Immunohematology
J O U R NA L O F B L O O D G RO U P S E RO L O G Y A N D E D U C AT I O N
VO L U M E 2 3 , N U M B E R 1 , 2 0 0 7
Immunohematology
J O U R NA L O F B L O O D G RO U P S E RO L O G Y A N D E D U C AT I O N
VO L U M E 2 3 , N U M B E R 1 , 2 0 0 7
C O N T E N T S
1
Dedication in memoriam
R.R.VASSALLO
2
A1/A2
HPA-1a/b(Pl
a/b
, Zw ): the odyssey of an alloantigen system
R.H.ASTER AND P.J. NEWMAN
9
Neonatal alloimmune thrombocytopenia: a 50-year story
C. KAPLAN
14
Platelet additive solutions: current status
H. GULLIKSSON
20
ABO and platelet transfusion therapy
L. COOLING
34
Scott Murphy’s contribution in the early years of posttransfusion purpura: a remembrance
M. KEASHEN-SCHNELL
38
Differences in ABO antibody levels among blood donors: a comparison between past and present
Japanese, Laotian, and Thai populations
T. MAZDA, R.YABE, O. NATHALANG,T.THAMMAVONG, AND K.TADOKORO
42
43
C O M M U N I C AT I O N
Missing Scott Murphy, a platelet “maestro”
P. REBULLA
IN MEMORIAM
Katherine M. Beattie
44
45
E R R AT U M
ANNOUNCEMENTS
46
50
ADVERTISEMENTS
I N S T RU C T I O N S F O R AU T H O R S
EDITORS-IN-CHIEF
MANAGING EDITOR
Sandra Nance, MS, MT(ASCP)SBB
Cynthia Flickinger, MT(ASCP)SBB
Philadelphia, Pennsylvania
Philadelphia, Pennsylvania
Connie M.Westhoff, MT(ASCP)SBB, PhD
Philadelphia, Pennsylvania
TECHNICAL EDITORS
SENIOR MEDICAL EDITOR
Christine Lomas-Francis, MSc
Geralyn M. Meny, MD
New York City, New York
Philadelphia, Pennsylvania
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Glen Allen, Virginia
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David Moolton, MD
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Philadelphia, Pennsylvania
Philadelphia, Pennsylvania
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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
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Washington, District of Columbia
Anne F. Eder, MD
John J. Moulds, MT(ASCP)SBB
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Washington, District of Columbia
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Lund, Sweden
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Bethesda, Maryland
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Immunohematology is published quarterly (March, June, September, and December) by the American Red Cross, National Headquarters,
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Copyright 2007 by The American National Red Cross
ISSN 0894-203X
Dedication in memoriam
This issue of Immunohematology is a very special
one.
It is dedicated to
Dr. Scott Murphy, a uniquely
gifted physician renowned for
his equanimity, who passed
away last year after a long and
courageous battle with lymphoma. After lengthy service on
Immunohematology’s editorial
board, Scott became its senior
medical editor in 2003, serving in
that capacity for almost 3 years, until his untimely
death in April 2006. He was a driving force behind a
bold new direction for the journal, beginning with its
20th anniversary celebration in 2004, marked by four
special issues devoted to review articles. As a result of
Scott’s efforts, the journal has a new look. Every issue
is packed with important reviews from leaders in
transfusion medicine along with the primary research
reports that have always made the journal a valuable
resource for blood group serologists and molecular
biologists.
After an internal medicine internship and
residency at the Peter Bent Brigham Hospital in Boston,
Scott returned to his native Philadelphia to complete a
research fellowship in hematology at the PresbyterianUniversity of Pennsylvania Medical Center. He
published his very first paper in the New England
Journal of Medicine the following year. A landmark
report, this research launched warm platelet storage,
making routine platelet support feasible and ushering
in an era of aggressive cancer treatment and complex
surgical procedures. Scott served on the medical staffs
of the Presbyterian-University of Pennsylvania Medical
Center and the Thomas Jefferson University Hospital,
finally accepting the position of chief medical officer at
the Penn-Jersey Region of the American Red Cross
Blood Services in 1994. Throughout his career, he
guided international efforts in platelet container
design, storage medium development, and platelet
viability assessment. Most recently, he developed
“Murphy’s Rule,” a proposal for an objective quality
standard for stored platelets adopted by the Food and
Drug Administration. The importance of Scott’s work
has been recognized with numerous honors, including
the AABB’s Karl Landsteiner Award and the American
Red Cross’ Charles R. Drew Award for Lifetime
Achievement. He was a member and past chairman of
the Biomedical Excellence for Safer Transfusion (BEST)
Collaborative, a prolific group of international
transfusion medicine leaders, an accomplishment of
which Scott was quite proud.
This issue, written by his peers and collaborators,
contains reviews of interest to platelet serologists and
hospital-based cellular therapists. Richard Aster and
Peter Newman recount the noteworthy history of the
discovery of the HPA-1 polymorphism that has resulted
in a better understanding of platelet alloimmunity and
improvements in the support of alloimmunized
patients. Cecile Kaplan takes this further with a skillful
description of the consequences of platelet alloimmunization in fetuses and neonates, reviewing the
common syndrome of fetomaternal/neonatal alloimmune thrombocytopenia. Hans Gulliksson provides
a fascinating perspective on platelet additive solutions
that promise to improve storage conditions and reduce
the allogeneic plasma content of apheresis and whole
blood–derived platelets. Laura Cooling writes elegantly
about the basic science underlying major and minor
ABO mismatches in platelet transfusion as well as the
clinical consequences of the use of out-of-group
platelets. Maryann Keashen-Schnell shares a personal
perspective as one of Scott’s coworkers, investigating a
perplexing case of posttransfusion purpura.
Finally, an original article and several noteworthy
communications complete this issue, dedicated to the
life and work of a dearly missed collaborator, mentor,
leader, and friend, Scott Murphy.
I M M U N O H E M A T O L O G Y, V O L U M E 2 3 , N U M B E R 1 , 2 0 0 7
Ralph R.Vassallo, MD, FACP
Associate Medical Editor and
Guest Editor of this issue
Chief Medical Officer
American Red Cross Blood Services
Penn-Jersey Region
700 Spring Garden Street
Philadelphia, PA 19123
1
HPA-1a/b (PlA1/A2, Zwa/b): the
odyssey of an alloantigen system
R.H.ASTER AND P.J. NEWMAN
The HPA-1a/b (PlA1/A2, Zwa/b) alloantigen system was
one of the first such systems to be identified on a cell
other than the RBC. From the discovery of HPA-1a in
1958 to the present day, studies of this antigen and its
allele, HPA-1b, have led to a remarkable number of
“firsts” in immunobiology. In this review, we shall trace
the history of the HPA-1a/b system and will highlight
selected observations made during the past 48 years
that have contributed not only to the field of platelet
immunology but also to immunohematology and
transfusion medicine in general.
Serologic Characterization of HPA-1a and 1b,
the First Recognized Platelet-Specific
Alloantigen System
It is likely that an HPA-1a-specific alloantibody was
first identified by Zucker and colleagues1 in 1956 or
1957 in studying a patient who developed profound
thrombocytopenia and hemorrhagic symptoms 5 days
after receiving a blood transfusion. The patient’s serum
contained a strong antibody that produced
agglutination (Fig. 1) and induced complementdependent lysis of platelets from four normal subjects
but not with the patient’s own platelets obtained after
recovery, about 4 weeks after the acute episode.
Although the antigen recognized by this antibody was
not characterized further, this case was almost certainly
the first example of a syndrome that was later
designated “posttransfusion purpura” (PTP) associated
with an antibody specific for HPA-1a. Shortly
thereafter, van Loghem and colleagues2 identified a
platelet isoagglutinin in serum from a transfused
patient and showed that it was specific for a marker
they designated “Zwa” that was present in about 98
percent of the Dutch population and was inherited as
a dominant trait. Their studies were facilitated by the
fact that van Loghem himself possessed the rare Zwanegative phenotype. At about the same time, Shulman
and colleagues3 at the National Institutes of Health in
2
Fig. 1.
The first anti-HPA-1a–specific antibody was detected on the basis
of its ability to cause complement-dependent lysis of normal
platelets. EDTA platelet-rich plasma was reconstituted with 0.1M
MgCl2. Patient serum (above) but not normal serum (below)
produced platelet fragmentation when the mixture was
incubated at 37°C.*
*Reprinted with permission from Zucker et al.1
Bethesda used platelet agglutination and quantitative
complement fixation to characterize two examples of
an alloantibody specific for a high-frequency
alloantigen they called “platelet antigen 1” (PlA1). It was
soon found that PlA1 and Zwa were identical.4 The
expected allele of Zw a (Zw b) was identified by van der
Weerdt et al.5 as the target for an IgM platelet
isoagglutinin found in a transfused patient. When a
unified nomenclature was developed for plateletspecific alloantigens, Zwa/PlA1 and Zwb/PlA2 were
designated “HPA-1a” and “HPA-1b,” respectively.6
Analysis of the allelic frequencies of this and other
platelet alloantigen systems suggests that the HPA-1a
epitope was present in the primordial allele of platelet
membrane glycoprotein (GP) IIIa (discussed in a later
I M M U N O H E M A T O L O G Y, V O L U M E 2 3 , N U M B E R 1 , 2 0 0 7
HPA-1a/b(PlA1/A2, Zwa/b) alloantigen system
Fig. 2.
Early mutations in the GPIIIa gene in Homo sapiens gave rise to
polymorphisms responsible for creation of platelet alloantigenic
epitopes.
section), traveled with early humans out of Africa
during migrations to Northern Europe approximately
40,000 years ago, and finally came to North America
with European colonists about 500 years ago (Fig. 2).
Today, the approximate gene frequencies of HPA-1a and
HPA-1b in the latter two populations are 0.86 and 0.14,
respectively. However, HPA-1b is much less common in
Amerindians7 and African Americans8 and is extremely
rare in Asians.8,9
Early Studies Led to Identification of Two
Thrombocytopenic Syndromes Associated
With Sensitization to HPA-1a
The two examples of anti-HPA-1a studied by
Shulman and his colleagues3 came from patients who
had received a blood transfusion and, 1 week later,
developed profound thrombocytopenia lasting about 3
weeks (Fig. 3). They coined the term “posttransfusion
purpura” (PTP) to define the combination of clinical
and serologic findings typical of this condition. By
1986, at least 75 well-documented cases of PTP, nearly
all associated with anti-HPA-1a antibodies, had been
described. Not long after their description of PTP,
Shulman et al.4 identified two more examples of antiHPA-1a in women who had given birth to infants with
severe neonatal alloimmune thrombocytopenia
(NATP), and provided the first description of that
condition involving maternal-fetal incompatibility for a
platelet-specific antigen. It is now recognized that
NATP occurs in about one of approximately every
1000 newborns, and that maternal-fetal incompatibility
of HPA-1a is responsible in the majority of cases.11
HPA-1a-Negative Platelets Were Used for the
First “Antigen-Compatible” Platelet
Transfusions
The recognition of NATP as a clinical entity raised
the issue of whether HPA-1a-negative (HPA-1b-positive)
platelets could be transfused successfully to infants
with thrombocytopenia caused by maternal anti-HPA1a. Because only 2 percent of the general population is
homozygous for HPA-1b and few blood banks were
capable of performing platelet typing in the 1960s,
platelets from HPA-1a-negative donors were not
generally available. However, Adner and colleagues12
showed that washed maternal platelets produced
satisfactory posttransfusion increments in an infant
with this condition. Because a mother’s platelets are
invariably compatible with her own antibody, this
approach has subsequently been used for treatment of
many infants born with NATP, regardless of the
specificity of the mother’s alloantibody.
Localization of HPA-1a to GPIIIa (the
Integrin β3 Subunit)
Fig. 3.
PTP occurring 6 days after a blood transfusion in a 43-year-old
woman. Prednisone was without effect on the platelet count, but
spontaneous recovery occurred after about 3 weeks. A
complement-fixing antibody was detected on Day 11 and
declined to undetectable levels at about the time of platelet
recovery.*
*Reprinted with permission from Shulman et al.3
From studies done using techniques available in
1960, Shulman and colleagues3 concluded that HPA-1a
might reside on a relatively abundant platelet
membrane protein. An important clue to the actual
localization of the antigen came from the finding by
Kunicki and Aster13 that platelets from patients with
type I Glanzmann thrombasthenia, known to lack
glycoproteins IIb and IIIa, were invariably HPA-1a
negative. Kunicki and Aster14 then used immunochemical methods to isolate GPIIIa, showed that it was
the carrier protein for HPA-1a, and speculated that the
I M M U N O H E M A T O L O G Y, V O L U M E 2 3 , N U M B E R 1 , 2 0 0 7
3
R.H.ASTER AND P.J. NEWMAN
antigen was held in a configuration suitable for
antibody recognition by one or more disulfide bonds.
Newman and colleagues15 soon thereafter localized the
HPA-1a epitope to a 17-kd tryptic fragment of the
GPIIIa and provided evidence that oligosaccharide
moieties were not required for immunogenicity.
Recognition that GPIIIa was one of the subunits of the
integrin αIIbβ316 enabled others to show that HPA-1a/b
alloantigens are not restricted to platelets, but can also
be expressed as part of the αvβ3 complex on
endothelial cells,17,18 macrophages, and fibroblasts.19
Genetic Basis for the HPA-1a/b
Polymorphism
Discovery of PCR in the mid-1980s made it possible
for the first time to identify nucleotide and amino acid
polymorphisms assumed to be responsible for the
creation of platelet alloantigens. Initially, it appeared
that this approach might not be applicable to “platelet
molecular biology” as platelets both lack a nucleus and
contain only vanishingly small amounts of mRNA. This
problem was circumvented by showing that mRNA
could be extracted from circulating platelets,
converted into cDNA, cloned, and then sequenced.20
Using this approach, Newman and colleagues21 then
sequenced cDNAs encoding GPIIIa from serologically
identified HPA-1a-positive and HPA-1a-negative
platelets and found a single nucleotide that accounted
for the generation of Leu33 (PlA1 = HPA-1a) and Pro33
(PlA2 = HPA-1b) allelic forms of GPIIIa. That the
Leu33Pro polymorphism was not only associated with
this alloantigen system but was directly responsible for
creating the alloantigenic epitope was later shown by
expressing the Leu33 and Pro33 forms of GPIIIa in
Chinese hamster ovary cells and demonstrating that
anti-HPA-1a human alloantisera bound only the Leu33
allelic isoform, whereas anti-HPA-1b sera reacted with
only the Pro33 form of the glycoprotein.22 This
combined approach was subsequently used by us and
others to characterize nucleotide and amino acid
substitutions responsible for the creation of other
clinically important platelet-specific alloantigens,23–25
making possible routine DNA typing for most of the
platelet antigen systems,26,27 an application that is now
commonplace in transfusion medicine.
PlA1 at Last! Structural Characterization of a
Platelet-Specific Alloantigen
The discovery that the HPA-1a and -1b alloantigen
system was controlled by a polymorphism at amino
4
Fig. 4.
Schematic diagrams from the early 1990s of the hypothetical
structure of the GPIIb-IIIa complex (upper left). The amino
terminus of GPIIIa, including a small disulfide-bonded loop that
was thought at that time to be formed by amino acids 26 and 38
and to encompass polymorphic amino acid residue 33 (PlA1/PlA2
controlling region) is shown in the upper right (encircled).
Bottom: an early molecular model of the Leu33 and Pro33 forms of
the AA 26–38 loop thought to encompass the PlA1 and PlA2
alloantigenic epitopes. Modeling was performed by Jack Gorski,
Blood Research Institute, BloodCenter of Wisconsin, using SYBIL
molecular modeling software running on a Silicon Graphics
Workstation.
acid 33 of GPIIIa, together with a complex biochemical
analysis of disulfide bond assignments within GPIIIa,28
facilitated generation of several models of the HPA-1a
and HPA-1b epitopes. One such early model is shown in
Figure 4, which depicts a circular peptide loop held
together by cysteine residues 26 and 38 that was
predicted to form the alloantigen. Frustratingly,
however, when cyclic peptides expected to contain
HPA-1a/b were synthesized, they were found to be
incapable of reacting with HPA-1a- and HPA-1b-specific
antibodies.29 An explanation for this apparent anomaly
was finally provided by Springer and colleagues,30 who
determined the actual crystal structure of the Nterminal plextrin-semaphorin-integrin (PSI) homology
domain of GPIIIa—the domain that encompasses
polymorphic residue 33. In addition to providing the
correct disulfide bond assignments, this landmark study
provided the coordinates of the PSI domain at the Nterminus of β3 integrin and made it possible to
visualize for the first time the region of GPIIIa that
controls formation of the HPA-1a and HPA-1b epitopes
(Fig. 5)—some 45 years after the initial description of
this platelet alloantigen system! More work remains,
however, because the actual surface in GPIIIa
recognized by HPA-1a-specific antibodies appears to be
complex, as Valentin and coworkers31 found that
I M M U N O H E M A T O L O G Y, V O L U M E 2 3 , N U M B E R 1 , 2 0 0 7
HPA-1a/b(PlA1/A2, Zwa/b) alloantigen system
Fig. 5.
PlA1 at last! The amino terminal PSI domain of GPIIIa, based on
the actual crystal structure of the molecule, is shown in four
different rotated views. Amino acid 33 is portrayed as a ball and
stick structure at the base of a disulfide-bonded loop formed by
residues 16 and 38. The structure is complicated by the presence
of two additional cysteine residues within this loop—one at
amino acid 23, and the other at residue 26—making mimicking
the epitope for potential therapeutic purposes an extremely
challenging task. Amino acid strands connecting the PSI domain
to the hybrid domain of GPIIIa are shown at the top of each
structure.
certain mutant forms of GPIIIa are recognized by some,
but not all, HPA-1a-specific antibodies, and devised the
terms “type 1” and “type 2” to describe those that
require a cysteine residue at amino acid residue 435
and those that do not, respectively. It seems likely that
type 1 antibodies recognize a peptide sequence
containing Leu33 plus a noncontiguous peptide
sequence near Cys435.
