REVIEW
Advances in alloimmune thrombocytopenia: perspectives on current
concepts of human platelet antigens, antibody detection strategies,
and genotyping
Tomoya Hayashi, Fumiya Hirayama
Japanese Red Cross Kinki Block Blood Centre, Osaka, Japan
Abstract
Alloimmunisation to platelets leads to the production
of antibodies against platelet antigens and consequently
to thrombocytopenia. Numerous molecules located on
the platelet surface are antigenic and induce immune-
mediated platelet destruction with symptoms that
can be serious. Human platelet antigens (HPA) cause
thrombocytopenias, such as neonatal alloimmune
thrombocytopenia, post-transfusion purpura, and platelet
transfusion refractoriness. Thirty-four HPA are classified
into 28 systems. Assays to identify HPA and anti-HPA
antibodies are critically important for preventing
and treating thrombocytopenia caused by anti-HPA
antibodies. Significant progress in furthering our
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understanding of HPA has been made in the last decade:
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new HPA have been discovered, antibody-detection
methods have improved, and new genotyping methods
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have been developed. We review these advances and
discuss issues that remain to be resolved as well as
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future prospects for preventing and treating immune
thrombocytopenia.
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Introduction
Immune and non-immune mechanisms can
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decrease the number of platelets, leading to bleeding
manifestations that range from petechiae and simple
bruising to intracranial haemorrhage and death1. Platelets
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interact with coagulation factors to arrest haemorrhage
and support vascular integrity2,3. At sites of vascular
injury, platelets change shape, adhere to the site, and
secrete cytokines that are essential for tissue repair1,4,5.
Platelets are indispensable for maintaining vascular
integrity. Molecules expressed by platelets that are
involved in arresting haemorrhage include integrin
αIIbβ3, GPIb/IX, and integrin α2β16-10.
The antigens that are recognised by alloantibodies
have been categorised by the Platelet Nomenclature
Committee, and 34 human platelet antigens (HPA)
have been defined11,12. Antibodies against HPA are
regarded as the principal cause of various reactions
elicited by platelet transfusion, such as platelet
transfusion refractoriness (PTR) and post-transfusion
purpura (PTP)1,13. Antibodies against HPA cause foetal/
neonatal alloimmune thrombocytopenia (FNAIT)14.
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HPA antibodies were detected after a peripheral blood
stem cell transplant between sisters with identical
HLA-A, -B, -DR, -DQ, -DP and ABO phenotypes
which was followed by persistent, severe isolated
thrombocytopenia resistant to platelet transfusions15.
The detection of these antibodies is required to diagnose,
treat, and prevent these disorders. It is also important
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to distinguish between thrombocytopenia induced by
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alloantibodies from drug-induced disease16.
The platelet surface membrane contains antigenic
molecules other than HPA, including ABO blood type
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antigens17, human leucocyte antigens (HLA)18, and the
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Naka antigen localised on CD3619. HLA alloantibodies
are the most important cause of PTR18; therefore, careful
screening for HLA antibodies is required even when
other antibodies are identified. Because CD36 deficiency
is rare in Caucasians but frequent in Asians and Africans,
Nak a antibodies are important for diagnosing and
treating thrombocytopenia in the latter populations.
Early diagnosis is essential to prevent severe disease in
a timely and effective manner20.
New HPA were discovered in the last decade, and they
have been implicated in immune thrombocytopenia11;
strategies for detecting antibodies and genotyping are
improving. The primary purpose of this review is to
describe the molecular properties of HPA and related
molecules (other than HLA and ABO) and the antibody-
detection systems that have been developed to enhance
the sensitivity and specificity of HPA detection. We
consider genotyping methods as well, which can be
performed quickly using multiple analytical techniques.
We conclude by discussing important issues that must
be resolved and offer our perspectives for the future
regarding the prevention and treatment of immune
thrombocytopenia.
Polymorphisms and functions of human platelet
antigens and CD36
According to the Immuno Polymorphism Database
(IPD - www.ebi.ac.uk/ipd/hpa/), the number of HPA has
reached 28 systems11. The variety of HPA is generated
by the substitution of a single amino acid residue and
by deletion of one amino acid residue from platelet
glycoproteins (Figure 1)11,12,21-23. Six of the systems
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Current concepts of human platelet antigens

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Figure 1 - Human platelet antigens (HPA) polymorphisms.

