Immunohematology

Journal of Blood Group Serology and Education

25
Celebrating

Years
Volume 25, Number 2, 2009
 Immunohematology
Journal of Blood Group Serology and Education
Volume 25, Number 2, 2009

Contents
39 Invited Review: Milestones Series
Milestones in laboratory procedures and techniques
M.E. Reid

44 Case Report
Anti-Kpa–induced severe delayed hemolytic transfusion reaction
R. Koshy, B. Patel, and J.S. Harrison

48 Review
The ABO blood group system revisited: a review and update
J.R. Storry and M.L. Olsson

60 Case Report
Clinical evaluation for lymphoproliferative disease prompted by
finding of IgM warm autoanti-IT in two cases
R.M. Leger, F. Lowder, M.C. Dungo, W. Chen, H.M. Mason, and G. Garratty

63 Original Report
Southeast Asian ovalocytosis is associated with increased expression
of Duffy antigen receptor for chemokines (DARC)
I.J. Woolley, P. Hutchinson, J.C. Reeder, J.W. Kazura, and A. Cortés

67 Review
Sickle cell disease: a review
S.D. Roseff

75 Report
Consortium for Blood Group Genes (CBGG): 2008 report
G.A. Denomme, C.M. Westhoff, L. Castilho, and M.E. Reid for the CBGG

81 Special Dedication
W. John Judd, FIBMS, MIBiol
D. Mallory

82 Letter from the Editors

83 Announcements

84 Advertisements

88 Instructions for Authors

 Editors-in-Chief Managing Editor
Sandra Nance, MS, MT(ASCP)SBB Cynthia Flickinger, MT(ASCP)SBB
Philadelphia, Pennsylvania Philadelphia, Pennsylvania

Connie M. Westhoff, PhD, MT(ASCP)SBB Technical Editor

Philadelphia, Pennsylvania Christine Lomas-Francis, MSc
New York City, New York
Senior Medical Editor
Geralyn M. Meny, MD
Philadelphia, Pennsylvania

Associate Medical Editors

David Moolten, MD Ralph R. Vassallo, MD
Philadelphia, Pennsylvania Philadelphia, Pennsylvania

Editorial Board

Patricia Arndt, MT(ASCP)SBB Brenda J. Grossman, MD Joyce Poole, FIBMS

Pomona, California St. Louis, Missouri Bristol, United Kingdom

James P. AuBuchon, MD W. John Judd, FIBMS, MIBiol Mark Popovsky, MD

Lebanon, New Hampshire Ann Arbor, Michigan Braintree, Massachusetts

Martha R. Combs, MT(ASCP)SBB Christine Lomas-Francis, MSc Marion E. Reid, PhD, FIBMS
Durham, North Carolina New York City, New York New York City, New York

Geoffrey Daniels, PhD Gary Moroff, PhD S. Gerald Sandler, MD

Bristol, United Kingdom Rockville, Maryland Washington, District of Columbia

Anne F. Eder, MD John J. Moulds, MT(ASCP)SBB Jill R. Storry, PhD

Washington, District of Columbia Shreveport, Louisiana Lund, Sweden

George Garratty, PhD, FRCPath Paul M. Ness, MD David F. Stroncek, MD

Pomona, California Baltimore, Maryland Bethesda, Maryland

Emeritus Editorial Board

Delores Mallory, MT(ASCP)SBB
Supply, North Carolina

Editorial Assistant Production Assistant

Judith Abrams Marge Manigly
Proofreader
Lucy Oppenheim
Copy Editor Electronic Publisher
Mary L. Tod Wilson Tang

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ISSN 0894-203X
Invited Review: Milestones Series
Milestones in laboratory procedures and
techniques
M.E. Reid
W
hen I left England in 1969, hemagglutination was
done by sedimentation. In New York, I was intro-
duced to centrifugation as a method to speed the
process. Both approaches accomplished similar goals, but
one was more time efficient. Use of other techniques, such
as Western immunoblotting, cloning genes, and polymerase
chain reaction, made it possible to gain much knowledge
about blood group antigens.
Immunohematology Beginnings
In 1975, Sandy Ellisor and I from Central California
Region of the American Red Cross (ARC) and Helen Glid-
den from ARC Headquarters volunteered to co-edit the first
American Red Cross Reference Laboratory Newsletter.
The purpose of this newsletter, which was for the technolo-
gists and by the technologists, was to share techniques and
technical tips, educate, and mentor in technical writing. The
first year (1976), the newsletter was low-tech; it was simply
photocopied. That year, the ARC Reference Laboratory
Committee ran a competition and by popular vote the news-
letter was renamed the Red Cell Free Press. And to improve
its appearance it was printed. This informal newsletter was
published for 8 years, until in 1984 it evolved into Immuno-
hematology under the able editorship of Delores Mallory.1
During the 25-year life of Immunohematology, techniques
evolved that allowed the gathering of an amazing amount
of knowledge about blood groups and the membrane com-
ponents on which they are carried. Despite this evolution,
to this day we still mainly rely on hemagglutination (direct
and indirect) for detection and identification of antibodies
to blood group antigens and for testing for incompatibility.
This amazing technique is hard to beat in terms of sensitivi-
ty and specificity that are appropriate for safe transfusions.
Early Immunohematology Testing Practices
In the era before the ARC Newsletter, it was common-
place to perform excessive testing (e.g., testing at room
temperature, use of enzyme-treated RBCs at temperatures
below 37ºC, absorbing eluates from autoimmune hemo-
lytic anemia cases, and performing minor crossmatches)
on a single blood sample. Fortunately, Eloise Giblett in the
early 1960s advocated stopping unnecessary testing; she
believed testing should be done as close to physiologic con-
ditions as possible, i.e., testing at 37ºC using plasma-sus-
pended RBCs. Her reasoning was scholarly and logical and
changed the way testing was (and is) done. Giblett’s labora-
IMMUNOHEMATOLOGY, Volume 25, Number 2, 2009
tory used plasma-suspended RBCs, which neutralized most
antibodies to Lewis and Ch/Rg antigens and thus they did
not spend time identifying these clinically insignificant an-
tibodies. Abandonment of unnecessary testing dramatically
improved the efficiency of testing by manual hemagglutina-
tion and did not have a noticeable negative impact on the
efficacy of transfusion. Other changes, which occurred with
the same goal, include using anti-IgG instead of the broad-
spectrum reagent in routine antiglobulin tests, eliminating
the routine direct antiglobulin test (DAT), and not prepar-
ing eluates from all samples whose RBCs were positive in
the DAT.
Also in the late 1970s, an extraordinary number of
crossmatches were performed. Indeed, the number was ex-
cessive. The concepts of a “type and screen”2 and a “maxi-
mum surgical blood order schedule”3 were introduced.
These approaches were quickly adopted, and they reduced
not only the number of crossmatches but also the number
of RBC components reserved for a particular patient. Soon
after, the suggestion to drop the antiglobulin crossmatch
for patients who had a negative antibody screen was un-
leashed.4–6 Despite concerns that antibodies to antigens
not expressed on screening RBCs (e.g., anti-Wra) would
be missed, this practice is now commonplace. Indeed, the
physical immediate-spin crossmatch is now frequently re-
placed by a computer crossmatch.7
Use of Enhancement Reagents and Nonserologic
Techniques
Old standby techniques such as saline and albumin fell
from favor and were largely replaced by low-ionic-strength
solution (LISS) methods in the mid-1970s8 and by LISS-ad-
ditive solutions shortly thereafter. LISS rapidly gained popu-
larity because it simultaneously reduced incubation time and
increased sensitivity. In 1984, the same year that the first vol-
ume of Immunohematology appeared, a solid-phase adher-
ence assay for detection of antibody-antigen reactions was
introduced. For a review of this technology, see Beck et al.9
Subsequently, other techniques were reported and quickly
became popular; two examples are the use of polyethylene
glycol (PEG) as a potentiator of antibody-antigen reactions
in 198710 and the column agglutination technology, using
a gel as the support medium, in 1990.11 Automated assays
based on hemagglutination and manual or automated solid-
phase adherence assays are now in use, but they have not
replaced hemagglutination tests in tubes in many settings.
39
M.E. Reid
Many reagents became commercially available, and as
compliance requirements became more stringent, labora-
tories reduced the number of reagents they prepared in-
house. Safety concerns prompted many changes. Favorite
methods fell from grace, such as the use of ether—as a re-
sult of concerns about its low flash point. Also, teratogenic,
carcinogenic, and neurotoxic chemicals became less read-
ily available. Awareness of the potential hazards of work-
ing with certain chemicals and with blood samples that may
contain various pathogens led to the strict requirement to
wear personal protective equipment such as gloves, labora-
tory coats, eye protection, and closed-toed shoes. The long-
standing practice of drinking, eating, and smoking in the
laboratory was abolished.
The value of proteolytic enzymes, notably ficin, for en-
hancing the reactivity of some antibody-antigen reactions
while weakening or ablating others has been appreciated
for many years.12 Other red cell modifications such as di-
thiothreitol (DTT), trypsin, and α-chymotrypsin13 were in-
troduced. However, it took some time before the full power
of using the combination (i.e., testing RBCs treated with
ficin or papain, trypsin, α-chymotrypsin, or DTT) of these
methods to aid in antibody identification was appreciated.
Although classic antibody identification approaches (and
appropriate controls) are still necessary to name an anti-
body specificity positively, a system of parallel testing of the
same RBCs untreated and treated with different reagents
provides a valuable tool in helping to guide us in the right di-
rection, without requiring a vast library of rare reagents. 14,15
When using enzymes for this purpose, it is important
to include a negative (or auto) control, as a proportion of
human sera contain antibodies that attach to RBCs treated
with a proteolytic enzyme.
Use of Monoclonal Reagents
For many years, we were dependent on polyclonal anti-
bodies from human or rabbit sera or from plant lectins such
as Vicia graminea, Ulex europeaus, and Dolichos biflorus
for blood grouping reagents. In 1975, Köhler and Milstein
described a technique to fuse murine myeloma cells with
antibody-secreting B cells to produce monoclonal antibod-
ies.16 This technique was embraced, and monoclonal an-
tibodies were produced to highly immunogenic antigens
such as to carbohydrate human blood group antigens (A
and B, Lewis, and P1) and high copy number glycoproteins
(M, N).17 It took several years before human B cells were
used in place of mouse cells. With this advancement came
the production of monoclonal anti-D in 1983.19 Monoclo-
nal antibodies with other specificities have been made in
the last 25 years.19 They are now the predominant source
of typing reagents, and three international workshops have
been held to study reaction characteristics of numerous
monoclonal antibodies. Monoclonal antibodies have had
a major impact on our ability to define variant RBCs, espe-
cially partial D antigens, and to optimize techniques such
as flow cytometry and Western immunoblotting. Molecular
40
biology techniques have made it possible to convert murine
IgG monoclonal antibodies to human IgG or directly agglu-
tinating IgM.20
Techniques to Predict Clinical Significance of Anti-
bodies
Hemagglutination detects antibodies, but in itself does
not predict their clinical significance. Criteria such as titer,
phase of reactivity, Ig class, IgG subclass, and specificity
(and the historic knowledge of the clinical significance of an
antibody in other patients) are useful indicators but are not
unequivocally good predictors of clinical significance. The
monocyte monolayer assay (MMA) is as close to conditions
in vivo as is possible to establish in vitro and provides in-
sight into the potential clinical significance of an antibody.
It has been applied to predicting the clinical relevance of
antibodies in transfusion21 and hemolytic disease of the
newborn,22–24 but is a highly specialized and technically dif-
ficult test that belongs in a small number of laboratories.
Another technique, flow cytometry, has been used to detect
minor RBC populations in a fetal maternal hemorrhage and
to follow the survival of transfused RBCs,25,26 to determine
the dosage of a blood group antigen on RBCs,27 and quanti-
tation of RBC-bound IgG.28
Numerous techniques have been used to identify al-
loantibodies underlying autoantibodies.29–32 All are time-
consuming, as are methods to separate patient RBCs from
those of transfused donor RBCs so that the patient’s RBCs
can be phenotyped.33 Washing peripheral blood RBCs from
transfused patients with sickle cell disease with a hypoton-
ic solution is a simple and clever technique to obtain the
patient’s RBCs for typing by hemagglutination.34 Although
these techniques have value, they also have limitations.
Molecular Testing
Although hemagglutination remains the predominant
test for detecting antibody-antigen interactions, unrelated
techniques have provided insight into RBC components
that carry blood group antigens. In the decade before
Immunohematology first appeared, the Western immuno-
blotting method was used extensively to study characteris-
tics of components carrying blood groups. This approach,
together with cloning and sequencing of the genes encoding
blood group antigens,35 has revealed sequence homology to
known proteins in other cell types and provided insights
into the structure and function of the RBC membrane com-
ponents carrying blood groups (Table 1).36
The cloning and sequencing of genes laid the ground-
work for analysis of many alleles encoding variant blood
group antigens and phenotypes and, thus, our ability to test
DNA by the polymerase chain reaction (PCR) to predict a
blood group. Subsequent manufacturing of machines to
automatically perform PCR amplification made it possible
to perform this technique in a clinical laboratory setting.
PCR-based tests on DNA to predict a blood type have several
applications that provide a substitute for phenotyping.37 The
IMMUNOHEMATOLOGY, Volume 25, Number 2, 2009
 Milestones in laboratory procedures and techniques

presence of donor RBCs in a patient’s blood sample or IgG Table 2. Potential uses of DNA-based assays
on RBCs (positive DAT) does not interfere with the tests. To type patients who have been recently transfused
RBCs are not required to type a fetus at risk for hemolytic To identify a fetus at risk for hemolytic disease of the newborn
disease of the newborn (Table 2). Thus, during the life of
To type patients whose RBCs are coated with immunoglobulin (positive
Immunohematology, we have had a reversal of phenotype DAT)
and genotype. We used to phenotype RBCs to predict the To type patients with AIHA* to select antigen-negative RBCs for absorp-
genotype; now we test DNA to predict the phenotype, both tion of autoantibodies when searching for underlying alloantibodies
with the understanding that variant and null alleles exist. To type donors, including mass screening for antigen-negative donors,
If high-throughput platforms were inexpensive enough, when appropriate antisera are not readily available
DNA testing and prediction of antigen types could be used To type donors for use on antibody identification panels when antisera are
as a screening method to increase antigen-negative inven- not available
tories. Where antisera are available, negative DNA screening To type patients who have an antigen that is expressed weakly on RBCs
results could be confirmed by hemagglutination methods,
To resolve blood group A, B, and D discrepancies
thereby conserving reagents. When antisera are not avail-
To study unusual and novel serologic reactions
able, (e.g., anti-Dob, anti-Hy) the predicted type is often
more reliable than the crossmatch. The availability of *AIHA = autoimmune hemolytic anemia
increased inventories of RBC components with various
combinations of antigen-negativity would make it possible Modifications of classic hemagglutination, which in-
to more precisely match a patient’s phenotype, before the creased its sensitivity and specificity and reduced the time
patient was immunized. The technique could be used to es- it takes to perform the test, have contributed to the safe
sentially eliminate repetitive time-consuming techniques transfusion practice as we know it today. Although West-
needed to identify underlying alloantibodies in cases of ern immunoblotting and PCR-based assays have led to an
warm autoimmune hemolytic anemia and antibodies to understanding of the function and structure of RBC membrane
high-prevalence antigens. components carrying blood groups, hemagglutination
remains the workhorse of immunohematology testing.
However, despite all our advances, errors in identification
and clerical errors are still the big problem,
Table 1. Structure and function of RBC membrance components and ABO incompatibility remains a primary
carrying blood group antigens
cause of preventable transfusion-related fatali-
Function Carbohydrate Single-pass Multi-pass GPI-linked Adsorbed ties.38–40 Approximately half of the errors occur
Glycocalyx ABO MNS LE when blood is transfused to the wrong patient.
P Sample collection error, i.e., drawing blood
H
I from the wrong patient or mislabeling the
GLOB sample, is also responsible for the blood being
Structural GE DI transfused to the wrong patient.41 Hemaggluti-
RH nation in tubes does not require sophisticated
XK equipment. It is simple, inexpensive (although
Transport CO antibodies are becoming expensive and tech-
DI nologist costs constantly rise), sensitive, spe-
GIL
cific, and reproducible. It remains the basic
JK
RH method for detecting reactions between RBCs
RHAG and antibodies.
XK
Adhesion/ IN FY JMH Acknowledgments
receptor LU RAPH I thank Steve Pierce for helpful sugges-
LW
MNS
tions and Robert Ratner for help in preparing
OK the manuscript.
SC
XG
Enzyme KEL DO
YT
Complement KN CR CH/RG
regulator MSN

IMMUNOHEMATOLOGY, Volume 25, Number 2, 2009 41

M.E. Reid
References
1. Mallory D. Milestones in Immunohematology 1984-
2009, Immunohematology 2009;25:6–8.
2. Boral LI, Henry JB. The type and screen: a safe alter-
native and supplement in selected surgical procedures.
Transfusion 1977;17:163–8.
3. Friedman BA, Oberman HA, Chadwick AR, Kingdon
KI. The maximum surgical blood order schedule and
surgical blood use in the United States. Transfusion
1976;16:380–7.
4. Oberman HA, Barnes BA, Friedman BA. The risk of ab-
breviating the major crossmatch in urgent or massive
transfusion. Transfusion 1978;18:137–41.
5. Garratty G. The role of compatibility tests. (Report of
a meeting sponsored by the Bureau of Biologics for
the Blood Products Advisory Committee). Transfusion
1982;22:169–72.
6. Garratty G. Abbreviated pretransfusion testing (edito-
rial). Transfusion 1986;26:217–19.
7. Butch SH, Judd WJ, Steiner EA, Stoe M, Oberman HA.
Electronic verification of donor-recipient compatibility:
the computer crossmatch. Transfusion 1994;34:105–9.
8. Moore HC, Mollison PL. Use of a low-ionic-strength
medium in manual tests for antibody detection. Trans-
fusion 1976;16:291–6.
9. Beck ML, Plapp FV, Sinor LT, Rachel JM. Solid-phase
techniques in blood transfusion serology (review). Crit
Rev Clin Lab Sci 1986;22:317–42.
10. Nance SJ, Garratty G. A new potentiator of red blood
cell antigen-antibody reactions. Am J Clin Pathol
1987;87:633–5.
11. Lapierre Y, Rigal D, Adam J, et al. The gel test: a new
way to detect red cell antigen-antibody reactions.
Transfusion 1990;30:109–13.
12. Morton JA, Pickles MM. The proteolytic enzyme test
for detecting incomplete antibodies. J Clin Pathol
1951;4:189–99.
13. Judson PA, Anstee DJ. Comparative effect of trypsin
and chymotrypsin on blood group antigens. Med Lab
Sci 1977;34:1–6.
14. Daniels G. Effect of enzymes on and chemical modifica-
tions of high-frequency red cell antigens. Immunohe-
matology 1992;8:53–7.
15. Reid ME, Lomas-Francis C. Blood group antigens and
antibodies: a guide to clinical relevance and technical
tips. New York: Star Bright Books, 2007.
16. Kohler G, Milstein C. Continuous cultures of fused cells
secreting antibody of predefined specificity. Nature
1975;256:495–7.
17. Barnstable CJ, Bodmer WF, Brown G, et al. Production
of monoclonal antibodies to group A erythrocytes, HLA
and other human cell surface antigens: new tools for
genetic analysis. Cell 1978;14:9–20.
42
18. Crawford DH, Barlow MJ, Harrison JF, Winger L,
Huehns ER. Production of human monoclonal anti-
body to rhesus D antigen. Lancet 1983;1:386–8.
19. Anstee DJ, Parsons SF, Mallinson G, Spring FA, Judson
PA, Smythe J. Characterization of monoclonal antibod-
ies against erythrocyte sialoglycoproteins by serological
analysis, immunoblotting and flow cytometry. Rev Fr
Transfus Immunohematol 1988;31:317–32.
20. Huang TJ, Reid ME, Halverson GR, Yazdanbakhsh K.
Production of recombinant murine-human chimeric
IgM and IgG anti-Jsb for use in the clinical laboratory.
Transfusion 2003;43:758–64.
21. Nance SJ, Arndt P, Garratty G. Predicting the clinical
significance of red cell alloantibodies using a monocyte
monolayer assay. Transfusion 1987;27:449–52.
22. Nance SJ, Nelson JM, Horenstein J, Arndt PA, Platt
LD, Garratty G. Monocyte monolayer assay: an effi-
cient noninvasive technique for predicting the severity
of hemolytic disease of the newborn. Am J Clin Pathol
1989;92:89–92.
23. Lucas GF, Hadley AG, Nance SJ, Garratty G. Predicting
hemolytic disease of the newborn: a comparison of the
monocyte monolayer assay and the chemiluminescence
test. Transfusion 1993;33:484–7.
24. Hunt JS, Beck ML, Wood GW. Monocyte-mediated
erythrocyte destruction: a comparative study of current
methods. Transfusion 1981;21:735–8.
25. Nance SJ, Nelson JM, Arndt PA, Lam HC, Garratty
G. Quantitation of fetal-maternal hemorrhage by flow
cytometry: a simple and accurate method. Am J Clin
Pathol 1989;91:288–92.
26. Nance SJ, Garratty G. Application of flow cytometry to
immunohematology. J Immunol Meth 1987;101:127–
31.
27. Oien L, Nance S, Arndt P, Garratty G. Determination of
zygosity using flow cytometric analysis of red cell anti-
gen strength. Transfusion 1988;28:541–4.
28. Garratty G, Nance SJ. Correlation between in vivo
hemolysis and the amount of red cell-bound IgG mea-
sured by flow cytometry. Transfusion 1990;30:617–21.
29. Reid ME, Ellisor SS. Absorption of warm autoantibod-
ies using glutaraldehyde-treated human red cells. Am J
Med Technol 1982;48:679–84.
30. Branch DR, Petz LD. A new reagent (ZZAP) having
multiple applications in immunohematology. Am J
Clin Pathol 1982;78:161–7.
31. Reid ME, Toy PTCY. Use of preserved autologous red
blood cells to absorb warm autoantibodies from the
serum of patients receiving blood transfusion therapy.
Vox Sang 1984;46:355–9.
32. Leger RM, Garratty G. Evaluation of methods for de-
tecting alloantibodies underlying warm autoantibod-
ies. Transfusion 1999;39:11–16.
IMMUNOHEMATOLOGY, Volume 25, Number 2, 2009
 Milestones in laboratory procedures and techniques

33. Reid ME, Toy PTCY. Simplified method for recovery

of autologous red blood cells from transfused patients.
Am J Clin Pathol 1983;79:364–6.
34. Brown DJ. A rapid method for harvesting autologous
red cells from patients with hemoglobin S disease.
Transfusion 1988;28:21–3.
35. Lögdberg L, Reid ME, Lamont RE, Zelinski T. Human
blood group genes 2004: chromosomal locations and
cloning strategies. Transf Med Rev 2005;19:45–57.
36. Reid ME, Mohandas N. Red blood cell blood group
antigens: structure and function. Semin Hematol
2004;41:93–117.
37. Reid ME. Applications of DNA-based assays in blood
group antigen and antibody identification. Transfusion Marion E. Reid, PhD, FIBMS
2003;43:1748–57. Editorial Board
38. Linden JV, Paul B, Dressler KP. A report of 104 Immunohematology
transfusion errors in New York State. Transfusion
1992;32:601–6.
39. Shulman IA, Kent D. Unit placement errors: a potential
risk factor for ABO and Rh incompatible blood transfu-
sions. Lab Med 1991;22:194–6.
40. Williamson LM, Lowe S, Love EM, et al. Serious haz-
ards of transfusion (SHOT) initiative: analysis of the
first two annual reports. BMJ 1999;319:16–19.
41. Sazama K. Reports of 355 transfusion-associated deaths:
1976 through 1985. Transfusion 1990;30:583–90.

Attention: State Blood Bank Meeting Organizers

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and Education, is printed on acid-free paper.
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IMMUNOHEMATOLOGY, Volume 25, Number 2, 2009 43

Case Report

Anti-Kpa–induced severe delayed hemolytic

transfusion reaction
R. Koshy, B. Patel, and J.S. Harrison

Kpa is a low-frequency antigen occurring in less than 2 percent rectal bleeding, nausea, weakness, and dizziness. Rectal ex-
of the Caucasian population. Mild to moderate delayed hemolytic amination confirmed the rectal fissures and bleeding. There
transfusion reactions (DHTR) and hemolytic disease of the fetus were no other remarkable findings noted on physical exam-
and newborn attributable to anti-Kpa have been reported. Severe ination. ECG showed sinus tachycardia at 106 beats/min.
overt DHTR has not been reported with anti-Kpa. A case of a se-
She was admitted for further workup and for RBC transfu-
vere DHTR attributed to anti-Kpa after multiple RBC transfusions
is being reported. A 52-year-old Caucasian woman received mul- sions.
tiple units of RBCs for a lower gastrointestinal bleed. She was re- Past history was negative for prior transfusions. Patient
ferred to our institution for hepatic and renal failure, which was has an adult son who was listed as a contact person, and no
supported by laboratory findings of peak LDH, bilirubin, BUN, other pregnancy history was available. She had left renal
and creatinine elevations. Hemoglobin had dropped on Day 10 angioplasty several years ago for left renal artery stenosis.
after transfusion. The DAT and antibody screen (ABS) were nega- She had a history of rectal fissures. She is a smoker (2 packs
tive. Initial workup and subsequent ABS were negative. Anti-Kpa per day) and drinks occasionally. She has a history of al-
was identified when an additional RBC panel was tested. One of lergy to amitriptyline.
the RBC units transfused was incompatible by antihuman globu-
lin (AHG) crossmatch with the patient’s plasma and typed positive
for Kpa. DHTR was confirmed after extensive workup. The patient
responded to supportive therapy and experienced an uneventful
recovery. DHTR may not be considered when DAT and ABS are
negative. However, correlation of recent transfusion with signs
and symptoms should alert the clinician to entertain and investi-
gate a DHTR that should include the AHG crossmatch of all impli-
cated RBC units. The severity of the reaction also raises concerns
as to when and what antigen specificity should be considered for
inclusion in the antibody screening cells.
Immunohematology 2009;25:44–47.

K
pa (KEL 3, Penney) is one of 31 antigens in the Kell
(ISBT symbol, KEL) blood group system. The Kell
antigens, encoded by KEL, located on chromosome
7q33, appear to be erythroid specific and are found in the
Fig. 1 Hemoglobin (Hb; g/dL) and hematocrit (Hct; %) values for day
fetal liver and in bone marrow cells.1 Kpa is a low-incidence
0 to day 13 are shown.
antigen found in less than 2 percent of Caucasians.1,2 The
antigen is resistant to the effects of enzyme treatment but is
sensitive to treatment with dithiothreitol and acid. Mild to
moderate delayed hemolytic transfusion reaction (DHTR)
and mild to moderate hemolytic disease of the fetus or
newborn (HDFN) have been reported as well as a case of
hydrops fetalis attributed to anti-Kpa.3 The antibody is an
IgG.4–6 Suppression of erythropoiesis by anti-Kpa has been
reported as the cause of decreased hemoglobin in HDFN.7
Reports of an overt DHTR attributable to several antibodies
missed in the antibody screen and immediate-spin cross-
match mention anti-Kpa as one of the antibodies.8 However,
there have been no reported cases of severe overt DHTR as
a result of anti-Kpa as per MEDLINE search.

Case Report Fig. 2 Alkaline phosphatase (Alk Phos), lactate dehydrogenase (LDH),
A 52-year-old Caucasian woman presented to the emer- and creatine phosphokinase (CPK) values for day 0 to day 14 are
gency room complaining of being light-headed and having shown.

