supplied by the a New Drug, Biologic or an Antibiotic Drug for Human Use,”
May 1999. Available at www.fda.gov/BiologicsBloodVaccines/
donor GuidanceComplianceRegulatoryInformation/Guidances/Blood/
b. Report the biological product deviation to CBER if ucm077087.htm.
the product has already been distributed 9. Changes to an Approved Application. Federal Register. 62 FR
c. Enter the donor in a record so that he can be iden- 39901, July 24, 1997.
tified and his product not be distributed while he is 10. FDA Guidance for Industry: Changes to an Approved Appli-
cation: Biological Products: Human Blood and Blood Compo-
deferred nents Intended for Transfusion or For Further Manufacture,
d. Notify the AABB July 2001. Available at www.fda.gov/BiologicsBloodVaccines/
GuidanceComplianceRegulatoryInformation/Guidances/Blood/
8. A patient dies following transfusion of ABO-incompatible ucm076729.htm.
blood. To whom should this event be reported? 11. Memorandum of Understanding between the Health Care
a. The Center for Biologics Evaluation and Research Financing Administration and the Food and Drug Administra-
b. Center for Medicare and Medicaid Services tion. FDA 225-80-4000, June 6, 1983. Available at www.fda.
gov/AboutFDA/PartnershipsCollaborations/Memorandaof
c. The AABB central office
UnderstandingMOUs/DomesticMOUs/ucm116313.htm.
d. The Occupational Safety and Health Administration 12. FDA Compliance Program Guidance Manual—7342.001: Inspec-
tion of Licensed and Unlicensed Blood Banks, Brokers, Reference
9. Which federal agency has the responsibility to routinely Laboratories, and Contractors. FDA Office of Regulatory Affairs,
inspect an unregistered transfusion service that does not Implementation Date: October 1, 2006. Available at www.fda.
collect blood? gov/BiologicsBloodVaccines/GuidanceComplianceRegulatory
a. Food and Drug Administration Information/ComplianceActivities/Enforcement/Compliance
Programs/ucm095226.htm.
b. Centers for Medicare and Medicaid Services
13. Chapter 2, Biologics, FDA Compliance Policy Guides, FDA
c. Occupational Safety and Health Administration Office of Regulatory Affairs. Available at www.fda.gov/ICECI/
d. State health department ComplianceManuals/CompliancePolicyGuidanceManual/
ucm116336.htm.
10. Which of the following is NOT one of the FDA layers of 14. Investigations Operations Manual, FDA Office Of Regulatory
safety? Affairs, 2009. Available at www.fda.gov/ICECI/Inspections/
a. Donor screening IOM/default.htm.
15. FDA Regulatory Procedures Manual. FDA Office of Regula-
b. Biologics License Application
tory Affairs, March 2009. Available at www.fda.gov/ICECI/
c. Investigation of manufacturing problems ComplianceManuals/RegulatoryProceduresManual/default.htm.
d. Infectious disease testing 16. Drugs: current good manufacturing practice in manufacture,
processing, packing, or holding. Federal Register. 28 FR 6385,
References June 20, 1963.
17. Human blood and blood products: collection, processing
1. Code of Federal Regulations, Title 21, FDA. US Government and storage. Federal Register. 40 FR 53531, November 18,
Printing Office, Washington, DC, April 1, 2010. Available at 1975.
www.gpoaccess.gov/CFR/.
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18. FDA Guideline for Quality Assurance in Blood Establish- 23. Biological product deviation annual summary for fiscal
ments, July 11, 1995. Available at www.fda.gov/BiologicsBlood year 2008. October 1, 2007, through September 30, 2008. Avail-
Vaccines/GuidanceComplianceRegulatoryInformation/ able at www.fda.gov/BiologicsBloodVaccines/SafetyAvailability/
Guidances/Blood/default.htm. ReportaProblem/BiologicalProductDeviations/ucm169990.htm.
19. FDA Compliance Policy Guide, Sec. 130.300 FDA access to re- 24. The Enforcement Story, FDA Office of Regulatory Affairs,
sults of quality assurance program audits and inspections. FDA March 2009. Available at www.fda.gov/ICECI/Enforcement
Office of Regulatory Affairs, June 2, 2007. Available at www. Actions/EnforcementStory/default.htm.
fda.gov/ICECI/ComplianceManuals/CompliancePolicy 25. FDA Guidance for Industry: Notifying FDA of Fatalities Related
GuidanceManual/ucm073841.htm. to Blood Collection or Transfusion, September 2003. Available at
20. Blood and blood products: amendment to allow for alternative www.fda.gov/BiologicsBloodVaccines/GuidanceCompliance
procedures; removal of a labeling requirement. Federal Regis- RegulatoryInformation/Guidances/Blood/ucm074947.htm.
ter. 55 FR 10420, March 21, 1990. 26. Fatalities Reported to FDA Following Blood Collection and
21. FDA Guidance for Industry: Cooperative Manufacturing Transfusion: Annual Summary for fiscal year 2008. October 1,
Arrangements for Licensed Biologics, November 2008. Available 2007, through September 30, 2008. Available at www.fda.gov/
at www.fda.gov/BiologicsBloodVaccines/GuidanceCompliance BiologicsBloodVaccines/SafetyAvailability/ReportaProblem/
RegulatoryInformation/Guidances/General/ucm069883.htm. TransfusionDonationFatalities/ucm113649.htm.
22. FDA Guidance for Industry: Biological Product Deviation 27. CBER’s Annual Report for Fiscal Year 2008. Available at
Reporting for Blood and Plasma Establishments, October 2006. www.fda.gov/AboutFDA/CentersOffices/CBER/ucm122880.htm.
Available at www.fda.gov/BiologicsBloodVaccines/Guidance
ComplianceRegulatoryInformation/Guidances/Blood/
ucm073455.htm.
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Chapter 26
Laboratory Information Systems
Ann Tiehen, MT(ASCP)SBB, Gregory Wright, MT(ASCP)SBB,
and Melissa Volny, MT(ASCP)SBB
OBJECTIVES
1. Explain the purpose of an information system in a blood bank.
2. Correlate the hardware components of a blood bank information system with their functions.
3. Describe a blood bank information system hardware configuration.
4. Describe the functions of the software components of a blood bank information system.
5. List the responsibilities of operating and maintaining a blood bank information system.
6. Identify regulatory and accrediting agency requirements pertaining to blood bank information systems.
7. Apply the functions of blood bank software applications to blood bank processes.
8. Explain the purpose of a truth table.
9. Construct a truth table for a routine blood bank test.
10. Identify the standard operating procedures (SOPs) needed to manage a blood bank information system.
11. Describe and justify the kinds of testing that should be included in the validation of a blood bank information system.
12. Interpret the results of a software validation test.
13. Identify the times at which validation testing must be performed.
14. Define control function and differentiate between process control and decision support.
15. Evaluate a validation test plan and identify its component parts.
556
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cannot recognize voice instructions. Commands and data applications depends on the quality of print desired. For
must be entered in a format that the computer can under- example, a bar-code label requires a high-resolution printing
stand. This requires tools that are connected to the system’s that bar-code readers can accurately interpret. Management
CPU such as keyboards, pointing devices, bar-code readers, reports, which are usually used internally, can be of lower
and testing instruments. Keyboards are used to type instruc- quality print.
tions that tell the computer what to do or to enter data such An example of a combined input and output device is a
as donor demographic information or patient test results. modem. Modems allow computer systems to communicate
Many systems also accept bar-coded information from with each other via network or telephone lines. Blood banks
sources such as blood component labels or test tubes. Blood can use this mechanism to connect remote facilities to the
component labels contain many pieces of bar-coded infor- main computer system so that all sites have access to the
mation, including the unit number, blood type, product type, system’s databases. This configuration is found in blood cen-
facility identification number, and expiration date. Entry of ters with several collection or testing locations or in a system
this information by a bar-code reader is much more accurate of hospital affiliates. The technical support staff of software
than when done by manual entry methods. Figure 26–2 vendors uses modems to investigate and solve system prob-
shows a blood component label with this bar-coded infor- lems and to transfer files to the customer.
mation. Scanning the bar codes with an optical or laser
device allows efficient entry of the blood component data. Information Storage Hardware
The most common output device is the monitor, which All computer systems have hardware that allows long-term
displays information as it is typed on the keyboard. Together, storage of data. This is sometimes referred to as memory but
the monitor and the keyboard make up the user terminal. should not be confused with RAM, which is only temporary
The monitor may also display historical information at the and active when the system is turned on. Long-term memory
user’s request. is data saved on a medium from which they can later be
Another commonly used output device is the printer, retrieved, such as a hard disk. Most systems contain a hard
which provides hard-copy output on paper. Printers can disk controlled by a hard drive. The disk is made of metal
produce labels, donor registration records, compatibility coated with a magnetic film and contains software applica-
tags, management reports, patient chart reports, and donor tions (programs) and user-entered data. The hard disk is
correspondence. The kind of printer chosen for any of these often contained in the main system unit, but it may also be
a peripheral device. The information on the disk is accessed
by entering commands, usually with a keyboard connected
to a display terminal. Other information storage hardware
may be used for archiving old information that has been
removed from the hard disk.
Software
Software tells the computer what to do with all of the infor-
mation it has received. Minimally, every computer system
has two kinds of software—operating system software and
application software. Some systems may also use interface
software, which allows the system to communicate with
other computer systems.
Application Software
An application is software that has been designed to perform
specific tasks. Personal computers (PCs) can be equipped
with application programs such as word processing, spread-
sheets, and databases. In a blood bank information system,
the application software allows users to perform tasks that
are specific to blood bank operations. In a donor setting,
some of these tasks might include entering donor demo-
graphic information and test results, confirming blood com-
ponent labeling, and generating donor recruitment lists. In
a transfusion service, the computer may help with tasks such
as searching for a blood component of a particular ABO
group, entering blood component modification information,
and issuing blood for transfusion. These tasks are distinctly
connected to blood banking and would be difficult, if not
impossible, to perform in an application not specifically
Figure 26–2. Bar codes on unit label. designed for blood bank use.
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One of the most important functions performed by blood It is a set of instructions that controls the computer’s
bank applications is maintaining a database of donors, blood hardware, manipulates the application software, and coordi-
components, and transfusion recipients. A database is an or- nates the flow of data to and from disks and memory. When
ganized set of information divided into files and then further new data are entered into one of the application programs,
subdivided into records. Files exist in one of two ways: they it is the operating system that places those data on the disk
may be static or dynamic. The dynamic files contain records for storage; when a request for those data is made through
related to a specific donor, patient, or blood component. the application program, the operating system retrieves them
These records are frequently changed as updated information and sends them to the application software for display on a
is added to them. For example, the status of a blood compo- monitor or printed report.
nent can go from “quarantined” to “transfused,” with many
intermediate statuses during its shelf life. Interface Software
The static files contain information that is updated infre- Frequently, different information systems must be allowed
quently, such as the list of blood products used in the facility. to share data with each other to take full advantage of their
These files, which look different in each blood bank, define functionality. Because different systems communicate in dif-
the terminology that the blood bank uses. When a new blood ferent computer languages, they need an interpreter that will
bank information system is installed, the static files must be allow data to flow between the two systems in a controlled
defined before the system can be put into use. One of the manner. The interpreter is another set of software called an
most important functions of the static files is to provide a interface. Interface software may be used to allow data to
“dictionary” of coded terms that the information system flow between a hospital information system (HIS) and the
can use to sort and organize the tremendous amount of data blood bank system or between the LIS and the blood bank.
entered into it. For example, two static files, one containing For example, an interface between the HIS and the blood
codes for physicians and the other codes for blood products, bank information system can allow patient demographic
can allow the system to sort information regarding the cross- information to flow from the HIS to the blood bank system
match-to-transfusion (C:T) ratio of each physician who and can allow test results and blood component information
ordered blood within a particular time period. to flow from the blood bank system to the HIS, where they
Figure 26–3 shows how the parts of a database relate to can be displayed on terminals in patient care areas.
each other. The database itself can be considered a file cabinet
containing folders or files that contain specific records. This People
illustration is of a dynamic file containing patient records. A
static file illustrated in this way—for example, for blood prod- The human components of a blood bank information system
uct codes—would list a separate file for each blood product are the users and at least one person designated as the system
code used in the facility. The associated records would include manager. Users have access to the technical applications
such information as the official name of the blood component, needed to perform daily blood bank operations. System
its maximum allowable shelf life, and whether it contains red managers require access to a wider range of applications,
blood cells (RBCs) and thus requires crossmatching. including system maintenance functions.
Etc.
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system can be used depends not only on its ease of use but telephone contact for recruitment, and notification in the event
also on the training provided to its users. Most systems are that abnormal laboratory testing results are obtained. In addi-
equipped with both a “live” or production database and a “test” tion, the system should have a means for preventing a donation
database. The static files in both databases are identical, but from a deferred individual or from one attempting to donate
their dynamic files contain different information. The produc- before the required waiting period between donations.
tion database contains real information, whereas the test
database consists of fictitious records. The test database offers Donor Registration. When a potential donor registers at a
the user an opportunity to practice the applications of the sys- donation facility, several pieces of demographic data must be
tem (and to make mistakes) without corrupting the database provided, such as name, date of birth, phone number, and
containing actual donor and patient information. Users should mailing address. When this information is entered into the
be trained in every application they will be expected to use. donor database, the system can search for a previous dona-
tion record from the same donor. If none is found, a new
System Managers donor record is created. If there is a record of a previous do-
The configuration and location of the information system will nation, the system can review the donor’s eligibility status.
determine the identity and quantity of people performing this This would include calculating the length of time elapsed
task. In a large community blood center, there may be an since the last donation and examining the record for a de-
entire staff dedicated to managing the system. In a hospital ferral because of medical history or disease testing results. If
blood bank or transfusion service that uses the blood bank the donor is ineligible because of an inadequate amount of
module of a complete clinical LIS, the laboratory system man- time since the last donation or a previous deferral, the system
ager is likely to have responsibility for the entire system, but can alert the registrar and prevent an unsuitable donation
there should also be a designated blood bank system manager. from occurring. If the registration is taking place at a location
In blood banks equipped with a stand-alone blood bank that does not have immediate access to the electronic data-
computer, designated system managers may also have other base, as may happen on a mobile blood drive, the registrars
supervisory, technical, or quality assurance duties. can be equipped with a printed list of eligible and ineligible
Whatever the duties of the system manager, he or she also donors. Alternatively, the database can be downloaded to a
oversees the maintenance of the system’s hardware and soft- portable PC and transported to the mobile site.
ware. Some specific duties include adding or deleting items
Donation Data. Information regarding the donation event can
from the static database files, assigning access codes to new
also be entered into the system. This data entry is usually
users, and implementing software upgrades from the vendor.
performed after the donation but can include such important
In addition, the system manager will be required to investi-
information as the unique identification number applied to
gate problems encountered by the users and to report them
the collection container, the type of donation made (e.g.,
to the system vendor.
whole blood or apheresis), the collection time, and the
Blood Bank Software Applications occurrence of a donor reaction. If the donation is intended
for a specific recipient, as in the case of an autologous or
The kinds of data that must be managed by a particular blood designated donation, data regarding the intended recipient
bank information system depend on the types of services pro- can be entered.
vided by the blood bank. Most community blood centers focus
on donations and distribution of the donated blood compo- Donor Recruitment. The capture of donor demographic infor-
nents to customer hospitals. In the hospital transfusion serv- mation at registration allows the collecting facility to recruit
ice, the primary concern is the transfusion of patients. the donor from a system-generated list of eligible donors.
However, a community blood center or a hospital blood bank Mailing labels, lists of telephone numbers or e-mail addresses
with both a transfusion service laboratory and a donation can be generated for recruitment efforts. Once laboratory
center must address issues of donor and patient management. testing is associated with the donor’s record, lists of donors
Application programs exist for every task, from scheduling meeting special needs can be printed. Such lists may include
donors to assigning a transfused status to a blood component. donors with a specific ABO group, other RBC antigen type,
Many of the typical blood bank information system applica- or cytomegalovirus (CMV) seronegativity.
tions currently available are discussed in this section.
Blood Component Management
Donation Facility Aspects of blood component management include compo-
nent preparation, laboratory testing, label application and
The applications utilized at a donor center enable the users verification, and inventory management and product shipping.
to manage the flow of donors and blood products from donor
recruitment to distribution of the final products to hospital Component Production. After a whole blood unit has been
customers. collected, it is usually delivered to a component-processing
laboratory where it is separated into different components,
Donor Management including RBCs, fresh frozen plasma, and platelets, and labeled
The system should allow the capture of donor demographic appropriately. Data on the new components created are entered
information necessary for definitive donor identification, into the system with the unique donation identification
2682_Ch26_556-570 22/05/12 12:06 PM Page 561
number assigned at the time of donation, new blood product ABO/Rh confirmation testing may be assigned an “available”
codes, time of preparation, and expiration dates. status on entry into the system.
Laboratory Testing. Samples of the donor’s blood that were Inventory Management. A computerized inventory makes it
collected at the time of donation and labeled with the same easy for blood bank staff members to monitor inventory
unique donation identification number are tested for ABO, Rh, levels so that levels do not drop below predefined minimums.
atypical antibodies, and markers of transfusion-transmitted Most systems sort the inventory by product code, ABO/Rh,
diseases such as hepatitis and HIV. The results of all these tests and expiration date, and output it to a display monitor or
are entered into the system so that they are associated with printed report. When a selection list of blood components is
the unique donation identification number and with the indexed in this way, the products that are closer to the
donor. In larger community blood centers, the results of test- end of their shelf lives can be chosen for patients with a high
ing performed on automated instruments can be sent directly likelihood of transfusion.
from the instrument to the system via an instrument interface.
Blood Component Modification. Many components require mod-
Label Application and Verification. After completion of component ification to meet the special transfusion needs of a particular
production and laboratory testing, the blood products are patient. Modifications include irradiation, leukocyte reduc-
labeled with the ABO/Rh type. This is a crucial step and must tion, aliquoting (dividing), washing, and pooling. As these
be controlled stringently so that no unsuitable blood prod- physical modifications are performed in the blood bank, the
ucts are released into the blood supply. After the label has steps associated with them are captured by the information
been applied, the unique donation identification number and system, either by changing the product name or by adding a
ABO/Rh type bar codes on the blood component label can special attribute to the component data. Some of these steps
be scanned into the system with a bar-code reader. This al- may also require shortening the component’s shelf life, and
lows the system to perform a final check on donor suitability the system can calculate and apply the new expiration date
and blood type. If the component “passes” this verification, and time, if needed. Attributes may also be added to com-
it can be placed in the available inventory. ponents that have undergone special testing, such as tests
for antibodies to CMV or specific RBC antigens.
Inventory Management and Product Shipping. The collection facil-
ity staff members who are responsible for product distribu- Component Status Tracking. After blood components are received
tion to customer hospitals must have access to the entire in the transfusion service, they are assigned various statuses,
inventory of available blood products. As transfusion serv- ending with a final disposition of “transfused” or “discarded.”
ices place requests for quantities and ABO/Rh types of blood Each blood component record in the information system
components, the distribution staff can monitor and control should contain a complete status history. Figure 26–4 illus-
distribution to optimize blood use within the community. trates the various statuses that a blood component may be
For example, blood products nearing their expiration date ascribed throughout its shelf life.
can be sent to active transfusion services where there is a
high probability of transfusion before expiration. As prod-
Unit received
ucts are shipped to transfusing facilities, their status is from blood center
Unit donated
updated in the system, along with the identification of the Unit
facility to which they were shipped. retyped
Unit discarded
Transfusing Facility Quarantine Available or
Unit shipped to
The applications utilized at a transfusing facility enable the tested another facility
user to maintain accurate histories of patient and donor unit
testing and ensure the release of compatible blood products Crossmatched
for transfusion.
Blood Component Management
Aspects of component management unique to the transfusion Reserved
for patient
service include product receipt and entry, inventory manage-
ment, blood component modification, and component status
tracking.
Returned Issued
Product Receipt and Entry. When the products are received at
the transfusing facility, they are entered into the blood bank
information system, where the entire inventory of blood
components is maintained. If ABO/Rh confirmation is Transfused
required, as in the case of components containing RBCs, a
status of quarantine will be assigned by the system until Figure 26–4. Various statuses of a blood component in a blood bank informa-
confirmation testing is completed. Components not requiring tion system.
2682_Ch26_556-570 22/05/12 12:06 PM Page 562
Figure 26–6. Display monitor showing entry of an invalid ABO group. In addition to the requirements specific to a collection or
transfusion facility, there are general applications that are
found in all blood bank information systems. These functions
Blood Component Reservation. The system can aid in selecting assist with system security, quality assurance, and management
blood components that will satisfy special transfusion assistance.
needs and that are compatible with the patient’s ABO/Rh.
Selections may be made by manual entry or bar-code entry Security
or from an indexed selection list. If autologous or desig- It is critical for only authorized users to have access to the
nated blood components are available for a patient, the sys- information contained in a blood bank computer system.
tem can alert the user. As blood components are selected Access is obtained through the use of assigned user codes
and either crossmatched or reserved for a patient, the sys- and passwords, and it is the users’ responsibility to keep their
tem links those products to the patient record and prints passwords confidential. A second level of security restricts
compatibility tags. some users to some of the system’s functions. This is impor-
A fairly recent application available on some systems tant for two reasons: donor/patient confidentiality and pro-
is the computer crossmatch. It allows electronic verifica- tection of data from accidental or unauthorized destruction
tion of recipient and donor compatibility and dispenses or modification. Sensitive information, such as test results
with the serologic crossmatch test. A patient is eligible for markers of transfusion-transmitted disease, should be
for the computer crossmatch when his or her records available only to the medical director and selected supervi-
indicate that two criteria have been met: (1) there is no sory personnel. The system manager has access to most or
current or past history of clinically significant antibodies, all of the functions, including those that allow modification
and (2) there are at least two concordant ABO grouping of the static database files. Technical staff members can
test results. access those functions that are specific to their jobs, such
In addition, the system must contain the donation iden- as test result entry, labeling, or component production.
tification number, component name, component ABO group Clerical staff members may be restricted to inquiry func-
and Rh type, the interpretation of the component ABO con- tions that allow them to answer questions from patients
firmatory test, and recipient information, including ABO group or physicians but not to enter or modify patient data. In
and Rh type. The system must process this information to addition to providing system security, user codes capture
alert the user when there are discrepancies between donor the identity of each person performing each step in the
unit labeling and blood group confirmatory test interpre- information system.
tation and when ABO incompatibilities exist between the
recipient and donor unit. Finally, the system must require Quality Assurance
verification of correct entry of data before blood compo- Examples of quality assurance activities performed by a
nents are released. These stringent requirements for a blood bank information system include control functions,
computer crossmatch must be in place to prevent the issue the generation of corrected and amended results, and tools
of ABO-incompatible components. that can be used to monitor blood product utilization.
Result Reporting. Information regarding patient test results and Control Functions. One of the most useful features that a blood
any products linked to the patient should be accessible to bank information system can offer is assistance to users in
the patient’s caregivers. This may be in the form of printed ensuring safe transfusion. When blood bank personnel depend
reports or monitor displays. The status of linked products on this assistance in making decisions at critical points in
2682_Ch26_556-570 22/05/12 12:06 PM Page 564
Donor management Preventing registration of permanently Warning of current ABO/Rh not matching
deferred donor previous results
Blood component Preventing unit release if testing Calculation of component expiration date
management: collection facility unacceptable
Blood component Preventing entry of donation Warning when entering an expiration date that exceeds
management: transfusing facility identification number that already the possible shelf life of a blood component
exists in the system
Patient management Preventing issue of outdated unit Warning of product not meeting special transfusion needs
2682_Ch26_556-570 22/05/12 12:06 PM Page 565
perform thorough validation testing before making software Backup of Software Programs and Data
available for purchase, and users must document validation Blood banks have come to depend heavily on their informa-
procedures on site before using such a system. tion systems, but systems are not infallible. Unexpected, and
Voluntary accreditation agencies, such as the Joint Com- sometimes inexplicable, failures of software or hardware can
mission, the American Association of Blood Banks (AABB), occur. Worse, a natural disaster such as a fire or flood can
and the College of American Pathologists (CAP), also have destroy parts or all of an information system. Software and
standards or inspection checklist items that emphasize the databases should be routinely copied to some storage
responsibilities of operating an information system. A blood medium, such as a backup hard drive. The copies can then
bank must have documented evidence of validation of its be used to restore any corrupted or lost information. The
system as well as written procedures for all aspects of system frequency with which this backup routine is performed
management so as to comply with regulatory and accredita- depends on each blood bank’s data volume and the recom-
tion requirements. mendations of the software vendor. The copies should be
stored in a safe location separate from the information sys-
System Management tem so that they will not be affected by disastrous events
such as a fire or flood. There should also be a procedure to
Many responsibilities are associated with the operation and
identify and restore any information that was not included
maintenance of a computerized blood bank information sys-
in the backup copies. This can be done by manually reentering
tem. The bulk of these responsibilities occur when a system
data that was input between the time of the most recent
is first installed as new SOPs are written, the system is
backup and the disaster.
validated, and personnel are trained. After a system is estab-
lished, routine maintenance procedures are performed to Computer Downtime
ensure the ongoing operation of the system.
With any computer system, there will be times when the sys-
tem is not available to users. These disruptions in availability
Standard Operating Procedures
are commonly referred to as downtime. Sometimes the
Written standard operating procedures (SOPs) for every downtime is planned, as when the system must undergo
blood bank operation are required by all regulatory and maintenance procedures, backup, or software enhancement.
accreditation agencies, and computer operations are no Other downtimes are unplanned and usually unpleasant,
exception. The computer-related tasks associated with each because the system is experiencing some difficulty such as a
of the blood bank’s technical operations can be incorporated power outage. In either case, there must be a written proce-
into each technical procedure or can be addressed in a dure that will allow blood bank operations to continue. The
separate section of the procedures manual. In addition, there procedure must address the need for retrieving historical
are several computer-specific SOPs that must also be avail- patient and donor data and for recording new data obtained
able. The subjects of these are included in the following during the downtime.
discussions. Transfusion service staff must be able to compare current
patient test results with previous records before blood com-
Archiving Data ponents are issued. Donor center staff must have access to
As the system’s databases grow in size, the hard disk becomes permanently deferred donor files. This essential information
crowded with this stored information. As a result, the com- may be in the form of paper records or may be available to
puter’s response time slows, and the hard disk is no longer personnel on a PC where the information has been previously
able to store new data. When this happens, additional hard downloaded from the blood bank information system. New
disks may be added to the system, or old data may be re- data generated during downtime—such as test results, blood
moved from the hard disk and placed in archival storage. donations, and distribution or issuance of blood products—
Removing old data frees up space on the hard disk so that can be recorded on hard-copy worksheets or forms and then
new data can be placed onto it. Old data may be archived “backloaded” into the computer when it becomes functional.
to long-term storage media as such as microfiche and flash
or external hard drives. A procedure for periodically Hardware Maintenance
assessing hard disk space should be instituted so that Information system hardware, like all pieces of equipment,
data can be archived in an orderly fashion before the hard requires periodic cleaning, lubrication, and replacement of
disk becomes full. Procedures for archiving must also be parts to ensure continued operation. Maintenance proce-
written and should adhere to applicable regulatory and dures usually require that the system be “taken down” or
standard-setting agency requirements for data retention. turned off for a period. For that reason, regular maintenance
For example, AABB standards require that each donor’s procedures should be scheduled and posted so that blood
ABO group and Rh type be retained for a minimum of bank staff members can prepare for downtime. When possi-
10 years, and patient records of adverse reactions to trans- ble, maintenance should be scheduled to minimize interrup-
fusion must be retained for 5 years. Written procedures tion of service, such as during usual periods of low activity.
for retrieving archived data must ensure that records As the system’s databases grow in size, the computer’s
can be accessed within a reasonable period to maintain response time may reach an undesirably slow pace. Proce-
patient care. dures should be established for removing the data from the
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disk. The software will contain programs for purging and SOPs, and people require that each blood bank perform
archiving older data. Vendors may also offer support services on-site validation testing to prove that the system will perform
that can examine the system disk(s) to identify potential appropriately under those unique conditions.
problems. Validation testing must be performed before a new sys-
tem can be implemented and whenever new software, data-
Security Maintenance bases, or hardware is added to the system. When validation
A procedure for adding users to a system must be estab- is to be performed on an existing system, the testing should
lished, including the basis on which security levels will be be done in the test database, where it will not affect the
assigned. For example, the list of functions or applications production data.
to which a user will have access can be created for each job
description. It is also important to have a method for deleting Risk Assessment
the access codes of users who leave the facility. An additional Proper validation of the system requires careful planning.
level of security can be obtained by requiring users to change Risk assessment is a required analysis tool that focuses
their passwords periodically; many security applications can validation efforts on system functions or settings that present
be programmed to require this at specified intervals. the greatest potential to harm the patient, donor, or business
in the event of a failure. Vendor descriptions of control func-
Tracking and Correcting Errors tions and on-site system configuration settings are reviewed
Error management, required by regulatory and accrediting and assigned a risk category (such as high, medium, or low)
agencies, has become an integral part of blood bank opera- by the system manager or validation team. A validation test
tions. It includes detecting, documenting, and investigating plan is then prepared, ensuring that the highest risk areas
errors; implementing corrective actions; and reporting to ap- receive an appropriate degree of validation.
propriate agencies. Errors in the operation of a blood bank
information system may be caused by inadequate training, Test Plan
incomplete or cumbersome written procedures, or system A test plan describes the series of exercises that will be used
problems. In any case, tracking and categorizing errors is to validate the blood bank software. Each system function
essential if corrective actions are to be taken. Identified infor- that the blood bank will use must be included in the test
mation system problems should be reported to the software plan. A test plan should be created for each function or
vendor so that other users can be notified and remedies can operation that the computer will perform. Risk analysis will
be included in a future software revision. determine the extent of testing required. Test plans should
Personnel Training include the following items:
The most elegant and user-friendly information system can • Control functions
be a disaster if staff members are inadequately trained in its • Data entry methods (keyboard, bar-code reader, instru-
use. Training programs should address every function and ment interface, LIS, or HIS interface)
procedure that each user will be expected to perform. Flow- • Specific test cases
charts and checklists are useful training tools. Training mod- • Documentation methods
ules can be designed that present users with the situations • Acceptance criteria (expected outputs)
that they will encounter in their work. The competence of • Result review
each staff person must be documented before allowing rou- • Corrective action (if necessary)
tine use of the computer. Competence assessment methods • Acceptance
can include direct observation, review of system-generated Most of these items will be different in each of the system’s
records, and written examinations. Once initial competence applications, so a separate test plan for each application may
has been demonstrated, periodic assessment must be done be necessary. Control functions should be defined by the
to document the continued competence of blood bank staff vendor, but data entry methods will be selected by the user;
to use the information system appropriately. As previously some applications may have multiple data entry methods.
discussed, it is most beneficial to allow users access to a test Specific tests cases are discussed below. Screen printouts,
database for training purposes. If one is not available, test written logs, or printed reports can be used to document the
data can be used in the production database. testing. After the testing is completed, the results should be
compared with acceptance criteria to determine acceptability.
Validation of Software
If unacceptable results are obtained, they should be investi-
Software validation is the establishment of documented evi- gated, and corrective action should be implemented. When
dence that provides a high degree of assurance that the sys- corrective action is necessary, testing should be repeated
tem will consistently function as expected. It is the until it is found acceptable.
responsibility of the software vendor to validate, to the ex-
tent possible, the functionality of a software product before Test Cases
it is marketed to blood banks. However, software vendors Validation test cases include the specific steps to be followed
cannot simulate the conditions in which each system will be by those performing the testing. Test cases should assess the
used. Differences in environment, hardware, databases, system under normal operating conditions but must also
2682_Ch26_556-570 22/05/12 12:06 PM Page 567
offer challenges to the system. The goal is to make sure that involves pushing the system to its physical limits. This might
the system repeatedly performs intended functions and does be accomplished by allowing large volumes of data to be en-
not perform unintended functions. The types of testing that tered into the system via all available input devices.
should be performed are: Not every type of test case may be appropriate for each
function to be tested. For example, boundary test cases will
• Normal testing
probably not be applicable in a donor registration function.
• Boundary testing
Another kind of validation testing is parallel testing. This
• Invalid test cases
involves running two systems in parallel and comparing the
• Special test cases
outputs of both. For a blood bank switching from a manual
• Stress testing
to a computerized system, every procedure would be per-
Normal testing uses typical blood bank inputs to produce formed manually and in the computer.
normal, or routine, outputs. Boundary testing involves Validating a blood bank information system is a very
forcing the system to evaluate data that are slightly below or lengthy and labor-intensive process, but it provides great
slightly above valid ranges. This kind of testing might be benefits if undertaken in a thorough manner. Extensive test-
used for disease test results. Invalid test cases assess the ing will identify potential problems in the way that the blood
system’s ability to recognize and reject incorrect inputs. Exam- bank intends to use the system. Sometimes workarounds
ples of invalid inputs include entering Q for an ABO inter- have to be created, but when it is done as part of validation
pretation or entering a blood product code that has not been testing, staff members can be properly trained before going
defined in the static database file. Special test cases are those “live” on the new system or software. The creation and
that make the system react to unusual inputs. A special performance of comprehensive and detailed validation test
case could be designed to see how the system responds when cases require allocation of significant resources to the project,
more than one person attempts to add or edit special trans- but the end result—a well-validated blood bank information
fusion instructions in the same patient’s record. Stress testing system—is worth the resource costs.
SUMMARY CHART
Blood bank information systems assist in the manage- A blood bank information system can have numerous
ment of data and can allow tracing of a blood component specific applications related to the management of
through all processing steps from donation through donors, patients, and blood components.
transfusion (final disposition). User codes and passwords prevent unauthorized use
A computer system is composed of three main compo- of the system and provide a means for capturing the
nents: hardware, software, and people. identity of each person who performs a task on the
Hardware components perform functions related to information system.
input and output, processing, and storage of data. Control functions assist in making decisions at critical
Software tells the computer what to do with the infor- points in the selection of donors and release of blood
mation it has received. components for transfusion.
Application software allows users to perform tasks that When results must be corrected or amended, they
are specific to blood bank operations. must be clearly designated as such on printed reports
Donor, patient, and blood component information is and monitor displays used by patient caregivers.
maintained in databases, which are divided into files A blood bank must have documented evidence of val-
and further subdivided into individual records. idation of its system as well as written procedures for
Static database files define the terminology that the all aspects of system management in order to comply
blood bank will use and provide a dictionary of coded with regulatory and accreditation requirements.
terms that the system can use to sort data. Blood bank SOPs must address computer tasks related
Operating system software controls the hardware, to blood bank technical duties and must address
manipulates the application software, and coordi- computer-specific procedures such as computer down-
nates the flow of information between the disks and time, backup of software programs and data, system
memory. maintenance, security, error management, and person-
nel training.
The system manager oversees the maintenance of the
system’s hardware and software, including adding or On-site software validation must provide documented
deleting items from the static database files, assigning evidence that provides a high degree of assurance that
access codes to new users, implementing software up- the system will function consistently as expected
grades from the vendor, and investigating and reporting under the unique combination of hardware, databases,
problems encountered by the users. environment, SOPs, and people at the site.
2682_Ch26_556-570 22/05/12 12:06 PM Page 568
12. During validation testing, a computer user entered the 3. The computer will beep and display **Unit Has Been
following results for an antibody screen test: Transfused** when a blood component that has already
been transfused is entered.
SCI IS 37 AHG CC Interpretation 4. The Transfusion History screen display will indicate the
patient has been transfused and will display the date of
Result 0 0 0 + Negative the last transfusion.
After the user verified the entries, the monitor displayed 5. Printed reports will indicate relevant units were
the following message: “Invalid test results.” What assigned transfused status
caused the error message to display? Name of Section C. ________________________________
a. An invalid entry was made in the check cells (CC) Section D
column. 1. Attempt to assign transfused status to the following
b. The truth table was set up incorrectly.
units:
c. The interpretation does not correlate with the test
a. Quarantined blood component
entries. b. Selected (but not issued) blood component
d. The interface to the laboratory computer system is
c. Transfused blood component
down. 2. Selection of units from issued inventory list
13. The following test plan has been created to validate 3. Manual entry of issued blood components
the blood bank computer function used to update the Name of Section D. ________________________________
status of blood units that have been transfused. The Section E
test plan contains each of the sections, lettered A through
1. Operator will input blood component information and
H, required for a thorough test plan. Evaluate each sec-
select blood components.
tion and, using the list below, assign a name to each
2. Operator will input selected patient information.
section.
3. Blood bank computer will update the patient and unit
Section Names record.
• Acceptance • Data entry methods Name of Section E. ________________________________
• Acceptance criteria • Documentation methods
Section F
• Control functions • Result review
• Test cases The following screen displays and printed reports will be
• Corrective action
verified for accuracy:
Test Plan Function: Assigning Transfused Status Screen Displays Printed Reports
Section A Patient Information Patient History Report
Description: This function is used to change the status of Unit Information Transfusion Listing
issued units to a transfused status. All records pertaining Unit History
to the unit and patient will be updated: Transfusion History
Name of Section A. ________________________________ Name of Section F. _________________________________
Section B Section G
1. Preventing the assignment of transfused status to a The acceptability of the results of each test case will be
quarantined blood unit. determined by the blood bank manager and documented
2. Preventing the assignment of transfused status to a on the validation documentation form.
blood unit that has already been transfused. Name of Section G. ________________________________
3. Preventing the assignment of transfused status to a Section H
blood component that has not been issued. If the software does not perform as expected, the problem
Name of Section B. ________________________________ must be recorded on a computer problem report and the
Section C supervisor alerted. A remedial action plan will be devised
1. The computer will beep and display **Unit Has Not with the assistance of the blood bank computer system
Been Issued** when a blood unit number that has not vendor.
been issued is entered. Name of Section H. ________________________________
2. The computer will beep and display **Unit Is in Blood bank director signature:____ Date:_____________
Quarantined Status** when a blood component that Comments: _____________________________________
is in quarantine status is entered.