The Immune Response to HPA-1a is Unique
As blood banks and other laboratories acquired the
ability to detect HPA-1a-specific antibodies, it soon
became apparent that not all HPA-1a-negative
individuals challenged with HPA-1a-positive platelets
became sensitized to this antigen. In a series of studies
begun in the 1980s,32,33 it was found that HPA-1a is
unique among blood group antigens in inducing an
immune response that is almost invariably linked to the
class II HLA marker DRB3*010134 or DQB1*02.35 In the
case of NATP, the relative risk for a fetus that is HPA-1a
incompatible with its mother is approximately 141 if
the woman is positive for DRB3*0101, roughly the
same as the risk of developing ankylosing spondylitis in
HLA-B27-positive individuals.36 The allele HPA-1b
induces antibodies much less often than HPA-1a, and
the response to this marker is not HLA-linked.37
A potential explanation for the remarkable
association between the immune response to HPA-1a
and HLA was provided by Gorski and associates.36,38 In
one series of studies, they showed that T cells from two
women who had produced anti-HPA-1a antibodies are
specifically stimulated by a cyclic peptide containing
the polymorphism that controls HPA-1a expression
(residues 27–37 of the HPA-1a allele of GPIIIa, β3
integrin) but not by the same set of peptides from the
HPA-1b allele (Leu to Pro at amino acid residue 33).36
Interestingly, a common complementarity determining
region motif was identified in responding T cells from
two different women, despite use of genes from
different V beta families to encode the peptide
recognition domain. These findings showed that the
response to HPA-1a is unusual in that the same peptide
is recognized by both B-cell and T-cell receptors. The
same group then found that a peptide containing Leu33
bound tightly to recombinant DRB3*1010 whereas the
one containing Pro33 was nonreactive, thus providing a
molecular explanation for the unidirectional nature of
the immune response to HPA-1 antigens.38
The HPA-1a/b Alloantigen System in
Hemostasis
A new and still evolving chapter in the HPA-1a/b
story began with observations by Weiss and
coworkers39 that persons admitted to an intensive care
unit with myocardial infarction were more likely to be
positive for HPA-1b than individuals from the general
population. This association appeared to be particularly significant in younger individuals. This report set
off a series of epidemiological and biochemical studies
that are remarkable for lack of consensus in nearly 100
published reports. For example, one study found that
fibrinogen binds more readily to GPIIb/IIIa on
activated HPA-1b-positive platelets than on HPA-1bnegative platelets,40 but no difference was found by
two other groups.41,42 Various reports suggested that
HPA-1b-positive individuals are at higher risk for
thrombosis or premature atherosclerosis42,43 and even
renal transplant rejection.44 However, a retrospective
analysis of DNA samples from almost 15,000
individuals found no association between HPA-1b and
thrombosis, atherosclerosis, or stroke.45 HPA-1bpositive platelets were found to be hypersensitive to
the agonist ADP in one study46 and resistant to
activation by thrombin receptor activating peptide in
another.47 Recent work has provided a certain amount
of objective biochemical evidence that HPA-1b-positive
I M M U N O H E M A T O L O G Y, V O L U M E 2 3 , N U M B E R 1 , 2 0 0 7
5
R.H.ASTER AND P.J. NEWMAN
platelets may be “hyperfunctional” in several
respects.48,49 Perhaps further work along these lines
will provide an explanation for apparently discordant
observations made at the clinical level.
Conclusion
Since the alloantigen system now designated HPA1a/b was discovered about 1960, serologic,
biochemical, epidemiologic, and molecular biological
studies of this diallelic alloantigen system have
contributed importantly to our understanding of
alloimmune thrombocytopenia, the cellular and
humoral basis of the alloimmune response, the
structural basis of alloantigenicity, and platelet
pathophysiology. As more is yet to be learned, it is to
be hoped that further examination of this remarkable
alloantigen system will be similarly rewarding.
Acknowledgment
Supported by Grants HL-13629 and HL-44612 from
the National Heart, Lung, and Blood Institute.
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Blood
1988;72:230–3.
I M M U N O H E M A T O L O G Y, V O L U M E 2 3 , N U M B E R 1 , 2 0 0 7
HPA-1a/b(P1A1/A2, Zwa/b) alloantigen system
19. Giltay JC, Brinkman HJ, von dem Borne AE, van
Mourik JA. Expression of the alloantigen Zwa (or
PlA1) on human vascular smooth muscle cells and
foreskin fibroblasts: a study on normal individuals
and a patient with Glanzmann’s thrombasthenia.
Blood 1989;74:965–70.
20. Newman PJ, Gorski J, White GC 2nd, Gidwitz S,
Cretney CJ, Aster RH. Enzymatic amplification of
platelet-specific messenger RNA using the
polymerase chain reaction. J Clin Invest 1988;82:
739–43.
21. Newman PJ, Derbes RS, Aster RH. The human
platelet alloantigens, PlA1 and PlA2, are associated
with a leucine33/proline33 amino acid polymorphism in membrane glycoprotein IIIa, and are
distinguishable by DNA typing. J Clin Invest
1989;83:1778–81.
22. Goldberger A, Kolodziej M, Poncz M, Bennett JS,
Newman PJ. Effect of single amino acid
substitutions on the formation of the PlA and Bak
alloantigenic epitopes. Blood 1991;78:681–7.
23. Lyman S, Aster RH, Visentin GP, Newman PJ.
Polymorphism of human platelet membrane
glycoprotein IIb associated with the Baka/Bakb
alloantigen system. Blood 1990;75:2343–8.
24. Wang R, Furihata K, McFarland JG, Friedman K,
Aster RH, Newman PJ. An amino acid
polymorphism within the RGD binding domain
of platelet membrane glycoprotein IIIa is
responsible for the formation of the Pena/Penb
alloantigen system. J Clin Invest 1992;90:2038–43.
25. Santoso S, Kalb R, Walka M, Kiefel V, MuellerEckhardt C, Newman PJ. The human platelet
alloantigens Br(a) and Br(b) are associated with a
single amino acid polymorphism on glycoprotein
Ia (integrin subunit alpha 2). J Clin Invest 1993;
92:2427–32.
26. McFarland JG,Aster RH, Bussel JB, Gianopoulos JG,
Derbes RS, Newman PJ. Prenatal diagnosis of
neonatal alloimmune thrombocytopenia using
allele-specific oligonucleotide probes. Blood 1991;
78:2276–82.
27. Skogen B, Bellissimo DB, Hessner MJ, et al. Rapid
determination of platelet alloantigen genotypes
by polymerase chain reaction using allele-specific
primers.Transfusion 1994;34:955–60.
28. Calvete JJ, Henschen A, Gonzalez-Rodriguez J.
Assignment of disulphide bonds in human platelet
GPIIIa. A disulphide pattern for the beta-subunits
I M M U N O H E M A T O L O G Y, V O L U M E 2 3 , N U M B E R 1 , 2 0 0 7
of the integrin family. Biochem J 1991;274(Pt
1):63–71.
29. Flug F, Espinola R, Liu LX, et al. A 13-mer peptide
straddling the leucine33/proline33 polymorphism
in glycoprotein IIIa does not define the PLA1
epitope. Blood 1991;77:1964–9.
30. Xiao T, Takagi J, Coller BS, Wang JH, Springer TA.
Structural basis for allostery in integrins and
binding to fibrinogen-mimetic therapeutics.
Nature 2004;432:59–67.
31. Valentin N, Visentin GP, Newman PJ. Involvement
of the cysteine-rich domain of glycoprotein IIIa in
the expression of the human platelet alloantigen,
PlA1: evidence for heterogeneity in the humoral
response. Blood 1995;85:3028–33.
32. Reznikoff-Etievant MF, Muller JY, Julien F, Patereau
C. An immune response gene linked to MHC in
man.Tissue Antigens 1983;22:312–4.
33. Mueller-Eckhardt C, Mueller-Eckhardt G, WillenOhff H, et al. Immunogenicity of and immune
response to the human platelet antigen Zwa is
strongly associated with HLA-B8 and DR3. Tissue
Antigens 1985;26:71–6.
34. Valentin N, Vergracht A, Bignon JD, et al. HLADRw52a is involved in alloimmunization against
PL-A1 antigen. Hum Immunol 1990;27:73–9.
35. L’Abbe D, Tremblay L, Goldman M, Decary F,
Chartrand P. Alloimmunization to platelet antigen
HPA-1a (Zwa): association with HLA-DRw52a is
not 100%.Transfus Med 1992;2:251.
36. Maslanka K, Yassai M, Gorski J. Molecular
identification of T cells that respond in a primary
bulk culture to a peptide derived from a platelet
glycoprotein implicated in neonatal alloimmune
thrombocytopenia. J Clin Invest 1996;98:1802–8.
37. Kuijpers RW, von dem Borne AE, Kiefel V, et al.
Leucine33-proline33 substitution in human
platelet glycoprotein IIIa determines HLADRw52a (Dw24) association of the immune
response against HPA-1a (Zwa/PlA1) and HPA-1b
(Zwb/PlA2). Hum Immunol 1992;34:253–6.
38. Wu S, Maslanka K, Gorski J. An integrin
polymorphism that defines reactivity with
alloantibodies generates an anchor for MHC class
II peptide binding: a model for unidirectional
alloimmune responses. J Immunol 1997;158:
3221–6.
39. Weiss EJ, Bray PF,Tayback M, et al.A polymorphism
of a platelet glycoprotein receptor as an inherited
7
R.H.ASTER AND P.J. NEWMAN
risk factor for coronary thrombosis. N Engl J Med
1996;334:1090–4.
40. Vijayan KV, Liu Y, Souza S, Thiagarajan P, Bray PF.
Fibrinogen and prothrombin binding is enhanced
to the Pro33 isoform of purified integrin
alphaIIbbeta3. J Thromb Haemost 2006;4:905–6.
41. Bennett JS, Catella-Lawson F, Rut AR, et al. Effect of
the Pl(A2) alloantigen on the function of beta(3)integrins in platelets. Blood 2001;97:3093–9.
42. Meiklejohn DJ, Urbaniak SJ, Greaves M. Platelet
glycoprotein IIIa polymorphism HPA 1b (PlA2): no
association with platelet fibrinogen binding. Br J
Haematol 1999;105:664–6.
43. Gardemann A, Humme J, Stricker J, et al.
Association of the platelet glycoprotein IIIa
PlA1/A2 gene polymorphism to coronary artery
disease but not to nonfatal myocardial infarction
in low risk patients. Thromb Haemost 1998;80:
214–7.
44. Salido E, Martin B, Barrios Y, et al. The PlA2
polymorphism of the platelet glycoprotein IIIA
gene as a risk factor for acute renal allograft
rejection. J Am Soc Nephrol 1999;10:2599–605.
45. Ridker PM, Hennekens CH, Schmitz C, Stampfer
MJ, Lindpaintner K. PlA1/A2 polymorphism of
platelet glycoprotein IIIa and risks of myocardial
infarction, stroke, and venous thrombosis. Lancet
1997;349:385–8.
46. Feng D, Lindpaintner K, Larson MG, et al. Increased
platelet aggregability associated with platelet
GPIIIa PlA2 polymorphism: the Framingham
Offspring Study. Arterioscler Thromb Vasc Biol
1999;19:1142–7.
47. Lasne D, Krenn M, Pingault V, et al. Interdonor
variability of platelet response to thrombin
receptor
activation: influence
of
PlA2
polymorphism. Br J Haematol 1997;99:801–7.
48. Vijayan KV, Liu Y, Dong JF, Bray PF. Enhanced
activation of mitogen-activated protein kinase and
myosin light chain kinase by the Pro33
polymorphism of integrin beta 3. J Biol Chem
2003;278:3860–7.
49. Vijayan KV, Liu Y, Sun W, Ito M, Bray PF. The Pro33
isoform of integrin beta3 enhances outside-in
signaling in human platelets by regulating the
activation of serine/threonine phosphatases. J Biol
Chem 2005;280:21756–62.
Richard H. Aster, MD, and Peter J. Newman, PhD,
Blood Research Institute of BloodCenter of Wisconsin,
Inc., and Departments of Medicine and Cell Biology,
Medical College of Wisconsin, Milwaukee, Wisconsin.
IMPORTANT NOTICE ABOUT MANUSCRIPTS
FOR
IMMUNOHEMATOLOGY
Please e-mail all manuscripts for consideration to Marge Manigly at mmanigly@usa.redcross.org
8
I M M U N O H E M A T O L O G Y, V O L U M E 2 3 , N U M B E R 1 , 2 0 0 7
Neonatal alloimmune
thrombocytopenia: a 50-year story
C. KAPLAN
Since the first description of the immunologic
mechanisms in neonatal and thrombocytopenic
purpura1 and the first report of a maternal antibody
directed against a platelet alloantigen inherited from
the father,2 in the 1950s, much has been learned
concerning fetal and neonatal alloimmune
thrombocytopenia (FNAIT), but questions are still
unanswered. FNAIT has been regarded as the platelet
counterpart of HDN, but, in contrast to HDN, the first
infant is affected in 50 percent of cases. This condition,
which has been estimated to have an incidence of 1 in
800 to 1 in 1000 live births,3,4 can cause severe
bleeding in the fetus and newborn, and all
incompatible fetuses will be at risk for subsequent
pregnancies. Therefore, it is important to diagnose
FNAIT and to manage the subsequent pregnancies to
prevent the consequences of severe fetal
thrombocytopenia.
1953–2007: the Platelet Alloantigen Story
Over the years, considerable progress has been
made in the characterization of platelet-specific
alloantigens. Improvements in serologic methods for
the detection of maternal alloantibodies—including
the use of antigen-capture assays, the monoclonal
antibody-specific immobilization of platelet antigens
technique,5 or monoclonal antigen capture ELISA6—
the development of immunochemical techniques, and
the advent of molecular biological techniques have led
to the description of 24 platelet-specific alloantigens.
A human platelet antigen (HPA) nomenclature was
adopted in 1990 to replace the lab-specific
nomenclature that was used previously. The antigenic
systems are numbered in order of the date of
discovery; the high-incidence allele is called “a,” and the
low-incidence allele is called “b” in the original
population in which the alleles were identified.7 Under
the auspices of the International Society of Blood
Transfusion and the International Society of
Thrombosis and Haemostasis, a Platelet Nomenclature
Committee has published tables that will be updated;
these include the list of the HPA antigens, the antigen
genetic basis, and the platelet antigen alleles defined by
sequencing. The HPA nomenclature will still be used
for clinical and scientific purposes.8 The reader is
referred to the table of numbered HPAs, their
glycoprotein location, and approximate Caucasian
phenotype frequencies in “Scott Murphy’s contributions in the early years of posttransfusion purpura: a
remembrance” in this issue of Immunohematology.
HPA antigens are expressed on different integrins
playing a role in cellular interactions. α2bβ3 (GPIIb−IIIa)
is the major platelet integrin and is restricted to
platelets, whereas the αvβ3 integrin is expressed more
widely. α2bβ1, also known as GPIa-IIa, is the second
most important platelet integrin and is also found on
lymphocytes. Antigens located on β3 (GPIIIa) have
been found on other cells, such as endothelial cells, and
on activated T lymphocytes when located on α2; this
may play a pathophysiologic role.
Frequencies of platelet antigens vary among
different populations. In Caucasians, HPA-1a is by far
the most common antigen implicated in FNAIT,9
followed at much lower frequency by HPA-5b,10 then
HPA-3.11 In contrast, in Asians, FNAIT is essentially
associated with HPA-4 and HPA-5b. FNAIT has been
reported involving rare or private antigens.8 Recent
studies have shown that these low-frequency antigens
are not restricted to single families,12 especially antiHPA-9bw, which could account for up to 2 percent of
confirmed cases13,14; therefore they must not be ignored
in the screening for FNAIT with a negative initial
laboratory investigation. The humoral maternal
response is not uniform in this condition and must be
taken into account when undertaking serologic
diagnosis.15 Further study of the characteristics of the
maternal alloantibodies and their relevance to the
clinical condition would be of interest.16
I M M U N O H E M A T O L O G Y, V O L U M E 2 3 , N U M B E R 1 , 2 0 0 7
9
C. KAPLAN
The genetic basis for maternal alloimmunization
has been investigated. Alloimmunization to the HPA-1a
antigen appears to be associated with HLA class II
alleles: DRB3*0101 and DQB1*0201 (odds ratio 24.9
and 39.7, respectively).17,18 An anti HPA-1b response
was not associated with either DRB3*0101 or any
known HLA class II molecules.19 This finding implies
the Leu33/Pro33 substitution on the platelet
glycoprotein IIIa plays a role in antigen presentation.
Data have shown that the binding of peptides from the
Leu33/Pro33 dimorphic region to HLA-DR3*0101 is
allele specific with stimulation of specific T cells20,21
providing help to B cells for generating alloantibodies;
however, the positive predictive value is only 35
percent, and utility for screening is therefore limited.
The immune response to HPA-5b antigen is
strongly associated with a particular DRB1 gene
sequence encoding residues Glu-Asp at positions 69
and 70 of the DRβ chain.22 For other antigens, because
of the low number of cases, statistical analyses are not
significant when compared with the general
population, as seen for HPA-6b and DRB1*1501,
DQA1*0102, DQB1*0602 haplotypes shared by
immunized mothers.23
1953–2007: the Fetal and Neonatal
Alloimmune Thrombocytopenia Story
Natural history
FNAIT has been known for decades as “neonatal
alloimmune thrombocytopenia” (NAIT). The usual
presentation is a full-term neonate exhibiting petechiae
or widespread purpura at birth, or a few hours after
birth, to a healthy primiparous mother. Otherwise, this
infant is well with no clinical signs of infection
(hepatosplenomegaly) or malformation (hemangioma,
absence of radii). Visceral hemorrhages such as
gastrointestinal bleeding or hematuria are less common
than purpura or hematoma. The thrombocytopenia is
isolated. Coexisting anemia is caused by hemorrhage.
Anti-HPA-1a and anti-HPA-3a immunization induce
severe neonatal thrombocytopenia with platelet
counts less than 50 × 109/L in most cases.9,11 NAIT
linked to HPA-5b incompatibility seems to be less
severe than HPA-1a NAIT.10 The most serious
complication is fetal or neonatal intracranial
hemorrhage (ICH; 25.5% of cases for HPA-1a, 24% for
HPA-3a, 15% for HPA-5b)24 leading to death in up to 10
percent or neurologic sequelae in up to 20 percent of
reported cases.
10
The risk of life-threatening hemorrhage
necessitates prompt diagnosis and effective therapy.
Phenotyped platelet transfusion is the best postnatal
management. Because of the logistic difficulties in
obtaining such platelets in emergency situations,
random platelet transfusions with or without IVIG have
been proposed.25 However, compatible platelets give
better results.26 On the other hand, thrombocytopenia
may be asymptomatic and pass unnoticed unless there
is a routine platelet count performed. Therefore,
unexpected or unexplained neonatal thrombocytopenia or severe early onset thrombocytopenia in
both preterm and term babies should raise the
possibility of NAIT and guide investigations
accordingly. The fetal thrombocytopenia tends to
worsen in subsequent incompatible pregnancies.