44 IMMUNOHEMATOLOGY, Volume 25, Number 2, 2009

 Anti-Kpa–induced DHTR

Table 1. Laboratory data from the referring hospital transfused. Urinalysis on Day 11
Tests Normal range Patient results showed evidence of increased free
Admission Day 2 Day 7 Day 10 Day 11 Day 12 hemoglobin, bilirubin, and uro-
Hemoglobin 4.5–11 g/dL 6.1 12 — 8.7 10.2 9.9
bilinogen. Computed tomographic
scan and magnetic resonance im-
Hematocrit 36%–48% 19.6 — — 26.9 31.2 29.3
aging to rule out acute cholecystitis
WBC 4.5–11.0 x 9.9 — — — — — revealed an enlarged gallbladder
109/dL
with thickened wall and inflamma-
Platelets 120,000– 628,000 — — — — —
tion of adjacent areas, reported as
450,000/µL
suspicious for cholecystitis. Liver,
Reticulocytes 0.9%–1.9% — — — — 4.1 —
spleen, and pancreas were within
PT 11.5–14.7 s 13.8 — — 13.5 15.2 18.1 normal limits. There was also evi-
Glucose 65–100 mg/dL 125 — — — dence of atrophic left kidney and
Alkaline phosphatase 30–133 U/L 138 142 203 211 422 393 hypertrophic right kidney with se-
BUN 7–21 mg/dL 18 — 18 36 46 51 vere stenosis of a segment of the
right renal artery close to its origin.
Creatinine 0.5–1.4 mg/dL 0.8 — 1.0 4.2 5.6 6.2
The patient received 3 additional
AST 5–39 U/L 31 — — 161 529 303
units of RBCs between Days 10 and
ALT 7–56 U/L 19 — — 113 168 120 11 for the drop in hemoglobin.
LDH 333–699 U/L — 521 3983 — 7905 2215 On the 13th day of her hospital
7637 course, she was admitted to the re-
CPK 35–230 U/L 87 425 — — — — ferral hospital, our institution, for
Total bilirubin 0.2–1.3 mg/dL 0.3 — — 1.8 23.5 18.3 investigation of acute liver and re-
Direct bilirubin 0.0–0.4 mg/dL — — — — 24.7 16.1 nal failure. DHTR was suspected,
ABO, D — O — — O O —
and a workup was ordered by the
D positive D positive D positive hematologist. Pigment nephropa-
Antibody screen — Negative — — Negative Negative —
thy attributable to intravascular
hemolysis was considered as the
DAT — — — — — Negative —
cause for the acute renal failure.
HBsAg, HBc antibody, HBs antibody, and hepatitis C antibody—all nonreactive The cause of the severe hepatotox-
Urinalysis — — — — — icity could not be ascertained.
Bilirubin Negative Moderate
Urobilinogen <1 mg/dL 4 mg/dL
Free hemoglobin Negative Large Materials and Methods
At the referral hospital, ABO and
ALT = alanine aminotransferase; AST = aspartate aminotransferase; BUN = blood urea nitrogen; D typings were performed using the
CPK = creatine phosphokinase; HBc antibody = hepatitis B core antibody; HBs antibody = Ortho A/B/D Monoclonal and Re-
hepatitis B surface antibody; HBsAg = hepatitis B surface antigen; PT = prothrombin time; WBC verse Grouping Card (Ortho-Clinical
= white blood cell count. Diagnostics, Inc., Raritan, NJ). Affir-
magen reagent RBCs (pooled cells),
Admission Hb was 6.1 g/dL. She was transfused with 0.8%, were used for reverse typing, and antibody screen was
5 units of type-specific, leukoreduced or washed (as per done by IgG gel card, 0.8% Surgiscreen (Johnson & Johnson,
hospital policy), immediate-spin, crossmatch-compatible New Brunswick, NJ). Antibody panel was performed using
RBC during the course of 24 hours of admission, which 0.8% Resolve Panel A (Ortho-Clinical Diagnostics, Inc.) and
raised her Hb to 12 g/dL. She remained hospitalized for Panocell 16, by Immucor, Inc. (Norcross, GA). The DAT was
further evaluation for gastrointestinal bleed. On the 10th performed using anti-IgG-C3d with check cells, anti-IgG and
posttransfusion day, her Hb dropped to 8.7 g/dL and her check cells, and anti-C3b/C3d with complement check cells
Hct was 26.9%. Her reticulocyte count was 4.1% (reference (Immucor, Inc.). Reagents used for phenotyping were from
range, 0.9 to 1.9%) on Day 11. There was no evidence of con- Immucor, Inc. Elution was performed with Gamma ELU-KIT
tinued rectal bleed. There was a gradual increase of several II (Gamma Biologicals, Inc., Houston, TX) following initial sa-
chemistry results, which peaked on the 10th and 11th post- line wash (Fisher Diagnostics, Middleton, VA) of patient cells.
transfusion days (Table 1; Figs. 1–4). The patient appeared ABO and D typings and a three-cell antibody screen were
severely jaundiced with signs of acute liver and renal fail- performed by gel technique and read through the MTS reader
ure, which was supported by her laboratory values. Investi- SA (Ortho-Clinical Diagnostics, Inc.). DAT was performed
gation for a suspected DHTR revealed a negative DAT and with polyclonal AHG, anti-IgG, and anti-C3d by tube method
a negative antibody screen. Investigation did not proceed and eluate panel by gel method using Ortho and Immucor
to antihuman globulin (AHG) crossmatch for all RBC units panel cells.

IMMUNOHEMATOLOGY, Volume 25, Number 2, 2009 45

R. Koshy et al.

Results
The DHTR investigation on the day of admission at the
referral hospital revealed a negative antibody screen with
the three-cell antibody screen. The DAT was negative (in-
cluding a 15-minute incubation) with polyspecific, IgG, and
C3b/C3d AHG. At the referral hospital, tests with a 16-cell
reagent RBC panel (Immucor, Inc.) revealed the presence
of anti-Kpa. All other clinically significant antibodies were
ruled out. Eluate prepared from DAT-negative RBCs was
negative against Kp(a+) RBC on Immucor Panocell 16. The
patient typed negative for Kpa. AHG crossmatch was done
Fig. 3 Total and direct bilirubin values for day 0 to day 20 are shown.
from samples obtained from donor segments of the trans-
fused RBC units received from the blood supplier. One of
the five RBC units transfused on Day 1 of admission was in-
compatible with the patient’s plasma and typed positive for
Kpa. Haptoglobin was 1 mg/dL (reference range, 34–200
mg/dL). A diagnosis of severe DHTR with intravascular
hemolysis attributable to anti-Kpa was confirmed. As the
patient’s renal and liver functions improved, she did not
undergo dialysis or consideration for further liver or renal
evaluation.
The patient was monitored and maintained on support-
ive therapy. Her hospital course was uneventful. All labora-
tory results returned to normal values (Figs. 1–4). She was
discharged 10 days after her admission to the referral hos- Fig. 4 Alanine aminotransferase (ALT) and aspartate aminotrans-
pital. ferase (AST) values for day 0 to day 20 are shown.
Table 2 presents the patient’s laboratory data on ad-
mission to the referral hospital on Day 13.
Table 2. Laboratory data on admission at the referral hospital, Day reside on the C-terminal domain of Kell in the structural
13 sequence N-terminal to the zinc-binding catalytic motif,
Test Result which is the major site for endothelin-3–converting enzyme
activity.
Haptoglobin (reference range 1 mg/dL
34–200 mg/dL) Kell antigens are important in transfusion medicine
owing to their strong immunogenicity, and the correspond-
ABO, D (by Ortho gel) O, D positive
ing antibodies are clinically significant.9 Anti-Kpa has been
Antibody screen (0.8% Surgiscreen) Negative
implicated in mild to moderate HDFN and DHTR. Severe
Panel cells, A Negative DHTR as presented in this case has not been reported.
Panocell 16 Positive (anti-Kpa identified) Clinical signs and the laboratory values presenting on Day 7
Kp typing of patient’s RBCs
a
Negative with peak laboratory values at Days 10 and 11 after transfu-
DAT—using polyclonal, IgG, and Negative sion of a unit of RBCs positive for Kpa support a diagnosis
C3b/C3d AHG in this case of a severe DHTR with intravascular hemoly-
Panel with eluate Negative sis attributable to anti-Kpa. The cause of the primary Kpa
exposure could not be determined from the patient’s his-
Crossmatch with 1 of 5 units trans- Incompatible, Kp(a+)
fused at referring hospital tory. AHG crossmatch of all implicated RBC units despite
Kpa typing of incompatible unit Kp(a+): 1+ by tube and 2+ by gel
the negative DAT or antibody screen would have positively
techniques identified the incompatible Kp(a+) RBC as the implicated
unit for the DHTR.
Discussion The patient under discussion experienced unusually
Kpa was first described by Allen and Lewis in 1957.2 The severe hepatotoxicity, and the reasons for this remain un-
Kpa antigen is a member of the Kell blood group system and clear: there was no prior history of liver disease. Severe
is carried on the Kell glycoprotein. The prototypical gene hemolysis with resultant high levels of free heme can cause
for the Kell protein family was cloned and characterized in undesirable toxicity, leading to organ, tissue, and cellu-
the early 1990s.9 As discussed by Lee and colleagues,9 the lar injury. The mechanism of action is through free heme
antigens of the Kell blood group system result from single that catalyzes the oxidation, covalent crosslinking, and ag-
nucleotide changes in the Kell protein. Most Kell antigens gregate formation of protein and its degradation to small

46 IMMUNOHEMATOLOGY, Volume 25, Number 2, 2009


peptides. It also catalyzes the formation of cytotoxic lipid
peroxide via lipid peroxidation and damages DNA through
oxidative stress. Heme, being a lipophilic molecule, inter-
calates in the membrane and impairs lipid bilayers and or-
ganelles, such as mitochondria and nuclei, and destabilizes
the cytoskeleton. It also causes endothelial cell injury, lead-
ing to vascular inflammation, and stimulates the expression
of intracellular adhesion molecules. As a proinflammatory
molecule, heme induces inflammation that results in toxic
effects on the kidney, liver, central nervous system, and car-
diac tissue.10 The severe heme toxicity may also be attribut-
able to the markedly compromised renal condition owing to
the severe stenosis of the right renal artery.
The severity of the DHTR in this case may warrant con-
sideration for inclusion of clinically significant low-frequency
antigens that may be missed by current screening cells for
detection of clinically significant RBC antibodies. How-
ever, the risk of overt DHTRs to low-incidence antigens is
estimated at 1 per 650,000 crossmatches.5 The calculation
was based on report of probability of acute overt hemolytic
transfusion reaction at 1 per 260,000 with immediate-spin
crossmatches of 1.3 million negative antibody screens.8 As
the risk of overt DHTR is extremely small, the cost benefit
should be considered for inclusion of low-frequency an-
tigens such as Wra, Cw, and Kpa in the antibody screening
cells.5 We concur with the previous observation. AHG phase
crossmatch must be included in the investigation of any
suspected DHTR irrespective of a negative DAT or nega-
tive antibody screen. Appropriate and timely patient care to
avert further patient harm depends on a thorough investi-
gation.
References
1. Roback, JD, ed. Technical Manual. 16th ed. Bethesda,
MD: American Association of Blood Banks, 2008.
2. Allen FH Jr, Lewis SJ. Kpa (Penney), a new antigen in
the Kell blood group system. Vox Sang 1957;2:81–7.
IMMUNOHEMATOLOGY, Volume 25, Number 2, 2009
Anti-Kpa–induced DHTR
3. Smoleniec J, Anderson N, Poole G. Hydrops fetalis
caused by a blood group antibody usually undetected
in routine screening. Arch Dis Child Fetal Neonatal Ed
1994;71:F216–7.
4. Pinkerton PH, Coovadia AS, Goldstein J. Frequency of
delayed hemolytic transfusion reactions following an-
tibody screening and immediate-spin crossmatching.
Transfusion 1992;32:814–17.
5. Garratty G. How concerned should we be about miss-
ing antibodies to low incidence antigens? Transfusion
2003;43:844–47.
6. Reid ME, Lomas-Francis C. The blood group antigen
factsbook. San Diego, CA: Academic Press, 1997:182–
4.
7. Tuson M, Koyal K, Desai R, et al. Probable suppression
of fetal erythropoiesis by anti-Kp (abstract). Transfu-
sion 2007;47(3 Suppl):166A.
8. Shulman IA. The risk of an overt hemolytic transfusion
reaction following the use of an immediate spin cross-
match. Arch Pathol Lab Med 1990;114:412–14.
9. Lee S, Zambas ED, Marsh WL, Redman CM. Molecular
cloning and primary structure of Kell blood group pro-
tein. Proc Natl Acad Sci U S A 1991;88:6353–7.
10. Kumar S, Bandyopadhyay U. Free heme toxicity and
its detoxification systems in human. Toxicol Lett
2005;157:175–88.
Ranie Koshy, MD (corresponding author), Director, Blood
Bank and Transfusion Services, and Bhishma Patel, SBB,
Blood Bank and Transfusion Safety Officer, Department
of Pathology and Laboratory Medicine, NJMS/UMDNJ,
C101, University Hospital, Newark, NJ 07103; and Jona-
than S. Harrison, MD, Department of Medicine, Hema-
tology, Robert Wood Johnson Medical School—Pisc/New
Brunswick, New Brunswick, NJ.
47
Review

The ABO blood group system revisited: a

review and update
J.R. Storry and M.L. Olsson

The antigens of the ABO system were the first to be recognized as
blood groups and actually the first human genetic markers known.
Their presence and the realization of naturally occurring antibod-
ies to those antigens lacking from the cells made sense of the
erratic failure of blood transfusion hitherto and opened up the
possibility of a safe treatment practice in life-threatening blood
loss. Although initially apparently simple, the ABO system has
come to grow in complexity over the years. The mass of knowl-
edge relating to carbohydrate chemistry, enzymology, molecular
genetics, and structural and evolutionary biology is now enormous
thanks to more than a century of research using ABO as a principal
model. This has provided us with data to form a solid platform
of evidence-based transfusion and transplantation medicine used
every day in laboratories and clinics around the globe. This review
aims to summarize key findings and recent progress made toward
further understanding of this surprisingly polymorphic system.
Immunohematology 2009;25:48–59.
Key Words: ABO, blood group, antigen, allele, genotype
Variation in A antigen expression was also recognized
very early in the twentieth century (reviewed in Race and
Sanger10), and the A blood group was divided into A1 and
A2.1,11 Other descriptions of weakened A antigen expression
followed, and the A blood group was subdivided further
based on characteristic reactivity with human polyclonal
antisera, i.e., strength of reactivity and presence of mixed
field agglutination; presence of anti-A1; and whether A or H
blood group substance was present in the saliva of secretor
subjects (Table 1). Weak forms of B antigen were also found
but are typically more difficult to define serologically into
specific categories although some subgroups (e.g., Bel) are
analogous to their A counterparts (Ael).
The frequency of the common ABO phenotypes (A1, A2,
B, A1B, A2B, and O) varies greatly among different popula-
tions.12,13 Populations with a high frequency of A phenotype
are found mainly in Northern and Central Europe, and the

T
Table 1. Subgroups of A—agglutination reaction patterns adapted
here have been many reviews of the ABO blood group
from textbooks
system written throughout the years, covering dif-
ferent aspects of this fascinating topic. A limited se- Sub- Reactions* with Sub- Anti-
group stances A1 in
lection of such reviews can be found in the reference list, Anti-A Anti-A,B Anti-A1 Anti-H
of A in saliva† serum
particularly for readers who want to focus more on the dis-
A1 ++++ ++++ ++++ 0 A, H No
covery,1 biochemistry,2,3 enzyme structure,4 and molecular
A2 ++++ ++++ 0 ++++ A, H Some-
genetics.5,6 Our intention is not to reproduce them but to times
follow the guidelines of this new series of blood group sys-
Aint ++++ ++++ ++(+) +++ A, H No
tematic reviews in Immunohematology to provide a brief
introduction to this amazingly complex blood group system. A3 ++(+) mf
++(+) mf
0 ++++ A, H Some-
times

History Ax 0/+ ++(+) 0 ++++ H Often

The discovery of the ABO blood group system ranges Aend + + 0 ++++ H Some-
from myth and folk legend all the way to the Nobel Prize. Karl times
Landsteiner, a Viennese pathologist, made the observation Am 0/+ 0/+ 0 ++++ A, H No
that when his serum and that of five colleagues were mixed Afinn + + 0 ++++ H Yes
individually with their saline-suspended RBCs, agglutina- Abantu +(+) +(+) 0 ++++ H Yes
tion was observed with some mixtures but not with others. A1ae 0‡
0 +++** ++++ H Yes***
He reported this as a footnote to a paper published in 19007
Ay 0‡ 0 0 ++++ A, H No
followed by a more comprehensive paper in 1901.8 Transla-
tions of both papers can be found in the review by Camp Ael 0‡
0 0 ++++ H Some-
times
and Ellis.1 In these early studies, he showed that two each
of the six sera discriminated among three blood groups: A, *A negative reaction is denoted by 0. Positive reactions are denoted
as from + (weak agglutination) to ++++ (maximal agglutination).
B, and C (later renamed O from the German ohne, meaning **Dolichos biflorus only.
without). Thus, Landsteiner demonstrated that a person’s ***Serum reactivity against both A1 and A2 RBC.
serum contained antibodies to the antigen(s) lacking from †Blood group ABO substances in saliva and other body fluids of
their RBCs. The fourth blood group, AB, was described a secretors.
year later by Decastello and Sturli9 in four individuals in a
‡
Despite lack of agglutination, anti-A can be adsorbed to and eluted
from cells in this subgroup.
larger study of 34 healthy subjects and 121 patients. mf = mixed field agglutination.

48 IMMUNOHEMATOLOGY, Volume 25, Number 2, 2009

 ABO blood group system

A2 phenotype reaches its peak among the Lapps in North-
ern Scandinavia but is very rare in Asia. The B phenotype
is most frequent in Central Asia and almost absent in Am-
erindians. Blood group O is the most frequent phenotype
in a global perspective, with Native American Indians be-
ing almost exclusively blood group O. Parts of Africa and
Australia also show high frequencies of blood group O. The
reason for the differences observed among populations is
not fully understood although several theories have arisen.
The concept of evolutionary selection based on pathogen-
driven blood group changes will be discussed later (see
section on pathogen interactions). The early importance of
ABO diversity is supported by reports in which the group
O-defining single base pair deletion at nucleotide position
261 (see section on molecular genetics) has been found in
both Neandertal people14 and ancient Egyptian mummies,15
suggesting that selection pressure and survival of the fittest
were indeed early features during our co-evolution with
pathogens like malaria parasites and many others.
Nomenclature
The ABO system was the first to be discovered and has
therefore been given the number 001 in the official Inter-
national Society of Blood Transfusion (ISBT) terminology.
Nomenclature for the ABO antigens is actually straightfor-
ward since the antigen status is determined by the pres-
ence or absence of specific carbohydrate molecules. This is
particularly true for the A and B antigens but may be less
clear for the two other antigens (A,B and A1) given official
antigen status by the ISBT. The A,B antigen is thought to
be an epitope not involving the A versus B–differentiating
surfaces, but consists of a common recognition motif16
found when either the A or B antigens are present. The de-
bate surrounding the real identity of the A1 antigen is still
ongoing,17,18 but the A1 phenotype reveals many differences
compared with A2, both quantitative and qualitative, so the
question is quite complex (see section on biochemistry).
Table 2 shows both the numerical and traditional nomen-
clature according to the ISBT Working Party on Terminol-
ogy for Red Cell Surface Antigens.19
Table 2. ABO nomenclature
System ABO antigen notation
ISBT number 001 ABO1 ABO2 ABO3 ABO4
(001001) (001002) (001003) (001004)
ISBT name ABO A B A,B A1
dbRBC Web site (http://www.ncbi.nlm.nih.gov/projects/
gv/rbc20). In this review, alleles will be referred to in the
traditional way but with dbRBC terminology given in brack-
ets, e.g., A1 [A101] and O1 [O01].
It can be specifically noted that it is important for clar-
ity and consistency to use subscripts, superscripts, and
italics appropriately. For example, A1, A1, and A1 mean the
antigen, the phenotype, and the allele, respectively.
Inheritance and Molecular Genetics
The A and B antigens are inherited as Mendelian char-
acteristics in a codominant autosomal fashion. In 1908, Ep-
stein and Ottenberg21 were the first to suggest in a short case
report that ABO blood groups might be inherited. This was
later proved by von Dungern and Hirszfeld in 1910 (trans-
lated by Pohlmann22). In fact, ABO inheritance was one of
the first genetic markers to be used in paternity testing and
in forensic medicine.23,24
Unlike the majority of blood groups, the antigens of the
six currently known carbohydrate systems are not coded
by genes directly. Instead, these blood group genes encode
glycosyltransferases that in turn synthesize the oligosac-
charide epitopes (Fig. 1). Thus, the A and B antigens are
made by A and B glycosyltransferase, respectively, encoded
by the ABO gene carried on the long arm of chromosome 9
(9q34). As with many of the blood group genes, the position
of the ABO locus was known for many years before the gene
was cloned.25 The genes encoding the A-synthesizing 3-α-N-
acetylgalactosaminyltransferase and the B-synthesizing
3-α-N-galactosaminyltransferase were cloned by Yamamo-
to and colleagues in 199026,27 after purification and partial
amino acid sequencing of A transferase from lung tissue.28
Probing cDNA libraries obtained from human adenocar-
cinoma cell lines of different ABO types, the main alleles
were defined.26 They determined that the B-specific mRNA
differed from the A-specific gene by only 7 of 1062 coding
nucleotides, of which 4 result in amino acid differences in
the enzyme product. The difference between the A1 [A101]
and O1 [O01] genes was shown to be a single guanosine (G)
deletion, which alters and severely truncates the open reading
frame (ORF) as shown in Figure 2. The A2 phenotype was
shown to depend on a cytosine deletion in the 5′ end of the
gene, resulting in elongation of the ORF.29 The organization
of the ABO locus30,31 is shown in Figure 3. The gene consists
O H O H

Fig. 1 Principal C H 2O H
O
C H 2O H
O
O H
O H

structure of the A, B, HO C H 2O H HO C H 2O H

Blood group allele nomenclature including ABO is un- O O

A cN H O H

and H oligosaccha- O O

der consideration by a subcommittee of the aforementioned rides. The difference

O O H O O H

CH 3

ISBT Working Party. In the absence of an officially agreed-

CH 3 O
O

between the A and HO

O H
HO
O H

on terminology, several different variants have evolved (see B structures is high- O H O H

O H

Table 7 in Chester and Olsson6). Typically, alleles are re- lighted by arrows.
A tr is a c c h a r id e B t r is a c c h a r id e
C H 2O H
O

ferred to by their serologic activity and a number. An un- HO

O O H

official but often used terminology is found at the Blood CH 3 O

O H

Group Antigen Gene Mutation Database, also known as the O H

HO

H d is a c c h a r id e

IMMUNOHEMATOLOGY, Volume 25, Number 2, 2009 49

J.R. Storry and M.L. Olsson
of seven exons (plus an alternative exon 1a located upstream
of exon 132), with the majority of the catalytic domain of the
enzyme encoded by exon 7.
Since the groundbreaking cloning paper, 215 ABO se-
quence entries have been submitted to the dbRBC Web
site20 (accessed on April 15, 2009) and curated by the dbR-
BC staff and experts, but new alleles are constantly being
identified. Figure 4 shows a breakdown of the 181 alleles
in the dbRBC categorized by their association with normal
or altered phenotypes: these include 65 different A alleles,
47 B alleles, 58 O alleles, and 11 “AB” alleles. The remain-
ing 34 sequences listed in the current dbRBC consist of 20
sequences encompassing the 5′ noncoding region including
the repetitive and polymorphic CCAAT box binding fac-
tor/nuclear factor Y (CBF/NF-Y) motif and 14 intronic or
overlap sequences. Of the A alleles, 6 and 11 encode nor-
mal A1 or A2 phenotypes on RBCs, respectively, whereas 48
describe mutated A alleles (Aweak) associated with different
variants of weak A antigen expression (Table 1); the B al-
leles include 9 normal and 38 weak alleles; and 11 alleles
261
261 467 467 526
526 703* *
703 1060
1060
A1
A2
B
O1
O2
Fig. 2 Schematic representation of predicted open reading frames
for some common ABO alleles. White rectangles represent trans-
lated A1 [A101] consensus; black rectangles represent translated
non-A1 consensus (nonsense). Vertical bars indicate nucleotide
(nt) positions (given above bars) of mutations leading to amino acid
changes. * indicates B [B01] allele is mutated at nt 796 and 803;
O2 [O03] allele at nt 802.
Fig. 3 Organization of the ABO gene. A: The seven exons and six
introns are not drawn to scale. The numerals above boxes represent
the first and last nucleotides (nt) of the coding region in each exon,
and those below boxes show the corresponding amino acid (a.a.)
numbers. The size of each intron is indicated with a thin oblique bar.
B: The ABO gene drawn to scale (except intron 1); exons are black
and introns gray.
50
have been described that encode glycosyltransferases ca-
pable of synthesizing easily detectable levels of both A and
B antigens. This group includes alleles conveying the two
unusual phenotypes cisAB and B(A). Fifty-eight alleles are
predicted to give rise to proteins without enzymatic activity
of which 45 contain 261delG, the mutation that alters the
translational ORF and predicts a shortened protein product
with no transferase activity. This large group of O alleles
includes four principally important evolutionary lineages:
O1 [O01], O1v [O02], Otlse09 or O1(467T;318T) [O09], and O1bantu
[O54].33,34 Numerous minor variants of these main alleles35
as well as hybrid O alleles combining A2 or B with different
261delG-containing O sequences36 have also been found,
most notably in individuals of African ancestry. Of the re-
maining 13 “nondeletional” alleles, 3 suffer similar fates to
that caused by 261delG because they contain nonsense mu-
tations resulting in altered ORFs caused by nucleotide in-
sertions. Another 3 have nonsense mutations introducing
immediate stop codons, thereby truncating the ORF at the
same codon where they occur. Finally, 7 recorded nonde-
letional O alleles are crippled by at least one missense mu-
tation each, giving rise to critical amino acid substitutions,
the most famous example of which is 802G>A resulting in
268Arg. For a recent review about this latter group of inter-
esting O alleles designated O2 [O03], see Yazer and Olsson.37
What is most intriguing about these nondeletional O alleles
is that they are all attributable to mutations in A allele back-
bone sequences. This has two main practical consequences:
first, virtually all ABO genotyping methods will signal the
70
60
13
50
alleles
40 48
Unusual/weak
Unusual/weakphenotype
alleles
phenotype
Common/expected
Commo/expected phenotype
Number ofof
phenotype
30
Number
38
Number of alleles
45
20
10
17
9 11
0
A B O AB
Blood
B lo o d blood
g ro u pspecificity
s pe cificity
Fig. 4 Graphic representation showing the number of ABO alleles
currently listed in the dbRBC database. The black bars represent al-
leles that encode a glycosyltransferase with normal activity; the white
bars represent alleles encoding glycosyltransferases that have altered
activity or specificity. The AB bar includes alleles encoding glycosyl-
transferases capable of synthesizing both A and B antigens, i.e., cisAB
and B(A). The group O bar has been divided into the 45 O alleles
that contain the mutation 261delG (black) and the 13 “nondeletional”
alleles without it (white). The latter group is often associated with weak
expression of A antigen.
IMMUNOHEMATOLOGY, Volume 25, Number 2, 2009