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Chapter 27
Medicolegal and Ethical Aspects of Providing
Blood Collection and Transfusion Services
Kathleen Sazama, MD, JD, MS, MT(ASCP)
OBJECTIVES
1. Describe the legal and ethical parameters for providing blood collection and transfusion services.
2. Describe the legal bases for liability for providing transfusion medicine services.
3. Explain the necessity of establishing and following standard operating procedures similar to those of other comparable facilities
throughout the United States.
4. Identify the evolving legal and ethical concerns that are likely to accompany the increasing complexity of providing blood during
the early 21st century.
5. List the two reasons why patients sue for transfusion injury.
6. Describe the steps that blood bank professionals can take to avoid or minimize litigation.
Introduction being found liable, when possible. Emerging concerns for the
21st century are also identified. This chapter is not intended
Patients who are injured during or as a result of transfusion as a substitute for legal advice, which is necessary in particular
may seek redress through legal channels, which is usually filed situations; rather, it is intended to provide the reader with
by their families. This has been of particular concern in blood some general principles and definitions so that ethically and
banking and transfusion medicine because of the possibility legally sound practices continue within transfusion medicine.
of disease transmission, especially from previously unknown
sources, and because mistakes may mean death. Basing prac- Focus of Current Legal Issues
tices on sound ethical principles provides a good foundation
to limit liability when these unforeseen events occur. This Legal issues for the transfusion medicine professional in the
chapter briefly describes the sources of law and theories of 21st century center around ABO errors, acute lung injury,
liability, including practical hints for reducing the likelihood of and patient privacy. There are also concerns about possible
571
2682_Ch27_571-580 22/05/12 12:07 PM Page 572
transfusion-transmitted diseases, including babesiosis and of federal law, state law can provide precedence for future
dengue fever or other exotic diseases that are emerging globally. decisions. However, states may choose to adapt, follow, or
Concern has also focused on issues regarding transfusion ignore decisions made in sister state courts, whereas federal
indications, informed consent, and other medically relevant law is applicable to all states. The details of how laws are to
topics. A new concept called patient blood management be put into action are provided in regulations. Regulations,
is gaining prominence in light of the new emphasis on both federal and state, can be applied only if they have been
evidence-based medical practice. New studies have been established according to a formal process called the Admin-
published questioning the safety of blood transfusions, both istrative Procedure Act (APA).3 Federal regulations that
allogeneic and autologous, with concerns regarding the apply specifically to blood banking are found in the Title 21,
age of the red blood cells at the time of transfusion and Code of Federal Regulations, Parts 600–699 and to some
recognition that patient anemia may play a larger role in extent in Parts 200–299 and 800–899, published annually
patient outcomes than previously appreciated. on April 1.4 Blood banks and transfusion services are also
However, even before transfusion-transmitted acquired federally regulated by provisions of the Clinical Laboratories
immunodeficiency syndrome (TTAIDS) became the basis for Improvement Act of 1988 (CLIA 88) and the Medicare
numerous lawsuits against blood centers, hospitals, and provisions of the Social Security Act. In addition, the 1996
physicians, there was litigation because of death and serious federal law protecting health information (Health Insurance
injury caused by transfusion-transmitted hepatitis B virus Portability and Accessibility Act [HIPAA]), including the
(HBV) and a few because of donor injury.1 (See Perlmutter v. 2008 modification (HITECH) that extends these protections
Beth David Hospital, 308 NY 100, 123 N.E.2d 792 [1954] in to electronic records and transmissions and the 2008 federal
which the court decided that transactions involving blood law protecting persons from discrimination on the basis of
were not sales but were incidental to the provision of medical genetic information (GINA), also apply.
services. This decision precludes the application of commercial State legislatures also enact laws and publish explanatory
law, particularly that of warranties, to blood transfusions.) regulations about blood banking, clinical laboratories, and
The HBV cases stimulated nearly every state legislature to transfusion practices, principally covering licensure of facilities
enact protection for blood banks through blood shield and personnel.
statutes.2 (California was the first state to enact such protection
for blood banks in 1955. Only New Jersey lacks such a law, but Case Law
it provides similar protection solely through judicial decisions.)
Because of these blood shield statutes, most of which extended Case law is established by court decisions, sometimes related
protection without amendment or modification for TTAIDS to interpretations and applications of statutes and regula-
and HBV, many TTAIDS lawsuits have been either dismissed tions. Patients generally believe that medical treatment
or unsuccessful for the person suing (the plaintiff). administered to them (after obtaining their informed consent)
Recently, however, this premise has come under new will, on balance, be beneficial. When transfusion causes
challenge in the courts. As most TTAIDS cases have been harm (e.g., through identification error, lung injury, and
unsuccessful in compensating plaintiffs (usually patients other causes), patients have understandably reacted by seeking
or their families), new theories of liability are increasingly redress in the courts. The legal bases for such suits are
being raised. When questions of appropriateness of trans- generally civil (not criminal) actions for tort. Tort is defined
fusion, availability of blood components, and informed as any wrongdoing for which action for damages may be
consent arise, the ethical bases of transfusion medicine brought.
practice are focused more sharply. United States civil law depends on each competent adult
Although blood shield statutes generally protect against in our society behaving reasonably (i.e., not negligently
application of strict liability, tort liability remains the basis and not aggressively) toward every other person, respecting
for most lawsuits. other people’s rights. Civil lawsuits arise because someone
disrespects another’s rights by:
Sources of Law 1. Striking or threatening to strike another person (battery
and assault)
Laws are created by society through legislation called
2. Being careless or reckless (negligence)
statutes (passed by either the U.S. Congress or by individual
3. Failing to complete an agreement (breach of contract)
state legislatures) or by court decisions, referred to as case
4. Intruding on another’s property or privacy
law (in federal courts, including the U.S. Supreme Court, or
5. Misbehaving in other similar ways
in state courts through their own highest state court).
Civil lawsuits can also arise because of violation of
Statutes and Regulations statutes or regulations that require certain types of actions.
Chapter 27 Medicolegal and Ethical Aspects of Providing Blood Collection and Transfusion Services 573
Intentional Tort of Battery way in which the history is obtained have emphasized more
face-to-face oral questioning, not just self-administration
Any touching without consent, including deliberate blows of these questionnaires, until the recent availability of infor-
and intentional striking, is the legal definition of battery. For mation technology to permit such activities. One part of
transfusion medicine, this concept is used when a donor the donation process has always been for donors to sign a
or a patient claims that he or she never agreed to have the statement of consent or assent to donate.
needle placed in his or her arm. If significant harm occurs Until the late 1980s, donors were protected from subpoena
as a result of the needle, this legal theory may be upheld. in cases of transfusion-transmitted diseases. However, an
Generally, however, the fact that the donor or patient allowed increasing number of plaintiffs in state courts are insisting
the needle to be placed is sufficient evidence that he or that the donor be subject to questioning regarding his or
she agreed to the procedure. Informed consent is generally her donation. This questioning may completely or partially
not an issue in battery but is fundamental in the tort of protect a donor’s identity or may require that donor to appear
negligence. in open court. Because no one donates blood expecting to
have to defend such an altruistic act at some future date, the
Doctrine of Informed Consent impact of these decisions on the future availability of blood
for transfusion is uncertain.
Particularly in the special circumstances of the practice of
medicine, the issue of whether a patient (or, in the case of
blood collection, a donor) agreed to undergo the procedure Negligence
actually performed, with full knowledge of the possible
benefits, alternatives, and harm that may accompany it, has Probably the most common tort is that of negligence. This
come to be known as the doctrine of informed consent. This is the basis for lawsuits when injury occurs.
doctrine protects the patient (or donor) by requiring that Elements
information be provided in a manner understandable to the
patient under circumstances that permit the patient to ask Liability for negligence is found when all of the following
questions or express concerns and to receive answers to elements are present:
them (Canterbury v. Spence, 464 F.2d 772 [DC Cir 1972]). 1. A duty was owed to the injured party.
For TTAIDS transfusion recipients, this issue first came 2. The duty was not met by the injuring party.
into sharp focus in the case of Kozup v. Georgetown University, 3. Because the duty was not met, the injured party was
663 F. Supp. 1048 (DC 1987), 851 F.2d 437 (DC Cir 1988), harmed.
906 F.2d 783 (DC Cir 1990). In this case, an infant brought 4. Failure to meet the duty owed was directly responsible
by his parents to Georgetown University Hospital for medical for or could have been predicted to cause the harm
care contracted TTAIDS and died. His parents argued that suffered by the injured party.
they were insufficiently informed about the harm of the trans- 5. Some measurable (compensable) harm (called damages)
fusions and did not specifically agree to transfusions as part occurred. To be successful in a negligence action, the
of the care given their child. The District of Columbia court plaintiff has the responsibility to prove all these factors
ruled that their actions in bringing the child to the hospital against the person being sued (the defendant).
and not objecting to transfusions that they observed at the
time of infusion amounted to tacit consent. The issue of Standard of Care
whether specific consent is required for transfusion and who There are two standards of care applicable to blood collection
should obtain such consent remains controversial. Generally, and transfusion services: ordinary and professional.
physicians have been held responsible (Ritter v. Delaney, 790
S.W.2d 29 [Tex. App. San Antonio, 1990] and Howell v. Ordinary Standard of Care. When the alleged negligence
Spokane, 785 P.2d 815 [Wash 1990]; Hoemke v. New York involves ordinary things that anyone may encounter in daily
Blood Center, 90–7182 [2d Cir. 1990] and Gibson v. Methodist life (e.g., injuries caused by traffic accidents, fistfights, etc.),
Hospital, 01-89-00645-CV [Tex. Ct. App. 1991]). However, a jury or judge can consider the facts and decide whether the
in 1996, the Joint Commission specifically required hospitals behavior of the defendant was reasonable—that is, did the
to obtain informed consent for transfusion in some situations.5 defendant meet the standard of care required in the situation?
Another arena in which the informed consent is the key For ordinary negligence, this standard of care depends
to resolving disputes is that of donor rights. With the onset only on what the average person (e.g., a juror) believes is
of AIDS, the language of donor histories has been subjected acceptable in our society, or the ordinary standard of care
to continuous revising and updating to include information (i.e., the jury decides whether a reasonable person, in the
about the disease, how it is acquired, and under what same circumstances as the defendant, would have acted
circumstances a person may donate blood. In the United the same way as the defendant did). If the defendant acted
States, unlike other countries, there is no right to donate reasonably in the circumstances, the plaintiff will be
blood or other tissues. Instead there has been increasing unsuccessful in the lawsuit and vice versa.
emphasis on educating donors about the restrictions on In situations in which larger organizations are involved,
blood donation. Federal and voluntary requirements for the the question of who is liable for the actions of employees has
2682_Ch27_571-580 22/05/12 12:07 PM Page 574
been resolved under the doctrine of respondeat superior. and distributing blood may be considered differently from
Under this doctrine, the actions of employees are attributable the crossmatching, issuance, and transfusion of blood to
to the employer or person who directs their actions. The individual patients. Redefining blood banking as not medical
person responsible for transfusion services has been defined practice removes the extra protection provided by the re-
by federal regulation and general practice to be a physician, quirement for expert medical testimony to establish the
a definition that has been reinforced by judicial decision in standard of care, leaving defendants to be judged by the
many states for blood centers as well as for hospitals. The ordinary negligence standard. There is little dispute that loss
advantage of having a physician as the responsible employer of medical professional stature would significantly alter
is that the principles and regulations related to medical the practice of blood banking. None of the protections
malpractice usually apply, including the requirement for of medical malpractice reform would be available, and
establishing a professional standard of care. blood centers may even find themselves subject to strict
liability.
Professional Standard of Care. When the negligence lawsuit
involves professionals such as physicians and scientists, Strict Liability
including laboratory professionals, nurses, or other allied
health practitioners, the definition of what is reasonable, the Manufacturers and distributors of goods used in everyday
“standard of care,” depends on expert testimony from other life have been defined by law to have certain responsibilities
professionals in the same field (e.g., physicians, nurses, in their activities to protect consumers. Among other
or scientists) about what should have been done by other requirements, there are certain warranties that the product
reasonable practitioners (usually of the same specialty). The purchased—for example, a television set—will actually work
law makes an extra requirement—in discharging his or her and will continue to do so for some fixed period, with small
duty to the plaintiff, the defendant must apply the special risk of harm from such things as electric shock or blowing
knowledge and ability he or she possesses by virtue of the up. These warranties, actual or implied, exist for virtually
profession. This increased professional standard of care anything a consumer buys and uses. If a product fails to
is not just what a reasonable person such as the judge or perform as expected or creates harm when none was
members or the jury would have done but also what other expected, the consumer has the right to have a replacement
professionals (the expert witnesses) testify should have been or, if the manufacturer denies responsibility, to sue for
done. The judge or jury is not permitted to decide what they negligent manufacturing or distribution.
would have done but must depend upon testimony by expert In addition, for some items (such as dynamite), the danger
witnesses of the same profession as the defendant who define from proper use is so great that manufacturers are legally
what that reasonable professional standard of care is. For the liable for all harm that occurs, which is called strict liability.
complex scientific, technical, and medical issues involved in This means that anyone who is harmed when properly using
TTAIDS litigation, this distinction has been a key element in dynamite does not have to prove that the manufacturer or
protecting blood bankers. distributor was negligent; he or she has only to show that he
or she was injured while properly using it. Imagine how rare
Voluntary and Mandatory Standards and expensive blood transfusions would become if these
practices were applied to it. Instead, nearly every state
The testimony of experts should generally be supportable by has enacted specific protection—the blood shield statutes
authorities such as statutes, regulations, or other bodies of described previously—to exclude harm from blood transfu-
published knowledge, including published scientific articles sions from suit under these legal theories. It is important
and texts. The existence of voluntary standards—particularly that blood bankers avoid implying or stating that blood
those provided by the AABB (formerly known as the American transfusion is completely safe, because such statements may
Association of Blood Banks)6 but also those from the College be construed as creating a warranty, invoking these theories
of American Pathologists (CAP), the American Association of liability.
of Tissue Banks (AATB), the American Society for Histocom-
patibility and Immunogenetics (ASHI), the Joint Commission Intentional Infliction of Emotional Distress
(TJC), and other organizations—are helpful in establishing the
professional standard of practice for transfusion medicine. In As the phrase intentional infliction of emotional distress sug-
fact, some state laws and federal regulations cross-reference the gests, a plaintiff must show that what the defendant did to
AABB standards specifically. Blood bankers and transfusion cause actual and severe emotional distress was intentional, usu-
services that can show that they acted in conformance with ally some extreme or outrageous conduct that was calculated
these standards are more likely to be found non-negligent than to deliberately cause harm to the plaintiff. This can take the
those that do not follow such guidelines. form the plaintiff’s relative claiming wrongful death, particu-
larly in some TTAIDS cases.
Blood Banking as a Medical Profession
Invasion of Privacy Under Civil Case Law
The question of whether blood banking is a medical profession
is being relitigated in courts today, with conflicting results.7 Health-care providers, including blood bankers, are required
One possible chilling result is that collecting, processing, to respect personal privacy and to maintain patient and
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Chapter 27 Medicolegal and Ethical Aspects of Providing Blood Collection and Transfusion Services 575
donor confidentiality. Plaintiffs may claim remuneration for Doctrine of Charitable Immunity
loss of privacy under four theories:
Historically, courts provided immunity for nonprofit organ-
1. Intruding upon the plaintiff’s seclusion or solitude or into izations such as hospitals from excess liability because they
his or her private affairs perform charitable acts. This is referred to as the doctrine of
2. Publicly disclosing embarrassing facts charitable immunity. Many state legislatures have enacted
3. Publicly placing plaintiff in a “false light” and continue to support statutes to provide protection for
4. Appropriating plaintiff’s name or likeness for defendant’s boards of directors and volunteers using this common law
benefit rationale. A 1996 decision in New Jersey redefined this
These categories protect a patient or donor from illegal or protection in that state (Snyder v. AABB, 144 N.J. 269
inadvertent disclosure of his or her personal information, of [1996]). Fortunately for blood bankers, other states have
particular concern with HIV because of the risk of loss been loathe to accept the Synder decision. Also, many
of employment, housing, insurance, and other benefits of health-care institutions rely less on this doctrine and more
society. When information is exciting or noteworthy, the on insurance for protection.
media may become aware of and publish private information.
Tort Reform
Basis of Liability: HIPAA
State legislatures, recognizing the need to protect some
In 1996, the U.S. Congress responded to the information specialties such as obstetrics, have been active in seeking
age by enacting a law specifically directed toward protecting limits on damages against physicians, protecting them from
personal health information (PHI): the Health Information abusive litigation. For transfusion practices, it is vital that
Portability and Accessibility Act (HIPAA). The regulations these protections be afforded.
implementing the provisions of this act occurred in segments
beginning in 1996, with the full provision for PHI effective Risk Management
on April 7, 2003. With this new federal requirement,
information about donors and patients must be kept in Avoiding liability for TTAIDS and other possible harm from
such a manner that inadvertent “use or disclosure” does not practicing transfusion medicine, whether in hospitals or
occur. Exceptions for health-care providers, insurers, and blood centers, depends on having well-established policies
government scrutiny have been specified, but safeguards and procedures that are consistent with quality principles
should be in place even for these exceptions. and that comply with recognized authorities, regulations, and
Although few if any cases have been filed, blood banks statutes and that have some measurement of how persons
and transfusion services will be involved in suits under engaged in all activities actually follow those procedures.
the preexisting civil cases if they are responsible (through Complying with accreditation requirements (e.g., AABB,
negligence or by intention) for releasing confidential data of TJC, CAP, and state laws) for quality systems will assist in
a donor or patient. Great care should be exercised by blood limiting risk. To avoid being negligent, one must behave
banking professionals to ensure that private information reasonably. Reasonable behavior for transfusion medicine
(whether about donors, patients, or relatives) be kept confi- practice includes continually obtaining and applying new
dential and not released without written authorization. knowledge from all possible sources that will safeguard the
Procedures to safeguard such release should include proper donor during collection, the component during handling and
use of copy and facsimile machines and direct electronic delivery, and the patient before and during transfusion.
transfer via information systems.
Specific Donor Issues
Restrictions on Plaintiff Suits
Although it is possible to sue another party for many reasons,
and Recovery
the most common ones are discussed here.
Even when harm or injury occurs, the plaintiff may be
Screening
unable to pursue legal redress for a number of reasons.
What the AIDS epidemic has taught us is that every person
Statutes of Limitations who volunteers to donate does not have an unqualified right
to do so. In fact, for several years before March 1985 (when
Some protection for defendants arises because of a statutorily a test for HIV antibodies in blood first became available), the
defined limit of time during which a lawsuit can be filed, best safeguard against TTAIDS was improved donor education
referred to as the statute of limitations. States often have and more pertinent questioning regarding behaviors that
different limits, depending on the legal requirements for might put that donor at risk for acquiring HIV. Although
initiating suits. Statutes of limitations for medical malpractice numerous lawsuits have been filed against blood collection
are generally shorter (approximately 2 years from the date agencies for improper donor screening, few have been
that the injury should have been discovered in adults) than successful when collection facilities could show that they
limitations for other kinds of negligence (2 to 6 years is had implemented written procedures and had properly
common). trained employees who followed those procedures and that
2682_Ch27_571-580 22/05/12 12:07 PM Page 576
proper documentation of each screen occurred. Problems and reagents, and establishing the necessary information
occurred when breaches in procedure, typically failures to service support, was seen as negligent by the jury (even with
follow SOPs or to properly document actual practice, were expert testimony to the contrary).
discovered. However, with several state courts demanding
release of donor identity or access to donors for questioning, Failure to Perform Surrogate Testing
further attention to the process of donor screening is Despite concerted efforts, rarely has a plaintiff prevailed
appropriate. Balancing the real threat to patient care that when alleging that blood collection facilities should have
would arise if blood components were not available with the performed more or different surrogate tests between 1983
serious nature of litigation will continue to challenge blood and 1985, when a specific HIV antibody test was first available
banking professionals. (Baker v. JK and Susie Wadley Research Institutes and Blood
Bank, aka The Blood Center at Wadley, 86-2728-C [Tex. Jud.
Donations Requested by Patients Dist. Ct. 1988] and Clark v. United Blood Services, CV
Several monetary settlements in the range of hundreds of 88–6981 [Nev. 2d Jud. Dist. Ct. 1990]).
thousands to millions of dollars have resulted from either
failing to offer directed donor services or from improperly Failure to Properly Perform Testing
characterizing them. With the advent of the HIPAA, patient- Testing personnel should be constantly alert to proper per-
requested donations from friends and family require close formance and documentation of all required testing of blood
attention to ensure that inadvertent or overt disclosure of components. There are multiple opportunities for errors to
protected health information does not occur. occur during the processes of collecting and transfusing
blood components. In addition to mistakes in doing the
Untimely Notification required testing, failure to document is as damning as failure
When recipients received notification in years, rather than to perform at all. The FDA has expended considerable effort
in weeks or months, after blood centers knew (or should to inform facilities and to enforce requirements for proper
have known) that the recipient had received an HIV-reactive testing for virally transmissible diseases in blood components.
unit, many of them or their families were angry enough to Likewise, private accrediting organizations emphasize proper
file suit on the basis that they should have been informed performance and documentation of these activities.
sooner. Procedures for look-back and recipient notification
were not well established for several years following Informed Consent
application of specific testing in most blood collection In TTAIDS cases arising in the early 1980s, it was frequently
organizations. It was not until 1996 that the federal gov- alleged that patients were insufficiently warned of the hazards
ernment, through the Center for Health Care Financing of transfusion because transfusion experts failed to warn
Administration (now called Centers for Medicaid and hospitals and ordering physicians about them. Few cases
Medicare Services, or CMS) and the FDA, issued specific were successful because the state of scientific knowledge,
regulations for HIV look-back notification by hospitals. established by expert testimony relying on published data,
Parallel requirements for HCV are in progress. was limited. Also, the early HBV cases had established a
record that supported the defense position that transfusions
Component Collection
were already known by hospitals and other physicians to be
Occasionally, lawsuits have occurred from injury that donors unavoidably unsafe, particularly for transfusion-transmitted
received during the collection process. Generally, the injuries viral diseases. Some states (e.g., California) enacted specific
are more severe than a simple bruise at the needle site and legislation about informed consent for transfusion.
involve such things as nerve damage, slip-and-fall incidents,
and other similar severe reactions. Also, although donor Medical Malpractice
deaths continue to be reported at a rate of approximately two
per year, these infrequently result in litigation. Several multimillion-dollar lawsuits, decided for the plaintiff,
resulted from successful allegations that either the patient
Processing, Labeling, and Distribution did not give adequate informed consent, did not need a
transfusion at all, could have waited until test-negative blood
Lack of Standard Protocol for Implementing Testing was available before receiving a transfusion, or required
Several successful TTAIDS lawsuits awarded millions of transfusion solely because of something the physician did.
dollars against blood-collecting organizations (Belle Bonfils In all these cases, the basis for fault was negligence by the
Memorial Blood Bank v. Denver District Court, 723 P.2d 1003 treating physician.
[Colo. 1988]) because they had no standard protocol
for implementing new testing to ensure that all available Ethics and Transfusion Medicine
components (including those distributed or in active
inventory) were test-negative before transfusion once the Biomedical ethical principles that must be balanced when
test kits and equipment were received by the collection considering appropriateness of and informed consent for
facility. The lack of a written plan to implement such testing, transfusion include autonomy, beneficence, and justice
including training of personnel, validating instrumentation (see definitions in Box 27–1).
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Chapter 27 Medicolegal and Ethical Aspects of Providing Blood Collection and Transfusion Services 577
by the news and storms out of the center. Two weeks later, 3. Can/should health-care organizations deny autologous
the donor sues the blood center for intentional infliction donor-patients access to their own test-reactive blood?
of emotional distress.
Case 27-6
1. Is the donor likely to be successful?
2. Are there other bases on which suit can be brought? On July 5, a Monday afternoon, two trauma victims, both
3. Are there established standards for preventing such an group O-negative, arrive in the same hospital emergency
occurrence? room. Both patients have massive injuries requiring
large-volume RBC transfusions. Because of the time of
Case 27-4 year, the hospital transfusion service is low on O-negative
RBCs. As the requests for RBCs rise, units crossmatched
A 59-year-old man was notified in 1999 that he had received
for tomorrow’s elective surgery are taken to be used for
a unit of RBCs in 1989 that was negative by first-generation
the trauma patients. The transfusion medicine physician
HCV antibody testing, but the donor was found to be positive
(TMP) contacts the department of surgery to cancel elective
when subsequently tested by second-generation methods.
surgery cases for Tuesday, July 6. The transfusion service
He was tested by the blood center in late 1999 and found
manager (TSM) puts in an emergency call to the regional
to be HCV-positive. He filed a lawsuit against the treating
blood center. The blood center informs the TSM that it,
physician, the hospital, and the blood center in 2002.
too, is short of O-negative RBCs but that it will canvas
1. What legal theory can this man use? the neighboring hospitals and put in an emergency
2. What is the likely outcome of this lawsuit? request to the blood exchange network.
As several hours pass, during which the trauma
Case 27-5 surgeons and OR team work diligently to correct the
injuries and restore vital organs, it is clear that there will
A 67-year-old woman, crippled by degenerative arthritis,
be sufficient RBCs available to adequately support only
requests that her own blood and blood from family
one patient. The chair of the hospital ethics committee
members be used during her hip replacement surgery.
is consulted for assistance in determining how to
The blood center describes its donation procedures,
proceed.
which do not permit directed donations. Although she
Additional information available includes this: One
is disappointed, she agrees to donate for herself. When
patient is a 55-year-old bank president who is the sole
her first unit is tested, it is reactive for hepatitis B surface
financial support for his spouse, two college-age children,
antigen and for antibody to hepatitis B core antigen. The
and widowed mother; the other is a 28-year-old gas
patient-donor is advised that she may no longer donate
service attendant who is unmarried. Both patients
for herself and that the unit she has already donated will
are critically injured, but with adequate transfusion
not be available for her surgery. Surgery proceeds. She
support, both are deemed likely to recover by their
receives 4 units of volunteer blood and develops TTAIDS
treating physician.
3 years later. She sues the blood-collecting organization.
1. Which ethical principle is being emphasized?
1. On what legal grounds can this suit be brought?
2. How will a decision favoring one patient over the
2. What is the current standard of care regarding
other be viewed ethically? Legally?
patient-directed donations?
SUMMARY CHART
Tort liability is the basis for most lawsuits. The elements of negligence include:
Federal regulations that apply specifically to blood • A duty was owed to the injured party.
banking are found in Title 21, Code of Federal • The duty was not met by the injuring party.
Regulations. • Because the duty was not met, the injured party was
Claims of the intentional tort of battery is used in harmed.
transfusion medicine when a donor or a patient claims • Failure to meet the duty owed was directly
that he or she never agreed to have the needle placed responsible for or could have been predicted to
into his or her arm. cause the harm suffered by the injured party.
The doctrine of informed consent protects the • Some measurable (compensable) harm occurred
(damages).
patient (or donor) by requiring that information be
provided in a manner understandable to the patient Under the doctrine of respondeat superior, the actions
under circumstances that permit the patient to ask of employees are attributable to the employer or person
questions and to receive answers to any questions or who directs their actions; for example, in transfusion
concerns. medicine, this would be a physician.
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Chapter 27 Medicolegal and Ethical Aspects of Providing Blood Collection and Transfusion Services 579
SUMMARY CHART—cont’d
The concept of product liability implies that a warranty Traditionally, hospitals and other nonprofit organiza-
exists for virtually any product a consumer buys, and tions are protected under the doctrine of charitable
if the product fails to perform or creates harm when immunity from excess liability because they perform
none was expected, the consumer has a right to a charitable acts.
replacement or, if denied a replacement, to sue for Biomedical ethical principles that must be balanced
negligent manufacturing or distribution. when considering appropriateness of and informed
Blood centers may be liable for invasion of privacy consent for transfusion include autonomy, benefi-
lawsuits if confidentiality is breached via public cence, and justice.
disclosure of embarrassing facts (e.g., HIV-positive
results) or for violation of federal law (the HIPAA).
Chapter 28
Tissue Banking: A New Role for the
Transfusion Service
Holli M. Mason, MD
OBJECTIVES
1. Name the principal modern organizations involved in standardization of tissue banks.
2. List examples of tissues contained within the scope of FDA CFR 1270 and 1271, and examples of tissues outside the scope of
these regulations.
3. State the minimum requirements for qualifying a tissue supplier and vendor.
4. List the minimum labeling requirements for autologous tissue and the conditions required to utilize autologous tissue without
registering with the FDA as a tissue manufacturing establishment.
5. Compare and contrast the similarities and differences between the various regulatory bodies in terms of regulations for tissue
banks, and identify voluntary accrediting agencies.
6. Compare the steps taken in an adverse event investigation with those taken in a recall.
581
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Chapter 28 Tissue Banking: A New Role for the Transfusion Service 583
mandate record keeping for traceability and the investigation Hematopoietic stem cells and progenitor cells are used
of adverse events.1 to repopulate bone marrow in patients who have undergone
chemotherapy and irradiation to destroy tumor cells. This
The Hospital Tissue Bank type of therapy destroys the native bone marrow as well,
which is fatal without the transplant of donor or autologous
The basic function of the hospital tissue bank is to provide stem cells following therapy.
surgeons and their patients with safe, quality tissue products Cornea transplants are used to restore sight in patients
while maintaining detailed records for each tissue handled with corneas that have been damaged through trauma, dis-
through final disposition. This includes AABB Hospital ease, or birth defect. Eye tissue, or the sclera, which consists
Tissue Management:8 of the “white” part of the eye, is composed of thick fibrous
tissue. Sclera can be used as a patch for eyes that have been
• Acquisition of safe and effective autografts and allografts
punctured or torn in a trauma.9 Sclera is also occasionally
• Oversight of use throughout the organization
used in dental procedures.
• Qualification of suppliers
The majority of the tissues described here have the ad-
• Conduction of inspections
vantage of being useful regardless of the donor’s or patient’s
• Provision of temperature-controlled, monitored storage
HLA type. No blood type or tissue match is required. Many
• Record keeping and ensuring traceability
of the tissues come in standardized shapes and sizes (e.g.,
• Quality assurance
bone screws) and can be used by any patient. Other tissues
• Selection of appropriate allografts
must be ordered in advance specifically for an individual
• Investigation of adverse outcomes
with a specific size, such as heart valves or whole bones.
• Administration of recalls and look-back investigations
Inventory should take into account not only the “off the
• Compliance with regulations and standards
shelf” utility of a particular product, but also its frequency
• Promotion of cost-effective and appropriate tissue use
of use and expiration date. Tissues that require specific sizes
Common tissues found in tissue banks and used in surgical may be ordered in smaller quantities or only ordered in an-
procedures include bones, ligaments, tendons, heart valves, ticipation of specific procedures, depending on the size of
skin, veins, cartilage, dura mater, hematopoietic stem and pro- the organization and turnover of product. Tissues such as
genitor cells, and corneas or eyes. Bone can be sterilized and cornea have such a short shelf life that it is best to schedule
all living tissue removed. Pieces of bone can be further worked patient surgeries as the tissue is available and issue the
into spacers, wedges, screws of various sizes, and “chips.” cornea as soon after arrival as possible.9
These have a variety of surgical uses, including decompression Surgeons should have access to the inventory lists and
of nerves in spinal surgery with donor bone spacers, screws may even want to look at individual tissue prior to surgery
to pin fractured bone together, or chips to aid in the incorpo- to avoid wasting tissues that do not fit as expected for sur-
ration of hip or knee replacements into the native bone. Once geries requiring tissue of a specific size. At times, surgeons
implanted into the patient, bone slowly incorporates into the may request a selection of tissues to choose from in the
living native bone and adds strength and structure. operating room when the patient’s situation is better under-
Heart valves are used for valve replacement, most com- stood. Clear guidelines specifying duration of time out of the
monly in children and women of childbearing age. The advan- tissue bank or regulated temperature ranges in order to reen-
tage of nonmechanical heart valves for this population is the ter the unused tissues into inventory must be made ahead of
nonreliance on anticoagulants that are mandatory for recipients time.10 These guidelines should be based upon the tissue
of mechanical valves. Drugs such as Coumadin put active manufacturer’s recommendations.
children in danger of serious bleeding and are teratogenic in In general, the scope of the tissue bank is limited to non-
pregnant women, causing birth defects. vascularized tissue. Excluded from Parts 1270 and 1271 of
Tendons and ligaments are often used in repairs of knee the FDA’s Code of Federal Regulations (CFR) are vascular-
and other orthopedic injuries, particularly in the replace- ized organs such as the liver, kidney, lung, pancreas, and
ment of the anterior cruciate ligament. Tendons and cartilage heart.11 In general, these tissues are immediately trans-
are also useful in repair of facial disfigurements. planted and storage is not necessary or desirable. However,
Skin is widely used for burn victims and often means the there are exceptions to this general rule. Renal transplants
difference between life and death. Not only are skin grafts may be stored, if properly packaged, for several days. Because
an aesthetic improvement, but they also provide the barrier kidneys are vascularized organs, and therefore fall outside
to the outside world that is essential to protect the body from the general scope of the hospital tissue bank, it is not neces-
pathogens. Skin grafts are also used in patients with any large sary to include such tissue in the tissue bank. However, hos-
loss of skin, such as pressure sores, nonhealing ulcers, au- pital administrators may opt for standardization and for
toimmune disorders, and surgical or traumatic loss of large ensuring proper storage and may ask to include organs, such
areas of skin. as kidneys, in tissue banks.1,12
Veins are commonly used in coronary artery bypass grafts. Other exclusions include autologous tissue, which may
They are also used frequently to bypass other vessels that have also be stored in the tissue bank for convenience and stan-
become blocked by severe atherosclerosis. Such bypasses often dardization; blood vessels recovered with organs and intended
make it possible for a patient to avoid limb amputation. for use in organ transplantation; minimally manipulated bone
2682_Ch28_581-600 22/05/12 12:07 PM Page 584
marrow; tissue intended for education or nonclinical research; coordinator is responsible for all administrative operations
xenografts; blood components and blood products; and of the tissue dispensing service, including handling regula-
secreted or extracted products such as human milk, collagen, tory issues and hospital policies. He or she defines policies
or cell factors.11,12 and procedures for the service, ensures competency and
training of the staff, manages acquisition and inventory, and
Tissue Bank Oversight prepares reports for the tissue committee, including adverse
event reporting. This person is often in the position of su-
The hospital tissue bank must have a physician-appointed pervisor in the blood bank or may be an additional person
medical director. In keeping with the blood bank model, the at the same level.
AABB requires that the medical director be a medical doctor
who investigates adverse outcomes, participates in look-back Acquisition of Allografts and Inventory
investigations, approves standard operating procedures with
any deviations, and communicates with the physicians using The hospital tissue bank serves as the clearing house for all
the end product.10 Hospital tissue banks within the blood tissues entering the hospital (excluding most or all vascular-
bank have the advantage of the presence of a medical direc- ized organs). It may be necessary or desirable to involve the
tor. If the institution chooses to delegate this responsibility surgical staff in decisions as to the standard inventory and day-
to another qualified physician other than the medical direc- to-day ordering; however, the tissue bank should be respon-
tor of the blood bank, this individual should participate in sible for approving tissue vendors and receiving all shipments.
the development and review of all policies, procedures, and Once a shipment of tissue is received in the tissue bank,
processes involving the handling of tissue. He or she should it should be inspected for package integrity and correct
also be involved in investigating adverse events, deviations labeling and reconciled with the packing invoice.13 Tissue
from standard operating procedures, and look-backs. An receipt records must contain, at a minimum, name and ad-
important part of the Joint Commission requirement from dress of tissue supplier, description of tissue and quantity,
2005 is that there must be a standard operating procedure date of tissue receipt, condition of tissue upon receipt, and
that assigns and designates responsibility for the oversight expiration date, if applicable (AATB L4.100).5 Each tissue
of the organization’s tissue-related activities.1 specimen must have a tissue identification number (TIN).
Development of a tissue committee is recommended with In addition, the name of the person conducting the incoming
the intent to ensure and promote best practices based on inspection should be recorded on the tissue receipt record.
evidence-based medicine. The tissue committee may be Joint Commission standards require that the individual re-
structured similarly to the transfusion committee, including ceiving the shipment must verify package integrity and en-
medical staff who are involved in the use of transplantable sure that the transport temperature range has been
human tissue. The chair may be the director of the blood controlled and acceptable, as evidenced by residual dry or
bank, but the committee may prefer to choose a chair from wet ice.1 The vendor should be able to present evidence of
the surgery department secondary to that physician’s knowl- validation of transport containers for maintenance of proper
edge regarding tissue transplant and use. temperature range during transport.
