Advances in alloimmune thrombocytopenia: perspectives on current concepts of human platelet antigens, antibody detection strategies, and genotyping

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 understanding of HPA has been made in the last decade: new HPA have been discovered, antibody-detection methods have improved, and new genotyping methods have been developed. We review these advances and discuss issues that remain to be resolved as well as future prospects for preventing and treating immune thrombocytopenia.

Introduction

Immune and non-immune mechanisms can decrease the number of platelets, leading to bleeding manifestations that range from petechiae and simple bruising to intracranial haemorrhage and death¹. Platelets interact with coagulation factors to arrest haemorrhage and support vascular integrity^2,3. At sites of vascular injury, platelets change shape, adhere to the site, and secrete cytokines that are essential for tissue repair^1,4,5. Platelets are indispensable for maintaining vascular integrity. Molecules expressed by platelets that are involved in arresting haemorrhage include integrin αIIbβ3, GPIb/IX, and integrin α2β1^6–10.

The antigens that are recognised by alloantibodies have been categorised by the Platelet Nomenclature Committee, and 34 human platelet antigens (HPA) have been defined^11,12. 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. 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 to distinguish between thrombocytopenia induced by alloantibodies from drug-induced disease¹⁶.

The platelet surface membrane contains antigenic molecules other than HPA, including ABO blood type antigens¹⁷, human leucocyte antigens (HLA)¹⁸, and the Nak^a 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 are biallelic (HPA-1, -2, -3, -4, -5, and -15), and others include 34 platelet-specific alloantigens defined according to the rules of the ISBT Platelet Working Party. The allelic frequencies of HPA differ among them, and racial differences are numerous. For the six biallelic systems, the most frequent HPA are defined as “a” alleles or “wild-type.” All alloantigen systems comprise genes encoding only six proteins. Among them, GPIIb [integrin alpha subunit IIb (αIIb), CD41] and GPIIIa [integrin beta subunit 3 (β3), CD61] form the GPIIb/GPIIIa (GPIIbIIIa, integrin αIIbβ3) complex and GPIbα GPIbβ and GPIX 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 (β1) form the GPIaGPIIa (GPIaIIa, integrin α2β1) complex.

Figure 1.

Human platelet antigens (HPA) polymorphisms.

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 (http://www.genenames.org). Sequences encoding HPA within the same exons are indicated by the same colours. Nucleotide variations: 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 amino acid residue number in the mature protein. The definitions of symbols (#, s, §,□, ‡, *, ¶ and †) are described under each residue.

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 haemostasis^25,26. Functional abnormalities and deficiency of GPIIbIIIa are present in patients with Glanzmann’s thrombasthenia, which is an autosomal recessive disorder caused by mutation of the genes encoding GPIIb or GPIIIa or is acquired as an autoimmune disorder that leads to an increased tendency to bleed²⁶.

Most HPA are localised to GPIIb and GPIIIa (Figure 1). GPIIIa and GPIIb comprise 16 and seven HPA systems, respectively^11,12. There are several genetic hot-spots in which the mutations that encode two to four HPA are present in the same exons (Figure 1).

Although all HPA present on GPIIbIIIa are associated with FNAIT, HPA-1a is strongly associated with FNAIT, particularly in Caucasians, and antibodies against HPA-4b and HPA-21b are present in Asians^27–29. 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 PTP^30–33. 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 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, respectively^11,12. Antibodies against HPA-2b and HPA-12bw are associated with FNAIT^37,38, 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 with Bernard-Soulier syndrome.

GPIa

GPIa (CD49b) associates with GPIIa (CD29) to form integrin α2β1 (GPIaIIa) complex, known as very late antigen (VLA-2). GPIaIIa is expressed by monocytes, T cells, B cells, NK cells, and platelets⁴¹. Platelet GPIaIIa is involved in adhesion at low flow rates by binding to collagen and participates in cell surface-mediated signalling leading to GPIIbIIIa activation. Approximately 800–2,800 GPIaIIa molecules are present on the platelet surface.

Four HPA systems (HPA-5, HPA-13, HPA-18, HPA-25) are located on GPIa^11,12. The HPA-13bw 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 race^42–44. 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-β-1-binding protein, C3 and PZP-like alpha-2-macroglobulin domain-containing protein 7) links to the platelet surface through a GPI anchor⁴⁵. 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-β 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.

CD109 is a component of only HPA-15^11,12. The detection of antibodies against CD109 is hampered, because platelets express low levels of CD109 on their surface and CD109 is unstable when platelets are 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 importance^48,49. 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 FNAIT^48,49. 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 Nak^a 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.

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.