Fetal blood sampling
In 1983, a major advance in the diagnosis of NAIT
was the use of ultrasound-guided fetal blood sampling
(FBS), which led to a better understanding of the fetal
status.27 The mean platelet count has been evaluated to
be more than 150 × 109/L by the end of the first
trimester of pregnancy in healthy fetuses and similar to
the adult platelet count later on. Therefore, thrombocytopenia has been defined in the fetus and the
neonate as a platelet count less than 150 × 109/L,
irrespective of the gestational age.28
In 1984, the first fetal alloimmune thrombocytopenia case was documented with FBS at 32 weeks
of gestation, and in utero maternal platelet transfusion
before delivery was proposed as therapy to avoid
perinatal ICH.29 Severe fetal thrombocytopenia was
then documented early during pregnancy when FBS,
as part of the antenatal management protocol, was
carried out at 21 weeks of gestation for subsequent
pregnancies in women with a previously affected
infant.30 A retrospective survey of 5194 fetal blood
samplings showed that fetal thrombocytopenia
resulting from maternal alloimmunization was the
most severe thrombocytopenia observed among the
different disorders encountered, including chromosomal malformations, infections, or maternal
autoimmune thrombocytopenia.31
Development of antenatal management
The rationale for antenatal management is the
high rate of recurrence for subsequent incompatible
fetuses who usually experience more severe thrombocytopenia. The first attempts to prevent severe
I M M U N O H E M A T O L O G Y, V O L U M E 2 3 , N U M B E R 1 , 2 0 0 7
Neonatal alloimmune thrombocytopenia
thrombocytopenia in the preterm period and thus ICH
during delivery were in utero platelet transfusions
before delivery.30 However, this management could not
prevent in utero ICH, which has been reported
primarily before 30 weeks of gestation.24 Because of
the short survival of transfused platelets, weekly fetal
platelet transfusions have been proposed and shown to
be effective in preventing ICH in a number of cases.32
The risks of in utero platelet transfusion, bleeding, and
fetal loss have been estimated to be 1 to 2 percent per
procedure and 8 percent per pregnancy.33 Fetal cardiac
arrhythmia (prolonged bradycardia) has also been
reported. Less invasive therapy has also been
proposed, involving maternal administration of IVIG,
steroids, or both.
The mechanism of action of IVIG is complex,
including inhibition of the transplacental passage of
maternal alloantibodies and modification of the
immune response. Corticosteroids may modulate the
maternal immune response. Different IVIG protocols
have been developed and differences in results were
reported mainly because of the definition of a
successful response.34–36 Low-dose corticosteroids, as
sole therapy, have been given to a limited number of
patients, and response to therapy appears highly
variable.35
The optimal management remains to be
determined, and an international forum in 2003
demonstrated the absence of standardization.37
The decision regarding therapy depends on
different factors, among which the fetal status plays a
central role. There is no reliable and sensitive
parameter for predicting which fetuses will be severely
affected and which ones will respond to therapy. The
only way to assess the fetal status is to perform FBS, but
the risk of serious adverse events from this technique
is high, up to 10 percent.38 Most protocols favor
maternal therapy with less invasive strategies39,40 with
stratification according to the previously affected
sibling’s status.41
Serial in utero platelet transfusions are considered
only as salvage therapy after failure of maternal
treatment.
at reducing the risk of neonatal disorders going
undiagnosed and untreated. Prospective studies have
shown that although 2 to 3 percent of women are at
risk for developing anti-HPA immunization, only 1 in
800 to 1000 newborns will be affected.3,4,43 It has been
found that 26 percent of infants born to immunized
mothers are nonthrombocytopenic. Until procedures
are found that predict which women will have an
affected fetus, maternal screening has a low sensitivity.
Only identifying thrombocytopenic newborns at birth
increases the sensitivity but will miss fetuses severely
affected during pregnancy who should have been
treated. Cost-effectiveness analysis indicated screening
newborns was more cost-effective than screening
primiparous women.3 A consensus on routine investigation and optimal antenatal management has yet to be
reached.
Future Directions
Although real progress has been achieved in the
more accurate diagnosis and management of FNAIT,
further studies must focus on improvements in
antenatal management and on mechanisms of
maternal sensitization. A recent study has shown that
high maternal anti-HPA-1a alloantibody concentrations
may provide an indication of severely affected fetuses44
and this may contribute to a less invasive antenatal
therapeutic strategy. However, the follow-up of maternal anti-HPA-1a concentration during pregnancy should
not be considered as an indicator of therapeutic
effectiveness.
A murine model has also been recently developed,
which may contribute to a better understanding of this
condition in the future.45
References
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No systematic screening for FNAIT exists at the
moment, and this condition is still underdiagnosed.42
Reduction of neonatal death and disability is a public
health issue, and recent progress in the prevention of
current causes of neonatal disorders has been efficient
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1. Harrington WJ, Sprague CC, Minnich V, et al.
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3. Durand-Zaleski I, Schlegel N, Blum-Boisgard C, et
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4. Williamson LM, Hackett G, Rennie J, et al. The
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18. L’Abbe D, Tremblay L, Filion M, et al.
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21. Wu S, Maslanka K, Gorski J. An integrin
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of feto-maternal alloimmune thrombocytopenia.
Br J Haematol 2003;122:275–88.
37. Engelfriet CP, Reesink HW, Kroll H, et al. Prenatal
management of alloimmune thrombocytopenia of
the fetus.Vox Sang 2003;84:142–9.
38. Paidas MJ, Berkowitz RL, Lynch L, et al.Alloimmune
thrombocytopenia: fetal and neonatal losses
related to cordocentesis. Am J Obstet Gynecol
1995;172:475–9.
39. Radder CM, Brand A, Kanhai HH. A less invasive
treatment strategy to prevent intracranial
hemorrhage in fetal and neonatal alloimmune
thrombocytopenia. Am J Obstet Gynecol 2001;
185:683–8.
40. Kanhai HHH, van den Akker ESA,Walther FJ, Brand
A. Intravenous immunoglobulins without initial
and follow-up cordocentesis in alloimmune fetal
and neonatal thrombocytopenia at high risk for
intracranial hemorrhage. Fetal Diagn Ther
2006;21:55–60.
41. Berkowitz RL, Kolb EA, McFarland JG, et al. Parallel
randomized trials of risk-based therapy for fetal
alloimmune thrombocytopenia. Obstet Gynecol
2006;107:91–6.
42. Turner ML, Bessos H, Fagge T, et al. Prospective
epidemiologic study of the outcome and costeffectiveness of antenatal screening to detect
neonatal alloimmune thrombocytopenia due to
anti-HPA-1a.Transfusion 2005;45:1945–56.
43. Dreyfus M, Kaplan C, Verdy E, et al. Frequency of
immune thrombocytopenia in newborns: a
prospective study. Blood 1997;89:4402–6.
44. Bertrand G, Martageix C, Jallu V, Vitry F, Kaplan C.
Predictive value of sequential maternal anti-HPA1a antibody concentrations for the severity of fetal
alloimmune thrombocytopenia. J Thromb
Haemost 2006;4:628–37.
45. Ni H, Chen P, Spring CM, et al. A novel murine
model of fetal and neonatal alloimmune
thrombocytopenia: response to intravenous IgG
therapy. Blood 2006;107:2976–83.
Cecile Kaplan, MD, Platelet Immunology Laboratory,
Institut National de la Transfusion Sanguine (INTS),
6 Rue Alexandre Cabanel, 75015 Paris, France.
I M M U N O H E M A T O L O G Y, V O L U M E 2 3 , N U M B E R 1 , 2 0 0 7
13
Platelet additive solutions:
current status
H. GULLIKSSON
During the last two decades, there has been great
interest in developing and using platelet additive
solutions (PASs) for the storage of platelets.1–5 At
present, such additive solutions are in use for
transfusion in several countries. PAS is generally used as
a substitute for plasma to (1) reduce the amount of
plasma transfused with platelets and to recover plasma
for other purposes, primarily fractionation into plasma
products; (2) avoid transfusion of large volumes of
plasma with possible adverse reactions and circulatory
overload; (3) make possible photochemical treatment
for the inactivation of bacteria and other pathogens in
platelets; and (4) improve storage conditions as was
discussed in a previous review.6
A basic principle is that aging of platelets after in
vitro storage at 22°C is significantly slower than aging
of platelets in vivo at 37°C, a situation that may make
extended storage of platelets possible.7 Three
approaches were suggested to be of specific importance to improve storage conditions of platelets6: (1)
reducing the activation of platelets during collection of
blood and the preparation and storage of platelets; (2)
reducing the metabolic rate in terms of glucose
consumption and lactate production; and (3) ensuring
that glucose will be available in the storage medium
during the entire storage period. The activation of
platelets can be counteracted either by the addition of
platelet activation inhibitors or of certain components
such as magnesium to the PAS.
The use of PAS offers the possibility of including
components with specific effects on platelets in the
storage solution that are not present in plasma or in the
anticoagulant. A number of effects have been observed
that can be assigned to certain components.4,5
Reducing platelet activation and inclusion of key
components in the platelet storage environment, such
as acetate, citrate, glucose, potassium, and magnesium,
were suggested to be useful tools to optimize platelet
storage conditions. The compositions of some present
PASs are presented in Table 1. The purpose of this
14
review is to describe some events of the last several
years as a complement to the knowledge presented in
previous reviews.1–6
In Vivo Characteristics
Results from a number of in vivo studies using PASs
have been published during the last decade. In the first
patient transfusion studies in the 1990s using PASs
such as PAS-II (T-Sol, Baxter) or Plasma Lyte A (Table 1),
the results were not consistent.8–14 In some studies,
satisfactory CCIs were found in patient transfusion
studies,9,11 in some studies significantly lower CCIs
were observed than for platelets stored in plasma.12,14
In a recent study, encouraging platelet recovery and
survival data were found, comparing platelets prepared
by apheresis and stored in PAS-II for 1 versus 7 days.15
Mean recovery was 69 percent and 53 percent, and
survival was 8.2 and 5.1 days at Days 1 and 7,
respectively. The ratios of Day 7 to Day 1 were 0.80
and 0.65 for recovery and survival, respectively. A
proposal by Murphy16 of a new standard of efficacy for
the evaluation of platelets for transfusion has created
considerable interest. This concept implies that
acceptably stored platelets on the last day of storage
would demonstrate at least two-thirds the recovery and
one-half the survival of platelets collected from the
same subject and then retransfused as reference “fresh”
platelets. The ratios in this study15 met the proposed
criteria for 7-day storage, although the use of a Day 1
control may not totally have fulfilled the requirements
of the reference indicated.16
Comprehensive patient transfusion studies using
pathogen-reduced platelets have added significant
knowledge about platelets stored in more recent PASs,
particularly PAS-III (InterSol, Baxter). A photochemical
treatment method using a novel psoralen, amotosalen
HCl, in combination with ultraviolet light has been
developed to inactivate viruses, bacteria, protozoa, and
Two major studies were
WBCs in platelets.17
conducted in Europe and in the United States, namely
I M M U N O H E M A T O L O G Y, V O L U M E 2 3 , N U M B E R 1 , 2 0 0 7
Platelet additive solutions
Removal of platelets from the circulation is
believed to be age-dependent and is
Plasma
PAS-II
PAS-III
Composol
PAS-III M
probably associated with failure to maintain
Lyte A
(T-Sol,
(InterSol,
(Fresenius)
(SSP+,
Baxter)
Baxter)
MacoPharma)
normal hemostasis. Transfusion of aging
platelets may be of little help to stop
NaCl
90
116
77
90
69
bleeding because they would be removed
KCl
5
–
–
5
5
from the circulation.
MgCl2
3
–
–
1.5
1.5
Studies comparing aging of platelets in
Na3-citrate
–
10
10
11
10
vivo with in vitro storage at 22°C, using
–
–
26
–
26
NaH2PO4/Na2HPO4
isotope labeling, suggested that aging of
Na-acetate
27
30
30
27
30
platelets after in vitro storage for 5 days at
Na-gluconate
23
–
–
23
–
22°C was similar to aging of platelets during
*The compositions of commercial solutions may be slightly different from basic compositions.
2.1 days in vivo at 37°C.7 The relative aging
the euroSPRITE trial in Europe and the SPRINT trial in
factor was found to be 0.42 (2.1 days divided by 5.0
the United States.18–21 In the euroSPRITE trial,
days). In a subsequent study, a similar relative aging
factor was found (0.44).7 These differences were
comparable platelet CIs and CCIs and a comparable
associated with a much higher turnover rate of ATP, the
safety profile for photochemically treated and
major energy carrier in platelets, at 37°C than at 22°C.
reference untreated pooled buffy-coat (BC) platelet
The calculated ATP turnover ratio of 0.48 at those two
units in 103 oncology patients with thrombocytopenia
temperatures was of the same magnitude as the
were demonstrated. In the SPRINT study, which
calculated relative aging factor, indicating that the
included 645 transfused patients, the hemostatic
relative decrease in aging of platelets at 22°C compared
efficacy of photochemically treated and reference
with that at 37°C is similar to the relative decrease in
untreated platelets was found to be equivalent.19
metabolic rate at this temperature. The rate of aging of
Transfusion reactions were significantly fewer, and
platelets during storage at 22°C may be less than half of
adverse events and overall safety profiles were
that found in vivo at 37°C. If this ratio is applied to the
comparable for treated and reference platelets.20 In a
normal lifespan of platelets, 9 to 10 days in the
second evaluation of data from the euroSPRITE patient
circulation would correspond to at least 18 to 20 days
transfusion study using BC-derived platelets, reference
22
of in vitro storage at 22°C. These data may provide
groups with untreated platelets were compared.
meaningful objectives to develop methods and storage
Reference platelets were stored in either T-Sol (n=35)
environments for extended storage of platelets.
or plasma (n=16). The results indicated that 1-hour and
24-hour mean platelet CIs and posttransfusion
hemostatic scores were not significantly different for
The Use of PAS in Combination With
patients receiving platelet components suspended in
Different Methods for the Preparation of
100 percent plasma and patients receiving platelets in
Platelets for Transfusion
a T-Sol environment. In a review in 2003, the
In general, the various PASs can be used for
preliminary conclusion had been that additional in vivo
apheresis as well as for BC platelets. The storage
data, from studies of clinical outcome after transfusion
medium normally is composed of a mixture of plasma
of platelets stored in either PAS or plasma, were
(generally 20–40%) and PAS (60–80%). The main
needed/required.6 Today, comprehensive clinical data
difference is that in apheresis platelets, ACD is often
support the routine use of PAS as an equivalent
preferred to CPD anticoagulant, because resuspension
alternative to plasma to suspend platelets.
of platelets after preparation is facilitated. In some
Table 1. Composition of some current PASs (in mmol/L) including commercial
designations*
In Vitro Platelet Aging at 22°C Versus In Vivo
Aging at 37°C
Because the average survival of human platelets in
the circulation, after release from megakaryocytes, is
known to be 9 to 10 days, it may be questioned
whether it is at all meaningful to extend the shelf life
of platelet concentrates up to or even beyond 7 days.23
apheresis equipment, platelets are kept in the
centrifugation chamber during the entire apheresis
cycle; in other equipment, platelets are continuously
transferred to a platelet storage container. These
differences may result in significant variation in platelet
activation. Although apheresis equipment originally
may have been designed for the preparation of
platelets suspended in plasma, most equipment can be
I M M U N O H E M A T O L O G Y, V O L U M E 2 3 , N U M B E R 1 , 2 0 0 7
15
H. GULLIKSSON
used for platelet collection in a small final resuspension
volume of plasma, which is necessary to allow the
addition of PAS. Satisfactory results were obtained by
Ringwald et al.24,25 when evaluating the suspension of
high concentrations of apheresis platelets (4000–5000
× 109 platelets/L) in PAS.
BC-derived platelets suspended in PAS are generally
prepared in pools from several donors. There are
primarily variations in the number of BCs included in
the pools (generally 3–7 BCs) and in the time of
holding of whole blood preceding the preparation of
BCs. Either BCs can be prepared on the day of
collection, generally within 8 hours, and stored
overnight for the preparation of platelets on the
following day, or whole blood can be stored overnight
for the preparation of BCs and platelets on the
following day.
The platelet rich plasma (PRP) method is generally
not used for the routine preparation of platelets in PAS.
However, in a recent study by Sweeney et al.,26 platelets
were prepared from individual donors by the PRP
method in a first step and then pooled as platelets from
several donors and suspended in either plasma or PAS.
Platelets were stored for 7 days for in vitro evaluation.
Good preservation of platelet quality in both
environments was reported.
Effects on Metabolism and Platelet Function
Associated With Components in PAS
The results of early studies by Holme et al.27 and
Gulliksson et al.28 on the effects of PAS indicated that
the presence of glucose during the entire platelet
storage period is crucial for platelet metabolism.
Effects observed after depletion of glucose involved
rapid decrease in adenine nucleotide levels, cessation
of lactate production, and finally disintegration of
platelets. Depletion of glucose is generally associated
with an increased rate of platelet metabolism and fall in
pH levels. The results suggested that depletion of
glucose, not the pH level alone, is detrimental to
platelets during storage. In contrast to plasma, the fall
in pH during storage of platelets in PAS will stop at a
significantly higher level than pH 6.0, often at about pH
6.5 as a result of the limited amount of glucose
generally available in PAS.28 Because the buffering
capacity of PAS is approximately half that of plasma,
PAS is more susceptible to increased production of
lactic acid by platelets.29,30 On the other hand,
metabolism of acetate present in PAS significantly
stabilizes the pH level. In parallel with glucose, acetate
16
is used as a substrate for the oxygen-dependent platelet
metabolism, and enters into the tricarboxylic acid
cycle, and is further oxidized in the respiratory
chain.29–32 The end products are carbon dioxide from
the first step and water from the second step. By
formation of bicarbonate from the carbon dioxide
produced by acetate, very stable pH levels are
maintained during platelet storage.29,30,32 A possible
third substrate for platelet metabolism may be fatty
acids.33
Generally, phosphate has two possible roles during
storage of platelets, as a stimulant of platelet glycolysis
to increase production of lactic acid and as a buffer to
prevent a fall in pH. These two effects theoretically
compete and may neutralize each other. There are no
indications of net utilization or production of
phosphate during storage of platelets.29
The new PASs designated Composol and PAS-IIIM
as well as the early Plasma Lyte A all contain
magnesium and potassium. Composol and PAS-IIIM
also contain citrate, in contrast to Plasma Lyte A. The
three components: magnesium, potassium, and citrate
are associated with complexity of effects and
interdependence. Effects on platelet membrane
function and platelet activation as well as rate of
glycolysis have been described and the various effects
may even be combined.