presence of A1 or A2 alleles unless specifically designed
to detect rare O alleles, which will result in the potentially
catastrophic prediction of group A in a group O person.
Second, the phenotype actually conveyed by inheritance of
one of these so-called O alleles is not always O but often
weak A or O-like but without anti-A present in plasma. The
reason for this is currently unclear, but there has recently
been a series of papers investigating the serologic pattern
and mechanisms behind this interesting phenomenon.38–42
There is currently no O allele described that is based on a B
sequence, but we predict that when such an allele is found,
it will show the opposite serology, i.e., either weak B expres-
sion or O-like without anti-B in plasma.
It is the molecular genetics that make this system so fas-
cinating because mutations ranging from single nucleotide
changes to more complex hybrid gene formations can alter
the specificity of the enzyme, the efficacy of the enzyme, or
both; changes that, at the phenotypic level, are manifested
simply by altered antigen expression. Good examples are
the various molecular bases of the Ax phenotype, which can
either result from a single missense mutation in exon 7 of
an A1 [A101] allele or be based on different hybrids, for in-
stance between the 5′ end of B and the 3′ end of O alleles.43,44
The B3 phenotype in the Taiwanese population has also
been shown to result from different molecular backgrounds,
one a missense mutation but more commonly, a principally
interesting mutation that was the first one shown to affect
splicing of ABO mRNA and cause exon skipping.45 In ad-
dition to altered specificity or enzymatic efficacy, aberrant
intracellular trafficking of ABO transferase may cause weak
subgroup phenotypes.46
As alluded to previously, this added layer of complexity,
in which mutations affect enzyme activity and not the anti-
gen directly, provides difficulties in designing genotyping
assays that will detect rare variants, which if misinterpreted
can have serious consequences in ABO group determina-
tion.47
There are problems associated with all the more than
30 ABO genotype screening methods published or commer-
cially available so far (reviewed in Olsson and Chester48).
The vast majority is only designed to determine between
three and six of the common alleles, although additional
alleles can often surface if their polymorphisms interfere
with the detection system. However, virtually all methods
will fail to predict the phenotype of a sample in the pres-
ence of most A/B subgroup alleles, rare nondeletional null
alleles, cisAB and B(A) alleles, or hybrid alleles, which can
lead to serious consequences. Because this is particularly
dangerous if blood or transplant recipients are typed (as
suggested, e.g., by Procter et al.49) we recently developed
and implemented an improved ABO genotype screening
method that addresses these problems and can be used in a
clinical laboratory setting.47 In summary, the complex genet-
ics of ABO is a major challenge for ABO genotyping efforts,
not least because of the disturbing fact that a certain allele
can lead to more than one phenotype and seemingly identi-
IMMUNOHEMATOLOGY, Volume 25, Number 2, 2009
ABO blood group system
cal phenotypes can have more than one molecular genetic
background.
The regulatory mechanism of the ABO gene has been
investigated extensively. An enhancer element located ap-
proximately 4 kbp upstream of exon 150 was found to con-
tain four 43-bp repeats in all alleles except A1 [A101] and the
infrequent O2 [O03], which have only one copy.51 This may
play a role in expression.52 The enhancer region contains
a CBF/NF-Y binding site; mutations in this site decrease
enhancer activity in a gastric cancer cell line,50 and altera-
tions in this region may even cause rare B subgroup pheno-
types.53 However, it was recently shown that A1 [A101] or A2
[A201] transcripts are virtually undetectable in peripheral
blood whereas B and O (including O2 [O03]) mRNA is read-
ily found.54,55 This appears to speak against a critical role for
CBF repeats in erythroid ABO regulation, but more work is
required. There is also an Sp1-binding site in the proximal
promoter that may be important for erythroid ABO regula-
tion.56
Although much is understood regarding the cause of
weak A/B antigen expression on RBCs when it comes to in-
herited weak ABO subgroups,5,6 less is known about altered
ABO expression in hematologic disorders. Erythrocytes los-
ing A, B, or H antigen have been noted in patients with he-
matologic malignancy, especially in the myeloid lineage.57,58
Very recently it was suggested that methylation of the ABO
proximal promoter is the reason for such leukemia-associated
downregulation.59 Reduced A and B antigen expression in
bladder and oral carcinomas is partially attributable to loss
of heterozygosity or hypermethylation.60,61 Furthermore,
urothelial tumor tissue contains decreased amounts of A/B
antigens that correlated to decreased levels of ABO mRNA
compared with cells from normal tissue.62 Transient depres-
sion of A antigens has also been observed in some pregnant
women,44 but the reason for this is still unknown.
Biochemistry
The biochemistry of the A and B antigens was elucidat-
ed by the astonishingly early and brilliant work from the
groups of Morgan and Watkins, and Kabat (reviewed by
Watkins2 and Kabat63). The A, B, and H determinants were
hypothesized to reside on water-soluble glycoproteins able
to inhibit agglutination of RBCs by antibodies or lectins.
A precursor substance, H, was hypothesized as a building
structure for A and B, and the terms O- (or H-) substance
and anti-H were introduced in 1948.64
Owing to difficulties in obtaining sufficient quantities of
blood group active material after extraction from RBCs, most
of the early studies were performed on ovarian cyst or animal
mucin, rich in secreted blood group substances. Before isola-
tion and chemical identification of these substances, inhibi-
tion with simple sugars indicated N-acetyl-d-galactosamine
(GalNAc), d-galactose (Gal), and l-fucose (Fuc) as the defin-
ing sugars in blood groups A, B, and O, respectively.65,66
Independent of the blood group of the individual
investigated, a surprisingly similar composition of carbo-
51
J.R. Storry and M.L. Olsson
hydrate residues (mainly Fuc, Gal, GalNAc and N-acetyl-
d-glucosamine [GlcNAc]) was found. This was taken as a
sign of large similar precursor molecules carrying small
residues that differentiate blood groups.67 The minimal
determinant structures were subsequently shown to be
trisaccharides as shown in Figure 1. The important concept
of precursor-product relationship between H and A/B was
formulated,67,68 and the critical role of nucleotide-bound
sugars as substrates in oligosaccharide synthesis was appre-
ciated.69 The biosynthetic pathway of the ABO blood group
structures is as outlined in Figure 5, and the presence of
A and B glycosyltransferases was first predicted70 and then
experimentally established.71–73 Thus, the A and B glycosyl-
transferases use UDP-GalNAc and UDP-Gal, respectively,
as substrates. Both require the H determinant as acceptor.
The O protein is nonfunctional, leaving the H-defining
terminal Fuc unaltered.
U D PUDP
- G a-lN
GaINAc
Ac
G D P- -Fuc
GDP Fu c GGDP
DP
GGalβ
a l β--R
-R
A transferase
A tr a n s fe r a s e
H Htrtransferase
a n s fe r a s e α2
G aGalβ
l β --RR α2
Fu c
Fuc BB tr a n s fe r a s e
transferase
H antigen
P rPrecursor
e c u rs o r H a n tig e n
UPD - Gal
U D P -G a l
UDP
UDP
Fig. 5. Schematic depiction of the biosynthetic pathways for con-
version of H determinants to A or B determinants. R represents the
core structure. See text for enzyme abbreviations.
Although the composition of the A and B antigens is
apparently straightforward, the biochemistry behind the
shared A,B antigen recognized by many group O plasmas
has only recently been elucidated experimentally. Bovin
and his colleagues16 synthesized a deacetylated A trisaccha-
ride structure and bound it to the precursor in such a way
as to expose what they hypothesized would be the common
A,B epitope. They were able to demonstrate binding of both
monoclonal and polyclonal anti-A,B to the synthetic struc-
ture.
Although it is well known that there are approximately
five times fewer A antigens on A2 than on A1 RBCs, the un-
derlying basis for the qualitative differences between them
is less well defined. The A2 transferase is 10 times less ef-
ficient than the A1 transferase and has a different pH opti-
mum and pI.74,75 The A2 enzyme is also less able to use chains
other than type 1 or 2 carbohydrate precursors like the ex-
tended type 3 (repetitive A) and type 4 (globo-A) chains on
RBC glycolipids.17,76 More recently, Svensson et al.18 have
produced somewhat contradictory data indicating that al-
though the A2 transferase can readily use H type 3 chains to
synthesize A antigen, the low levels of type 4 chains remain
unconverted.
The ABH sugars are found on glycolipids (approximate-
ly 10%) and glycoproteins (approximately 90%) on the RBC
as well as on many different tissues and cell types, including
52
epithelial cells that line the lumen of the gastrointestinal,
respiratory, and reproductive tracts as well as in salivary
glands and skin. This wide distribution is a common fea-
ture for many of the carbohydrate blood groups, which has
resulted in the term histo-blood group often being used to
reflect this wide distribution. A and B antigen synthesis
occurs during normal glycosylation of proteins and lipids
in the Golgi compartment.77 The precursor H substance is
synthesized by one of two fucosyltransferases depending on
the acceptor substrate used. The FUT1 gene that encodes
the 2-α-fucosyltransferase (α2FucT1) is responsible mainly
for the synthesis of the H antigen on type 2 (and type 4) car-
bohydrate precursors found on RBCs.78 The closely related
FUT2 gene encodes a very similar 2-α-fucosyltransferase
(α2FucT2) that is expressed in epithelial cells and synthe-
sizes H antigen mainly on type 1 and type 3 chains.77 The
βα
major precursor types present in different tissues and
UUDP
DP
secretions are shown in Table 3.
GaINAc α 3Galβ - R
G a lN A c α 3 G a l β - R
A antigen α2
α2
Table 3. Peripheral core structures and their principal tissue
A a n tig e n
distribution (modified from Clausen and Hakomori3)
Fuc
Fuc
B antigen
B a n tig e n G Galα3Galβ
a l α 3 G a-l Rβ - R
Peripheral core type Structure Distribution
α2
α2 Type 1 Galβ1→3GlcNAcβ1→R Endodermal, secretions,
F Fuc
uc
plasma
Type 2 Galβ1→4GlcNAcβ1→R Ecto- and mesodermal
(e.g., erythrocytes)
Type 3 Galβ1→3GalNAcα1→R O-linked mucin-type,
repetitive A
Type 4 Galβ1→3GalNAcβ1→R Glycolipids in kidneys
(and erythrocytes)
R = inner core structure or linkage.
In the Bombay phenotype (Oh), a silenced FUT1 gene is
present together with a silenced FUT2 gene. Because H an-
tigen is the precursor substrate for both A and B antigens,
neither antigen can be synthesized without α2FucT activity,
independent of the ABO genotype. The para-Bombay phe-
notype results from either (1) a silenced FUT1 gene present
together with an active FUT2 gene, which permits the syn-
thesis of H type 1 (and therefore A/B antigens) that may be
adsorbed onto the RBC from the plasma; or (2) a mutated
FUT1 gene in which the encoded enzyme activity is greatly
diminished, so that very low amounts of H antigen (and
A/B antigen) are produced. It may be present with or with-
out an active FUT2 gene. In both cases, H antigen (and A/B
antigen) is very weakly expressed and is often only detected
by adsorption and elution tests with the appropriate blood
group reagents. The H blood group system (ISBT 018) will
be the subject of another review later in this Immunohematology
series.
The A and B glycosyltransferases are type II membrane
proteins located in the Golgi compartment,79,80 although
soluble forms are found in plasma and other body fluids.
The enzyme consists of a short transmembrane domain, a
stem region, and a catalytic domain that extends into the
Golgi lumen (Fig. 6). The crystal structure elucidated by
IMMUNOHEMATOLOGY, Volume 25, Number 2, 2009

Fig. 6 A: The topology of the transferase is shown as a Golgi-local-
ized, membrane-bound enzyme with a cytoplasmic N-terminus and
catalytic C-terminal domain (modified from Paulson and Colley79).
B and C: The three-dimensional surface model (B) was created
with the Deep View Swiss Pdb Viewer version 3.7, and the three-
dimensional ribbon structure (C) was generated using SETOR and
SetoRibbon (unpublished). The vertical bar to the left illustrates
the approximate exon usage for the different glycosyltransferase
domains.
Patenaude and colleagues81 showed that the catalytic site
is divided into two domains; the N-terminal domain rec-
ognizes the nucleotide sugar donor substrate (UDP-Gal or
UDP-GalNAc), whereas the acceptor substrate is held by
the C-terminal domain. The DXD motif (DVD in these en-
zymes) that serves to capture Mn2+, essential to the reac-
tion because of its ability to bind the UDP part of the donor
substrate, lies between the two domains. In addition, two
so-called disordered loops have been identified by the crys-
tal structural studies. One is located at the C-terminal of the
enzyme. The other disordered loop lies close to the catalytic
site of the enzyme. Its role is unclear4; however, mutations
in this region have been shown to reduce enzyme activity
and are often associated with weak subgroup phenotypes.82
Antibodies in the System
Anti-A and anti-B are naturally occurring antibodies
that are produced by immunocompetent individuals from
the age of approximately 6 months. The widely held dogma
is that these antibodies are in fact mimicking antibodies
produced against terminal carbohydrates on bacterial cell
walls as a response to our normal intestinal microbial flora,
and that these glycotopes share structural homology with A
and B antigens.83 In a modern follow-up to this original con-
cept, three children with different congenital immune de-
fects were studied after apparent ABO grouping discrepan-
cies.84 These patients (age range, 1.7 to 8 years) were limited
to total parenteral nutrition and tube feeding. Their RBCs
typed as group A, but each child lacked the expected anti-B
by routine testing, although anti-B was weakly detectable in
the serum of one of them after prolonged incubation at 4°C.
IMMUNOHEMATOLOGY, Volume 25, Number 2, 2009
ABO blood group system
The authors concluded that the absence of dietary exposure
to bacteria prevented the production of anti-B despite nor-
mal immunoglobulin levels in the patients’ sera. Recogni-
tion of self prevents an individual from making antibodies
to antigens shared by the bacteria and may also partially
explain disease susceptibility of one blood group over an-
other (discussed briefly in the next section).
Anti-A and anti-B are predominantly IgM antibodies
although IgG or IgA components are often found.85,86 Class-
switching to IgG does not occur unless there is a “hyperim-
munizing” event such as an ABO-incompatible pregnancy
or transfusion. Absence of the expected antibodies occurs
rarely, although antibody titers vary considerably among
individuals and have been shown to diminish with age. Pa-
tients who have immunoglobulin deficiencies will also lack
anti-A or anti-B. Otherwise, most often the absence of an
agglutinin in a healthy person should be taken as an indi-
cation that there may be a weak antigen or even (micro)
chimerism present, perhaps detectable by adsorption and
elution or by flow cytometry.
Individuals of the A2 and A2B phenotypes as well as
those whose RBCs carry certain A subgroups (Table 1) can
also produce anti-A1, generally reactive at room tempera-
ture and below. Although ABO antibodies of IgG type can
cross the placenta, severe ABO-related hemolytic disease
of the newborn is not common. The mechanisms behind
this are discussed in more detail in the section on clinical
consequences.
Pathogen Interactions
Bacteria, viruses, and parasites have been proposed as
important driving forces for the geographic distribution of
ABO blood group phenotypes because different pathogens
demonstrate blood group–identical or –cross-reactive mol-
ecules on their surfaces. These are the probable targets for
“blood group” antibodies, and their existence is the lead-
ing hypothesis as to why we make naturally occurring an-
tibodies against the carbohydrate blood groups we lack. In
addition, many pathogens show selective binding to blood
group carbohydrate moieties via lectins (reviewed by Gar-
ratty87). More recent but perhaps anecdotal evidence for
this theory was provided after severe hemolytic transfusion
reactions in two group B patients receiving apheresis plate-
lets from the same group A platelet donor.88 The donor had
donated regularly for a period of 20 years, during which
time no adverse effects of transfusion had been observed,
but had recently begun to supplement his diet with high-
dose probiotics and his anti-B titer was shown to be greater
than 8000. Inhibition studies performed with plasma from
random group A donors and solubilized probiotic tablets
demonstrated a reduction in titer, hinting at a role for ABO
antibodies in neutralizing pathogens. This line of thinking
is supported further by data from studies on viral glycosy-
lation in host cells. HIV cultured in vitro with peripheral
blood mononuclear cells (PBMC) from donors of different
53
J.R. Storry and M.L. Olsson
ABO groups demonstrated specific neutralization with
anti-A of those isolates grown in group A PBMCs but not
those cultured with group B or group O cells.89 Measles vi-
rus, when cocultured in a system expressing ABH glycosyl-
transferases (to mimic an in vivo environment), expressed
A or B epitopes, or not, according to the enzymes expressed.
Viral particles could then be neutralized by normal poly-
clonal anti-A or anti-B sera in the presence of complement
if the corresponding glycans were present.90 Conversely,
the A and B (and H) antigens are also used as receptors for
pathogen invasion by attachment to host cells as mentioned
previously, so the equilibrium between invasion and eva-
sion is a fine balance on both sides.
Furthermore, growing evidence for a leading role of
Plasmodium falciparum as the major force shaping the hu-
man genome including the distribution of the blood groups
has emerged.91,92 The parasite-induced RBC surface protein
pfEMP1 is known to bind to the A antigen trisaccharide,93
and it has been shown that the severity, including mortality,
of malarial disease is significantly lower in group O children
than in other groups, mainly through the mechanism of re-
duced rosetting.94,95 It is the focus on pediatric disease that
is thought to make this pathogen particularly effective in
exerting a selection pressure on our genome.
The general theme here is the concept of herd immu-
nity, i.e., the fact that differences in the population will keep
at least a fraction of all individuals protected from most if
not all pathogens for humanity to survive. ABO differences
as part of our innate immunity appear to be one of the bet-
ter examples of this idea.96
Clinical Significance
Of all antibodies to RBC blood group antigens, anti-A
and anti-B are arguably the most clinically important. On
the other hand anti-A1 and anti-H are very seldom found
to correlate with hemolytic adverse events but often show
lower thermal optima. Before the discovery of ABO, trans-
fusion between humans had been attempted with mixed
success. In some cases, the patient survived and got bet-
ter. In others the patient died rapidly, which we now re-
alize was attributable to rapid intravascular hemolysis of
ABO-incompatible RBCs (reviewed by Mollison et al.97).
It has been reported that transfusion of as little as 30 mL
of ABO-mismatched blood can result in a rapid fatal in-
travascular transfusion reaction (reviewed in Issitt and
Anstee98). Together with transfusion-related acute lung
injury (TRALI) and bacterial contamination of blood com-
ponents, ABO incompatibility is still one of the main risks
for major morbidity and mortality associated with transfu-
sions, after erroneous transfusions.99,100 The latest FDA re-
port on transfusion-associated fatalities (found at http://
www.fda.gov/cber/blood/fatal08.htm) shows that blood
group incompatibility was judged as the cause of death in
37 percent of all cases during 2008, with TRALI at 35 per-
cent. Among the hemolytic fatalities ABO was the cause in
54
59 percent. Surprisingly, however, many reports show that
approximately 50 percent of patients who are inadvertently
transfused with a major ABO-mismatched unit of blood tol-
erate the blood without any apparent signs of a transfusion
reaction.101 The mechanisms underlying the ability of some
individuals to tolerate ABO-mismatched blood are not well
understood but are very interesting as identification of
resistance markers could be exploited in transplantation
therapy. Despite our relative sophistication in providing ap-
propriately ABO-matched blood for patients, there is recent
clinical evidence that even the concept of ABO-compatible
(as opposed to ABO-identical) products may not be as safe
as it would appear. A study that followed the posttransfu-
sion mortality among more than 86,000 patients receiving
plasma showed that exposure to ABO-compatible but non-
ABO-identical plasma was associated with an increased risk
of death.102 Whether this is associated with immune com-
plexes between anti-A/B and soluble A/B antigen in AB
plasma, for instance, remains to be studied. Furthermore,
reports of the usage of platelet concentrates in cardiac
surgery has also demonstrated less favorable outcomes in
those patients receiving ABO-mismatched products.103 The
authors hypothesized that the formation of immune com-
plexes may trigger cellular and inflammatory changes that
adversely affect patient outcome.
Hemolytic disease of the fetus and newborn (HDFN) as
a result of ABO incompatibility between mother and baby
is a relatively common event in group O mothers carrying a
group A or group B fetus. However, the disease is generally
mild and rarely requires treatment, most often by photo-
therapy,97 although inhibition of antibodies by administra-
tion of soluble A or B trisaccharides has been used success-
fully.104 Mild HDFN is a consequence of the comparatively
low levels of IgG ABO antibodies (which are often mainly
IgG2 or IgG4) capable of crossing the placenta and also is
related to the immaturity of the glycosylated structures on
fetal RBCs. In addition, ABH antigens are present on many
other cell types so the antibody concentration on RBCs is
limited and the DAT often only weakly positive.
The clinical significance of anti-A and anti-B extends
beyond transfusion medicine and is important in both solid
organ and hematopoietic transplantation. Although ABO-
mismatched hematopoietic stem cell transplantation is
standard practice with a favorable outcome in most cases,
transplantation of ABO-incompatible solid organs has been
established only relatively recently with moderately suc-
cessful outcome.105 Children younger than 3 years of age
have been shown to tolerate mismatched organs better, and
West and colleagues have demonstrated good posttrans-
plant survival in infants undergoing ABO-incompatible
heart transplants,106 showing that the relatively immature
B-cell response in these patients can be exploited.107 In a
study of 46 patients undergoing ABO-mismatched renal
transplants, Tobian et al.108 demonstrated that therapeu-
tic apheresis to reduce anti-A and anti-B titers to less than
IMMUNOHEMATOLOGY, Volume 25, Number 2, 2009
 ABO blood group system

16 together with standard immunosuppression protocols we recently introduced the first microarray-based ABO typ-
results in successful and stable engraftment. Progress ing system that takes the first steps toward this goal on a
continues in this field, driven by a shortage of appropriate higher throughput system.110 Efforts to renew methods for
organs for transplant. Yazer and Triulzi109 conclude that serologic typing are also ongoing. For instance, the young
immune hemolysis remains a major complication in ABO- field of microfluidics holds promise for alternative blood
mismatched transplantation of solid organs and to a lesser grouping technology being more rapid, using smaller vol-
extent hematopoietic progenitor cells. However, passenger umes of reactants, and being applicable to broader testing
lymphocyte syndrome attributable to ABO or other blood platforms.111
group antibodies can now often be ameliorated by mono- The temptation to create ABO-universal blood for
clonal antibody therapy, once clinicians realize the prob- transfusion purposes has kept scientists busy since the
lem. early 1980s when the first successful report of deliberate
transfusion with modified RBCs across the ABO barrier was
Summary and Future Perspectives published.112 Progress from those early days, using a poorly
Despite the relative simplicity of the A and B antigens, effective B-converting exoglycosidase from green coffee
perhaps especially considering the minor biochemical dif- beans, has continued with the aims of eliminating ABO-
ference between them, the ABO blood group system remains associated hemolytic transfusion reactions and simplify-
one of the most interesting, both clinically and scientifi- ing blood logistics and inventory management. Not only
cally, dividing the world’s population including patients has the process taken important strides toward becoming
and donors into four groups irrespective of origin or creed. a reality with both A- and B-degrading enzymes recombi-
Figure 7 summarizes the four principal levels at which the nantly available and necessary clinical trials in progress or
ABO system can be considered and shows how immunohe- being designed, but the project has also brought with it the
matologists have been able to exploit the steadily increasing scientifically exciting discovery of a large new class of bac-
knowledge of this system by devising tests taking advan- terially derived exoglycosidase enzymes unknown hitherto
tage of each of the different levels. It also shows some of the and without clear homology or resemblance to any other
natural consequences generated and their relation to the group of molecules.113,114 It is unknown at this point why so
microbes surrounding us. many different bacterial species have developed these blood
Analytic modalities Natural consequences
group–converting enzymes. Speculation includes that it
Analytic modal ities Natural conse quences
would simply be a means of digesting potential nutrient
Genetic level
Genotyping
Genotypin
Genotypingg
(DNA, mRNA )
Polymorphic population saccharides, but it also may be yet another way for microbes
to make sure they are able to attach to the host cell surface,
Counteracting enzymes
Enzymic activity
Enzym ic activity Enzyme level (exoglycosidases)
Counteracting developed
enzymes (exoglyco - even if the histo-blood group of the current host happens
(glycosyltransferase)
(glyco syltransferase) by bacteria
sidas es) developed by bacteria not to fit the bacterial lectins initially. It is actually quite
Forward
Forward typing
typing
Glycoconjugate level Cell surface receptor variation likely that yet other glycosidase specificities will be found
Adsorption/elution
Adsorption/ elution Cell surface receptor variation
Flow cytomery
Flow cytometry
(carbohydrate antigens) Neutralization by secreted antigens
Neutralization by secreted antigens in this extended arsenal of newly discovered enzymes. A
Saliva
Saliva inhibi tion
inhibition
recently exploited example is the Galα3Gal (also known as
Reverse typing Immunological level
Neutralizatiom
the straight B, fucose-less B, or Galili antigen) -degrading
Reverse typing Neutralizatio
Neutralizationn byby immunoglobulins
immunoglobu lins
(antibodies )
Titration
Titration enzyme subfamily of galactosidases that can, for instance,
Fig. 7 Principles of the ABO system at four molecular levels and be used to degrade xenograft antigenicity in pig tendons for
their modes of interaction are schematically represented by arrows use in knee surgery.115
in black. On the left side, the laboratory analytical modalities cur- Finally, it is easy to make the mistake of sitting back
rently exploited at each level are listed; the right side shows some of and looking at the ABO system as close to complete when
the innate mechanisms used between host and pathogen. it comes to knowledge and discovery. We predict that both
the antigenic diversity and the way we look at antibody
specificities in this and related systems will change dramat-
A whole array of ABO-related research and other proj- ically during the next few years based on recent and future
ects is currently in progress. Even if a surprising number findings made possible by new technologies. ABO has for a
of ABO alleles has been discovered already, no doubt there long time served as a great model for genetics, enzymology,
are more to come. ISBT is currently working to facilitate and biochemistry thanks to our deep understanding of the
communication and reporting by introducing an official al- interindividual variation in this system, and there is noth-
lele nomenclature. Although ABO genotyping cannot yet be ing to suggest that it will stop now when high-throughput
used as a stand-alone analysis to support clinical decisions, genetics and glycotope arrays are here to help us. On the
improvement of the current status is highly desirable for contrary, it should be expected that there are still more sur-
its use as an independent addition to serology in the refer- prises waiting around the corner.
ence laboratory. Together with colleagues across Europe,

IMMUNOHEMATOLOGY, Volume 25, Number 2, 2009 55

J.R. Storry and M.L. Olsson
Acknowledgments
Some of the content and figures in this review have been
modified from the doctoral theses of M.L.O. (Molecular Ge-
netic Studies of the Blood Group ABO Locus in Man; Lund
University, 1997) and Dr. Bahram Hosseini-Maaf (Genetic
Characterisation of Human ABO Blood Group Variants with
a Focus on Subgroups and Hybrid Alleles; Lund University,
2007).
References
1. Camp FR, Ellis FR. Selected contributions to the litera-
ture of blood groups and immunology. Fort Knox, KY:
US Army Medical Research Laboratory, 1966.
2. Watkins WM. Biochemistry and genetics of the ABO,
Lewis and P blood group systems. In: Harris H,
Hirschhorn K, eds. Advances in human genetics. New
York: Plenum Press, 1980:1–136.
3. Clausen H, Hakomori S. ABH and related histo-blood
group antigens; immunochemical differences in carrier
isotypes and their distribution. Vox Sang 1989;56:1–
20.
4. Yazer MH, Palcic MM. The importance of disordered
loops in ABO glycosyltransferases. Transfus Med Rev
2005;19:210–16.
5. Yamamoto F. Molecular genetics of ABO. Vox Sang
2000;78:91–103.
6. Chester MA, Olsson ML. The ABO blood group gene:
a locus of considerable genetic diversity. Transfus Med
Rev 2001;15:177–200.
7. Landsteiner K. Zur Kenntnis der antifermentativen,
lytischen und agglutinierenden Wirkungen des Blutse-
rums und der Lymphe. Zbl Bakt 1900;27:357–62.
8. Landsteiner K. Über Agglutinationserscheinungen nor-
malen Menschlichen Blutes. Wien Klin Wochenschr
1901;14:1132–4.
9. Decastello A, Sturli A. Ueber die isoagglutinine im ge-
sunder und kranker menschen. Munch Med Wochen-
schrift 1902;49:1090–5.
10. Race RR, Sanger R. Blood groups in man. 6th ed. Ox-
ford, UK: Blackwell Scientific Publications, 1975.
11. von Dungern E, Hirzsfeld L. Uber die gruppens-
pezifische strukturen des blutes III. Immun Forsch
2191;8:526–62.
12. Mourant AE, Kope AC, Domaniewska K. The distribu-
tion of human blood groups and other polymorphisms.
New York: Oxford University Press, 1976.
13. Roychoudhuri AK, Nei M. Human polymorphic genes
world distribution. Oxford: Oxford University Press,
1988.
14. Lalueza-Fox C, Gigli E, de la Rasilla M, Fortea J, Rosas
A, Bertranpetit J, Krause J. Genetic characterization of
the ABO blood group in Neandertals. BMC Evol Biol
2008;8:342.
15. Crainic K, Durigon M, Oriol R. ABO tissue antigens of
Egyptian mummies. Forensic Sci Int 1989;43:113–24.
56
16. Korchagina EY, Pochechueva TV, Obukhova PS,
Formanovsky AA, Imberty A, Rieben R, Bovin NV.
Design of the blood group AB glycotope. Glycoconj J
2005;22:127–33.
17. Clausen H, Levery SB, Dabelsteen E, Hakomori S.
Blood group ABH antigens: a new series of blood group
A-associated structures (genetic regulation and tissue
distribution). Transplant Proc 1987;19:4408–12.
18. Svensson L, Rydberg L, de Mattos LC, Henry SM. Blood
group A and A revisited: an immunochemical analysis.
Vox Sang 2009;96:56–61.
19. Daniels GL, Fletcher A, Garratty G, et al. Blood group
terminology 2004: from the International Society of
Blood Transfusion committee on terminology for red
cell surface antigens. Vox Sang 2004;87:304–16.
20. Blumenfeld OO, Patnaik SK. Allelic genes of blood
group antigens: a source of human mutations and cS-
NPs documented in the Blood Group Antigen Gene
Mutation Database. Hum Mutat 2004;23:8–16.
21. Epstein AA, Ottenberg R. Simple method of performing
serum reactions. Proc N Y Pathol Soc 1908;8:117–23.
22. von Dungern E, Hirszfeld L. Concerning hered-
ity of group specific structures of blood. Transfusion
1962;2:70–2.
23. Bernstein F. Zusammenfassande betrachtungen über
die erblichen Blutstrukturen des Menschen. Z Indukt
Abstamm u VererbLehre 1925;37:237–70.
24. Crow JF. Felix Bernstein and the first human marker
locus. Genetics 1993;133:4–7.
25. Ferguson-Smith MA, Aitken DA, Turleau C, de Grouchy
J. Localisation of the human ABO: Np-1: AK-1 linkage
group by regional assignment of AK-1 to 9q34. Hum
Genet 1976;34:35–43.
26. Yamamoto F, Clausen H, White T, Marken J, Hakomori
S. Molecular genetic basis of the histo-blood group ABO
system. Nature 1990;345:229–33.
27. Yamamoto F, Marken J, Tsuji T, White T, Clausen H,
Hakomori S. Cloning and characterization of DNA
complementary to human UDP-GalNAc:Fuc-alpha1–
2Gal alpha1–3GalNAc transferase (histo-blood group
A transferase) mRNA. J Biol Chem 1990;265:1146–51.
28. Clausen H, White T, Takio K, et al. Isolation to homo-
geneity and partial characterization of a histo-blood
group A defined Fuc-alpha1–2Gal alpha1–3-N-acetyl-
galactosaminyltransferase from human lung tissue. J
Biol Chem 1990;265:1139–45.
29. Yamamoto F, McNeill PD, Hakomori S. Human histo-
blood group A2 transferase coded by A2 allele, one of
the A subtypes, is characterized by a single base deletion
in the coding sequence, which results in an additional
domain at the carboxyl terminal. Biochem Biophys Res
Commun 1992;187:366–74.
30. Yamamoto F, McNeill PD, Hakomori S. Genomic orga-
nization of human histo-blood group ABO genes. Gly-
cobiology 1995;5:51–8.
IMMUNOHEMATOLOGY, Volume 25, Number 2, 2009