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This figure is constructed using data from the IPD web site (http://www.ebi.ac.uk/ipd/hpa/table2.html) and United States National
Center for Biotechnology Information (NCBI). HPA are located on the following six molecules, GPIIIa (integrin β3), GPIIb (integrin
α2b), GPIa (integrin α2), CD109, GPIbα, and GPIbβ, and are encoded by ITGB3, ITGA2B, ITGA2, CD109, GP1BA, and GP1BB,
respectively. The gene symbols are those approved by the Human Genome Organisation (HUGO) Gene Nomenclature Committee

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(http://www.genenames.org). Sequences encoding HPA within the same exons are indicated by the same colours. Nucleotide variations:
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nucleotide numbers are taken from the reference sequence in the NCBI database, which may differ from those given in the original
publication describing the mutation. Nucleotide and protein variations are shown as changes from the more common (a) to the less
common (b) forms. The number of amino acid variations is derived from the precursor protein, and the numbers in parenthesis indicate
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amino acid residue number in the mature protein. The definitions of symbols (#, s, §, , ‡, *, ¶ and †) are described under each residue.
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are biallelic (HPA-1, -2, -3, -4, -5, and -15), and are present in patients with Glanzmann's thrombasthenia,
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others include 34 platelet-specific alloantigens defined which is an autosomal recessive disorder caused by
according to the rules of the ISBT Platelet Working mutation of the genes encoding GPIIb or GPIIIa or is
Party. The allelic frequencies of HPA differ among them, acquired as an autoimmune disorder that leads to an
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and racial differences are numerous. For the six biallelic increased tendency to bleed26.
systems, the most frequent HPA are defined as "a" alleles Most HPA are localised to GPIIb and GPIIIa
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or "wild-type." All alloantigen systems comprise genes (Figure 1). GPIIIa and GPIIb comprise 16 and seven
encoding only six proteins. Among them, GPIIb [integrin HPA systems, respectively11,12. There are several genetic
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alpha subunit IIb (IIb), CD41] and GPIIIa [integrin beta hot-spots in which the mutations that encode two to four
subunit 3 (β3), CD61] form the GPIIb/GPIIIa (GPIIbIIIa, HPA are present in the same exons (Figure 1).
integrin IIbβ3) complex and GPIb GPIb and GPIX Although all HPA present on GPIIbIIIa are associated
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and GPV form the GPIb-IX-V complex. GPIIIa also
forms a complex with αV and is expressed by platelets
and other cells including osteoclasts and endothelium
cells24. GPIa (α2) and GPIIa (β) form the GPIaGPIIa
(GPIaIIa, integrin α2β) complex.
GPIIb/GPIIIa (integrin αIIbβ3, the fibrinogen
receptor)
GPIIb and GPIIIa form the GPIIbIIIa complex,
although GPIIIa forms a complex with αV as well24.
The GPIIbIIIa complex is the most abundant molecule
on the surface of platelets. At the site of endothelial
damage, GPIIbIIIa is activated and plays a central role
in the formation of an occlusive thrombus; it binds
to fibrinogen, von Willebrand factor, fibronectin, and
vitronectin, which are required for haemostasis25,26.
Functional abnormalities and deficiency of GPIIbIIIa
with FNAIT, HPA-1a is strongly associated with FNAIT,
particularly in Caucasians , and antibodies against HPA-
4b and HPA-21b are present in Asians27-29. Antibodies
against HPA-1a, HPA-1b, and HPA-3b cause PTR, and
antibodies against HPA-1a, HPA-1b, HPA-3a, HPA-
3b, and HPA-4a cause PTP30-33. PTR can be caused by
isoantibodies against GPIIbIIIa in patients with type 1
Glanzmann's thrombasthenia34.
GPIb
GPIbα (CD42b) and GPIbβ (CD42c) bind covalently
to form a complex and associate non-covalently with
GPIX (CD42a). Dimers of these three molecules further
associate with the GPV (CD42d) molecule to form
GPIbIXV (CD42). CD42 (approximately 25,000 copies
of GPIb/IX and 12,000 copies of GPV molecules per
cell) is the second most abundant molecule on the platelet