Increased
concentration
of
extracellular
magnesium ions significantly inhibits exposure of Pselectin, decreases binding of fibrinogen to ADPactivated platelets, and significantly decreases ADPinduced platelet aggregation.34 Magnesium may also
modify calcium influx into the platelets. In addition,
magnesium, calcium, and the concentration of citrate
have an effect on potassium permeability of the
platelet membrane and the intracellular concentration
Citrate also heightens platelet
of potassium.35
responsiveness to some activating agents such as ADP.36
In addition, effects on the rate of glucose consumption
and lactate production related to the concentration of
citrate and the presence of potassium and magnesium
in PAS have been observed. Platelets stored in medium
with a citrate concentration of 8 mmol/L produced
only half the quantity of lactate produced by platelets
in a similar medium with a citrate concentration of 14
to 26 mmol/L.37 Inasmuch as no negative effects on
adenine nucleotide levels were observed, the results
suggested that synthetic media preferably should
include citrate at low concentrations to avoid excessive
lactate production and an acid pH. On the other hand,
I M M U N O H E M A T O L O G Y, V O L U M E 2 3 , N U M B E R 1 , 2 0 0 7
Platelet additive solutions
increased production of lactate associated with higher
concentrations of citrate can be neutralized by the
addition of acetate.37 Again, this situation illustrates the
complexity of effects and interdependence associated
with those components. In parallel, the combination of
magnesium and potassium ions present in PAS has a
similar effect on platelets, i.e., significantly reduced
metabolic rate in terms of glucose consumption and
lactate production and also reduced platelet
activation.38–40
When platelets are shipped between different
centers, agitation may be interrupted for a considerable
period of time. A previous study using a plasma
environment suggests that possible storage time
without agitation is strongly affected by platelet
concentration.41 The level of pH should be kept above
6.5 to avoid negative effects on functional in vitro
factors such as hypotonic shock response (HSR).41 In a
recent study, the effects of interruption of agitation on
platelets stored in two PAS alternatives, namely PASIIIM and Composol, were evaluated.42 At a platelet
concentration of approximately 1000 × 109/L, platelets
could be stored for 4 days in PAS-IIIM with
maintenance of HSR and pH. This was not possible
with Composol, suggesting that the presence of
phosphate is of importance to maintaining pH and
other in vitro characteristics during interruption of
agitation. Similar effects were not observed during
continuous agitation.
References
Future Perspectives
To conclude, present knowledge and experience
support platelet storage only at 22°C. The use of PAS
instead of plasma as the platelet storage environment
would provide benefits to patients and facilitate the
inclusion of certain components with proven favorable
effects on platelets that are present in neither plasma
nor anticoagulant. The present knowledge of effects
associated with different approaches discussed above
suggests that reducing platelet activation in
combination with the inclusion of key components in
the platelet storage medium, such as acetate, citrate,
glucose, potassium, magnesium, and additional possible
future ingredients, should be the tools to optimize the
storage conditions and maintain the function of
platelets. Additional in vivo studies of recovery and
survival as well as count increments in patients with
thrombocytopenia will be needed.
I M M U N O H E M A T O L O G Y, V O L U M E 2 3 , N U M B E R 1 , 2 0 0 7
1. Holme S. Effect of additive solutions on platelet
biochemistry. Blood Cells 1992;18:421–30.
2. Holme S. Platelet storage in liquid environment.
Transfus Sci 1994;15:117–30.
3. Högman CF. Aspects of platelet storage. Transfus
Sci 1994;15:351–5.
4. Murphy S. The efficacy of synthetic media in the
storage of human platelets for transfusion.
Transfus Med Rev 1999;13:153–63.
5. Gulliksson H. Additive solutions for the storage of
platelets for transfusion. Transfus Med 2000;
10:257–64.
6. Gulliksson H. Defining the optimal storage
conditions for the long-term storage of platelets.
Transfus Med Rev 2003;17:209–15.
7. Holme S, Heaton WA. In vitro platelet ageing at
22°C is reduced compared to in vivo ageing at
37°C. Br J Haematol 1995;91:212–8.
8. Oksanen K, Ebeling F, Kekomäki R, et al. Adverse
reactions to platelet transfusions are reduced by
use of platelet concentrates derived from buffy
coat.Vox Sang 1994;67:356–61.
9. van Rhenen DJ,Vermeij K, Kappers-Klunne R, et al.
Evaluation of a new citrate-acetate-NaCl platelet
additive solution for the storage of white cellreduced platelet concentrates obtained from halfstrength CPD pooled buffy coats. Transfusion
1995;35:50–3.
10. Eriksson L, Kristensen J, Olsson K, et al. Evaluation
of platelet function using the in vitro bleeding
time and corrected count increment of transfused
platelets:
comparison
between
platelet
concentrates derived from pooled buffy coats and
apheresis.Vox Sang 1996;70:69–75.
11. Strindberg J, Berlin G. Transfusion of platelet
concentrates: clinical evaluation of two
preparations. Eur J Haemotol 1996;57:307–11.
12. Turner VS, Mitchel SG, Hawker RJ. More on the
comparison of Plasma-Lyte A and PAS-2 as platelet
additive solutions.Transfusion 1996;36:1033–4.
13. Högman CF, Eriksson L,Wallvik J, et al. Clinical and
laboratory experience with erythrocyte and
platelet preparations from a 0.5 CPD Erythro-sol
Opti System.Vox Sang 1997;73:212–9.
14. de Wildt-Eggen J, Nauta S, Schrijver JG, et al.
Reactions and platelet increments after
transfusion of platelet concentrates in plasma or
an additive solution: a prospective, randomised
study.Transfusion 2000;40:398–403.
17
H. GULLIKSSON
15. Shanwell A, Diedrich B, Falker C, et al. Paired in
vitro and in vivo comparison of apheresis platelet
concentrates stored in platelet additive solution
for 1 versus 7 days.Transfusion 2006;46:973–9.
16. Murphy S. Radiolabeling of PLTs to assess viability:
a proposal for a standard. Transfusion 2004;44:
131–3.
17. Lin L, Cook DN, Wiesehahn GP, et al.
Photochemical inactivation of viruses and
bacteria in platelet concentrates by use of a novel
psoralen and long-wavelength ultraviolet light.
Transfusion 1997;37:423–35.
18. van Rhenen, D, Gulliksson H, Cazenave JP, et al.
Transfusion of pooled buffy coat platelet
components prepared with photochemical
pathogen inactivation treatment: the euroSPRITE
trial. Blood 2003;101:2426–33.
19. McCullough J, Vesole DH, Benjamin RJ, et al.
Therapeutic efficacy and safety of platelets treated
with a photochemical process for pathogen
inactivation. The SPRINT Trial. Blood 2004;104:
1534–41.
20. Snyder E, McCullough J, Slichter JS, et al. Clinical
safety of platelets photochemically treated with
amotosalen HCl and ultraviolet A light for
pathogen inactivation: the SPRINT trial.
Transfusion 2005;45:1864–75.
21. Janetzko K, Cazenave JP, Klüter H, et al.
Therapeutic efficacy and safety of photochemically treated apheresis platelets processed
with an optimized integration set. Transfusion
2005;45:1443–52.
22. van Rhenen, DJ, Gulliksson H, Cazenave JP, et al.
Therapeutic efficacy of pooled buffy-coat platelet
components prepared and stored with a platelet
additive solution.Transfus Med 2004;14:289–95.
23. Gewirtz AM, Schick B. Platelet production and
function: megakaryocytopoiesis. In: Colman RW,
Hirsh J, Marder VJ, et al., eds. Hemostasis and
thrombosis. 3rd ed. Philadelphia: JB Lippincott,
1994:353–96.
24. Ringwald J, Walz S, Zimmermann R, et al.
Hyperconcentrated platelets stored in additive
solution: aspects on productivity and in vitro
quality.Vox Sang 2005;89:11–8.
25. Ringwald J, Duerler T, Frankow O, et al. Collection
of hyperconcentrated platelets with Trima Accel.
Vox Sang 2006;90:92–6.
18
26. Sweeney J, Kouttab N, Holme S, et al. Storage of
platelet-rich plasma-derived platelet concentrate
pools in plasma and additive solution.Transfusion
2006;46:835–40.
27. Holme S, Heaton WA, Courtright M. Platelet
storage lesion in second-generation containers:
correlation with platelet ATP levels. Vox Sang
1987;53:214–20.
28. Gulliksson H, Sallander S, Pedajas I, et al. Storage of
platelets in additive solutions: a new method for
storage using sodium chloride solution.
Transfusion 1992;32:435–40.
29. Shimizu T, Murphy S. Roles of acetate and
phosphate in the successful storage of platelet
concentrates prepared with an acetate-containing
additive solution.Transfusion 1993;33:304–10.
30. Bertolini F, Murphy S, Rebulla P, et al. Role of
acetate during platelet storage in a synthetic
medium.Transfusion 1992;32:152–6.
31. Guppy M, Whisson ME, Sabaratnam R, et al.
Alternative fuels for platelet storage: a metabolic
study.Vox Sang 1990;59:146–52.
32. Murphy S. The oxidation of exogenously added
organic anions by platelets facilitates maintenance
of pH during their storage for transfusion at 22°C.
Blood 1995;85:1929–35.
33. Cesar J, DiMinno G, Alam I, et al. Plasma free fatty
acid metabolism during storage of platelet concentrates for transfusion. Transfusion 1987;27:
434–7.
34. Gawaz M, Ott I, Reininger AJ, et al. Effects of
magnesium on platelet aggregation and adhesion.
Thromb Haemost 1994;72:912–8.
35. Weis-Fogh US. The effect of citrate, calcium, and
magnesium ions on the potassium movement
across the human platelet membrane. Transfusion
1985;25:339–42.
36. Kinlough-Rathbone RL, Packham MA, Mustard JF.
Platelet aggregation. In: Harker LA, Zimmerman
TS, eds. Methods of hematology: measurements of
platelet function. New York: Churchill Livingstone,
1984:64–91.
37. Gulliksson H. Storage of platelets in additive
solutions: the effect of citrate and acetate in in
vitro studies.Transfusion 1993;33:301–3.
38. de Wildt-Eggen J, Schrijver JG, Bins M, et al. Storage
of platelets in additive solutions: effects of
magnesium and potassium. Transfusion 2000;42:
76–80.
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Platelet additive solutions
39. Gulliksson H, AuBuchon JP, Vesterinen M, et al.
Storage of platelets in additive solutions: a pilot
study of the effects of potassium and magnesium.
In vitro studies.Vox Sang 2002;82:131–6.
40. Gulliksson H, AuBuchon JP, Cardigan R, et al.
Storage of platelets in additive solutions: a
multicentre study of the in vitro effects of
potassium and magnesium. Vox Sang 2003;85:
199–205.
41. Hunter S, Nixon J, Murphy S. The effect of the
interruption of agitation on platelet quality during
storage for transfusion. Transfusion 2001;41:
809–14.
42. van der Meer PF, Gulliksson H, AuBuchon JP, et al.
Interruption of agitation of platelet concentrates:
effects on in vitro parameters. Vox Sang 2005;
88:227–34.
Hans Gulliksson, PhD, Department of Clinical
Immunology and Transfusion Medicine C2 66,
Karolinska University Hospital, Huddinge, SE-141 86
Stockholm, Sweden.
Phone, Fax, and Internet Information: If you have any questions concerning Immunohematology,
Journal of Blood Group Serology and Education, or the Immunohematology Methods and Procedures
manual, contact us by e-mail at immuno@usa.redcross.org. For information concerning the National Reference
Laboratory for Blood Group Serology, including the American Rare Donor Program, please contact Sandra
Nance, by phone at (215) 451-4362, by fax at (215) 451-2538, or by e-mail at snance@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 3 , N U M B E R 1 , 2 0 0 7
19
ABO and platelet transfusion
therapy
L. COOLING
It is appropriate to take steps to optimize the efficacy
and safety of transfusion therapy. Perhaps we should
study the role of ABO compatibility in platelet
transfusion therapy more carefully so that further
improvements may be made.
Scott Murphy, Transfusion Editorial 19881
The relative importance of ABO compatibility and
platelet transfusion has been a matter of debate for
more than 50 years. Since the early studies of Richard
Aster,2 it has been recognized that transfusion of ABOincompatible platelets can be associated with decreased
platelet increments after transfusion. In the last 10 to 15
years, there has been increasing discussion and concern
regarding minor-incompatible platelet transfusions that
have been linked to acute hemolytic transfusion
reactions, venoocclusive disease, and increased
morbidity in allogeneic transplants.3–5 This review will
briefly summarize the biochemistry, regulation, clinical
practice, and considerations surrounding ABO
compatibility and platelet transfusions.
ABO Glycoconjugates on Platelets
ABO antigens are expressed on several endogenous
platelet glycoproteins and glycolipids. Platelet-specific
glycoproteins (GP) known to express ABH antigens
include GPIIb/IIIa, GPIb/IX, GPIa/IIa, GPIc, GPIV, GPV,
CD31 (PECAM), and CD109.6–10
GPIIb/IIIa, in
particular, appears to be a significant contributor and
determinant of ABH expression on platelets. The
GPIIb/IIIa complex numbers nearly 250,000 molecules
per platelet and possesses between five and eight biand triantennary N-glycans capable of displaying ABH
epitopes.10,11 GPIIb/IIIa is the major target of human
anti-A and anti-B in vitro.7 Likewise, a linear correlation
between A antigen on GPIIb and intact platelet
membranes (R = 0.91) was reported by Cooling et al.10
Translocation and ongoing synthesis of GPIIb/IIIa may
also contribute to the increased antigenicity of
20
platelets during in vitro storage. Julmy et al.12 reported
a nearly 50 percent increase in ABH expression on
platelets during routine storage, accompanied by
evidence of platelet activation and translocation of αgranule proteins (including GPIIb/IIIa) to the platelet
membrane. More recent studies in platelet proteomics
and the platelet transcriptome suggest ongoing
translation and synthesis of GPIIb/IIIa during storage as
well.13
Platelets also express a small population of
glycosphingolipids with ABH activity. Early studies
showed the ability of platelets to adsorb and elute
soluble type 1 chain ABH and Lewis antigens from
plasma, leading to the speculation that all ABH-active
glycolipids were entirely of exogenous origin.14
Subsequent studies using chain-specific monoclonal
antibodies showed that platelets expressed type 2
chain glycolipids and glycoproteins, indicating that
most ABH-active glycolipids were of megakaryocyte or
endogenous origin.6,10,15,16 This was confirmed by
Cooling et al.10 who identified ABH-active glycolipids in
platelets from Le(a+b–) individuals who should lack
soluble type 1 chain antigen. The ABH-active
glycosphingolipids of platelet are predominantly
simple, linear structures (6–8 oligosaccharides)
although a few complex sialylated structures are
present.10,17,18 In group A1 donors, type 4 chain or
globo-ABH structures have also been identified.16,19
This is in sharp contrast to RBCs, which express a rich
variety of ABH-active glycolipids, including complex,
The
highly branched polyglycosylceramides.19,20
contribution of ABH-active glycolipids to overall ABH
expression on platelets appears to be minor.10
ABO Expression on Megakaryocytes
Although recent studies indicate some residual
protein synthesis in young platelets,13 the majority of
ABH synthesis occurs at the level of the megakaryocyte.
ABH antigens have been identified on immortalized
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ABO and platelets
megakaryoblastic cell lines and cultured bone marrow
In the latter, ABH exhibits
megakaryocytes.21,22
developmental and distinct clonal variation, with
individual megakaryocyte colonies differing widely in
the amounts of A, B, and H antigens expressed.21 Clonal
variation may underlie the heterogeneity observed on
circulating platelets.10,21 In addition, there is a direct
correlation between A and H on megakaryocytes, with
colonies either positive or negative for both H and A
antigens. Cooling et al.10 subsequently showed a direct,
linear relationship between H and A antigens on
circulating platelets by two-color flow cytometry (R =
0.95). On the basis of their findings, Dunstan et al.21 and
Cooling et al.10 speculated that H antigen synthesis is a
rate-limiting step regulating ABH expression in
megakaryocytes and platelets.
H antigen appears to be developmentally regulated
in early hematopoiesis. H, LeY, and FUT1 mRNA are
expressed by CD34+ cells and likely early megakaryocyte progenitors.22–24 During megakaryocytic
differentiation, both H and LeY antigens are
progressively lost with increasing ploidy and
proplatelet formation.22 The presence of H antigen on
early megakaryocyte progenitors may facilitate
adhesion to stromal fibroblasts,25 a necessary step for
megakaryocytic preservation and expansion.26,27
Schmitz and colleagues25 were able to inhibit
megakaryocyte adhesion to stromal fibroblasts with
fucosylated bovine albumin, soluble H antigen, and
fucose-specific lectins. Although the precise lectinligand pair involved in fucose-mediated adhesion is not
yet identified, several megakaryocyte proteins critical
to megakaryocyte development and adhesion can
express ABH antigens, including α4β1, α5β1 GPIIb/IIIa,
GPIb, and PECAM.28,29 H antigen on integrins may be
particularly important: H and LeY expression are
associated with increased fibronectin binding, cell
adhesion, and resistance to apoptosis in epithelial
tissues.30–32 An increase in fucose-mediated adhesion,
with delayed megakaryocyte maturation, may account
for delayed platelet engraftment in some group O
autologous transplant patients.33
Platelet ABO and Genetic Variation
There are dramatic differences in the amount of
ABO expressed on platelet membranes between
individual donors. Genetic differences in ABO
glycosyltransferase alleles underlie some of these
differences. In group A donors, expression of A antigen
on platelet membranes, glycoproteins, and glycolipids
is linked to an A1 RBC phenotype.7,10,16,34,35 In contrast,
A2 donors express little or no A antigen and can be
considered group O compatible.10,35 Lewis and
secretor phenotype appear to play little role in either
the presence or strength of ABO antigens on platelets:
A2, Le(a–b+) donors are also negative for A antigen by
flow cytometry.10 Because they lack A antigen on
platelets,A2 individuals can develop an anti-A1 and ABHspecific refractoriness.34
Among group A1 donors, there is a wide variation in
platelet A expression (Fig. 1E).7,10,35 In individual
donors, the percentage of circulating platelets positive
for A antigen by two-color flow cytometry (CD41+,
HPA+) can range from less than 5 percent to 87
percent (population average = 40%).10 Even within a
single donor, there is distribution of positive and
negative platelets (Fig. 1B). Despite the latter, the
average percentage of platelets positive for A antigen is
relatively stable over time and may represent a unique,
donor-specific characteristic.10 As shown in Figure 1F,
the percentage of A antigen–positive platelets, in paired
samples (S1, S2) collected from the same donor at two
separate times, is nearly identical.
