Platelet-specific Alloantibodies Research Articles

Generation of iPSC-Derived Human Megakaryocytes for Flow Cytometric Detection of Platelet-Specific Alloantibodies

Antibodies that form against human platelet alloantigens (HPAs) are responsible for several clinically important alloimmune bleeding disorders, including fetal and neonatal alloimmune thrombocytopenia, posttransfusion purpura, and multitransfusion platelet refractoriness. Some of HPAs are relatively rare in the population, and difficult to obtain for purposes of transfusion therapy and diagnostic testing. In addition, HPA alloantisera often contain antibodies against human leucocyte antigen (HLA) class I, thereby limiting antibody detection to glycoprotein (GP)-specific assays such as the modified antigen capture enzyme-linked immunosorbent assay (MACE) and the monoclonal immobilization of platelet antigen (MAIPA), which are tedious and require solubilization of platelet GPs that may cause the loss of epitopes. In this study we aimed to generate gene-edited, HPA-specific, megakaryocytes (MKs) derived from human induced pluripotent stem cells (iPSCs) that could be used for simple flow cytometric detection of specific HPA alloantibodies present in patient sera. The HPA-3a/HPA-3b alloantigen system, also known as Baka/Bakb, is caused by a single T13809G nucleotide substitution in the ITGA2B gene, resulting in an Ile874Ser amino acid polymorphism near the C terminus of the integrin αIIb subunit (GPIIb). Here we targeted HPA-3 system because alloantibodies targeting HPA-3 are often hard to detect with current detection methods, in part due to the requirement for cell type-specific glycosylation. To prevent interference of anti-A or anti-B antibodies in patient sera, a blood type O iPSC line (OT1-1) was generated from human peripheral blood mononuclear cells derived from a healthy donor using integration-free episomal vectors. The gene for β2 microglobulin (B2M) was first ablated from the OT1-1 iPS cell line using CRISPR/Cas9 to prevent binding of HLA class I alloantibodies. The resulting B2M knockout (B2MKO) cells were then additionally gene edited to convert the endogenous HPA-3a alloantigenic epitope present on B2MKO OT1-1 cells to HPA-3b. Two different guide RNAs targeting sequences that flank exon 26 of the ITGA2B gene were designed such that the entire exon harboring the HPA-3 polymorphic site was removed. A plasmid harboring a template replacing exon 26 with the G13809 mutation, flanked by 600 bp homology arms, was cotransfected into the B2MKO OT1-1 iPSCs together with the two CRISPR/Cas9 guide RNA constructs. iPSC clones containing the desired targeted T13809G mutation were identified by a diagnostic MfeI digestion specific for the G13089-bearing HPA-3b allele. Sequence analysis confirmed conversion of T13089 to G in these HPA-3b clones. Flow cytometric analysis showed the HPA-3a iPSCs, when differentiated into CD41+/CD42b+ MKs, specifically reacted with HPA-3a, but not HPA-3b, patient sera, while the HPA-3b iPSC-derived MKs lost reactivity with HPA-3a patient serum, and gained the reactivity with HPA-3b patient sera. Taken together, we have established genetically modified iPSC-derived MKs expressing specific HPAs that are suitable for simple flow cytometry-based detection of HPA alloantibodies in patient sera, with low non-specific background binding. This system provides intact antigens on the cell surface with carbohydrate moieties that likely mimic those found on human platelets, thus facilitating the detection of HPA alloantibodies that are normally hard to detect with current methods. Application of this strategy to genetically edit this and other clinically-important HPAs holds great potential for producing Designer Platelets for diagnostic, investigative and ultimately therapeutic use. DisclosuresNo relevant conflicts of interest to declare.