31. Bennett EP, Steffensen R, Clausen H, Weghuis DO, van
Kessel AG. Genomic cloning of the human histo-blood
group ABO locus. Biochem Biophys Res Commun
1995;206:318–25.
32. Kominato Y, Hata Y, Takizawa H, et al. Alternative
promoter identified between a hypermethylated up-
stream region of repetitive elements and a CpG island
in human ABO histo-blood group genes. J Biol Chem
2002;277:37936–48.
33. Hosseini-Maaf B, Smart E, Chester MA, Olsson ML.
The Abantu phenotype in the ABO blood group system
is due to a splice-site mutation in a hybrid between a
new O1-like allelic lineage and the A2 allele. Vox Sang
2005;88:256–64.
34. Calafell F, Roubinet F, Ramirez-Soriano A, Saitou N,
Bertranpetit J, Blancher A. Evolutionary dynamics of
the human ABO gene. Hum Genet 2008;124:123–35.
35. Roubinet F, Kermarrec N, Despiau S, Apoil PA, Dug-
oujon JM, Blancher A. Molecular polymorphism of O
alleles in five populations of different ethnic origins.
Immunogenetics 2001;53:95–104.
36. Olsson ML, Guerreiro JF, Zago MA, Chester MA. Mo-
lecular analysis of the O alleles at the blood group ABO
locus in populations of different ethnic origin reveals
novel crossing-over events and point mutations. Bio-
chem Biophys Res Commun 1997;234:779–82.
37. Yazer MH, Olsson ML. The O2 allele: questioning the
phenotypic definition of an ABO allele. Immunohema-
tology 2008;24:138–47.
38. Hosseini-Maaf B, Irshaid NM, Hellberg A, et al. New
and unusual O alleles at the ABO locus are implicated
in unexpected blood group phenotypes. Transfusion
2005;45:70–81.
39. Seltsam A, Das GC, Wagner FF, Blasczyk R. Nondele-
tional ABO*O alleles express weak blood group A phe-
notypes. Transfusion 2005;45:359–65.
40. Lee HJ, Barry CH, Borisova SN, et al. Structural basis
for the inactivity of human blood group O2 glycosyl-
transferase. J Biol Chem 2005;280:525–9.
41. Wagner FF, Blasczyk R, Seltsam A. Nondeletional
ABO*O alleles frequently cause blood donor typing
problems. Transfusion 2005;45:1331–4.
42. Yazer MH, Hult AK, Hellberg A, Hosseini-Maaf B, Palcic
MM, Olsson ML. Investigation into A antigen expres-
sion on O2 heterozygous group O-labeled red blood cell
units. Transfusion 2008;48:1650–7.
43. Olsson ML, Chester MA. Heterogeneity of the blood
group Ax allele: genetic recombination of common al-
leles can result in the Ax phenotype. Transfus Med
1998;8:231–8.
44. Olsson ML, Irshaid NM, Hosseini-Maaf B, et al. Ge-
nomic analysis of clinical samples with serologic ABO
blood grouping discrepancies: identification of 15 novel
A and B subgroup alleles. Blood 2001;98:1585–93.
IMMUNOHEMATOLOGY, Volume 25, Number 2, 2009
ABO blood group system
45. Yu LC, Twu YC, Chou ML, Chang CY, Wu CY, Lin M.
Molecular genetic analysis for the B(3) allele. Blood
2002;100:1490–2.
46. Seltsam A, Gruger D, Just B, et al. Aberrant intracellu-
lar trafficking of a variant B glycosyltransferase. Trans-
fusion 2008;48:1898–905.
47. Hosseini-Maaf B, Hellberg A, Chester MA, Olsson ML.
An extensive PCR-ASP strategy for clinical ABO blood
group genotyping that avoids potential errors caused
by null, subgroup and hybrid alleles. Transfusion
2007;47:2110–25.
48. Olsson ML, Chester MA. Polymorphism and recombi-
nation events at the ABO locus: a major challenge for
genomic ABO blood grouping strategies. Transfus Med
2001;11:295–313.
49. Procter J, Crawford J, Bunce M, Welsh KI. A rapid molec-
ular method (polymerase chain reaction with sequence-
specific primers) to genotype for ABO blood group and
secretor status and its potential for organ transplants.
Tissue Antigens 1997;50:475–83 [published erratum
appears in Tissue Antigens 1998;51:319].
50. Kominato Y, Tsuchiya T, Hata N, Takizawa H, Yama-
moto F. Transcription of human ABO histo-blood group
genes is dependent upon binding of transcription fac-
tor CBF/NF-Y to minisatellite sequence. J Biol Chem
1997;272:25890–8.
51. Irshaid NM, Chester MA, Olsson ML. Allele-related
variation in minisatellite repeats involved in the tran-
scription of the blood group ABO gene. Transfus Med
1999;9:219–26.
52. Yu LC, Chang CY, Twu YC, Lin M. Human histo-blood
group ABO glycosyltransferase genes: different enhanc-
er structures with different transcriptional activities.
Biochem Biophys Res Commun 2000;273:459–66.
53. Seltsam A, Wagner FF, Grüger D, Gupta CD, Bade-Do-
eding C, Blasczyk R. Weak blood group B phenotypes
may be caused by variations in the CCAAT-binding fac-
tor/NF-Y enhancer region of the ABO gene. Transfu-
sion 2007;47:2330–5.
54. Twu YC, Hsieh CY, Yu LC. Expression of the histo-
blood group B gene predominates in AB-genotype cells.
Transfusion 2006;46:1988–96.
55. Thuresson B, Chester MA, Storry JR, Olsson ML. ABO
transcript levels in peripheral blood and erythropoietic
culture show different allele-related patterns indepen-
dent of the CBF/NF-Y enhancer motif and multiple
novel allele-specific variations in the 5′- and 3′-noncod-
ing regions. Transfusion 2008;48:493–504.
56. Hata Y, Kominato Y, Yamamoto FI, Takizawa H. Char-
acterization of the human ABO gene promoter in eryth-
roid cell lineage. Vox Sang 2002;82:39–46.
57. Bianco T, Farmer BJ, Sage RE, Dobrovic A. Loss of red
cell A, B, and H antigens is frequent in myeloid malig-
nancies. Blood 2001;97:3633–9.
57
J.R. Storry and M.L. Olsson
58. Hosoi E, Hirose M, Hamano S, Kuroda Y. Detection of
histo-blood group ABO mRNA in human chronic my-
eloid leukemia cell lines using reverse transcription-
polymerase chain reaction (RT-PCR). Cancer Lett
1998;133:191–6.
59. Bianco-Miotto T, Hussey DJ, Day TK, O’Keefe DS, Do-
brovic A. DNA methylation of the ABO promoter under-
lies loss of ABO allelic expression in a significant propor-
tion of leukemic patients. PLoS ONE 2009;4:e4788.
60. Chihara Y, Sugano K, Kobayashi A, et al. Loss of blood
group A antigen expression in bladder cancer caused
by allelic loss and/or methylation of the ABO gene. Lab
Invest 2005;85:895–907.
61. Dabelsteen E, Gao S. ABO blood-group antigens in oral
cancer. J Dent Res 2005;84:21–8.
62. Orntoft TF, Meldgaard P, Pedersen B, Wolf H. The
blood group ABO gene transcript is down-regulated in
human bladder tumors and growth-stimulated urothe-
lial cell lines. Cancer Res 1996;56:1031–6.
63. Kabat EA. Blood group substances. Their chemistry and
immunohistochemistry. New York: Academic Press,
1956.
64. Morgan WT, Watkins WM. The detection of a product
of the blood group O gene and the relationship of the
so-called O-substance to the agglutinates A and B. Br J
Exp Pathol 1948;29:159–73.
65. Morgan WT, Watkins WM. The inhibition of the hae-
magglutinins in plant seeds by human blood group sub-
stances and simple sugars. Br J Exp Pathol 1953;34:94–
103.
66. Watkins WM, Morgan WT. Inhibition by simple sug-
ars of enzymes which decompose the blood-group sub-
stances. Nature 1955;175:676–7.
67. Watkins WM, Morgan WT. Possible genetical pathways
for the biosynthesis of blood group mucopolysaccha-
rides. Vox Sang 1959;4:97–119.
68. Ceppellini R. Physiological genetics of human blood
group factors. In: Wolstenholme GEW, O’Connor CM,
eds. Ciba Foundation symposium on biochemistry of
human genetics. London: Churchill, 1959:242–61.
69. Leloir LF. Nucleoside diphosphate sugars and saccha-
ride synthesis. Biochem J 1964;91:1–8.
70. Watkins WM. Gene-enzyme relationships of the A, B,
H and Le blood group genes (abstract). Transfusion
1967;7:367.
71. Hearn VM, Smith ZG, Watkins WM. An a-N-acetyl-D-
galactosaminyltransferase associated with the human
blood-group A character. Biochem J 1968;109:315–17.
72. Race C, Ziderman D, Watkins WM. An alpha-d-galacto-
syltransferase associated with the blood-group B char-
acter. Biochem J 1968;107:733–5.
73. Schenkel-Brunner H, Tuppy H. Enzymes from human
gastric mucosa conferring blood-group A and B speci-
ficities upon erythrocytes. Eur J Biochem 1970;17:218–
22.
58
74. Schachter H, Michaels MA, Tilley CA, Crookston MC,
Crookston JH. Qualitative differences in the N-acetyl-d-
galactosaminyltransferases produced by human A1 and
A2 genes. Proc Natl Acad Sci U S A 1973;70:220–4.
75. Topping MD, Watkins WM. Isolectric points of the hu-
man blood group A-1, A-2 and B gene- associated gly-
cosyltransferases in ovarian cyst fluids and serum. Bio-
chem Biophys Res Commun 1975;64:89–96.
76. Clausen H, Levery SB, Nudelman E, Tsuchiya S, Hako-
mori S. Repetitive A epitope (type 3 chain A) defined
by blood group A1-specific monoclonal antibody TH-
1: chemical basis of qualitative A1 and A2 distinction.
Proc Natl Acad Sci U S A 1985;82:1199–203.
77. Varki A, Cummings R, Esko J, Freeze H, Hart G, Marth
J. Essentials of glycobiology. Cold Spring Harbor, NY:
Cold Spring Harbor Laboratory Press, 1999.
78. Oriol R, Candelier JJ, Mollicone R. Molecular genetics
of H. Vox Sang 2000;78(Suppl 2):105–8.
79. Paulson JC, Colley KJ. Glycosyltransferases. Structure,
localization, and control of cell type-specific glycosyla-
tion. J Biol Chem 1989;264:17615–18.
80. Clausen H, Bennett EP, Dabelsteen E. Carbohydrates
of the cell surface: molecular aspects of glycosyltrans-
ferases and their genes. APMIS Suppl 1992;27:9–17.
81. Patenaude SI, Seto NO, Borisova SN, et al. The struc-
tural basis for specificity in human ABO(H) blood group
biosynthesis. Nat Struct Biol 2002;9:685–90.
82. Hosseini-Maaf B, Letts JA, Persson M, et al. Structural
basis for red cell phenotypic changes in newly identi-
fied, naturally occurring subgroup mutants of the hu-
man blood group B glycosyltransferase. Transfusion
2007;47:864–75.
83. Springer GF, Williamson P, Readler BL. Blood group
active gram-negative bacteria and higher plants. Ann N
Y Acad Sci 1962;97:104–10.
84. Cooling LW, Sitwala K, Dake LR, Judd WJ, Davenport
R. ABO typing discrepancies in children requiring long-
term nutritional support: it is the gut after all! Transfu-
sion 2007;47:10A.
85. Kunkel HG, Fudenberg H, Ovary Z. High molecular
weight antibodies. Ann NY Acad Sci 1960;86:966-73.
86. Kunkel HG, Rockey JH. β2A and other immunoglobu-
lins in isolated anti-A antibodies. Proc Soc Exp Biol
Med 1963;113:278.
87. Garratty G. Blood group antigens as tumor markers,
parasitic/bacterial/viral receptors, and their associa-
tion with immunologically important proteins. Immu-
nol Invest 1995;24:213–32.
88. Daniel-Johnson J, Lee-Stroka A, Schechterly C, et al.
Probiotic-associated high-titer anti-B in a group A
platelet donor as a cause of severe hemolytic transfu-
sion reactions. Transfusion 2008;48S(Suppl 2):260A.
IMMUNOHEMATOLOGY, Volume 25, Number 2, 2009

89. Arendrup M, Hansen JE, Clausen H, Nielsen C, Math-
iesen LR, Nielsen JO. Antibody to histo-blood group A
antigen neutralizes HIV produced by lymphocytes from
blood group A donors but not from blood group B or O
donors. AIDS 1991;5:441–4.
90. Preece AF, Strahan KM, Devitt J, Yamamoto F, Gustafs-
son K. Expression of ABO or related antigenic carbohy-
drates on viral envelopes leads to neutralization in the
presence of serum containing specific natural antibod-
ies and complement. Blood 2002;99:2477–82.
91. Cserti CM, Dzik WH. The ABO blood group sys-
tem and Plasmodium falciparum malaria. Blood
2007;110:2250–8.
92. Uneke CJ. Plasmodium falciparum malaria and ABO
blood group: is there any relationship? Parasitol Res
2007;100:759–65.
93. Chen Q, Heddini A, Barragan A, Fernandez V, Pearce
SF, Wahlgren M. The semiconserved head structure of
Plasmodium falciparum erythrocyte membrane pro-
tein 1 mediates binding to multiple independent host
receptors. J Exp Med 2000;192:1–10.
94. Loscertales MP, Owens S, O’Donnell J, Bunn J, Bosch-
Capblanch X, Brabin BJ. ABO blood group phenotypes
and Plasmodium falciparum malaria: unlocking a piv-
otal mechanism. Adv Parasitol 2007;65:1–50.
95. Rowe JA, Handel IG, Thera MA, et al. Blood group O
protects against severe Plasmodium falciparum ma-
laria through the mechanism of reduced rosetting. Proc
Natl Acad Sci U S A 2007;104:17471–6.
96. Gagneux P, Varki A. Evolutionary considerations in re-
lating oligosaccharide diversity to biological function.
Glycobiology 1999;9:747–55.
97. Mollison PL, Engelfriet CP, Contreras M. Blood trans-
fusion in clinical medicine. 10th ed. London: Blackwell
Scientific, 1997.
98. Issitt PD, Anstee DJ. Applied blood group serology. 4th
ed. Miami: Montgomery Scientific Publications, 1998.
99. Sazama K. Reports of 355 transfusion-associated deaths:
1976 through 1985. Transfusion 1990;30:583–90.
100. Stainsby D, Jones H, Asher D, et al. Serious hazards of trans-
fusion: a decade of hemovigilance in the UK. Transfus Med
Rev 2006;20:273–82.
101. Linden JV, Wagner K, Voytovich AE, Sheehan J. Trans-
fusion errors in New York State: an analysis of 10 years’
experience. Transfusion 2000;40:1207–13.
102. Shanwell A, Andersson TM, Rostgaard K, et al. Post-trans-
fusion mortality among recipients of ABO-compatible but
non-identical plasma. Vox Sang 2009;96:316–23.
103. Blumberg N, Heal JM, Hicks GL Jr, Risher WH. Asso-
ciation of ABO-mismatched platelet transfusions with
morbidity and mortality in cardiac surgery. Transfusion
2001;41:790–3.
IMMUNOHEMATOLOGY, Volume 25, Number 2, 2009
ABO blood group system
104. Romano EL, Soyano A, Montano RF, et al. Treatment of
ABO hemolytic disease with synthetic blood group trisac-
charides. Vox Sang 1994;66:194–9.
105. Rydberg L. ABO-incompatibility in solid organ transplan-
tation. Transfus Med 2001;11:325–42.
106. West LJ, Pollock-Barziv SM, Dipchand AI, et al. ABO-in-
compatible heart transplantation in infants. N Engl J Med
2001;344:793–800.
107. West LJ. B-cell tolerance following ABO-incompat-
ible infant heart transplantation. Transplantation
2006;81:301–7.
108. Tobian AA, Shirey RS, Montgomery RA, Tisch DJ, Ness
PM, King KE. Therapeutic plasma exchange reduces ABO
titers to permit ABO-incompatible renal transplantation.
Transfusion 2009 Feb 6 [Epub ahead of print].
109. Yazer MH, Triulzi DJ. Immune hemolysis following ABO-
mismatched stem cell or solid organ transplantation.
Curr Opin Hematol 2007;14:664–70.
110. Avent ND, Martinez A, Flegel WA, et al. The BloodGen
project: toward mass-scale comprehensive genotyping
of blood donors in the European Union and beyond.
Transfusion 2007;47(Suppl):40S–6S.
111. Kline TR, Runyon MK, Pothiawala M, Ismagilov RF.
ABO, D blood typing and subtyping using plug-based
microfluidics. Anal Chem 2008;80:6190–7.
112. Goldstein J, Siviglia G, Hurst R, Lenny L, Reich L.
Group B erythrocytes enzymatically converted to group
O survive normally in A, B, and O individuals. Science
1982;215:168–70.
113. Liu QP, Sulzenbacher G, Yuan H, et al. Bacterial gly-
cosidases for the production of universal red blood
cells. Nat Biotechnol 2007;25:454–64.
114. Olsson ML, Clausen H. Modifying the red cell surface:
towards an ABO-universal blood supply. Br J Haematol
2008;140:3–12.
115. Liu QP, Yuan H, Bennett EP, et al. Identification of a
GH110 subfamily of alpha 1,3-galactosidases: novel en-
zymes for removal of the alpha 3Gal xenotransplanta-
tion antigen. J Biol Chem 2008;283:8545–54.
Jill R. Storry, PhD, FIBMS (corresponding author), Coor-
dinator, Special Serology, Department of Clinical Immu-
nology and Transfusion Medicine, University and Regional
Laboratories, and Martin L. Olsson, MD, PhD, Division of
Haematology and Transfusion Medicine, Department of
Laboratory Medicine, Lund University, Lund, Sweden.
59
Case Report
Clinical evaluation for lymphoproliferative
disease prompted by finding of IgM warm
autoanti-IT in two cases
R.M. Leger, F. Lowder, M.C. Dungo, W. Chen, H.M. Mason, and G. Garratty
Anti-IT is an unusual specificity originally described as a naturally
occurring cold agglutinin. The antibody reacts strongly with cord
RBCs, weakly with adult I RBCs, and most weakly with the rare
adult i RBCs. IgG anti-IT in patients with hemolytic anemia has
been associated with Hodgkin’s lymphoma. Difficulties in blood
grouping tests and the presence of a warm reactive agglutinin in
samples from two patients with hemolytic anemia led to further
serologic studies and the identification of anti-IT. In both cases,
the anti-IT was a rarely encountered IgM warm reactive aggluti-
nin; in one case, the IgG component was also anti-IT, whereas in
the second case the IgG antibody was broadly reactive. The un-
usual serologic finding of anti-IT prompted further clinical evalu-
ation for lymphoproliferative disease in these two patients.
Immunohematology 2009;25:60–62.
Key Words: IgM warm autoantibody, autoanti-IT,
hemolytic anemia
A
nti-IT was originally described as a benign, naturally
occurring, IgM cold agglutinin in Melanesians1 and
later in Venezuela Indians.2 The anti-IT agglutinin
did not demonstrate classical I or i specificity but reacted
strongly with cord RBCs, weakly with normal adult I RBCs,
and most weakly with the rare adult i RBCs. It was thought
the agglutinin recognized a transition state of i to I, thus the
designation IT (T for transition).1 Subsequent data do not
support this hypothesis; IT is expressed nearly as well or as
well on fetal RBCs ranging in age from 11 to 16 weeks as on
cord RBCs.3 The biochemical structure of IT is still unclear.
The first four examples of IgG anti-IT were in sera of
patients with autoimmune hemolytic anemia (AIHA) and
Hodgkin’s lymphoma.3,4 Others reported a similar associa-
tion.5 Hafleigh et al.6 found three examples of IgG autoanti-
IT in patients who did not have Hodgkin’s disease or AIHA.
One example of IgM warm anti-IT and one IgM cold plus
IgG anti-IT in patients with AIHA were not associated with
Hodgkin’s disease, but one of the cases was associated with
non-Hodgkin’s lymphoma.7,8 We report an unusual 37°C
reactive IgM agglutinin plus an IgG anti-IT9 and a 37°C re-
active IgM agglutinating anti-IT plus broadly reactive IgG
autoantibody in two patients with hemolytic anemia who
were subsequently evaluated for lymphoproliferative dis-
ease owing to the specificity of the antibodies reported.
60
Case Reports
Case 1
A 69-year-old Hispanic woman presented with pancy-
topenia (WBC = 2.3 × 109/L; Hb = 6.9 g/dL; Hct = 19.1%;
platelets = 64,000/µL), splenomegaly but no lymphade-
nopathy, and hemolytic anemia (spherocytosis; LDH = 672
U/L [normal 355–630]; total bilirubin = 2.8 mg/dL [normal
0.1–1.2]; direct bilirubin = 0.7 mg/dL [normal 0–0.4]). The
direct antiglobulin test (DAT) was positive. Lymphoma was
not suspected until after the anti-IT was identified. Comput-
ed tomography (CT) scans showed evidence of large lym-
phoid infiltrates composed of a mixture of small and large
lymphocytes. Bilateral bone marrow aspiration showed in-
creased lymphocytes but was not diagnostic of lymphoma.
The patient was empirically treated with steroids and had
improved hemoglobin and sense of well-being. Steroids
were tapered. Two and a half months later the patient re-
turned for a splenectomy, which was pathologically nega-
tive for lymphoma. Sections showed lymphoid hyperplasia
with a benign phenotype consisting of T and B cells with no
aberrant expression of CD5, CD23, or bcl-2. Serologic stud-
ies at that time showed similar reactivity of anti-IT. Three
months later, the patient exhibited mild thrombocytopenia
with slightly elevated LDH. A repeat bone marrow showed
lymphocytosis suggestive of low-grade B-cell lymphopro-
liferative disease. A year later, after rituximab and other
chemotherapy, the bone marrow showed atypical lymphoid
infiltrates. The infiltrates were composed primarily of small
mature lymphocytes with occasional larger lymphocytes
noted, most likely representing a lymphoproliferative dis-
order. Flow cytometry showed an entire B-cell population
that did not express CD20. Less than 3 years after original
presentation, the patient was transfusion-dependant and
diagnosed with aplastic anemia. She was discharged to hos-
pice care with end-stage liver disease, lymphoproliferative
disorder, and hemolytic anemia.
Case 2
A previously healthy 19-year-old Korean man presented
to the emergency room with insidious onset of fatigue, short-
ness of breath, dyspnea on exertion, and pallor. Laboratory
data revealed thrombocytopenia and hemolytic anemia:
IMMUNOHEMATOLOGY, Volume 25, Number 2, 2009
 Two cases of IgM warm autoanti-IT