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surface. The levels of the glycoprotein Ib-IX-V complex
are higher in neonatal cord blood than in adult blood.
The binding of GPIbIXV to von Willebrand
factor mediates platelet adhesion under fast flow
conditions and plays an important role in maintaining
haemostasis at sites of vascular injury. GPIbIXV binds
to the complement receptor of macrophages Mac-1
(macrophage-1 antigen or integrin αMβ2), which
comprises CD11b (integrin αM) and CD18 (integrin
β2), and mediates the phagocytosis of platelets,
indicating that GPIbIXV plays an important role in
platelet turnover35. Quantitative or qualitative defects of
GPIbIXV expressed by platelets occur in patients with
Bernard-Soulier syndrome, which is characterised by no
platelet agglutination in response to ristocetin, prolonged
bleeding and abnormal platelet size and turnover36.
The HPA-2 and HPA-12 systems are located on
the alpha and beta chains of GPIb, respectively11,12.
Antibodies against HPA-2b and HPA-12bw are
associated with FNAIT37,38, and the former are sometimes
detected in the patients with PTR39. Combinations
of antibodies against HPA-1b and HPA-2a induced
moderate thrombocytopenia in a patient with FNAIT40.
Isoantibodies against GPIb can cause PTR in patients
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with Bernard-Soulier syndrome.
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GPIa
GPIa (CD49b) associates with GPIIa (CD29) to form
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integrin α2β1 (GPIaIIa) complex, known as very late
antigen (VLA-2). GPIaIIa is expressed by monocytes,
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T cells, B cells, NK cells, and platelets41. Platelet
GPIaIIa is involved in adhesion at low flow rates by
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binding to collagen and participates in cell surface-
mediated signalling leading to GPIIbIIIa activation.
Approximately 800-2,800 GPIaIIa molecules are present
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on the platelet surface.
Four HPA systems (HPA-5, HPA-13, HPA-18,
HPA-25) are located on GPIa 11,12. The HPA-13bw
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mutation is unusual in that it alters the function of GPIa,
and platelets from HPA-13bw-positive individuals
have a reduced response to collagen, as revealed by
aggregation studies, and a reduced ability to spread on
a collagen surface. Antibodies against HPA-5a, HPA-5b,
HPA-13bw, HPA-18bw, and HPA-25bw cause FNAIT,
and antibodies against HPA-5a are not limited to patients
of a particular race42-44. Antibodies against HPA-5a and
HPA-5b are present in patients with PTR, and the former
causes PTP as well.
CD109
GPI-anchored alpha 2 macroglobulin-related protein
(CD109, 150 kDa TGF--1-binding protein, C3 and PZP-
like alpha-2-macroglobulin domain-containing protein
7) links to the platelet surface through a GPI anchor45.
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Hayashi T, Hirayama F
CD109 is expressed as a 205-kDa glycoprotein, which
is cleaved by furin (furinase) in the Golgi apparatus into
180-kDa and 25-kDa subunits46. CD109 is associated
with the growth of carcinomas and keratinocytes
through the regulation of TGF- signaling46. CD109 may
play a role in the interactions of T cells with antigen-
presenting cells or in T- and B-cell interactions46. The
amounts of CD109 on the platelet surface differ by as
much as a factor of >100 among individuals. However,
the expression levels of CD109 are generally lower
than those of HPA-associated glycoproteins47. Surface
expression of CD109 on platelets is decreased by cooling
and freezing47. Although the precise function of platelet
CD109 is unknown, its identification as a member
of thioester-containing proteins suggests that it may
mediate covalent binding of cells to substrates as well as
intercellular interactions46. Further studies are required
to define the function of platelet CD109 in detail.
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CD109 is a component of only HPA-1511,12. The
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detection of antibodies against CD109 is hampered,
because platelets express low levels of CD109 on
their surface and CD109 is unstable when platelets are
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cooled or frozen. Panels of platelets must, therefore,
be carefully selected according to CD109 levels and
stored under appropriate conditions. The development
of new methods for detecting anti-HPA antibodies may
accelerate our understanding of their importance48,49.
This subject is considered in the section "Assays for
detecting HPA antibodies". Anti-HPA-15 antibodies are
detected in patients receiving multiple transfusions and
mothers with FNAIT48,49. Although antibodies against
HPA-15a and HPA-15b are associated with FNAIT,
they are usually detected together with anti-HLA
antibodies49. The incidence of HPA-15 alloimmunisation
is significantly higher in patients who undergo multiple
transfusions than in mothers with FNAIT48.
CD36
CD36 (also called GPIIIB, PAS IV, PAS-4, fatty acid
translocase, glycoprotein IIIb, platelet glycoprotein 4,
platelet glycoprotein IV, or Naka antigen) is expressed
on various cells, including platelets, and is not an
HPA component. Because CD36 mediates immune
thrombocytopenia50, it is relevant to this review. CD36
is one of four major glycoproteins on the platelet surface
and serves as a receptor in cells other than platelets51.
CD36 binds diverse molecules, including collagen,
anionic phospholipids, oxidized low density lipoproteins,
and thrombospondin. It directly mediates cytoadherence
of Plasmodium falciparum to erythrocytes, binds long-
chain fatty acids, and may regulate or directly mediate
the transport of fatty acids. CD36 is, therefore, attracting
considerable attention from researchers in the fields of
obesity and diabetes.
Blood Transfus 2015; 13; 380-90 DOI 10.2450/2015.0275-14
Current concepts of human platelet antigens
The expression or lack of expression of CD36
among cell types, particularly by blood cells, is
noteworthy (Table I)52. Briefly, in patients with type I
CD36 deficiency, CD36 is not expressed by platelets or
monocytes, whereas in patients with type II deficiency
only platelets fail to express CD36. Type II deficiency
comprises types 2a and 2b, and CD36 deficiency
restricted to platelets is designated type 2a. When CD36
is absent from erythroblasts, the phenotype is classified
as type 2b53. Multiple alternatively spliced transcripts
encoding CD36 are present in various tissues and may
account for its complex pattern of expression.
Certain mutations in CD36 cause type I deficiency;
therefore, the antibody against CD36 is defined as an
isoantibody, not as an alloantibody. CD36 is expressed
in almost 100% of white Europeans and is not detectably
expressed by 2% of sub-Saharan Africans and 10% of
Asians54-56. Because anti-CD36 antibodies target diverse
tissues, patients display a broad range of symptoms.
For example, antibodies that react with platelets may
lead to immune thrombocytopenia, those that react
with platelets and monocytes lead to transfusion-related
acute lung injury as well as life-threatening transfusion
reactions, and those that react with erythrocytes lead to
hydrops foetalis57.
Assays for detecting antibodies against human
platelet antigens
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Several methods that use platelets as target cells
are available for the detection of antibodies against
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HPA, such as the monoclonal antibody-specific
immobilisation of platelet antigens (MAIPA) assay58, the
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platelet-antigen capture (PAC) assay59, the mixed passive
haemagglutination test (MPHA)60, flow cytometric
analysis61, a modified antigen-capture enzyme-linked
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immunosorbent assay (ELISA) 62 and a Luminex
bead assay63,64. The properties of these methods are
summarised in Table II.
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Anti-HLA antibodies create problems for "whole
platelet" antibody detection methods such as MPHA
and flow cytometric analysis but not for glycoprotein-
specific assays such as MAIPA. Because the MAIPA
assay is highly sensitive and specific, it is considered
the gold standard65,66. These tests, including the MAIPA,
do have some disadvantages. First, the preparation of
Table I - Types and frequencies of CD36 deficiency.
Deficiency Expression
type
Platelets Monocytes RBC progenitors
I absent absent
II absent
#depending on the presence or absence on RBC progenitors; the deficiency is classified into type II-a and type II-b, respectively.
Blood Transfus 2015; 13; 380-90 DOI 10.2450/2015.0275-14
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a well-characterised panel of platelets is necessary for
detecting antibodies against HPA. Unfortunately, it is
difficult to prepare such a panel. Second, the serum
antibodies and the monoclonal antibody may compete67.
This risk can be limited to some extent by using multiple
mouse monoclonal antibodies reactive with different
epitopes; however, developing monoclonal antibodies
is expensive. To overcome these problems, new assay
systems were developed that substitute target platelets
with transfected cell lines or recombinant peptides
(Table III).
Panel of transfected cell lines
Techniques using cells transfected with cDNA
encoding specific HPA typically employ CHO and 293T
cells and serve as alternatives to unavailable platelet
panels. Recently, we established various K562 cell lines
such as Hayashi's platelet-associated (HP) cells that
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express various HPA, do not express HLA, HNA, or
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HPA, and show low non-specific reactivity with human
sera. These test systems are highly specific and sensitive
for detecting antibodies against HPA68-72. Briefly, the cell
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lines express one of the molecules as follows: CD36,
wild-type GPIIb/GPIIIa (HPA-1a) as well as HPA-1b,
-3b, -4b, -5b, -6b, -7b, -7 variant, -13b, -15a, -15b,
-18b, or HPA-21b. We recently established cell lines
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expressing wild-type GPIb and GPIb (HPA-2a and
HPA-12a) and HPA-2b (unpublished data).
These cell lines overcome the difficulty of preparing
a platelet panel. They also detect anti-HPA antibodies in
the presence of anti-HLA antibodies. More recently, we
developed transfected cell lines that employ an antigen-
capture assay system for detecting antibodies against
CD36 without using monoclonal antibodies against
CD3673. Since this cell line-dependent system does not
need mouse antibodies, it can avoid binding competition
between human and mouse antibodies. Moreover, the
receiver operating characteristic curve is superior to
that of the MAIPA system, and the transfectants allow
monoclonal antibodies against CD36 to be omitted.
We provided HP-15a and HP-15b cells to the ISBT
Platelet Immunology Working Party. They recently
reported the usefulness of paraformaldehyde-fixed
HP-15 cells at the meeting of the ISBT held in June
2014, Seoul, Korea. In addition, we have provided some
Frequency of deficiency
Endothelial cells Caucasian Sub-Sahara Asian
African
absent absent <0.3% 2.50% 0.5-1%
# 3-10%
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 Hayashi T, Hirayama F