Peer review, evidence-based clinical indications, and Manufacturers’ instructions for storage should be rigor-
audits ensuring compliance with standard operating proce- ously followed and the tissue entered manually or electron-
dures should be major goals of the committee. The commit- ically into an inventory list. If necessary, the storage site
tee should review operations, usage trends, shortages, location should be included in the inventory for ease of
wastages, and errors. All reported adverse outcomes should locating the tissue upon issue or inspection. Surgeons who
be brought to the tissue committee and investigated fully. transplant tissue should have access to the inventory so they
The committee may also be involved in selecting tissue ven- are aware of what tissues are available to them before they
dors and in educating the hospital staff in the appropriate begin surgical procedures.
use of tissue. Often the tissue committee can be successfully Detailed and accurate inventory management is cost-
incorporated into the transfusion committee with committee saving and will reduce wastage of products stored until ex-
members fulfilling both roles. piration or will prevent inadvertently reordering when the
Additional important roles within the hospital tissue bank product is already in inventory. Some products, such as
are the quality assurance coordinator and the hospital tissue corneas, have a very short shelf life, and careful coordination
coordinator. The quality assurance coordinator, often known between the surgical staff and the tissue bank allow for
as the compliance officer, is responsible for the quality plan ordering and acquiring the appropriate tissue in time for the
for the transfusion service as mandated by the AABB and surgery, but not so far in advance that the product expires
CAP.10 He or she assesses processes, policies, and procedures and must be wasted. Duration of storage time will vary
to make sure they are in compliance with the applicable greatly between the different types of tissue. Therefore, a
accrediting agencies. Depending on the size of the organiza- system, preferably electronic, must be in place to anticipate
tion, the job description may include applying these duties replacement of outdated tissues or management of those
to the entire laboratory or to discrete parts. The quality tissues to avoid outdating.13
assurance coordinator should make recommendations to the To avoid waste, some tissue vendors will agree to ex-
medical director regarding operations. The hospital tissue change tissue that is close to outdate with similar tissue that
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Chapter 28 Tissue Banking: A New Role for the Transfusion Service 585
has an expiration date further in the future. This is not true integrity of the product being stored. The most important
in all cases and must be worked out between individual ven- function of the alarm is to allow blood and tissue bank per-
dors and hospital tissue banks. Typically vendors who are sonnel to be aware of an equipment problem before the prod-
willing to exchange late-dated products have a network of uct is compromised by a temperature that is too high or too
hospital tissue banks to which they can transfer the product low for proper storage. Alarm systems must be set to activate
for quick use. Wastage rate for each tissue type should be under conditions that will allow action to be taken before
tracked and examined by the tissue committee for ways to products reach unacceptable conditions. Procedures should
improve the process. Changes in the surgical staff, or focus be in place to instruct the blood and tissue bank personnel
of the surgical center, may result in unanticipated wastage on actions to take when the alarm is activated, including cor-
if the same tissue inventory is kept despite changes in rective action and possible relocation of products to another
tissue use. Tracking wastage and communicating with other monitored storage device. An alarm activation must be im-
departments using tissue can keep wastage to a minimum. mediately investigated to find the cause of the malfunction.
Storage conditions vary between the different tissue types. Good record keeping is essential in the hospital tissue bank
Even the same tissue, such as bone or eye, can have different to demonstrate that standard operating procedures are
temperature and duration requirements depending upon its followed (Box 28–1). Also important is the ability to track
processing and packaging.14 Therefore it is critical to care- the tissue bidirectionally, from donor to recipient or disposal
fully follow the recommendations and instructions from the and back again. Each change in the status of the product—
tissue vendor or supplier for each specimen. Failure to do so including receipt into the hospital tissue bank, change in
may result in tissue that does not function as intended and location within the tissue bank, issue, return, transplantation,
may interfere with the tissue’s functional and mechanical or disposal—must be documented concurrent with the ac-
properties, making it unsuitable for transplant.14 Special tion. The identity of the person documenting each step and
attention should be paid to products stored at “room” or the date should be included. Records must be accurate, leg-
“ambient” temperature. These words may vary in definition ible, written in indelible ink, and as detailed as possible to
between tissue suppliers, and it is advisable to clarify the provide a clear history.
definition with the vendor. A written response to any such Traceability becomes especially important during recalls.
question should be kept on file in the hospital tissue bank. Occasionally a tissue donor is found to have had a disease
The Joint Commission and AATB require that tissue stor- or disease exposure that was not apparent at the time of
age shall conform to guidelines established by the distribut- donation or may be suspected because one of the tissue
ing tissue bank5 and that organizations use standardized recipients became sick following transplant. In cases such as
procedures to store tissues.7 The Joint Commission further this, all of the tissues that originated from that donor must
requires the hospital to continuously monitor storage refrig- be investigated and tracked down to prevent transmission
erators and freezers with records showing daily storage tem- of disease to other recipients. Often, transplant has already
peratures.1 AABB takes a more detailed approach to storage. occurred and the recipient can be tested or treated if neces-
Standard 3.0 deals with equipment and specifically names sary. Poor record keeping makes it impossible to track
tissue that requires the blood bank to identify equipment potentially dangerous tissues or to determine who may need
that is critical to providing blood and tissue services.13 The
blood bank is required to maintain policies, processes, and
procedures to ensure that calibration, maintenance, and
monitoring of equipment conforms to the blood bank or BOX 28–1
tissue bank standards.5 Standard Operating Procedure (SOP) Checklist
AABB standard 3.6 states that storage devices must have
The following SOPs should be written and included in the hospital
the capacity and design to ensure proper temperature is tissue bank:
maintained.13 The temperature of refrigerators, freezers, and • Assignment of responsibility for the oversight of the tissue program
platelet incubators must be monitored. Storage units using • Validation and qualification of tissue suppliers/vendors
liquid nitrogen must have liquid nitrogen levels checked or • Tissue ordering
the temperature monitored. Tissues that can be stored at • Tissue receipt
room or ambient temperature do not require a monitored • Tissue storage and temperature control
storage device, such as a platelet incubator, as long as the • Issuance and final inspection of tissue
definition of room temperature does not include a narrow • Temperature alarms and emergency backup equipment
temperature range.13 However, many tissue banks will store • Documentation of abnormalities, including instructions on actions
these products in a monitored device for uniformity and such as quarantine
convenience. • Quarantine of tissues that are questionable
Alarm systems have been used in blood banks for decades • Documentation of decisions not to use tissue
and are an important assurance that storage temperatures • Disposal/disposition of expired tissue
have been consistently maintained, thereby ensuring the
2682_Ch28_581-600 22/05/12 12:07 PM Page 586
treatment or increased surveillance due to the transplant of operating procedures and standards and regulations are
a tissue from a donor suspected of transmitting disease. The being followed.
bidirectional traceability is important so that if a recipient
becomes ill after transplant, the tissue bank and supplier can Qualification of Vendors
cooperate to trace the source of the tissue in order to initiate
a recall. Tissue vendors or suppliers must meet a variety of standards.
Records must be maintained for a minimum of 10 years First, the tissue supplied must be safe and effective and of
after the expiration of the tissue product.5 If no expiration use to the physician who transplants it. The tissue must be
date applies to a specific tissue specimen, records must be readily available in a useful time frame, and its cost must fall
maintained for 10 years following issue.5 Each tissue speci- within budgetary guidelines and be within the customary
men must have a unique TIN, and upon receipt by the hos- price range. The surgeons who will use the tissues should be
pital tissue bank, must include the name and address of the involved in selecting the tissue vendor based on the tissue’s
tissue supplier, a description of the tissue and quantity re- usefulness to them. Most often, a surgeon will request a spe-
ceived, date of receipt, condition upon receipt, and expira- cific tissue from a specific vendor because of its utility. It is
tion date along with the name of the receiving individual. then up to the medical director of the hospital tissue bank
When a tissue is dispensed to be used for transplant, to determine whether the vendor qualifies as a supplier of
dispensing records must be created. The dispensing record quality, safe, and effective tissues.
must include the name, address, and telephone number of Sales representatives must be compelled to seek approval
the tissue bank; the type and quantity of tissue; the unique and qualification with the hospital tissue bank prior to
TIN; the recipient’s name, medical record number, or social demonstrating any new tissue products in the operating
security number; transplantation site; date and time of re- room. It is also important to consider other physicians
lease; name of the ordering physician or other health pro- within the organization who may also utilize tissue for pa-
fessional; name of the person dispensing the tissue; and tient care in a nonsurgical setting, such as a wound care
name of the person preparing the tissue if further prepara- clinic, and solicit their input as well.
tion is necessary prior to transplant.5 The dispensing Before the qualification of a vendor can commence, the
records must be maintained in a log format in the hospital hospital tissue bank must have written guidelines and crite-
tissue bank. It must also be included as part of the recipi- ria for the acceptance of a vendor or supplier.10 Some ven-
ent’s medical record to permit tracing to the appropriate dors may be part of a larger entity in which different
individual. Tracing forms must also be filled out and returned establishments participated in different parts of the process
to the tissue supplier or vendor for their records.5 The of supplying a tissue product. For example, one establish-
records should clearly specify the final disposition of ment may recover the tissue and pass it on to another that
the tissue. The tissue will have been implanted, meaning the processes and packages the tissue for distribution. The writ-
tissue was implanted in the patient; discarded, meaning ten policy should reflect if each of the intermediaries should
the tissue was discarded in the operating room and not also be qualified by the hospital tissue bank. Theoretically,
implanted into the patient; or explanted, meaning the tissue qualification of the supplier directly supplying the tissue
came into contact with the patient but was removed either should be sufficient, as that supplier must have, in turn,
immediately during surgery or at a later date. qualified its suppliers. However, some medical directors may
feel more secure in verifying this for themselves. The medical
Quality Assurance director may also take into account such things as the trans-
parency and willingness to provide information, method of
Quality is the goal of virtually any blood or tissue bank. A processing and disinfecting, the involvement of the supplier’s
quality plan is essential for good tissue practices and excel- medical director, and the ability of the supplier to meet spe-
lent patient care. The goals of the quality plan are to maintain cial needs. Table 28–1 outlines the required documentation
safe, functional, and effective products that meet the needs for tissue vendor and supplier qualification.
of the patient and the surgeon. Preventing contamination The first step in qualifying any vendor is to verify the sup-
and having an error-free process is critical. To achieve these plier’s FDA registration. On a yearly basis, the tissue bank
goals, the quality plan must include written procedures, val- must verify that its suppliers are registered with the FDA as
idation of processes, and investigation of deviations and er- required by 21 CFR 1271.15 This can be done by using the
rors. Errors will occur even in the best-run tissue banks. FDA’s online database: www.fda.gov/cber/tissue/tissregdata
Well-written standard operating procedures that conform to .htm. In January 2001, the FDA published a rule requiring
the regulations and are easy to follow in actual practice de- establishments that supply human cells, tissues, and cellular
crease errors. However, analysis of deviations and errors, as and tissue-based products (HCT/Ps) to register with the
well as development of corrective action to improve process agency and list their HCT/Ps.15 The final rule, 21 CFR
and prevent future errors, builds quality over time. Part 1271, became effective in April 2001 for human tissues
The quality plan should also verify supplier qualifications, intended for transplant that are regulated under section 361
ensure proper training of personnel, and monitor compli- of the Public Health Safety (PHS) Act and 21 CFR 1270.16
ance in the tissue bank. Periodic quality audits should be In March 2001, the requirement became effective for all
performed by the tissue service to ensure that standard other HCT/Ps except human dura mater and heart valves,
2682_Ch28_581-600 22/05/12 12:07 PM Page 587
Chapter 28 Tissue Banking: A New Role for the Transfusion Service 587
Tissue supplier’s state license and registration When suppliers are located in states that require licensure by the state.
which became effective when the remaining parts of 21 agencies when required by state law in the physical location
CFR 1271 were implemented in 2005.16 of the organization.1 Not all states require licensure; in fact,
Establishments that meet the requirements for registration most do not. As of this writing, six states require licensure:
with the FDA must do so within 5 days after beginning oper- New York, Florida, New Jersey, California, Georgia, and
ations.15 Registrations must be updated annually in December Maryland (Table 28–2).
in conjunction with an update of the HCT/P list unless there
is no change. Upon acceptance of the establishment’s regis- Autologous Tissue
tration form, the FDA will assign each location a permanent
number.15 This registration number does not imply that the The term autologous tissue refers to any tissue that is removed
establishment is in compliance with applicable rules and reg- from a patient with the intent to reimplant it at a later date and
ulations or is approved by the FDA. However, the establish- during a different procedure than its removal. Strictly speaking,
ment, once registered, must be available for public inspection a bone flap that is elevated and placed in sterile saline during
by the FDA (CFR 1271).15 A yearly review of FDA registra- a procedure and then replaced at the end of that same proce-
tion of tissue vendors is recommended in the first quarter of dure does not qualify as being autologous tissue as defined in
each year because of the requirement that the establishments this chapter.17 In order for the regulations and standards
update their registration each December. pertaining to autologous tissue to apply, that bone flap would
A copy of the form used to register with the FDA (Form require additional processing, storage, and reimplantation at
FDA 3356) should be obtained from the vendor and kept on another time within the same facility.1,17 Ironically, the Joint
file in the hospital tissue bank.15 The registration form con- Commission defines this as the “same procedure,” as it is done
tains the following information: federal registration number, by the same surgeon in the same facility on the same patient,
physical location of the establishment, reporting official’s in- despite the intervening storage in the tissue bank.1
formation, the establishment’s function (e.g., recovery, pack- Autologous tissue can be logistically problematic for a hos-
aging, processing, etc.), product information, and proprietary pital tissue bank. First, qualifying the supplier of the tissue is
names. Additionally, the hospital tissue bank’s medical direc- changed in this circumstance. If a tissue is to be used within
tor may request copies of any observations made during FDA
inspection (Form 483), including responses and corrective
action.16 Warning letters to an establishment are available Table 28–2 State Laws Governing Tissue
through the Freedom of Information Act and may be found Banking
on the FDA website (www.fda.gov).16
Some tissue vendors will become voluntarily accredited by STATE APPLICABLE LAWS
a recognized organization. This is not required but may weigh California California Health and Safety Code, Chapter 4.1 and 4.2
in the medical director’s decision to qualify a vendor for his
or her tissue bank. This should also be spelled out in the writ- Florida Chapter 893, Florida statutes
ten guidelines. Organizations that accredit tissue establish- Georgia State law requires tissue banks to be licensed as
ments are the American Association of Tissue Banks (AATB)5 clinical laboratories.
and the Eye Bank Association of America (EBAA).9 Similar
Maryland State law requires tissue banks to be licensed as
to the FDA website, these organizations list their accredited clinical laboratories.
establishments on their websites, which can be found at
www.aatb.org and www.restoresight.org, respectively. New Jersey New Jersey Statute 26:2A-1
Finally, the Joint Commission requires that hospital tissue New York New York State Public Health Law, Art. 43-B
banks ensure that tissue establishments are licensed by state
2682_Ch28_581-600 22/05/12 12:07 PM Page 588
the same institution and not shipped to an off-site facility under and recommendations for autologous tissue recovery that
different management for any further processing or storage, maintain aseptic techniques and prevent contamination.21
there is no requirement to register as a tissue establishment The recommended guidelines include:
with the FDA. It is important to note that if an autologous
1. Removal of tissue. The surgeon removes the tissue and
tissue is transferred to any other facility, such as a different hos-
hands it off to an assistant in an aseptic manner. The as-
pital because the patient was transferred, the hospital tissue
sistant places the tissue in a sterile container. More than
bank would be required to register with the FDA as a tissue
one piece of tissue may be placed in the container if re-
manufacturing establishment and would be subject to FDA in-
moved from the same site at the same time. Otherwise,
spection.16 In order to remain exempt from the requirement to
separate containers should be used.
register with the FDA, the hospital tissue bank must retain the
2. Culturing of tissue. If the procedure requires tissue cul-
tissue until reimplantation at the same facility.
ture, complete written instructions should be available.
Common autologous tissues include skull bone flaps
Culturing should include swabbing the entire tissue
(most common), ribs, iliac crests, and endocrine tissue such
surface, including edges. Culture swabs are labeled with
as the pancreas and parathyroid.18 Occasionally, other tissues
at least two unique patient identifiers and sent to the
such as skin and connective tissue will require storage. The
microbiology laboratory. Note: Culture should be done
advantage of autologous tissue is the perfect immunological
before any antibiotic is used as additive.
match. Additionally, the size may be ideal, such as is the case
3. Addition of storage solution. If the tissue requires addi-
for the skull bone flap that is exactly the right size for the
tion of storage media or antibiotics, it should be added
hole that must be patched. The risk of infectious disease
after the culture step. Written protocols for each tissue
transmission is diminished. However, one of the main dis-
type must clearly establish which tissues require additives
advantages to autologous tissue is the possibility of reinfec-
and which additives are required. Because of the possi-
tion of bacteria or fungus or the reintroduction of malignant
bility of allergic reactions, hospital personnel must con-
cells.19 The age and health of the donor-patient may impact
firm that the patient does not have an allergy to an
the functionality of the tissue as well.
additive antibiotic. The container must be labeled with
the name of the antibiotic used.
Preparation
4. Containers for storage. Containers may vary depending
The first step in the preparation for harvesting autologous on the tissue and the institution. AORN recommends
tissue is to define and develop clearly written policies and sterile plastic bags or sterile Nalgene jars. The Nalgene
procedures for both the surgical service and the hospital jars are useful because they are able to withstand temper-
tissue bank.18 These policies and standard operating proce- atures to –40°C for up to 5 years and can be used with ir-
dures require collaboration between the two services in order radiation sterilization procedures.21
to create a seamless operation and to ensure full agreement 5. Wrapping the container. The sterile container is further
on all conditions that must apply. The hospital tissue com- protected with at least two sterile outer wraps.
mittee may be involved in this process. 6. Labeling the container. The outer wrap of the container
The surgical service should include, as part of its proce- must be labeled with two unique patient identifiers, patient
dures, a notification step to alert the tissue bank of a planned age, date of collection, type of tissue, name and address of
procedure that will result in autologous tissue. Clear guide- the facility, physician’s name, time of collection and expi-
lines should be incorporated to instruct operating room per- ration date, if applicable. It may require further biohazard
sonnel on the steps required both administratively and labels according to FDA CFR 1271.90 and must contain a
medically. Informed consent must be obtained from the donor- label stating “FOR AUTOLOGOUS USE ONLY” and “NOT
patient. This is best done by the surgeon treating the patient. EVALUATED FOR INFECTIOUS SUBSTANCES.”16
The tissue bank should include a procedure to ascertain 7. Tissue storage. The tissue must be placed immediately
whether the donor-patient is being treated for bacteremia or into a temperature-monitored storage unit. If there is a
is at high risk for HIV or hepatitis.20 In general, it is not ad- delay, the tissue should be transported on wet ice.
visable to use autologous tissue for reimplant if the patient 8. Tissue processing. Tissue that is to be processed is done
was bacteremic at the time of harvest because of the risk of in a controlled environment.
reinfection. However, this is not true in every case, and pro- The Joint Commission also adds a requirement to “track and
cedures for sterilization exist, so each case must be approved identify materials used to prepare or process tissues and
individually by the tissue bank medical director and the sur- instructions used for preparation.”1 (JC Standard TS. 3.2.1)
geon jointly. The tissue bank should keep a donor-patient Careful documentation of materials, including name, lot
record on file for each autologous tissue. Any deviations, number, expiration date, and amount should be made in the
test results, adverse reactions, and the disposition should medical record.
be documented and retained in this file.
Within the operating room, the preparation of the autol- Storage
ogous tissue falls under the regulatory standards of the Joint
Commission, the AATB, and the Association of periOperative Ideally, the tissue bank will be expecting the arrival of the
Registered Nurses (AORN). AORN has developed guidelines autologous tissue, having been informed by the surgical staff
2682_Ch28_581-600 22/05/12 12:08 PM Page 589
Chapter 28 Tissue Banking: A New Role for the Transfusion Service 589
of the procedure ahead of time. Prior to tissue recovery, the deceased donor-patient. This is permissible as long as regu-
surgical team and the tissue bank should confer on the pro- lations are followed and documentation is complete.
cedure and verify that written standard operating procedures
address any individual issues that may be present for each Sterilization and Testing
donor-patient.1 Once the tissue arrives at the tissue bank,
the tissue bank personnel must inspect the label to make Sterilization is not required and not possible with many tis-
sure all elements are present (Box 28–2). The surgical team sues. A medical director in conjunction with the surgeon
should be immediately involved to solve any discrepancies may choose a sterilization technique. For skull bone flaps, a
or unclear labeling. common and popular method of sterilization has been high
As per written standard operating procedure and recom- heat by using an autoclave. This has the advantage of killing
mendation on the label, the tissue is placed in the proper bacteria and even removing malignant cells; however, the
storage site to ensure the correct temperature is main- disadvantage is that the high heat denatures proteins and col-
tained. A separate area should be reserved for autologous lagen in the bone, making it less suitable for reimplant.
tissues to keep them away from allogeneic tissue, as is Steam has also been utilized but has the same denaturing
done with autologous blood. Tissue from donor-patients effect. Many surgeons will opt for irradiation as a method to
who are known to have infectious diseases should be stored destroy bacteria. This is useful if there is an irradiator on-
in a quarantine area. site at the hospital and clear guidelines are worked out for
A common complaint from tissue banks who store autol- exposure times and radiation amount. Note: If the tissue is
ogous tissues is what to do with tissues that never get used. taken to another facility under different management for
It is surprisingly common for surgeons to decide not to use sterilization techniques, the hospital tissue bank must regis-
autologous tissue after it has been harvested. This can be ter as a tissue manufacturing establishment with the FDA
solved with good communication with the surgical staff and and become subject to FDA inspection.15
agreement of a reasonable expiration date for all tissues. If Testing is optional for autologous tissue, as it is for au-
agreement cannot be reached and an expiration date is as- tologous blood. Culture of autologous tissue is also optional
signed by the tissue bank, care should be taken to be sure and should be clearly spelled out in the operating proce-
the container is suitable for the temperature and duration of dures as to what tissues, if any, require culture. If the facility
storage. The surgeon should be informed of the expiration opts to culture or test the tissue donor-patient for infectious
date at the time it is assigned. According to AATB standards, diseases, the same testing should be performed as for
autologous tissues should have the same shelf life as a similar blood donors (HBsAg, anti-HBc, anti-HCV, HCV NAT,
allogeneic tissue under the same storage conditions.5 anti-HIV-1/2, HIV NAT, anti-HTLV I/II, syphilis, and West
Discard of autologous tissue should require the medical Nile virus NAT).22 If any testing or culture is positive, the
director’s approval. As with blood products and allogeneic tissue should be released only with the medical director’s
tissue, proper documentation of discard is required. Docu- approval. The surgical team should be notified of any positive
mentation must clearly outline the manner and location of tests, and the medical director may approve or disapprove
disposal according to local, state, and federal regulations for based on consultation with the transplanting surgeon. If any
medical waste.16 Occasionally, a request will be made that test is positive or if donor-patient screening indicates an
the tissue be sent to a funeral home for burial with the increased risk for infectious disease, a biohazard label is
mandatory for the tissue container. If the facility opts not
to test the autologous tissue, a label stating “NOT EVALU-
ATED FOR INFECTIOUS SUBSTANCES” must be affixed
BOX 28–2 to the container.16
Requirements for Autologous Tissue Labels
• Must read “For Autologous Use Only”
Implantation
• Name and address of the tissue bank Issuing or dispensing autologous tissue is much the same as
• Two unique patient identifiers for allogeneic tissue. There are a few differences, however,
• Complete description of tissue and the tissue bank that is located within the blood bank can
• Date of collection utilize familiarity with autologous blood products to con-
• Name of surgeon struct standard operating procedures that cover all of the
• Date of expiration according to validation of packaging and necessary requirements. As with autologous blood, the blood
tissue type
bank and tissue bank personnel should be made aware of the
• Disinfection or sterilization steps (if performed)
expected surgical date for reimplantation. Prior to the sur-
• Presence and name of any additives
gery date, the tissue bank must perform a thorough record
• Biohazard label if testing is positive or screening is positive for risk
factors review and all deviations and discrepancies resolved before
• “NOT EVALUATED FOR INFECTIOUS SUBSTANCES” unless the tissue is needed for surgery. Suitability should be docu-
complete donor testing has been done mented in the donor-patient file. The issuing procedure
• Recommended storage temperatures should be the same as for allogeneic tissue and must include
a final visual inspection of the outer wrapping for integrity
2682_Ch28_581-600 22/05/12 12:08 PM Page 590
and inspection of the label to verify all required elements are more than the others in one area but less in another, depend-
present. If any problems or deviations are discovered, the ing upon the focus of the organization. However, they over-
tissue should be placed into quarantine until the medical lap significantly and it is not difficult to develop an
director resolves the matter in consultation with the trans- organization that is in compliance with them all. It is impor-
planting surgeon. tant to note that compliance with AABB Standards often ex-
ceeds requirements from other agencies, another important
Regulatory Overview reason to house the tissue bank within the blood bank.13
While the author has attempted to summarize the relevant
The number of organizations involved in tissue regulations standards and regulations for each of the major organizations
and standards can be daunting to a facility intending to set listed here, it is recommended that a full review of each
up a hospital tissue bank. It can be overwhelming to look at regulation is done prior to developing a hospital tissue bank
the various documents to be sure that all requirements are structure or standard operating procedures (SOP) designed
carried out and satisfied. Table 28–3 is a “crosswalk” that for compliance.
compares each of the major regulatory agencies, matching
the categories of regulations for easy cross-reference and Federal Laws
comparison.
In taking a broad overview of the regulatory agencies, it The Code of Federal Regulations, Title 21 was established
is clear that they have evolved in parallel. One may require for food and drugs, and part 1271 deals with human cells,
Table 28–3 “Crosswalk” for HCT/P-Related Standards of the AABB, AATB, EBAA, and the Joint
Commission*
AABB1 AATB2 EBAA3 Joint Commission4
Chapter 28 Tissue Banking: A New Role for the Transfusion Service 591
Table 28–3 “Crosswalk” for HCT/P-Related Standards of the AABB, AATB, EBAA, and the Joint
Commission*—cont’d
AABB1 AATB2 EBAA3 Joint Commission4
*Horizontal alignment represents only general relationships among standards. This table represents only selected highlights of AABB HCT/OP-related standards. See Chapters 9 through 12 for
more detail on the above standards and organizations; see Frizzo5 for a fuller treatment of all relevant AABB standards and those of the AATB, the College of American Pathologists, and the
Joint Commission.
1Price, TH (ed): Standards for Blood Banks and Transfusion Services, 25th ed. AABB, Bethesda, MD, 2008.
2Pearson, K, Dock, N, and Brubaker S (eds): Standards for Tissue Banking, 12th ed. American Association of Tissue Banks, McLean, VA, 2008.
3Eye Bank Association of America: Medical Standards. EBAA, Washington, DC, 2008.
4The Joint Commission: Comprehensive Accreditation Manual for Hospitals. PC.17.10–PC.17.30. Joint Commission, Oakbrook Terrace, IL, 2008.
5Frizzo, WL: Tissue Management Self-Assessment Tool for Transfusion Services and Hospitals. AABB, Bethesda, MD, 2008.
Key: AABB = American Association of Blood Banks; AATB = American Association of Tissue Banks; EBAA = Eye Bank Association of America; HCT/P = human cells, tissues, and cellular and
tissue-based products
Reprinted with permission from Eisenbrey, AB, and Eastlund, T (eds): Hospital Tissue Management: A Practitioner’s Handbook, 1st ed. AABB, Bethesda, 2008.
tissues, and cellular and tissue-based products. Part 1271 is manipulated; it is intended for and labeled for homologous
subdivided into subparts A through F:16 use only; it is not combined with another vehicle except for
water, crystalloids, and sterilizing and preserving agents, unless
• A: General provisions
such additives compromise the clinical safety of the HCT/P;
• B: Procedures for registration and listing
and the HCT/P does not have a systemic effect and is not de-
• C: Donor eligibility
pendent upon the metabolic activity of living cells for its pri-
• D: Current good tissue practices
mary function.6 Alternatively, the HCT/P may have a systemic
• E and F: Additional requirements, inspection, and enforce-
effect or may depend on the metabolic activity of living cells
ment of establishments covered in 1271.10.
for primary function if it is for autologous use, allogeneic use
Section 361 of the PHS Act refers specifically to regulations in a first- or second-degree blood relative, or reproductive use.
to control communicable diseases (42 USC, section 264).23 Tissues that are not considered HCT/Ps include vascular-
ized human organs for transplantation; whole blood or blood
General Provisions components or blood derivative products; secreted or ex-
The purpose of part 1271 was to create a system to register and tracted human products (except for semen), such as milk, col-
list establishments that manufacture HCT/Ps.16 Additionally, it lagen, and cell factors; minimally manipulated bone marrow
is meant to establish guidelines for donor eligibility and incor- for homologous use and not combined with another
porate principles of good tissue practice in order to prevent the article, except for additives for preservation and storage; an-
introduction, transmission, and spread of communicable dis- cillary products used in the manufacture of HCT/P; cells, tis-
eases through tissue transplant. An establishment is defined as sue, and organs derived from animals; in vitro diagnostic
a business, which may include any individual, partnership, products; and blood vessels recovered with an organ intended
corporation, or association, under single management, in a sin- for transplant and labeled “For use in organ transplant only.”5
gle location that engages in the manufacture of HCT/Ps as well Although these items are not HCT/Ps, they may be found as
as facilities who contract manufacturing services. part of a hospital tissue bank at the discretion of the hospital
Human cells, tissues, or cellular tissue-based products administration and for the sake of uniformity in handling
are defined as articles containing or consisting of human biological items for human transplant, transfer, or infusion.
cells or tissues intended for transplantation or otherwise There are a few exceptions to who must comply with the
transferred into a human recipient. Some examples include requirements of part 1271. HCT/Ps used solely for nonclinical
bone, ligament, skin, hematopoietic stem cells derived from or educational purposes are exempt.16 Entities that merely
peripheral and cord blood, manipulated autologous cells, receive, carry, and deliver HCT/Ps and entities that do not re-
epithelial cells on a synthetic matrix, semen, and other re- cover, screen, test, process, label, package, or distribute but
productive tissue.7 This list is by no means exhaustive and only receive and store tissues for use on patients only in that
represents a small number of examples of HCT/Ps. facility are also exempt.16 The latter type of institution is the
HCT/Ps that are regulated under section 361 of the PHS usual model for hospital tissue banks. Thus, the requirements
Act meet the following criteria: The tissue is minimally set forth in 21 CFR 1271 are not regulations that hospital
2682_Ch28_581-600 22/05/12 12:08 PM Page 592
tissue banks that only store and issue HCT/Ps must meet. The required documentation includes a distinct identifica-
However, it is important that the tissue banker be aware of tion code that links the HCT/P to the donor. Except in the
the requirements of its suppliers, how the tissues are handled, cases of autologous tissues or HCT/Ps donated directly to a
and what tests are routinely run. Hospital tissue banks that first- or second-degree relative, the identification code
participate in harvesting, processing, packaging, or distribu- should not include the donor’s name, social security number,
tion of tissues must comply with the Code of Federal Regu- or medical record number. A statement that the donor has
lations (21 CFR 1271) and become registered establishments.15 been determined “eligible” or “ineligible,” along with a sum-
Distribution is defined as the transfer of HCT/Ps to another mary of the records used to make this determination, is re-
facility for use on a patient. It does not include the return of quired. In addition, a statement that disease testing was
tissue to a supplier. performed by a CLIA-certified laboratory or equivalent, a list
of tests performed with results, and the name and address of
Procedures for Registration and Listing the establishment determining eligibility are required.
Establishments that meet the requirements for registration Donor eligibility determination is not required in some
with the FDA must do so within 5 days after beginning circumstances. Examples of this include cells and tissues for
operations using Form FDA 3356.15 Registrations must be autologous use, reproductive cells or tissue donated by a sexu-
updated annually in December in conjunction with an up- ally intimate partner of the recipient, or cryopreserved cells for
date of HCT/P list, unless there is no change. If a change in reproductive use (when possible, measures should be taken to
the HCT/P list occurs between December and June, the test the semen and oocyte donors before transfer of an embryo).
change must be updated in June.15 In these cases, the tissue must be clearly labeled “FOR AUTOL-
In addition to the annual renewal and notifications of OGOUS USE ONLY” or, for allogeneic tissue, “NOT EVALU-
change in HCT/P list within 6 months, any change in loca- ATED FOR INFECTIOUS SUBSTANCES” and “WARNING:
tion or ownership of the establishment must be amended on Advise recipient of communicable disease risks.”16
the registration within 5 days of the change.15 Upon accept-
ance of the establishment’s registration form, the FDA will Current Good Manufacturing Practice
assign each location a permanent number.15 This registration Subpart D sets forth current good tissue practice (CGTP)
number does not imply that the establishment is in compli- requirements.16 These requirements are intended to prevent
ance with applicable rules and regulations or is approved by the introduction, transmission, or spread of communicable
the FDA. However, the establishment, once registered, must diseases by HTC/Ps by ensuring that they do not contain
be available for public inspection by the FDA.16 disease agents, are not contaminated, and do not become con-
taminated during manufacturing. The CGTP requirements
Donor Eligibility govern all steps in the manufacture of HCT/Ps, specifically
Establishments that manufacture HCT/Ps must develop and donor eligibility, screening, and testing. Core CGTP require-
maintain procedures for all steps in testing, screening, ments cover facility control, environment control, equipment,
donor eligibility, and all other requirements.16 The proce- supplies and reagents, recovery, processing, labeling, storage,
dures are subject to review and approval of a responsible in- receipt, distribution, and donor eligibility.16 Contracts between
dividual. Donor eligibility is determined by screening tissue suppliers are addressed here. Establishments that
relevant medical records to ensure that the donor is free recover and process tissue must be in compliance with sub-
from risk factors and clinical evidence of relevant commu- parts C and D as they apply to the operations performed at
nicable diseases, that there are no communicable disease that establishment.16 If another establishment performs some
risks associated with xenotransplantation, and that results of the operations, such as sterilization, that establishment is
of donor testing for relevant disease agents are negative or also required to conform to the regulations for the operations
nonreactive. Relevant communicable diseases include HIV, performed at that facility. Before entering into a contract, the
types 1 and 2; hepatitis B virus; hepatitis C virus; human primary establishment is responsible for verifying that the
transmissible spongiform encephalopathy; Creutzfeldt- second establishment is in compliance. If at any time the pri-
Jakob disease; and Treponema pallidum.24 For leukocyte- mary establishment becomes aware of noncompliance at the
rich tissue, there should be additional testing for human secondary establishment, the contract or agreement must be
T-lymphotropic virus, types 1 and 2. Cells and tissue that terminated.
are reproductive in nature must also be tested for Chlamydia Also part of CGTP is the establishment and maintenance
trachomatis and Neisseria gonorrhea.24 of a quality program. Functions of the quality program must
Relevant medical records that must be reviewed include include having appropriate procedures relating to core
a current donor medical history interview; a current report CGTP requirements and procedures for receiving, evaluat-
of the physical assessment of a cadaveric donor or a physical ing, and documenting core CGTP requirements; ensuring
examination of a living donor, if available; laboratory test that appropriate corrective actions relating to core CGTP
results in addition to those outlined above; medical records; requirements are done, including reaudits of deficiencies
coroner or autopsy reports; and records from any source and verification that corrective actions are effective; ensur-
pertaining to risk factors for communicable diseases. ing proper training and education of personnel; establishing
Once the donor is deemed eligible, records pertaining to appropriate monitoring systems; and investigating and doc-
the donor eligibility must accompany the tissue at all times. umenting deviations and trends.16
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Chapter 28 Tissue Banking: A New Role for the Transfusion Service 593
1.3 Policies, Processes, and Procedures Quality SOPs to ensure the requirements of the Standards are satisfied
4.0 Supplier and Customer Issues Supplier qualification, testing in AABB or equivalent accredited laboratory, registered with FDA
4.3 Incoming Receipt, Inspection, and Testing Inspection, testing as necessary, label verification
5.0 Process Control SOPs to ensure quality of tissue, derivatives, and services
5.1 Identification and Traceability Documentation of each step and identification of person who performed it, traceability, no
more than two donation identifications per container, handling, storage, and transportation
6.0 Documents and Records SOPs for documents to be identified, reviewed, approved, and archived
6.2.3 Record system shall make it possible to trace any tissue from source to final disposition
7.0 Deviations, Nonconformances, SOPs to capture, assess, investigate, and monitor deviations, responsibility for review,
and Adverse Events prevention of issue, quarantine, and notification
7.1.4 Released Nonconforming Tissue Determination of effect, notification of customer if necessary, records created
7.2 Fatality Reporting Fatalities related to blood shall be reported to outside agencies as required
7.4.4 Adverse Events Related to Tissue SOP for investigating and reporting adverse events
10.3 Discarded Tissue Minimize potential for human exposure to infectious agents
(Data from: Price, T, ed: Standards for Blood Banks and Transfusion Services, 26th ed. AABB, Bethesda, 2009.)