WBC = 6.8 × 109/L; Hb = 4.6 g/dL; Hct = 12%; platelets Results

= 20,000/µL; spherocytes on the peripheral smear; total Case 1
bilirubin = 2.9 mg/dL; direct bilirubin = 0.4 mg/dL; and The DAT on DTT-treated RBCs was 3+ with anti-IgG
LDH = 539 U/L. The haptoglobin was less than 5.8 mg/ and anti-C3; the 6% albumin control was nonreactive. The
dL. There was no splenomegaly or lymphadenopathy. The initial titer and thermal amplitude studies showed a 37°C
DAT was positive. An autoanti-IT agglutinin plus a broadly agglutinin that reacted to a higher titer with cord RBCs than
reactive IgG autoantibody were identified. After a few days with adult I RBCs. Titration results with adult I, cord i, and
on pulse dexamethasone (Decadron) therapy the hemoglo- adult i RBCs (to determine specificity) are shown in Table 1.
bin improved only mildly, and the patient was started on Anomalous results (unexplained high titer) were obtained
a 4-day course of IVIG with recovery of his hemoglobin to with DTT-treated autologous RBCs: titers of 512 at 37°C,
9.3 g/dL. Because of the association of lymphoma with an- 1024 at 30°C, 2048 at 22°C, and 8000 at 4°C. An apparent
ti-IT the patient underwent CT scans, bone marrow biopsy, anti-I was detected at 4°C (adult I RBC titer of 256, cord
and positron emission tomography. There was no evidence RBC titer of 64). An acid eluate prepared from the patient’s
of malignancy or lymphoproliferative process. During the RBCs reacted more strongly with cord RBCs than with adult
period of a year, the patient had multiple relapses of the I RBCs at 37°C. After treatment with DTT, the patient’s se-
hemolytic anemia (but not thrombocytopenia), necessi- rum and the eluate did not agglutinate cord RBCs, but re-
tating further treatment with steroids. Rituximab, given acted 1+ and 4+, respectively, at the antiglobulin test with
for the hemolytic process, had no sustained benefit. The anti-IgG. The dilution control (for the abolished aggluti-
patient underwent a splenectomy, and both the hemoglo- nation) reacted 3½ to 4+ with the serum and 1½ + with
bin and platelet count remain normal. There has been no the eluate. Thus, IgM plus IgG anti-IT was demonstrated in
evidence of Hodgkin’s disease, lymphoma, or other malig- both the serum and the eluate. In the AIHA serum screen,
nancy in several years of follow-up. agglutination was observed at 22°C and 37°C similar to that
in the titration and thermal amplitude studies; a hemolysin
Materials and Methods was also observed with enzyme-treated RBCs at both tem-
The DAT was performed with anti-human IgG (Ortho peratures, but without a clear preference for either temper-
Clinical Diagnostics, Raritan, NJ) and anti-human C3 (in- ature.
house reagent). A 6% bovine albumin control was tested
in parallel. The in-house anti-C3 was prepared by inject- Case 2
ing rabbits with purified proteins and standardized as The DAT on DTT-treated RBCs was 4+ with anti-IgG
previously described. Before testing, the patients’ RBCs
10 and anti-C3; the 6% albumin control was nonreactive. In the
were treated with 0.01 M dithiothreitol (DTT) to break initial titer and thermal amplitude studies, an agglutinin re-
IgM bonds that cause spontaneous agglutination. Eluates
11 acted at 37°C with cord RBCs; adult I RBCs did not react at
were prepared from the patients’ RBCs using a commercial 37°C or 30°C. Titration results with adult I, cord i, and adult i
acid elution kit (Gamma Elu-Kit II, Gamma Biologicals, RBCs are shown in Table 1. Sufficient autologous RBCs were
Houston, TX); cold LISS was substituted for the kit wash not available. DTT treatment of the patient’s serum abolished
solution. 12 agglutination with cord RBCs; however, dilutions of DTT-
For immunoglobulin classification, samples were treat- treated serum tested against adult I and cord RBCs did not
ed with 0.01 M DTT. The agglutinin titer and thermal am- show a difference in reaction strength of the IgG component.
plitude was determined at 37°C (prewarmed), 30°C, 22°C, Thus, an IgM anti-I T
plus a broadly reactive IgG antibody was
and 4°C with group O adult I, cord, and adult i RBCs, and
10 demonstrated in the serum. The eluate contained an IgG an-
for Case 1, with DTT-treated autologous RBCS. The tests at tibody suggestive of anti-I T
(titer/score versus adult I RBCs
each temperature were set up separately (i.e., using sepa- was 32/42 and versus cord RBCs was 128/55). In the AIHA
rate sets of dilutions) to eliminate potential carryover of serum screen, untreated RBCs were weakly agglutinated at
agglutination. Patients’ sera were also tested by a previous- 22°C and weakly sensitized at 37°C; enzyme-treated RBCs
ly described serum screen method10 used to characterize were agglutinated and hemolyzed at both temperatures.
antibodies in AIHA. Briefly, serum Discussion
was tested with and without
acidification and the addition Table 1. Titer and thermal amplitude of two examples of anti-IT
of fresh normal serum as a RBCS Case 1 titer (score) at: Case 2 titer (score) at:
source of complement, against 37°C 30°C 22°C 4°C 37°C 30°C 22°C 4°C
untreated and enzyme-treated Adult I 1 (4) 1 (6) 2 (11) 256 (78) 0 0 1 (4) 8 (28)
RBCs at 37°C (prewarmed) and
Cord i 32 (37) 128 (61) 256 (68) 64 (56) 64 (54) 64 (52)-128 64 (49) 32 (48)
20°C. Agglutination results (60)
were graded and scored as pre-
Adult i 1 (6) 2 (10) 2 (10) NT* 0 1 (4) 1 (5) NT
viously described.10
*NT = not tested

IMMUNOHEMATOLOGY, Volume 25, Number 2, 2009 61

R.M. Leger et al.
IgM warm autoagglutinins reactive at 37°C are an
unusual subcategory of warm autoimmune hemolytic ane-
mia.10,13 Typically, the patient’s RBCs are spontaneously
agglutinated, requiring treatment with a sulfhydryl reagent
such as DTT to resolve ABO and Rh typing and to interpret
the DAT. The two cases presented here were referred to the
immunohematology research laboratory for further diag-
nostic testing and characterization of the agglutinin when
these characteristics were noted by our reference labora-
tory. In both cases the warm agglutinin demonstrated IT
specificity. Both of our cases also demonstrated an IgG au-
toantibody component; in Case 1, the IgG autoantibody was
anti-IT.
We have no explanation for the high titers obtained af-
ter DTT-treatment of the group O autologous RBCs in Case
1. This may indicate that the patient’s RBCs had greatly
increased IT expression. When group O untreated, DTT-
treated, and ficin-treated cord RBCs were tested in parallel
with dilutions of this patient’s anti-IT and another sample
of anti-IT, no enhancement of reactivity was observed with
DTT treatment whereas reactivity with ficin-treated RBCs
was increased as expected by three to four dilutions (data
not shown). Thus, the DTT treatment should not have af-
fected the autologous RBC test results. The previous sample
of warm IgM anti-IT associated with AIHA did not aggluti-
nate that patient’s untreated autologous RBCs at 37°C (the
optimal temperature of reactivity).7
Anti-IT is an unusual specificity and may not even be
suspected unless dilutions of the sera are tested with cord i
and adult i RBCs in parallel with adult I RBCs. In our cases,
additional titrations to identify the specificity were only
pursued after the initial increased reactivity with cord RBCs
was observed. Examples of anti-IT have been reported to be
IgM cold agglutinins or IgG antibodies optimally reactive
at 37°C. We are only aware of one reported example of IgM
warm anti-IT, and that case was not associated with Hodg-
kin’s disease or lymphoma.7 In more than 20 years, we have
encountered only two other examples of IgM warm reactive
anti-IT; neither had an IgG component, but both were as-
sociated with a history of lymphoma before the serologic
workup.
Determining the specificity of autoantibodies is typical-
ly of academic interest only. In general, serologic results do
not necessarily prompt a clinical evaluation for lymphoma.
In both of these cases, the unusual RBC antibody and the
historic association of this specificity with lymphoma, to-
gether with the presenting findings of AIHA, led to the lym-
phoma workup. For patients who do not progress to lym-
phoma, one might speculate that rituximab (anti-CD20)
given for the AIHA could have eradicated a malignant clone
of B cells.
62
References
1. Booth PB, Jenkins WJ, Marsh WL. Anti-IT: a new an-
tibody of the I blood group system occurring in certain
Melanesian sera. Br J Haematol 1966;12:341–4.
2. Layrisse Z, Layrisse M. High incidence cold autoag-
glutinins of anti-IT specificity in Yanomama Indians of
Venezuela. Vox Sang 1968;14:369–82.
3. Garratty G, Petz LD, Wallerstein RO, Fudenberg HH.
Autoimmune hemolytic anemia in Hodgkin’s disease
associated with anti-IT. Transfusion 1974;14:226–31.
[Correction note: Transfusion 1974;14:630.]
4. Garratty G, Hafleigh B, Dalziel J, Petz LD. An IgG an-
ti-IT detected in a Caucasian American. Transfusion
1972;12:325–9.
5. Levine AM, Thornton P, Forman SJ, et al. Positive
Coombs test in Hodgkin’s disease: significance and im-
plications. Blood 1980;55:607–11.
6. Hafleigh EB, Wells RF, Grumet FC. Nonhemolytic IgG
anti-IT. Transfusion 1978;18:592–7.
7. Freedman J, Newlands M, Johnson CA. Warm IgM
anti-IT causing autoimmune haemolytic anaemia. Vox
Sang 1977;32:135–42.
8. Freedman J, Cheng LF, Murray D, Myers R. An unusual
autoimmune hemolytic anemia in a patient with immu-
noblastic sarcoma. Am J Hematol 1983;14:175–84.
9. Leger RM, Nguyen N, Garratty G, Lowder F, Feldman
N. An unusual IgM warm + IgG autoanti-IT associated
with lymphoma, pancytopenia and hemolytic anemia
(abstract). Transfusion 2003;43:100A.
10. Petz LD, Garratty G. Immune hemolytic anemias. 2nd
ed. Philadelphia: Churchill Livingstone, 2004.
11. Reid ME. Autoagglutination dispersal utilizing sulphy-
dryl compounds. Transfusion 1978;18:353–5.
12. Leger RM, Arndt PA, Ciesielski DJ, Garratty G. False-
positive eluate reactivity due to the low-ionic wash so-
lution used with commercial acid-elution kits. Transfu-
sion 1998;38:565–72.
13. Arndt PA, Leger RM, Garratty G. Serologic findings
in autoimmune hemolytic anemia associated with im-
munoglobulin M warm autoantibodies. Transfusion
2009;49:235–42.
Regina M. Leger, MSQA, MT(ASCP)SBB, Research Associ-
ate, American Red Cross Blood Services, Southern Califor-
nia, 100 Red Cross Circle, Pomona, CA 91768; Frederick
Lowder, CLS,MT(ASCP)SBB, Olive View-UCLA Medical
Center, Sylmar, CA; Maria C. Dungo, MD, Assistant Clini-
cal Professor, David Geffen School of Medicine at UCLA,
Harbor-UCLA Medical Center, Torrance, CA; Weip Chen,
MD, Harbor-UCLA Medical Center, Torrance, CA; Holli
M. Mason, MD, Assistant Clinical Professor of Pathol-
ogy, David Geffen School of Medicine at UCLA, Director
of Transfusion Medicine and Serology, Harbor-UCLA
Medical Center, Torrance, CA; and George Garratty, PhD,
FRCPath, Scientific Director, American Red Cross Blood
Services, Southern California, Pomona, CA.
IMMUNOHEMATOLOGY, Volume 25, Number 2, 2009
Original Report

Southeast Asian ovalocytosis is associated

with increased expression of Duffy antigen
receptor for chemokines (DARC)
I.J. Woolley, P. Hutchinson, J.C. Reeder, J.W. Kazura, and A. Cortés

The Duffy antigen receptor for chemokines (DARC or Fy glyco-
protein) carries antigens that are important in blood transfusion
and is the main receptor used by Plasmodium vivax to invade
reticulocytes. Southeast Asian ovalocytosis (SAO) results from
an alteration in RBC membrane protein band 3 and is thought to
mitigate susceptibility to falciparum malaria. Expression of some
RBC antigens is suppressed by SAO, and we hypothesized that
SAO may also reduce Fy expression, potentially leading to reduced
susceptibility to vivax malaria. Blood samples were collected from
individuals living in the Madang Province of Papua New Guinea.
Samples were assayed using a flow cytometry assay for expres-
sion of Fy on the surface of RBC and reticulocytes by measuring
the attachment of a phycoerythrin-labeled Fy6 antibody. Reticu-
locytes were detected using thiazole orange. The presence of the
SAO mutation was confirmed by PCR. There was a small (approxi-
mately 10%) but statistically significant (p=0.049, Mann-Whitney
U test) increase in Fy expression on SAO RBC compared with RBC
from individuals without this polymorphism: mean Fy expres-
sion (mean fluorescence intensity [MFI]) was 10.12 ± 1.22 for SAO
heterozygotes versus an MFI of 8.95 ± 1.1 for individuals without
SAO. For reticulocytes the MFI values were 27.61 ± 19.12 for SAO
heterozygotes and 16.47 ± 3.81 for controls. SAO is associated with
increased and not decreased Fy6 expression so that susceptibility
to P. vivax infection is unlikely to be affected.
Immunohematology 2009;25:63–66.
Key Words: Duffy, Southeast Asian ovalocytosis, antigen
expression
RBC morphology.3 SAO is widespread in several popula-
tions of Papua New Guinea (PNG), where its prevalence
correlates with malaria endemicity and altitude.4 In these
populations, homozygosity is apparently incompatible with
full development of the fetus inasmuch as no persons ho-
mozygous for the band 3 mutation have yet been described,
but heterozygosity confers strong protection against cere-
bral malaria.5,6 Although early studies had suggested that
the SAO trait may afford some protection against vivax or
falciparum parasitemia,7–9 other studies did not support
this hypothesis.10 The mechanism of protection against
cerebral malaria is not completely understood,11,12 but the
specific protection against cerebral malaria suggests that it
may operate by means of alterations in sequestration of in-
fected RBC. However, SAO confers limited or no protection
against placental malaria, which is also linked to sequestra-
tion of infected RBCs.13,14
Individuals whose RBCs are negative for expression of
Duffy antigens illustrate homozygote advantage for vivax
malaria in that their RBCs are completely refractory to in-
vasion by Plasmodium vivax.15 Historic serologic data sug-
gest expression of some antigens on human RBCs was sup-
pressed by the SAO trait, but expression of Duffy antigens
did not appear suppressed as determined by the less sensi-
tive serologic instruments available at that time.16 In those
studies, antibodies of varying specificities were placed in

I
tubes using serial dilution, and results were read macro-
n 1949 Haldane proposed that malaria had a dramatic
scopically with the aid of an eyepiece using a standard-
influence on the human genome, by selecting for ge-
ized scoring system. In this study, we used molecular and
netic polymorphisms that were known or suspected to
cytometric techniques to diagnose SAO and to reexamine
protect against malaria.1 Although some of these polymor-
whether there was a difference in expression of Duffy an-
phisms can be detrimental when individuals are homozy-
tigen receptor for chemokines (DARC) between SAO and
gous, e.g., sickle cell anemia, their apparent selection in hu-
control individuals in the context of possible differential
man populations living in malaria-endemic areas is evidence
susceptibility to infection with different Plasmodium spe-
that they offer some protection against death from malaria,
cies
an infectious disease that continues to kill more than 1 mil-
lion persons annually.2 Critical to this theory is the concept
Materials and Methods
of heterozygote advantage. That is, carriage of the mutant
Blood was collected by venipuncture in CPD anticoag-
gene on one member of a chromosomal pair is beneficial
ulant or by finger prick using a BD Microtainer (Becton
whereas homozygosity is deleterious. An extreme example
Dickinson, San Jose, CA) with EDTA anticoagulant from
of heterozygote advantage in Melanesian populations is
healthy volunteers from two villages, Sempi and Bemlon,
Southeast Asian ovalocytosis (SAO), a hereditary form of
on the north coast of the Madang Province, PNG. Oral in-
ovalocytosis that is caused by a 27-base pair deletion in the
formed consent was obtained after approval for the study
gene encoding the major integral RBC membrane protein
by the Medical Research Advisory Committee of the PNG
band 3 (AE1, SLC4A1), leading to a distinctive change in

IMMUNOHEMATOLOGY, Volume 25, Number 2, 2009 63

I.J. Woolley et al.
Ministry of Health. After collection, samples were kept on
ice or refrigerated at 4°C. Within 1 week, flow cytometric
studies were undertaken with antibody to Fy6 (kindly pro-
vided by John Barnwell, CDC, Atlanta, GA). This is a mouse
monoclonal antibody that attaches to the first extracellular
domain of the Fy glycoprotein, in an area thought to cor-
respond to the binding site of P. vivax merozoites.17,18 The
antibody was conjugated directly to a phycoerythrin (PE)
label (Prozyme, San Leandro, CA) according to the manu-
facturer’s instructions. A 5-µL aliquot of blood was washed
three times in 50 µL of PBS and pelleted. RBCs were incu-
bated with 50 µL of a 1 in 50 dilution of Fy6 antibody for 15
minutes at 37°C. Cells were then washed twice in PBS and
resuspended in thiazole orange (TO) solution according to
the manufacturer’s specifications (Becton Dickinson). The
samples were analyzed on a flow cytometer (Mo-Flo, Da-
koCytomation, Fort Collins, CO) equipped with a 70-mW
air-cooled argon ion laser at 488 nm. TO fluorescence was
read with a 525-nm bandpass optical filter and PE fluores-
cence with a 570-nm bandpass filter. Compensation was
applied, and negative fluorescence signals were adjusted
until they were orthogonal. Forward-scatter, side-scatter,
and fluorescence data were analyzed. A count of 5000 TO-
positive cells was taken per sample. This was approximately
1 to 2 percent of the total number of cells assayed for each
individual. Beads of known immunofluorescence (Immu-
no-Brite, Coulter, Puerto Rico) were used to standardize
experiments undertaken at different times. Detection of the
SAO mutation was undertaken using previously described
PCR methods.19 Experienced microscopists from the PNG
Institute of Medical Research performed light microscopic
inspection of Giemsa-stained blood films for malaria para-
sites. Statistical analysis was undertaken by using SPSS
(Statview 4.51, Abacus Concepts, Berkeley, CA). We have
used nonparametric testing because we do not have evi-
dence for normal distribution of DARC abundance in the
surface of SAO erythrocytes.
Results
Samples from 19 SAO heterozygotes and 20 non-SAO
donors were examined. Mean age of donors did not dif-
fer significantly between the two groups (mean age, 29.32
± 13.70 years for SAO heterozygotes versus 26.91 ± 11.80
years for non-SAO individuals). Four individuals had ma-
laria infection based on inspection of blood smears. One
SAO and one non-SAO individual had Plasmodium malari-
ae, and two non-SAO donors had Plasmodium falciparum.
SAO RBC showed increased expression of Fy glycoprotein
relative to non-SAO RBC as measured by binding of the Fy6
monoclonal antibody (Table 1). The level of Fy6 antigen
was also higher in SAO reticulocytes compared with control
reticulocytes, but the difference was not statistically sig-
nificant (Table 1). Subsequent analysis of the data did not
suggest the differences were caused by a systemic bias at-
tributable to sex, village of origin, or method of obtaining
blood (data not shown).
64
Table 1. Mean fluorescence intensity (± standard deviation) of Fy6
antibody binding to erythrocytes and reticulocytes
All Subjects All Subjects
(Reticulocytes) (Erythrocytes)
SAO heterozygotes (N = 19) 27.61 ± 19.12 10.12 ± 1.22
Non-SAO individuals (N = 20) 16.47 ± 3.81 8.95 ± 1.1
P value for difference (Mann- 0.65 0.0487
Whitney U test)
Discussion
The main finding of this study was that the SAO trait
does not result in reduced expression of Fy antigen. In fact,
we observed a 10 percent increase (p=0.049) in Fy antigen
expression by SAO RBCs when compared with RBCs from
non-SAO individuals from the same area. This difference
was significant when expression by all RBC populations
was evaluated but not when only SAO reticulocytes were
compared with non-SAO reticulocytes (although a sugges-
tive trend existed). As anticipated, reticulocyte expression
of Fy6 antigen exceeded RBC expression.20 The reasons for
the increased RBC Fy6 antigen expression in SAO are un-
clear at present, but it may reflect either a decreased rate of
loss of Fy6 antigen from the surface of SAO RBC relative to
control RBC, perhaps in relation to the increased rigidity of
the SAO cell wall, or differences in the surface area of ovalo-
cytes versus non-SAO RBC. Because there was only a small
increase in Fy6 antigen expression on SAO RBC and there
was no statistically significant increase in the reticulocyte
subpopulation, which is the only subpopulation susceptible
to infection by P. vivax, it is unlikely that this alteration af-
fects susceptibility to this parasite, although there is other
in vitro evidence that alterations in numbers of Fy antigens
expressed may alter that susceptibility.21 It is not surprising
that the difference in Fy levels was not shown in the paper
of Booth et al.16 because they were using less sensitive se-
rologic methods or because Fy6 antigen is more available
or more expressed in SAO ovalocytosis over the antigens
tested in the previous study. However, their findings with
respect to other antigens, including Rh polypeptides, have
subsequently been confirmed by molecular techniques.22
Fy antigen negativity caused by homozygosity for a pro-
moter mutation that silences transcription of the FY gene23
is observed in many ethnic groups in sub-Saharan Africa,
and presumably accounts for the relative lack of vivax ma-
laria in this part of the world. The apparently independent
emergence of heterozygosity for this same promoter muta-
tion has also been described in the Wosera area of PNG,
where SAO is rare or nonexistent. There is a need for further
evaluation of whether other common RBC polymorphisms
affect Fy antigen expression, especially those that are as-
sociated with changes in RBC shape and surface area. For
example, α-thalassemia is common in PNG, and in many
areas, including the Madang Province, nearly all individuals
have the typical α-globin mutation seen in Melanesians.24 It
IMMUNOHEMATOLOGY, Volume 25, Number 2, 2009

seems unlikely that this common polymorphism would act
as a confounding factor in our study on the grounds of bio-
logic plausibility, low likelihood of linkage disequilibrium,
and very high prevalence in the region (assuming therefore
more or less equal distribution between SAO and control
individuals within the population studied). Finally, it will
be important to determine whether environmental vari-
ables extant in malaria-endemic populations might change
Fy antigen expression. Another example would be iron defi-
ciency, which is prevalent in PNG and many other malaria-
endemic regions of the world.
There is evidence that the SAO trait affects several steps
of the biologic cycle of P. falciparum that may be relevant
for the selective advantage conferred by this trait against
malaria. In particular, SAO RBCs are partly resistant to in-
vasion in culture by merozoites of Plasmodium knowlesi
and several lines of P. falciparum,25,26 and the SAO trait
also results in altered adhesion of infected RBCs to CD36.
As with ABO,27 it is likely that the evolutionary pressure ex-
erted by survival with SAO is related to survival of children
from cerebral malaria before reproductive age. Although Fy
expression probably affects P. vivax infection,28 this evolu-
tionary pressure is almost certainly caused by P. falciparum
through its relationship to RBC adhesion.
Acknowledgment
This study was funded in part by a Covance Fellowship
from the Royal Australasian College of Physicians (I.W.).
References
1. Haldane JBS. Disease and evolution. Ric Sci Suppl A
1949;19:68–76.
2. Maitland K, Bejon P, Newton CR. Malaria. Curr Opin
Infect Dis 2003;16:389–95.
3. Jarolim P, Palek J, Amato D, et al. Deletion in erythro-
cyte band 3 gene in malaria-resistant Southeast Asian
ovalocytosis. Proc Natl Acad Sci U S A 1991;88:11022–
6.
4. Mgone CS, Koki G, Paniu MM, et al. Occurrence of the
erythrocyte band 3 (AE1) gene deletion in relation to
malaria endemicity in Papua New Guinea. Trans R Soc
Trop Med Hyg 1996;90:228–31.
5. Genton B, al-Yaman F, Mgone CS, et al. Ovalocytosis
and cerebral malaria. Nature 1995;378:564–5.
6. Allen SJ, O’Donnell A, Alexander ND, et al. Prevention
of cerebral malaria in children in Papua New Guinea by
Southeast Asian ovalocytosis band 3. Am J Trop Med
Hyg 1999;60:1056–60.
7. Fowkes FJ, Michon P, Pilling L, et al. Host erythrocyte
polymorphisms and exposure to Plasmodium falcipar-
um in Papua New Guinea. Malar J 2008;7:1.
8. Cattani JA, Gibson FD, Alpers MP, Crane GG. Heredi-
tary ovalocytosis and reduced susceptibility to malar-
ia in Papua New Guinea. Trans R Soc Trop Med Hyg
1987;81:705–9.
IMMUNOHEMATOLOGY, Volume 25, Number 2, 2009
SAO and DARC
9. Serjeantson S, Bryson K, Amato D, Babona D. Malaria
and hereditary ovalocytosis. Hum Genet 1977;37:161–
7.
10. Foo LC, Rekhraj V, Chiang GL, Mak JW. Ovalocytosis
protects against severe malaria parasitemia in the Ma-
layan aborigines. Am J Trop Med Hyg 1992;47:271–5.
11. Cortes A, Benet A, Cooke BM, Barnwell JW, Reeder JC.
Ability of Plasmodium falciparum to invade Southeast
Asian ovalocytes varies between parasite lines. Blood
2004;104:2961–6.
12. Cortes A, Mellombo M, Mgone CS, Beck HP, Reeder JC,
Cooke BM. Adhesion of Plasmodium falciparum–
infected red blood cells to CD36 under flow is enhanced
by the cerebral malaria-protective trait South-East Asian
ovalocytosis. Mol Biochem Parasitol 2005;142:252–7.
13. O’Donnell A, Raiko A, Clegg JB, Weatherall DJ, Allen
SJ. Southeast Asian ovalocytosis and pregnancy in a
malaria-endemic region of Papua New Guinea. Am J
Trop Med Hyg 2007;76:631–3.
14. Benet A, Khong TY, Ura A, et al. Placental malaria in
women with South-East Asian ovalocytosis. Am J Trop
Med Hyg 2006;75:597–604.
15. Miller LH, Mason SJ, Clyde DF, et al. The resistance
factor to Plasmodium vivax in blacks. The Duffy blood
group genotype, FyFy. N Engl J Med 1976;295:302–4.
16. Booth PB, Serjeantson S, Woodfield DG, Amato D. Se-
lective depression of blood group antigens associated
with hereditary ovalocytosis among Melanesians. Vox
Sang 1977;32:99–110.
17. Nichols ME, Rubenstein P, Barnwell J, et al. A new hu-
man Duffy blood group specificity defined by a murine
monoclonal antibody. Immunogenetics and association
with susceptibility to Plasmodium vivax. J Exp Med
1987;166:776–85.
18. Wasniowksa K, Blanchard D, Janvier D, et al. Identifi-
cation of the Fy6 epitope recognised by two monoclonal
antibodies in the N-terminal extracellular portion of
the Duffy antigen receptor for chemokines. Mol Im-
munol 1996;33:917–23.
19. Jarolim P, Palek J, Amato D, et al. Deletion in erythro-
cyte band 3 gene in malaria-resistant Southeast Asian
ovalocytosis. Proc Natl Acad Sci U S A 1991;88:11022–
6.
20. Woolley IJ, Hotmire KA, Sramkoski RM, Zimmerman
PA, Kazura JW. Differential expression of the Duffy
antigen receptor for chemokines according to RBC age
and FY genotype. Transfusion 2000;40:949–53.
21. Michon P, Woolley I, Wood EM, Kastens W, Zimmer-
man PA, Adams JH. Duffy-null promoter heterozygosi-
ty reduces DARC expression and abrogates adhesion of
the P. vivax ligand required for blood-stage infection.
FEBS Lett 2001;495:111–14.
65
I.J. Woolley et al.

22. Beckmann R, Smythe JS, Anstee DJ, Tanner MJ. Coex-
pression of band 3 mutants and Rh polypeptides: dif-
ferential effects of band 3 on the expression of the Rh
complex containing D polypeptide and the Rh complex
containing CcEe polypeptide. Blood 2001;97:2496–
505.
23. Tournamille C, Colin Y, Cartron JP, Le Van Kim C. Dis-
ruption of a GATA motif in the Duffy gene promoter
abolishes erythroid gene expression in Duffy-negative
individuals. Nat Genet 1995;10:224–8.
24. Cockburn IA, Mackinnon MJ, O’Donnell A, et al. A
human complement receptor 1 polymorphism that re-
duces Plasmodium falciparum rosetting confers protec-
tion against severe malaria. Proc Natl Acad Sci U S A
2004;101:272–7.
25. Kidson C, Lamont G, Saul A, Nurse GT. Ovalocytic
erythrocytes from Melanesians are resistant to invasion
by malaria parasites in culture. Proc Natl Acad Sci U S
A 1981;78:5829–32.
26. Hadley T, Saul A, Lamont G, Hudson DE, Miller LH,
Kidson C. Resistance of Melanesian elliptocytes (ovalo-
cytes) to invasion by Plasmodium knowlesi and Plas-
modium falciparum malaria parasites in vitro. J Clin
Invest 1983;71:780–2.
27. Cserti CM, Dzik WH. The ABO blood group sys-
tem and Plasmodium falciparum malaria. Blood
2007;110:2250–8.
28. Kasehagen LJ, Mueller I, Kiniboro B, et al. Reduced
Plasmodium vivax erythrocyte infection in PNG Duffy-
negative heterozygotes. PLoS ONE 2007;2:e336.
Ian J. Woolley (corresponding author), Department of
Infectious Diseases, Monash Medical Centre and Monash
University, Clayton, Victoria, Australia, Paul Hutchinson,
Centre for Inflammatory Diseases, Monash Medical Cen-
tre, Clayton, Victoria, Australia, John C. Reeder, Papua
New Guinea Institute of Medical Research, Goroka, Papua
New Guinea (current affiliation: Burnet Institute, Prah-
ran, Victoria, Australia), James W. Kazura, Center for
Global Health & Diseases, Case Western Reserve Univer-
sity, Cleveland, Ohio, USA, and Alfred Cortés, Papua New
Guinea Institute of Medical Research, Madang, Papua
New Guinea (current affiliation: ICREA and Institute for
Research in Biomedicine, Barcelona, Spain).