Unlike A1 donors, most group B donors weakly
express ABO antigens on platelets.7,10,36 In our own
studies, only 20 percent of platelet donors were
positive for B antigen by flow cytometry.10 Similar
findings are observed with platelet crossmatching, in
which 83 to 100 percent of group B platelet donors
were crossmatch-compatible with group O and group
A serum, respectively.36 Despite these results, group B
antigen can be identified on platelet glycoproteins and
glycosphingolipids in most group B donors (80–90%).10
The density or antigenicity on individual platelet
glycoproteins, however, may be decreased relative to
most A1 donors (< 50%), on the basis of solid phase and
radioimmunoassay studies with human anti-B.7
High-Expressor Phenotype
Approximately 4 to 8 percent of A1 and B donors
are ABO high expressors (HXP), defined as a 2- to 3-fold
increase in platelet ABO expression (> 2 SD).10,34,37 ABO
HXPs can be further subclassified as type 1 and type 2
on the basis of their flow cytometry profiles.10,35 Type
1 donors demonstrate a bimodal population of strongly
positive and moderately to weakly positive platelets,
akin to the bimodal population of positive and negative
platelets observed in most A1 donors (Fig. 1B). In
contrast, type 2 donors have an essentially uniform
population of strongly positive platelets (Fig. 1C).
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21
L. COOLING
Fig. 1.
Donor-specific differences in ABO expression. A–C: Sample histograms of platelets from A2 (A),A1 (B), and an A1 high-expressor donor (A1-HXP, C).
Platelets were dual labeled with a platelet-specific monoclonal antibody (CD41) and the anti-A lectin, Helix pomatia (HPA). For analysis, platelets
were gated on CD41 and the percent CD41+, HPA+ platelets determined by two-color flow cytometry. D and E: Expression of A antigen on
platelets in A2 (D) and A1 donors (E). Note the wide distribution of A antigen-positive platelets in A1 donors, including 5 percent with high
expression (A1-HXP). Little or no A antigen was detected in A2 and 6 percent A1 donors. F: Stable expression of A antigen on platelets.The percent
HPA+ platelets in individual donors was compared in two different samples (S1, S2) at 1- to 4-month intervals.
Transfusion of ABO-HXP platelets to a group O
recipient has resulted in profound transfusion failures,
even with HLA-matched platelets.37 Platelets from ABHHXP donors should be reserved for ABO-identical
recipients only.
The molecular origin of the ABO-HXP phenotype is
still unknown. Family studies indicate that the trait is
autosomal dominant.37 There is no relation between
the ABO-HXP and a secretor phenotype.10,37 Elevated
ABO glycosyltransferase activity has been noted in the
sera of some donors35,37; however, sequencing studies of
the ABO gene have not revealed any mutations.35
Cooling et al.10 suggested that the ABO-HXP might
represent clonal upregulation of the H or FUT1 gene.
Future studies focusing on transcriptional regulation of
H and ABO genes in megakaryocytes, including
22
epigenetic phenomena, may eventually unravel the
genetic mystery behind the ABO-HXP phenotype.
ABO Compatibility and Platelet Transfusion
Response
The influence of platelet ABO and the posttransfusion response is highly variable, reflecting both
donor and recipient factors. Recipient factors that
may influence the transfusion response include ABO
type, sex, and parity; isohemagglutinin titers; and
immunosuppressive drugs. Platelet and donor factors
can include ABO type (A1, A2, B), donor-specific
differences in ABO strength, platelet activation, and
storage time. As noted in the previous section, ABO
expression varies widely between individual donors
and ABO types.10,35
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The earliest studies examining the effect of ABO
and platelet transfusion were performed by Richard
Aster in 1965.2 Using radiolabeled platelets, Aster et al.
reported a 60 to 90 percent decrease in the 1-hour
posttransfusion platelet increments after transfusion of
group A and AB platelets to group O recipients. This
was substantiated in an elegant study by Jimenez et
al.,38 who compared the posttransfusion recovery in
paired recipients of split apheresis products from the
same donor. The authors found similar platelet
recoveries when both recipients were ABO compatible
with the donor (R = 0.8); however, transfusion to an
ABO-incompatible recipient was accompanied by
marked, significant decreases in posttransfusion
recovery at 1 (p < 0.001), 4 (p < 0.004), and 24 hours
(p < 0.04). Improved platelet recovery with ABOidentical and ABO-compatible platelets has also been
observed in large clinical trials, including the Trial to
Reduce Alloimmunization to Platelets.39,40
There are several examples of platelet
refractoriness caused by ABO in the literature.34,37,41,42
In a small randomized trial of ABO-identical and ABOunmatched platelets, the vast majority of patients
receiving ABO-unmatched platelets had a decrease in
posttransfusion recovery, with 37 percent having clear
evidence of ABO-specific refractoriness.43 In a second
study, clinical refractoriness was significantly higher in
patients receiving ABO-mismatched platelet transfusions (69% vs. 8%) and was typically heralded by a
sudden acute rise in isoagglutinin titers.44 In a more
recent study, ABO-incompatible platelet transfusions
stimulated isohemagglutinin titers in 40 to 50 percent
of patients after only one to two transfusions.45 Not
surprisingly, patients with documented ABO refractoriness often have high titer isohemagglutinins
(> 1:500–1:1000). Platelet-associated antibody in these
patients can approach 30,000 molecules of IgG per
platelet after transfusion of an ABO-incompatible
platelet.7 In contrast, IgM binding to platelets is
minimal (< 350 molecules/platelet).7
Although most case reports and studies have
focused on ABO major-incompatible transfusions, poor
platelet recoveries and clinical refractoriness can also
be observed with ABO minor-incompatible or out-ofgroup transfusions.39,46,47 According to one study,
plasma-incompatible transfusions were associated with
an 18 percent decrease in posttransfusion recovery
when compared with ABO-identical transfusions.39 The
adverse effect of incompatible-plasma infusion may be
caused by the formation of immune complexes
composed of donor ABO antibodies and soluble ABO
substances in blood.46,47 These immune complexes may
then bind to either complement or Fc receptors on
platelets, leading to accelerated immune clearance.46
Among refractory patients, 40 percent have elevated
levels of circulating immune complexes. Among group
A patients transfused with ABO-mismatched platelets,
80 percent had evidence of immune complexes
containing IgG anti-A of donor origin.47
Alloimmunization
Routine transfusion of ABO-incompatible platelets
can promote HLA alloimmunization. In randomized
trials, patients routinely transfused with ABOmismatched platelets were more likely to develop both
HLA and platelet-specific antibodies, accompanied by
an earlier onset and higher incidence of clinical
platelet refractoriness.44,48 ABO incompatibility can
also act synergistically with HLA and crossmatch
compatibility to further decrease the posttransfusion
response.36,39,49
In a retrospective study of 51 refractory patients,
Blumberg and colleagues39 demonstrated decreased
platelet recoveries in 60 percent of all ABO-incompatible
transfusions, regardless of platelet crossmatch results.
Among patients receiving crossmatch-compatible
platelets, an ABO mismatch was associated with a 40
percent decrease in posttransfusion recovery. The
impact was more dramatic with ABO-mismatched,
crossmatch-incompatible platelets (85% decrease).
Overall, ABO-identical platelets were clinically equivalent or better than ABO-incompatible, HLA-matched,
or crossmatch-compatible platelets.
The synergism between ABO, HLA, and clinical
refractoriness can have direct economic consequences. The University of Rochester reported a 20
percent decrease in platelet utilization and a 40
percent decrease in HLA-matched platelets after
instituting a policy requiring ABO-identical platelets
for their leukemic patients.50–52 This policy has led to
improved survival and decreased morbidity based on
the results of a small randomized trial (n = 40 patients).
Leukemia patients who received only ABO-identical
platelets had longer remissions and improved survival
relative to patients transfused with ABO-nonidentical
platelets.53 There was also a decrease in major bleeding
episodes (5%) with 70 percent of patients having no
evidence of clinical bleeding.54
The impact and cost effectiveness of ABO
compatibility in other patient populations is still
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23
L. COOLING
debated. In a retrospective study of cardiovascular
surgery patients, Blumberg et al.55,56 found that patients
transfused with ABO-identical platelets had decreased
morbidity and mortality, as measured by fever,
antibiotic usage, transfusion support, and hospital
admission days. These differences corresponded to an
average savings of $10,000 to $15,000 in direct costs
and patient charges. These findings have been
challenged by Lin et al.,57 who found no significant
differences in either morbidity or mortality in a similar
group of cardiovascular patients. In the latter study, the
authors concluded that the use of ABO-nonidentical
platelets is “an acceptable and safe practice” in surgical
patients.
plasma compatible with the donor in the immediate
perioperative period. However, transfusion of ABO
major-incompatible platelets could also present
problems, particularly postoperatively. As discussed in
a previous section, ABO-incompatible platelets can
stimulate isoagglutinin titers in some recipients.44,45 In
one small study,ABO-incompatible platelet transfusions
from the intended kidney donor were used to test the
immune responsiveness of the recipient.45 Of seven
patients, one developed HLA antibodies and three
demonstrated a rise in isohemagglutinin titers. Only
patients with little or no response to platelet
transfusion underwent transplantation with good
outcomes 4 to 7 years after transplant.
ABO, Platelets, and Transplantation
Acute Hemolytic Transfusion Reactions
There is increasing concern regarding platelets and
ABO compatibility in bone marrow transplant patients,
particularly in patients receiving ABO-incompatible
allogeneic transplants who have complex transfusion
needs.58–60 Because of the adverse effect of ABO
incompatibility on erythroid engraftment, it is a
practice to transfuse plasma and platelets that are
compatible with the stem cell or marrow donor.61 In
most instances, this may require transfusion of an ABO
major-incompatible or minor-incompatible platelet.
Several studies have linked increased platelet
transfusions and transfusion of plasma-incompatible
platelets with increased morbidity, venoocclusive
disease, multiorgan failure, and death.4,5,62 Organ
dysfunction usually follows a 1- to 2-week period of
rising platelet transfusion requirements, suggesting a
causal relationship.62
It is hypothesized that donor ABO antibodies may
result in immune complex formation in the host,
leading to systemic inflammation. Alternatively, passively infused antibodies may recognize and bind ABO
antigens on endothelial cells, leading to an increase in
treatment-related toxicity and microvasculature
damage.5,62 Some transplant centers provide ABOcompatible, plasma-reduced products for their ABOmismatched, allogeneic transplant patients.4,5
Little is known regarding the effect of ABOincompatible platelets in the setting of solid organ
transplantation, particularly ABO-incompatible kidney
and heart transplants. Current regimens require several
rounds of plasmapheresis or immunoadsorption
combined with immunosuppression to decrease isoagglutinin titers.63,64 To avoid passive transfusion of
isoagglutinins harmful to the graft, platelets should be
Currently, the greatest discussion around ABO and
platelet transfusion is the risk of acute hemolytic
transfusion reactions (HTR) with ABO minorincompatible or out-of-group platelets (Table 1).65–88
Although most queried transfusion services provide
ABO-identical platelets or plasma-compatible platelets
when available,89,90 it is estimated that 10 to 40 percent
of transfusions are plasma incompatible with the
recipient.73,76 The most common reasons for transfusing out-of-group platelets are limited inventory of
ABO type-specific platelets and HLA-matched platelets
and to minimize product wastage.90 To date, only a
handful of severe HTRs with platelet transfusion have
been reported in the literature and Internet chat
rooms.
Despite the transfusion of more than 2 million
platelet concentrates per year (including potentially
200,000 to 400,000 out-of-group transfusions), the true
risk and incidence of hemolysis with plasmaincompatible platelet transfusions is still a matter of
conjecture. Facility-specific rates for platelet-associated
HTR range from less than 1 in 1000 to less than 1 in
25,000 for all platelet transfusions and less than 1 in
100 to less than 1 in 9000 for minor-incompatible
platelet trans-fusions.73,76,91,92 This wide range may
reflect heterogeneity in the type of platelet products
transfused (apheresis vs. random), index of clinical
suspicion, and severity of the reaction. On the basis of
the yearly number of transfusions in the United States,
it is clear that severe HTRs after out-of-group platelet
transfusions are relatively rare events. However, mild
hemolysis may be more common than appreciated.
Oza et al.92 found mild to moderate evidence of
hemolysis with elution of ABO antibodies in 1 to 2
24
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Table 1. Acute hemolytic transfusion reactions with platelets
Donor
Reference
ABO type
Platelet donor and product
↓Hb (%)
Age*
Sex
Diagnosis
Saline
AHG
Product
Volume
Zoes65
44
F
AML-M4
AB
O
Anti-A:256‡
Anti-B:64
NR
Random-pool
500 mL
NR
McLeod66
45
M
AML-M6
A
O
O
1280
640
10,240
10,240
Random-pool
Random-pool
50
50
6 (43%)
Conway67
15
F
Transplant
A
O
8192
NR
Apheresis-HLA
200
NR
2.5
58
F
F
ALL
Angioplasty
A
B
O
O
512
512
32,000
16,384
Apheresis
Random
200
50
5.8 (50%)
6.1 (43%)
AML
A
O
256
>4000
Random
50
2.6
Pierce
68
Ferguson69
Patient Donor(s)
Outcome†
66
M
70
Reis
56
M Aplastic anemia
B
O
NR
4096
Apheresis
NR
6.1 (46%)
Murphy71
30
F
AML
A
O
512
256
2048
1024
Apheresis-HLA
Apheresis-HLA
225§
448§
4.0 (40%)
5.4 (47%)
Chow72
18
F
AML-M4
AB
O
1024‡
520‡
Apheresis, pooled
1200¶
5.5 (50%)
28
M Neuroblastoma
A
O,A,AB
128
NR
Apheresis
225
2.6 (31%)
72
F Cardiac surgery
AB
O,B,A
NR
NR
Apheresis
3380¶

F
73
Mair
McManigal74
76
Larsson
44
A
O
Anti-A:16384
NR
Apheresis
371
2.3 (30%)
Duguid75
0.1
0.02
M Cardiac surgery
F Cardiac surgery
A
A
O
O
NR
NR
NR
NR
Random-pool
Random-pool
100
100
4.5 (33%)
NR
51
16
F Breast cancer
F Aplastic anemia
A
A
O
>8000
NR
Double apheresis
(dry platelet)
37
37
3.5 (40%)
3.2 (37%)
35
M
Transplant
B
O
4096
NR
Apheresis
526
5.3 (50%)
Anonymous
36
45
F
M
Hodgkin’s
AML
A
A
O
O
NR
NR
2048
4096
Apheresis
Apheresis
214
241
2.5 (22%)
1.6 (15%)
Gresens80
29
M
Trauma
A
O
1024
1024
Apheresis
260
NR
Valbonesi77
Sauer-Heilborn78
79
82
AML
Ozturk
21
M
RAEB
A
O
NR
NR
Apheresis
600
Yeast81
0**
NR
NAIT
B
O
NR
NR
Apheresis
15
10 (83%)
Anonymous83
NR
NR
NR
AB
O
NR
NR
Apheresis
NR
5.3 (53%)
Josephson84
Adult
Adult
NR
NR
Leukemia
Leukemia
A
A
O
O
256
NR
8192
1024
Apheresis
Apheresis
50
50
3
3
Angiolillo85
0.7
M
Histiocytosis
A
O
128
NR
Apheresis
107
3.5 (47%)
86
5.7 (36%)
Sapatnekar
2
F Medulloblastoma
A
O
2048
16384
Apheresis
145
3.9 (32%)
Sadani87
65
F
A
O
160
1280
Apheresis
NR
3.8 (49%)
350
5.8
82%-Apheresis
15–3380
1.6–10
AML-M0
Mean
32.5
2403
7162
Range
0–72
128–16,384
1024–32,000
AHG = antihuman globulin; ALL = acute lymphocytic leukemia; AML = acute myelocytic leukemia; NAIT = neonatal alloimmune thrombocytopenia; NR = not reported; RAEB =
refractory anemia with excess blasts.
* Age in years.
† Fall in Hb by g/dL and percent decrease (%) from pretransfusion levels.
‡ Isoagglutinin titers in the recipient after transfusion of ABO minor-incompatible platelets.
§ Same HLA-matched donor, transfusions 1 month apart.
¶ Total volume of incompatible plasma infused.
 Active bleeding.
** Intrauterine transfusion.
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25
L. COOLING
percent of all ABO-mismatched apheresis platelets,
which accounted for 6.7 percent of all transfusion
reactions associated with platelets. An increased rate of
adverse reactions with ABO minor-incompatible
transfusions was also noted by Wagner and Adamo.93
Out-of-group platelet transfusions, however, have a
minimal impact on RBC utilization. Two studies have
found no difference in the level of anemia or RBC
utilization in oncology patients receiving plasmaincompatible transfusions.73,94
A summary of severe HTRs associated with plasmaincompatible platelets is shown in Table 1. In nearly all
cases, hemolysis followed the transfusion of group O
platelets to a non-group O patient. Clinically, the
unfortunate recipient displayed classic symptoms of an
acute HTR immediately or shortly after transfusion,
leading to rapid recognition and diagnosis. In a few
instances, the diagnosis was only suspected hours or
days later after the onset of hemoglobinemia,
hemoglobinuria, or unexplained anemia.66,68,75,77,85,87
Although most patients recovered with hydration and
supportive care, a handful of deaths are known,
including at least five deaths in the United States.68,78 In
very rare cases, plasma exchange, RBC exchange with
group O RBCs, or both were performed to stem the
hemolysis.77
Laboratory studies in these patients were all
consistent with an acute HTR. Patients’ RBCs typically
became strongly positive in the DAT (IgG, C3) with
elution of ABO antibodies from the RBCs. Laboratory
evidence of intravascular hemolysis was always
present, including an abrupt drop in hemoglobin,
hemoglobinemia, hemoglobinuria, decreased haptoglobin, spherocytes, hyperbilirubinemia, and elevated
lactate dehydrogenase. Disseminated intravascular
coagulation has been reported in two cases with fatal
outcomes.68,78 The degree of hemolysis can be
impressive, with a drop in hemoglobin ranging from 2
to 10 g/dL. In many cases, the patient will show an
appropriate increment in platelet count after
transfusion.70,74,81 An investigation of the implicated
platelet donors revealed unusually high isoagglutinin
titers in most cases. Several of the group O donors
in published reports were women, and included
a directed donation from a group O mother to her 2year-old daughter.68,77 Women with a history of a prior
ABO-incompatible pregnancy are at increased risk for
developing high-titer isoagglutinins.95
Several factors may raise the risk for an HTR after
an out-of-group platelet transfusion. Apheresis
26
platelets, which contain 200 to 400 mL of plasma from
a single donor, carry a higher risk of causing hemolysis
because of a high-titer donor than do pooled platelets.