Table II - Summary of the properties of antibody detection assays.

Whole platelet methods

Method Principle Advantage Disadvantage Additional remarks

MPHA The test serum is added onto a microplate coated with platelet Simple procedure Possible insufficient High-titre HLA
SPRCA extracts and indicator sheep red blood cells coated with anti- Low cost sensitivity and antibodies hamper
human IgG are added. The presence of the human antibody is Detector not required specificity the detection of HPA
judged by agglutination patterns. antibodies.
PSIFT The patient's serum is added to a platelet suspension and an Simple procedure Possible insufficient HLA antibodies
anti-human IgG conjugated to a fluorescent dye is added. NonHPA platelet sensitivity and hamper the detection
Fluorescence emission is analysed using a flow cytometer or antibodies are specificity of HPA antibodies.
fluorescence plate reader. detectable as well instrumentation
(FACS) is expensive

Glycoprotein specific methods

Method Principle Advantage Disadvantage Additional remarks

MAIPA Immune complexes composed of the target HPA, human-specific High sensitivity and High cost Need to be careful
PAC antibody, and mouse monoclonal antibody are captured by anti- high specificity Complicated procedure about the antibody
mouse IgG coated on a plate. The human antibody is detected competition
using enzyme-linked anti-human IgG and a colorimetric detector.