Table I.

Types and frequencies of CD36 deficiency.

Deficiency type	Expression				Frequency of deficiency
	Platelets	Monocytes	RBC progenitors	Endothelial cells	Caucasian	Sub-Sahara African	Asian
I	absent	absent	absent	absent	<0.3%	2.50%	0.5–1%
II	absent		#				3–10%

^#depending on the presence or absence on RBC progenitors; the deficiency is classified into type II-a and type II-b, respectively.

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 Asians^54–56. 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

Several methods that use platelets as target cells are available for the detection of antibodies against HPA, such as the monoclonal antibody-specific immobilisation of platelet antigens (MAIPA) assay⁵⁸, the platelet-antigen capture (PAC) assay⁵⁹, the mixed passive haemagglutination test (MPHA)⁶⁰, flow cytometric analysis⁶¹, a modified antigen-capture enzyme-linked immunosorbent assay (ELISA)⁶² and a Luminex bead assay^63,64. The properties of these methods are summarised in Table II.

Table II.

Summary of the properties of antibody detection assays.

Whole platelet methods
Method	Principle	Advantage	Disadvantage	Additional remarks
MPHA SPRCA	The test serum is added onto a microplate coated with platelet extracts and indicator sheep red blood cells coated with anti-human IgG are added. The presence of the human antibody is judged by agglutination patterns.	Simple procedure Low cost Detector not required	Possible insufficient sensitivity and specificity	High-titre HLA antibodies hamper the detection of HPA antibodies.
PSIFT	The patient’s serum is added to a platelet suspension and an anti-human IgG conjugated to a fluorescent dye is added. Fluorescence emission is analysed using a flow cytometer or fluorescence plate reader.	Simple procedure NonHPA platelet antibodies are detectable as well	Possible insufficient sensitivity and specificity instrumentation (FACS) is expensive	HLA antibodies hamper the detection of HPA antibodies.
Glycoprotein specific methods
Method	Principle	Advantage	Disadvantage	Additional remarks
MAIPA PAC	Immune complexes composed of the target HPA, human-specific antibody, and mouse monoclonal antibody are captured by anti-mouse IgG coated on a plate. The human antibody is detected using enzyme-linked anti-human IgG and a colorimetric detector.	High sensitivity and high specificity	High cost Complicated procedure	Need to be careful about the antibody competition
MACE	Monoclonal antibodies coated onto a solid phase are used to capture the target HPA. The human antibody is detected using enzyme- linked anti-human IgG and a colorimetric detector.	High specificity	High cost Complicated procedure	Need to be careful about the antibody competition
ICFA	Microarray beads are separately coupled with recombinant GP fragments or monoclonal antibodies specific for HPA and HLA. Luminex xMAP technology adapts MAIPA onto the Luminex platform by using a secondary antibody conjugated to a fluorescent dye.	High specificity Simultaneous analysis of HLA and HPA antibodies in one well	High cost Expensive instrumentation (FACS or Luminex)	Need to be careful about the antibody competition when using antibody-coupled microspheres.
SPR	Measures binding of antibody onto an antigen-coated planar metal surface. Bound molecules change the local index of refraction, which is generated by the antigen-antibody interaction that changes the resonance of the surface plasmon waves.	High sensitivity. Detects low-avidity antibodies. Washing and labelling are not required	Requires highly purified antigens and immunoglobulins. Requires specialised equipment.	Since the sensitivity is so high that the clinical significance of the detected antibodies are addressed by in-vivo experiments, such as those using the NOD/SCID mouse.
HP-IPA	Immune complexes composed of the target HPA, human specific antibody, and enzyme-linked mouse-anti-human IgG are captured by anti-mouse IgG coated on a plate, and the human antibody is detected after adding the substrate.	High sensitivity Eliminates antibody competition	High cost Complicated procedure Requires a cell panel

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.

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 standard^65,66. These tests, including the MAIPA, do have some disadvantages. First, the preparation of 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).

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 (integrin αIIb and β3) (CD41 and CD61)	Wild type, HPA-1b, HPA-3b, HPA-4b, HPA-6b, HPA-7b, HPA-7variant, HPA-21b	HPA-1a, 3a*, 4b, 6b, 7variant, 21b	MAIPA, MACE, IFT, HP-IPA, ICFA (xMAP)
	GPIa (integrin α2, CD49b)	HPA-5b, HPA-13b, HPA-18b	HPA-5a, 5b	MAIPA, MACE, IFT, ICFA (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	Δβ3 peptide (wild type), Super rare peptide, 1a, 1b, 4b, 8b	HPA-1a, 1b, 4a, 4b, 6bw, 7bw, 8bw, 11bw*, 16bw	ICFA (xMAP), SPR
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.