2682_Ch28_581-600 22/05/12 12:08 PM Page 594
American Association of Tissue Banks tissue bank, the AATB’s intention was to provide greater safety
and traceability to final disposition, whether implanted into a
The AATB was founded in 1976 by a group of scientists and patient or disposed.
doctors from the U.S. Navy Tissue Bank.4 These individuals The section in the AATB Standards that is most applicable
recognized that the scope of tissue banking should be to the hospital tissue bank is the L1.000 series for tissue
widened to include the entire nation. It is the only national dispensing services.5 Standards that apply to autologous
tissue bank organization and accredits over 100 tissue sup- tissue can be found throughout. See Table 28–5 for a list of
pliers nationwide. AATB’s Standards was first published in standards relevant to the hospital tissue bank. A comparison
1984. The Standards, now in its 12th edition, is a compre- of Tables 28–4 and 28–5 shows the similarity between the
hensive and detailed guide to tissue banking.5 This guide has Standards for Blood Banks and Tissue Services and the Standards
been used internationally as a framework for other national for Tissue Banking as they each pertain to tissue banking.
tissue banking organizations. At least six states (including
California, Georgia, and Maryland) require that tissue- The Joint Commission
manufacturing establishments be accredited by the AATB. In
1988, the organization initiated a certification program, The Joint Commission is a nonprofit accrediting agency that
Certified Tissue Banking Specialist (CTBS), for individual accredits and certifies over 17,000 health-care organizations
workers in the tissue banking industry. It currently certifies in the United States. Its mission is “to continuously improve
over 4,000 people. health care for the public, in collaboration with other stake-
In the 1990s, the AATB expanded its scope from tissue holders, by evaluating health care organizations and inspir-
manufacturing establishments to include hospital tissue ing them to excel in providing safe and effective care of the
banks. This era also saw the beginnings of a partnership and highest quality and value.”25 Its history reaches back to
collaboration between the AABB and the AATB. In 1993, a 1910, when Ernest Codman, MD, proposed the “end result
new section appeared in AATB’s Standards entitled “Medical system of hospital standardization.”26 He felt that if every
Facility Tissue Storage and Issuance.” Subsequently, several hospitalized patient could be tracked long enough to deter-
more sections that apply to the hospital tissue bank were mine effectiveness of treatment, medicine would benefit. The
added, including “Records Management,” “Release and Trans- benefit would come primarily in determining for whom
fer of Tissues,” “General Operations,” and “Quality Assurance treatment was not effective, determining why, and changing
and Quality Control Programs.” Following AABB’s change to practices for future patients. In 1917, a one-page list of
the ten QSEs, the AATB Standards were reorganized in the minimum standards for hospitals was published, and the fol-
same format. In widening its scope to include the hospital lowing year, the organization began inspections based on
L2.300 Labeling Tissue shall not be relabeled; labels shall not be altered
L3.100 Dispensing Physician’s order required, all associated written material is to be available to the end user
L3.200 Release to Intermediary All original written materials, label, and tissue identification number must accompany, documentation of transfer
to another facility
L3.300 Tissue Disposal Minimize hazard to staff or environment, comply with applicable laws and regulations
L3.400 Return of Tissue Cryopreserved reproductive tissue may not be redistributed for use, except as required by local or state
regulations
L4.000 Records Concurrently record all steps for traceability, maintain records for 10 years after expiration
L4.100 Tissue Receipt Records Each specimen must have a unique tissue identification number, minimum requirements for receipt records
L4.2 Dispensing Records Minimum requirements for disposition records, maintained in log format
L5.000 Adverse Outcomes Potential or suspect adverse outcomes, directly or indirectly related to tissue shall be reported to tissue processor,
investigated, and documented
(Data from: Pearson, K, Dock, N, and Brubaker, S (eds): Standards for Tissue Banking, 12th ed. American Association of Tissue Banks, McLean, VA, 2008.)
2682_Ch28_581-600 22/05/12 12:08 PM Page 595
Chapter 28 Tissue Banking: A New Role for the Transfusion Service 595
these minimum standards.26 Of the 692 hospitals surveyed EP 3: Records must identify who accepted the tissue, who
that year, only 89 met the minimum standards.26 prepared the tissue, and the date and time of preparation.
By 1950, the original document grew significantly, and EP 4: Documentation in the recipient’s clinical record of
there were as many as 3,200 accredited hospitals.26 In 1951, tissue use, including unique identifier of tissue.
the American College of Physicians (ACP), the American EP 5: Maintenance of records for at least 10 years following
Hospital Association (AHA), the American Medical Associ- expiration.
ation (AMA), and the Canadian Medical Association (CMA) EP 6: Documentation of minimum required elements in
joined with the original American College of Surgeons (ACS) records maintained for at least 10 years.
to create the Joint Commission on Accreditation of Hospitals EP 7: Compliance with completion and return of tissue usage
(JCAH).26 information cards requested by the source facility.
In 1965, Congress passed the Social Security Amendments
Hospital Standard PC.17.30 (Laboratory Standard
with the provision that hospitals accredited by JCAH had
QC.5.320) concerns the process of investigating adverse reac-
deemed status with the Medicare Conditions of Participation
tions to tissue or donor infections. This standard is in five parts:
for Hospitals and could, therefore, participate in Medicare
and Medicaid programs.26 In 1978, JCAH joined forces with EP 1: Having SOPs to investigate recipient adverse events
the College of American Pathologists (CAP) to further eval- related to tissue use.
uate laboratories. JCAH adopted the name Joint Commission EP 2: Reporting to the source facility any post-transplant
on Accreditation of Healthcare Organizations (JCAHO) in infections and adverse events.
1987 to reflect its expanding scope of activities.26 EP 3: Quarantining tissue identified by the source facility
Effective July 1, 2005, JCAHO adopted the tissue storage as possibly contaminated.
and issuance standards for the ambulatory care, office-based EP 4: Informing and tracking patients whose donors are sub-
surgery, critical access hospitals, and hospital accreditation sequently found to have a transmissible infectious disease.
programs that store and/or issue human tissue. These were EP 5: Following procedures for real or suspected adverse
in the form of standards PC.17.10, PC.17.20, and PC17.30. events
In 2007, JCAHO officially shortened its name to “Joint
A review of the tissue standards from the Joint Commission
Commission.”26
reveals striking similarity to regulations from AABB, AATB,
Hospital Standard PC.17.10 (Laboratory Standard
and FDA. The most notable addition is the requirement for
QC.5.300) addresses the use of standardized procedures to
source facilities to comply with applicable state laws for
receive, store, and issue tissues. This standard is broken
registration and accreditation.
down into ten parts, or elements of performance (EP):26
EP 1: Assign responsibility for overseeing the tissue program Eye Bank Association of America
throughout the hospital.
EP 2: Validate that source facilities are registered with the The EBAA accredits facilities that provide eye tissue for sur-
FDA. gery, education, or research.9 It is a nonprofit organization
EP 3: Coordinate tissue ordering, receipt, storage, and that also certifies eye bank technicians. The organization was
issuance throughout the hospital. established in 1961 by the American Academy of Ophthal-
EP 4: Transport, handle, store, and use tissues according to mology’s Committee on Eye Banks.9 It is the oldest national
manufacturer’s instructions. tissue transplant organization in the United States, and its
EP 5: Log in all incoming tissue. mission is to “restore sight through the promotion and
EP 6: Maintain continuous temperature monitoring of advancement of eye banking.” In addition to accreditation and
storage equipment. certification, EBAA also develops uniform medical standards
EP 7: Maintain daily records of appropriate storage and procedures for its member banks to maintain proficiency
temperature. in eye tissue recovery, storage, preservation, and transplan-
EP 8: Make sure there are functional alarms and emergency tation.9 These medical standards are reviewed and approved
backup for storage equipment. semiannually by the American Academy of Ophthalmology.
EP 9: Comply with state and federal regulations when acting The EBAA requires facilities providing tissue to be registered
as a source facility. with the FDA.9 The source facility may voluntarily be accredited
EP 10: Verify package integrity and transport temperature by EBAA; however, the Joint Commission does not require this
at receipt. as a condition for hospital tissue banks to receive tissue from
an establishment. The hospital tissue bank medical director may
Hospital Standard PC.17.20 (Laboratory Standard QC.5.310)
select suppliers of ocular tissue from EBAA-accredited facilities
is concerned with record keeping, particularly in the trace-
as an added level of safety and security.9
ability from donor to final disposition. There are seven parts
to this standard:
Adverse Events
EP 1: Records must permit tracing of any tissue.
EP 2: Records must track and identify material used to An adverse event is a negative change, unexpected outcome,
prepare or process tissue. or reaction to the tissue implant. Adverse events can be due
2682_Ch28_581-600 22/05/12 12:08 PM Page 596
to infectious disease, failure of the allograft to function as director should be certain to make transplanting surgeons
expected, or transmission of malignancy. It is important to aware of bacterial and fungal risks.
discover and report adverse events primarily for the protec- Dura mater has been known to transmit Creutzfeldt-
tion of other patients who may receive tissue from the same Jakob disease (CJD) in more than 100 cases of transplant.24
donor.2,7 CJD is caused by a prion, which is neither virus, bacteria, or
fungus but rather is a self-replicating protein known to
Infection invade neurological tissue and cause death. Incubation times
vary, and patients have shown symptoms as early as
Transmission of infectious disease through allograft trans- 6 months following transplant and as late as 16 years.
plantation is relatively rare. Many of the recorded cases of Another possibility for introduction of infectious agents
disease transmission occurred in years prior to the current, is through perimortem blood transfusions. The author is
comprehensive standards and improved donor testing. In- currently investigating several donors whose blood was
fections are most often transmitted by tissues that are fresh transfused to a trauma victim who died and subsequently
or cryopreserved. These methods of storage allow some in- became a tissue donor. A recipient has contracted parvovirus
fectious entities to survive and cause disease in the new B19. Appropriate investigation of the tissue donor has been
host. Fortunately, the most common type of tissue used, unrevealing. Because blood is not routinely tested for par-
freeze-dried bone, has not been seen to transmit disease vovirus B19, the donors of the units transfused at death are
readily. Often the biggest cause of disease transmission is being investigated. Fortunately, transfusion-transmitted
human error. Inadequate donor screening, inadequate ster- disease is rare.
ilization, and other system failures allow potentially con-
taminated tissue to remain in the system. For these reasons, Malignancy
the above regulations have been developed.
Malignancy transmitted through tissue transplant is ex-
Similar to blood donation, the first step in ensuring safe
tremely rare. Two cases of carcinoma transmitted through
tissue is to search the donor population for individuals with
corneal transplant have been recorded.19 Almost all other
low risk of disease. This can be a challenging task for man-
known cases of malignancy transmission have been
ufacturing facilities. Medical records may be incomplete, and
through organ transplant and include a number of adeno-
if family of a deceased donor is available for interview, they
carcinomas, melanoma, sarcoma, lymphoma, glioblastoma,
may not be aware of all the details that may be important. It
and choriocarcinoma.19 Tissues, such as bone, tendon, and
may also be unclear what caused the donor’s death. A mis-
ligament, have few viable cells present at transplant and
diagnosis of a fatal infection could result in that infection
undergo several sterilization steps. Heart valves and vascu-
passing on to a tissue recipient. This was the case for a rural
lar tissue rarely harbor malignancies and are therefore less
man who died with “chest numbness and back pain” in
likely to transmit it. Of note, there has never been a
1979, reported by Houff and colleagues.27 His corneas were
recorded incident of transmission of malignancy through
transplanted into an individual who died six weeks later, fol-
blood transfusion. Both tissue and blood donors are ex-
lowing development of headaches, eye pain, and respiratory
cluded from donation on the basis of malignancy present
failure. Studies on both donor and recipient revealed a rabies
at time of donation.
infection in both individuals. Had the clinical history of the
deceased been more accurate, the transplant and subsequent
Allograft Failure
second death could have been avoided. Infections may lie
dormant for years. There may be only limited mention in The allograft may fail to function as expected or intended.
medical records, or none at all for a virus that is dormant In many cases, this may be because of the patient’s under-
with an undetectable antibody titer. Antibody titers may also lying condition. However, it is important to investigate
be unreliable and in the “window period” shortly after initial other causes not related to the patient. Failure of the tissue
infection. For these reasons, RNA testing for HIV and manufacturer to faithfully adhere to standard operating
Hepatitis C has become standard practice. procedures can cause changes that may affect the stability
Bacterial and fungal infections transmitted by tissue and durability of the tissue product. Discovering the
transplant are getting more attention in recent years due to source of tissue failure, either intrinsic to the donor or
increased awareness and regulatory oversight. It is likely because of an irregularity in manufacturing, will lead to
that many adverse events due to fungal and bacterial con- recall of other products that may have potential to harm
tamination went undetected, usually with the assumption other patients.
that the recipient acquired the infection de novo. Investi-
gation into the source of these types of adverse events have Reporting and Investigation
shown that the organism was often not part of the pre-
mortem condition in the donor but was introduced during Reporting and investigating suspected adverse outcomes
the surgical harvest of the tissue or the subsequent process- is important for many reasons. This area of medicine has
ing. Many tissue products undergo minimal processing recently received a great deal of attention with efforts to
without sterilization steps. These include heart valves, create a national biovigilance network. Biovigilance, as a
tendons, ligaments, and cartilage. The tissue bank medical structured, region-wide entity, has been in practice for many
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Chapter 28 Tissue Banking: A New Role for the Transfusion Service 597
years in Europe. The backbone of a quality program is the should be opened on each case, and all documents gener-
ability to capture errors and unexpected outcomes and then ated in the investigation should be kept together in the
investigate them to determine, as much as possible, the root file. Immediately following a complaint, the personnel in
cause. This information is of little value if it is not shared the tissue bank should look for and quarantine any tissue
with everyone involved in the process so that meaningful from the same donor, pending outcome of the investiga-
change can take place. In the individual case, it is crucial to tion. The medical director must be notified as soon as
alert the tissue supplier of a suspected disease transmission possible. The tissue supplier must also be notified as soon
or contamination so that the supplier may track down all as possible so that steps may be taken to quickly sequester
other tissue harvested from the same donor to prevent other any other tissue from that donor that may exist at different
adverse events. Often, tissue tracked from the same donor facilities.
may have already been transplanted. In that case, it is equally The medical director must then confirm the complaint
important to know of possible contamination so that the and begin to determine what other causes may be responsi-
patient may be appropriately tested or treated. ble. He or she should look into the possibility of nosocomial
The most basic, and arguably the most important, step in infection, other potential routes of infection, and risk factors
the reporting process is recognizing an adverse event by the independent of tissue transplant and should examine patient
clinical staff. Postoperative infections are not uncommon, records for any irregularities associated with tissue transplant
and a true adverse reaction may be dismissed as a routine procedures. If warranted, corrective action should be imple-
postoperative complication. Some adverse reactions may mented depending on the result of the investigation. The
take weeks, months, or even years to express themselves, tissue bank must cooperate fully with the tissue supplier’s
such as hepatitis C infection or CJD. The medical director investigation.
of the tissue bank should communicate regularly with the Other ancillary investigation should include testing of
surgeons, especially on this topic. It may be helpful to sched- any unused tissue from the same donor and investigation
ule grand rounds or other educational talks to ensure that into the postoperative course of other patients who re-
all surgical staff are aware of the risks and what to look for. ceived tissue from the donor. The results of the investiga-
Some clinical staff may be falsely assured by sterile packaging tion must be reported to all parties involved, including the
or disinfecting steps in processing. While contamination is transplanting surgeon and the tissue supplier, as well as
rare, it is more common in tissues with viable cells, such as the hospital risk management department and the tissue
valves and vessels. Disinfection steps may not be effective committee. Although not required, the tissue bank is en-
for every organism. Additionally, steps may be missed due couraged to report the findings to the Joint Commission.
to human error. If the event is severe or fatal, the Joint Commission recog-
Contamination by bacteria or fungus should be suspected nizes this as a sentinel event requiring root cause analysis
based on the type of organism cultured, especially if the in- and corrective action. Although not required, the event
fection is deep and not a superficial wound infection. Un- may be reported to the FDA through MedWatch. Reporting
usual organisms for the wound or tissue should also be to the state is necessary for those cases involving re-
suspect. Although the time frame of onset can be factored portable disease. A final report should be placed in the file
into the overall decision to report the infection as a suspected created for the case, and another copy should be kept in
adverse event, it is less reliable, as it may vary greatly de- the tissue vendor’s qualification file.
pending on the organism. Once the surgeon has sufficient It is important to remember to keep the patient informed
information to suspect that the transplant is the reason for as well. The Code of Medical Ethics and the Joint Commis-
the infection, he or she should immediately report the sus- sion are clear that the patient has a right to know when
pected event to the tissue bank. errors or unanticipated outcomes occur, particularly if the
Suspicion of viral or prion transmission by tissue should adverse event is severe or considered a sentinel event. It is
similarly be reported as soon as the physician becomes aware. best if the treating surgeon conveys this information because
Obvious other causes of infection should be sought at the a relationship and level of trust already exists. If another
time of diagnosis. For example, a patient newly diagnosed physician must inform the patient, care should be taken not
with hepatitis C at some point following tissue transplant to alienate the patient and treating physician, and the treat-
should be questioned about IV drugs, tattoos, needle sticks, ing physician should be fully informed beforehand. When
and household contacts as possible sources of infection. informing a patient of an error or allograft-related infection,
The surgeon may also report the suspected disease trans- it should be done compassionately, factually, and without
mission to the FDA through the MedWatch program blame. Expressions of concern and regret are appropriate,
(www.fda.gov/medwatch). However, it is important that the and there should be a plan for how to proceed. An offer of
hospital tissue bank is not bypassed in the process, as an im- assistance in further treatment as appropriate should be
portant function of the medical director is to report to the made as well.
tissue supplier and investigate the case.
In the tissue bank, there are several important steps to Recalls
the investigation process. First, every complaint or suspi-
cion should be taken seriously, even if there is a low Recalls are almost always voluntary and may be initiated
chance that the event is related to tissue transplant. A file at virtually any step in the process from the donor to the
2682_Ch28_581-600 22/05/12 12:08 PM Page 598
recipient. The recall is initiated after an error is discovered, found to be positive for HIV, human T-cell lymphotropic
even if it is months to years after the tissue has been virus (HTLV) type I or II, viral hepatitis, or other known
processed. An irregularity may be discovered in an FDA infectious agents transmissible by tissue must be notified.
inspection that initiates the recall, or a problem may be This would include making reasonable attempts to notify
reported by workers or doctors who discover it in the course the implanting physician and request that he or she notify
of using or handling the tissue. When a problem is discov- the patient. If the surgeon cannot be reached or does not
ered in the course of an investigation, the FDA may compel wish to notify the patient, it becomes the tissue bank’s respon-
the establishment to initiate a recall. sibility and reasonable attempts to reach the patient must be
There are three classes of FDA recalls (Box 28–3). Class I made. Reasonable attempts to notify should occur in less
is the most severe and serious. These recalls are reserved for than 12 weeks of the recall. The explanation to the patient
products that may cause serious harm, health problems, or should include information regarding the reason for the
death if used. Class II is intermediate and is for products that recall, possible effects to him or her, and the opportunity to
may cause temporary health problems or less serious injury. make a decision regarding further testing or counseling with
Class III is likely the most common type of recall and per- a list of testing locations.
tains to products in which a manufacturing irregularity or
violation in labeling is discovered, but the product is unlikely
to cause harm if used.
Typically, the manufacturing establishment where the CASE STUDIES
tissue was originally processed notifies its customers of
recalls. The FDA also disseminates information on recalls Case 28-1
of all types. These recalls can be found on the FDA website A new orthopedic surgeon has just been hired at the hos-
through MedWatch, the FDA’s safety information and pital. He has a great deal of experience using a particular
adverse event reporting tool. Hospital tissue banks may type of processed bone that can be acquired from only one
also subscribe to a national recall reporting service such tissue vendor. He requests that the hospital tissue bank
as the National Recall Alert Center, although that is not begin ordering this product for his upcoming scheduled
required. surgeries. When the approved vendor list is consulted,
Just as there are steps in reporting adverse events, noti- the vendor is not on the list.
fication of a recall has several important steps. Upon receiv-
ing notification of a recall, the hospital tissue bank must 1. What do you tell the surgeon who is expecting the
verify that the product is or was in inventory, determine the tissue next week?
disposition of any and all tissue included in the recall, and 2. What is the minimum requirement to qualify the new
quarantine any unused tissue. All tissues that have been vendor?
transplanted must be tracked to the recipient. If any tissues
have been sold, transferred, or returned, the receiving Case 28-2
facility must be notified of the recall. Just as in the adverse A neurosurgeon calls the hospital tissue bank in advance
event investigation, a file should be created for any inves- of a planned craniotomy. He explains that he will not be
tigation into the disposition of recalled tissue and main- able to replace the bone flap at the completion of the sur-
tained for 10 years. gery planned for the next day, but within 1 to 2 weeks,
Regulations on notification of the recipient have been he would like to close the head wound with the patient’s
addressed by the Joint Commission and AABB. Both require own bone. He further states that it is possible that once
that recipients of tissue from donors who are subsequently stabilized, the patient may be moved to another facility
for the second procedure. He requests that the autolo-
gous tissue be stored in the tissue bank and inquires as
to the procedure for transferring the tissue with the
BOX 28–3 patient.
FDA Recall Classes 1. What are the minimum requirements for labeling
• Class I recall: Potential to cause serious injury or death autologous tissue?
• Class II recall: Potential to cause temporary or less serious health 2. Under what condition could the hospital tissue bank
problems transfer the tissue?
• Class III recall: Unlikely to cause harm but violates FDA regulations 3. Give two examples of procedural steps that should be
or labeling discussed prior to the surgery.
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Chapter 28 Tissue Banking: A New Role for the Transfusion Service 599
SUMMARY CHART
In 2005, the Joint Commission ruled that hospitals packaging invoice, and must receive verification that
must assign responsibility for overseeing the tissue temperature conditions have been maintained appro-
program throughout the organization, including stor- priate to the tissue delivered.
age and issuance activity (PC17.10A1). The hospital tissue bank must maintain continual
AABB and AATB recommend a centralized model of monitoring of storage refrigerators and freezers.
tissue banking, and AABB states that the ideal location Record keeping must detail each step or change in
for the tissue bank is in the hospital blood bank. status of the tissue with notation of the person who
The FDA requires that any tissue-manufacturing estab- performed it and the date to allow for bidirectional
lishment must register yearly with the FDA and submit tracking from donor to final disposition.
to inspection. Records must be maintained for 10 years following the
Hospital tissue banks that receive, store, and issue expiration of a tissue product or 10 years after issue if
tissue do not need to register with the FDA unless their there is no expiration date.
function also involves procurement, processing, or Each tissue product must have a unique tissue identi-
distribution. fication number (TIN).
Transfer of tissue to another facility is considered The hospital tissue bank must verify any supplier and
distribution. vendor’s FDA registration on a yearly basis.
Vascularized organs, such as liver, kidney, lung, pancreas, The Joint Commission requires that hospital tissue
and heart are excluded from FDA CFR 1270 and 1271. banks ensure that suppliers are licensed by state agen-
The tissue bank should have a physician-appointed cies as the laws apply for the state in which the vendor
medical director in line with the AABB’s model for or tissue bank is located.
blood bank. Autologous tissue, used within the same facility with
A tissue committee should be established that is simi- no intervening shipments off-site for further process-
lar to, or part of, the transfusion committee. ing, may be stored and issued with no requirement
Tissue delivered to the tissue bank must be inspected to register with the FDA as a tissue manufacturing
for package integrity and labeling and reconciled with establishment.
7. The medical director for the tissue bank can be: 8. Eisenbrey, AB, and Eastlund, T (eds): Hospital Tissue Management:
A Practitioner’s Handbook, 1st ed. AABB, Bethesda, 2008.
a. Any individual appointed by the hospital medical
9. Eye Bank Association of America: Medical Standards. Washington,
director. DC: EBAA, 2008. Available at www.restoresight.org/aboutus.
b. The lead supervisor in the blood bank. Accessed January 25, 2010.
c. The head nurse/transplant coordinator from surgical 10. Eastlund, T: Tissue and organ transplantation and the hospital
nursing. transplantation service. In Roback, J, Combs, MR, Grossman,
B, and Hillyer, C (eds): Technical Manual, 16th ed. AABB,
d. A qualified physician involved in tissue transplant or
Bethesda, pp 833–864, 2008.
blood banking. 11. Code of Federal Regulations: Title 21 CFR Part 1270 and 1271.
Washington, DC: US Government Printing Office, 2009 (re-
8. Notification of a recipient of tissue that has been vised annually). Available at www.accessdata.fda.gov/scripts/
recalled because of possible contamination should be cdrh/cfdocs/cfCFR/CFRSearch.cfm?CFRPart=1271. Accessed
conducted by: January 25, 2010.
a. The tissue bank director only. 12. Hillyer, CD, and Josephson, CD: Tissue oversight in hospitals:
The role of the transfusion services (editorial). Transfusion
b. The patient’s transplanting surgeon.
47:185–187, 2007.
c. The patient does not need to be told unless he devel- 13. Price, T (ed): Standards for Blood Banks and Transfusion
ops an infection. Services, 26th ed. AABB, Bethesda, 2009.
d. The informed consent covers this contingency and 14. Frizzo, W: Tissue Management Self-Assessment Tool for Trans-
no further notification is necessary. fusion Services and Hospitals. AABB, Bethesda, 2008.
15. Code of Federal Regulations: Tissue Establishment Registra-
9. Tissue receipt records must include all of the following tion. Title 21 CFR Part 1270 and 1271. Available at www.fda.
EXCEPT: gov/BiologicsBloodVaccines/GuidanceComplianceRegulatory
Information/EstablishmentRegistration/TissueEstablishment
a. Unique tissue identification number. Registration/default.htm. Accessed January 25, 2010.
b. Name and address of tissue supplier. 16. Code of Federal Regulations: Current Good Tissue Practice
c. Expiration date. for Human Cell, Tissue, and Cellular and Tissue-Based Product
d. Tissue supplier’s FDA registration number. Establishments; Inspection and Enforcement. Title 21 CFR
Parts 16, 1270, and 1271; Final Rule. Federal Register
10. Records that must be reviewed to determine donor 69:68612, 2004.
eligibility by the tissue manufacturer include: 17. Shulman, IA (forum ed and moderator): Should Blood Banks
Store Autologous Bone Flaps in Their Freezers? California Blood
a. Donor family history. Bank Society e-Network Forum. 2001. Available at www.cbbsweb.
b. Records from any source pertaining to risk factors for org/enf/2001/bonestorage.html. Accessed January 25, 2010.
communicable diseases. 18. Shulman, IA (forum ed and moderator): Management and En-
c. Interview of next-of-kin. suring Compliance of “Tissue Banking/Tissue Dispensing Serv-
ices.” California Blood Bank Society e-Network Forum. 2006.
d. Consent to harvest tissue.
Available at www.cbbsweb.org/enf/2006/tissue_mgmt.html.
Accessed November 18, 2011.
19. Gandhi, MJ, and Strong, DM (eds): Donor derived malignancy
following transplantation: A review. Cell Tissue Bank 8:267–286,
References 2007.
20. Southeast Tissue Alliance: The Truth About Tissue Donation.
1. Joint Commission: Tissue Storage and Issuance Standards: 2007. Available at www.donorcare.org/pdfs/ceu_the_truth_about_
PC.17.10, PC.17.20, PC.17.30, QC.5.300, QC.5.310, QC.5.320. tissue_donation_web.pdf. Accessed January 25, 2010.
Joint Commission, Oakbrook Terrace, IL, 2008. 21. Association of periOperative Registered Nurses: Standards,
2. Eastlund, T, and Eisenbrey, AB (eds): Guidelines for Managing Recommended Practices, and Guidelines. AORN Publications,
Tissue Allografts in Hospitals. AABB, Bethesda, 2006. Denver, CO, 2006.
3. New York Organ Donor Network: Transplantation—the 22. Centers for Disease Control and Prevention: West Nile virus
history, 2009. Available at www.donatelifeny.org/all-about- infections in organ transplant recipients—New York and Penn-
transplantation/tissue-transplant-history/. Accessed November sylvania, August–September, 2005. MMWR Morb Mortal Wkly
17, 2011. Rep 54:1021–1023, 2005.
4. Strong, DM: The US Navy Tissue Bank: 50 Years on the Cutting 23. United States Code: Regulations to Control Communicable
Edge. Cell Tissue Bank 1:9–16, 2000. Diseases. Section 361 Public Health Service Act, 42 USC §
5. Pearson, K, Dock, N, and Brubaker, S (eds): Standards for 264(a). Washington, DC. US Government Printing Office, 2006.
Tissue Banking, 12th ed. American Association of Tissue 24. Eastlund, T, and Strong, DM: Infectious disease transmission
Banks, McLean, VA, 2008. through tissue transplantation. In Phillips, GO (ed): Advances
6. Public Law 98-507. October 19, 1984. The National Organ in Tissue Banking. Vol 7. World Scientific Publishing,
Transplant Act. Available at www.history.nih.gov/research/ Singapore, 2003, pp 51–131.
downloads/PL98-507.pdf. Accessed November 17, 2011. 25. Joint Commission: About The Joint Commission. Available at
7. Food and Drug Administration: Guidance for Industry: Med- www.jointcommission.org/about_us/about_the_joint_commis-
Watch Form FDA 3500A: Mandatory Reporting of Adverse sion_main.aspx. Accessed 2011
Reactions Related to Human Cells, Tissues, and Cellular and 26. Joint Commission: History of The Joint Commission. Available at
Tissue-Based Products (HCT/Ps), 2009. Rockville, MD: CBER www.jointcommission.org/about_us/history.aspx. Accessed 2011.
Office of Communication, Training, and Manufacturers Assis- 27. Houff, SA, Burton, RC, Wilson, RW, et al: Human-to-human
tance. Available at www.fda.gov/Safety/MedWatch/HowToReport/ transmission of rabies virus by corneal transplant. N Engl
ucm085568.htm. Accessed January 25, 2010. J Med 300:603–604, 1979.
2682_Answer Key_601-612 22/05/12 5:40 PM Page 601
Answer Key
Chapter 1 Chapter 5
Review Questions Case 5-1
1. c 6. c 11. b 16. a 1. No. Adding check cells to all tubes negative at the AHG
2. c 7. d 12. d 17. d phase provides a quality-control measure to the test. The
3. b 8. d 13. d 18. c expected result is positive agglutination. A negative result
4. c 9. a 14. c 19. c deems our test invalid.
5. d 10. a 15. c 20. a 2. The blood bank tech is required to repeat the test, starting
from step 1.
3. The most likely cause is failure to adequately wash cells
after the incubation phase.
Chapter 2 4. Failing to add AHG reagent is also common; AHG reagent
that has failed QC or is expired should never be used for
Review Questions patient testing.
1. b 5. d 9. c 13. a Case 5-2
2. b 6. d 10. a 14. a
3. c 7. b 11. b 15. c 1. Yes.
4. d 8. a 12. a 2. The initial antibody screen gel reaction is positive but
may be demonstrating a method-dependent antibody.
Some antibodies, both specific and nonspecific, have the
ability to demonstrate method dependency. The results
Chapter 3 show reactivity in one method but not others. The nega-
tive reactions in the LISS panel confirm the prediction of
Review Questions the method-dependent antibody.
3. The use of PCR technology to genotype the patient might
1. a 7. b 13. d 19. a
be useful in situations of method-dependent antibodies.
2. c 8. c 14. c 20. b
It should also be noted in the patient’s record that future
3. b 9. d 15. a 21. d
testing should be conducting using the LISS method.
4. d 10. c 16. b
5. d 11. a 17. c Review Questions
6. c 12. a 18. d
1. d 5. b 9. b 13. a
2. c 6. b 10. a 14. b
3. a 7. d 11. c 15. c
Chapter 4 4. a 8. a 12. c 16. b
Review Questions
1. a 5. d 9. c 13. c
2. c 6. a 10. b
3. c 7. d 11. b
4. b 8. d 12. d
601
2682_Answer Key_601-612 22/05/12 5:40 PM Page 602
13. d 18. b 23. c 28. b 3. When working up any antibody identification, it is good
14. c 19. c 24. d 29. d practice to get the patient’s history of transfusions, trans-
15. c 20. d 25. c 30. c plantations, and, if female, pregnancies. A list of medica-
16. b 21. b 26. d tions and the patient’s diagnosis may prove helpful.
17. c 22. a 27. b Knowing the patient’s ethnicity may give clues as to the
identity of the antibody, as antigen frequencies vary
among races. One example of a high-prevalence antigen
associated with a specific ethnicity is the U antigen,
Chapter 9 which is present in virtually all whites and all but approx-
imately 1% of blacks. Anti-U is produced mainly in
Case 9-1 multiparous black females or those patients who have
1. There is inconsistent reactivity (1+ to 3+). The pattern of been repeatedly transfused.
reactivity appears to fit anti-Fyb; however, one homozy-
gous cell (cell 2) reacts 3+ while others (cells 8 and 9) Case 9-3
react only 2+. This inconsistency is seen in the cells with 1. A positive reaction was seen with cell 4 only, which is Jsa
heterozygous antigen expression as well. Cell 7 reacts 2+, positive. All other antibody specificities have been elim-
while cells 1, 6, and 11 react only 1+. inated except for anti-Lua (an antibody to another other
2. Anti-C, anti-Cw, anti-K, anti-Kpa, anti-Jsa, anti-Fyb, anti- low-prevalence antigen).
Jka, and anti-Lua have not been excluded. Fyb antigens 2. Testing with additional antigen-positive cells will be
would be destroyed or diminished after treatment with required to confirm the specificity. Panel cells that are
enzymes. C, Cw, Jka, and Lua would have enhanced antigen positive for these rare antigens are normally indicated on
expression. K, Kpa and Jsa would be unaffected by routine the panel profile sheet or listed on the extended typing
enzymes. form. Testing two other cells positive for the Jsa antigen
3. The specificity of the antibodies was unclear after the ini- to satisfy the 3 and 3 rule and phenotyping the patient
tial panel. After repeating the panel using ficin-treated for the Jsa antigen should be done to complete the antibody
cells, it appears that the antibodies present are anti-K and identification workup. If additional cells are not readily
anti-Fyb. The enzyme treatment removed the Fyb anti- available, consult a reference laboratory.
gens, thereby eliminating the anti-Fyb reactivity, which
allows the anti-K to present clearly. Anti-C and anti-Jka Case 9-4
were also not eliminated by the initial panel. However, 1. All cells (except the I-negative cord blood cells) are positive
one would expect these antibodies to demonstrate en- at the immediate spin phase of testing, and the autocontrol
hanced reactivity with the ficin-treated cells. Anti-Jka may is positive. The reactivity does not persist into the AHG
be excluded using cell 8 of the ficin panel. Whereas phase of testing.
anti-C cannot be excluded using a homozygous cell, the 2. The use of cord blood cells, which lack the I antigen, con-
pattern of reactivity and lack of response to enzyme treat- firms the presence of anti-I in this sample.
ment suggest it is not present in this sample. 3. Many laboratories avoid detection of cold autoantibodies
4. Testing a C+, c–, K–, Fyb– cell would be necessary for by omitting the immediate spin phase of the antibody
exclusion. screen (and panel) and by using monospecific anti-IgG
Coombs’ reagent.
Case 9-2 One of the least complex methods used to prevent cold
1. The positive reactions on this panel are all the same autoantibodies from interfering in antibody detection or
strength. Cell 10, the only cell that failed to react with identification tests is the prewarm technique. In this
the patient’s serum, has been identified as being Yta neg- procedure, the serum being tested and the screen or panel
ative. As Yta is a high-prevalence antigen, one may be sus- cells are heated to 37°C in separate test tubes. Once at
picious that this is the antibody present in this specimen. 37°C, a drop of warm RBCs is added to each of the tubes
2. For this case, the ideal solution is to test two additional containing serum, and the test proceeds with incubation,
Yta-negative cells, which would provide 95% confidence of washing, and AHG steps. The wash step uses saline that
correct antibody identification and allow for additional ex- has also been maintained at 37°C so that at no time is the
clusions. If antigen-negative cells are not readily available, test system allowed to drop below that temperature, thus
it may be necessary to consult a reference laboratory that avoiding the optimal temperature range of the autoanti-
maintains a stock of rare cells. Any other antibodies not body. Because this method does not usually have enhance-
excluded following this testing may require patient phe- ment media in the system, it is possible to fail to detect
notyping or absorption studies to confirm their presence some weak significant antibodies. Also, the warm saline
or absence. The services of a reference laboratory may be may result in the dissociation of some significant antibodies
required to antigen type the patient for the Yta antigen. from the cells.
2682_Answer Key_601-612 22/05/12 5:40 PM Page 604
A more complex method, discussed previously, is ad- ZZAP to enhance expression of certain antigens and facili-
sorption. The patient’s autologous cells may be incubated tate autoantibody removal. These treatment and adsorption
with the patient’s serum at 4°C to remove the autoanti- steps are time-consuming. There is an alternative method,
bodies before antibody detection steps are performed. in which PEG is used to enhance antibody removal, cutting
This method provides autoantibody-free serum for anti- the processing time approximately in half while still pro-
body detection and identification procedures and for viding for adequate autoantibody removal.50,51
compatibility testing. If the autoantibody is particularly 4. The pattern of reactivity in the “absorbed serum” column
strong or if the patient has been recently transfused, RESt of Figure 9–14 matches that of anti-K. Positive reactions
may be used to perform the adsorption instead of the in the autoabsorbed serum were found with cells 2, 6, and
patient’s RBCs. RESt-adsorbed serum is not suitable for 10, which were positive for the K antigen. All other
crossmatch, as anti-B may be removed. antibody specificities could be ruled out except for Cw,
Sulfhydryl compounds, such as DTT and 2-mercap- Kpa, Jsa, and Lua. These are low-prevalence antigens and
toethanol (2-ME), are known to break the disulfide bonds generally do not need to be excluded. Additional pheno-
in IgM. Treating the patient’s serum with such a reagent typing strategies should be employed to determine if the
before the antibody detection test will denature the cold patient is negative for the K antigen.
autoantibody. IgG antibodies are not affected by these 5. Transfusion requirements include RBC units negative for
reagents and will remain detectable. A control of saline the K antigen that appear to be less incompatible with the
and serum is tested in parallel with the treated serum to warm autoantibody than the patient’s own cells (least
ensure that the autoantibody was truly denatured, not incompatible) when tested with the antiglobulin cross-
merely diluted. match method using unabsorbed serum.
Review Questions
1. d 3. d 5. a 7. c Chapter 14
2. a 4. c 6. d 8. d
Case 14-1
1. Top five possible diagnoses for this case:
Case 16-3
1. Laboratory workup for the diagnosis of TAS must rule Chapter 18
out hemolysis and perform a gram stain and culture
Review Questions
of the implicated component and patient. The culture
must be obtained from the container and not the seg- 1. a 8. b 15. a 22. c
ments attached to the product. Blood cultures from the 2. b 9. d 16. c 23. a
patient should be drawn from a site other than the site of 3. d 10. c 17. a 24. d
transfusion. 4. a 11. c 18. d 25. b
2. Symptoms implicated with TAS include a fever with 5. c 12. d 19. c
greater than 2°C increase in body temperature, rigors, and 6. b 13. a 20. d
hypotension. 7. c 14. d 21. b
Review Questions
1.
2.
c
c
5.
6.
c
b
9.
10.
d
d
13. b
14. d
Chapter 19
3. b 7. c 11. d 15. d Case 19-1
4. b 8. b 12. a 1. The most probable cause is that the mother received
prophylactic Rh immune globulin at 28 weeks gestation.