Manuscripts
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66 IMMUNOHEMATOLOGY, Volume 25, Number 2, 2009

Review
Sickle cell disease: a review
S.D. Roseff*
S
ickle cell disease (SCD) is described as the first identi-
fied “molecular” disease since its manifestations stem
from a substitution of valine for glutamic acid in the
structure of the β chain hemoglobin molecule.1 As a result
of this change, RBCs form characteristic “sickle” shapes and
the surface of these RBCs attract each other, polymerizing
when in a low oxygen environment. This seemingly “small”
variation in the structure of the RBC causing polymerization
leads to manifestations such as chronic occlusion of blood
vessels (vaso-occlusion), reduced blood flow to vital organs
(ischemia), and alterations of the immune system. In ad-
dition, the abnormal sickle cells are prematurely removed
from circulation, resulting in hemolytic anemia. Transfu-
sion is a vital component of the treatment of some of the
complications of SCD. It is also a modality used to prevent
some of these complications from occurring. Patients with
SCD are unique because those who are transfused usually
require chronic transfusion, resulting in exposure to many
different blood donors over the course of treatment. In
addition, most patients with SCD in the United States are
African American, and most donors are Caucasian from
Western European descent. As a result of this difference,
patients with SCD are exposed to RBC antigens that they
lack, putting them at risk for forming alloantibodies, de-
fined as RBC alloimmunization.2 Therefore, it is important
to understand the issues involved in the safe and effective
transfusion of patients with SCD.
Defining Hemoglobinopathies
Hemoglobin (Hgb) is made up of iron (heme) and 4
globin chains. The type of globin chains determines the type
of hemoglobin (see Table 1).3
Since SCD is characterized by the presence of an abnor-
mal or variant hemoglobin, hemoglobin S, it is characterized
as a hemoglobinopathy. The composition of hemoglobin for
patients with homozygous SS is presented in Table 2.
There are a variety of other abnormal hemoglobins that
may be present with or without Hgb S. The type of hemo-
globin a person has is based on patterns of inheritance. If
each parent contributes hemoglobin S, the child can inherit
two copies and is designated as Hgb SS, or is homozygous
for hemoglobin S. If a child only inherits one copy from one
parent and a copy of normal hemoglobin, Hgb A, they are
designated as Hgb AS, or heterozygous. These individuals
are usually asymptomatic, and only develop manifestations
under rare circumstances, where they become hypoxic,
such as at high altitudes. Therefore, not every person with
an abnormal hemoglobin develops or exhibits signs and
symptoms. Although Hgb S is found worldwide, it is most
*Reprinted with permission of the College of American Pathologists.
IMMUNOHEMATOLOGY, Volume 25, Number 2, 2009
commonly found in western Africa. About one in every 400–
500 African Americans, or 80,000, has SCD. About 9000
African Americans, or one in 12, have sickle cell trait.4
On the other hand, patients who have manifestations
of their sickle hemoglobin are considered to have SCD. This
includes patients who are homozygous for Hgb SS, as de-
scribed previously. In addition, some patients who inherit
Hgb S from one parent and another abnormal hemoglobin
from the other parent can also have SCD. Common exam-
ples are designated as Hgb SC and Sβ-thalassemia. These
individuals can have a milder clinical course than that of
individuals who are homozygous for Hgb S.
Since RBCs with Hgb S are abnormal, they are removed
from circulation in the spleen more rapidly than normal
RBCs. This leads to a reduced life in the circulation of 16–
20 days in comparison to 120 days for normal RBCs.5 The
premature destruction of RBCs, with the accompanying
decrease in hemoglobin, is classified as a hemolytic anemia.
Table 1. Composition of normal adult hemoglobin
Hemoglobin Composition Percent (%)
Hgb A α2β2 96–98
Hgb A2 α2δ2 1.5–3.5
Hgb F α2γ2 <1
Table 2. Hemoglobin SS (-α2β2S)
Hemoglobin Percent (%)
Hgb S 80
Hgb A2 2–4.5
Hgb F 120
Typical Laboratory Findings in Sickle Cell Disease
Patients with SCD are commonly diagnosed after new-
born screening. In the past, young children presented with
painful swellings of the feet and hands, a consequence of
vaso-occlusion (see discussion on Clinical Manifestations
of Disease).
Many patients with SCD have anemia, with hemoglobin
levels between 6 and 8 g/dL. Characteristic sickle shaped
cells are seen on peripheral smear (Fig. 1). Patients do not
always have symptomatic anemia because the body com-
pensates for the peripheral RBC destruction by increasing
the rate of RBC production (erythropoiesis) in the bone mar-
row. Consequently, the bone marrow contains an increase
in the number of RBC precursors. As a result, immature
67
S.D. Roseff
precursors, suggestive of reticulocytes, are released from
the marrow prematurely. Therefore, the peripheral smear
will also show these immature RBCs, which are larger than
mature RBCs and have a slightly blue color since they are
not fully hemoglobinized.3 The term reticulocytes will be
used in this education activity, and refers to these RBCs.
Fig. 1 The arrowed cell is a classic example of a sickle cell. Anoth-
er sickle cell is located in the upper left corner. There is a nucleated
red blood cell in the lower right corner. The large cell in the center
is a polychromatophilic red cell or reticulocyte. (Source: Figure
HE-42. In: Glassy EF, ed. Color Atlas of Hematology. Northfield, IL:
College of American Pathologists, 1998:97.)
Another consequence of the high rate of hemolysis and
erythropoiesis, or increased RBC turnover, is the accumula-
tion of unconjugated or indirect bilirubin. As a result, some
patients will have yellowing of the white of the eyes, scleral
icterus, and skin, or jaundice. The increase in bilirubin also
puts patients at increased risk for gallstones, and the pa-
tient may have to have their gallbladder removed. Table 3
lists the common laboratory findings in SCD and their
etiologies.
Early diagnosis is essential in order to treat and even
prevent some of the complications of SCD. In the past,
approximately 25% of children between the ages of four
months and five years with SCD died of pneumonia. The
use of prophylactic penicillin in children with SCD in the
setting of comprehensive care reduced the risk of death in
childhood to less than 3%.6–8 Therefore, in order to be able
Table 3. Common laboratory findings in sickle cell disease
Laboratory value Etiology
Low hemoglobin and hematocrit Hemolysis
High mean cellular volume (MCV) Reticulocytes
High white blood cell (WBC) Inflammation and increased marrow
count production
High platelet count Increased marrow production
High lactate dehydrogenase Hemolysis
(LDH)
Low haptoglobin Hemolysis
High total and indirect bilirubin Hemolysis
High alkaline phosphatase until Elevated bone marrow activity
puberty
68
to refer these patients to specialists and provide state-of-
the-art patient care, identification of children with SCD at
birth is an important public health intervention. Screening
can be done in the neonatal period using fetal DNA during
pregnancy, or more commonly, performing DNA analysis
soon after birth. Prenatal testing can be done between 14–
16 weeks of gestation in pregnancies where there is a chance
that the baby will inherit Hgb S from both parents.1,3,4
More commonly, newborn infants are screened through
mandatory programs required by most states, soon after
birth.1,3,4 Several methods exist for detecting abnormal he-
moglobins. These include:
• High performance liquid chromatography (HPLC)—
Considered one of the best methods to be able to detect
abnormal hemoglobins and to be able to differentiate
them from normal hemoglobin. HPLC uses reactions in
columns of reagents. This technique also measures the
quantity of the various hemoglobins.
• Hemoglobin electrophoresis—Separates different he-
moglobins according to their characteristic migration
across a gel of either cellulose acetate at an alkaline
pH or citrate agar at an acid pH, based on charge. The
different types of hemoglobin will migrate at different
rates, occupying a characteristic position on the gel.
• Isoelectric focusing—If further separation is necessary,
then isoelectric focusing can be performed. An electric
current is passed through the gel that has areas of dif-
ferent pH. The identity of the hemoglobin can be made
by its pattern of migration.
• Solubility test—Taking advantage of the fact that re-
duced Hgb S is insoluble in certain solutions, a simple
solubility test can be used to detect the presence of Hgb
S. After lysis, RBCs are reduced by sodium hydrosulfite.
If Hgb S is present, the solution will be turbid; if not
present, then the hemoglobin remains in solution and
the suspension is clear. Since this test requires a certain
amount of whole blood, it is not routinely used for new-
born screening. Note that this test will just detect the
presence of Hgb S, so patients who have sickle cell trait
(heterozygous for Hgb S) will also be positive.
When performing patient testing, it is always important to
follow the package insert and ensure that proficiency test-
ing is performed in compliance with the Clinical Laboratory
Improvement Amendments of 1988 (CLIA). CLIA regula-
tions require that laboratories enroll in a Centers for Medi-
care and Medicaid Services (CMS) approved proficiency
testing (PT) program for all regulated tests that the labora-
tory performs.
Clinical Manifestations of Sickle Cell Disease
The function of a normal RBC is dependent upon its
ability to flow freely through blood vessels and to be flexible
enough to move in and out of vessels and tissue where oxygen
delivery occurs. Due to the rigidity of the sickle RBC, these
properties are lost. Interestingly, when a patient with SCD
has a higher percentage of hemoglobin F, the course of their
IMMUNOHEMATOLOGY, Volume 25, Number 2, 2009

disease is milder and their life expectancy is longer. This is
probably the result of the lower percentage of Hgb S in these
patients, and that RBCs with Hgb F do not form polymers.9
These chronic physiologic changes can lead to problems in
all organ systems; heart, lungs, and kidneys. SCD can be
seen as a disease based on vaso-occlusion (VOC), chronic
hemolytic anemia, and immunologic impairment.
Occlusive Disease
There are a variety of ways by which sickle cells cause
obstruction. One minor mechanism is whereby irreversibly
sickled cells obstruct small capillaries. More commonly,
sickle cells are trapped in small venules and capillaries.1,10
The process of sickling and occlusion involves a complex in-
teraction between chemical mediators, as well as elements
other than the RBC itself. There is increasing understanding
that the walls of blood vessels are involved. There appears to
be increased adherence of sickle cells to the vascular walls,
due to properties of the RBCs, as well as changes in the en-
dothelial cells of blood vessels. The lipid bilayer of RBCs is
disrupted in sickle cells, causing their early removal from
circulation. Additionally, changes in their interactions with
other cells within the blood stream, such as sticky white
blood cells (WBCs) and activated platelets, contribute to
vaso-occlusion.10,11 These changes can also cause the acti-
vation of coagulation, leading to an increased risk of deep
venous thrombosis.10,12
Vaso-occlusion manifests itself as one of the most com-
mon findings in a patient with SCD, pain crisis. Prior to
routine screening of newborns, children were commonly
diagnosed with SCD prior to the age of two, with occlu-
sion in the hands and feet presenting as painful swelling,
or dactylitis.4,5 This would not occur until six months of age
because the higher level of fetal hemoglobin prior to six
months is protective. Most patients will often have severe
pain due to occlusion of blood flow to bones, bone marrow,
muscles, or organs, and in their arms, legs, back, abdomen,
and chest. Treatment consists of analgesia (many patients
with SCD require chronic medication) and hydration, since
dehydrated RBCs are more likely to sickle. The cause of cri-
sis can vary between patients and between episodes in the
same patient, but may be related to infection, extremes of
temperature, medication, or any other physiologic changes.
Occlusion is also associated with bony infarcts leading to
osteonecrosis, skin ulcers, organ occlusion (including the
spleen, resulting in functional asplenia or autosplenecto-
my), acute chest syndrome, and cerebrovascular accidents
or strokes.
Stroke
Stroke in any patient is a devastating event, with ap-
proximately 11 percent of patients with Hgb SS having a
stroke by the age of 20.13 As blood vessels become occluded,
their diameter gets smaller and the rate of blood flow in-
creases, thereby increasing the risk of stroke. Using a type
of ultrasound, transcranial Doppler (TCD), to measure
IMMUNOHEMATOLOGY, Volume 25, Number 2, 2009
A review of topics in SCD
blood flow through the internal carotid and middle cerebral
arteries of the brain, a study found that one could predict
patients at risk for a first stroke.14 Chronic transfusion ther-
apy has been used successfully to prevent primary stroke
and then subsequent stroke (see Treatment of Sickle Cell
Disease).15,16
Acute Chest Syndrome
Acute chest syndrome (ACS) is the leading cause of
death in adults with SCD and a major cause of morbidity
in patients of all ages. It is characterized by the onset of
worsening respiratory status, fever, and new infiltrate on
chest x-ray. The inciting event can be respiratory infection;
however, since a majority of cases of acute chest syndrome
in adults follow pain crisis, it is believed that an embolus
of fatty tissue from the bone marrow (the site of the initial
vaso-occlusion) into the lungs is the culprit. Patients need
enhanced oxygenation, so supplemental oxygen is admin-
istered. Transfusion is an essential component in the treat-
ment of acute chest syndrome, as will be discussed in more
detail later.17
Of interest, there are many investigators looking at the
role of inflammatory chemicals in the pathophysiology of
SCD, as well as their potential for developing new treat-
ments. For example, there is a reduction in levels of an ad-
hesion molecule (VCAM-1) in patients with SCD on chronic
transfusion protocols.18 Therefore, chronic transfusion pro-
tocols might prevent vaso-occlusion. Another recent study
found that the increases in the inflammatory mediator,
secretory phospholipase A2, can predict acute chest syn-
drome. Transfusing when levels are elevated can prevent
acute chest syndrome.19
Hemolytic Anemia
The abnormal shape and surface of sickle cells result
in their shortened life span in the circulation and lead to
the characteristic anemia. As RBCs are cycled through the
spleen, the site of their ultimate destruction, they also injure
the tissue of the spleen. Their rigidity impairs their ability to
flow smoothly through the sinusoids, and their sharp edges
cause them to be stuck, and to damage splenic tissue. In
children under the age of five, blood can pool in the spleen,
which becomes a site of sequestration of RBCs. When this
occurs, the child has a painful, enlarged spleen accompa-
nied by a drop in hemoglobin. Splenic sequestration can be
life-threatening since there is also a drop in blood volume in
the vasculature, or hypovolemia. Transfusion management
is essential in these cases. Many of these patients eventu-
ally need to have their spleen removed. As a patient gets
older, the damage to the spleen is chronic, and the spleen
actually becomes smaller, eventually shriveling and losing
function.1,4,5
In order to keep up with this chronic destruction, the
body attempts to compensate by increasing RBC produc-
tion. This chronic hemolysis may also create an inflamma-
tory state.1,10,11 Also, the free hemoglobin that is released
69
S.D. Roseff
from hemolyzed RBCs causes additional damage to the lin-
ing of blood vessels.10,13 The marrow is very active, as evi-
denced by immature RBCs or reticulocytes in the peripheral
blood. As the marrow increases production of RBCs, plate-
let and WBC production also increases. As a consequence of
increased erythropoiesis, many patients do not suffer from
signs and symptoms of anemia, even though they may have
a low hemoglobin. As long as the individual can continue
to compensate, the anemia does not present a problem.
When the marrow cannot keep up with production (i.e., a
lack of folic acid), or has its production impaired by viral ill-
ness, such as parvovirus B-19, an aplastic crisis may occur.
Parvovirus B-19 is a common childhood virus responsible
for Fifth’s Disease. This disease causes a rash, the classic
“slapped-cheek” appearance, and high fevers. Parvovirus
can suppress bone marrow production by destroying the
RBC precursor cells in the bone marrow. In these cases of
severe anemia, transfusion therapy is necessary.5
Immunologic and Infectious Manifestations
The spleen can be affected in SCD due to occlusive forc-
es, but it is also damaged by the pointed, inflexible sickle
cells that travel through it and are stuck. The spleen is an
important immunologic organ, helping to fight infections
with encapsulated organisms (S. pneumoniae, H. influ-
enzae, and N. meningitidis). Therefore, it is important to
recognize children with SCD and immunize them. As stated
earlier, the use of prophylactic penicillin has been found to
reduce death in children.9,20 All infections should be treated
aggressively.
Treatment of Sickle Cell Disease
Prevention of early complications, such as infection, is
important in the overall treatment plan for patients with
SCD. As more is being learned about the basic mechanisms
of the disease, new treatments are being developed. Anal-
gesics, including opioids, are a mainstay of treatment for
people suffering with pain crisis. In addition, it is impor-
tant to give patients with SCD vitamins, such as folic acid,
which are essential to the production of RBCs. Hydration is
also key, since RBCs sickle when dehydrated. In addition,
hydration improves the viscosity of the patient’s blood and
allows the RBCs to move more easily. As hemoglobin rises,
the viscosity of the bloodstream increases and there is an
increased risk of occlusive disorders, as well as an increased
risk of pain crisis.21 There is no data to show that transfu-
sion should be part of the routine treatment for crisis.5,20
The role of nitric oxide is also being investigated as a thera-
peutic modality, since nitric oxide plays an important role
in the physiology of RBCs.1,4
Patients of all ages who have higher concentrations
of Hgb F have milder disease and lower mortality than
patients with lower levels of Hgb F. Increasing the pro-
duction of Hgb F seems like a reasonable therapeutic in-
tervention. In the laboratory, Hgb F has been shown to
interfere with the polymerization of deoxygenated Hgb S.
70
The chemotherapeutic agent, hydroxyurea (HU), increases
the amount of Hgb F that is made by the bone marrow.
RBCs with Hgb F lack the β chain, which is responsible for
the sickling seen in Hgb S RBCs. Therefore, these RBCs
do not sickle, nor can they polymerize. This is one of the
mechanisms considered to be responsible for the improved
outcomes. In addition, HU has been found to reduce WBC
production, thereby reducing the number of circulating in-
flammatory cells, capable of adhering to blood vessel walls.
Platelet counts also drop and this may inhibit one of the
factors responsible for vaso-occlusion. HU also influences
nitric oxide metabolism. Nitric oxide leads to the dilation
of blood vessels and seems to reduce the adhesion between
RBCs and blood vessel walls.22
HU has been found to reduce mortality due to an induc-
tion of Hgb F and a reduction in vaso-occlusive events. In
addition, it reduces the incidence of acute chest syndrome,
transfusion requirements, episodes of hospitalization,
pain crisis, and reduces blood flow through intracranial
blood vessels, as measured by transcranial Doppler in chil-
dren.22–24 Currently, there is controversy about whether or
not HU can be used to prevent repeat strokes as effectively
as chronic transfusion in children.13,24,25 Since HU is a che-
motherapeutic agent, there is concern that it will have long
term side effects. It is not used in women who are preg-
nant or planning to become pregnant. In addition, some
patients have to discontinue therapy because their WBC
count becomes too low. The effects of hydroxyurea are not
immediate, generally taking a few months to be effective.23
Therefore, it does not provide rapid response during acute
illness.
Bone marrow transplant, peripheral blood stem cell
transplant, and umbilical cord blood transplant are all
strategies that can cure SCD. Due to the morbidity and
mortality secondary to transplant, it is important to care-
fully choose patients with severe disease with a high risk
of morbidity and mortality, such as those with recurrent
strokes. Therefore, trials have centered on treating children
or young adults, and there are some encouraging results.
Unfortunately, one impediment is the low availability of
matched sibling donors.26–28
Transfusion Management
Transfusion therapy should only be initiated in patients
with signs and symptoms of anemia. As mentioned, most
patients with SCD, though anemic, do not generally have
daily signs and symptoms of anemia. Therefore, RBC trans-
fusion should only be used for specific indications. RBC
transfusion, when necessary in patients with SCD, provides
some additional benefits. While increasing the patient’s he-
moglobin, transfusion dilutes the Hgb S with Hgb A. The
RBCs with Hgb A have longer survival than the RBCs with
Hgb S and neither sickle nor polymerize. In addition, trans-
fusion will suppress the patient’s own erythropoiesis, or
RBC production. As a consequence, they will produce less
of their own Hgb S RBCs.4,5,29
IMMUNOHEMATOLOGY, Volume 25, Number 2, 2009

The decision to transfuse should take into account the
known benefits in the face of risks. All blood products are
capable of transmitting certain infectious diseases and
causing transfusion reactions. In addition, the recipient is
exposed to certain inflammatory mediators that accumu-
late in transfused blood and to foreign antigens from the
donor. Exposure to foreign RBC antigens has important im-
plications for patients with sickle cell disease.
RBCs can be transfused as a simple transfusion or via
exchange transfusion (erythrocytopheresis).5,29 During
simple transfusion, one or two units of RBCs are transfused
through a peripheral IV. Exchange transfusion is usually
performed using an automated machine that is designed to
remove whole blood from the patient, separate into its vari-
ous components, and then discard the patient’s Hgb S RBCs.
RBCs from the blood bank are then used as replacement.
Typically, an RBC exchange exchanges one to two RBC vol-
umes (total blood volume × hematocrit = 1 blood volume).
A larger bore, stiff-walled central venous catheter is usually
required due to the flow requirements of the automated in-
strument. In the absence of automated equipment, manual
exchange transfusion can be done. This is not optimal, since
this can create blood pressure and volume changes during
the alternating removal of whole blood through a peripheral
vein with subsequent reinfusion of banked RBCs. Each type
of transfusion has its own risks and benefits, as outlined in
Table 4 and in Table 5.
Table 4. The benefits and risks of simple transfusion
Benefits Risks
Technical ease—requires only peripheral Increases viscosity
IV access Risk of iron overload
Low donor exposure—only 1 or 2 units
of RBCs necessary
Dilution of Hgb S
Table 5. The benefits and risks of exchange transfusion
Benefits Risks
Produces rapid reduction in Hgb S Requires large gauge IV, usually
No increase in viscosity central venous catheter
No risk of iron overload—some Requires expertise and special
observe reductions in serum equipment—may require transfer of
ferritin over time patient to another facility
Higher donor exposure—at least 4
units of RBCs for an adult, usually
more
Higher expense
Indications for Transfusion in Sickle Cell Disease
Indications for transfusion in SCD include:
Aplastic Crisis: When RBC production in the bone marrow
is interrupted, the delicate balance to maintain RBC pro-
duction during chronic hemolysis is disrupted. Therefore,
if the patient has a drop in their hemoglobin and becomes
symptomatic, transfusion is required until the underlying
process abates.5,29
IMMUNOHEMATOLOGY, Volume 25, Number 2, 2009
A review of topics in SCD
Splenic Sequestration: When a child’s hemoglobin
drops and they become symptomatic, transfusion is neces-
sary, in this setting. Of interest, transfusion increases the
hemoglobin beyond what’s expected, so it is important to
transfuse slowly to avoid over-transfusion. Hepatic seques-
tration can also occur.5,29
Pregnancy: In uncomplicated pregnancies, there is no im-
provement in outcomes in women who are transfused. Pa-
tients with other complications of SCD should be transfused
accordingly.5,29,30
Priapism: Priapism is the painful engorgement of the
penis, as a result of vaso-occlusion. Supportive therapy
consisting of hydration and analgesia is recommended, and
a urologist should be consulted. When priapism does not
resolve, simple transfusion or exchange transfusion should
be considered. There are no studies to direct transfusion
therapy. In addition, exchange transfusion has been associ-
ated with adverse neurologic sequelae, such as seizures and
increases in intracranial pressure, so there is a reluctance
to perform exchange until the patient is refractory to other
treatments.5,29,31
Presurgical Prophylaxis: Patients with SCD are at high
risk for complications when undergoing major surgery.
Currently, some practitioners recommend that patients
be transfused to a hemoglobin of 10 g/dL prior to surgery.
A study done comparing exchange transfusion to simple
transfusion found that exchange transfusion is unneces-
sary.5,29,32 There are investigations underway to determine
whether or not a hemoglobin lower than 10 g/dL is safe for
certain surgeries.33
Acute Chest Syndrome (ACS): Transfusion, either sim-
ple or exchange, implemented early in the course, improves
oxygenation and alleviates organ dysfunction. For patients
who are stable, simple transfusion should be performed. If
the patient deteriorates, does not improve, or has a rapidly
evolving course, exchange transfusion is recommended.17,34
Simple transfusion should only be performed until the he-
moglobin reaches about 10 g/dL. Beyond that, there is con-
cern for vaso-occulsion.5,29
Stroke: Due to the ease of simple transfusion in pediat-
ric patients, they usually undergo simple transfusion. For
patients who have an initial stroke, exchange transfusion
is used to rapidly reduce the amount of Hgb S that can be
recruited and extend the immediate damage to the brain.
Once a patient has a stroke, they are at risk for additional
strokes. By performing a monthly transfusion, either sim-
ple or exchange, this risk of recurrence is reduced. A recent
study showed that stopping monthly transfusion after the
return of normal flow by transcranial Doppler results in
recurrent strokes and the return of abnormal TCDs.13,15,16,35
Of interest, during studies to reduce the risk of first stroke,
patients on a chronic transfusion protocol also had a reduc-
tion in the risk of acute chest syndrome and a reduction in
the number of pain crises.15,16
71
S.D. Roseff
Adverse Consequences of Transfusion of Patients with
Sickle Cell Disease
Adverse consequences of transfusion of patients with
SCD include:
Alloimmunization: Due to the disparity between do-
nors and patients with SCD in the United States, patients
with SCD are among those most frequently alloimmmu-
nized. Studies have shown that the most common antibod-
ies formed in this population are C, E, and K1.2,36 There are
also other antibodies that are formed due to donor–recipi-
ent disparity. Therefore, in order to prevent alloimmuniza-
tion, some centers routinely perform RBC phenotypes on
patients with SCD and only transfuse RBCs that lack C, E,
and K1, if the patient is negative for the antigen. This strat-
egy reduces the rate of antibody formation in these at-risk
patients.36 Despite these findings, practice is variable.
Analyzing the results of the 2003 J-C College of American
Pathologists Proficiency Testing Survey, Osby and Shul-
man found that only 37 percent of North American hospi-
tals routinely perform antigen typing on the RBCs of non-
alloimmunized patients with SCD. In the 439 laboratories
that do perform RBC phenotyping, C, E, and K1 were the
most frequent antigens that were matched.37 In a survey of
50 academic medical centers in the US and Canada, 73 per-
cent of centers reported that they performed routine phe-
notyping with 89% of those centers also matching for C, E,
and K1.38 There is controversy about providing RBCs that
lack additional antigens. As you match for additional anti-
gens, it becomes more difficult to find compatible units. It is
argued that it is a better use of resources to save those rare
units for patients with existing antibodies.39 Another strat-
egy has been to use RBCs from donors who are ethnically
similar to the patient, thereby creating a better chance that
the donor and patient will match more closely.40 Whenever
a patient develops an antibody, they will also receive RBCs
lacking the corresponding antigen.41
Hyperhemolytic Syndrome: A serious type of hemo-
lytic transfusion reaction, called the “hyperhemolytic syn-
drome,” can occur in the setting of transfusion. During these
episodes, the patients are typically being transfused, and
instead of rising, their hemoglobin falls with subsequent
transfusion. It is felt that there is a “bystander” hemolysis of
the patient’s own RBCs, as well as destruction of transfused
RBCs. Of interest, the units transfused are crossmatch com-
patible, and no new alloantibodies are identified at the time
of transfusion. Further transfusion compounds the prob-
lem. Therefore, it is important to recognize the syndrome
and, if possible, stop transfusing. If transfusion is neces-
sary, intravenous immunoglobulin (IVIG) and intravenous
steroids have been found to be effective. If transfusion is
required because of life-threatening anemia, it should be
done cautiously, using IVIG and steroids concurrently.42–44
Iron Overload: Each unit of transfused RBCs contains
about 200–250 mg of iron. With chronic transfusion, this
iron accumulates and can be deposited into organs such
as the heart, liver, and endocrine glands. In order to pre-
72
vent this, medications are used to remove, or chelate, iron.
Intravenous chelators have been used, but their efficacy
is hindered by its half-life and poor patient compliance.
A new generation of oral chelators is effective since there
is increased compliance, and mode of actions results in a
more continuous chelation. Serum ferritin is monitored in
patients on chronic transfusion protocols who have SCD.45
The aim of treatment is to reduce serum ferritin and to
remove iron from organs.
Transfusion Recommendations
When a patient develops complications from SCD, it
also makes sense not to transfuse them with RBCs with ad-
ditional Hgb S. Therefore, many laboratories will do a sim-
ple solubility test, as described earlier, and select units that
lack Hgb S. Realize that individuals with SCD are anemic
and cannot serve as blood donors, however, there are active
donors with sickle cell trait.41
Leukoreduction (LR) of blood products has been prov-
en to reduce the risk of cytomegalovirus (CMV) transmis-
sion, reduce the risk of febrile non-hemolytic transfusion
reactions, and reduce the risk of human leukocyte antigen
(HLA) alloimmunization.41 There are also studies, though
controversial, that show there are deleterious immunologic
sequelae of transfusion that are prevented by reducing the
load of WBCs in transfused blood products. Since patients
with SCD are chronically transfused, many believe that these
patients should receive LR blood products. Of note, there is
also a study that shows reduced rates of RBC alloimmuniza-
tion in patients who receive LR blood products.46 Transfu-
sion recommendations for patients with SCD are:
• Blood products that are sickle hemoglobin negative
• Blood products that are leukoreduced
• Blood products that are negative for C, E, and K1 anti-
gens if the patient lacks these antigens
• Blood products that are negative for any additional an-
tigens against which the patient has antibody
• Blood products that are from African American donors,
if a program exists
Summary
The substitution of one amino acid in the hemoglobin
molecule results in sickle hemoglobin. As a result, RBCs
sickle in low oxygen states causing occlusion of blood ves-
sels, increased viscosity, and inflammation. These RBCs
are prematurely removed from the circulation, resulting in
a chronic hemolytic anemia. With newborn screening and
early treatment, the death rate among children with SCD
has declined. In addition, a variety of treatments are being
introduced to help manage the various manifestations of
disease. Transfusion, simple or exchange, is a mainstay of
therapy, since it reduces the amount of Hgb S in circulation
and suppresses erythropoiesis. Transfusion is indicated
for symptomatic anemia and specifically to prevent stroke
(first or recurrent), during acute stroke, and for acute chest
syndrome. Unfortunately, transfusion carries risks
IMMUNOHEMATOLOGY, Volume 25, Number 2, 2009