This is substantiated by a review of the literature, in
which 23 of 28 (82%) platelet-associated HTRs were
associated with apheresis platelets. This risk is
substantially lessened with pooled platelets because of
the smaller volume of plasma per donor (50 mL), which
is subsequently diluted 4- to 6-fold in the final product.
The use of pooled platelets does not eliminate the risk
of an HTR: four HTRs caused by plasma-incompatible
pooled random platelets have been reported, in two
neonates75 and two adults.65,66 In the latter, two hightiter donors were pooled and infused.66 The total
volume of ABO-incompatible plasma must also be
considered, including residual plasma present in
transfused group O RBCs.74,87,96 McManigal et al.74
reported severe hemolysis in a patient who received
multiple out-of-group platelet transfusions, resulting in
the transfusion of 3380 mL of incompatible plasma
(106% of patient’s blood volume) during a period of 4
days.74
Patient factors can also contribute to the risk of an
HTR after an ABO-incompatible transfusion. As shown
in Table 1, there is an increased risk of hemolysis when
high-titer isoagglutinins are transfused to group A and
AB recipients (23 of 28)—a finding not surprising to
blood bank staff. Very young children may also be at
increased risk because of their relatively small blood
volume.75,81,85,87 It has been surmised that young
children and nonsecretors may also have an increased
susceptibility because ABO substances capable of
adsorbing and neutralizing ABO antibodies are
decreased in or absent from their blood. Among
published cases, only two investigated the Lewis type
of the recipient.65,78 One recipient was a nonsecretor
[Le(a+b–)])65; however, the second patient was
Le(a–b+),78 suggesting that soluble ABO antigens may
provide only limited protection. Finally, other illdefined patient factors could also play a role. Lookback investigations of two high-titer group O apheresis
platelet donors failed to identify any additional HTRs in
70 prior transfusion recipients.76,78
Approaches to Minimize Hazards of PlasmaIncompatible Platelet Transfusions
The increased use of apheresis platelets, in which
all the plasma is from a single donor, has heightened
awareness and concern regarding out-of-group
platelet transfusions. Several strategies have been
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ABO and platelets
implemented. Most transfusion services have policies
dictating the transfusion of ABO-identical or plasmaMore
compatible platelets when available.89,90
proactive strategies are discussed in the following
sections.
Identification of “dangerous donors”
Although only 2 percent of U.S. centers routinely
screen group O donors,90 the practice is relatively
common in Europe.89 With rare exceptions, donors
identified as high titer are still permitted to donate;
however, their products are segregated and labeled as
high titer with a warning to only transfuse to group O
patients. The cost effectiveness of prospective
screening is still unproven, given the relative rarity of
HTR with out-of-group platelets. Sadani et al.87 recently
reported a severe HTR in a group A patient after
transfusion of group O apheresis platelets that initially
tested as low titer by automated testing. Subsequent
testing revealed a high-titer anti-A IgG (Table 1).87 In
contrast, the New York Blood Center, which routinely
screened group O apheresis donors until 1998, has had
no reports of acute HTRs in the 5 years, and more than
25,000 platelet transfusions, since screening was
discontinued.91
A persistent problem that plagues widespread
donor screening is the absence of a recognized
standard method for testing. Several manual and
automated methods are currently used for donor
screening (tube, gel, and solid phase) with a variety of
end point measurements: saline titers, AHG titers, or
hemolysis.84,87,89,97 Even with a single method, there is a
wide range in results (Fig. 2A). Improvements and
increased use of automated blood group testing, such
as gel and solid phase methods, may eliminate much of
the perceived variation in donor isoagglutinin testing
with minimal add-on cost to the final product. This has
been demonstrated in England, where high-throughput
testing is performed using a single plasma or serum
dilution (1 in 100) on an automated blood analyzer.89 A
cost analysis by Emory University, which implemented
testing of all group O apheresis donors by gel method,
estimated an additional cost of $1.20 per apheresis
platelet.84
A second problem with donor screening is
determining a critical titer for “safe” versus high-titer,
“dangerous” donors. As shown in Figure 2A, the
percentage of donors classified as high titer can range
from 3 to 50 percent, depending on the population
tested and the method and critical titer used.76,84,89,97 A
Fig. 2.
ABO titers in platelet donors. A: Distribution and variability in
“critical” titers (anti-A) among group O donors. Critical titers by
saline (black circles) and with AHG (squares) for several studies
are shown. B and C: Distribution of anti-A titers in a random
sampling of group O platelet donors (hatched columns) and
group O donors implicated in acute HTRs (black, Table 1). Data
for normal donors taken from Josephson et al.84 by gel. Note that
38 percent of normal donors had titers less than the initial titer
of 32.84 Shown are saline titers (B) and after AHG, C.
method and critical titer that classifies 40 to 50 percent
of group O donors as high titer could significantly
hamper platelet availability to non-group O patients,
particularly during times of severe shortages. A survey
of current practice shows a cutoff titer of 32 to 200 for
saline testing and 250 to 512 for AHG (Table 2). For
I M M U N O H E M A T O L O G Y, V O L U M E 2 3 , N U M B E R 1 , 2 0 0 7
27
L. COOLING
Table 2. Strategies for screening apheresis platelet donors for high-titer
isoagglutinins
Screen
donors
Method
Critical
titer
%
Donors
United States84,90,91
No (2%)90
Tube, gel
1:50–1:200
3–28%
89
England
Yes
Automated
Tube
1:100
1:128
3–10%
Scotland87
Yes
89
Yes
Gel
IAT
1:64
1:256
Yes
Tube, saline
1:64
Czech Republic
Yes
Tube, saline
1:64
Norway89
Yes
IAT
1:250
Sweden89
Yes
Tube, saline
IAT
1:100
1:400
Switzerland89
Yes
Hemolysis
1:16
Finland89
Yes
Tube, saline
1:32
Yes
IAT
1:512
Country
Italy
Germany89
89
89
Japan
1:50
5%
5.7%
this review, we compared the distribution of
isoagglutinin titers from normal group O apheresis
donors84 and group O donors implicated in HTRs
(Table 1). As shown in Figures 2B and 2C, many of the
cutoffs used (32–64) are quite conservative.
Plasma reduction and platelet additive solutions
A popular method to reduce the risk of ABO HTR
caused by plasma-incompatible transfusions combines
plasma reduction with resuspension in either additive
solutions or group AB donor plasma. In the most
common method, platelet concentrates are prepared
and stored in plasma (30–40%) diluted with a platelet
additive storage solution. Although not licensed in the
United States, a selection of storage solutions are
commercially available or undergoing trials in
Europe.98,99 The use of additive solutions has several
attractive advantages for increasing transfusion safety
including a decreased risk of HTRs as well as TRALI
and allergic and febrile reactions caused by residual
proteins, cytokines, and antibodies present in donor
plasma.93,98–100 In Germany, Wagner and Alamo93 noted
a marked decrease in transfusion reactions with
plasma-reduced platelet concentrates (0%) compared
with routine pooled platelets (27%). Plasma-reduced
platelets reportedly have equivalent posttransfusion
recovery at 1 hour although there is reduced 24-hour
survival.89,98
Plasma reduction and use of platelet additive
solutions may reduce, but will not completely
eliminate, the risk of an acute HTR caused by a hightiter donor. An enlightening case was recently
published by Valbonesi et al.,77 who described severe
28
HTRs in two recipients of a split, group O apheresis
unit (Table 1). Apheresis platelets were collected “dry”
with only minimal plasma carryover (74 mL), followed
by resuspension in 400 mL of a platelet additive (T-Sol).
An investigation identified extremely high isoagglutinin
titers (> 8000) in the female donor. The authors noted
that this was the first severe HTR encountered in more
than 16,000 “dry” platelet collections in the last 10
years.
Limiting plasma infusion
Some transfusion services monitor and limit the
total volume of incompatible plasma that a recipient
Policies vary between
can receive.79,83,89,90,101
institutions, ranging from 300 to 500 mL of plasma per
day to 1000 mL of plasma per week (Table 3). In the
United States, approximately 10 percent of polled
institutions (255) limited the amount of incompatible
plasma transfused to adult patients.
In neonates and young children with small blood
volumes, there is a greater concern regarding the risk
of out-of-group platelets.102 As a consequence, most
U.S. hospitals serving neonatal populations provide
only ABO-identical or plasma-compatible platelets
(60%). Very few institutions (1%, 29 institutions) have
policies limiting incompatible plasma infusion to
neonates and children.
Identification of A2 donors as “universal platelet
donors”
Because A2 donors have little or no A antigen on
platelet membranes, they are compatible with group A
and O donors. Donor screening to identify group A2
donors could increase the inventory of group O
compatible from 44 to 52 percent, increasing the
number of apheresis platelets available for platelet
crossmatching,10 which requires ABO compatibility
between donor and patient.36 It could also benefit
selection and survival of HLA-matched platelets.39,40 To
our knowledge, only Norway has policies for
identifying A2 platelet donors as “universal donors.”89
A2 platelets are also an ideal component for patients
undergoing ABO-mismatched allogeneic bone marrow
transplantation. In ABO major-incompatible transplants,
present guidelines recommend that transfused platelets
be plasma compatible with the marrow or stem cell
donor to minimize delays in erythroid engraftment.46 As
a consequence, platelets will often be ABO
incompatible with the patient’s isohemagglutinins. In
group A (donor) to group O (patient) mismatched
I M M U N O H E M A T O L O G Y, V O L U M E 2 3 , N U M B E R 1 , 2 0 0 7
ABO and platelets
Table 3. Transfusion strategies for minimizing HTR caused by
incompatible-plasma infusion
ABO-identical platelets
ABO plasma-compatible platelets
Plasma-reduced
Resuspension in storage additive solution, group AB plasma
Pooled platelets
Screened “low-titer” apheresis platelets
Plasma volume limits
Adults79,83,101
4 plasma-incompatible platelet transfusions per week79
1000 mL incompatible plasma per week79
2 plasma-incompatible apheresis platelets per 72 hours83
300–500 mL incompatible plasma per day101
Children79
>40 kg:
25–39 kg:
5–24 kg:
Neonates:
600 mL per week
400 mL per week
100 mL per week
0 mL, plasma-compatible only
transplants, this incompatibility can be overcome by
transfusion of A2 platelets, which are ABO compatible
with the patient’s isoagglutinins and plasma compatible
with the donor graft.10 A2 platelets might also be a
product of choice in group O (donor) to group A
(patient) minor-mismatch transplants. Likewise, A2
platelets are an ideal product for group O patients
receiving group A-incompatible solid organ transplants.
In these patients, transfusion of incompatible plasma
and platelets should be avoided because of the risk of
acute humoral rejection and immune stimulation,
respectively.45,60 It is my fervent wish that A subtyping
of apheresis platelet donors become a standard of
practice by blood collection centers.
transplant patients, who are at increased risk for
significant morbidity and mortality because of ABO
incompatibility.
ABO-identical platelets should be provided
whenever possible, particularly for patients requiring
long-term transfusion support. In patients undergoing
ABO-mismatched transplants, provision of A2 platelets
may be preferable. In patients requiring HLA-antigen
negative and HLA-matched platelets, HLA matching has
priority; however, the ABO compatibility of HLA
platelets should be recorded and considered when
assessing the success or failure of HLA platelets. ABO
compatibility should always be considered when
evaluating and monitoring patients with clinical
refractoriness, particularly group O patients who have
higher mean isohemagglutinin titers.
In neonates and young children, only ABO-identical
or plasma-compatible platelets should be transfused.102
In surgical and other patients with short-term
transfusion needs, ABO compatibility is less of a
concern. ABO-identical or plasma-compatible platelets
should be transfused if available; however, an actively
bleeding patient should never be denied a platelet
transfusion because of ABO compatibility. The risks of
bleeding and the need to achieve hemostasis outweigh
the potential adverse consequences of transfusing an
ABO-incompatible platelet. One potential exception is
ABO-incompatible organ transplantation, in which
plasma containing donor-reactive isoagglutinins should
be avoided. In the postoperative period, donorcompatible platelets may be considered to minimize
immune stimulation of recipient isoagglutinins.
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is heterogeneous and determined by genetic and
epigenetic factors. Routine transfusion of ABOincompatible platelets can be associated with
cumulative adverse effects including decreased
posttransfusion recovery, increased platelet utilization,
incompatible platelet crossmatches, HLA alloimmunization, and ABH-specific refractoriness. Out-ofgroup platelet transfusion can also be associated
with adverse effects including decreasing platelet
increments and, rarely, severe HTRs. Out-of-group
platelet transfusion may have more severe
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91. Schwartz J, Delpalma H, Kapoor K, Hamilton T,
Grima K. Anti-A titers in group O single donor
platelets: to titer or not to titer (abstract).
Transfusion 2003;43(Suppl):SP247.
92. Oza KK.ABO mismatched platelet transfusion and
acute intravascular hemolysis (abstract). Transfusion 2002;42(Suppl):SP308.
93. Wagner F, Adamo W. Reduced rate of adverse
reactions to plasma reduced pooled platelet units:
possible role of minor incompatible transfusion
(abstract).Vox Sang 2006;91(Suppl 3):P508.
94. Shanwell A, Ringden O, Wiechel B, Rumin S,
Akerblom O. A study of the effect of ABO
incompatible plasma in platelet concentrates
transfused to bone marrow transplant recipients.
Vox Sang 1991;60:23–7.
95. Mollison PL, Engelfriet CP, Contreras M, ed. Blood
transfusion in clinical medicine. 10th ed. Oxford:
Blackwell Science, 1997.
96. Boothe G, Brecher ME, Root M, Robinson J, Haley
R. Acute hemolysis due to passively transfused
high-titer anti-B causing spontaneous in vitro
agglutination. Immunohematology 1995;11:43–5.
97. The practice of transfusing out-of-type platelets.
CBBS, e-network forum. Available at: http://www.
cbbsweb.org/enf/2002/plttx_aboincompat.html.
98. de Wildt-Eggen J, Gulliksson H. In vivo and in vitro
comparison of platelets stored in either synthetic
media or plasma.Vox Sang 2003;84:256–64.
99. Ringwald J, Zimmerman R, Eckstein R. The new
generation of platelet additive solution for storage
at 22°C: development and current experience.
Transfus Med Rev 2006;20:158–64.
100. de Wildt-Eggen J, Nauta S, Schrijver JG, van Marwijk
Kooy M, Bins M, van Prooijen HC. Reactions and
platelet increments after transfusion of platelet
concentrates in plasma or an additive solution: a
prospective, randomized study. Transfusion
2000;40:398–403.
101. References on patient outcomes following
transfusion of ABO incompatible platelets. CBBS,
e-network forum. Available at: http//www.
cbbsweb.org/enf/outcome_aboinc_plt.html.
102. Chambers LA. Transfusion of plasma and platelets
to neonates. In: Herman JH, Manno CS, eds.
Pediatric transfusion therapy, 2002. Bethesda, MD:
AABB Press, 2002:93–107.
Laura Cooling, MD, MS, Assistant Professor,
Pathology, Associate Medical Director, Transfusion
Medicine, University of Michigan Hospitals, 2F225
UH, Box 0054, 1500 East Medical Center Drive, Ann
Arbor, MI 48109-0054.
I M M U N O H E M A T O L O G Y, V O L U M E 2 3 , N U M B E R 1 , 2 0 0 7
33
Scott Murphy’s contribution in
the early years of posttransfusion
purpura: a remembrance
M. KEASHEN-SCHNELL
Dr. Scott Murphy always enjoyed a medical mystery
and his passion for the syndrome of posttransfusion
purpura (PTP) was one of his favorite topics. This
“perfect storm” of immunologic events is characterized
by profound thrombocytopenia approximately 7 days
after transfusion and includes the development of a
platelet-specific alloantibody which destroys any
antigen-positive transfused platelets as well as the
patient’s own platelets, which lack the corresponding
antigen. Dr. Murphy’s interest in this syndrome began
in the early years of his research career. He also
provided our laboratory with the first antiserum, which
we used to develop testing procedures and to
investigate numerous cases of PTP. In the next few
pages of this edition dedicated in his honor, I would
like to provide a brief trip down memory lane to
remember his valued contribution to the Platelet
Serology Laboratory at the Penn-Jersey Region of the
American Red Cross (ARC) in Philadelphia, to outline
some of the many advances that have taken place in
the investigation of platelet antibodies, and to
summarize the results of our laboratory investigation of
PTP at Penn-Jersey.
I first had the pleasure of meeting Scott Murphy in
the late 1970s while working as a research technologist
at the Penn-Jersey Region of the ARC. At that time, we
were in the process of developing a procedure to type
donor platelets for the PlA1 antigen. Dr. Miriam Dahlke,
our medical director, informed me that Dr. Murphy had
offered to share a supply of anti-PlA1, which was the
result of a plasma exchange performed on a patient
diagnosed with PTP. Not wanting to wait for him to
change his mind, I headed to his laboratory at the other
end of town on that very hot, late spring day, armed
with a box and a small supply of ice.
I was greeted by a bearded 1970s version of our
own Scott Murphy, complete with heavy cotton lab
34
coat emblazoned with his name and “Cardeza” in bright
red embroidery. He appeared supremely happy in his
crowded research lab as he presented me with a bag
containing nearly a full liter of plasma. I remember his
response after I thanked him and asked if our
laboratory could do anything to return the favor. He
said, “just type a lot of platelets for our patients.” I
would later learn that this was his way, always a
gracious gentleman who held his patients and others’
as his first priority.
As I headed back with my precious cargo, I noticed
a small split in the bag and was faced with the dilemma
of making it back to the ARC building before it thawed
(and leaked all over Center City Philadelphia). The justin-time arrival of an available taxi cab allowed me to
beat the clock and the weather. I have to admit that
this was, unquestionably, the only time in my career
that, while working late into the evening, the usually
boring task of preparing a thousand tiny 1-mL vials was
pure pleasure.
In 1975, while on the staff of the Hematology
Research Laboratory at the Presbyterian-University of
Pennsylvania Medical Center in Philadelphia, Dr.
Murphy coauthored a seminal publication calling
attention to the variety of responsible antibodies and
patient profiles in the relatively newly described and
clinically mysterious syndrome of PTP.1
Before the publication of this paper in 1975, all of
the 14 described cases of PTP were attributed to the
presence of anti-PlA1 produced by PlA1-negative female
patients. Two of the three patients described in this
paper (one PlA1-negative man who produced anti-PlA1
and a PlA1-positive woman with a demonstrable antiplatelet antibody of unknown specificity) served to
expand the definition of PTP to include both male and
female patients with antibody specificities not limited
to anti-PlA1.