The antibody screen was negative at 28 weeks gestation
Chapter 17 prior to the administration of Rh immune globulin. The
antibody titer of anti-D due to Rh immune globulin is
Case 17-1 typically less than 1:4.4
2. Yes, unless the newborn infant is Rh(D)-negative.
1. The blood group for RBC transfusion is O, which is the
only blood group compatible with both the donor and Case 19-2
recipient. If A is chosen, the cells will be hemolyzed
1. The maternal history, the same father, and the father
or have a shortened life span because of the preexisting
homozygous for the D antigen suggest that the infant is
anti-A in the patient. If B is chosen, engraftment will be
Rh(D)-positive.
difficult to determine.
2. The titer should be repeated in 6 weeks (16 weeks’
2. Group AB should be selected for FFP and platelet trans-
gestation).
fusion. If group A is selected, the anti-B in the plasma
3. The infant is only 8 weeks gestational age, so it is too
is incompatible with the recipient’s RBC and tissue
early in the pregnancy to perform any intervention
antigens (B). The anti-A in group B products is incom-
(MCA-PSV, cordocentesis, intrauterine transfusion). The
patible with the donor RBCs and, if selected, may contribute
earliest an intervention can take place is 16 weeks.
to delayed engraftment of RBC precursors.
4. The patient should be scheduled for MCA-PSV at 16 to
3. On day 117, the interpretation is B, as B cells and anti-A
18 weeks gestation to determine if the test is positive.
are present.
4. On day 200, the interpretation is group A, because A cells Case 19-3
are present, and B cells and anti-A are absent. On day 156,
1. The most probable cause is ABO HDFN. Group O mothers
the forward type is group O (remember that the patient
produce IgG anti-A, which can cross the placenta.
is receiving group O RBC transfusions), but the reverse
2. No. Anti-Lea does not cause HDFN because Lewis anti-
type still has anti-A; thus, the interpretation is indetermi-
gens are poorly developed at birth.
nate. The source of the anti-A may be small amount of
plasma in RBC transfusions or anti-A in ABO-incompatible Case 19-4
platelet transfusions.
1. The blood types of the cord blood and heel-stick speci-
Review Questions mens are different—A-negative versus O-negative. The
severely anemic infant could have HDFN, although the
1. a 4. b 7. c 10. a cord blood results do not indicate severe disease (DAT +/–).
2. e 5. c 8. c 11. b On the other hand, a large fetomaternal hemorrhage
3. d 6. d 9. c 12. c could have occurred.
2682_Answer Key_601-612 22/05/12 5:40 PM Page 608
the guidelines are based should be widely disseminated were unwilling to change their practice patterns. Following
and readily available to clinical staff. review by the transfusion committee, the antiquated order
5. Any of the types of review (retrospective, concurrent, sets were removed and a discontinuous prospective review
targeted, or discontinuous prospective) may be utilized was adopted, focusing on plasma orders for coronary
depending on the facility. However, for a medium- or care patients. In addition, educational measures were
small-sized facility, retrospective review is most likely to employed, in the form of several short lectures on current
be the primary method, due to resource limitations. Large plasma guidelines presented to the coronary unit’s physi-
facilities such as academic institutions are more likely to cians and nursing staff.
have the available personnel to conduct concurrent and 4. Over the following three quarters, the transfusion safety
prospective review actions. Targeted or discontinuous officer continued the discontinuous prospective review
prospective review may be appropriate for smaller facilities, of the coronary care patients in addition to the monthly
since the benefits of “real-time” intervention are balanced retrospective reporting. At the end of the third quarter,
by the limited scope. blood utilization appeared to be at the desired state and
the labor-intensive discontinuous prospective review
Case Study 24-2 strategy was retired. It is important to always reassess and
1. The first step is to determine the gap between the current revaluate the blood utilization management process to
and desired state. This gap defines the scope of the prob- determine the effectiveness of corrective strategies and to
lem and should be qualified and quantified as succinctly identify new areas of waste.
as possible. Review of the value stream will help identify
the source of the problem. In this case, review of the nurs- Review Questions
ing units and physician services show that the plasma 1. d 4. c 7. b 10. b
trend appears to be related to a recent expansion in the 2. a 5. d 8. d
hospital’s coronary care unit. This is further supported by 3. b 6. b 9. a
the observation that the increased usage and waste trends
vanish when the data from the coronary unit are excluded
from the utilization report. Review of the timing of the
blood orders showed that the majority of the increased
utilization appeared just prior to the performance of Chapter 25
cardiac procedures and surgeries. Review Questions
2. Further auditing revealed multiple types of waste with
plasma usage among patients awaiting cardiac surgery 1. b 4. d 7. d 10. b
and procedures. In nearly every case, there was an excess 2. c 5. c 8. a
of plasma ordered in advance of the procedure that was 3. d 6. a 9. b
thawed by the blood bank but was either never dispensed
or never transfused. Regarding the dispensed plasma,
most of these patients had an average of 2 units of unused
thawed plasma. In a few cases, the product was improp-
erly returned and had to be discarded. This overproduc-
Chapter 26
tion type of waste increased the number of expired Review Questions
plasma components, resulting in increased inventory
waste. Further waste was identified through a detailed 1. c 4. b 7. d 10. c
medical chart audit, which demonstrated that nearly 2. a 5. a 8. a
half of the plasma transfused was likely inappropriately 3. d 6. b 9. b
administered when compared to the institution’s transfu- 11. Phase SCI SCII Interpretation
sion guidelines. IS 0 0 Negative
3. Solutions and improvements arise from the collaborative 37ºC 0 0
input of multiple sources such as the medical director AHG 0 0
of the blood bank, the blood bank supervisor, members CC + +
of the transfusion committee, and appropriate members
from administration. If a specific unit or specialty is 12. b
identifiable, as in this case, input should also be requested 13. A. Control functions
from clinical staff in that area. In this case, after investi- B. Acceptance Criteria
gation by the medical director, the new coronary unit was C. Test Cases
employing antiquated order sets copied from another D. Data entry methods
institution. These order sets included template standing E. Documentation methods
plasma orders that were not appropriate for a large insti- F. Result review
tution with readily available thawed plasma. Unfortunately, G. Corrective action
in spite of discussion with the involved practitioners, many H. Acceptance
2682_Answer Key_601-612 22/05/12 5:40 PM Page 611
Chapter 27
2. The supplier’s facility and all intermediaries must be
registered with the FDA and hold state licenses, if appli-
cable by state.
Cases 27-1 through 27-6
Editor’s Note: Because of the nature of these cases, an- Case 28-2
swers are not provided. Answers to these questions would 1. See Box 28–2.
be decided by a judge or jury. 2. The hospital tissue bank must register with the FDA as a
tissue distributer.
Review Questions 3. A decision should be made as to whether the tissue should
1. d be cultured and if so, with which method. The storage con-
2. b tainer and appropriate temperature for storage should also
3. d be decided. (Any of the AORN recommendations for harvest
4. c of autologous tissue are correct answers to this question.)
5. a
Review Questions
1. d 4. b 7. d 10. d
Chapter 28 2. b
3. c
5. b
6. c
8. b
9. d
Case 28-1
1. This tissue manufacturer must be qualified by the medical
director of the tissue bank prior to ordering any tissue.
The surgeon may consider postponing surgeries until
the vendor has been qualified or use tissue from a vendor
currently qualified.
2682_Answer Key_601-612 22/05/12 5:40 PM Page 612
2682_Glossary_613-636 22/05/12 12:10 PM Page 613
Glossary
Abruptio placentae Premature detachment of normally Agglutinogen A substance that stimulates the production
situated placenta. of an agglutinin, thereby acting as an antigen.
Absorbed anti-A1 If serum from a group B individual that Agranulocytosis An acute disease in which the white
contains anti-A plus anti-A1 is incubated with A2 cells, blood cell count drops to extremely low levels, and
the anti-A will adsorb onto the cells. Removal of the neutropenia becomes pronounced.
cells yields a serum containing only anti-A1; thus, it is Albumin Protein found in the highest concentration in
absorbed anti-A1. human plasma; used as a diluent for blood typing anti-
Absorption Removal of an unwanted antibody. sera and a potentiator solution in serologic testing to
Acid-citrate-dextrose (ACD) An anticoagulant and preser- enhance antigen-antibody reactions.
vative solution that was once used routinely for blood Aldomet See Methyldopa.
donor collection but now used only occasionally. Alkaline phosphatase (ALP) A red blood cell enzyme
Acid phosphatase (ACP) A red blood cell enzyme used used as an identification marker in paternity testing
as an identification marker in paternity testing and and criminal investigation.
criminal investigation. Allele One of two or more different genes that may
Adenosine deaminase (ADA) A red blood cell enzyme occupy a specific locus on a chromosome.
used as an identification marker in paternity testing Allo- Prefix indicating differences within a species (e.g.,
and criminal investigation. an alloantibody is produced in one individual against
Adenosine triphosphate (ATP) A compound composed of the red blood cell antigens of another individual).
adenosine (nucleotide containing adenine and ribose) Allogeneic Transplant donor who is related or unrelated
and three phosphoric acid groups, which, when split by to the recipient.
enzyme action, produces energy that can be used to
Allograft A tissue transplant between individuals of the
support other reactions.
same species.
Adenylate kinase (AK) A red blood cell enzyme used as
Allosteric change A change in conformation that exposes
an identification marker in paternity testing and crimi-
a new reactive site on a molecule.
nal investigation.
Alpha-adrenergic receptor A site in autonomic nerve
Adjuvant One of a variety of substances that, when com-
pathways wherein excitatory responses occur when
bined with an antigen, enhance the antibody response
adrenergic agents such as norepinephrine and epineph-
to that antigen.
rine are released.
Adsorption Providing an antibody with its corresponding
Alum precipitation A method for obtaining an enhanced
antigen under optimal conditions so that the antibody
response when producing an antibody; see also Adjuvant.
will attach to the antigen, thereby removing the anti-
body from the serum; often used interchangeably with Aminoacyl-tRNA synthetase An enzyme involved in
absorption. protein synthesis by attaching a specific amino acid to
transfer RNA (tRNA), based on a three-nucleotide se-
Agammaglobulinemia A rare disorder in which gamma
quence present in the loop area of the tRNA called anti-
globulin is virtually absent.
codon. Once the amino acid is attached to the tRNA, it
Agarose Seaweed extract used to make gels used in elec- may be joined in the ribosome with the growing peptide
trophoresis. Molten agarose (0.5% to 3.5%, depending chain.
on application) is poured into a mold where a plastic
Amniocentesis Transabdominal puncture of the amniotic
comb is suspended. As it cools, the agarose hardens to
sac, using a needle and syringe, in order to remove am-
form a porous gel slab.
niotic fluid. The material may then be studied to detect
Agglutination The clumping together of red blood cells or genetic disorders or fetomaternal blood incompatibility.
any particulate matter resulting from interaction of anti-
Amniotic fluid Liquid or albuminous fluid contained in
body and its corresponding antigen.
the amnion.
Agglutinin An antibody that agglutinates cells.
613
2682_Glossary_613-636 22/05/12 12:10 PM Page 614
614 Glossary
Amorph A gene that does not appear to produce a Anti-dl An antibody implicated in warm autoimmune
detectable antigen; a silent gene, such as Jk, Lu, O. hemolytic anemia, which reacts with all Rh cells,
Amplicon The product of polymerase chain reaction. including Rhnull and Rh-deleted cells.
Anamnestic response An accentuated antibody response Antigen A substance recognized by the body as being for-
following a secondary exposure to an antigen. Antibody eign, which can cause an immune response. In blood
levels from the initial exposure are not detectable in the banking, antigens are usually, but not exclusively, found
patient’s serum until the secondary exposures, when a on the red blood cell membrane.
rapid rise in antibody titer is observed. Antihemophilic factor See Hemophilia A.
Anaphylaxis An allergic hypersensitivity reaction of the Antihemophilic globulin See Hemophilia A.
body to a foreign protein or drug. Antihistamine Drug that opposes the action of histamine.
Anastomosis A connection between two blood vessels, Anti-H lectin A reagent anti-H produced from the seeds of
either direct or through connecting channels. the plant Ulex europaeus.
Anemia A condition in which there is reduced oxygen Antihuman globulin or antiglobulin serum See Antihu-
delivery to the tissues; may result from increased de- man serum.
struction of red blood cells, excessive blood loss, or
Antihuman globulin or antiglobulin test (AGT) Test to
decreased production of red blood cells.
ascertain the presence or absence of red blood cell coat-
Angina pectoris Severe pain and constriction about the ing by immunoglobulin G (IgG) or complement or
heart caused by an insufficient supply of blood to the both; uses a xenoantibody (rabbit antihuman serum)
heart. or monoclonal antibody (to IgG or complement) to act
Anion An ion carrying a negative charge. as a bridge between sensitized cells, thus yielding agglu-
Annealing Process in which single strands of DNA anneal tination as a positive result. Direct antihuman globulin
to each other by formation of hydrogen bonds, under test (DAT): Used to detect in vivo cell sensitization.
certain temperature conditions, due to complementarity. Indirect antihuman globulin test (IAT): Used to detect
Annealing, renaturation, and hybridization are chemi- antigen-antibody reactions that occur in vitro.
cally the same processes. Two strands of DNA, separated Antihuman serum An antibody prepared in rabbits or
by heat denaturation, will start annealing (renaturation) other suitable animals that is directed against human im-
upon cooling to approximately 5°C (or more) below munoglobulin or complement or both; used to perform
DNA melting point (Tm). This phenomenon is used the antihuman globulin or Coombs’ test. The serum may
in polymerase chain reaction when oligonucleotide be either polyspecific (anti-IgG plus anticomplement) or
primers must anneal to denatured DNA prior to starting monospecific (anti-IgG or anticomplement).
DNA synthesis by the enzyme polymerase. Anti-M lectin A reagent anti-M serum produced from the
Antecubital In front of the elbow, at the bend of the plant Iberis amara.
elbow; usual site for blood collection. Anti-nl An antibody implicated in warm autoimmune
Antenatal Occurring before birth. hemolytic anemia, which reacts with all normal Rh cells
Anti-A1 lectin A reagent anti-A1 serum produced from the except Rhnull cells and deleted Rh cells.
seeds of the plant Dolichos biflorus; reacts with A1 cells Anti-N lectin A reagent anti-N serum produced from the
but not with A subgroup cells, such as A2, A3, and so plant Vicia graminea.
on; reacts weakly with Aint cells. Anti-pdl An antibody implicated in warm autoimmune
Anti-B lectin A reagent anti-B serum produced from the hemolytic anemia, which reacts with all normal Rh cells
seeds of the plant Bandeiraea simplicifolia. and deleted Rh cells but not with Rhnull cells.
Antibody A protein substance secreted by plasma cells Antipyretic An agent that reduces fever.
that is developed in response to, and interacting specifi- Antiserum A reagent source of antibody, as in a commer-
cally with, an antigen. In blood banking, it is found in cial antiserum.
serum, from either a commercial manufacturer or a
Antithetical Referring to antigens that are the product of
patient.
allelic genes (e.g., Kell [K] and Cellano [k]).
Antibody screen Testing the patient’s serum with group O
Apheresis A method of blood collection in which whole
reagent red blood cells in an effort to detect atypical
blood is withdrawn, a desired component separated and
antibodies.
retained, and the remainder of the blood returned to the
Anticoagulant An agent that prevents or delays blood donor. See also Plateletpheresis and Plasmapheresis.
coagulation.
Aplasia Failure of an organ or tissue to develop normally.
Anticodon A sequence of three nucleotides in the loop
Aplastic anemia Anemia caused by aplasia of bone
region of the transfer RNA (tRNA), which aligns in the
marrow or bone marrow’s destruction by chemical
ribosome with the corresponding mRNA codon and
agents or physical factors.
attaches the amino acid to the growing peptide chain.
2682_Glossary_613-636 22/05/12 12:10 PM Page 615
Glossary 615
Apoptotic Adjective of apoptosis, the programmed (non- Bandeiraea simplicifolia See Anti-B lectin.
traumatic) cell death. A natural process occurring in Bar-code reader An optical input device that reads and
senescent cells. interprets data from a bar code for entry into a computer
Arachis hypogaea A peanut lectin used to differentiate system.
T polyagglutination from Tn polyagglutination. Beta globin chain A structural peptide subunit of hemo-
Asphyxia Condition caused by insufficient intake of globin molecule that consists of two beta globin and two
oxygen. alpha globin chains. Alpha and beta chains are coded for
Asthma Paroxysmal dyspnea accompanied by wheezing by different genes located on different chromosomes.
caused by a spasm of the bronchial tubes or by swelling Bilirubin The orange-yellow pigment in bile carried to the
of their mucous membrane. liver by the blood; produced from hemoglobin of red
Atypical antibodies Any antibody other than anti-A, anti-B, blood cells by reticuloendothelial cells in bone marrow,
or anti-A,B. spleen, and elsewhere. Direct bilirubin: The conjugated
water-soluble form of bilirubin. Indirect bilirubin: The
Australia antigen Old terminology referring to the hepatitis
unconjugated water-insoluble form of bilirubin.
B–associated antigen.
Bilirubinemia Pathological condition in which excessive
Auto- Prefix indicating self (e.g., an autoantibody is reac-
destruction of red blood cells occurs, increasing the
tive against one’s own red blood cell antigens); usually
amount of bilirubin found in the blood.
associated with a disease state.
Binding constant The “goodness of fit” in an antigen-
Autoabsorption A procedure to remove a patient’s anti-
antibody complex.
body, using the patient’s own cells.
Biphasic Reactivity occurring in two phases.
Autoimmune hemolytic anemia Shortened RBC survival
mediated through the immune response of humoral anti- Blood bank information system Computer system that
body directed at “self” antigenic determinants. Acquired has been developed specifically to assist blood bank
disorder characterized by premature erythrocyte destruc- professionals in the management of the patient, donor,
tion owing to abnormalities in the individual’s immune and blood component information.
system. Blood gases Determination of pH, PCO2, PO2, and HCO3;
Autologous Donor and recipient are the same person. performed on a blood gas analyzer.
Autologous control Testing the patient’s serum with his Blood group genotyping DNA typing, aimed at genes
or her own cells in an effort to detect autoantibody coding for blood group antigens.
activity. Blood group–specific substances (BGSSs) Soluble
Autologous transfusion blood taken from a patient to be antigens present in fluids that can be used to neutralize
used for the same patient. their corresponding antibodies; systems that demon-
strate BGSSs include ABO, Lewis, and P blood group
Autoradiography An image recorded on a photographic
systems.
film or plate produced by the radiation emitted from
a specimen, such as DNA labeled with radioactive Blood order sets A means of standardization to clarify in-
phosphorus isotope. dications and enforce guidelines during physician order
entry.
Autosomal-recessive pattern An inheritance pattern in
which a condition or disease determined by a gene Blood utilization management A process incorporating
located on an autosomal (nonsex) chromosome occurs blood utilization review with particular intervention
when both genes on the homologous chromosomes are strategies to reduce inappropriate transfusion and
affected. Individuals with only one gene affected are improve patient safety.
considered carriers and do not have symptoms due to Blood utilization review A focused audit of a population
proper function of the unaffected gene on the other or subpopulation of transfused patients to determine
chromosome. the appropriateness of transfusion.
Autosome Any chromosome other than the sex (X and Y) Blunt ends The ends of a DNA sequence resulting from
chromosomes. restriction enzymes that cut both strands in the middle
Bacterial artificial chromosomes (BACs) In recombinant of the target sequence, leaving no “overhangs.” Blunt
DNA technology, vectors capable of carrying DNA ends may be rejoined using enzyme ligase.
fragments (inserts) of up to 350 kb. Bombay Phenotype occurring in individuals who possess
Bactericidal Destructive to or destroying bacteria. normal A or B genes but are unable to express them
because they lack the gene necessary for production
Bacteriophage A virus that infects and reproduces in
of H antigen, the required precursor for A and B. These
bacterial cells. Often carries genes coding for antibiotic
persons often have a potent anti-H in their serum,
resistance. Basic tools used in molecular biology (vec-
which reacts with all cells except other Bombays. Also
tors) frequently used to introduce and clone (multiply)
known as Oh.
foreign genes or their fragments inside of a bacterium.
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616 Glossary
Glossary 617
Citrate-phosphate-dextrose-adenine (CPDA-1) The anti- bonds between the nitrogen containing bases in the
coagulant preservative solution in current use. It has nucleotides on the opposite strands of the double helix.
extended the shelf life of blood from 21 days (ACD Due to this process, adenine (A) always pairs by two
and CPD) to 35 days. hydrogen bonds with thymine (T) or uracil (U), and
Clone A group of genetically identical cells. guanine (G) always pairs by three hydrogen bonds
with cytosine (C).
Codominant A pair of genes in which neither is dominant
over the other—that is, they are both expressed. Complementary DNA (cDNA) An in vitro enzymatically
synthesized DNA from an RNA template. A product of
Codon A sequence of three nucleotides (bases) in the
reverse transcription.
strand of DNA that provides the genetic code for specific
amino acid. The complementary triplets are found in the Complement fixation (CF) An immunologic test.
messenger RNA (mRNA), which is synthesized based on Component therapy Transfusion of specific components
the DNA template in the nucleus, and then proceeds to (e.g., red blood cells, platelets, plasma) rather than
the ribosomes for protein synthesis, where the transfer whole blood to treat a patient. Components are sepa-
RNA (tRNA), upon alignment with the mRNA, attaches rable by physical means such as centrifugation.
the corresponding amino acid to the growing peptide Compound antibody An antibody whose corresponding
chain. antigen is an interaction product of two or more
Colloid A gluelike substance, such as protein or starch, antigens.
whose particles, when dispersed in a solvent to the Compound antigen Two or more antigens that interact
greatest possible degree, remain uniformly distributed and are recognized as a single antigen by an antibody.
and fail to form a true solution.
Computer crossmatch Comparison of recent serologic
Colony-forming unit committed to erythropoiesis (CFU-E) results and interpretations on computer file for both the
A progenitor cell that is committed to forming cells of donor and the recipient being matched, establishing
the red blood cell series. compatibility based on the comparison.
Colony-forming unit—culture (CFU-C) Generation of Concurrent review A blood utilization review that occurs
stem cells using tissue culture methods. Current syn- shortly after transfusion events.
onym is CFU-GM, which is a colony-forming unit com-
Configuration The physical layout and design of the
mitted to the production of myeloid cells (granulocytes
central processing unit and the peripheral devices of an
and monocytes).
information system.
Colostrum Thin yellowish breast fluid secreted 2 to 3 days
Conglutinin A substance present in bovine serum that
after birth but before the onset of true lactation; it con-
will agglutinate sensitized cells in the presence of
tains a great quantity of proteins and calories as well as
complement.
antibodies and lymphocytes.
Constant region The portion of the immunoglobulin
Compatibility test A series of tests to determine if a par-
chain that shows a relatively constant amino acid se-
ticular blood component is appropriate for transfusion
quence within each class of immunoglobulin. Both
to a particular patient. This series of tests usually in-
light and heavy chains have these constant portions,
cludes the ABO/Rh of the component and the intended
which originate at the carboxyl region of the molecule.
recipient, antibody detection of the recipient, and a
crossmatch (serologic or electronic) of the recipient’s Convulsion Involuntary muscle contraction and relaxation.
serum/plasma with the RBCs of the donor unit. Coombs’ serum See Antihuman serum.
Competent cells Cells (usually various strains of E. coli) Coombs’ test See Antihuman globulin test (AGT).
that possess easily altered cell membrane and readily Cord cells Fetal cells obtained from the umbilical cord at
incorporate foreign DNA (are prone to transformation). birth; may be contaminated with Wharton’s jelly.
Competence may be achieved by exposure to calcium
Cordocentesis Umbilical cord blood sampling performed
chloride and heat shock.
by ultrasound-guided needle insertion through the
Complement A series of proteins in the circulation that, uterine wall.
when sequentially activated, causes disruption of
Cosmids Vectors that can accept DNA 28 to 45 kb long.
bacterial and other cell membranes. Activation occurs
They have a small region of bacteriophage lambda (λ)
via one of two pathways; once activated, the compo-
necessary for packaging viral DNA into λ particles.
nents are involved in a great number of immune
They are useful for producing large-insert genomic
defense mechanisms, including anaphylaxis, chemo-
libraries.
taxis, and phagocytosis. Red blood cell antibodies
that activate complement may be capable of causing Coumarin (Coumadin) A commonly employed anticoagu-
hemolysis. lant that acts as a vitamin K antagonist to prolong pro-
thrombin time.
Complementarity A phenomenon occurring in nucleic
acids, resulting from formation of noncovalent hydrogen Counterelectrophoresis (CEP) An immunologic procedure.
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618 Glossary
Crossmatch The testing of the patient’s serum with the Deletion The loss of a portion of chromosome.
donor red blood cells, including an antiglobulin phase Denaturation In molecular biology, a term that describes
or simply an immediate spin phase to confirm ABO separation of two DNA strands upon thermal or chemi-
compatibility. cal treatment, which destroys the noncovalent hydrogen
Cross-reacting antibody Antibody that reacts with anti- bonds between the complementary nitrogen-containing
gens functionally similar to its specific antigen. bases. Thermal denaturation is reversible (see renatura-
Cryoprecipitate A concentrated source of coagulation fac- tion). The temperature at which a given double-
tor VIII prepared from a single unit of donor blood; it stranded DNA denatures is proportional to the number
also contains fibrinogen, factor XIII, and von Willebrand’s of hydrogen bonds in the molecule and increases with
factor. the increase of G-C pair content. In reference to DNA,
denaturation is a chemical or thermal process of break-
Cryopreservation Preservation by freezing at very low
ing hydrogen bonds between the opposite nucleotides
temperatures.
in the double helix, resulting in reversible separation
Cryoprotectant A substance that protects blood cells from of the two strands. Denaturation is the first step of
damage caused by freezing and thawing. Glycerol and polymerase chain reaction (PCR).
dimethyl sulfoxide are examples.
Deoxyribonucleic acid (DNA) The chemical basis of
Cryptantigens Hidden receptors that may be exposed heredity and the carrier of genetic information for all
when normal erythrocyte membranes are altered by organisms, except RNA viruses. Structured as a double
bacterial or viral enzymes. helix of polymers of nucleotides, each consisting of one
Crystalloid A substance capable of crystallization; oppo- of the nitrogen-containing bases (A, T, C, and G), sugar
site of colloid. deoxyribose, and a phosphate.
Cyanosis Slightly bluish or grayish skin discoloration Deoxyribonucleoside triphosphates (dNTPs) Chemical
resulting from accumulations of reduced hemoglobin compounds (nucleotides) used during DNA synthesis.
or deoxyhemoglobin in the blood caused by oxygen They consist of one of the nitrogen-containing bases
deficiency or carbon dioxide buildup. (A, T, C, and G), sugar deoxyribose, and three
Cycle sequencing A variation of Sanger DNA sequencing phosphates. They become monophosphates once
conducted in a thermocycler and during the elongation they are built into the DNA molecule.
(extension) step of a modified polymerase chain reaction. Dexamethasone A topical steroid with anti-inflammatory,
Cytapheresis A procedure performed using a machine by antipruritic, and vasoconstrictive actions.
which one can selectively remove a particular cell type Dextran A plasma expander that may be used as a substi-
normally found in peripheral blood of a patient or tute for plasma; can be used to treat shock by increasing
donor. blood volume. Rouleaux may be observed in the recipi-
Cytokines A family of signaling molecules, such as inter- ent’s serum or plasma.
ferons or interleukins, secreted by certain cell types and Diagnosis-related group (DRG) Classification system that
triggering various physiological processes by interacting organizes short-term, general hospital inpatients into
with receptors located either on cell membranes or statistically stable groups based on age and illness.
inside of cells. Diaphoresis Profuse sweating.
Cytomegalovirus (CMV) One of a group of species-specific Diastolic pressure The point of least pressure in the arte-
herpesviruses. rial vascular system; the lower or bottom value of a
Cytotoxicity Ability to destroy cells. blood pressure reading.
Cytotoxicity testing Procedure commonly used in HLA Dideoxyribonucleoside triphosphates (ddNTPs) Deoxyri-
typing and crossmatching. bonucleoside triphosphates deprived of an oxygen atom
Dane particle Hepatitis B virion. located on the third sugar in the deoxyribose molecule.
Lack of this oxygen prevents polymerization of nu-
Database An organized group of files in which informa-
cleotides during DNA synthesis. Used as terminators of
tion is stored in an information system.
DNA synthesis in the process of DNA sequencing.
Degenerate In molecular biology, the quality of the ge-
Dielectric constant A measure of the electrical conductiv-
netic code that refers to each amino acid being encoded
ity of a suspending medium.
by more than one nucleotide triplet (codon) in messen-
ger RNA (mRNA) corresponding to a triplet in DNA. Differential count Counting 100 leukocytes to ascertain
Degeneracy is also called redundancy of the genetic the relative percentages of each.
code. For example, the amino acid leucine is coded by 2,3-Diphosphoglycerate (2,3-DPG) An organic phosphate
UUA, UUG, CUU, CUC, CUA, or CUG codons. in red blood cells that alters the affinity of hemoglobin
Deglycerolization Removal of glycerol from a unit of red for oxygen. Blood cells stored in a blood bank lose
blood cells after thawing has been performed; required 2,3-DPG, but once infused, the substance is resynthe-
to return the cells to a normal osmolality. sized or reactivated.
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Glossary 619
Diploid Having two sets of 23 chromosomes, for a total Domain Portions along the immunoglobulin chain that
of 46. show specific biological function.
Direct antiglobulin test (DAT) Test that detects in vivo Dominant A trait or characteristic that will be expressed
coating of an individual’s red blood cells with IgG or in the offspring even though it is carried on only one of
complement (C3). the homologous chromosomes.
Direct transfusion Transfer of blood directly from one Donath-Landsteiner test A test usually performed in
person to another. the blood bank to detect the presence of the Donath-
Discontinuous prospective review A temporary, focused Landsteiner antibody, which is a biphasic immunoglob-
prospective blood utilization review strategy that is per- ulin G antibody with anti-P specificity found in patients
formed on a combination of particular product types, suffering from paroxysmal cold hemoglobinuria.
DRGs, clinical services, or individual clinicians that can Donor An individual who donates a pint of blood.
be employed in areas of increased inappropriate blood Dopamine A catecholamine synthesized by the adrenal
utilization. gland, used especially in the treatment of shock.
Disk drive A hardware device in an information system Dosage A phenomenon whereby an antibody reacts more
that contains a disk on which data are stored; provides strongly with a red blood cell carrying a double dose
for quick access to storing or retrieving data. (homozygous inheritance of the appropriate gene) than
Disseminated intravascular coagulation (DIC) Clinical with a red blood cell carrying a single dose (heterozy-
condition of altered blood coagulation secondary to a gous inheritance) of an antigen.
variety of diseases. Dot blotting In molecular biology, a technique in which
Dithiothreitol (DTT) A sulfhydryl compound used to dis- the nucleic acid extracted from specimen is immobi-
rupt the disulfide bonds of immunoglobulin M, yielding lized on a membrane (blot) in a form of a dot. This
monomeric units rather than the typical pentameric procedure does not require electrophoresis like other
molecule. blotting techniques (Southern, Northern, or Western).
Diuresis Secretion and passage of large amounts of urine. The membrane is subsequently incubated in a solution
with labeled probes, complementary to sequences, ex-
Diuretic An agent that increases the secretion of urine, ei-
pected to be present in the tested specimen. Microarrays
ther by increasing glomerular filtration or by decreasing
are an example of a modified (reversed) dot blotting
reabsorption from the tubules.
technique.
Dizygotic twins Twins who are the product of two fertil-
Drug-induced immune hemolytic anemia Shortened RBC
ized ova (also called fraternal twins).
survival caused by an antibody to a therapeutic agent
DMSO Dimethyl sulfoxide, a cryoprotectant used for (e.g., antibiotic) that has become bound to the RBC
hematopoietic progenitor cells. membrane or that has altered the RBC membrane.
DNA See Deoxyribonucleic acid. Dyscrasia An old term now used as a synonym for disease.
DNA fingerprinting DNA typing of multiple loci simulta- Ecchymosis A form of macula appearing in large irregu-
neously in order to establish a genetic profile unique larly formed hemorrhagic areas of the skin; first blue-
for every individual. A method initially developed by black, then changing to greenish brown or yellow.
Alex Jeffreys and performed using restriction enzymes
Edema A local or generalized condition in which the body
and DNA probes complementary to short repetitive
tissues contain an excessive amount of tissue fluid.
sequences. Later, improved by the polymerase chain
reaction (PCR). Electrolyte A substance that in solution conducts an electric
current; common electrolytes are acids, bases, and salts.
DNA libraries Collections of clones (cells produced in
molecular cloning) that contain all genomic or mRNA Electrophoresis The movement of charged particles
sequences of a particular cell type. through a medium (paper, agar gel) in the presence of
an electrical field; useful in the separation and analysis
DNA polymerase An enzyme that catalyzes the template—
of proteins.
dependent on synthesis of DNA. Also known as the
HBeAg of the hepatitis B virion. Eluate See Elution.
DNA polymerase III Bacterial enzyme synthesizing a new Elution A process whereby cells that are coated with anti-
DNA strand using deoxyribonucleoside triphosphates body are treated in such a manner as to disrupt the
(dNTPs) as substrates, double-stranded DNA as tem- bonds between the antigen and antibody. The freed anti-
plate, and magnesium as an enzyme cofactor. body is collected in an inert diluent such as saline or 6%
albumin. This antibody serum then can be tested to
DNA typing Also called genetic profiling, this is a process
identify its specificity using routine methods. The mech-
in which it is established which polymorphic version or
anism to free the antibody may be physical (heating,
variant of a gene (allele, locus) is present in an individ-
shaking) or chemical (ether, acid), and the harvested
ual. Typing of multiple loci is called DNA fingerprinting.
antibody-containing fluid is called an eluate.
Dolichos biflorus See Anti-A1 lectin.
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620 Glossary
Embolism Obstruction of a blood vessel by foreign Exchange transfusion Transfusion and withdrawal of
substances or a blood clot. small amounts of blood, repeated until blood volume
Embolus A mass of undissolved matter present in a blood is almost entirely exchanged; used in infants born with
or lymphatic vessel, brought there by the blood or hemolytic disease.
lymph circulation. Exclusion An exclusion occurs when a child has inherited
Endemic A disease that occurs continuously in a particu- a genetic marker not detected in an alleged parent.
lar population but has a low mortality rate; used in Exclusions can be direct or indirect, which are distin-
contrast to epidemic. guished in the following way: A direct exclusion refers
to the presence of a genetic marker in the child that is
Endogenous Produced or arising from within a cell or
not detectable in the alleged parent. An indirect exclu-
organism.
sion occurs when the alleged parent has a marker that
Endothelium A form of squamous epithelium consisting should be transmitted to all his or her children, but the
of flat cells that line the blood and lymphatic vessels, child in question lacks that genetic marker. Indirect
the heart, and various other body cavities; derived from exclusions can result when mutations occur during
mesoderm. meiosis or when the gene transmitted to the child is
Endotoxemia The presence of endotoxin in the blood; silent or “null.”
endotoxin is present in the cells of certain bacteria Exogenous Originating outside an organ or part.
(e.g., gram-negative organisms).
Exon The coding part of a gene in eukaryotes. Only this
Engraftment The successful establishment, proliferation, part of the gene is translated into protein product. In
and differentiation of transplanted hematopoietic stem genomic DNA, exons are separated by introns.
cells.
Expression library A DNA library generated in a vector
Enzyme A substance capable of catalyzing a reaction; pro- that not only replicates itself, but also drives protein
teins that induce chemical changes in other substances synthesis in E. coli (an expression vector).
without being changed themselves.
Extracorporeal Outside of the body.
Enzyme-linked immunosorbent assay (ELISA) An
Extravascular Outside of the blood vessel.
immunologic test.
Factor assay Coagulation procedure to assay the concen-
Enzyme treatment A procedure in which red blood cells
tration of specific plasma coagulation factors.
are incubated with an enzyme solution that cleaves some
of the membrane’s glycoproteins, then washed free of the Factor VIII concentrate A commercially prepared source
enzyme and used in serologic testing. Enzyme treatment of coagulation factor VIII.
cleaves some antigens and exposes others. Febrile reaction A transfusion reaction caused by
Epistaxis Hemorrhage from the nose; nosebleed. leukoagglutinin that is characterized by fever; usually
observed in multiply transfused or multiparous patients.
Epitope The portion of the antigen molecule that is
directly involved in the interaction with the antibody; Fetal and neonatal alloimmune thrombocytopenia
the antigenic determinant. (FNAIT) A rare condition that occurs when a mother
becomes sensitized to a foreign antigen of paternal
Equivalence zone The zone in which antigen and anti-
origin that is present on fetal thrombocytes (platelets).
body concentrations are optimal and lattice formation is
These platelet antigens provoke production of maternal
most stable.
antibodies that cross the placenta and destroy fetal
Erythroblast A precursor form of nucleated red blood cell platelets.
that is not normally seen in the circulating blood.
Fibrin A whitish filamentous protein or clot formed by
Erythroblastosis fetalis See Hemolytic disease of the fetus the action of thrombin on fibrinogen, converting it to
and newborn (HDFN). fibrin.
Erythrocyte The blood cell that transports oxygen and Fibrinogen A protein produced in the liver that circulates
carbon dioxide; a mature red blood cell. in plasma. In the presence of thrombin, an enzyme pro-
Ethidium bromide (EB) A fluorescent dye intercalating duced by the activation of the clotting mechanism,
between the grooves of the double-stranded DNA. Used fibrinogen is cleaved into fibrin, which is an insoluble
for staining of DNA in electrophoretic gels for UV visu- protein that is responsible for clot formation.
alization. Mutagenic and carcinogenic is being replaced Fibrinolysin The substance that has the ability to dissolve
by safer SYBR green dye. fibrin; also called plasmin.