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 express various HPA, do not express HLA, HNA, or HPA, and show low non-specific reactivity with human sera. These test systems are highly specific and sensitive for detecting antibodies against HPA^68–72. Briefly, the cell 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 expressing wild-type GPIbα and GPIbβ (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 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 antigen, is recognised by HPA-1a alloantibodies with very high sensitivity⁷⁷. Because aptamers are usually designed according to structure or affinity, their specific antigenicity limits applying them to each HPA. Future studies are, therefore, required to develop assays using aptamers.

Limitations of serological systems for detecting antibodies against human platelet antigens

Standard control sera

Although positive control antibodies are important 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 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-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 both⁸². 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 antigen antibodies and thrombocytopenia

At present, low affinity anti-HPA antibodies can be detected using highly sensitive methods such as surface plasmon resonance. However, detection of 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 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.

Table IV.

Genotyping methods.

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 denaturing polyacrylamide gel electrophoresis. It distinguishes mobility shifts of type a and b caused by a change in the conformation of single-stranded amplicons.	Low cost	Lengthy procedure	Ambiguous data
PCR-RFLP83,85	Digestion using restriction enzymes	High specificity and low cost	Lengthy procedure	Occasionally leaves ambiguities
HRM86,88,89	The melting temperatures of DNA depend on their base composition. PCR is performed using DNA intercalating dyes, such as SYBR® green. As the sample is heated and the two strands of the DNA separate (melt), the concentration of double-stranded DNA decreases, reducing fluorescence. The instrument generates a melting curve.	Simple and quick. Applicable for high- throughput analysis	Expensive detector	Occasionally amplicons with different sequences melt at the same temperature
Real-time PCR-SSP85,87,88,89	Uses allele-specific primers to amplify allele-specific DNA and intercalating agents such as SYBR® green are used.	Simple and quick. Applicable for high- throughput analysis	Expensive detector

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.

Numerous techniques available for determining single nucleotide polymorphisms can be applied to HPA genotyping^83,84. HPA genotypes are widely determined using sequence-specific primer-polymerase chain reaction (PCR-SSP), denaturing gradient gel electrophoresis (PCR-DGGE)⁸⁵, and restriction fragment length polymorphism (PCR-RFLP) techniques. However, these methods are generally specialised to determine one polymorphism in a single test and are therefore applicable in the analysis of patients with FNAIT. In contrast, a high-throughput method is required for donor screening and frequency analysis.

For example, the high-resolution melting (HRM) method is cost-effective. Several groups reported that HRM is useful for HPA genotyping of a large donor pool, for determining genotype frequency, or both^86–89. HRM determines the melting point of DNA, which is influenced by the abundance of C-G base pairs. Most HPA polymorphisms are generated by replacing C-G 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.

Pitfalls of genotyping

Although genotyping is a powerful tool for definitive diagnosis of FNAIT, genotype does not always correlate with phenotype, as in a propositus heterozygous for type I Glanzmann’s thrombathenia or Bernard-syndromes. A propositus can be genotypically heterozygous for a particular HPA but phenotypically homozygous, because one of the allele is not expressed on the platelet surface^36,92.

Future study

Predicting the severity of thrombocytopenia is likely important for efficacious treatment of patients with FNAIT, autoimmune thrombocytopenia, or alloimmune thrombocytopenia⁹³. Alloantibodies destroy platelets and eliminate them from the circulation through antibody-induced immune-mediated phagocytosis and activation of complement^59,93–100. 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 antibodies^59,93. 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 normal⁵⁹. 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, immunoglobulin subclasses should be kept in mind.

Glycosylation pattern of immunoglobulin heavy chain

Immunoglobulin molecules comprise heavy and light chains. The Fab and Fc domains of antibodies are important for recognition of antigen and binding to Fc receptors, respectively. Furthermore, the glycosylation pattern of the Fc domain affects its binding affinity for Fc receptors. Structural studies of Fc domains show that N297 glycans stabilise an “open” Fc conformation recognised by Fcγ receptors⁹⁵. Okazaki showed that depleting fucose from the oligosaccharide moiety of human IgG1 increases the moiety’s affinity for the Fcγ 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 model⁹⁸. 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 Fcγ receptor-dependent platelet destruction. B2G1Δnab 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 antibodies^99,100.

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 with antibody-dependent thrombocytopenia, there should be antibodies against already-known epitopes present on the six molecules GPIIb, GPIIIa, GPIbα, GPIbβ, GPIa, or CD109. Otherwise, antibodies against 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.