I M M U N O H E M A T O L O G Y, V O L U M E 2 3 , N U M B E R 1 , 2 0 0 7
Scott Murphy’s contribution to PTP
PTP is a relatively rare, but well-defined, syndrome
first described by Shulman et al. in 1961.2 The classic
patient profile describes an older, multiparous woman
with a precipitous, antibody-mediated drop in platelet
count occurring 7 to 10 days after transfusion. Unlike
the maternal antibodies responsible for neonatal
alloimmune thrombocytopenia, the antibodies
produced by patients diagnosed with PTP cause severe
thrombocytopenia in the antibody producer. PTP is
self-limiting, but carries a significant risk of fatal
hemorrhage. Similarities have been noted between
PTP and the hemolysis of autologous RBCs during
delayed hemolytic transfusion reactions caused by
some RBC antibodies.3 To date, there is no definitive
explanation for the destruction of autologous cells in
either situation.
In the years after Dr. Murphy’s 1975 publication
and since the first description of a platelet-specific
antigen by van Loghem in 1959,4 much has been
learned about the nature of platelet antigens. The
nomenclature for platelet-specific antigens was
standardized by a Working Party on Platelet Serology5
(Table 1) and adopted by the International Society of
Blood Transfusion. In this more orderly, numeric
system, the platelet antigens are designated as HPA
(human platelet antigens) and are numbered (with
Arabic numbers) according to discovery date, followed
by a lower case “a” or “b,” which denotes the high (a) or
low (b) frequency member of the antigen pair. As an
example of the need for simplification, the first antigen
reported was referred to as Zwa in Europe and PlA1 in
the United States. Under the new nomenclature, it is
now called HPA-1a. Its lower frequency allele (PlA2 or
Zwb) is now HPA-1b.
We now know that six platelet-specific antigen
systems (HPA-1 through 5 and HPA-15) are biallelic
with one high frequency and one low frequency
antigen or allele.6 For the remaining 10 antigens (HPA6 through 14 and HPA-16), alloantibodies against the
low frequency but not the high frequency antigen have
been observed. The molecular basis and location on
the platelet membrane has been resolved for antigens
listed in Table 1. With the exception of HPA-14, the
differences in the alleles are the result of a single amino
acid substitution at a specific location on the gene
encoding for the membrane glycoprotein. The
isoantigen, Naka,7 is now known to be located on GPIV
(CD36).8,9
These advances, as well as the availability of
monoclonal antibodies, which recognize specific
Table 1. Human platelet antigen nomenclature
New
nomenclature
Old
nomenclature
Glycoprotein
location
Phenotypic
frequency
(approx. %
Caucasians)*
HPA-1a
PlA1 (Zwa)
IIIa (CD61)
98
HPA-1b
PlA2 (Zwb)
IIIa (CD61)
HPA-2a
Kob
Ib
(CD42b)
> 99
HPA-2b
Koa (Siba)
Ib
(CD42b)
13
HPA-3a
Baka (Leka)
IIb (CD41)
81
HPA-3b
Bakb
IIb (CD41)
70
b
a
29
HPA-4a
Yuk (Pen )
IIIa (CD61)
> 99
HPA-4b
Yuka (Penb)
IIIa (CD61)
< 0.1
HPA-5a
Brb (Zavb)
Ia (CD49b)
99
HPA-5b
Bra (Zava, Hca)
Ia (CD49b)
20
HPA-6bw
Caa,Tua
IIIa (CD61)
0.7
a
HPA-7bw
Mo
IIIa (CD61)
0.2
HPA-8bw
Sra
IIIa (CD61)
< 0.1
HPA-9bw
Maxa
IIb (CD41)
0.6
IIIa (CD61)
< 1.6
IIIa (CD61)
< 0.3
a
HPA-10bw
La
HPA-11bw
Groa
a
HPA-12bw
Iy
HPA-13bw
Sita
Ib
HPA-14bw
Oea
(CD42c)
0.4
Ia (CD49b)
0.3
IIIa (CD61)
< 0.2
HPA-15a
b
Gov
CD109
74
HPA-15b
Gova
CD109
81
HPA-16bw
Duva
IIIa (CD61)
<1
*After Norton A, Allen DL, Murphy MF. Review: platelet alloantigens and antibodies
and their clinical significance. Immunohematol 2004;20:89–99.
glycoprotein locations on the platelet surface, have led
to the development of serologic testing techniques
with increased sensitivity10–14 as well as to the
development of molecular techniques15 for platelet
antigen typing.
Other specificities have since been identified as
pathogenic antibodies in PTP using a variety of
investigative techniques. These include anti-PlA2 (HPA1b),16 anti-Baka (HPA-3a),17 anti-Bakb (HPA-3b),18 antiPena (HPA-4a),19 anti-Bra (HPA-5b),20 and anti-Brb (HPA5a).21,22
In 1992, Dr. Murphy became the medical director of
the Penn-Jersey Region, and we had the honor of
working with him for the next 14 years. His fascination
with the syndrome of PTP and his concern for the
patients involved never waned. He was never too busy
to make time in his schedule to learn the details of each
new case. He would then consult directly with the
patient’s clinician to ensure that our center could
provide appropriate testing, consultation, and
transfusion support. Options for transfusion support,
I M M U N O H E M A T O L O G Y, V O L U M E 2 3 , N U M B E R 1 , 2 0 0 7
35
M.A. KEASHEN-SCHNELL
years ago remains in use in our laboratory and continues
to help so many of “our patients.”
References
Fig. 1.
Penn-Jersey ARC Platelet Serology Laboratory results: antibody
specificities of PTP investigations 1983–2007.
when needed, would vary from case to case and might
include antigen-negative single-donor platelets or
washed or deglycerolized RBCs.
Since 1983, our laboratory has investigated 115
patient samples submitted to rule out PTP. Samples
were tested for the presence of platelet-specific and
HLA antibodies using a variety of methodologies. Of
115, 33 (28.6%; 31 from women, 2 from men)
contained platelet-specific antibody(ies) and these
patients were diagnosed with PTP based on this
finding along with clinical history. Of these, 23
demonstrated anti-HPA-1a, 4 anti-HPA-1b, 3 anti-HPA-3a,
1 anti-HPA-1b and anti-HPA-3a, 1 anti-HPA-5b, and 1 antiHPA-5a (Fig. 1). Molecular testing was used to confirm
the antigen-negative status in five cases. Nineteen were
confirmed using serologic techniques on postrecovery
platelet samples. Nine were not submitted or were
unavailable for confirmation. Of note is the fact that 23
of the 33 (69.6%) sera also demonstrated HLA class I
antibodies in combination with platelet-specific
antibodies, indicating the necessity to use glycoproteinbased as well as standard whole-cell methods as part of
the investigative protocol to ensure detection of
coexisting antibodies. Of the remaining 82 samples,
the majority either were negative or demonstrated HLA
antibody only. These results, along with other
published data, support Dr. Murphy’s early report of the
heterogeneous nature of PTP.
Thank you, Scott Murphy, for your support, sustained
enthusiasm, and undiminished desire to help patients in
need. You are missed by everyone here at the ARC. As I
anticipate further advances in the challenging fields of
platelet testing and transfusion, I will not forget that
happy, bearded researcher of many years ago, and I am
delighted to report that the plasma handed to me 29
36
1. Zeigler Z, Murphy S, Gardner F. Post-transfusion
purpura: a heterogeneous syndrome. Blood 1975;
45:529–36.
2. Shulman NR, Aster RH, Leitner A, Hiller MC.
Immunoreactions involving platelets. V. Posttransfusion purpura due to a complement-fixing
antibody against a genetically controlled platelet
antigen. A proposed mechanism for thrombocytopenia and its relevance in “autoimmunity.” J
Clin Invest 1961;40:1597–620.
3. Garratty G. Review: platelet immunology—
similarities and differences with red cell
immunology. Immunohematol 1995;11:112–24.
4. van Loghem JJ, Dorfmeijer J,Van Hart M, Schrueder
F. Serological and genetical studies on a platelet
antigen (Zw).Vox Sang 1959;4:161–9.
5. von dem Borne AE, DeCary F. Nomenclature of
platelet-specific antigens (letter). Transfusion
1990;30:477.
6. Metcalfe P, Watkins NA, Ouwehand WH, et al.
Nomenclature of human platelet antigens. Vox
Sang 2003;85:240–5.
7. Ikeda H, Mitani T, Ohnuma M, et al.A new plateletspecific antigen, Naka, involved in the refractoriness of HLA-matched platelet transfusion. Vox
Sang 1989;57:213–7.
8. Yamamoto N, Ikeda H,Tandon NN, et al. A platelet
membrane glycoprotein (GP) deficiency in
healthy blood donors: Naka-platelets lack
detectable GPIV (CD36). Blood 1990;76:
1698–703.
9. Greenwalt DE, Lipsky RH, Ockenhouse CF, et al.
Membrane glycoprotein CD36: A review of its
roles in adherence, signal transduction, and
transfusion medicine. Blood 1992;80:1105-15.
10. von dem Borne AE, Verheught FW, Oosterhof F, et
al. A simple fluorescence test for the detection of
platelet antibodies. Br J Haematol 1978;39:
195–207.
11. Rachel JM, Sinor LT,Tawfik OW, et al. A solid-phase
red cell adherence test for platelet crossmatching. Med Lab Sci 1985;42:194–5.
12. Nordhagen R, Flaathen ST. Chloroquine removal of
HLA antigens for platelets for the platelet
immunofluorescence test. Vox Sang 1985;48:
156–9.
I M M U N O H E M A T O L O G Y, V O L U M E 2 3 , N U M B E R 1 , 2 0 0 7
Scott Murphy’s contribution to PTP
13. Kiefel V, Santoso S,Weisheit M, Mueller-Eckhardt C.
Monoclonal antibody-specific immobilization of
platelet antigens (MAIPA): a new tool for the
identification of platelet-reactive antibodies. Blood
1987;70:1722–6.
14. Furihata K, Nugent DJ, Bissonette A, et al. On the
association of the platelet specific alloantigen,
Pena, with glycoprotein IIIa. Evidence for
heterogeneity of glycoprotein IIIa. J Clin Invest
1987;80:1624–30.
15. McFarland JG, Aster RH, Bussel JB, et al. Prenatal
diagnosis of neonatal alloimmune thrombocytopenia using allele-specific oligonucleotide
probes. Blood 1991;78:2276–82.
16. Taaning E, Killmann SA, Morling N, et al. Posttransfusion purpura (PTP) due to anti-Zwb (-PlA2):
the significance of IgG3 antibodies in PTP. Br J
Haematol 1986;64:217–25.
17. Keimowitz RM, Collins J, Davis K, Aster RH. Posttransfusion purpura associated with alloimmunization against the platelet-specific antigen, Baka.
Am J Hematol 1986;21:79–88.
18. Kickler TS, Herman JH, Furihata K, et al.
Identification of Bakb, a new platelet-specific
antigen associated with posttransfusion purpura.
Blood 1988;71:894–8.
19. Simon TL, Collins J, Kunicki TJ, et al. Posttransfusion purpura associated with alloantibody
specific for the platelet antigen Pen(a). Am J
Hematol 1988; 29:38–40.
20. Christie DJ, Pulkrabek S, Putnam JL, et al. Posttransfusion purpura due to an alloantibody
reactive with glycoprotein Ia/IIa (anti-HPA-5b).
Blood 1991;77:2785–9.
21. Anolik JH, Blumberg N, Snider J, Francis CW.
Posttransfusion purpura secondary to an alloantibody reactive with HPA-5a (Br(b)). Transfusion
2001;41:633–6.
22. Ziman A, Klapper E, Pepkowitz S, et al. A second
case of post-transfusion purpura caused by HPA5a antibodies: successful treatment with intravenous immunoglobulin. Vox Sang 2002;83:
165–6.
Maryann Keashen-Schnell, BS, Supervisor, Platelet/
Neutrophil Serology Laboratory, Manager, Process
Improvement, American Red Cross Blood Services,
Heritage Division, Penn-Jersey Region Department of
Technical Services, 700 Spring Garden Street,
Philadelphia, PA 19123.
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I M M U N O H E M A T O L O G Y, V O L U M E 2 3 , N U M B E R 1 , 2 0 0 7
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immuno@usa.redcross.org
37
Differences in ABO antibody
levels among blood donors: a
comparison between past and
present Japanese, Laotian, and
Thai populations
T. MAZDA, R.YABE, O. NATHALANG,T.THAMMAVONG, AND K.TADOKORO
Passively transfused blood group antibodies cause clinical
problems. High titers of anti-A and anti-B seem to be one reason for
hemolytic transfusion reactions and for ABO HDN. In Japan, anti-A
and anti-B titers notably decreased in the 15 years between 1986
and 2001. At present, titers of more than 100, as measured using the
saline method, are rare. Differences in the level of anti-A and anti-B
among ethnic populations have been reported; these differences
were found to be the result of environmental factors rather than
hereditary factors. In the present study, the anti-A and anti-B titers
of random donors in three Asian populations are compared. In
Thailand, the IgM anti-A and anti-B titers are low and are similar to
the Japanese titers reported in 2001, but the IgG anti-A and anti-B
titers are high and are similar to the Japanese titers reported in
1986. In the Lao People’s Democratic Republic, both the IgM and
the IgG anti-A and anti-B titers are high and are similar to those
reported in Japan in 1986. In addition, anti-A and anti-B titers of
different sex donors and of various age groups were also compared.
High titers were found in 8.8 percent of the female donors in the
younger than 30 age group and in 36.7 percent of the female
donors in the older than 50 age group. Immunohematology
2007;23:38–41.
Key Words: ABO blood groups, anti-A, anti-B
Passively transfused blood group antibodies cause
clinical problems.1 The effects of anti-A and anti-B in
ABO-mismatched platelet transfusions, especially in
HLA-matched platelet transfusions, have long been
debated. Although reports are rare, intravascular
hemolytic transfusion reactions have been known to
occur.2,3 These hemolytic transfusion reactions seem to
be caused by high titers of anti-A and anti-B. Such
reactions have been seen in groups A, B, and AB
recipients receiving group O plasma.
Anti-A and anti-B levels have been reported to differ
among ethnic populations.4 Many previous studies
have suggested that ethnicity is a risk factor for ABO
38
HDN. However, the differences among populations
were found to be the result of environmental factors
rather than hereditary factors.5,6 Redman et al.6
addressed the question of variations in ABO antibody
levels in persons with different ethnic backgrounds by
studying antibody levels in Asian, Caucasian, and
African blood donors, all of whom were living in the
north London area of the United Kingdom. Although
the highest levels of IgG anti-A and anti-B titers were
found among group O African female donors, these
levels were not significantly higher than those in the
other group O donors who were tested.
In this report, we investigated the changes in the
anti-A and anti-B titers in the Japanese population
during the last 19 years. Both anti-A and anti-B titers
decreased greatly during this period. We also
compared these titers with the anti-A and anti-B titers
in Laotian and Thai populations.
Materials and Methods
Sera were obtained from Japanese blood donors in
1986 (n = 106), 2001 (n = 107), and 2005 (n = 93) in
Tokyo, Japan. In 2001, sera were obtained from blood
donors in Vientiane, Lao People’s Democratic Republic
(Lao PDR; n = 58); in 2005, sera were obtained from
blood donors in Bangkok,Thailand (n = 93). All serum
samples were obtained randomly from the general
population of volunteer unremunerated blood donors.
The sera were stored at –30°C until tested.
Sera from group O Japanese blood donors classified
according to age (16–29 years old and 51–69 years old)
I M M U N O H E M A T O L O G Y, V O L U M E 2 3 , N U M B E R 1 , 2 0 0 7
ABO antibody levels in Asians
Table 1. Distribution of anti-A and anti-B IgM titers in three populations
Anti-A titer
2
4
8
16
32
Laos 2001*
–
–
–
–
–
Thailand 2005*
–
6
8
17
14
Japan 1986*
–
–
–
–
–
Japan 2001*
–
–
–
2
16
Japan 2005*
–
9
18
27
14
Japan (group O)
16–29 male
16–29 female
51–69 male
51–69 female
2
3
3
10
9
14
24
9
28
32
32
13
32
31
23
9
Anti-B titer
2
4
8
Laos 2001*
–
–
–
Thailand 2005*
–
3
Japan 1986*
–
–
64
128
256
512
1024
Total (n)
–
2
36
8
6
52
12
11
7
–
–
75
6
3
67
7
3
86
51
8
1
1
–
79
7
–
–
–
–
75
26
20
12
6
5
2
–
2
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
102
102
94
49
16
32
64
128
256
512
1024
Total (n)
–
–
–
–
36
3
1
40
5
17
11
8
12
6
–
–
62
–
–
–
6
8
68
2
4
88
Japan 2001*
–
–
–
11
69
9
2
2
–
–
93
Japan 2005*
2
5
17
27
7
4
–
–
–
–
62
Japan (group O)
16–29 male
16–29 female
51–69 male
51–69 female
–
3
11
8
12
37
19
10
43
30
49
21
23
23
14
4
19
7
1
5
5
2
–
1
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
102
102
94
49
* Data include group O and nongroup O donors
and sex were also collected for
the present study. These serum
samples were separate from
those collected from the
general population in 1986,
2001, and 2005.
The sera were titrated using
a 1 to 1 dilution with 0.01M
PBS, pH 7.2, containing 2% BSA.
The IgM antibody titers were
determined using the saline
method with incubation at
room temperature for 15
minutes. The IgG antibody
titers were determined by an
IAT using LISS and an
incubation of 30 minutes at
37°C. Before the IAT, the sera
were treated with 2-ME for 15
minutes at 37°C using the
method described in the AABB
this
Technical
Manual 7;
treatment is used to destroy IgM
antibodies in the sera.
Testing was performed by
the same person to avoid
variations in technique and test
interpretation.