for infectious disease transmission, as well as immunologic
and inflammatory sequelae. For patients with SCD who may
be chronically transfused, iron overload occurs frequently.
In addition, due to differences in RBC antigens between do-
nors and recipients, these patients are at increased risk for
development of RBC alloantibodies, which can complicate
further transfusion. It is, therefore, important to prevent
alloimmunization by transfusing leukoreduced RBCs that
match the patient for the C, E, and K1 antigens. Human
progenitor cell (from bone marrow, peripheral blood stem
cells, or umbilical blood) transplant can cure the disease,
and is used for patients with severe disease for whom con-
ventional therapy may not be effective.
References
1. Frenette PS, Atweh GF. Sickle cell disease: old discov-
eries, new concepts, and future promise. J Clin Invest
2007;117:850–8.
2. Vichinsky EP, Earles A, Johnson RA, et al. Alloimmu-
nization in sickle cell anemia and transfusion of racially
unmatched blood. N Engl J Med 1990;322:1617–21.
3. Elghetany MT, Banki K. Erythrocytic disorders. In:
McPherson RA, Pincus MR, eds. Henry’s clinical diag-
nosis and management by laboratory methods. 21st ed.
Philadelphia, PA: Saunders Elsevier, 2007:504–44.
4. Ogedegbe HO. Sickle cell disease: an overview. Lab Med
2002;33:515–43.
5. Ohene-Frempong K. Indications for red cell transfusion
in sickle cell disease. Semin Hematol 2001;38:5–13.
6. Gaston MH, Verter JI, Woods G, et al. Prophylaxis with
oral penicillin in children with sickle cell anemia. A ran-
domized trial. N Engl J Med 1986;314:1593–9.
7. Leikin SL, Gallagher D, Kinney TR, et al. Mortality in chil-
dren and adolescents with sickle cell disease. Cooperative
study of sickle cell disease. Pediatrics 1989;84:500–8.
8. Gill FM, Sleeper LA, Weiner SJ, et al. Clinical events in
the first decade in a cohort of infants with sickle cell dis-
ease. Blood 1995;86:776–83.
9. Platt OS, Brambilla DJ, Rosse WF, et al. Mortality in
sickle cell disease—life expectancy and risk factors for
early death. N Engl J Med 1994;330:1639–44.
10. Telen MJ. Role of adhesion molecules and vascular en-
dothelium in the pathogenesis of sickle cell disease. He-
matol Am Soc Hematol Educ Program 2007;84–90.
11. Kupyers FA. Membrane lipid alterations in hemoglobin-
opathies. Hematology Am Soc Hematol Educ Program
2007;68–73.
12. Key NS, Slungaard A, Dandelet L, et al. Whole blood tis-
sue factor procoagulant activity is elevated in patients
with sickle cell disease. Blood 1998;91:4216–23.
13. Wang WC. The pathophysiology, prevention, and treat-
ment of stroke in sickle cell disease. Curr Opin Hematol
2007;14:191–7.
IMMUNOHEMATOLOGY, Volume 25, Number 2, 2009
A review of topics in SCD
14. Adams RJ, McKie VC, Nichols F, et al. The use of
transcranial ultrasonography to predict stroke in sick-
le cell disease. N Engl J Med 1992;326:605–10.
15. Adams RJ, McKie VC, Hsu L, et al. Prevention of a first
stroke by transfusions in children with sickle cell ane-
mia and abnormal results on transcranial Doppler ultra-
sonography. N Engl J Med 1998;339:5–11.
16. Adams RJ, McKie VC, Brambilla D, et al. Stroke pre-
vention trial in sickle cell anemia. Control Clin Trials
1998;19:110–29.
17. Vichinsky EP, Neumayr LD, Earles AN, et al. Causes and
outcomes of the acute chest syndrome in sickle cell dis-
ease. National Acute Chest Syndrome Study Group. N
Engl J Med 2000;342:1855–65.
18. Sakhalkar VS, Rao SP, Weedon J, Miller ST. Elevated
plasma sVCAM-1 levels in children with sickle cell dis-
ease: impact of chronic transfusion therapy. Am J He-
matol 2004;76:57–60.
19. Styles LA, Abboud M, Larkin S, Lo M, Kuypers FA.
Transfusion prevents acute chest syndrome predicted
by elevated secretory phospholipase A2. Br J Haematol
2007;136:343–4.
20. Claster S, Vichinsky EP. Managing sickle cell disease.
BMJ 2003;327:1151–5.
21. Platt O, Thorington B, Brambilla D, et al. Pain in sickle
cell disease. N Engl J Med 1991;325:11–16.
22. Steinberg MH, Barton F, Castro O, et al. Effect of hy-
droxyurea on mortality and morbidity in adult sickle cell
anemia. Risks and benefits up to 9 years of treatment.
JAMA 2003;289:1645–51.
23. Charache S, Terrin ML, Moore RD, et al. Effect of hy-
droxyurea on the frequency of painful crises in sickle cell
anemia. N Engl J Med 1995;332:1317–22.
24. Ware RE, Zimmerman SA, Shultz WH. Hydroxyurea as
an alternative to blood transfusion for the prevention
of recurrent stroke in children with sickle cell disease.
Blood 1999;94:3022–6.
25. Zimmerman SA, Schultz WH, Burgett S, et al. Hydroxyu-
rea therapy lowers transcranial Doppler flow velocities in
children with sickle cell anemia. Blood 2007;110:1043–7.
26. Locatelli F, Rocha V, Reed W, et al. Related umbilical
cord blood transplantation in patients with thalassemia
and sickle cell disease. Blood 2003;101:2137–43.
27. Bernaudin F, Socie G, Kuentz M, et al. Long-term results
of related myeloablative stem-cell transplantation to
cure sickle cell disease. Blood 2007;110:2749–56.
28. Woodard P, Jeng M, Handgretinger R, et al. Sum-
mary of symposium: the future of stem cell transplan-
tation for sickle cell disease. J Pediatr Hematol Oncol
2002;34:512–17.
29. Josephson CD, Lu LL, Killyer KL, Hillyer CD. Transfu-
sion in the patient with sickle cell disease: a critical review
of the literature and transfusion guidelines. Transfus
Med Rev 2007;21:118–33.
73
S.D. Roseff
30. Koshy M, Burd L, Wallace D, et al. Prophylactic red-cell
transfusions in pregnant patients with sickle cell disease.
N Engl J Med 1988;319:1447–52.
31. Rackoff WR, Ohene-Frempong K, Month S, et al. Neuro-
logic events after partial exchange transfusion for pria-
pism in sickle cell disease. J Pediatr 1992;120:882–5.
32. Vichinsky EP, Haberkern CM, Neumayr L, et al. A com-
parison of conservative and aggressive transfusion regi-
mens in the perioperative management of sickle cell dis-
ease. N Engl J Med 1995;333:206–13.
33. Buck J, Casbard A, Llewelyn C, Johnson T, Davies SC,
Williamson LM. Preoperative transfusion in sickle cell
disease: a survey of practice in England. Eur J Haematol
2005;75:14–21.
34. Platt OS. The acute chest syndrome of sickle cell disease.
N Engl J Med 2000;342:1904–7.
35. Adams RJ, Brambilla D; Optimizing Primary Stroke
Prevention in Sickle Cell Anemia (STOP 2) Trial Inves-
tigators. Discontinuing prophylactic transfusions used
to prevent stroke in sickle cell disease. N Engl J Med
2005;353:2769–78.
36. Vichinsky EP, Luban NL, Wright E, et al. Prospective
RBC phenotype matching in a stroke-prevention trial in
sickle cell anemia: a multicenter transfusion trial. Trans-
fusion 2001;41:1086–92.
37. Osby M, Shulman IA. Phenotype matching of donor red
blood cells units for non alloimmunized sickle cell dis-
ease patients: a survey of 1182 North American laborato-
ries. Arch Pathol Lab Med 2005;129:190–3.
38. Afenyi-Annan A, Brecher ME. Pre-transfusion pheno-
type matching for sickle cell disease patients. Transfu-
sion 2005;44:619–20.
39. Ness PM. To match or not to match: the question for
chronically transfused patients with sickle cell anemia.
Transfusion 1994;34:558–60.
40. Hillyer KL, Hare VW, Josephson CD, et al. Partners for
life: the transfused program for patients with sickle cell
disease offered at the American Red Cross Blood Services,
Southern Region, Atlanta, Georgia. Immunohematology
2006;22:108–11.
Attention SBB and BB Students
You are eligible for a free 1-year subscription to
Immunohematology.
Ask your education supervisor to submit the name and
complete address for each student and the inclusive
dates of the training period to immuno@usa.redcross.
org.
74
41. Brecher ME. Technical Manual. 15th ed. Bethesda, MD:
American Association of Blood Banks, 2005.
42. King KE, Shirey RS, Lenkiewica MW, et al. Delayed
hemolytic transfusion reactions in sickle cell disease:
simultaneous destruction of recipients’ red cells. Trans-
fusion 1997;37:376–81.
43. Petz LD, Calhoun L, Shulman IA, et al. The sickle cell
hemolytic transfusion reaction syndrome. Transfusion
1997;37:382–92.
44. Win N, Yeghen T, Needs, et al. Use of intravenous immuno-
globulin and intravenous methylprednisolone in hyper-
haemolysis syndrome in sickle cell disease. Hematology
2004;9:433–6.
45. Porter JB. Concepts and goals in the management of trans-
fusional iron overload. Am J Hematol 2007;82:1136–9.
46. Blumberg N, Heal JM, Gettings KF. Leukoreduction
of red cell transfusion is associated with a decreased
incidence of red cell alloimmunization. Transfusion
2003;43:945–52.
Additional Resources
Mayo Clinic. Sickle cell anemia. Available at: http://www.
mayoclinic.com/health/sickle-cell-anemia/DS00324. Ac-
cessed July 20, 2009.
Sickle Cell Disease Association of America, Inc. Available
at: http://www.sicklecelldisease.org/. Accessed July 20,
2009.
March of Dimes. Available at: http://www.marchofdimes.
com/. Accessed July 20, 2009.
Lab Tests On Line. Available at: http://www.labtestsonline.
org/. Accessed July 20, 2009.
Susan D. Roseff, MD, Medical Director, Transfusion Medi-
cine, Professor, Virginia Commonwealth University, De-
partment of Pathology, PO Box 980662, Richmond VA
23298-0662.
Notice to Readers
All articles published, including communications and
book reviews, reflect the opinions of the authors and do
not necessarily reflect the official policy of the American
Red Cross.
IMMUNOHEMATOLOGY, Volume 25, Number 2, 2009
Report

Consortium for Blood Group Genes (CBGG):

2008 report
G. A. Denomme, C.M. Westhoff, L. Castilho, and M.E. Reid, on behalf of the CBGG Members*

The Consortium for Blood Group Genes is a worldwide organiza-
tion whose goal is to have a vehicle to interact, establish guide-
lines, operate a proficiency program, and provide education for
laboratories involved in DNA and RNA testing for the prediction
of blood group, platelet, and neutrophil antigens.
Immunohematology 2009;25:75–78.
Key Words: Blood group alleles, Consortium for Blood
Group Genes, proficiency, target alleles
of appropriately worded reports of molecular analyses for
both blood donors and transfusion recipients. Members
continue to support the current recommendation that blood
components should not be labeled with molecular test re-
sults as the sole means of antigen identification. Molecular
data continue to be a source of information in the resolution
of complex serologic problems, and are not intended as the
sole means for patient transfusion management decisions.

T
Target Alleles
he Consortium for Blood Group Genes (CBGG) is a
The list of target alleles prepared by the CBGG after
nonprofit organization whose mission is “To estab-
meeting in 2007 was adopted. The preferred current termi-
lish guidelines, to provide education, and to provide
nology is listed in Table 1 of this report under the heading
a proficiency exchange for laboratories involved in DNA or
Target antigen (target allele). However, the final naming
RNA testing for the determination of blood group, platelet,
of alleles relies on the decision of the International Soci-
and neutrophil antigens.” The consortium was established
ety for Blood Transfusion. When referring to a particular
at the inaugural meeting on October 23, 2004, in Balti-
single-nucleotide polymorphism, nucleotide changes fol-
more, Maryland, by a group of people with scientific or in-
low the designated position, e.g., 125G>A; intronic nucle-
dustry experience and interest in the field. The consortium
otide changes represented in the lower case, e.g., –33t>c.
is coordinated by Marion Reid with assistance of country
The associated amino acid substitutions (not shown) flank
coordinators: Lilian Castilho for Brazil, Gregory Denomme
the designated position, e.g., Pro103Ser or P103S. Because
(with Maryse St. Louis as successor in 2009) for Canada,
gene numbering systems vary, and to avoid ambiguity in
and Connie Westhoff for the United States. All members are
the location of nucleotide changes, the CBGG has adopted
expected to interact and participate. New members are en-
GenBank gene reference sequences (RefSeqGene) and ref-
couraged to refer to the previously published information
erence SNP numbers (rs#) for blood group genes and nucle-
for background and progress information.1–4 The exchange
otides (Table 1). These numbers provide the set of common
of information is mainly accomplished through electronic
references used to communicate or report molecular testing
mailings, proficiency evaluation exercises, and a yearly
results.
meeting.
Guidelines for Molecular Testing
The CBGG Document
The CBGG has developed guidelines for free and gener-
The CBGG Document outlines the function of the CBGG.
al distribution. In 2007, the CBGG guidelines were shared
This document contains information on the structure, or-
with the AABB Molecular Testing Standards Program Unit
ganizational rules and bylaws, regulatory compliance plan,
(SPU). In addition, five CBGG members are committee
preferred terminology, and progress on the working parties
members of the AABB Molecular Testing SPU, and one
including proficiency exchange program, guidelines of prac-
member (MER) holds liaison status with the committee.
tice, DNA repository, funding, forms and disclaimers, and
The AABB Standards for Molecular Testing for Red Cell,
proposed Web site. It is highly recommended that CBGG
Platelet, and Neutrophil Antigens was published in 2008.5
members refer to this document for the aforementioned
The intent of the CBGG guidelines is to maintain an inde-
duties and activities. Meetings are held annually, in part,
pendent forum and voice for proposed molecular testing
for members to discuss outstanding issues and to provide
standards in ISO format for use by international laborato-
the opportunity to give input and accept amendments to
ries. The members will update, modify, or otherwise amend
the document. The CBGG Document is distributed to mem-
the CBGG guidelines by process of discussion and consen-
bers and is available to nonmembers on request.
sus. The CBGG guidelines will not become standards as
such to reflect the fact that the CBGG is not responsible for
Template Disclaimers
laboratory inspection or accreditation.
CBGG members continue to recognize the importance

* Full listing of the members of the CBGG can be found in the appendix.

IMMUNOHEMATOLOGY, Volume 25, Number 2, 2009 75

G.A. Denomme et al.
Proficiency Program
An exchange of samples among several blood centers,
transfusion services, and reference laboratories serves as
one mechanism to evaluate proficiency in molecular test-
ing. The proficiency programs involve the sending out of a
single sample (either DNA or whole blood) by a member
in the spring and fall of each year. In the fall of 2008, the
CBGG blood group program provided its first whole blood
sample for proficiency exchange, which was coordinated
through the Canadian Blood Services, Network Center for
Applied Development (NetCAD) laboratory. Before any dis-
tribution, the target antigen must be tested by a PCR-based
method and be confirmed by serology, with the additional
caveat that the proficiency exercise will not involve rare al-
leles. A limited number of antithetical blood group antigens
are evaluated, which are based on the assays performed by
all participating members. As of spring 2008, the alleles are
as follows: RHCE*E/RHCE*e, GYPB*S/GYPB*s, KEL*1/
KEL*2, FY*A/FY*B, FY*–33C/T GATA, and JK*A/JK*B.
The cost of sample preparation and shipping the DNA
sample is borne in turn by each submitting laboratory. To
participate in the sample exchange, proficiency program
members must agree to provide a sample for distribution
in a subsequent year through a predetermined rotation.
Although participation in the CBGG proficiency program
mandates that samples are discarded after the results have
been validated, proficiency program members must ensure
they comply with regulatory bodies. Thus, before joining
the proficiency exchange program and committing to sup-
ply a sample for the exchange, new members should address
their institutional requirements on informed consent.
To prevent communication errors because of different
reporting mechanisms, a report form has been developed
specifically for the CBGG proficiency program, a copy of
which is contained in the CBGG 2008 Document. Commu-
nication of results between the submitting and testing labo-
ratories is done by using this form. Presently, proficiency
reports for FY or GYPB*S/s should include appropriate
disclaimers in the comment section for those laboratories
that do not evaluate common modifying and silencing nu-
cleotide changes for these genes. A summary of the results
from the participating laboratories for each proficiency ex-
ercise is compiled by the submitting laboratory and submit-
ted to the New York Blood Center for archival purposes.
The proficiency evaluations for platelets and neutro-
phils are distributed to a specific group of members.
Important Notice About Manuscripts
for
Immunohematology
Please e-mail all manuscripts for consideration to
immuno@usa.redcross.org
76
Serologic confirmation is not mandated by this group owing
to the lack of regulated antisera. The first exchange for plate-
let genotyping was coordinated by Vagner Castro (Platelet
Immunology Laboratory of Hematology and Hemotherapy
Center of the State University of Campinas, UNICAMP).
This sample was previously genotyped by PCR-RFLP and
PCR-SSP to the two HPA systems most frequently involved
in platelet alloimmunization: HPA-1 and HPA-5.
Conclusions
The CBGG is a self-help, not-for-profit organization
designed as an interactive collaborative for members to
learn from each other and to strive to achieve excellence in
molecular testing of blood group, platelet, and neutrophil
antigens. Anyone interested and willing to contribute intel-
lectually is welcome to join. To become a member contact
Marion Reid (mreid@nybloodcenter.org), Lilian Castilho
(castilho@unicamp.br), Maryse St. Louis (maryse.st-louis@
hema-quebec.qc.ca), Greg Denomme (greg.denomme@
bcw.edu), or Connie Westhoff (WesthoffC@usa.redcross.
org).
Acknowledgments
We thank Robert Ratner for help in the preparation of
this manuscript and Lakshmi Gaur and Tanya Kerrigan for
their help assembling the GenBank gene and SNP reference
accession codes. The findings and conclusions in the article
should not be construed to represent any agency determi-
nation or policy.
References
1. Denomme G, Reid M. Inaugural meeting of the Consor-
tium for Blood Group Genes (CBGG): a summary report.
Immunohematology 2005;21:129–31.
2. Reid ME. Consortium for Blood Group Genes. Transfu-
sion 2007;47(Suppl):98S–100S.
3. Reid ME, Westhoff CM, Denomme G, Castilho L. Con-
sortium for Blood Group Genes (CBGG): Miami 2006
report. Immunohematology 2007;23:81–4.
4. Reid ME, Westhoff C, Denomme G, Castilho L. Consor-
tium for Blood Group Genes (CBGG): 2007 report.
Immunohematology 2007;23:165–8.
5. Guidance for standards for molecular testing for red cell,
platelet, and neutrophil antigens. Bethesda, MD: AABB
Press, 2008.
Immunohematology is on the Web!
www.redcross.org/immunohematology
For more information, send an e-mail message
to immuno@usa.redcross.org
IMMUNOHEMATOLOGY, Volume 25, Number 2, 2009
 CBGG: 2008 report

Table 1. List of blood group target antigens and alleles

ISBT system name Target antigen Gene* Target nucleotide number Controls and comments
(symbol) number (target allele) (RefSeqGene) (GeneBank SNP rs#) Control each nt analysis
with the three genotypic
classes when appropriate †
ABO (ABO) 001 A (ABO*A1) ABO nt 1061ΔC (rs56392308) Many A/B alleles have been
A2 (ABO*A2) (NG_006669.1) described, multiple targets are
necessary for identification.
A (ABO*A1) nt 526C>G (rs7853989)
Targets for non-261delG Group O
B (ABO*B1) nt 703G>A (rs8176743)
alleles not listed.
nt 796C>A (rs8176746)
nt 803G>C (rs8176747)

A (ABO*A1) nt 261G/ΔG (rs8176719)

O (ABO*01)
MNS (MNS) M (GYPA*M) GYPA nt 59C>T;71G>A;72T>G nt 72 is the 3rd nt of codon 24
002 N (GYPA*N) (NG_007470.2) (rs7682260;7687256;7658293)

S (GPB*S) GYPB (NG_007483.1) nt 143T>C (rs7683365) Disclaimer required if null alleles not
s (GPB*s) tested.

S silenced nt 230C>T † (see note below)

intron 5+5g>t

Rh (RH) D RHD (NG_007494) Exon 4 & 7 Targets may vary as there are many
004 RHDΨ Ex 4 37 bp insert approaches

C (RHCE*C) RHCE (NG_009208) intron 2 insert Insert present in RHCE*C

c (RHCE*c)
nt 307T>C (rs676785)

E (RHCE*E) nt 676C>G (rs609320)

e (RHCE*e)

Cw nt 122A>G †
C x
nt 106G>A †
V & VS nt 733C>G (rs1053361) †
V (VS-) nt 1006G>T †
Lutheran (LU) Lua (LU*01) LU (NG_007480.1) nt 230A>G (rs28399653)
005 Lub(LU*02)
Kell (KEL) K (KEL*01) KEL (NG_007492.1) nt 578T>C (rs8176058)
006 k (KEL*02)
Kpa (KEL*03) nt 841T>C (rs8176059)
Kpb (KEL*04)
Jsa (KEL*06) nt 1790C>T (rs8176038)
Jsb (KEL*07)
Duffy (FY) Fya (FY*A) FY (NC_000001.9) nt 125G>A (rs12075)
008 Fyb (FY*B)
Fyx nt 265C>T (rs34599082) †
Fy null (RBC) nt -33t>c (rs2814778) Promoter GATA-1 box
Kidd (JK) Jk (JK*A)
a
JK (NC_000018.8) nt 838G>A (rs1058396) Testing for null alleles may be ap-
009 Jkb (JK*B) propriate.
Diego (DI) Dia DI (NG_007498.1) nt 2561T>C (rs2285644)
010 Dib
Yt (YT) Yta YT (NG_007474.1) nt 1057C>A (rs1799805)
011 Ytb
Scianna (SC) Sc1 SC (NG_008749.1) nt 169G>A (rs56025238) †
013 Sc2

IMMUNOHEMATOLOGY, Volume 25, Number 2, 2009 77

G.A. Denomme et al.

Dombrock (DO) Doa DO (NG_007477.1) nt 793A>G (rs11276)

014 Dob
Hy nt 323G>T (rs28362797)
Joa nt 350C>T (rs28362798)
Colton (CO) Co a
CO (NG_007475.1) nt 134C>T (rs28362692) †
015 Cob
Landsteiner-Wiener (LW) LWa LW (NG_007728.1) nt 308A>G †
016 LWb
Cromer (CR) Cra CROM (NG_007465.1) nt 679G>C (rs60822373)
021
Knops (KN) Kna KN (NG_007481.1) nt 4681G>A (rs41274768)
022 Knb
McCa nt 4768A>G (rs17047660) †
McCb
Sla nt 4801A>G (rs17047661) †
Vil
Indian (IN) Ina IN (NG_008937.1) nt 252C>G
023 Inb
OK (OK) Oka OK (NG_007468.1) nt 274G>A †
024 Heterozygote not required
* NG_number.n represent the GenBank reference gene sequence (RefSeqGene) accession code for each blood group system gene.
# The rs number in brackets represents the GenBank SNP reference sequence (SNP rs) accession code for the given nucleotide (nt).
† Homozygous rare alleles are not required for molecular testing.

Appendix Daniel B. Bellissimo, PhD

Members of CBGG at end of 2008: Molecular Diagnostics Laboratory
BloodCenter of Wisconsin
Coordinator: 638 N. 18th Street
Marion E. Reid, PhD Milwaukee, WI 53233
Laboratory of Immunochemistry and Laboratory
of Immunohematology Imelda M. Bromilow
New York Blood Center DIAMED SA
310 East 67th Street Cressier-sur-Morat, Switzerland
New York, NY 10065
Tony S. Casina
Country Coordinators: Ortho-Clinical Diagnostics, Inc.
Connie Westhoff, PhD 1001 Highway 202
Molecular Blood Group and Platelet Antigen Testing Laboratory Raritan, NJ 08869
American Red Cross-Penn-Jersey Region
700 Spring Garden Street Vagner Castro
Philadelphia, PA 19123 Hematology and Hemotherapy Center
Hemocentro-State University of Campinas (UNICAMP)
Maryse St-Louis, PhD Rua Carlos Chagas 480 Barão Geraldo
Operational Research, Hema-Quebec Campinas, São Paulo, 13081-970, Brazil
1070, Avenue des Sciences-de-la-Vie
Quebec, Quebec G1V 5C3, Canada Laura Cooling, MD
University of Michigan Hospitals
Lilian Maria de Castilho, PhD 1500 East Medical Center Drive
Laboratory of Immunohematology Ann Arbor, MI 48109-0054
Hemocentro-State University of Campinas (UNICAMP) Ana Paula Cozac, MD
Rua Carlos Chagas 480 Barão Geraldo Centro Regional de Hemoterapia de Ribeirão Preto
Campinas, São Paulo, 13081-970, Brazil Ribeirão Preto, S.P. CEP 14051-140, Brazil

Members: Daniel de la Vega, PhD

Bai Yu, MD, PhD Servicio de Medicina Transfusional
Department of Pathology and Laboratory Medicine Hospital Italiano Garibaldi
University of Texas Health Science Center at Houston Virasoro 1249 Rosario (2000)
6431 Fannin Street, MSB 2.222 Argentina
Houston, TX 77030

78 IMMUNOHEMATOLOGY, Volume 25, Number 2, 2009

 CBGG: 2008 report

Gregory Denomme, PhD

Immunohematology Reference Laboratory Mariza A. Mota, PhD
Blood Center of Wisconsin Departamento de Hemoterapia
638 N 18th Street Hospital Israelita Albert Einstein
Milwaukee, WI 53201-72178 Av. Albert Einstein, 627 - 4° andar, Morumbi
São Paulo, SP CEP 05651-901 Brazil
Helene M. DePalma
Immunohematology Laboratory Joann M. Moulds, PhD
New York Blood Center Clinical Immunogenetics
310 East 67th Street LifeShare Blood Centers
New York, NY 10065 8910 Linwood Avenue
Shreveport, LA 71106
Lakshmi K. Gaur, MSc, PhD
Puget Sound Blood Center John J. Moulds, MT(ASCP)SBB
921 Terry Avenue Scientific Support Services
Seattle, WA 98104-1256 LifeShare Blood Centers
8910 Linwood Avenue
Ghazala P. Hashmi, PhD Shreveport, LA 71106
Bioarray Solutions
35 Technology Drive, Suite 100 Sandra Nance, MS, MT(ASCP)SBB
Warren, NJ 07059 Technical Services
American Red Cross Penn-Jersey Region
Kim Hue-Roye 700 Spring Garden Street
Laboratory of Immunochemistry Philadelphia, PA 19123
New York Blood Center
310 East 67th Street Marcia Zago Novaretti, MD, PhD
New York, NY 10065 Division of Immunohematology and Transfusion
Fundaçao Pro-Sangue/Hemocentro de Sangue
Susan T. Johnson, MSTM, MT(ASCP)SBB Av. Angélica, 2.261
Manager, Immunohematology Services Sao Paulo, Brazil
BloodCenter of Wisconsin
638 North 18th Street Joseph Ong
Milwaukee, WI 53233 Blood Centers of the Pacific
270 Masonic Avenue
Hallie Lee-Stroka San Francisco, CA 94118
Department of Transfusion Medicine
Clinical Center Jordão Pellegrino, Jr.
National Institutes of Health Rua Sampaio Ferraz 750 ap 41 B Campinas, São Paulo, Brazil CEP
10 Center Drive MSC 1184 13024-431
Bethesda, MD 20892
Maria Rios, PhD
Christine Lomas-Francis, MS, FIBMS DETTD/OBRR/CBER
Immunohematology Laboratory 29 Lincoln Drive
New York Blood Center NIH Campus Building 29, Room B24
45-01 Vernon Blvd. Bethesda, MD 20892
Long Island City, NY 11101
Dennis Roscetti
Tanya Kanigan GTI Diagnostics
BioTrove Incorporated 20925 Crossroads Circle, Suite 200
12 Gill Street Waukesha, WI 53186
Woburn, MA 01801 Dawn M. Rumsey, ART(CSMLT)
Kirk D. Kitchen PEPFAR Team, Global Development
Clinical Laboratories American Association of Blood Banks
Blood Systems Laboratories 8101 Glenbrook Road
2424 West Erie Drive Bethesda, MD 20814
Tempe, AZ 85282
Gayle Teramura
Gandhi J. Manish, MD Puget Sound Blood Center
Laboratory Medicine and Pathology 921 Terry Avenue
Mayo Clinic Seattle, WA 98104-1256
200 First Street, S.W.
Rochester, MN 55905

IMMUNOHEMATOLOGY, Volume 25, Number 2, 2009 79

G.A. Denomme et al.