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MACE Monoclonal antibodies coated onto a solid phase are used to capture High specificity High cost Need to be careful
the target HPA. The human antibody is detected using enzyme- Complicated procedure about the antibody
linked anti-human IgG and a colorimetric detector. competition
ICFA Microarray beads are separately coupled with recombinant GP High specificity High cost Need to be careful

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fragments or monoclonal antibodies specific for HPA and HLA. Simultaneous analysis
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Luminex xMAP technology adapts MAIPA onto the Luminex of HLA and HPA instrumentation competition when
platform by using a secondary antibody conjugated to a fluorescent antibodies in one well (FACS or Luminex) using antibody-
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dye. coupled microspheres.
SPR Measures binding of antibody onto an antigen-coated planar metal High sensitivity. Requires highly Since the sensitivity is
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surface. Bound molecules change the local index of refraction, Detects low-avidity purified antigens and so high that the clinical
which is generated by the antigen-antibody interaction that changes antibodies. Washing immunoglobulins. significance of the
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the resonance of the surface plasmon waves. and labelling are not Requires specialised detected antibodies are
required equipment. addressed by in-vivo
experiments, such as
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those using the NOD/

SCID mouse.
HP-IPA Immune complexes composed of the target HPA, human specific High sensitivity High cost
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antibody, and enzyme-linked mouse-anti-human IgG are captured Eliminates antibody Complicated procedure
by anti-mouse IgG coated on a plate, and the human antibody is competition Requires a cell panel
detected after adding the substrate.
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MPHA: mixed passive haemagglutination61; SPRCA: solid phase red cell adherence61; PSIFT: platelet suspension immuno-fluorescence test58; MAIPA:
monoclonal antibody-specific immobilisation of platelet antigens; PAC: platelet antigen capture immunoassays60; MACE: modified antigen capture ELISA62;
ICFA: immuno-complex capture fluorescence analysis63,64; SPR: surface plasmon resonance75,76; HP-IPA: HP cell-based monoclonal antibody-independent
Immobilisation of Platelet Antigen73.
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Table III - Platelet-independent methods for detecting anti-HPA antibodies.

Sources of antigens Glycoproteins Established molecules (peptides) Detected antibodies Applicable methods
(reported)
HP cell lines GPIIb and GPIIIa Wild type, HPA-1b, HPA-3b, HPA- HPA-1a, 3a*, 4b, 6b, MAIPA, MACE, IFT,
(integrin αIIb and β3) 4b, HPA-6b, HPA-7b, HPA-7variant, 7variant, 21b HP-IPA, ICFA (xMAP)
(CD41 and CD61) HPA-21b
GPIa HPA-5b, HPA-13b, HPA-18b HPA-5a, 5b MAIPA, MACE, IFT, ICFA
(integrin α2, CD49b) (xMAP)
GPIbα (CD42b) HPA-2a, HPA-2b HPA-2b MAIPA, MACE, IFT
CD109 HPA-15a, HPA-15b HPA-15a, 15b MAIPA, MACE, IFT, ICFA
(xMAP)
CD36 CD36 Naka MAIPA, MACE, IFT,
HP-IPA, ICFA (xMAP)
Recombinant peptide GPIIIa ∆β3 peptide (wild type), Super rare HPA-1a, 1b, 4a, 4b, 6bw, ICFA (xMAP), SPR
peptide, 1a, 1b, 4b, 8b 7bw, 8bw, 11bw*, 16bw
Aptamer non glycoprotein Specific for HPA-1a antibody HPA-1a MPHA, SPRCA, ELISA
*Some antibodies are not detected. MAIPA: monoclonal-specific immobilization of platelet antigens; MACE: modified antigen capture ELISA; IFT: immuno
fluorescence test; HP-IPA: HP cell-based monoclonal antibody-independent Immobilisation of Platelet Antigen; ICFA (xMAP): immuno-complex capture
fluorescence analysis; SPR: surface plasmon resonance; MPHA:mixed passive hemagglutination; SPRCA: solid-phase red cell adherence assay.