Ethylenediaminetetraacetic acid (EDTA) An anticoagu- Fibrinolysis Dissolution of fibrin by fibrinolysin, caused
lant useful in hematologic testing and preferable when by the action of a proteolytic enzyme system that is con-
direct antihuman globulin testing is indicated. tinually active in the body but that is increased greatly
Euglobulin lysis Coagulation procedure testing for by various stress stimuli.
fibrinolysin.
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Glossary 621
Fibroblast Cells found throughout the body that synthe- Gamete A mature male or female reproductive cell.
size connective tissue. Gamma globulin A protein found in plasma and known to
Ficin A proteolytic enzyme derived from the fig. be involved in immunity.
Ficoll A macromolecular additive that enhances the agglu- Gamma marker Allotypic marker on the gamma heavy
tination of red blood cells. chain of the IgG immunoglobulin.
Ficoll-Hypaque A density-gradient medium used to sepa- Gel test A blood group serology test method that uses a
rate and harvest specific white blood cells, most com- microtube containing gel (with or without antisera or
monly lymphocytes. antiglobulin sera) that acts as a reaction vessel for
Fluorescent antibody Antibody reaction made visible by agglutination.
incorporating a fluorescent dye into the antigen-antibody Gene A unit of inheritance within a chromosome.
reaction and examining the specimen with a fluorescent Gene chips Microarrays.
microscope.
Gene expression A term generally describing the
Fluorescent in situ hybridization (FISH) A technique in processes of transcription and translation. A gene that is
molecular biology in which fluorescent DNA probes are not transcribed (not expressed) is considered “silent.”
used to detect homologous DNA or RNA sequences in
Gene therapy An introduction of new genetic material
preserved chromosomes or intact cells.
into the cells of an organism for therapeutic purposes.
Fluorescent resonance energy transfer (FRET) probes A
Genetic code A key according to which a sequence of sets
type of DNA probe used in real-time PCR. Instead of a
of three DNA nucleotides within a gene is transcribed
quencher, attached to the TaqMan probes or molecular
into a sequence of sets of three nucleotides within
beacons, in the FRET system the fluorescence “donor”
mRNA (codon) and then translated into amino acids,
and “acceptor” are attached to two separate hybridiza-
the building blocks of a peptide or protein.
tion probes designed to bind to adjacent DNA se-
quences within the amplified region. Until there is not Genetic transformation Process of foreign DNA uptake by
any PCR product (amplicon) present in the tube, these a cell, usually enforced by use of chemicals, electricity,
probes may not get close, as there is nothing for them to or liposomes that enable penetration of cell membrane.
bind to. Once the PCR product starts to accumulate in Genotype An individual’s actual genetic makeup.
the tube, the probes bind to the sequences within the Gestation In mammals, the length of time from concep-
product in close proximity and the energy from the tion to birth.
donor is transferred to the reporter, which results in
Globin A protein constituent of hemoglobin. There are
emission of light that may now be detected.
four globin chains in the hemoglobin molecule.
Formaldehyde A disinfectant solution.
Glomerulonephritis A form of nephritis in which the
Forward grouping Testing unknown red blood cells with lesions involve primarily the glomeruli.
known reagent antisera to determine which ABO anti-
Glutamic pyruvate transaminase A liver enzyme used to
gens are present.
monitor liver function; also called serum glutamic pyru-
Frameshift mutation A change in which a message is vate transaminase (SGPT) or alanine transferase (ALT).
read incorrectly either because a base is missing or an
Gluten enteropathy A condition associated with malab-
extra base is added, which results in an entirely new
sorption of food from the intestinal tract.
polypeptide because the triplet sequence has been
shifted one base. Glycerol A cryoprotective agent.
Fresh frozen plasma (FFP) A frozen plasma product Glycerolization Adding glycerol to a unit of red blood
(from a single donor) that contains all clotting factors, cells for the purpose of freezing.
especially the labile factors V and VIII; useful for clot- Glycine soja Soybean extract or lectin used to differentiate
ting factor deficiencies other than hemophilia A, different forms of polyagglutination.
von Willebrand’s disease, and hypofibrinogenemia. Glycophorin A A major glycoprotein of the red blood cell
Freund’s adjuvant Mixture of killed microorganisms, usu- membrane. MN antigen activity is found on it.
ally mycobacteria, in an oil-and-water emulsion. The Glycophorin B An important red blood cell glycoprotein:
material is administered to induce antibody formation SsU antigen activity is found here.
and yields a much greater antibody response.
Glycoprotein A protein with linked carbohydrates.
Furosemide (Lasix) An oral diuretic.
Glycosyl transferase A protein enzyme that promotes the
G-CSF Granulocyte-colony stimulating factor, filgrastim. attachment of a specific sugar molecule to a predeter-
GM-CSF Granulocyte macrophage–colony stimulating mined acceptor molecule. Many blood group genes code
factor, sargramostim. for transferases, which reproduce their respective
G6PD (glucose-6-phosphate dehydrogenase) A red cell antigens by attaching sugars to designated precursor
enzyme involved in the glycolytic pathway. substances.
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622 Glossary
Goodpasture’s syndrome A disease entity that represents a Hemodialysis Removal of chemical substances from the
rapidly progressive glomerulonephritis associated with blood by passing it through tubes made of semiperme-
pulmonary lesions. Usually the patients possess an anti- able membranes that are continually bathed by solu-
body to the basement membrane of the renal glomeruli. tions that selectively remove unwanted material; used
Graft-versus-host (GVH) disease A disorder in which the to cleanse the blood of patients in whom one or both
grafted tissue attacks the host tissue. kidneys are defective or absent and to remove excess
accumulation of drugs or toxic chemicals in the blood.
Granulocytopenia Abnormal reduction of granulocytes in
the blood. Hemodilution An increase in the volume of blood plasma,
resulting in reduced relative concentration of red blood
Griffith’s transformation A famous genetic experiment
cells.
performed in 1928 by Frederick Griffith using two strains
of Streptococcus pneumoniae. Griffith demonstrated that a Hemoglobin The iron-conjugated protein in the red blood
heat-resistant “transforming principle” from one strain cells whose function is to carry oxygen from the lungs
could change the virulence of another strain. This was to the tissues. This protein contains heme plus globin.
the first event that eventually led to discovery of DNA as Hemoglobinemia Presence of hemoglobin in the blood
carrying the genetic information on molecules. plasma.
Hageman’s factor Synonym for coagulation factor XII. Hemoglobin-oxygen dissociation curve The relationship
Half-life The time that is required for the concentration of between the percent saturation of the hemoglobin mole-
a substance to be reduced by one half. cule with oxygen and the environmental oxygen tension.
Haploid Possessing half the normal number of chromo- Hemoglobinuria The presence of hemoglobin in the urine
somes found in somatic or body cells; seen in germ cells freed from lysed red blood cells, which occurs when
(sperm and ova). hemoglobin from disintegrating red blood cells or from
rapid hemolysis of red blood cells exceeds the ability of
Haplotype A term used in HLA testing to denote the five
the blood proteins to combine with the hemoglobin.
genes (HLA-A, HLA-B, HLA-C, HLA-D, HLA-DR) on the
same chromosome. Hemolysin An antibody that activates complement, lead-
ing to cell lysis.
Haptene The portion of an antigen containing the group-
ing on which the specificity depends. Hemolysis Disruption of the red blood cell membrane and
the subsequent release of hemoglobin into the suspend-
Haptoglobin A mucoprotein to which hemoglobin re-
ing medium or plasma.
leased into plasma is bound; it is increased in certain
inflammatory conditions and decreased in hemolytic Hemolytic anemia Anemia caused by hemolysis of red
disorders. blood cells, resulting in reduction of normal red blood
cell life span.
Hardware Components of an information computer sys-
tem that are the tangible, physical pieces of equipment, Hemolytic disease of the fetus and newborn (HDFN) A
such as the central processing unit, cathode ray tube, disease, characterized by anemia, jaundice, enlargement
and keyboard. of the liver and spleen, and generalized edema (hydrops
fetalis), that is caused by maternal IgG antibodies
HBcAg Hepatitis core antigen, referring to the nucleocap-
crossing the placenta and attacking fetal red blood
sid of the virion.
cells when there is a fetomaternal blood group incom-
HBeAg Hepatitis DNA polymerase of the nucleus of the patibility (usually ABO or Rh antibodies). Synonym is
virion. erythroblastosis fetalis.
HBsAg Hepatitis B surface antigen. Hemolytic transfusion reaction (HTR) A reaction
Hemangioma A benign tumor of dilated blood vessels. from red blood cell destruction caused by patient’s anti-
Hemarthrosis Bloody effusion into the cavity of a joint. body(ies) directed to donor red blood cell antigen(s).
Hematinic Pertaining to blood; an agent that increases the Hemophilia A A hereditary disorder characterized by
amount of hemoglobin in the blood. greatly prolonged coagulation time (↑PTT). The blood
fails to clot and bleeding occurs; caused by inheritance
Hematocrit The proportion of red blood cells in whole
of a factor VIII deficiency, it occurs almost exclusively
blood, expressed as a percentage.
in males.
Hematoma A swelling or mass of blood confined to an
Hemophilia B “Christmas disease,” which is a hemophilia-
organ, tissue, or space and caused by a break in a blood
like disease caused by a lack of factor IX.
vessel.
Hemopoiesis Formation of blood cells. Synonym is
Hematopoietic progenitor cell Stem cells that are commit-
hematopoiesis.
ted to produce blood cells.
Hemorrhage Abnormal internal or external bleeding; may
Hematuria Blood in the urine.
be venous, arterial, or capillary; from blood vessels into
Heme The iron-containing protoporphyrin portion of the the tissues or out of the body.
hemoglobin wherein the iron is in the ferrous (Fe2+) state.
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Glossary 623
Hemorrhagic diathesis Uncontrolled spontaneous Hybrid gene A gene that results from combining two
bleeding. different genes.
Hemosiderin An iron-containing pigment derived from Hybridization Formation of hydrogen bonds between
hemoglobin from disintegration of red blood cells; a complementary strands of nucleic acids to form either
method of storing iron until it is needed for making DNA:DNA or DNA:RNA hybrids. Chemically, a process
hemoglobin. identical with renaturation or annealing. Nucleic acid
Hemostasis Arrest of bleeding; maintaining blood flow hybridization is a fundamental tool in molecular genet-
within vessels by repairing rapidly any vascular break ics that takes advantage of the ability of individual
without compromising the fluidity of the blood. single-stranded nucleic acid molecules to form double-
stranded molecules—that is, to hybridize to each other.
Hemotherapy Blood transfusion as a therapeutic measure.
The interacting single-stranded molecules must have
Heparin An anticoagulant used for collecting whole blood a sufficiently high degree of base complementarity.
that is to be filtered for the removal of leukocytes. Standard nucleic acid hybridization assays involve
Hepatitis Inflammation of the liver. using a labeled nucleic acid probe to identify related
Hepatitis-associated antigen (HAA) Older terminology DNA or RNA molecules—ones with a significantly
currently replaced by HBsAg. high degree of sequence similarity—within a complex
mixture of unlabeled nucleic acid molecules, the target
Hepatitis B immunoglobulin (HBIg) An immune serum
nucleic acid.
given to individuals exposed to the hepatitis B virus.
Hybridization protection (HPA) A molecular detection
Hereditary spherocytosis An inherited anemia character-
technology, in which ssDNA probes labeled with chemi-
ized by fragile, spherical RBCs prone to hemolysis. The
luminescent molecules are added to form hybrids with
condition occurs due to mutations within genes coding
the amplified RNA molecules produced during tran-
for cytoskeleton proteins like spectrin, ankyrin, or others.
scription mediated amplification (TMA). Light is emit-
Heterogenous RNA (hnRNA) See pre-mRNA. ted upon hybridization of the probes to the RNA and
Heterozygote An individual with different alleles on a captured by a luminometer.
gene for a given characteristic. Hybridoma A hybrid (cross) between a plasmacytoma cell
Heterozygous Possessing different alleles at a given gene and a spleen (or Ab-producing) cell that produces a
locus. monoclonal antibody, resulting in a cell line that can
High-prevalence antigen Also known as high-incidence grow indefinitely in culture and can produce high quan-
antigen; antigen whose frequency in the population is tities of Ab. This antibody is monoclonal because only
98% to 99%. one Ab-producing cell combined with the plasmacy-
toma cell is present.
High protein anti-D Reagent anti-D consisting of human
source IgG anti-D with potentiators (bovine albumin Hydatid cyst fluid Source of P1 substance.
and macromolecular additives) to enhance antibody Hydrocortisone A corticosteroid with anti-inflammatory
reactivity on direct testing. properties.
Histocompatibility The ability of cells to survive without Hydrogen bonds Noncovalent, heat-labile chemical bonds
immunologic interference; especially important in blood that occur between hydrogen atom and either nitrogen
transfusion and transplantation. or oxygen atom of the adjacent molecule. In DNA,
HLA Human leukocyte antigen. hydrogen bonding holds together the two strands of
double helix by formation of two bonds between ade-
Homeostasis State of equilibrium of the internal environ-
nine and thymine and three bonds between cytosine
ment of the body that is maintained by dynamic
and guanine.
processes of feedback and regulation.
Hydrops fetalis See Hemolytic disease of the fetus and
Homozygote An individual developing from gametes with
newborn (HDFN).
similar alleles and thus possessing like pairs of genes for
a given hereditary characteristic. Hydroxyethyl starch (HES) A red blood cell sedimenting
agent used to facilitate leukocyte withdrawal during
Homozygous Possessing a pair of identical alleles.
leukapheresis.
Hormone A substance originating in an organ or gland
Hypertension Increase in blood pressure.
that is conveyed through the blood to another part of
the body, chemically stimulating it to increase func- Hyperventilation Rapid breathing that results in carbon
tional activity and increase secretion. dioxide depletion and that accompanies hypotension,
vasoconstriction, and fainting.
Hoshin kanri In Lean, a total quality management princi-
ple to prioritize the elimination of waste via policy Hypogammaglobulinemia Decreased levels of gamma
deployment and control. globulins seen in some disease states.
Hyaluronidase An enzyme found in the testes; present in Hypotension Decrease in blood pressure.
semen. Hypothermia Having a body temperature below normal.
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624 Glossary
Hypovolemia Diminished blood volume. in almost every immune response during the early
Hypoxia Deficiency of oxygen. period of the reaction.
Iberis amara See Anti-M lectin. Immunologic memory The development of T and B mem-
ory cells that have been sensitized by exposure to an
Icterus A condition characterized by yellowish skin,
antigen and that respond rapidly under subsequent
whites of the eyes, mucous membranes, and body fluids
encounters with the antigen.
caused by increased circulating bilirubin resulting from
excessive hemolysis or from liver damage due to hepati- Immunologic unresponsiveness Development of a toler-
tis. Synonym is jaundice. ance to certain antigens that would otherwise evoke an
immune response.
Idiopathic Pertaining to conditions without clear patho-
genesis, or disease without recognizable cause, as of Immunoprecipitin An antigen-antibody reaction that
spontaneous origin. results in precipitation.
Idiopathic thrombocytopenic purpura (ITP) Bleeding Inclusion The opposite of an exclusion, in which all of
owing to a decreased number of platelets; the etiology the genetic information inherited by the child from the
is unknown, with most evidence pointing to platelet parent whose identity is in question, is present in the
auto-antibodies. alleged parent.
Idiopathic thrombocythemia An increase in blood Incubation In vitro combination of antigen and antibody
platelets of unknown etiology. under certain conditions of time and temperature to
allow antigen-antibody complexes to occur.
Idiotype The portion of the immunoglobulin variable
region that is the antigen-combining site, which inter- Indirect transfusion Transfusion of blood from a donor to
acts with the antigenic epitope. a suitable storage container and then to a patient.
Immune response The reactions of the body to substances Initiation The deposition of N-formylmethionine on the
that are foreign or are interpreted as being foreign. ribosome, which begins the synthesis of all proteins.
Cell-mediated or cellular immunity pertains to tissue In Lu A rare dominant gene that inhibits the production
destruction mediated by T cells, such as graft rejection of all Lutheran antigens as well as i, P1, and Aua
and hypersensitivity reactions. Humoral immunity (Auberger). The quantity of antigen on the red blood
pertains to cell destruction response during the early cell is markedly reduced in the presence of In Lu; it
period of the reaction. may be virtually undetectable.
Immune serum globulin Gamma globulin protein fraction Interface Software that allows a computer system to send
of serum-containing antibodies. data to or receive data from another computer system.
Immunoblast A mitotically active T or B cell. Intergenic recombination Recombination of genetic mate-
Immunoblotting A technique for detecting proteins rial in noncoding regions of DNA.
extracted from cells, separated by polyacrylamide gel Intracellular Occurs within a cell.
electrophoresis, transferred from the gel onto a filter Intraoperative salvage A procedure to reclaim a patient’s
membrane and then incubated with a specific labeled blood loss from an operation by reinfusion.
antibody.
Intrauterine transfusion Transfusion of blood into a fetus
Immunodeficiency A decrease from the normal concentra- in utero.
tion of immunoglobulins in serum.
Intravascular Within the blood vessel.
Immunodominant sugar In reference to glycoprotein or
Intron A noncoding part of the gene. This fragment of the
glycolipid antigens, the sugar molecule that gives the
gene is transcribed into RNA but is excised out during
antigen its specificity (e.g., galactose, which confers
the process of splicing so it is not translated into a pro-
B antigen specificity).
tein product.
Immunogen Any substance capable of stimulating an
In utero Within the uterus.
immune response.
Inversion The breaking of a chromosome during division,
Immunogenicity The ability of an antigen to stimulate an
with subsequent reattachment occurring in an inverted
antibody response.
or upside-down position.
Immunoglobulin (Ig) One of a family of closely related
In vitro Outside the living body, as in a laboratory setting.
though not identical proteins that are capable of acting
as antibodies: IgA, IgD, IgE, IgG, and IgM. IgA is the In vivo Inside the living body.
principal immunoglobulin in exocrine secretions such Ion exchange resin Synthetic organic substances of high
as saliva and tears. IgD may play a role in antigen recog- molecular weight. They replace certain positive or nega-
nition and the initiation of antibody synthesis. IgE, tive ions, which they encounter in solutions.
produced by the cells lining the intestinal and respira- Ionic strength Refers to the number of charged particles
tory tracts, is important in forming reagin. IgG is the present in a solution.
main immunoglobulin in human serum. IgM is formed
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Glossary 625
Ir genes Immune response genes found within the region infect E. coli in order to produce multiple copies of the
of the major histocompatibility complex. Ir genes in fragment.
humans are likely to exist; preliminary evidence shows Lean A systematic approach for identifying actions within
genes at the D-related locus may be analogous to the a process that create value, then optimizing the align-
Ir genes of mice. ment of these actions to the customer.
Iron-deficiency anemia Anemia resulting from a greater Lectin Proteins present in plants (usually seeds), which
demand on stored iron than can be met. bind specifically to carbohydrate determinants and
Irradiation Gamma or electron treatment of a cellular agglutinate erythrocytes through their cell surface of
blood product for protection against graft-versus-host oligosaccharide determinants.
disease. Leukemia Malignant proliferation of leukocytes, which
Ischemia Local and temporary deficiency of blood supply spill into the blood, yielding an elevated leukocyte count.
caused by obstruction of the circulation to a cell, tissue, Leukoagglutinin Antibodies to white blood cells.
or organ.
Ligases A class of enzymes that catalyze the linkage of two
Isoagglutinins The ABO antibodies anti-A, anti-B, and molecules, generally utilizing ATP as the energy donor.
anti-A,B. In recombinant DNA technology, these are enzymes
Isoimmune An antibody produced against a foreign anti- linking the adjacent nucleotides between two DNA frag-
gen in the same species. ments being joined after restriction enzyme digestion.
Isotype The subclasses of an immunoglobulin molecule. Ligature Process of binding or tying; a band or bandage; a
Jaundice See Icterus. thread or wire for tying a blood vessel or other structure
in order to constrict it.
Kaizen In Lean, a continuous improvement process
required for long-term success. LightCycler A special type of thermocycler with built-in
fluorescence detector, designed for real-time PCR
Karyotype A photomicrograph of a single cell in the
applications.
metaphase stage of mitosis that is arranged to show the
chromosomes in descending order of size. Linkage The association between distinct genes that oc-
cupy closely situated loci on the same chromosome, re-
kb (or kbp) A measure of DNA length. One thousand
sulting in an association in the inheritance of these
(“kilo”) nucleotides (or “bases” for simplification) in
genes.
single-stranded DNA. In double-stranded DNA, this is
referred to as one thousand base pairs. Linkage disequilibrium Genes associated in a haplotype
more often than would be expected on the basis of
Kernicterus A form of icterus neonatorum occurring in
chance alone.
infants, developing at 2 to 8 days of life; prognosis poor
if untreated. This condition is due to an increase in Locus The site of a gene on a chromosome.
unconjugated bilirubin. Low ionic-polycation test A compatibility test that incor-
Kinin A group of polypeptides that have considerable bio- porates both glycine (low ionic) and protamine (polyca-
logical activity (e.g., vasoactivity). tion) in an effort to obtain maximal sensitivity and to
minimize the need for antibody screening.
Kleihauer-Betke technique Quantitative procedure used
to determine the amount of fetal cells present in the ma- Low ionic strength solution (LISS) A type of potentiating
ternal circulation. medium in use for serologic testing. Reducing the ionic
strength of the red blood cell–suspending medium in-
Km Light chain marker on the kappa light chains of IgG
creases the affinity of the antigen for its corresponding
(formerly known as InV).
antibody such that sensitivity can be increased and
Knockout transgenic animal A model to study the conse- incubation time decreased. LISS contains glycine or
quences of lack of function of a gene that is blocked, or glucose in addition to saline.
“knocked out” (e.g., by RNA interference) and introduced
Low melting point agarose Agarose specially formulated
into the egg using transgenic technology (a microinjection
to melt in temperatures much lower than normal, which
of external/modified genetic material). The egg, upon im-
allows for easy dissolving of the gel in order to isolate
plantation into a surrogate mother, develops into geneti-
and purify DNA that was run in it.
cally modified organism missing the desired structural or
functional protein. Low-prevalence antigen Also known as low-incidence anti-
gen; antigen whose frequency in a random population is
Labile Capable of deteriorating rapidly upon storage.
very low—less than 10%.
Lambda (λ) vectors Vectors that contain the part of the
Luminometer An instrument that detects and quantifies
bacterial virus Lambda genome necessary for lytic
the amount of light emitted as a result of chemical reac-
replication in E. coli and one or more restriction sites
tion (chemiluminescence).
for insertion of the DNA fragment of interest. They can
carry foreign DNA 5 to 14 kb long. The recombinant Lymphocyte A type of white blood cell involved in the
DNA is “packaged” into viral particles and used to immune response. Lymphocytes normally total 20% to
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626 Glossary
45% of total white blood cells. T lymphocytes mature Melting point (Tm) The temperature at which half of all
during passage through the thymus or after interaction hydrogen bonds in the double-stranded DNA molecule
with thymic hormones; these cells function both in are broken, while the other half are still intact. It deter-
cellular and humoral immunity. Subsets include helper mines how much energy is needed to keep the strands
T-cells (Th), which enhance B-cell antibody production, of the helix apart. As a general rule, temperatures above
and suppressor T-cells (Ts), which inhibit B-cell anti- the Tm promote the separation of the strands. Knowing
body production. B-lymphocyte cells are not processed the Tm is necessary for calculating temperatures for
by the thymus. Through morphologic and functional hybridization-based assays and for annealing primers in
differentiation, they mature into plasma cells that the polymerase chain reaction. The primers generally
secrete immunoglobulin. anneal to DNA at about 5°C (or more) below the Tm.
Lymphoma A solid tumor of lymphocyte cells. If the temperature is decreased more, they will start to
anneal to sequences with only partial complementarity,
Lysosomes Part of an intracellular digestive system that
which affects the specificity of the reaction. For short
exists as separate particles in the cell. Even though
DNA sequences, the simplified formula to calculate the
their importance in health and disease is certain, all
Tm is 2AT + 4GC, where AC is the total number of
the precise ways lysosomes effect changes are not
adenines and thymines, and GC is the total number of
understood.
guanines and cytosines in the molecule. Based on this
Macroglobulinemia Abnormal presence of high-molecular- formula, it is evident that GC-rich molecules will have
weight immunoglobulins (IgM) in the blood. higher Tm than AT-rich sequences, because there are
Macrophages End-stage development for the blood mono- always three hydrogen bonds between guanines and
cyte; these cells can ingest (phagocytose) a variety of cytosines, while there are only two bonds between
substances for subsequent digestion or storage and are adenines and thymines.
located in a number of sites in the body (e.g., spleen, Mendel’s laws The classical principles of inheritance
liver, lung), existing as free mobile cells or as fixed cells. defined by the Moravian monk Gregor Mendel in 1866.
Functions include elimination of senescent blood cells The first law of genetics (the law of segregation) stated
and participation in the immune response. that the hereditary characteristics are determined by par-
Major ABO incompatibility ABO antibody in the recipient ticulate units (presently called genes) that occur in an
that is incompatible with the donor. individual as pairs (diploid), but in the formation of
Major histocompatibility complex (MHC) Present in all germ cells/gametes they segregate so the gamete contains
mammalian and ovarian species; analogous to HLA only one member of the pair (haploid). The second law
complex. HLA antigens are within the MHC at a locus (the law of independent assortment) stated that the par-
on chromosome 6. ticulate units (genes) that determine different character-
istics are inherited independently of other units (now we
Malaria An acute and sometimes chronic infectious disease
know that if the genes are close to one another on the
caused by the presence of a parasite within red blood
chromosome, they will be inherited together).
cells. The parasite is Plasmodium (P. vivax, P. falciparum,
P. malariae, P. ovale), which is introduced through bites Menorrhagia Excessive menstrual bleeding, in number of
of infected female Anopheles mosquitoes or through days or amount of blood, or both.
blood transfusion. 2-Mercaptoethanol (2-ME) A sulfhydryl compound used
Maternal antibody Antibody produced in the mother and to disrupt the disulfide bonds of immunoglobulin M,
transferred to the fetus in utero. yielding monomeric units rather than the typical
pentameric units.
Megaloblastic anemia Anemia in which megaloblasts are
found in the blood. Messenger RNA (mRNA) See Ribonucleic acid.
Meiosis Type of cell division of germ cells in which two Metastasis Movement of bacteria or body cells, especially
successive divisions of the nucleus produce cells that cancer cells, from one part of the body to another;
contain half the number of chromosomes present in change in location of a disease or of its manifestations or
somatic cells. transfer from one organ or part of another not directly
connected. Spread is by the lymph or blood circulation.
Melting curve analysis A postamplification analysis of the
amplicon produced in the real-time PCR by slowly Methemoglobin An abnormal form of hemoglobin
increasing the temperature and monitoring the decrease wherein the ferrous (Fe2+) iron has been oxidized to
of fluorescence as a result of DNA strand separation ferric (Fe3+) iron.
from the probes used in the reaction. The fluorescent Methyldopa (Aldomet) Common drug used to treat hy-
probes detach from the PCR product at lower tempera- pertension; frequently the cause of a positive direct
tures if the product has a mutation. This method may Coombs’ test result.
be used to distinguish between homo- and heterozygos- Microaggregates Aggregates of platelets and leukocytes
ity underlying certain conditions (the presence of the that accumulate in stored blood.
mutation in one or both homologous chromosomes).
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Glossary 627
Microarrays (gene chips) Devices consisting of glass Mosaic An antigen composed of several subunits, such as
slides or membrane filters with fragments of DNA the Rh0(D) antigen. A mixture of characteristics that
printed by a robot in small spots at high density. These may result from a genetic crossover or mutation.
DNA fragments serve as multiple probes that will bind Multiparous Having borne more than one child.
(hybridize with) the complementary regions of DNA
Multiple myeloma A neoplastic proliferation of plasma
(or RNA) present in the analyzed specimen applied to
cells, which is characterized by very high immunoglob-
the chip. The chips are scanned with a laser to identify
ulin levels of monoclonal origin.
the regions detected by the probes.
Multiplex assay In molecular biology, refers primarily to
Microglobulin (β2) A protein synthesized by all nucleated
multiplex polymerase chain reaction (PCR). Multiplex
cell types; an integral part of the class I MHC antigens.
PCR is a concurrent amplification of several DNA frag-
Microsatellites DNA regions composed of back-back- ments from the same DNA target using several sets of
repeated units of 1-8 nucleotides (also called short primers that must be carefully designed so that they
tandem repeats; STRs). don’t interfere with each other.
Microspherocytes Red blood cells, small and spherical, in Mutation A change in a gene potentially capable of being
certain kinds of anemia (i.e., hereditary spherocytosis). transmitted to offspring.
Miniprep A rapid method for making a small preparation Myelofibrosis Replacement of bone marrow by fibrous
of purified plasmid DNA from 1 to 5 mL of bacterial tissue.
culture.
Myeloproliferative An autonomous, purposeless increase
Minisatellites DNA regions composed of back-to-back in the production of the myeloid cell elements of the
repeated units ranging in size from 9 to 80 bp (also bone marrow, which includes granulocytic, erythrocytic,
called VNTR; variable number of tandem repeats). and megakaryocytic cell lines as well as the stromal
Minor ABO incompatibility ABO antibody in the donor connective tissue.
that is incompatible with the recipient. N-acetylneuraminic acid (NANA) See Sialic acid.
Mitosis Type of cell division in which each daughter cell Naturally occurring antibody Antibody present in a pa-
contains the same number of chromosomes as the par- tient without known prior exposure to the correspon-
ent cell. All cells except sex cells undergo mitosis. ding red blood cell antigen.
Mixed-field agglutination A type of agglutination pattern Neonatal alloimmune neutropenia (NAN) A rare condi-
in which numerous small clumps of cells exist amid a tion that occurs when a mother becomes sensitized to
sea of free cells. This usually occurs when there is more a foreign antigen of paternal origin that is present on
than one population of RBCs present in the sample fetal granulocytes. These fetal granulocyte antigens pro-
(e.g., in a recently transfused individual). voke antibody production. The maternal IgG antibody
MLC Mixed lymphocyte culture. crosses the placenta and destroys fetal granulocytes.
MLR Mixed lymphocyte reaction. This neutropenia is typically self-limiting and lasts for
several weeks but can persist for as long as 6 months.
Modem Hardware device that provides the ability to attach
During this period, neonates are at high risk of develop-
to a computer system via telephone communication
ing infections.
lines.
Neonate A newborn infant up to 4 months of age.
Molecular beacons Fluorescent DNA probes structured
like a hairpin loop with reporter and quencher, used in Network Configuration of personal computers linked to-
real-time PCR. The emission of fluorescent light from gether with cables; allows all of the personal computers
the reporter, indicating the presence of an amplicon, to access common data and software located on a file
occurs upon unfolding of the loop structure, which server.
physically separates the quencher. Neuraminidase An enzyme that cleaves sialic acid from
Molecular cloning Reproduction of recombinant DNA the red blood cell membrane.
molecules in host cells that, due to universality of the Neutralization Inactivating an antibody by reacting it with
genetic code, replicate the foreign DNA along with its an antigen against which it is directed.
own. The daughter cells of a single cell carrying the Neutrophil A leukocyte that ingests bacteria and small
recombinant molecule propagate to produce a clone. particles and plays a role in combating infection.
Monoclonal Antibody derived from a single ancestral Nondisjunction Failure of a pair of chromosomes to sepa-
antibody-producing parent cell. rate during meiosis.
Monocytes See Macrophage. Nonresponder An individual whose immune system does
Monospecific antiglobulin reagent Antiserum specific for not respond well in antibody formation to antigenic
one type of immunoglobulin (e.g., anti-IgG). stimulation.
Monozygotic twins Two offspring that develop from a Normal serum albumin See Albumin.
single fertilized ovum.
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628 Glossary
Northern blotting A variation of Southern blotting where, Palindromic sequences Four to 8 nucleotide DNA se-
instead of DNA, RNA is run on the electrophoretic gel quences that read the same in the 5⬘ to 3⬘ direction on
and transferred onto a blot for detection of specific RNA both strands of the double helix—for example,
sequence. 5⬘AAATTT3⬘ or 5⬘GAATTC3⬘.
Nucleic acid testing (NAT) General term used to describe Pallor Paleness; lack of color.
diagnostic tests (predominately amplification tests: Panagglutinin An antibody capable of agglutinating all red
NAATs) based on detection of nucleic acid sequences blood cells tested, including the patient’s own cells.
unique for the organism (pathogen) in question.
Pancytopenia A reduction in all cellular elements of the
Nucleosides Compounds consisting of one of nitrogen blood, including red blood cells, white blood cells, and
containing bases (A, T or U, C and G) and sugar de- platelets.
oxyribose or ribose. Depending on the base (adenine,
Panel A large number of group O reagent red blood cells
thymine, uracil, cytosine, and guanine), the nucleosides
that are of known antigenic characterization and are
are named: adenosine, thymidine, uridine, cytidine,
used for antibody identification.
and guanosine. Upon phosphorylation, they become
nucleotides and are building blocks of nucleic acids: Papain A proteolytic enzyme derived from papaya.
DNA or RNA. Paragloboside The immediate precursor for the H and P
Nucleotides Compounds consisting of one of nitrogen antigens of the red blood cell.
containing bases (A, T or U, C and G), sugar deoxyri- Parentage testing The analysis of one or more genetic
bose or ribose, and one, two, or three phosphates. markers in a group of individuals thought to be related
Chemically, they are referred to as nucleoside phosphates. as parent and child. The group normally consists of a
Nucleotides are the building blocks of nucleic acids: known parent (often a mother), a child, and an alleged
DNA or RNA. parent (often an alleged father). The goal of the testing
Oh See Bombay. is to confirm or refute the alleged relatedness of the
tested individuals as parents and child.
Oligo-dT primers Universal primers starting cDNA syn-
thesis from messenger RNA (mRNA) in the process of Paroxysm A sudden, periodic attack or recurrence of
reverse transcription. These primers consist of several symptoms of a disease.
thymine nucleotides that are complementary to the Paroxysmal cold hemoglobinuria (PCH) A type of cold
poly-A tail of mRNA. These primers will not start cDNA autoimmune hemolytic anemia usually found in chil-
synthesis from rRNA or tRNA because these molecules dren suffering from viral infections in which a biphasic
are missing the poly-A tail. immunoglobulin G antibody can be demonstrated with
Oligonucleotide A short synthetic segment of DNA, ap- anti-P specificity. See also Donath-Landsteiner test.
proximately 20 nucleotides in length, used as a probe. Paroxysmal nocturnal hemoglobinuria (PNH) An intrin-
Oliguria Diminished amount of urine formation. sic defect in the red blood cell membrane, rendering it
more susceptible to hemolysins in an acid environment;
Opsonin A substance in serum that promotes immune ad-
characterized by hemoglobin in the urine following
herence and facilitates phagocytosis by the reticuloen-
periods of sleep.
dothelial system.
Partial D Red blood cells that are missing normal RhD
Origin of replication (Ori) In DNA, the sequence that sig-
epitopes, resulting in a qualitative difference in the
nals the beginning of replication. In plasmids, they
RhD protein. Individuals with partial D can make
drive the replication of the foreign DNA fragments
anti-D.
along with their own.
Passenger lymphocytes Donor lymphocytes in the trans-
Orthostatic Concerning an erect position.
planted organ or HPC (hereditary progenitor cell)
Osmolality The osmotic concentration of a solution deter- product.
mined by the ionic concentration of dissolved sub-
Paternity index Term that refers to a statement of “weight”
stances per unit of solvent.
concerning the probability a tested individual, who can-
Ouchterlony diffusion An immunologic procedure in not be excluded as the parent of the child, is the true
which antibody and antigen are placed in wells of a gel parent. The paternity index represents a likelihood ratio
medium plate and allowed to diffuse in order to visual- that compares two mutually exclusive hypotheses. The
ize the reaction by a precipitin line. numerator of the ratio reflects the probability the tested
Oxyhemoglobin The combined form of hemoglobin and alleged parent is the true parent of the child and the de-
oxygen. nominator reflects the probability someone random and
P50 The partial pressure of oxygen or oxygen tension at unrelated to the alleged parent is the true parent.
which the hemoglobin molecule is 50% saturated with Perfusion Supplying an organ or tissue with nutrients and
oxygen. oxygen by passing blood or another suitable fluid
PAGE Polyacrylamide gel electrophoresis. through it.
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Glossary 629
Perioral paresthesia Tingling around the mouth occasion- platelets adhere to each other and to the edges of the
ally experienced by apheresis donors, resulting from the injury, forming a “plug” that covers the area and
rapid return of citrated plasma, which contains citrate- initially stops the blood loss.
bound calcium and free citrate. Platelet concentrate Platelets prepared from a single unit
Peroxidase An enzyme that hastens the transfer of oxygen of whole blood or plasma and suspended in a specific
from peroxide to a tissue that requires oxygen; this volume of the original plasma; also known as random-
process is essential to intracellular respiration. donor platelets.
Phagocytosis Ingestion of microorganisms, other cells, Plateletpheresis A procedure using a machine to remove
and foreign particles by a phagocyte. only platelets from a donor or patient.
Phenotype The outward expression of genes (e.g., a blood Platelet refractoriness Failure to yield an increase in recip-
type). On blood cells, serologically demonstrable anti- ient’s platelet count on transfusion of suitably preserved
gens constitute the phenotype, except those sugar sites platelets. HLA alloimmunization is a common cause.
that are determined by transferases. Point mutation A change in a base in DNA that can lead
Phenylthiocarbamide (PTC) A chemical used in studying to a change in the amino acid incorporated into the
medical genetics to detect the presence of a marker polypeptide; identifiable by analysis of the amino
gene. About 70% of the population inherits the ability acid sequences of the original protein and its mutant
to taste PTC, which tastes bitter; the remaining 30% offspring.
finds PTC tasteless. The inheritance of this trait is due Polarity (3⬘ and 5⬘ ends) Also called DNA “directionality,”
to a single dominant gene of a pair. determines the direction of DNA replication and tran-
Phlebotomy The procedure used to draw blood from a scription. Results from the antiparallel way that the two
person. strands of nucleotides ran in opposite directions. The
Phosphoglyceromutase A red blood cell enzyme. labels 3⬘ and 5⬘ refer to the number assigned by conven-
tion to the deoxyribose’ carbon atom linked to either
Phototherapy Exposure to sunlight or artificial light for
hydroxyl or phosphate group in DNA molecule.
therapeutic purposes.