Table 2. Distribution of anti-A and anti-B IgG titers in three populations
Anti-A titer
2
4
8
16
32
64
128
256
512
1024
Total (n)
Laos 2001*
–
–
–
–
–
13
15
16
6
2
52
Thailand 2005*
1
8
8
9
23
15
5
3
3
–
75
Japan 1986*
–
–
–
1
3
39
21
20
2
–
86
Japan 2001*
–
2
16
51
8
1
1
–
–
–
79
Japan 2005*
2
22
24
21
6
–
–
–
–
–
75
Japan (group O)
16–29 male
16–29 female
51–69 male
51–69 female
2
2
–
–
14
22
6
2
33
23
18
18
28
34
35
14
14
9
32
4
11
11
3
6
–
1
–
2
–
–
–
3
–
–
–
–
–
–
–
–
102
102
94
49
Anti-B titer
8
16
32
64
128
256
512
1024
2048
4096
Total (n)
Laos 2001*
–
–
–
–
–
1
23
11
3
2
40
Thailand 2005*
1
1
1
7
12
19
15
5
1
–
62
Japan 1986*
–
–
–
–
1
2
28
32
22
3
88
Japan 2001*
–
11
69
9
2
2
–
–
–
–
93
Japan 2005*
–
17
20
16
8
1
–
–
–
–
62
Japan (group O)
16–29 male
16–29 female
51–69 male
51–69 female
3
1
2
–
28
26
15
4
37
39
40
15
27
17
29
4
5
6
8
3
2
4
–
5
–
4
–
10
–
5
–
8
–
–
–
–
–
–
–
–
102
102
94
49
* Data include group O and nongroup O donors
I M M U N O H E M A T O L O G Y, V O L U M E 2 3 , N U M B E R 1 , 2 0 0 7
Results
In the Japanese population,
anti-A and anti-B titers in 1986
were distributed between 64
and 1024 (mode 256) for IgM
(Table 1) and between 64 and
2048 (mode 256–512) for IgG
(Table 2). In 2001, they markedly decreased to between 16
and 512 (mode 32–64) for both
IgM and IgG. In 2005, the IgM
titers further decreased to
between 2 and 64 (mode 8–16).
In the Laotian population in
2001, the IgM titers were similar
to those of the 1986 Japanese
population; the distribution
was between 128 and 1024
(mode 256), whereas the IgG
titers were slightly higher than
those of the 1986 Japanese
39
T. MAZDA ET AL.
population: between 256 and 4096 (mode 512–1024).
In the Thai population in 2005, IgM titers were similar
to those of the 2001 Japanese population: the distribution was between 4 and 256 (mode 16), and IgG
titers were similar to those of the 1986 Japanese
population: the distribution was between 8 and 2048
(mode 128–256).
In the Japanese group O blood donors, IgM titers of
anti-A and anti-B were low and no differences were
noted between the sexes. However, IgG titers of anti-A
and anti-B showed differences between the sexes and
an increase with donor age. High titers (> 512) of antiA, anti-B, or both were found in 8.8 percent (9 in 102)
of the female donors in the younger than 30 age group
and in 36.7 percent (18 in 49) of the female donors in
the older than 50 age group, determined by the
antihuman globulin test. No male donors were found
with titers more than 256.
Discussion
In the Japanese population, the anti-A and anti-B
titers markedly decreased within the 15-year period
between 1986 and 2001. At present, the Japanese antiA and anti-B titers are relatively low, compared with
those seen in the Laotian and Thai populations.
Schwartz et al.8 showed that only 3.3 to 3.6 percent of
American group O blood donors had high anti-A and
anti-B titers (> 100). People with high ABO system
antibody titers are now rare among the Japanese
population, and the mean titers are lower than those of
blood donors in New York.
The mechanisms responsible for the reductions in
Japanese anti-A and anti-B titers are unknown.
However, previous studies have suggested that
environmental factors may influence anti-A and anti-B
titers.5,6 Enteric bacteria and other parasites may affect
the production of anti-A and anti-B.4,6 Environmental
factors can affect the prevalence, counts, and susceptibility of enteric bacteria.9,10 Because environmental
factors are thought to affect anti-A and anti-B titers, we
compared the anti-A and anti-B titers in three Asian
countries: Japan, Lao PDR, and Thailand. Japan has long
been a developed country, Lao PDR is a developing
country, and Thailand has recently been given
developed country status.
The environment in Japan continues to change.
Recently, the Japanese lifestyle has become more
westernized, especially with regard to food.11 The
prevalence of allergic diseases, heart disease, diabetes,
and cancer has been increasing in Japan. In contrast,
40
Lao PDR remains relatively undeveloped, and Laotians
usually eat natural foods, whereas Thais and the
Japanese eat more processed foods.
These
environmental differences may explain, at least in part,
why the Laotian anti-A and anti-B titers were similar to
the titers observed in Japanese donors in 1986,
whereas the IgM and IgG antibody titers among the
Thais were similar to those observed in the Japanese
donors in 2001 and 1986, respectively. Our data
support the concept that anti-A and anti-B titers are
affected by environmental factors. Lao PDR is rapidly
developing and becoming more westernized. We plan
to continue our comparison of Laotian and Thai
antibody titers in the future.
Acknowledgment
We thank Dr. W.L. Gyure, Seton Hall University, for
his support in the preparation of this manuscript.
References
1. Garratty G. Problems associated with passively
transfused blood group alloantibodies. Am J Clin
Pathol 1998;109:769–77.
2. McManigal S, Sims KL. Intravascular hemolysis
secondary to ABO incompatible platelet products:
an underrecognized transfusion reaction. Am J
Clin Pathol 1999;111:202–6.
3. Larsson LG, Welsh VJ, Ladd DJ. Acute intravascular
hemolysis secondary to out-of-group platelet
transfusion.Transfusion 2000;40:902–6.
4. Issitt PD, Anstee DJ. Applied blood group serology.
4th ed. Durham: Montgomery Scientific Publications, 1998:188.
5. Toy PTCY, Reid ME, Papenfus L,Yeap HH, Black D.
Prevalence of ABO maternal-infant incompatibility
in Asians, Blacks, Hispanics and Caucasians. Vox
Sang 1988;54:181–3.
6. Redman M, Malde R, Contreras M. Comparison of
IgM and IgG anti-A and anti-B levels in Asian,
Caucasian and Negro Donors in the North West
Thames Region.Vox Sang 1990;59:89–91.
7. Brecher ME, ed. Method 2.11. Use of sulfhydryl
reagents to disperse autoagglutination. Technical
Manual, 15th ed. Bethesda: American Association
of Blood Banks 2005:744–5.
8. Schwartz J, Depalma H, Kapoor K, Hamilton T,
Grima K. Anti-A titers in group O single donor
platelets: to titer or not to titer (abstract).
Transfusion 2003;43(Suppl):115A.
I M M U N O H E M A T O L O G Y, V O L U M E 2 3 , N U M B E R 1 , 2 0 0 7
ABO antibody levels in Asians
9. Björkstén B, Naaber P, Sepp E, Mikelsaar M. The
intestinal microflora in allergic Estonian and
Swedish 2-year-old children. Clin Exp Allergy
1999;29:342–6.
10. Björkstén B, Sepp E, Julge K, Voor T, Mikelsaar M.
Allergy development and the intestinal microflora
during the first year of life. J Allergy Clin Immunol
2001;4:516–20.
11. McCurry J. Japanese people warned to curb
unhealthy lifestyles. Lancet 2004;363:1126.
Toshio Mazda, PhD, (corresponding author) and
Kenji Tadokoro, MD, PhD, Japanese Red Cross Central
Blood Institute, Tatsumi 2-1-67, Koto-ku, Tokyo 1358521, Japan; Ryuichi Yabe BS, Japanese Red Cross
West Tokyo Blood Center, Tokyo, Japan; Oytip
NaThalang, PhD, Department of Pathology,
Phramongkutklao College of Medicine, Bangkok,
Thailand; Te Thammavong, MD, Lao Red Cross Blood
Transfusion Centre, Vientiane, Lao PDR.
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 Immunohematology, P.O. Box 40325, Philadelphia, PA 19106.
I M M U N O H E M A T O L O G Y, V O L U M E 2 3 , N U M B E R 1 , 2 0 0 7
41
COMMUNICATION
Missing Scott Murphy, a platelet “maestro”
The most recent occasion I had to see Scott before his untimely death was at the 29th BEST meeting, held in
Rome in March 2005. Early spring in Rome is unique, as the local environment provides an ideal link between the
past—witnessed by its gorgeous monuments—and future dreams and expectations inspired by the Ponentino
breeze caressing the seven hills of Rome.
I met Scott as he exited his hotel on the via Veneto. I knew his disease had started a new aggressive phase, but
his optimistic smile overcame my concerns when he addressed me with a warm “Buongiorno!” The meeting was
scientifically very successful, as usual for all BEST gatherings, but most notable was Scott’s piano performance
during dinner, when he played four-hand Diabelli’s Sonatine with Georges Andreu, another musically gifted
transfusion medicine professional. This performance was the second in a series begun at an earlier BEST meeting
in Paris, where Scott played Mozart’s Twinkle,Twinkle Little Star with the devoted help of his wife Joanie playing
the triangle during a dinner at the Musée D’Orsay.
While everyone knows of Scott’s scientific accomplishments, it was his blend of science and art that truly
made him a platelet “maestro.” Our scientific interests crossed on several occasions, mainly surrounding the
European method to produce platelets for transfusion—the buffy-coat technique—as compared with the “plateletrich-plasma” procedure used in the United States. In particular, he was instrumental in studies developed by
Francesco Bertolini in his laboratory and ours, elucidating the role of acetate in platelet storage.
Finally, remembering Scott without mentioning his deep love and dedication to his wife, their children, and
grandchildren would be forgetting one of his most important qualities, his humanity. We, his friends, along with
the many patients who have and will continue to benefit from his work, will miss the scientist, the gentleman, and
the maestro.
Paolo Rebulla, MD
Director
Center of Transfusion Medicine, Cellular Therapy and Cryobiology
Ospedale Maggiore Policlinico, Mangiagalli e Regina Elena
Via Francesco Sforza 35
20122 Milano MI
Italy
42
I M M U N O H E M A T O L O G Y, V O L U M E 2 3 , N U M B E R 1 , 2 0 0 7
IN MEMORIAM
Katherine M. Beattie, BS, MT(ASCP)SBB 1922–2007
Kay Beattie received her BS in medical technology from Wayne State University
in Detroit, Michigan, in 1943 and began her career at Children’s Hospital in Detroit.
She became the supervisor of the Blood Bank at Children’s in 1954. In 1957, she
received her SBB certification, and in 1961, she became the co-director of the
Michigan Community Blood Center in Detroit. In 1975, she joined the Southeastern
Michigan region of the American Red Cross (ARC) Blood Services as director of the
reference laboratory and education, a position she held until she retired to North
Palm Beach, Florida, in 1988.
During her 45-year career, Kay was active at the local, state, and national levels,
and was honored often by her peers. She was a member of the Ontario Antibody
Club from 1966 to 1988; the Michigan Blood Council, as chairman of the Reference Laboratory Committee
from 1977 to 1988; the Invitational Conference of Investigative Immunohematologists from 1976 to 1988;
and the ARC Accreditation Reference Committee from 1984 and as chairman from 1987 to 1988. As a
member of the Michigan Association of Blood Banks (MABB) since its founding in 1956, Kay had served
as chairman of two committees and in all offices, including the presidency. She also served on the AABB
Committees on Education, Reference Laboratories, and Quality Control as vice chairman; Scientific Section
Coordinating Committee as secretary; as an inspector in the Inspection Program; and on the Committee
on Self-Testing and Review.
Among her awards from the AABB, she received the Ivor Dunsford Memorial Award in 1972; the
prestigious Emily Cooley Lectureship Award in 1980, which was given for the first time to a medical
technologist; and the Chapman-Franzmier Memorial Award in 1998. The MABB has honored Kay four
times, with the Merit Award in 1963, the Technical Award in 1971, the Founders Award in 1985, and the
Kay Beattie Lecture, a lecture given in her honor each year at the MABB meeting. Kay received the
Distinguished Alumni Award in 1982 from Wayne State University.
Kay published 44 scientific papers and was the co-editor of the ARC Immunohematology Methods
and Procedures Manual. Her final paper, on resolving ABO discrepancies, was published in 1993 in the
Methods Manual.
She was a superb reference technologist and had a phenomenal memory for patients and the
antibodies they made. Those of us who knew her will remember her as a mentor who always encouraged
us to try harder and do better. For example, she got us on committees and asked us to give talks. She
introduced us to famous people at meetings and could always explain something new in blood banking.
Kay was always a great friend with a ready joke. When she retired, she did not look back. She took her
golf clubs and went at golf like she went at antibodies. When she had it figured out, she was the women’s
club champion for many years. Her humor, intelligence, generosity, friendship, honesty, and jokes will be
greatly missed.
Delores Mallory
I M M U N O H E M A T O L O G Y, V O L U M E 2 3 , N U M B E R 1 , 2 0 0 7
43
ERRATUM
Vol. 22, No. 4, 2006; page 176
Review: molecular basis of MNS blood group variants
The author has informed the editors of Immunohematology that there is an error on page 176, second column,
second paragraph, third sentence. The sentence should read “Some of the classes of Miltenberger did not react
with anti-Mia but reacted with one or more of the other three specific antisera, e.g., GP.Hil (Mi.V) RBCs did not
react with anti-Mia but did react with anti-Hil.”
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without charge. Deadlines for receipt of these items are as follows:
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contact Cindy Flickinger, Managing Editor, 4 months in advance, by fax or e-mail at (215) 451-2538 or
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44
I M M U N O H E M A T O L O G Y, V O L U M E 2 3 , N U M B E R 1 , 2 0 0 7
ANNOUNCEMENTS
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, -Fy3, Wrb, -Xga, -CD99, -Dob, -H, -Ge2, -CD55, -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, -Kpa, and -Fya). Some are directly
agglutinating (Anti-M, -Wrb and -Rh17) and one has been humanized into the IgM isoform (Anti-Jsb). The antibodies
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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; or visit the Web site at http://www.nybloodcenter.org >research
>immunochemistry >current list of monoclonal antibodies available.
Meetings!
June 6–8
Blood Banks Association of New York State (BBANYS)
The 56th annual meeting of the Transfusion Medicine Symposium, Blood Banks Association of New York State
(BBANYS), cosponsored by the New York State Department of Health, will be held June 6 through 8, 2007, at the
Desmond Hotel and Conference Center in Albany, New York. For more information, contact Kevin Pelletier at
(518) 485-5341or meeting06@bbanys.org, or refer to the Web site at http://www.bbanys.org.
June 7–8
Heart of America Association of Blood Banks (HAABB)
The spring meeting of the Heart of America Association of Blood Banks (HAABB) will be held June 7 and 8, 2007,
at the Embassy Suites in Kansas City, Missouri. For more information, refer to the Web site at http://www.haabb.org.
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.
I M M U N O H E M A T O L O G Y, V O L U M E 2 3 , N U M B E R 1 , 2 0 0 7
45
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49
Immunohematology
JOURNAL OF BLOOD GROUP SEROLOGY AND EDUCATION
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I M M U N O H E M A T O L O G Y, V O L U M E 2 3 , N U M B E R 1 , 2 0 0 7
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
Supervisors of Reference Laboratories
Quality Assurance Officers
Why be an SBB?
Professional growth
Managers of Blood Centers
Research Scientists
Technical Representatives
Job placement
Job satisfaction
LIS Coordinators Educators
Consumer Safety Officers
Reference Lab Specialist
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
Fact #1: In recent years, the average SBB exam pass rate is only 38%.
Fact #2: In recent years, greater than 73% of people 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:
P ROGRAM
C ONTACT N AME
C ONTACT I NFORMATION
Walter Reed Army Medical Center
William Turcan
202-782-6210;
William.Turcan@NA.AMEDD.ARMY.MIL
Transfusion Medicine Center at Florida Blood Services
Marjorie Doty
727-568-5433 x 1514; mdoty@fbsblood.org
Univ. of Illinois at Chicago
Veronica Lewis
312-996-6721; veronica@uic.edu
Medical Center of Louisiana
Karen Kirkley
504-903-2466; kkirkl@lsuhsc.edu
NIH Clinical Center Department of Transfusion Medicine
Karen Byrne
301-496-8335; Kbyrne@mail.cc.nih.gov
Johns Hopkins Hospital
Christine Beritela
410-955-6580; cberite1@jhmi.edu
ARC-Central OH Region, OSU Medical Center
Joanne Kosanke
614-253-2740 x 2270; kosankej@usa.redcross.org
Hoxworth Blood Center/Univ. of Cincinnati Medical Center
Catherine Beiting
513-558-1275; Catherine.Beiting@uc.edu
Gulf Coast School of Blood Bank Technology
Clare Wong
713-791-6201; cwong@giveblood.org
Univ. of Texas SW Medical Center
Barbara Laird-Fryer
214-648-1785; Barbara.Fryer@UTSouthwestern.edu
Univ. of Texas Medical Branch at Galveston
Janet Vincent
409-772-4866; jvincent@utmb.edu
Univ. of Texas Health Science Center at San Antonio
Bonnie Fodermaier
Linda Smith
SBB Program: 210-358-2807,
bfodermaier@university-health-sys.com
MS Program: 210-567-8869; smithla@uthscsa.edu
Blood Center of Southeastern Wisconsin
Lynne LeMense
414-937-6403; Irlemense@bcsew.edu
Additional information can be found by visiting the following Web sites: www.ascp.org, www.caahep.org, and www.aabb.org
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Musser Blood Center
700 Spring Garden Street
Philadelphia, PA 19123-3594
(Place Label Here)
Immunohematology
The Journal of Blood Group Serology and Education
published quarterly by The American National Red Cross
2007 Subscription Application
■ United States—$30 per year*
■ Outside United States—$35 per year*
SBB/BB students free for 1 year with letter of validation
NAME ________________________________________________________________
DEGREE(S) ______________________
ORGANIZATION __________________________________________________________________________________________
DEPT./DIV.
____________________________________________________________________________________________
STREET __________________________________________________________________________________________________
CITY, STATE, ZIP CODE ____________________________________________________________________________________
■ Check if home address used
■ Check enclosed
■ VISA Acct. No. ______________________________________ Exp. Date: ________________
■ MasterCard Acct. No. ________________________________ Exp. Date: ________________
*Make check payable in U.S. dollars to THE AMERICAN RED CROSS. Mail this card in an envelope addressed to:
American Red Cross, Musser Blood Center, 700 Spring Garden Street, Philadelphia, PA 19123-3594. THIS FORM
MUST ACCOMPANY PAYMENT.
Immunohematology
Methods and Procedures
■ $70 United States*
■ $60 students and orders of 5 or more (United States)*
NAME
■ $85 foreign*
DEGREE(S)
ORGANIZATION
DEPT./DIV.
STREET
CITY
STATE
COUNTRY (FOREIGN)
ZIP CODE
■ Check enclosed.
■ VISA Acct. No. __________________________________________________ Exp. Date: __________
■ MasterCard Acct. No. ____________________________________________ Exp. Date: __________
*Make check payable in U.S. dollars to THE AMERICAN RED CROSS. Mail this card in an envelope addressed to:
American Red Cross, Musser Blood Center, 700 Spring Garden Street, Philadelphia, PA 19123-3594. THIS FORM
MUST ACCOMPANY PAYMENT.