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Current concepts of human platelet antigens
HP cell lines, including HP-15a and HP-15b, to several
blood centres.
Recombinant peptide techniques and highly sensitive
methods
Another approach that does not require platelet
panels involves the use of HPA peptides. Stafford
et al.22 published unique methods to detect integrin
β3-associated anti-HPA antibodies using recombinant
integrin β3 peptides that contained seven rare HPA,
termed SuperRare peptides that are suitable for detecting
anti-HPA antibodies. However, because the antigens
used in peptide techniques do not have a natural three-
dimensional structure or carbohydrate chains, this
system still requires careful evaluation using larger
numbers of positive control samples in the future74.
Although peptides are often used in surface plasmon
resonance assays for detecting antibodies, purified
glycoproteins have been used to detect anti-HPA
antibodies. Peterson et al.75 and Blackhoul et al.76
reported highly sensitive systems that detect low-avidity
anti-HPA-1a antibodies using GPIIbIIIa purified from
platelets.
A peptide aptamer, which mimics the HPA-1a
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antigen, is recognised by HPA-1a alloantibodies with
very high sensitivity77. Because aptamers are usually
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designed according to structure or affinity, their specific
antigenicity limits applying them to each HPA. Future
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studies are, therefore, required to develop assays using
aptamers.
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Limitations of serological systems for detecting
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antibodies against human platelet antigens
Standard control sera
Although positive control antibodies are important
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for every method, the availability of antisera is limiting.
Methods are being developed using a patient's B cells to
continuously produce specific anti-HPA antibodies. In
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the meantime, collaboration among laboratories expert
in platelet immunology is required to share the limited
amount of control antisera.
Anti-HPA-3a antibodies
Certain detection systems fail to detect anti-HPA-3a
antibodies. Harrison et al. reported that some anti-HPA-
3a antibodies are detected only in whole platelets but not
when the platelet lysate is used78. Kataoka et al. reported
that one of the anti-HPA-3b antibodies was detected
only using fresh but not fixed platelets79. Socher et al.
reported that some anti-HPA-3a antibodies were detected
only using fresh but not stored platelets, and other
anti-HPA-3a antibodies were not detected by western
blotting analysis of recombinant GPIIb and GPIIIa
expressed by CHO cells80. We found that some anti-
Blood Transfus 2015; 13; 380-90 DOI 10.2450/2015.0275-14
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HPA-3a antibodies were detected only using MPHA,
but not with recombinant GPIIbIIIa expressed by K562
cells71. Furthermore, Leon et al. reported that certain
anti-HPA-3a antibodies were not detected using the
HPA-3a peptide81. Taken together, these findings indicate
that the methods for detecting antibodies against HPA-3a
vary in sensitivity and that using only one method may
increase the risk of not detecting them.
Allen et al. indicated that detection of anti-HPA
antibodies is influenced by cation-chelating compounds
such as EDTA, and suggested that the results reported
by different laboratories might depend on the buffer,
antibody concentrations, or both 82. We, therefore,
suggest that it is important to consider the advantages
and disadvantages of each method and to use them in
combination.
Causal relationship between anti-human platelet
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antigen antibodies and thrombocytopenia.
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At present, low affinity anti-HPA antibodies can
be detected using highly sensitive methods such as
surface plasmon resonance. However, detection of
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anti-HPA antibodies does not necessarily indicate that
the antibodies cause thrombocytopenia. Clinically, it is
obviously important to determine whether the antibodies
cause thrombocytopenia. Some fascinating reports
describe that a very low titre of antibodies can decrease
platelet counts in animal models75. Indeed, there is
evidence that antibodies may cause thrombocytopenia
even if the antibody level is lower than the detection
limit of conventional methods; however, only a few
laboratories are capable of performing highly sensitive
assays.
Because none of the current tests is perfect,
depending on the circumstances of each laboratory,
the tests should be performed together according to
their characteristics summarized in Table II. Moreover,
genotype information would be useful for generating
reliable data, as described in the next section.
Importance of genotyping for patients, their
parents, and the platelet panel
Genotyping patients, their parents, or both
support antibody testing to diagnose alloimmune
thrombocytopenia. Although HPA phenotyping is
important, reference sera are precious and limited in
number, so that HPA phenotyping is sometimes difficult.
In contrast, genetic technologies provide powerful tools
to identify HPA. Techniques using genomic DNA for
HPA genotyping are relatively easy to perform, because
genomic DNA can be extracted from any cell type,
including leucocytes, except from erythrocytes and
platelets. Moreover, commercial materials and reagents
are available for the analysis of single nucleotide
385
 Hayashi T, Hirayama F