Polyacrylamide gel A polymer of acrylamide, used as a
Plasma The liquid portion of whole blood, containing
matrix for gel electrophoresis that provides better reso-
water, electrolytes, glucose, fats, proteins, and gases.
lution than agarose electrophoresis.
Plasma contains all the clotting factors necessary for
coagulation but in an inactive form. Once coagulation Polyagglutination A state in which an individual’s red
occurs, the fluid is converted to serum. blood cells are agglutinated by all sera, regardless of
blood type.
Plasma cell A B lymphocyte–derived cell that secretes
immunoglobulins or antibodies. Polyagglutinins Naturally occurring immunoglobulin anti-
bodies that are found in most normal human adult sera.
Plasmapheresis A procedure using a machine to remove
only plasma from a donor or patient. Poly-A tail A long chain of adenine nucleotides that is
added in the reaction of polyadenylation to a messenger
Plasma protein fraction (PPF) Also known as Plasmanate;
RNA (mRNA) molecule during RNA processing to in-
sterile pooled plasma stored as a fluid or freeze-dried
crease the stability of the molecule immediately after a
and used for volume replacement.
gene in a eukaryotic cell is transcribed.
Plasmid Bacterial circular genetic element, 2 to 4 kb long,
Polybrene A positively charged polymer that causes
that replicates independently from the chromosome.
normal red blood cells to aggregate spontaneously by
Used as vectors in recombinant DNA technology to
neutralizing the negative surface charge contributed by
carry up to 15 kb foreign DNA into host cells. A vast
sialic acid.
selection of plasmids are commercially available that are
useful for different purposes, such as DNA sequencing, Polyclonal Antibodies derived from more than one
protein expression in bacteria, and protein expression in antibody-producing parent cell.
mammalian cells. Polycythemia vera A chronic life-shortening myeloprolif-
Plasminogen A protein in many tissues and body fluids erative disorder involving all bone marrow elements,
important in preventing fibrin clot formation. characterized by an increase in red blood cell mass and
hemoglobin concentration.
Plasmodium See Malaria.
Polylinker In plasmid, a region with a series of recogni-
Plasmodium knowlesi A parasite that causes malaria in
tion sequences for different restriction endonucleases
monkeys.
that may be used to introduce a foreign DNA fragment
Platelet A round or oval disk, 2 to 4 µm in diameter, that cut out of its original source with the corresponding
is derived from the cytoplasm of the megakaryocyte, a enzymes.
large cell in the bone marrow. Platelets play an impor-
Polymer Combination of two or more molecules of the
tant role in blood coagulation, hemostasis, and blood
same substance.
thrombus formation. When a small vessel is injured,
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630 Glossary
Polymerase chain reaction (PCR) An in vitro method of are designed to anneal to complementary DNA se-
amplifying a specific fragment of DNA using ther- quences at the 3⬘ end of each strand of the DNA frag-
mostable DNA polymerase enzyme and short synthetic ment desired to be amplified and extended toward the
primers that attach to separated strands of DNA and are 5⬘ end. The forward primer anneals to the antisense
extended by the addition of deoxyribonucleotides in strand, and the reverse primer anneals to the sense
numerous cyclic changes of temperature. Each PCR strand.
cycle consists of denaturation, annealing, and extension Private antigen An antigenic characteristic of the red
(elongation). blood cell membrane that is unique to an individual or a
Polymorphism A genetic system that possesses numerous related family of individuals and therefore is not com-
allelic forms, such as a blood group system. monly found on all cells (usually less than 1% of the
Polyspecific Coombs’ sera A reagent that contains antihu- population).
man globulin sera against immunoglobulin G and C3d. Probability of exclusion (PE) Refers to the strength of the
Polyvinylpyrrolidone (PVP) A neutral polymeric sub- test battery to exclude a falsely accused alleged parent.
stance used to increase blood volume in patients Probability of parentage The probability of parentage is
with extensive blood loss; also used to enhance antigen- produced from the likelihood ratio (paternity index for
antibody reactions in vitro. example) using Bayesian statistical logic and reflects the
Portal hypertension Increased venous pressure in the por- level of conviction that the included alleged parent is
tal vein as a result of obstruction of the flow of blood the true parent of the child.
through the liver. Probe A fragment of DNA that is labeled and hybridized
Postpartum Occurring after childbirth. to diagnostic material to locate a complementary strand
of DNA.
Potentiator A substance that, when added to a serum and
cell mixture, will enhance antigen-antibody interactions. Prodrome A symptom indicative of an approaching disease.
Precipitation The formation of a visible complex (precipi- Propositus The initial individual whose condition led to
tate) in a medium containing soluble antigen (precip- investigation of a hereditary disorder or to a serologic
itinogen) and the corresponding antibody (precipitin). evaluation of family members. Feminine form is
proposita. Synonyms are proband and index case.
Precipitin An antibody formed in the blood serum of an
animal by the presence of a soluble antigen, usually a Prospective blood utilization review A blood utilization
protein. When added to a solution of the antigen, it review that occurs prior to transfusion events and is
brings about precipitation. The injected protein is the often used to determine the appropriateness of a trans-
antigen; the antibody produced is the precipitin. fusion order request.
Precursor substance A substance that is converted to an- Prospective validation Validation testing of software; done
other substance by the addition of a specific constituent before implementation of the computer system.
(e.g., a sugar residue). Prosthesis An artificial substitute for a missing part, such
Pre-mRNA Immature messenger RNA right after synthesis as an artificial extremity.
in the nucleus. Also called heterogeneous RNA Protamine A polycation with applications similar to those
(hnRNA) because it consists of sequences transcribed of polybrene.
from both: exons and introns. The introns (the noncod- Protamine sulfate A substance used to neutralize the
ing fragments) are removed during mRNA processing effects of heparin.
(maturation) by excision (splicing).
Prothrombin complex A concentrate of coagulation fac-
Preseroconversion window A period of time during tors II, VII, IX, and X in lyophilized form.
which a person may be infected with a viral pathogen
Provirus A DNA form of a retrovirus, upon integration
(HIV, HCV, HBV, etc.) but does not yet produce levels of
into the genome of the infected (host) cell.
antibodies detectable by serologic methods.
Prozone Incomplete lattice formation caused by an excess
Pretransfusion compatibility testing Series of testing pro-
of antibody molecules relative to the number of antigen
cedures and processes with the ultimate objective of
sites, resulting in false-negative reactions.
ensuring the best possible results of a blood transfusion,
including recipient and donor identification, ABO PRP Platelet-rich plasma.
testing, clerical checks, and so on. Public antigen An antigen characteristic of the red blood
Primer A short segment of single-stranded DNA, usually cell membrane found commonly among individuals,
17 to 25 nucleotides long, used to initiate DNA replica- usually more than 98% of the population.
tion in PCR. Pulmonary artery wedge pressure Pressure measured in
Primers (forward and reverse) Short, synthetic segments the pulmonary artery at its capillary end.
of single-stranded DNA, usually 15 to 25 nucleotides Pulse pressure The difference between the systolic and
long, used to initiate DNA replication in PCR. Primers the diastolic pressures.
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Glossary 631
Quality assurance (QA) A set of planned actions to pro- Relationship testing See Parentage testing.
vide confidence that systems and elements that influ- Renaturation See Annealing.
ence the quality of the product or service are working as
Replication The process of DNA synthesis based on the
expected, individually and collectively.
existing DNA molecule sequence (template) by enzyme
Radioimmunoassay (RIA) A very sensitive method for DNA polymerase, which recognizes the 3⬘ end of the
determining substances present in low concentrations template and starts adding the nucleotides, synthesizing
in serum or plasma by using specific antibodies and the new strand in the 5⬘ to 3⬘ direction (see polarity).
radioactively labeled or tagged substances. The place where the two original strands are separating
Random man not excluded (RMNE) Refers to the oppo- is called a replication fork—one strand is synthesized
site of probability of exclusion, which is the probability in a continuous manner and is called a leading strand,
that someone who is falsely accused of being the parent while the other strand’s synthesis occurs in short frag-
would not be excluded by a particular genetic test. The ments called Okazaki fragments, named after a Japanese
relationship between PE and RMNE is, PE + RMNE = 1. researcher. This strand is called a lagging strand because
Random primers (random hexamers) Six-nucleotide-long it is formed slightly slower due to the polymerase con-
sequences used as cDNA (complementary DNA) syn- stantly jumping toward the fork. DNA replication is said
thesis primers in the process of reverse transcription. to be semiconservative.
Random primers will attach to any RNA molecule Respiratory distress syndrome (RDS) A condition, for-
present in the reaction tube. The resulting cDNA repre- merly known as hyaline membrane disease, accounting
sents the total RNA isolated from the cell, not just for more than 25,000 infant deaths per year in the
mRNA, which is reverse transcribed using oligo-dT United States. Clinical signs, including delayed onset of
primers. respiration and low Apgar score, are usually present at
Rapid passive hemagglutination assay (RPHA) A third- birth.
generation procedure used in hepatitis testing. Restriction endonucleases Bacterial enzymes that cleave
Rapid passive latex assay (RPLA) A second-generation DNA at specific sequences and allow scientists to cut
procedure used in hepatitis testing. DNA in a controlled and predictable way.
Raynaud’s disease A peripheral vascular disorder charac- Restriction enzyme mapping Finding the sequences in a
terized by abnormal vasoconstriction of the extremities given DNA recognized by restriction enzymes and es-
upon exposure to cold or emotional stress. A history of tablishing the distance between such sites.
symptoms for at least 2 years is necessary for diagnosis. Restriction fragment length polymorphism (RFLP)
Real-time PCR A variation of polymerase chain reaction Refers to a specific molecular tool that can be used to
in which the product formed during each cycle of am- demonstrate nucleotide sequence polymorphisms in
plification is detected by fluorescence at the same time chromosomal DNA. A single nucleotide polymorphism
that it is produced, instead of being detected after the can alter the spatial arrangement of restriction enzyme
reaction is finished. In addition to primers, the real-time recognition sequences in chromosomal DNA that will
reaction mixture contains DNA probes complementary be revealed following restriction enzyme digestion as an
to the region between the primers (so called TaqMan altered pattern of restriction fragments.
or molecular beacons or FRET probes), labeled with flu- Reticulocyte Also known as neocyte, the last stage of
orophores that emit fluorescent light when binding to development before becoming a mature erythrocyte.
the newly synthesized amplicon. The reticulocyte has lost its nucleus but retains some
Recessive A type of gene that, in the presence of its domi- residual RNA in its cytoplasm, which is stainable by
nant allele, does not express itself; expression occurs special techniques. It may be slightly larger than the
when it is inherited in the homozygous state. mature red blood cell.
Recipient A patient who is receiving a transfusion of Reticuloendothelial system (RES) The fixed phagocytic
blood or a blood product. cells of the body, such as the macrophage, having the
ability to ingest particulate matter.
Recombinant DNA technology A process of recombining
two DNA fragments from different species and inserting Retrospective blood utilization review A blood utilization
such recombinant molecule into a host organism in review that occurs after the transfusion events.
order to produce new genetic combinations that are of Retrospective validation Validation testing of software,
value in medicine, science, and industry. which is done after the computer system has been im-
Recombinant proteins Proteins produced by translation of plemented.
recombinant genes (created by the recombinant DNA Retrovirus A virus in which the genetic material consists
technology). of two identical molecules of RNA that, upon entering
Refractory Obstinate; stubborn; resistant to ordinary treat- the infected cell, are converted into DNA by the enzyme
ment; resistant to stimulation (said of a muscle or reverse transcriptase to form a “provirus” that may be
nerve). integrated into the host’s own genomic DNA.
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632 Glossary
Reverse genetics Technology in which genes can be inac- Caenorhabditis elegans. Also referred to as gene silenc-
tivated (“knocked out”) in the germ line of a mouse or ing. Interfering RNAs prevent mRNA from being
other animal model in order to study systemic conse- translated into protein.
quences of lack of function of the protein coded by that RNase RNA endonuclease, a ubiquitous enzyme that
gene. The resulting model animal is a “knockout trans- destroys RNA.
genic animal.”
Rouleaux Coinlike stacking of red blood cells in the pres-
Reverse grouping Testing a patient’s serum with commer- ence of plasma expanders or abnormal plasma proteins.
cial or reagent A and B red blood cells to determine
Saline anti-D A low-protein (6% to 8% albumin) im-
which ABO antibodies are present.
munoglobulin M anti-D reagent.
Reverse transcription An enzymatic process, naturally
Salvia horminum Plant lectin used in the differentiation of
occurring in retroviruses, in which RNA sequence is
various forms of polyagglutination.
transcribed into the DNA sequence. In vitro reverse
transcription may be performed using isolated or syn- Salvia sclarea Plant lectin with anti-Tn activity, used in
thetic enzymes (reverse transcriptases) to obtain a com- the differentiation of various forms of polyagglutination.
plementary DNA (cDNA). Screening cells Group O reagent red blood cells that are
Rh immunoglobulin (RhIg) A concentrated, purified used in antibody detection or screening tests.
anti-Rh0(D) prepared from human serum (of immu- SD Serologically defined antigens.
nized donors) that is given to Rh0(D)-negative mothers SDS-PAGE An electrophoretic technique using a polyacry-
after they have given birth to an Rh0(D)-positive baby lamide gel (PAGE), in which proteins are denatured by
or after abortion or miscarriage. It acts to prevent the negatively charged detergent sodium dodecyl sulfate
the mother from becoming immunized to any (SDS), which masks their intrinsic electrical charge so
Rh0(D)-positive fetal cells that may have entered that they are separated according to size, not electrical
her circulation and thereby prevents formation of charge.
anti-Rh0(D) by the mother.
Secretor An individual who is capable of secreting soluble,
Rhmod Rare Rh phenotype resulting from mutations in the glycoprotein ABH-soluble substances into saliva and
RHAG gene, it can result in missing or significantly al- other body fluids.
tered (reduced) RhD and RhCE antigen expression.
Semiconservative Term referring to the DNA replication
Rh-negative Red blood cells lacking the D antigen. model (proved by Meselson and Stahl), in which each
Rhnull Rare Rh phenotype in which no Rh antigens are ex- double-stranded DNA molecule upon replication con-
pressed on the red blood cell; results from a mutation in tains one parental and one newly synthesized comple-
the RHAG gene (regulator-type Rhnull) or by a deletion mentary strand.
of the RHD gene (the amorphic type) and a mutation in Sensitization A condition of being made sensitive to a spe-
RHCE genes. cific substance (e.g., an antigen) after the initial exposure
Rh-positive Red blood cells possessing one particular Rh to that substance. This results in the development of im-
antigen, the D antigen. munologic memory that evokes an accentuated immune
Ribonucleic acid (RNA) A nucleic acid that controls pro- response with subsequent exposure to the substance.
tein synthesis in all living cells. There are three different Sepsis Pathological state, usually febrile, resulting from
types, and all are derived from the information encoded the presence of microorganisms or their toxins in the
in the DNA of the cell. Messenger RNA (mRNA) carries bloodstream.
the code for specific amino acid sequences from the Septicemia Presence of pathogenic bacteria in the blood.
DNA to the cytoplasm for protein synthesis. Transfer
Sequence-specific oligonucleotide probe (SSOP)
RNA (tRNA) carries the amino acid groups to the ribo-
hybridization A molecular method used in HLA
some for protein synthesis. Ribosomal RNA (rRNA),
testing, in which the individual’s DNA is immobilized
which exists within the ribosomes, is thought to assist
on a membrane as a dot-blot and incubated with a
in protein synthesis.
labeled complementary DNA probe designed to detect
Ribosome A cellular organelle that contains ribosomal specific HLA sequence.
RNA and protein and functions to synthesize protein.
Sequence-specific PCR (SSP) A polymerase chain reac-
Ribosomes may be single units or clusters called polyri-
tion method variant in which one of the primers is sup-
bosomes or polysomes.
posed to align exactly where the anticipated mutation is
Rickettsia Any of the microorganisms belonging to the located (sequence-specific primer).
genus Rickettsia.
Sequence-specific primers (1) In reverse transcription:
Ringer’s lactated injection An aqueous solution suitable sequences used as cDNA (complementary DNA) synthesis
for intravenous use. primers when only a specific region of cDNA is desired
RNA interference Gene-regulating enzymatic activity (not cDNA representing total RNA or messenger
of RNA molecules first discovered in nematode (mRNA). (2) In PCR: primers designed to anneal to the
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Glossary 633
specific mutated region of the DNA that is supposed to useful in genetic variation studies relevant to crime or
be amplified. In absence of mutation, the PCR product kinship investigations.
will either not be produced at all or will have a different Sodium dodecyl sulfate (SDS) An anionic detergent
length than the product resulting from the mutated DNA. that renders a net negative charge to substances it
Sequencing A manual or automated process of decipher- solubilizes.
ing the order of nucleotides in DNA to find various Software Written instructions for a computer, which result
polymorphisms (mutations) relevant in biotechnology in information being stored, manipulated, and retrieved.
or clinical applications.
Solid phase test A blood group serology test method that
Serologic test for syphilis (STS) First developed in 1906 uses red blood cell adherence on an endpoint instead of
by Wassermann, present tests are of three main types agglutination.
based on complement fixation, flocculation, and detec-
Somatic gene therapy Gene therapy in which the cells
tion of specific antitreponemal antibodies.
into which a new genetic material is introduced are the
Serotonin A chemical present in platelets that is a potent somatic cells (not the gametes) so that the material is
vasoconstrictor. not transmitted into the next generation.
Serum The fluid that remains after whole blood has Southern blotting Technique, developed by E. M. Southern,
clotted. in which DNA is cut with one or more restriction en-
Sex chromosome Chromosomes associated with determi- donucleases, the fragments are separated by agarose
nation of sex. gel electrophoresis, transferred onto a membrane
Sex linkage A genetic characteristic located on the X or (blot), and incubated with a labeled detection probe
Y chromosome. (a short fragment of DNA complementary to the
desired region).
Shelf life The amount of time blood or blood products
may be stored upon collection. Specificity The affinity of an antibody and the antigen
against which it is directed.
Shock A clinical syndrome in which the peripheral blood
flow is inadequate to return sufficient blood to the heart Spectrin A dimeric structural protein of the red blood cell
for normal function, particularly transport of oxygen to (RBC) cytoskeleton that maintains the shape of the cell
all organs and tissues. Shock may be caused by a variety by forming a network with other proteins such as actin
of conditions, including hemorrhage, infection, drug re- and ankyrin. Spectrin defects lead to hereditary ellipto-
action, trauma, poisoning, myocardial infarction, or de- cytosis or spherocytosis.
hydration. Symptoms include paleness of skin (pallor), Splenomegaly Enlargement of the spleen.
a bluish gray discoloration (cyanosis), a weak and rapid Splicing The process of excision of introns (noncoding
pulse, rapid and shallow breathing, or blood pressure sequences) from the immature, newly synthesized RNA
that is decreased and perhaps immeasurable. (pre-mRNA) in eukaryotes.
Short tandem repeats (STR) A type of genetic polymor- Spontaneous agglutination Direct agglutination of
phism demonstrated as back-to back repeats of sets of antibody-coated RBCs without the presence of antiserum;
1 to 8 nucleotides. The number of times each set is re- this usually occurs when RBCs are heavily coated with
peated is characteristic for each individual and is useful IgM or IgG autoantibody. Spontaneous agglutination
in kinship and crime investigations and in monitoring differs from cold agglutination in that it is not dispersed
of graft success or rejection. See also microsatellites. when the sample is warmed to 37°C.
Sialic acid A group of sugars found on the red blood cell Steatorrhea Increased secretion of the sebaceous glands.
membrane attached to a protein backbone; the major
Stem cell An unspecialized cell, capable of self-renewal,
source of the membrane’s net negative charge.
that gives rise to a group of differential cells, such as the
Sickle cell anemia Hereditary, chronic hemolytic anemia hematopoietic cells.
characterized by large numbers of sickle-shaped red
Steroid hormones Hormones of the adrenal cortex and
blood cells occurring almost exclusively in black
the sex hormones.
people.
Stertorous Pertaining to laborious breathing.
Sickle trait Blood that is heterozygous for the gene coding
for the abnormal hemoglobin of sickle cell anemia. Sticky (cohesive) ends The ends of DNA sequence result-
ing from restriction enzymes that cut both strands leav-
Siderosis Increase of iron in the blood that can lead to
ing “overhangs” that are complementary to any end cut
organ damage.
by the same enzyme. These ends will spontaneously join
Single-donor platelets Platelets collected from a single with complementary ends and do not require DNA ligase.
donor by apheresis.
Storage lesion A loss of viability and function associated
Single nucleotide polymorphisms (SNP) Single-nucleotide with certain biochemical changes that are initiated
differences between DNA sequences. The vast majority of when blood is stored in vitro.
these polymorphisms have no biological effect and are
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634 Glossary
STR Small tandem repeat, or short tandem repeat. The Tetany A nervous affliction characterized by intermittent
name describes the molecular nature of the DNA that spasms of the muscles of the extremities.
harbors this type of genetic marker, consisting of tetra- Thalassemia major The homozygous form of deficient
or pentanucleotide sequences that are tandemly repeated beta-chain synthesis, which is very severe and presents
a variable number of times in unrelated individuals. itself during childhood. Prognosis varies; however, the
Stroma The red blood cell membrane that is left after he- younger the child at disease onset, the less favorable the
molysis has occurred. outcome.
Subgroup Antigens within the ABO group that react less Thermal amplitude The range of temperature over which
strongly with their corresponding antisera than do A an antibody demonstrates serologic and/or in vitro
and B antigens. activity.
Survival studies A measure of the in vivo survival of Thermocycler (or thermal cycler) A programmable heat-
transfused blood cells; usually performed with radioac- ing and cooling machine used to conduct polymerase
tive isotopes. Normal red blood cells survive approxi- chain reaction.
mately 100 to 120 days in circulation. Thrombin An enzyme that converts fibrinogen to fibrin so
SYBR Green Fluorescent stain used in UV visualization of that a soluble clot can be formed.
nucleic acids in electrophoretic gels or in real-time PCR Thrombocytopenia A reduction in platelet count below
to detect the amplified product of the reaction. the normal level, which is associated with spontaneous
Syncytiotrophoblast The outermost fetal component of hemorrhage.
the placenta that allows for exchange of nutrients Thrombotic thrombocytopenic purpura (TTP) A coagula-
between the fetus and the mother. tion disorder characterized by (1) increased bleeding
Syngeneic Possessing identical genotypes, as monozygotic owing to a decreased number of platelets, (2) hemolytic
twins. anemia, (3) renal failure, and (4) changing neurological
Synteny Genes that are closely situated on a chromosome signs. The characteristic morphological lesion is throm-
but cannot be shown to be linked. botic occlusion of small arteries or capillaries in various
organs.
Systemic lupus erythematosus (SLE) A disseminated
autoimmune disease characterized by anemia, thrombo- Thymidine An essential ingredient used in DNA synthesis
cytopenia, increased immunoglobulin G levels, and the and incorporated by T lymphocytes undergoing blast
presence of four immunoglobulin G antibodies: antinu- transformation in response to foreign HLA-D antigens
clear antibody, antinucleoprotein antibody, anti-DNA in the mixed lymphocyte culture test.
antibody, and antihistone antibody; believed to be Titer A measure of the strength of an antibody by testing
caused by suppressor T-cell dysfunction. its reactivity at increasing dilutions against the appro-
System manager A specially trained person who is respon- priate antigen. The reciprocal of the highest dilution
sible for the maintenance of an information system. that shows agglutination is the titer.
Systolic pressure Maximum blood pressure that occurs at Titer score A method used to evaluate more precisely than
ventricular contraction; upper value of a blood pressure simple dilution by comparing the titers of an antibody.
reading. Agglutination at each higher dilution is graded on a
continuous scale; the total is the titer score.
Tachycardia Abnormally rapid heart action, usually de-
fined as a heart rate greater than 100 beats per minute. Trait A characteristic that is inherited.
Tachypnea Abnormally rapid respirations. Trans The location of two or more genes on opposite
chromosomes of a homologous pair.
TaqMan probes Also called cleavage or hydrolysis DNA
probes used to detect the amplicon produced in real-time Transcription The synthesis of RNA based on the sequence
PCR. These probes, labeled with reporter and quencher of DNA by the enzyme RNA polymerase. Process based
molecules, bind to their complementary area in a single on the principle of complementarity. The first stage of
strand of DNA region between the primers and get in the deciphering of the genetic code.
way of the Taq polymerase synthesizing the new strand. Transcription factor A protein that binds to DNA at a spe-
The polymerase, in order to continue the synthesis, de- cific nucleotide sequence (a promoter or enhancer se-
grades the probe and the reporter and quencher molecules quence) to influence the process of DNA transcription.
become separated, which results in emission of light. Transcription mediated amplification (TMA) Developed
Targeted review A type of prospective blood utilization initially at Gen-Probe, TMA is a molecular method of
review. detecting an organism by isothermal amplification of its
Template bleeding time The elapsed time a uniform inci- RNA using enzyme reverse transcriptase to produce
sion made by a template and blade stops bleeding, complementary DNA (cDNA), which is subsequently
which is a test of platelet function, assuming a normal transcribed into multiple RNA molecules, which then
platelet count. undergo another reverse transcription. This cycle
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Glossary 635
repeats several times, resulting in abundance of RNA to Value stream In Lean, identification of all the steps neces-
the levels that may be detected by chemiluminescent sary to bring a finished product to the customer.
DNA probes. Valvular Relating to or having a valve.
Transferase An enzyme that catalyzes the transfer of Variable number of tandem repeats (VNTR) DNA regions
atoms or groups of atoms from one chemical compound composed of back-to-back repeated units ranging in size
to another. from 9 to 80 bp. Like other polymorphisms, the VNTRs
Transfer RNA (t-RNA) See Ribonucleic acid. are useful in genetic fingerprinting performed in kin-
Transfuse To perform a transfusion. ship and crime investigations. See also minisatellites.
Transfusion The injection of blood, a blood component, Variable region That portion of the immunoglobulin
saline, or other fluids into the bloodstream. light and heavy chains where amino acid sequences
vary tremendously, thereby permitting the different
Transfusion guidelines Documentation of indications
immunoglobulin molecules to recognize different anti-
and thresholds for transfusion that is approved by
genic determinants. In other words, the variable region
the hospital or facility and based on evidence-based
determines the antigen against which the antibody will
literature.
react, thus providing each antibody molecule with its
Transfusion reaction An adverse response to a transfusion. unique specificity. The variable region is located at the
Transfusion safety officer A specialized member of the amino terminal region of the molecule.
blood utilization management team involved with coor- Vasculitis Inflammation of a blood or lymph vessel.
dination, audits of transfusion records, quality assur-
Vasoconstriction Constriction of blood vessels.
ance, and educational functions.
Vasodilation Dilation of blood vessels, especially small
Translation The production of protein, according to the
arteries and arterioles.
genetic code. An enzymatic process occurring in the
ribosomes, during which a codon within messenger Vasovagal syncope Syncope resulting from hypotension
RNA (mRNA) is aligned with an anticodon from the caused by emotional stress, pain, acute blood loss, fear,
transfer RNA (tRNA), which attaches a corresponding or rapid rising from a recumbent position.
amino acid into the growing peptide chain. Vector In recombinant DNA technology, a molecule of
Translocation Transfer of a portion of one chromosome to known nucleotide sequence that is used to carry a
its allele. foreign DNA fragment into a host organism in order
to produce multiple copies using the host’s replication.
Transmembrane protein A protein that crosses the red
Most common vectors are extrachromosomal, circular
blood cell membrane and is present on both sides of the
DNA plasmids, or bacterial viruses (bacteriophages).
membrane.
Venesection See Phlebotomy.
Transposition The location of two genes on opposite
chromosomes of a homologous pair. Venipuncture Puncture of a vein for any purpose.
Trypsin A proteolytic enzyme formed in the intestine. Veno-occlusive disease Disease involving the veins of the
liver associated with GVHD.
Type and screen Testing a patient’s blood for ABO, Rh,
and unexpected antibodies (antibody screen). If no Venule A tiny vein continuous with a capillary.
abnormalities exist in the ABO and Rh and no unex- Viability Ability of a cell to live or to survive for a reason-
pected antibodies are detected in the antibody screen, ably normal life span.
then the recipient blood sample is retained in the Vicia graminea See Anti-N lectin.
event that subsequent serologic crossmatching is
Virion A complete virus particle; a unit of genetic material
necessary.
surrounded by a protective coat that serves as a vehicle
Ulex europaeus See Anti-H lectin. for its transmission from one cell to another.
Ultracentrifugation Rapid and prolonged centrifugation, VNTR Variable number of tandem repeats.
used to separate by density gradient those substances of
von Willebrand’s disease A congenital bleeding disorder.
various specific gravities.
von Willebrand’s factor Coagulation factor VIII.
Urticaria A vascular reaction of the skin similar to hives.
WAIHA Warm autoimmune hemolytic anemia. A he-
Vaccine A suspension of infectious organisms or compo-
molytic anemia caused by the patient’s autoantibody
nents of them that is given as a form of passive immu-
that reacts at 37°C.
nization to establish resistance to the infectious disease
caused by that organism. Waste Also called Muda, as used within Lean, an action
that does not create value as defined by the customer.
Validation A systematic process of testing the hardware,
software, and user components of an information Weak D A general term used to describe individuals with
system to ensure that they are functioning correctly weakened expression of RhD. It represents a specific
for their intended purpose. classification used to describe mutations in the RHD
2682_Glossary_613-636 22/05/12 12:10 PM Page 636
636 Glossary
gene that cause changes in amino acids present in Xenogeneic Transplantation between species.
the transmembrane or intracellular region of the Yaws An infectious nonvenereal disease caused by the
RhD protein, thus causing quantitative differences in spirochete Treponema pertenue and found mainly in
the RhD protein. humid equatorial regions.
Western blotting See Immunoblotting. Zeta potential The difference in charge density between
Wharton’s jelly A gelatinous intercellular substance the inner and outer layers of the ionic cloud that sur-
consisting of primitive connective tissue of the rounds red blood cells in an electrolyte solution.
umbilical cord. Zygosity testing The process through which DNA se-
X chromosome The chromosome that determines quences are compared to assess whether individuals
female sex characteristics. The normal female has born from a multiple gestation (twins, triplets, etc.) are
two X chromosomes, and the normal male has an monozygotic (identical) or dizygotic (fraternal); often
X and a Y chromosome. used to identify a suitable donor for organ transplanta-
Xeno- Prefix indicating differing species. For example, a tion or to estimate disease susceptibility risk if one
xenoantibody is an antibody produced in one species sibling is affected.
against an antigen present in another species. Synonym
is hetero-.
2682_Index_637-648 22/05/12 12:11 PM Page 637
Index
637
2682_Index_637-648 22/05/12 12:11 PM Page 638
638 Index
phase of reactivity of, 222–223, 224t Antiglobulin test (AGT), 101–118 Autoimmune diseases, in blood bank serologic
polyclonal, 61–62, 103 case studies on, 115 testing, 73
as probes for proteins, 92–93 direct, 107–109, 108t, 109t Autoimmune hemolytic anemia(s) (AIHAs), 439–473
properties of, 63–64, 64f direct method for, 113 antibody characteristics in, 466t
reagent. See Antiserum(a). enzyme-linked, 112–113 blood group typing in patients with, 97
Antibody detection and identification, warm factors affecting, 110–111 case studies on, 466–469
autoantibodies and, 453 gel, 113, 114t DAT negative, 460, 461t
Antibody identification, 223–231. See also Antibody history of, 102 with IgM warm autoantibodies, 460
screen. indirect, 109–110, 110t, 113–114. See Indirect mixed-type, 458–460
adsorption in, 229–231, 230f, 231f antiglobulin test (IAT). warm, 450–460. See also Warm autoimmune
benign cold autoantibodies and, 444–445, 445t low ionic polybrene technique for, 112 hemolytic anemia (WAIHA).
cell panels in, 227, 228f methodologies of, compared, 113–114, 114t Autologous control, in antibody screen, 222
enzymes in, 227–228, 228t principles of, 107, 108f Autologous HPC donors, 393
evaluation of panel results in, 225–227, 225f solid phase technology for, 113, 114t Autologous tissue, 587–590
exclusion in, 224–225 sources of error in, 111–112, 112b implantation of, 589–590
in HDFN diagnosis, 431 techniques for, modified and automated, 112–113 preparation of, 588
investigations of, 267 Antihemophilic factor, cryoprecipitated, preparation sterilization and testing of, 589
neutralization in, 228–229, 229f, 229t of, 318–319, 319b storage of, 588–589, 589b
patient history in, 224 Antihuman globulin (AHG), 102 Autologous transfusion, 10, 360–361, 360f
reagents in, 224, 225f addition of, in antiglobulin test, 111 acute normovolemic hemodilution in, 302–304
resolving, additional techniques for, 227–231 antibodies specific to, 105–106, 106t intraoperative collection for, 304
warm autoantibodies and, 453 monospecific, 103 postoperative blood salvage for, 304
Antibody screen, 217–223. See also Antibody polyspecific, 103 preoperative collection for, 302, 303f, 304f
identification. preparation of, 103–105, 104f, 105f testing modifications for, 253
benign cold autoantibodies and, 444–445, 445t Antihuman globulin (AHG) reagents, 102–103, 103t transfusion reactions in, 385
in donor unit processing, 310 agglutination reactions and, 70 Automated cell washers, 261–262, 262f
gel method in, 220, 220f Antiserum(a), 61 Automated fluorescent cycle sequencing method, of
interpretation of, 221–222, 223f monoclonal, 122 DNA sequencing, 90, 90f
limitations of, 222–223, 224t polyclonal, 122 Automation, in blood bank laboratory, 273–274
in pretransfusion compatibility testing, Antithrombin III concentrates, preparation of, 322 Autonomy, 577, 577b
246, 246t Apheresis, 261, 331–351 Autosomal inheritance, 30–31, 30f, 31t
in screening for HDFN, 430–431 adverse effects of, 346–347, 346b
solid phase adherence method in, case study on, 347–348 B
220–221, 221f component collection by, 337–340. See also B antigen
tube method in, 217–220, 218f. See also Tube Plasmapheresis; Plateletpheresis. formation of, 124, 125f
antiglobulin test. definition of, 331 soluble, formation of, 126, 126f
Antibody titration, 232, 232t donation frequency for, 337, 337t B cells, 51, 54
in HDFN diagnosis, 431 donor criteria for, 305–306 B subgroups, in ABO blood group system,
Anticoagulant preservative solutions, in red blood cell in hematopoietic progenitor cell collection, 345 weak, 133–134, 134t
preservation, 8, 8t, 9t history and development of, 332, 332f, 332t, 333f Babesiosis
Anticoagulation, in apheresis, 333 immunoadsorption/selective absorption in, in donor history, 299–300
Anticodon, 80 345–346, 346f transfusion-associated, 417–418
Antigen(s) instruments used for, 338t Bacterial artificial chromosomes, in DNA cloning, 86
Ata, 208 methodology of, 334–337 Bacterial contamination
blood group, immunogenicity of, 62t centrifugation methods in, 334–336, 334f, 335f, of platelet components, screening of donor unit
characteristics of, 61, 61b 336f, 337f for, 312
Duffy, 195–196, 197 membrane filtration in, 336–337 of platelets, 17–19, 18b
high-prevalence, antibodies to, case study on, physiology of, 333 transfusion-transmitted diseases from, 416–417
234, 234f therapeutic, 340–345 Bacterial organisms, in transfusion-associated sepsis,
I and i, 183t, 185 general considerations for, 340–343 374, 374t
Jra, 208 indication categories for, 340–341, 341–342t Bacteriophages, 80
Kell system, 191–192t, 192–193, 195, 195t physiological considerations for, 343 Bar-code reader, 557f, 558
Kidd, 197 plasmapheresis as, 343, 343b Bar codes, on unit label, 558, 558f
low-prevalence, antibodies to, case study on, plateletpheresis as, 343–344 Battery, 573
234, 235f transfusion reactions with, 384 Behavioral influences, on utilization management,
Luke, 183 vascular access for, 341, 343 534
M and N, 186, 188f Apheresis platelets, 3 Beneficence, 577, 577b
MNS, 186, 187–188t, 188f Autoadsorption, in antibody identification, 230, 230f Bilirubin metabolism, hemolytic disease of fetus/
RBC, parentage testing based on, 497–498 Autoadsorption technique, in cold reactive newborn and, 430, 430t
S and s, 186, 188f autoantibody removal, 443 Biologics (biological products)
Sda, 208 Autoagglutinins, 440. See also Autoantibody(ies). definition of, 542–543, 543t
Vel, 208 Autoantibody(ies), 63, 440–460 deviation reporting requirements for, 551, 551t
Antigen-antibody ratio, agglutination reactions and, characterization of, 440–441 drugs compared with, 543t
66–67, 66f cold, 441–450. See also Cold autoantibody(ies). manufacturers/manufacturing of
Antigen-antibody reactions, 46, 46b definition of, 440 Current Good Manufacturing Practice
characteristics of, 63–64, 64t formation of, in drug-induced immune hemolytic regulations for, 549–550
RBC, detection of, 64–65 anemia, 464–465, 465t definition of, 542
Antigen-presenting cells (APCs), 52 to M and N, 191 regulatory oversight of, 546, 547t
Antigenic determinant, 47 warm, 450–460. See also Warm requirements for, 550–552, 551t, 552t
Antiglobulin crossmatch, 248 autoantibody(ies). product recalls of, 551–552
2682_Index_637-648 22/05/12 12:11 PM Page 639
Index 639
640 Index
Complementary DNA, 81 DIIHA. See Drug-induced immune hemolytic anemia hematopoietic progenitor cell, selection of, 393
Compliance, quality management versus, 510 (DIIHA). screening of, in risk management, 575–576
Computer crossmatch, 250–251, 251f, 265 2,3-Diphosphoglycerate (2,3-DDPG), in red blood cell testing of, for transfusion-transmitted diseases,
Confrontational strategies, in utilization metabolism, 6 403–404, 404t
management, 534 Direct antiglobulin test (DAT), 107–109, 113 Doppler ultrasound, color, in middle cerebral artery
Coombs’ serum, polyvalent, 220 in acute hemolytic transfusion reaction peak systolic velocity measurement in HDFN
Coombs’ test, 102. See also Antiglobulin investigation, 372–373 diagnosis, 432
test (AGT). in antibody detection and identification, 231–232 Dosage effect, 66
Cord blood. See Umbilical cord blood. benign cold autoantibodies and, 443 Double RBC pheresis
Cordocentesis, in HDFN diagnosis, 432 on cord blood, 266 collection procedure for, 338
Corticosteroids, for warm autoimmune hemolytic history of, 102 donor criteria for, 306
anemia, 458 negative, in autoimmune hemolytic anemia, 460, 461t Drug-induced immune hemolytic anemia (DIIHA),
Cosmids, in DNA cloning, 85–86 positive 460–466, 461f
Crawford antigen, 164 evaluation of, 108–109, 109t autoantibody formation in, 464–465, 465t
Creutzfeldt-Jakob disease (CJD) investigations of, 267 hapten mechanism in, 461–462, 462f, 462t, 464t,
exposure risk of donor to, 298, 299b principle and application of, 107, 108t 465t
transfusion-associated, 420 temperature-dependent methods of, 231 immune complex mechanism in, 463, 463f, 464t,
Cromer blood group system, 206 warm autoantibodies and, 453 465t
Cross reactivity, in HLA loci, 482, 482f, 483f Direct antiglobulin test (DAT) panel, 107–108, 108t membrane modification in, 463–464, 463f, 464t,
Crossmatch testing Discontinuous prospective review, in utilization 465t
antiglobulin, 248 management, 533 treatment of, 465–466
computer, 250–251, 251f, 265 Disease susceptibility, HLA antigens and, 487, 488t Drugs, biological products compared with, 543t
electronic, 265 Disseminated intravascular coagulation (DIC), 362 Duffy antigen group, on red blood cells, 54
immediate spin, 248 Disulfide bonding, in immunoglobulins, 56, 57f Duffy blood group system, 195–197, 196f, 196t
interpretation of results of, 248–249 DNA (deoxyribonucleic acid), 34–39
pretransfusion, 247–251 bases in, 34–35, 34f, 35f E
serologic, 248 complementary, 81 Education, in utilization management, 534–535
positive results in, causes of, 249–250, 250t fetal, testing of, in HDFN diagnosis, 431 Electronic crossmatch, 265
resolution of incompatibilities in, 249 as genetic material, 78–79, 78f, 79f Electrophoresis, gel, in DNA cloning, 84–85, 85f
Cruz-Gregurek model, of optimization, 528, 528f isolation of, 38–39 Elution techniques, in antibody identification, 231–232
Cryoprecipitate mutations of, 37–38, 37t Emergencies, pretransfusion testing in, 251–252
characteristics of, 324t profiling or fingerprinting of, in studying gene Emergency transfusions, 361
pooled, transfusion of, indications for, 353t polymorphism, 94 Emotional distress, intentional infliction of, 574
transfusion of, 357 recombinant, 82–90. See also Recombinant DNA. Endonucleases, restriction, in DNA cloning, 83–84,
Cryoprecipitated antihemophilic factor, preparation repair of, 36–37, 36b 84t
of, 318–319, 319b replication of, 35–36, 36f Enhancement media, agglutination reactions and, 67,
Cryoprecipitation, 3 sequencing of, 89–90, 90f 69t, 70f
Cryopreservation, in tissue banking, 582 in studying gene polymorphism, 93–94 Enhancement reagents, in tube antiglobulin test,
Crystalloids, 321 DNA microarrays, in nucleic acid hybridization, 91–92 219–220, 219f
colloids compared to, 322t DNA polymorphisms Enzyme(s)
Current blood utilization review, 532 polymerase chain reaction amplifying, parentage antibody identification, 227–228, 228t
Cw antigen, 163 testing based on, 500–501, 500f, 501b proteolytic, agglutination reactions and, 69–70
Cytokines, 48, 49–50t restriction fragment length, parentage testing RBC, parentage testing based on, 498
in immune response, 54 based on, 496, 496f, 498–500, 499f, 499t Enzyme-linked antiglobulin test (ELAT), 112–113
Cytomegalovirus (CMV) Doctrine of charitable immunity, 575 postpartum, 266
transfusion-associated, 414–415 Domains, of immunoglobulins, 56 Enzyme-linked immunosorbent assay (ELISA)
transmission of, in transfusion therapy for HPC Dombrock blood group system, 203–204 in HLA antibody detection, 485
transplantation, 399 Dominant traits, 30–31, 30f, 31t solid-phase, 280–282, 282f
Cytomegalovirus-negative cellular blood Donath-Landsteiner test, 449, 450t Epitope, 47
components, transfusion of, 359 Donation facility, information software applications Epstein-Barr virus (EBV), transfusion-associated, 415
for, 560–561 Erythroblastosis fetalis. See Hemolytic disease of
D Donation process, 3–4, 3b fetus and newborn.