Rebecca Thomas Liaison Members:

Memorial Blood Centers Sheryl A. Kochman
737 Pelham Blvd Devices Review Branch
St. Paul, MN 55114 Center for Biologics Evaluation and Research
Office of Blood Research and Review
Antonio Sergio Torloni, MD Division of Blood Applications
Mayo Clinic Food and Drug Administration
13400 East Shea Boulevard 1401 Rockville Pike, HFM-390
Scottsdale, AZ 85259 Rockville, MD 20852-1448

Sunitha Vege, MS Jill Storry, PhD

Molecular Blood Group and Platelet Antigen Testing Laboratory Blodcentralen Skåne-University Hospital
American Red Cross SE-221 85
700 Spring Garden Street Lund, Sweden
Philadelphia, PA 19123

Rita Fontão Wendel

Instituto de Hemoterapia Sirio-Libanes
Rua D. Adma Jafet, 91 - 2 Andar
Sao Paulo, CEP 01308-050 Brazil

Mark Yazer, MD
RBC Serology Reference Laboratory
Centralized Transfusion Service
University of Pittsburgh
3636 Boulevard of the Allies
Pittsburgh, PA 15213

For information concerning For information concerning the National

Immunohematology, Journal of Blood Reference Laboratory for Blood Group
Group Serology and Education, or Serology, including the American Rare
the Immunohematology Methods and Donor Program, please contact Sandra
Procedures manual, contact us by e-mail at Nance, by phone at (215) 451-4362, by fax at
immuno@usa.redcross.org (215) 451-2538, or by e-mail at snance@usa.
redcross.org

Free Classified Ads and Announcements

Immunohematology will publish classified ads and announcements (SBB schools, meetings, symposia, etc.)
without charge. Deadlines for receipt of these items are as follows:

Deadlines
1st week in January for the March issue
1st week in April for the June issue
1st week in July for the September issue
1st week in October for the December issue

E-mail or fax these items to immuno@usa.redcross.org or (215) 451-2538.

80 IMMUNOHEMATOLOGY, Volume 25, Number 2, 2009

Speical Dedication
W. John Judd, FIBMS, MIBiol
D. Mallory
W
. John Judd,
FIBMS, MIBiol,
is being honored
by Immunohematology,
Journal of Blood Group
Serology and Education
for his many contribu-
tions to the journal and to
the field of blood banking.
John served on the edito-
rial board of Immunohe-
matology for 7 years, from
2002 through 2008. We
thank him for giving gen-
erously of his time and
ideas to improve the scope
and concept of the journal.
This included contributing nine articles published between
1979 and 2005 that were significant to the interest of the
readers of Immunohematology. He also was a dedicated,
knowledgeable, and responsive peer reviewer of submitted
articles from the beginning of the publication and during
the length of his very important career.
John retired in 2008 after 34 years as director of a first-
rate AABB-accredited reference laboratory at the Univer-
sity of Michigan. His career is a tribute to his determination
to investigate the unusual and the undiscovered. In 1972,
he studied at the Sir John Cass College in London, England,
where he was elected a Fellow of the Institute of Biomedical
Sciences (FIBMS). He came to the University of Michigan
in 1974 and was the director of the reference laboratory and
professor of immunohematology in the Department of Pa-
thology until his retirement in 2008. He is now emeritus
professor of immunohematology, Department of Pathol-
ogy, at the University of Michigan. John was awarded the
usual blue and gold embossed chair at retirement to prove
it! John and his wife, Jane, then moved to North Carolina to
relax and watch the golfers go by.
John has received many awards, including the Ivor
Dunsford Memorial Award and the John Elliott Memo-
rial Award from the American Association of Blood Banks
(AABB); the Founders Award and Kay Beattie Lecture from
the Michigan Association of Blood Banks (MABB); the Pet-
taway-Sheppard Award of the North Carolina Association
of Blood Banks; L. Jean Stubbins Memorial Lecture of the
UTMB Medical Branch; Ronald Dubin Memorial Lecturer,
IMMUNOHEMATOLOGY, Volume 25, Number 2, 2009
NYABB; and Suzanne Leiden Memorial Lecturer, CABB
Society. In North Carolina, the extra room over the garage
is called the FROG (furnished room over garage); however,
in John’s case, it is called a TROG (trophy room over ga-
rage) for all of his well-deserved awards!
John received these awards for his many investigative
studies, research reports, review papers, presentations, and
publications. In 2008, John coauthored the third edition of
Judd’s Methods in Immunohematology, a major reference
methods manual for laboratory workers. This edition con-
tains more than 150 methods and procedures documents.
During the span of his career, he has had more than 80
scientific papers published in peer-reviewed journals. Ad-
ditionally, John is the author of review articles on lectins,
polyagglutination, elution studies, pretransfusion studies,
Rh blood groups, MNS-system antibodies, and investiga-
tion of the positive DAT. These are just some of the topics
that he studied and investigated in depth.
A popular speaker, John gave numerous presenta-
tions at local, state, national, and international meetings.
One could always count on John to talk! In fact, he and the
late John Case, of Gamma Biologicals, Inc., were famous
for their “chats” on the AABB SSCC forum and were well
known for debating, in depth, many questions concerning
blood group serology and its complexities.
In addition to this impressive scientific career, John
also managed to be active on many committees of the AABB,
MABB, and International Society of Blood Transfusion. He
was also on the board of the AABB for 4 years and was on
the board and president of the MABB.
The editors, authors, and reviewers of Immunohematology,
Journal of Blood Group Serology and Education, would
like to thank John for his contributions to the field of blood
banking during his 34 years at the bench as an investigator,
and as an author, educator, and mentor. We would particu-
larly like to acknowledge his contributions during the past
several years as an editor, peer reviewer, and contributing
author to Immunohematology. John, you may be gone
from the field, but you cannot be forgotten. Like the impor-
tant scientists before you, you have left a large and lasting
legacy of knowledge and respect. Thank you, and may you
and Jane have a long, healthy, and happy retirement.
Delores Mallory, MT(ASCP)SBB
Emeritus Editorial Board
Immunohematology
81
Letter from the Editors

Blood Group Reviews—A New Feature

T
he editors of Immunohematology present a new feature that is extremely exciting. Starting with this issue, Immunohe-
matology will publish a series of 28 “Blood Group Reviews” in upcoming issues. Experts in the field have agreed to put
their considerable knowledge and energy into writing a thorough review of each of the 28 selected areas. Readers will
then have comprehensive reviews of all the major blood group systems and other topics at their fingertips. Of additional value
will be the references for each article to which the reader can refer if interested in reading the original work. The authors will
present each blood group review in the following format: history, nomenclature, genetics, molecular basis, biochemistry, an-
tibodies in system, and clinical significance. The topics that will appear in sequential issues are detailed in the table below.

When all the topics have been published, Immunohematology will publish the entire collection as a stand-alone resource.
This series provides an excellent opportunity for experienced staff to obtain continuing education, and it will be an educa-
tional tool for new staff. The editors envision a copy of the collected reviews as a must-have for transfusion medicine facilities,
libraries, and personal bookshelves everywhere.

ABO FY H CR JMH
MNS JK DO KN P/Globoside
Rh and RHAG DI CO IN Lows
LU YT LW I GIL
KEL and Kx XG Ch/Rg OK
LE SC GE RAPH

Sandra Nance
Connie M. Westhoff
Editors-in-Chief
Immunohematology

82 IMMUNOHEMATOLOGY, Volume 25, Number 2, 2009

 Announcements

Masters (MSc) in Transfusion and Transplantation Sciences

at
The University of Bristol, England

Applications are invited from medical or science graduates for the Master of Science (MSc)
degree in Transfusion and Transplantation Sciences at the University of Bristol. The course
starts in October 2009 and will last for 1 year. A part-time option lasting 2 or 3 years is also
available. There may also be opportunities to continue studies for PhD or MD following the
MSc. The syllabus is organized jointly by The Bristol Institute for Transfusion Sciences and the
University of Bristol, Department of Pathology and Microbiology. It includes:

• Scientific principles of transfusion and transplantation

• Clinical applications of these principles
• Practical techniques in transfusion and transplantation
• Principles of study design and biostatistics
• An original research project

Application can also be made for Diploma in Transfusion and Transplantation Science or a
Certificate in Transfusion and Transplantation Science.

The course is accredited by the Institute of Biomedical Sciences.

Further information can be obtained from the Web site:

http://www.blood.co.uk/ibgrl/MscHome.htm

For further details and application forms please contact:

Dr Patricia Denning-Kendall
University of Bristol, Paul O’Gorman Lifeline Centre, Department of Pathology and
Microbiology, Southmead Hospital, Westbury-on-Trym, Bristol BS10 5NB, England
Fax +44 1179 595 342, Telephone +44 1779 595 455, e-mail: p.a.denning-kendall@bristol.
ac.uk.

IMMUNOHEMATOLOGY, Volume 25, Number 2, 2009 83

Announcements, cont.
Meetings

September 25
Illinois Association of Blood Banks (ILABB)
The Illinois Association of Blood Banks (ILABB) Fall Meeting will be held September 25, 2009, at the Lisle/Naperville Hil-
ton in Lisle, Illinois. For additional details please visit the Web site at www.ilabb.org or contact Kristi Williams at (309)
745-8999 or WilliaKr@usa.redcross.org.

Specialist in Blood Bank (SBB) Program

The Department of Transfusion Medicine, National Institutes of Health, is accepting applications for its 1-year Specialist
in Blood Bank Technology Program. Students are federal employees who work 32 hours/week. This program introduces
students to all areas of transfusion medicine, including reference serology, cell processing, HLA, and infectious disease
testing. Students also design and conduct a research project. NIH is an Equal Opportunity Organization. Application
deadline is December 31, 2009, for the July 2010 class. See www.cc.nih.gov/dtm > education for brochure and application.
For further information contact Karen M. Byrne at (301) 451-8645 or KByrne@mail.cc.nih.gov.
Monoclonal antibodies available at no charge
The New York Blood Center has developed a wide range of monoclonal antibodies (both murine and humanized) that are
useful for donor screening and for typing RBCs with a positive DAT. These include anti-A1, -M, -s, -U, -D, -Rh17, -K, -k,
-Kpa, -Jsb, -Fya, -Fy3, -Fy6, Wrb, -Xga, -CD99, -Dob, -H, -Ge2, -Ge3, -CD55 (both SCR2/3 and SCR4), -Oka, -I, and anti-
CD59. Most of the antibodies are murine IgG and require the use of anti-mouse IgG for detection (anti-K, k, and -Kpa).
Some are directly agglutinating (anti-A1, -M, -Wrb and -Rh17) and a few have been humanized into the IgM isoform (anti-
Jsb). The antibodies are available at no charge to anyone who requests them. Please visit our Web site for a complete list of
available monoclonal antibodies and the procedure for obtaining them.

For additional information, contact: Gregory Halverson, New York Blood Center, 310 East 67th Street, New York, NY
10021; e-mail: ghalverson@nybloodcenter.org; phone: (212) 570-3026; fax: (212) 737-4935; or visit the web site at http://
www.nybloodcenter.org >research >immunochemistry >current list of monoclonal antibodies available.
Advertisements
National Platelet Serology Reference Laboratory
Diagnostic testing for:
• Neonatal alloimmune thrombocytopenia (NAIT)
• Posttransfusion purpura (PTP)
• Refractoriness to platelet transfusion
• Heparin-induced thrombocytopenia (HIT)
• Alloimmune idiopathic thrombocytopenia purpura (AITP)
Medical consultation available
Test methods:
• GTI systems tests
— detection of glycoprotein-specific platelet antibodies
— detection of heparin-induced antibodies (PF4 ELISA)
• Platelet suspension immunofluorescence test (PSIFT)
• Solid phase red cell adherence (SPRCA) assay
• Monoclonal immobilization of platelet antigens (MAIPA)
• Molecular analysis for HPA-1a/1b
For further information, contact
Platelet Serology Laboratory (215) 451-4205
Maryann Keashen-Schnell (215) 451-4041 office
mschnell@usa.redcross.org
Sandra Nance (215) 451-4362
snance@usa.redcross.org
American Red Cross Blood Services
Musser Blood Center
700 Spring Garden Street
Philadelphia, PA 19123-3594
National Neutrophil Serology Reference
Laboratory
Our laboratory specializes in granulocyte antibody detection
and granulocyte antigen typing.
Indications for granulocyte serology testing include:
• Alloimmune neonatal neutropenia (ANN)
• Autoimmune neutropenia (AIN)
• Transfusion related acute lung injury (TRALI)
Methodologies employed:
• Granulocyte agglutination (GA)
• Granulocyte immunofluorescence by flow cytometry (GIF)
• Monoclonal antibody immobilization of neutrophil antigens
(MAINA)
TRALI investigations also include:
• HLA (PRA) Class I and Class II antibody detection
For further information contact:
Neutrophil Serology Laboratory (651) 291-6797
Randy Schuller(651) 291-6758
schullerr@usa.redcross.org
American Red Cross Blood Services
Neutrophil Serology Laboratory
100 South Robert Street
St. Paul, MN 55107

CLIA licensed CLIA licensed

84 IMMUNOHEMATOLOGY, Volume 25, Number 2, 2009

 Advertisements, cont.

Reference and Consultation Services IgA/Anti-IgA Testing

Antibody identification and problem resolution IgA and anti-IgA testing is available to do the
HLA-A, B, C, and DR typing following:

HLA-disease association typing • Identify IgA-deficient patients

Paternity testing/DNA • Investigate anaphylactic reactions
• Confirm IgA-deficient donors

For information, contact
Mehdizadeh Kashi at (503) 280-0210, or write to:
Pacific Northwest Regional Blood Services
ATTENTION: Tissue Typing Laboratory
American Red Cross
3131 North Vancouver
Portland, OR 97227
CLIA licensed, ASHI accredited
Our ELISA for IgA detects protein to 0.05 mg/dL.
For additional information contact Cindy Flickinger at
(215) 451-4909, or e-mail: flickingerc@usa.redcross.org,
or write to:
American Red Cross Blood Services
Musser Blood Center
700 Spring Garden Street
Philadelphia, PA 19123-3594
ATTN: Cindy Flickinger

National Reference Laboratory

for Blood Group Serology

Immunohematology Reference Laboratory CLIA licensed

AABB, ARC, New York State, and CLIA licensed

Donor IgA Screening
(215) 451-4901— 24-hr. phone number
(215) 451-2538— Fax
• Effective tool for screening large volumes of
American Rare Donor Program donors
• Gel diffusion test that has a 15-year proven track
(215) 451-4900— 24-hr. phone number record:
(215) 451-2538— Fax Approximately 90 percent of all donors
ardp@usa.redcross.org identified as IgA deficient by are confirmed by
the more sensitive testing methods
Immunohematology
Journal of Blood Group Serology and Education For additional information, call Kathy Kaherl at:
(860)678-2764, e-mail: kaherlk@usa.redcross.org
(215) 451-4902— Phone, business hours
(215) 451-2538— Fax or write to:
immuno@usa.redcross.org
Reference Laboratory
Quality Control of Cryoprecipitated-AHF American Red CrossBiomedical Services
Connecticut Region
(215) 451-4903— Phone, business hours 209 Farmington Ave.
(215) 451-2538— Fax Farmington, CT 06032

CLIA licensed

IMMUNOHEMATOLOGY, Volume 25, Number 2, 2009 85

Advertisements, cont.

Blood Group
Antigens &
Antibodies
A guide to clinical relevance
& technical tips
by Marion E. Reid & Christine Lomas-Francis
This compact “pocketbook” from the authors of the Blood
Group Antigen FactsBook is a must for anyone who is involved
in the laboratory or bedside care of patients with blood
group alloantibodies.
The book contains clinical and technical information about
the nearly 300 ISBT recognized blood group antigens and
their corresponding antibodies. The information is listed in
alphabetical order for ease of finding—even in the middle of
the night. Included in the book is information relating to:
• Clinical significance of antibodies in transfusion and HDN.
• Number of compatible donors that would be expected Ordering Information
to be found in testing 100 donors. Variations in different The book, which costs $25, can be
ethnic groups are given. ordered in two ways:
• Characteristics of the antibodies and optimal technique(s) • Order online from the publisher
for their detection. at: www.sbbpocketbook.com
• Technical tips to aid their identification. • Order from the authors, who
• Whether the antibody has been found as an autoantibody. will sign the book. Send a check,
made payable to “New York Blood
Center” and indicate “Pocket­
Pocketbook Education Fund book” on the memo line, to:
The authors are using royalties generated from the sale of
this pocketbook for educational purposes to mentor people Marion Reid
in the joys of immunohematology as a career. They will Laboratory of Immunochemistry
accomplish this in the following ways: New York Blood Center
• Sponsor workshops, seminars, and lectures 310 East 67th Street
• Sponsor students to attend a meeting New York, NY 10065
• Provide copies of the pocketbook Please include the recipient’s
(See www.sbbpocketbook.com for details to apply for funds) complete mailing address.

86 IMMUNOHEMATOLOGY, Volume 25, Number 2, 2009

 Advertisements, cont.
Becoming a Specialist in Blood Banking (SBB)
What is a certified Specialist in Blood Banking (SBB)?
• Someone with educational and work experience qualifications who successfully passes the American Society for Clinical Pathology (ASCP)
board of registry (BOR) examination for the Specialist in Blood Banking.
• This person will have advanced knowledge, skills, and abilities in the field of transfusion medicine and blood banking.

Individuals who have an SBB certification serve in many areas of transfusion medicine:
• Serve as regulatory, technical, procedural and research advisors
• Perform and direct administrative functions
• Develop, validate, implement, and perform laboratory procedures
• Analyze quality issues preparing and implementing corrective actions to prevent and document issues
• Design and present educational programs
• Provide technical and scientific training in blood transfusion medicine
• Conduct research in transfusion medicine

Who are SBBs?

Supervisors of Transfusion Services Managers of Blood Centers LIS Coordinators Educators
Supervisors of Reference Laboratories Research Scientists Consumer Safety Officers
Quality Assurance Officers Technical Representatives Reference Lab Specialist

Why be an SBB?
Professional growth Job placement Job satisfaction Career advancement

How does one become an SBB?

• Attend a CAAHEP-accredited Specialist in Blood Bank Technology Program OR
• Sit for the examination based on criteria established by ASCP for education and experience

However: In recent years, a greater percentage of individuals who graduate from CAAHEP-accredited programs pass the SBB exam.

Conclusion:
The BEST route for obtaining an SBB certification is …
to attend a CAAHEP-accredited Specialist in Blood Bank Technology Program

Contact the following programs for more information:

Program
Contact Name Phone Contact Email Contact Website On site
or On line
Program
Walter Reed Army Medical Center William Turcan 202-782-6210 William.Turcan@NA.AMEDD.ARMY. www.militaryblood.dod.mil On site
MIL
American Red Cross, Southern California Region Michael Coover 909-859-7496 CooverM@usa.redcross.org none On site

ARC-Central OH Region Joanne Kosanke 614-253-2740 x 2270 kosankej@usa.redcross.org none On site

Blood Center of Southeastern Wisconsin Lynne LeMense 414-937-6403 Lynne.Lemense@bcw.edu www.bcw.edu On site

Community Blood Center/CTS Dayton, Ohio Nancy Lang 937-461-3293 nlang@cbccts.org http://www.cbccts.org/education/ On line
sbb.htm
Gulf Coast School of Blood Bank Technology Clare Wong 713-791-6201 cwong@giveblood.org www.giveblood.org/education/ On line
distance/htm
Hoxworth Blood Center, Univ. of Cincinnati Susan Wilkinson 513-558-1275 susan.wilkinson@uc.edu www.hoxworth.org On site
Indiana Blood Center Jayanna Slayten 317-916-5186 jslayten@indianablood.org www.indianablood.org On line

Johns Hopkins Hospital Jan Light 410-955-6580 jlight5@jhmi.edu http://pathology2.jhu/ On site

department/divisions/trnafusion/
sbb.cfm
Medical Center of Louisiana Karen Kirkley 504-903-3954 kkirkl@lsuhsc.edu none On site
NIH Clinical Center Dept.. of Transfusion Medicine Karen Byrne 301-496-8335 Kbyrne@mail.cc.nih.gov www.cc.nih.gov/dtm On site
Rush University Veronica Lewis 312-942-2402 Veronica_Lewis@rush.edu www.rushu.rush.edu/health/dept. On line
html
Transfusion Medicine Center at Florida Blood Marjorie Doty 727-568-5433 x 1514 mdoty@fbsblood.org www.fbsblood.org On line
Services
Univ. of Texas Health Science Center at San Antonio Linda Myers 210-731-5526 lmyers@bloodntissue.org www.uthscsa.edu On site
Univ. of Texas Medical Branch at Galveston Janet Vincent 409-772-3055 jvincent@utmb.edu www.utmb.edu/sbb On line

Univ. of Texas SW Medical Center Barbara Laird- 214-648-1785 barbara.fryer@UTSouthwestern.edu http://telecampus.utsystem.edu On line
Fryer
Additional Information can be found by visiting the following Web sites: www.ascp.org, www.caahep.org and www.aabb.org Revised August 2007
IMMUNOHEMATOLOGY, Volume 25, Number 2, 2009 87
 Immunohematology
Immunohematology
Journal
JOURNAL of
OF Blood
BLOOD Group SerologyAND
GROUP SEROLOGY andEDUCATION
Education

Instructions
Instructions tofor
theAuthors
Authors
I. GENERAL INSTRUCTIONS b. Use short headings for each column needed and capitalize first letter
Before submitting a manuscript, consult current issues of of first word. Omit vertical lines.
Immunohematology for style. Double-space throughout the manuscript. c. Place explanation in footnotes (sequence: *, †, ‡, §, ¶, **, ††).
Number the pages consecutively in the upper right-hand corner, beginning 8. Figures
with the title page. a. Figures can be submitted either by e-mail or as photographs (5″ × 7″
II. SCIENTIFIC ARTICLE, REVIEW, OR CASE REPORT WITH glossy).
LITERATURE REVIEW b. Place caption for a figure on a separate page (e.g. Fig. 1 Results of...),
A. Each component of the manuscript must start on a new page in the ending with a period. If figure is submitted as a glossy, place first
following order: author’s name and figure number on back of each glossy submitted.
1. Title page c. When plotting points on a figure, use the following symbols if
2. Abstract possible: � � � � � �.
3. Text 9. Author information
4. Acknowledgments a. List first name, middle initial, last name, highest degree, position held,
5. References institution and department, and complete address (including ZIP
6. Author information code) for all authors. List country when applicable.
7. Tables
8. Figures III. EDUCATIONAL FORUM
B. Preparation of manuscript A. All submitted manuscripts should be approximately 2000 to 2500
1. Title page words with pertinent references. Submissions may include:
a. Full title of manuscript with only first letter of first word capitalized 1. An immunohematologic case that illustrates a sound investigative
(bold title)
approach with clinical correlation, reflecting appropriate collaboration
b. Initials and last name of each author (no degrees; all CAPS), e.g., M.T.
JONES, J.H. BROWN, AND S.R. SMITH to sharpen problem solving skills
c. Running title of ≤40 characters, including spaces 2. Annotated conference proceedings
d. Three to ten key words B. Preparation of manuscript
2. Abstract 1. Title page
a. One paragraph, no longer than 300 words a. Capitalize first word of title.
b. Purpose, methods, findings, and conclusion of study b. Initials and last name of each author (no degrees; all CAPs)
3. Key words 2. Text
a. List under abstract a. Case should be written as progressive disclosure and may include the
4. Text (serial pages): Most manuscripts can usually, but not necessarily, following headings, as appropriate
be divided into sections (as described below). Survey results and i. Clinical Case Presentation: Clinical information and differential
review papers may need individualized sections
diagnosis
a. Introduction
ii. Immunohematologic Evaluation and Results: Serology and
Purpose and rationale for study, including pertinent background
references molecular testing
b. Case Report (if indicated by study) iii. Interpretation: Include interpretation of laboratory results,
Clinical and/or hematologic data and background serology/molecular correlating with clinical findings
c. Materials and Methods iv. Recommended Therapy: Include both transfusion and
Selection and number of subjects, samples, items, etc. studied and nontransfusion-based therapies
description of appropriate controls, procedures, methods, equipment, v. Discussion: Brief review of literature with unique features of this
reagents, etc. Equipment and reagents should be identified in case
parentheses by model or lot and manufacturer’s name, city, and state. vi. Reference: Limited to those directly pertinent
Do not use patient’s names or hospital numbers. vii. Author information (see II.B.9.)
d. Results viii. Tables (see II.B.7.)
Presentation of concise and sequential results, referring to pertinent
tables and/or figures, if applicable IV. LETTER TO THE EDITOR
e. Discussion A. Preparation
Implication and limitations of the study, links to other studies; if 1. Heading (To the Editor)
appropriate, link conclusions to purpose of study as stated in
2. Title (first word capitalized)
introduction
3. Text (written in letter [paragraph] format)
5. Acknowledgments: Acknowledge those who have made substantial
contributions to the study, including secretarial assistance; list any grants. 4. Author(s) (type flush right; for first author: name, degree, institution,
6. References address [including city, state, Zip code and country]; for other authors:
a. In text, use superscript, Arabic numbers. name, degree, institution, city and state)
b. Number references consecutively in the order they occur in the text. 5. References (limited to ten)
7. Tables 6. Table or figure (limited to one)
a. Head each with a brief title; capitalize the first letter of first word (e.g.,
Table 1. Results of . . .) use no punctuation at the end of the title. Send all manuscripts by e-mail to immuno@usa.redcross.org

I M M U N O H E M A T O L O G Y, V O L U M E 2 2 , N U M B E R 4 , 2 0 0 6 215
88 IMMUNOHEMATOLOGY, Volume 25, Number 2, 2009
 FIRST CLASS
U.S. POSTAGE
PAID
SOUTHERN MD
Musser Blood Center PERMIT NO. 4144
700 Spring Garden Street
Philadelphia, PA 19123-3594