polymorphisms. Thus, technological innovation paves
the way to platelet typing of the foetus in suspected cases
of FNAIT and enables matched platelets to be found to
prevent PTR. Although determining the specificities
of antibodies is important in either case, the requisites
vary slightly between them. In the former case, platelet
typing predicts disease and indicates the requirement
for medical attention, while in the latter case it is used
to provide matched platelet products for treatment.
Further, allelic frequency is important to predict the
incidence of incompatibility between an infant and
mother as well as between patients and platelet donors.
Various genotyping methods work in these situations.
Genotyping technologies are summarized in Table IV.
Numerous techniques available for determining
single nucleotide polymorphisms can be applied
to HPA genotyping83,84. HPA genotypes are widely
determined using sequence-specific primer-polymerase
with A-T base pairs. HRM is, therefore, useful for
distinguishing HPA-b alleles from wild-type ones.
Furthermore, because the HRM method simultaneously
detects one to four polymorphisms, it is advantageous
for determining the frequency of rare HPA86; however
an expensive detector is required. The fluoPCR-reverse
sequence-specific oligonucleotide probe technique,
using a fluorescent hybridisation probe, simultaneously
determines several polymorphisms90. Many multiplex
PCR techniques are commercially available.
A limitation of genotyping techniques is that if the
target sequence is located in a genetic hot-spot, the
results may be greatly affected and it might be difficult to
design an appropriate primer and probe91. Thus, none of
the methods described above is perfect, and the method
of choice will depend on the purpose of the analysis
and the identity of the HPA. Use of two genotyping
techniques using different primer sets can limit this risk.

l
chain reaction (PCR-SSP), denaturing gradient gel

Sr
electrophoresis (PCR-DGGE)85, and restriction fragment Pitfalls of genotyping
length polymorphism (PCR-RFLP) techniques. Although genotyping is a powerful tool for definitive
However, these methods are generally specialised to diagnosis of FNAIT, genotype does not always correlate

i
determine one polymorphism in a single test and are
therefore applicable in the analysis of patients with
v iz
with phenotype, as in a propositus heterozygous for type
I Glanzmann's thrombathenia or Bernard-syndromes.
FNAIT. In contrast, a high-throughput method is A propositus can be genotypically heterozygous for
required for donor screening and frequency analysis. a particular HPA but phenotypically homozygous,
er

For example, the high-resolution melting (HRM) because one of the allele is not expressed on the platelet
method is cost-effective. Several groups reported that surface36,92.
S

HRM is useful for HPA genotyping of a large donor

pool, for determining genotype frequency, or both86-89. Future study
TI

HRM determines the melting point of DNA, which is Predicting the severity of thrombocytopenia is likely
influenced by the abundance of C-G base pairs. Most important for efficacious treatment of patients with
M

HPA polymorphisms are generated by replacing C-G FNAIT, autoimmune thrombocytopenia, or alloimmune

Table IV - Genotyping methods.

SI

Method Principle Advantage Disadvantage Additional

Remarks
PCR-SSP83 Sequence-specific primers amplify allele-specific DNA. Low cost Poor resolution
©

of amplicons
PCR-DGGE 84 The target sequence is amplified and separated using Low cost Lengthy procedure Ambiguous data
denaturing polyacrylamide gel electrophoresis. It
distinguishes mobility shifts of type a and b caused by a
change in the conformation of single-stranded amplicons.
PCR-RFLP83,85 Digestion using restriction enzymes High specificity and Lengthy procedure Occasionally leaves
low cost ambiguities
HRM86,88,89 The melting temperatures of DNA depend on their base Simple and quick. Expensive detector Occasionally
composition. PCR is performed using DNA intercalating Applicable for high- amplicons with
dyes, such as SYBR® green. As the sample is heated and the throughput analysis different sequences
two strands of the DNA separate (melt), the concentration melt at the same
of double-stranded DNA decreases, reducing fluorescence. temperature
The instrument generates a melting curve.
Real-time Uses allele-specific primers to amplify allele-specific DNA Simple and quick. Expensive detector
PCR-SSP85,87,88,89 and intercalating agents such as SYBR® green are used. Applicable for high-
throughput analysis
PCR-SSP: sequence specific primer; PCR-DGGE: denaturing gradient gel electrophoresis; PCR-RFLP: restriction fragment length polymorphism;
HRM: high-resolution melting method; SYBR® green: N',N'-dimethyl-N-[4-[(E)-(3-methyl-1,3-benzothiazol-2-ylidene)methyl]-1-phenylquinolin-1-ium-
2-yl]-N-propylpropane-1,3-diamine: SYBR Green is a cyanine dye and preferentially binds to double-stranded DNA.