D antigen, 150 Donor(s) in hemolytic disease of fetus/newborn, 429
expression of, variations of, 158–160, 159f, blood Erythrocytapheresis, therapeutic, 344
160f, 160t autologous, 301–304. See also Autologous Erythrocytes. See Red blood cell(s).
DAT. See Direct antiglobulin test (DAT). transfusion. Ethical issues, in transfusion medicine, 576–577
DCE terminology for Rh system, 150–151, 150f, 151t, blood from, processing of, 310–312 Ethidium bromide, in gel electrophoresis, 84
153t collecting samples from, 244–245 Ethnicity, RHD gene and, 157
Deglycerolization, in thawing of frozen red blood current statistics on, 3 Euchromatin, 31
cells, 314–315, 315t, 316b extensive blood group typing of, for Eukaryotic organisms, 31
Delayed serologic/hemolytic transfusion reaction alloimmunized patients, 97 Exchange transfusion, in HDFN management, 432
(DSHTR), 379–381, 380f, 381f identification of, for whole blood collection, 307 Exons, 83
Denaturation, in molecular testing, 79 informed consent from, 301, 301f Eye Bank Association of America, 595
Deoxyribonucleic acid (DNA), 34–39. See also DNA postdonation instructions for, 308, 309f
(deoxyribonucleic acid) entries. processing of unit from, 263–264, 264b F
Dextran, characteristics of, 325t reactions of, to blood collection, 308, 310 f antigen, 163–164
DIC (disseminated intravascular coagulation), 362 records on, 310, 310t Factor III concentrates, preparation of, 319–320
Diego blood group system, 202 selection of units from, 246–247 Factor VII
Differential adsorption, in antibody identification, whole blood collection from, 306–310, 307b, activated, preparation of, 319
230–231 308b, 309f recombinant human activated, transfusion of, 358
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Index 641
Factor VIII, transfusion of, 357–358, 357t solid-phase red cell adherence compared with, Hapten (drug-adsorption) mechanism, in
Factor VIII concentrates, characteristics of, 324t 282–284, 282t, 283t drug-induced immune hemolytic anemia,
Factor IX concentrates Gel tests, antiglobulin, 113, 114t 461–462, 462f, 462t
characteristics of, 324t Gene(s), 31 Hardware for blood bank information systems,
preparation of, 320 cloned, expression of, 87–88, 88b 557–558, 557f, 558f
Factor XIII concentrates, preparation of, 320–231 coding sequence of, 83 maintenance of, 565–566
Fatalities Rh, 156–157, 157f HDFN. See Hemolytic disease of fetus/newborn
from apheresis, 347 Gene expression, 78 (HDFN).
transfusion-related, reporting of, 552, 552t Gene polymorphism, techniques for studying, Health Insurance Portability and Accessibility Act
FC receptors, immunoglobulin, 58–59 93–94 (HIPAA), liability based on, 575
FDA. See Food and Drug Administration (FDA). Gene therapy, 88 Heart transplantation, HLA matching in, 491
FDA (U.S. Food and Drug Administration), in donor Genetic basis, of blood groups, 95, 96t Hemagglutination reactions, in transfusion laboratory
screening regulation, 290 Genetic code, 35, 35f testing, 65
Febrile nonhemolytic transfusion reaction, 375 Genetic information, expression of, 80–81 Hematomas, from blood donation, 310
Federal Food, Drug, and Cosmetic (FD&C) Act, Genetic systems, used in parentage testing, 497–591, Hematopoietic progenitor cell (HPC) transplantation
541–542 497b, 497t ABO incompatibility in, 397, 397t
Federal regulation Genetics transfusion therapy and, 399, 400t
of biological products, 540–555 basic, 26–44 adverse recipient reactions to, 398
enforcement actions in, 548, 549t cellular, 31–33, 32f, 32t, 33f case study, on, 400
inspection process in, 547–548 classical, 27 categories of, 392–393, 392b
registration and licensure in, 545–547, 545t, of Duffy blood group system, 196–197 disease relapse after, 398
547t Hardy-Weinberg principle in, 29–30, 29b, 29f donor/patient selection for, 393, 393b
regulatory process for, 542–544, 543t, 544b of I system, 184 engraftment after, 398
reporting requirements in, 550–552, 551t, 552t of immune system, 54–55 graft rejection after, 398
of tissue banking, 590–591t, 590–592 inheritance patterns in, 30–31, 30f, 31t graft-versus-host disease after, 398, 398t
Fetal DNA typing, 95 introduction to, 26–27 hematopoietic progenitor cell collection for,
Feto-maternal hemorrhage, postpartum evaluation of Kell blood group system, 194 393–394, 394b
for, 266 of Kidd blood group system, 198 HLA matching in, 490
Fetus, hemolytic disease of, 427–438. See also of Lewis system, 179–180, 179f, 180t indications for, 394–395, 395b
Hemolytic disease of fetus/newborn (HDFN). of Lutheran blood group system, 201 infusion of hematopoietic progenitor cells in, 397
Ficin, 70 Mendelian, 27–29, 28f, 29f outcomes of, 398
Filtration, membrane, in apheresis, 336–337 in ABO gene inheritance, 122 purpose and goals of, 392
Flow cytometry modern techniques of, steps in, 42 recipient conditioning for, 397
in HLA antibody detection, 485–486 molecular, 34–41. See also DNA (deoxyribonucleic transfusion therapy for, 398–399, 400t
in HLA crossmatching, 486–487, 487f acid) entries; Molecular genetics. Hematopoietic progenitor cell(s) (HPCs), 392
postpartum, 266 of P blood group system, 182 collection of, 393–394, 394b
in study of immunologic reactions, 71 population, 27–31 by apheresis, 345
Fluid replacement, in therapeutic cytapheresis Genotype, 31 detection and measurement of, 394
procedures, 344–345 Genotyping, red blood cell, 94–97, 96t freezing of, preparation for, 395
Fluid shifts, in apheresis, 333 clinical applications of, 95, 97 infusion of, 397
Fluorescent in situ hybridization, 92 Gerbich blood group system, 205, 205t processing of, 395–396, 396b
Food and Drug Administration (FDA) Gill blood group system, 207 storage and shipping of, 396–397
in biologics regulation, 542 Glycerol, in red blood cell freezing, 10–11, 10t thawing and preparing for transplant, 397
inspection process of, 547–548 Glycerolization, in freezing of red blood cells, uses of, 583
tissue recalls by, 597–598, 598b 314–315, 315t Hematopoietic stem cells, 392
Frameshift mutation, 38 Glycosyltransferases, in ABH antigen formation, 123, uses of, 583
Freezing 124t Hemizygous, 31
of hematopoietic progenitor cells, 396 Graft-versus-host disease (GVHD) Hemoglobin
preparation for, 395 in hematopoietic progenitor cell transplantation, bovine, in hemoglobin-based oxygen carriers,
of platelets, 21 398, 398t 13, 13t
of red blood cells, 10–11, 10t protection against, irradiation of blood stroma-free, ultra-purified, 12–13
glycerolization in, 314–315, 315t components in, 263 Hemoglobin-based oxygen carriers, 12–13,
thawing after, deglycerolization in, 314–315, transfusion-associated, 381–382, 382b, 382f 13t, 14t
315t, 316b in transfusion therapy for HPC transplantation, Hemoglobin oxygen dissociation curve, 6–7, 7f
Fresh frozen plasma (FFP), characteristics of, 324t 399 Hemoglobinemia, in acute hemolytic transfusion
Frozen/deglycerolized red blood cells, transfusion of, Granulocytes reaction, 370, 373f
354–355 collected by pheresis, 339–340 Hemoglobinuria
transfusion of, 356 in acute hemolytic transfusion reaction,
G in immune system, 53 370, 373f
G antigen, 164 irradiated, characteristics of, 325t paroxysmal cold, 449–450, 450f, 450t
Gatekeeping, in utilization management, 534 transfusion of, indications for, 353t autoanti-P associated with, 182–183
Gel electrophoresis, in DNA cloning, 84–85, 85f Griffith’s transformation, 78–79, 78f treatment for, 450
Gel low ionic antiglobulin test (GLIAT), 113 GVHD. See Graft-versus-host disease (GVHD). Hemolysis
Gel method of antibody screening, 220, 220f in hemolytic disease of fetus/newborn, 429
Gel technology, 274–277 H intravascular, ABO antibodies in, 122
advantages and disadvantages of, 276–277 H antigen RBC, in warm autoimmune hemolytic anemia,
applications of, 274 in A and B antigen formation, 123 451–452, 452b
automation of, 277, 277f formation of, 123, 124f in transfusion laboratory testing, 65
history of, 274 soluble, formation of, 126, 126f Hemolytic anemia, autoimmune, 439–473. See also
principle of, 274–275, 274f, 275f, 276f Haplotypes, HLA, 479, 479f Autoimmune hemolytic anemia(s).
2682_Index_637-648 22/05/12 12:11 PM Page 642
642 Index
Hemolytic disease of fetus/newborn (HDFN), 57, HLA crossmatch techniques, in histocompatibility innate, 46–47
427–438 testing, 486–487, 487f maturation of, 52, 53f
ABO incompatibility in, 435–436, 435t HLA gene products, 480–482, 481f, 482f Immune serum globulin (ISG)
in blood bank serologic testing, 73 HLA genetic region, 476 characteristics of, 325t
case studies on, 436–437 HLA matching, for HPC transplantation, 392, 392b preparation of, 321
diagnosis and management of, 430–435 HLA system, 475–494 Immune system
amniocentesis in, 432 clinical significance of, 487–491 blood product transfusions and, 73
color Doppler middle cerebral artery peak in disease association, 487, 488t cell lineages and markers of, 52–54
systolic velocity in, 432 in paternity testing, 487 cells of, 50–52
cordocentesis in, 432 in platelet transfusion, 487–489, 489t genetics of, 54–55
exchange transfusion in, 432 in transfusion-related acute lung injury, 489 major mechanisms of, comparison of, 47t
intrauterine transfusion in, 432 in transplantation, 489–491 organs of, 52–54, 52t
intravenous immune globulin in, 432 historical perspective on, 476 overview of, 46
newborn transfusions in, 433 nomenclature for, 476–479, 476f, 477–478t, 479t Immunity
phototherapy in, 432 parentage testing based on, 498 acquired, 47, 48t, 50
RhIG in, 433–435, 434b, 434f HLA typing, crossing over complicating, cellular, 47, 48t
serologic testing of mother in, 430–432, 430f 479–480, 480f humoral, 47, 48t
serologic testing of newborn in, 432–433 Homologous adsorption, in antibody identification, 230 innate, 47–50, 48t
etiology of, 428 Hospital information system, 559 Immunoadsorption, in apheresis, 345–346, 346f
pathogenesis of, 429–430, 430t Hospital tissue bank, 583–586 Immunodeficiency disorders
Rh antibodies causing, 163 Host cells, in DNA cloning, 86 in blood bank serologic testing, 72, 72b
Rh causing, 150 Host factors, in immune response, 64, 64b classification of, 72b
Rh incompatibility in, 428–429, 428f, 429t HPCs. See Hematopoietic progenitor cell(s) (HPCs). Immunodominant sugars, 123, 124t
Hemolytic transfusion reaction(s) Human immunodeficiency virus (HIV) antibody, Immunogen, 102
acute (AHTR), 370, 372–374, 372f, 373f screening of donor unit for, 311 Immunoglobulin(s), 47
delayed (DSHTR), 379–381, 380f, 381f Human immunodeficiency virus (HIV) infection in blood banking, 56–58, 57f
fatal, ABO incompatible, 120b in donor history, 299, 300b characteristics of, 55–59, 55t
Hemophilia, 362 transfusion-associated, 409–411, 409f, 409t, FC receptors of, 58–59
Hepatitis 410f, 411f IV, for warm autoimmune hemolytic anemia, 458
in donor history, 299 Human leukocyte antigen (HLA), 475–494. See also structure of, 55–56, 56f, 57f
transfusion-associated, 404–409 HLA entries; HLA system. type of, agglutination reactions and, 67, 68t
Hepatitis A, transfusion-associated, 404–405, 405f, Human T-cell lymphotropic virus (HTLV) antibody, variations in, 58
405t screening of donor unit for, 311 Immunoglobulin A (IgA), in transfusion medicine, 58
Hepatitis B, transfusion-associated, 405–406, 405t, Human T-cell lymphotropic virus (HTLV), Immunoglobulin allotypes, parentage testing based
407t, 406f, 408f transfusion-associated, 411–412, 413f on, 498
Hepatitis B surface antigen, screening of donor unit Humoral immunity, 47, 48t Immunoglobulin E (IgE), in allergic reactions, 58
for, 310–311 Hybrid formation, 38 Immunoglobulin G (IgG), molecules of, chemical
Hepatitis B virus core antigen, screening of donor unit Hybridization, nucleic acid, 90–92 reduction of, agglutination reactions and,
for, 311 Hybridization-based assays, 80 70–71
Hepatitis C, transfusion-associated, 407t, 406–408 Hydrogen bonding Immunoglobulin G (IgG) antibodies, in transfusion
Hepatitis C virus antigen, screening of donor unit for, in DNA molecule, 79–80 medicine, 56–58, 57f, 58t
311 in immunoglobulins, 56 Immunoglobulin M (IgM), molecules of, chemical
Hepatitis D, transfusion-associated, 408 Hydrophilic bonds, 63 reduction of, agglutination reactions and,
Hepatitis E, transfusion-associated, 407t, 408 Hydrophobic bonds, 63 70–71
Hepatitis G, transfusion-associated, 408–409 Hydrops fetalis, in hemolytic disease of Immunoglobulin M (IgM) antibodies, in transfusion
Hereditary spherocytosis, 78 fetus/newborn, 429–430 medicine, 56–57, 57f
Herpesvirus, human, transfusion-associated, 415–416 Hypersensitivity disorders, in blood bank serologic Immunohematology, molecular, events leading to
Hershey-Chase phage experiment, 79, 79f testing, 72–73 establishment of, 82, 82t
Heterochromatin, 31 Hypogammaglobulinemia, in leukemias, 136 Immunologic reactions, flow cytometry in study of, 71
Hh gene interaction with ABO genes, 123–125, 124f, Immunology
125f, 126f I fundamentals of, 45–76
High-concentration glycerol technique, in red blood I system, 183–185, 183t, 184f, 185t introduction to, 46
cell freezing, 10, 10t IAT. See Indirect antiglobulin test (IAT). Immunoregulatory molecules, 54
HIS antibody detection techniques, 484–486 Idiotypic variations, in immunoglobulins, 58 Immunosuppressive drugs, for warm autoimmune
Histocompatibility testing techniques, 483–487 IgM warm autoantibodies, warm autoimmune hemolytic anemia, 458
HLA antibody detection as, 484–486 hemolytic anemia with, 460 Incubation time, in antiglobulin test, 111
HLA antigen detection as, 483–484, 483f Immediate spin crossmatch, 248 Indian blood group system, 206
HLA crossmatch techniques as, 486–487, 487f Immune antibodies, 62 Indirect antiglobulin test (IAT), 109–110, 110t, 113–114
HLA molecular techniques as, 484, 484f Immune complex (innocent bystander) mechanism, use of polyspecific versus monospecific
Histones, 31, 34 in drug-induced immune hemolytic anemia, antihuman globulin in, 106–107
HLA alleles 463, 463f, 464t Infants. See also Neonates.
in inheritance, 479, 479f, 480f Immune globulin, intravenous, in HDFN compatibility testing for, 252–253, 252b
linkage disequilibrium between, 480, 480t management, 432 Infection
nomenclature for, 476, 477–478t Immune hemolytic anemia cold autoantibodies related to, 449–450, 450f,
HLA alloimmunization, in hematopoietic progenitor definition of, 440 450t
cell transplantation, 399 drug-induced, 460–466, 461f. See also Drug- in tissue transplantation, 596
HLA antibodies, 479–482, 482 induced immune hemolytic anemia (DIIHA). Infectious diseases, donor, screening tests for, 4, 4t
HLA antigens, 479–482 Immune response Inflammation, in innate immunity, 50
cross reactivity of, 482, 482f, 483f complement system in, 59–61 Inflammatory mediators, in acute hemolytic
detection of, 483–484, 483f host factors in, 64, 64b transfusion reaction, 370, 372f
2682_Index_637-648 22/05/12 12:11 PM Page 643
Index 643
Information processing, 556–557 Kidney transplantation, HLA matching in, 490–491 Licensure, of biological products, 545–546
Information systems Kleihauer-Betke test, 266 Linkage disequilibrium, between HLA alleles, 480,
blood bank, 556–570 Knops blood group system, 206 480t
acronyms for, 557t Liver transplantation, HLA matching in, 491
hardware component of, 557–558, 557f, 558f L Locus, 31
maintenance of, 565–566 Laboratory, blood bank, 260–272 Low ionic polybrene (LIP) technique, for antiglobulin
human component of, 559–560 blood component preparation and storage in, test, 112
management of, 565–567 261–263, 261t, 262f, 262t, 263b, 263f Low ionic strength solution (LISS)
regulatory and accreditation requirements for, cord blood studies in, 266 in antiglobulin test, 110
564–565 donor processing area of, 263–264, 264b as potentiator in antibody detection, 219
software component of, 558–559, 559f equipment for, 263, 263f Low ionic strength solution (LISS) media,
applications of, 560–564 information systems in, 556–570. See also agglutination reactions and, 68–69
validation of, 566–567 Information systems, in blood banks. Luke antigen, 183
standard operating procedures for, 565–567 in issue of blood products, 266, 266b, 267f Lung injury, acute, transfusion-related, 376–378,
types of, 557 main, 264–266, 265f, 266b 376b, 377f
hospital, interface between blood bank organization of, 260–268 HLA antibodies in, 489
information system and, 559 personnel requirements for, 268, 268f transplantation and, 484
Information technology, in utilization management postpartum evaluation in, 266 Lung transplantation, HLA matching in, 491
policy deployment, 536 prenatal evaluation in, 266 Lutheran blood group system, 199–202, 200t, 201f,
Informed consent product labeling in, 264, 264f, 264t 202t
doctrine of, 573 quality management system for, 511–522. See antigens of, 199, 200t
in risk management, 576 also Quality management system(s), for blood biochemistry of, 201, 201f
Inheritance. See also Genetics. bank laboratories. genetics of, 201
Mendel’s laws of, 27–29, 28f, 29f reference, 266–268 Lu(a-b-) phenotypes of, 201–202, 202t
patterns of, 30–31, 30f, 31t routine testing in, 265–266 Lymph nodes, in immune system, 52, 52t
Innate immunity, 47–50, 48t sample collection and acceptance in, 264–265 Lymphocytes
“Innocent bystander” (immune complex) standard operating procedures for, 268 in acquired immunity, 50–52
mechanism, in drug-induced immune in transfusion process oversight, 268–270, 269f, B, 51, 54
hemolytic anemia, 463, 463f, 464t 270f, 271f of immune system, 53–54
Intermolecular binding forces, in antigen-antibody type and crossmatch in, 265 T, 51–52, 54
reactions, 63 type and screen in, 265 Lymphocytotoxicity, in HLA crossmatching, 486
International Society of Blood Transfusion (ISBT) Lambda vectors, in DNA cloning, 85 Lymphoid cells, of immune system, 52–53
blood group collections of, 175, 177t Landsteiner-Weiner (LW) blood group system, 165, Lymphokines, in immune response, 54
blood group systems of, 175, 176t 204–205, 204t
low- and high-prevalence antigen series of, 175, Lean approach, to blood utilization management, M
177, 177t 527–530, 528f, 528t, 529f, 530f M phase, 33
terminology for Rh surface antigens of, 153–155, Lectins, in blood banking, 129, 129b Macrophages, factors affecting activity of, 452b
154–155t Legal issues Major histocompatibility complex (MHC), 476, 476f
Intrauterine transfusion in parentage testing, 506 in immune recognition, 55
compatibility testing for, 252 in transfusion medicine, 571–579. See also molecules of, 51
in HDFN management, 432 Medicolegal issues in transfusion medicine. Malaria
Introns, 83 Leishmaniasis, exposure risk of donor to, 298 in donor history, 299
Invasion of privacy, 574–575 Leukapheresis exposure risk of donor to, 298
Iron overload, complicating transfusion, 383 donor criteria for, 306 transfusion-associated, 419–420
Irradiated cellular blood components, transfusion for hematopoietic progenitor cell collection, 394, Malignancy, transmitted through tissue
of, 359 394b transplantation, 596
Irradiation therapeutic, 344 Malpractice, medical, 576
of blood components, 263, 323t, 324t, 325t Leukemia, serologic reactions typical of, 138t Massive transfusions, 361, 361t
of red blood cells, 314, 323t Leukocyte-reduced cellular blood components, adverse events associated with, 378–379
ISBT. See International Society of Blood Transfusion transfusion of, 359 testing before, 253
(ISBT). Leukoreduced platelets McLeod phenotype and syndrome, 194–195, 195t
ISG. See Immune serum globulin (ISG). characteristics of, 324t Medical malpractice, 576
Island cell transplantation, HLA matching in, 491 preparation of, 317–318 Medicolegal issues in transfusion medicine,
Isoagglutinins, ABO, ABO discrepancy caused by, transfusion of, indications for, 353t, 355t 571–579
141, 142t Leukoreduced red blood cells, 314 current, 571–572
Isotype variation, in immunoglobulins, 58 characteristics of, 323t liability as
transfusion of, 354 HIPAA as basis of, 575
J indications for, 353t, 355t torts as basis of, 572–575
John Milton Hagen blood group system, 207 Lewis antibodies, 178–179 restrictions on plaintiff suits and recovery as, 575
Joint Commission, tissue banking standards of, 594–595 Lewis antigens, 178, 178t, 180t risk management as, 575–576
Justice, in transfusion medicine, 577, 577b development of, 180 sources of law on, 572
Lewis blood group system, 177–180, 178t, 179f, 180t. Meiosis, 32–33, 32t, 33f
K See also Lewis system. Melting point, DNA, 80
Kell blood group system, 191–192t, 191–195, 194f, 195t Lewis system, 177–180 Membrane, red blood cell, 4–5
antibodies of, 193 genetics and biosynthesis of, 179–180, 179f, 180t Membrane attack complex (MAC), 60–61
antigens of, 191–192t, 192–193, 195, 195t phenotypes of, 178, 178t, 179, 180t Membrane filtration, in apheresis, 336–337
biochemistry of, 193–194, 194f Liability in transfusion medicine Mendelian genetics, 27–29, 28f, 29f
genetics of, 194 HIPAA as basis of, 575 in ABO gene inheritance, 122
McLeod phenotype and syndrome and, 194–195, strict, 574 Messenger RNAs (mRNAs), 80
195t torts as basis of, 572–575 MHC. See Major histocompatibility complex (MHC).
2682_Index_637-648 22/05/12 12:11 PM Page 644
644 Index
Index 645
Polyclonal antisera, 122 recipient identification for, 242–243, 243f Reagent antibodies. See Antiserum(a).
Polyclonal gammopathies, in blood bank serologic Rh typing in, 245–246 Real-time PCR, in nucleic acid detection, 92
testing, 73 sample collection for, 243–245 Recessive traits, 30, 30f, 31t
Polyclonal reagents, 70, 71 sample handling for, 242 Recombinant DNA, 82–90
Polyethylene glycol in special circumstances, 251–253, 252b libraries of, in DNA cloning, 87
agglutination reactions and, 69 standards for, 241–242, 242t polymerase chain reaction and, 88–89, 88f, 89f
in antiglobulin test, 110–111 Privacy, invasion of, 574–575 sequencing of, 89–90, 90f
as potentiator in antibody detection, 220 Probability Recombinant factor VIII, preparation of, 320
Polylinkers, in DNA cloning, 85 of exclusion, 504–505 Recombinant human activated factor VII, transfusion
Polymerase chain reaction (PCR), 80 of parentage, 504 of, 358
allele-specific probes and, 93, 93f Proficiency testing, in process management, 515–516 Recombinant proteins, in clinical use, 87–88, 88b
in DNA replication, 88–89, 88f, 89f Prokaryotic organisms, 31 Red blood cell alloimmunization, complicating
parentage testing based on, 496–497, 500–501, Prolonged clotting time, specimens with, transfusion, 384
500f, 501b pretransfusion testing in, 253 Red blood cell pheresis, double
real-time, in nucleic acid detection, 92 Prospective blood utilization review, 532–533 collection procedure for, 338
reverse transcriptase, in nucleic acid detection, 92 Protein(s) donor criteria for, 306
Polymorphism(s) antibodies as probes for, 92–93 Red blood cell (RBC) antigens, parentage testing
at ABO locus, 128 recombinant, in clinical use, 87–88, 88b based on, 497–498
DNA serum, RBC, parentage testing based on, 498 Red blood cell (RBC) enzymes, parentage testing
polymerase chain reaction amplifying, synthesis of, 40–41, 40f, 41f based on, 498
parentage testing based on, 500–501, 500f, Protein media, agglutination reactions and, 68 Red blood cell (RBC) serum proteins, parentage
501b Proteolytic enzymes, agglutination reactions and, testing based on, 498
restriction fragment length, parentage testing 69–70 Red blood cell(s) (RBCs)
based on, 496, 496f, 498–500, 499f, 499t Provirus, 81 aliquotted
single nucleotide, parentage testing based on, 501 Prozone effect, 66, 66f characteristics of, 323t
Polyspecific antihuman globulin Pseudogenes, 38 preparation of, 313–314
monospecific antihuman globulin versus, in Public Health Service Act, 541 antibody-coated, complement binding by, 61
indirect antiglobulin test, 106–107 Purpura, post-transfusion, 382–383 antigen-antibody reactions in, detection of, 64–65
preparation of, 104–105, 104f biology of, 4–7
Polyspecific antihuman globulin reagents, 103 Q characteristics of, 323t
Porcine factor VIII, preparation of, 320 Quality creation of, in laboratory, 12
Postpartum evaluation, 266 building blocks of, 510–511, 510f deformability of, 4, 5f
Postzone effect, 66, 66f cost of, 523 deglycerolized, characteristics of, 323t
Potentiators, 219 definition of, 510 exchange of, 344
Precipitation reaction, in transfusion laboratory Quality assurance, 510f, 511, 511b freezing of, 10–11, 10t
testing, 65 information software applications for, 563–564, frozen
Prenatal serologic testing, 266 564f, 564t characteristics of, 323t
Preservation in tissue banking, 586 deglycerolized, preparation of, 314–315, 315t,
of platelets, 14–21, 16–20 Quality control, 510, 510f 316b
additive solutions in, 19–20, 20b Quality control unit, for biologics manufacturers, genotyping of, 94–97, 96t
bacterial contamination and, 17–19, 18b 549–550 clinical applications of, 95, 97
at 1°C to 6°C, new approaches for, 20–21 Quality management, 509–525 hemolysis of, in warm autoimmune hemolytic
freezing in, 21 compliance versus, 510 anemia, 451–452, 452b
history of, 16 Quality management system(s), 510f, 511, 512b, 512f irradiated
pathogen reduction and inactivation in, 20 for blood bank laboratories characteristics of, 323t
platelet substitute development in, 20 assessments in, 519, 520f preparation of, 314
research on, current trends in, 19–21 continual improvement in, 519–522, 521b, 521f leukoreduced
in second-generation containers, 16–17, 17b customer focus in, 512–513 characteristics of, 323t
for 7 days at 20°C to 24°C, 19 documentation in, 516, 516f preparation of, 314
without agitation for limited times, 17 equipment in, 513–514 membranes of, 4–5
of red blood cells essentials of, 511–522, 512b, 512f modification of, in drug-induced immune
additive solutions in, 8–10, 9b, 9t facilities and safety in, 513 hemolytic anemia, 463–464, 463f, 464t
improved, 11 information management in, 517 zeta potential of, 67
anticoagulant preservative solutions in, 8, 8t, 9t nonconformance management in, 517–518, metabolic pathways of, 5–7, 6f
freezing in, 10–11, 11t 517t, 518f O-type, formation of, 11–12
history of, 2–3 organization in, 512 permeability of, 4–5
pathogen reduction/inactivation procedures personnel in, 513, 513b preparation of, 313, 313b
in, 11 process management in, 514–516, 514f, 515f preservation of, 7–11. See also Preservation, of
research on, current trends in, 11–13 purchasing and inventory in, 513 red blood cells.
Pretransfusion compatibility testing, 241–259 records management in, 516–517 rejuvenation of, 11
ABO typing in, 245 importance of, 523 substitutes for, 12–13, 12b, 12t, 13t, 14t
antibody screening in, 246, 246t for medical laboratories, 522–523, 522f transfusion of, 354
in blood inventory management, 253–254 Quality reviews, for biologics manufacturers, 550 indications for, 353t, 355t
case studies on, 256 washed, characteristics of, 323t
crossmatch testing in, 247–251. See also R washing of, in antiglobulin test, 111
Crossmatch testing. Raph blood group system, 207 Red blood cell storage lesion, 7, 7t
donor unit selection in, 246–247 RBC reagents, in tube antiglobulin test, 218–219, 218f, Red cell agglutination reactions, in transfusion
future of, 255–256 219b, 219f, 219t laboratory testing, 65
limitations of, 253, 254t RBCs. See Red blood cell(s) (RBCs). Reference laboratory, 266–268
protocols for, 245–246, 246t Reaction medium, in antiglobulin test, 110–111 Registration, of blood establishments, 545, 545t
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646 Index
Index 647
Kidd
Jkb JK2 73 43 strong RBC only yes Enz. ↑
MNS
Lutheran
Lub LU2 99.8 99.9 poor RBC only yes Enz. → AET → ZZAP ↓
LU3 >99.8
*This chart is to be used for general information only. Please refer to the appropriate chapter for more detailed information.
AET = 2-aminoethylisothiouronium bromide; ↑ = enhanced reactivity; → = no effect; ↓ = depressed reactivity; occ = occasionally; HDN = hemolytic disease of the newborn;
HTR = hemolytic transfusion reaction; NRBC = non-red blood cell; RBC = red blood cell; WBC = white blood cell; ZZAP = dithiothreitol plus papain. PCH = paroxysmal cold
hemoglobinuria; ↑ = enhanced reactivity;
†In the P system, phenotype P1 contains both P1 P, and Pk antigens; phenotype P2 contains only P antigens; phenotype p lacks both P1 P, and Pk antigens.
‡The Pk antigen is typically converted to P; therefore there is no Pk antigen detectable on adult cells. There are rare individuals (P1k, P2k) where pk antigen is not converted to P.
2682_IBC 22/05/12 12:10 PM Page 3
ANTIBODIES
Immunoglobin Clinical
Serology Comp. Class Optimum Significance
Stimulation Saline AHG Binding IgM IgG Temperature HTR HDN Comments
RBC no yes yes no yes warm yes yes Kidd antibodies seem to disappear
rapidly both in vivo and in vitro.
They are often associated with
delayed HTR.
RBC no yes yes no yes warm yes yes Jk(a-b-) RBCs are resistant to lysis by
2M urea.
RBC rare yes yes no yes warm yes yes
NRBC Yes some yes yes occ cold rare no Sometimes a pattern of unusual
agglutination sheeting occurs.
NRBC yes some yes yes occ cold no no Antigen expression for the Lewis
system may be lost during pregnancy.
NRBC yes no some yes rare cold yes no Commonly occurring antibody in
P2 individuals.
NRBC yes occ yes most few cold yes rare Anti-P may occur “naturally” in Pk
individuals; it may occur as an
auto-antibody in PCH, in which case
it is a cold reactive IgG antibody and
causes in vivo hemolysis.
NRBC yes yes yes cold rare Anti-PP1Pk may occur in p individuals
and can cause HTR and HDN.
NRBC yes some rare yes occ cold rare rare M–N–cells may also be En(a–), in
which case the cells are resistant to
invasion by P. falciparum merozoites.
NRBC yes no rare yes occ cold rare rare Anti–N–like antibodies may be
produced in renal dialysis patients
where the dialysis machine has
been sterilized with formaldehyde.
RBC some yes some occ yes warm yes yes
RBC rare yes occ occ yes warm yes yes
RBC no yes no no yes warm yes yes
NRBC yes some some yes occ cold no v.mild Lu(a–b–) cells may result from
inheritance of the recessive Lu gene
or from the dominant In(Lu)gene.
These cells are labile and hemolyze
easily on storage. The antibody
demonstrates a characteristic loose
mixed-field agglutination pattern.
RBC occ yes some yes yes warm yes mild