Blood Transfus 2015; 13; 380-90 DOI 10.2450/2015.0275-14

386

All rights reserved - For personal use only

No other uses without permission
Current concepts of human platelet antigens
thrombocytopenia93. Alloantibodies destroy platelets
and eliminate them from the circulation through
antibody-induced immune-mediated phagocytosis and
activation of complement59,93-100. The characteristics of
the alloantibodies and the titre of IgG may, therefore,
contribute to the severity of symptoms.
Immunoglobulin subclasses
The ability to activate complement and the binding
affinity for HPA antibodies of the Fc receptor on
phagocytic cells differ depending on the subclasses of
antibodies. Data are available on the immunoglobulin
subclasses of HPA antibodies59,93. Although evidence
indicates that IgG2 is not as effective as IgG1 and
IgG3 for inducing phagocytosis and complement-
mediated cell lysis, Kelsch pointed out that IgG2
causes platelet dysfunction and leads to clinically
significant bleeding even when platelet counts are
normal 59 . The IgG3 subclass contributes to the
pathogenesis of PTP93. Because multiple subclasses
are often detected in patients with thrombocytopenia93
and the causality of immunisation seems to have a
low impact on the repartition of subclasses, subclass
analysis is not considered clinically relevant94; however,
v iz
immunoglobulin subclasses should be kept in mind.
er
Glycosylation pattern of immunoglobulin heavy chain
Immunoglobulin molecules comprise heavy and
S
light chains. The Fab and Fc domains of antibodies are
important for recognition of antigen and binding to Fc
TI
receptors, respectively. Furthermore, the glycosylation
pattern of the Fc domain affects its binding affinity for
M
Fc receptors. Structural studies of Fc domains show
that N297 glycans stabilise an "open" Fc conformation
recognised by Fc receptors95. Okazaki showed that
SI
depleting fucose from the oligosaccharide moiety of
human IgG1 increases the moiety's affinity for the Fc
receptor. Other studies show that modulation of core-
©
fucosylation of IgG may exert a profound effect on
disease severity and prognosis of rheumatic diseases96
as well as the antibody-dependent cytotoxicity of
alloantibodies against HPA97.
This concept has been exploited in the manufacture
of recombinant medical products and antibody
drugs. Although still at the level of basic research,
in the field of platelet transfusion, Bakchoul et al.
reported that a deglycosylated monoclonal mouse anti-
HPA-1a antibody prevented anti-HPA-1a-mediated
platelet destruction in a mouse model 98. Further,
Ghevaert et al. constructed a recombinant high-affinity
anti-HPA-1a IgG antibody, B2G1Δnab, comprising
the variable region of a high-affinity antibody against
HPA-1a and constant region modified to minimise Fc
receptor-dependent platelet destruction. B2G1Δnab
Blood Transfus 2015; 13; 380-90 DOI 10.2450/2015.0275-14
All rights reserved - For personal use only
No other uses without permission
inhibits the binding of maternal anti-HPA-1a antibodies
harvested from patients with FNAIT to a patient's
platelets and abrogates monocyte responses to anti-HPA-
1a-coated platelets in vitro. Furthermore, the authors
reported that in mice and humans, B2G1Δnab prevents
the removal from the circulation of platelets that express
HPA-1a by destructive HPA-1a antibodies99,100.
Other antigen systems
Although many high-sensitivity methods for
detecting antibodies are available, as described in
this review, there are patients with thrombocytopenia
with no detectable anti-HPA antibodies. For example,
approximately 50% of patients with FNAIT in
Japan do not have detectable anti-HPA antibodies.
Alloantibody-dependent and alloantibody-independent
thrombocytopenia may occur, including drug-induced
and autoantibody-induced thrombocytopenia. In patients
l
with antibody-dependent thrombocytopenia, there
Sr
should be antibodies against already-known epitopes
present on the six molecules GPIIb, GPIIIa, GPIb,
GPIb, GPIa, or CD109. Otherwise, antibodies against
i
new epitopes on the six molecules or against molecules
other than these six molecules should be present. A large
number of molecules are expressed by platelets as well
as by other blood cells. Antibodies to these molecules
may cause thrombocytopenia. We should, therefore,
pay attention to non-HPA molecules that are expressed
by platelets. When thombocytopenias of non-immune
origin are discarded by clinical or other diagnosis, we
must assume that a causative antibody is present that
eludes detection by the available analytical techniques.
Conclusion
Improvements in technology help to identify the
antibodies and antigens that mediate alloimmune
thrombocytopenia, and management strategies are
under development. However, illuminating the
detailed mechanisms of this disease is a daunting
task. Success in meeting this challenge is essential
for the health of patients and to use an increasingly
scarce resource effectively. The underlying complex
problems described here should be overcome in the
future; however, strenuous efforts are required to
improve patients' care. Given the common goals but
limited resources and considering how difficult it is
for a single laboratory to obtain a large number of
specimens, international collaboration is required to
meet these challenges.
Acknowledgements
We thank the staff of the Japanese Red Cross
Kinki Block Blood Centre for invigorating discussions
about platelet alloimmunisation. We also thank Drs.
387

E. Amakishi, K. Yasui, N. Matsuyama, R.A. Furuta,
and S. Tanaka for their valuable comments during the
preparation of this review.
Keywords: human platelet antigen, antibody detection,
genotyping.
The Authors declare no conflict of interest.
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Arrived: 29 October 2014 - Revision accepted: 15 February 2015
Correspondence: Tomoya Hayashi
Japanese Red Cross
Kinki Block Blood Center
7-5-17 Saito Asagi
Ibaraki, Osaka, Japan
e-mail: to-hayashi@kk.bbc.jrc.or.jp
Blood Transfus 2015; 13; 380-90 DOI 10.2450/2015.0275-14