Platelet reactive alloantibodies responsible for immune thrombocytopenia in Malay population

Background: Alloantibodies against human platelet alloantigens (HPAs) are responsible for the development of platelet transfusion refractoriness (PTR) in patients receiving random platelets and bleeding disorder in babies with fetal neonatal alloimmune thrombocytopenia (FNAIT). Recently, our results based on the analysis of the allelic distribution of HPAs indicated that immunization may occur among Malay. In this study, we sought to analyze the frequencies of platelet reactive alloantibodies responsible for FNAIT and PTR in Malaysia.Methods: Sera from suspected FNAIT (n = 295) and PTR (n = 74) were collected in five years period (2008-2013) and tested for the presence of platelet reactive antibodies by the use of antigen capture assay.Results: In 5/74 (5.41%) platelet specific antibodies against HPA-2b (n = 1), HPA-5a (n = 1), HPA-5b (n = 1), HPA-15b (n = 2) could be identified in our PTR cohort. In FNAIT cohort, platelet specific alloantibodies could be detected in 18 sera (6.10%) consisting anti-HPA-1a (n = 1), anti-HPA-3a (n = 3), anti-HPA-5a (n = 6), anti-HPA-5b (n = 6), anti-HPA-15a (n = 1), and anti-HPA-15b (n = 1).Conclusion: Our study indicates that anti-HPA-3, -HPA-5 and -HPA-15 antibodies seems to be the most platelet specific antibodies involved in FNAIT and PTR cases in Malaysian population. Since similar HPA allelic distribution among Malaysian and Indonesian populations have been observed, immunization against these three HPA systems are expected to be the most potential risk of alloimmune mediated platelet disorders in Indonesia.

Establishment of platelet donor registry improves the treatment of platelet transfusion refractoriness in Guangzhou region of China

Platelet transfusion refractoriness (PTR) is the major complication of long-term platelet supportive care. To improve the effectiveness of platelet transfusion therapy in PTR patients, we aimed to establish a platelet donor registry in our region (Guangzhou, China) by typing the human leukocyte antigen (HLA) and human platelet antigen (HPA). Blood donors (n = 864) from our population were genotyped for HLA-A, HLA-B and HPA systems by polymerase chain reaction amplification with sequence-specific primer(PCR-SSP) techniques. Using this cohort, we compared the results of platelet transfusions (matched vs. random) in 23 patients with PTR. Matched platelets were selected either by HLA antigen matching or by HLA antibody matching, as predicted by antibody specificity prediction (ASP) analysis. Significantly higher platelet recovery (PPR) values were obtained with HLA-matched platelets in comparison with random platelets. No significant difference in PPR was observed between HLA matching and ASP methods. In two patients, platelet-specific alloantibodies (alloabs) (anti-HPA-3b and anti-HPA-5b) were detected besides HLA class I alloabs. Transfusion with HLA- and HPA-compatible platelets in both the patients resulted in significantly higher PPR when compared with HLA-compatible platelet transfusion alone. In this study, we demonstrated that the establishment of an HLA- and HPA-typed platelet aphaeresis donor registry is useful to improve the treatment outcome of PTR patients and to maintain a long-term platelet transfusion strategy.

Platelet immunology – an update

The human platelet allo (HPA) antigen system has been well described with more than 16 HPA epitopes identified on platelet glycoproteins. There have been 6 bi-allelic systems characterised, HPA 1–5 and 15, which show variation in genotype frequency across populations. The very low frequency epitopes (HPA 6–14, 16, 17) may be a unique single nucleotide polymorphism (SNP) and have been identified through the characterisation of the specific antibody to the high frequency antigen. Although thought to be platelet specific, some of the HPA antigens have been identified on other cells and tissues. For example, GPIIIa has been detected on endothelial cells and fibroblasts. Allo-antibodies to the HPA system have been shown to be clinically significant in neonatal alloimmune thrombocytope-nia (NAIT) causing severe thrombocytopenia in the neonate due to a maternal antibody against the fetal paternally derived HPA antigen. Antibodies to the HPA 1a antigen are most commonly implicated in NAIT (75%), followed by antibodies to HPA 5,¹ but all HPA systems have been reported. NAIT has been the main source of HPA allo-antibodies identifying the majority of the HPA epitopes. This presentation will review the HPA allo-antigen systems and methods used to identify the platelet specific allo-antibodies.

An International Reference Reagent (minimum sensitivity) for the detection of anti‐human platelet antigen 1a

The platelet-specific alloantibody anti-human platelet antigen (HPA) 1a is involved in feto-maternal alloimmune thrombocytopenia, post-transfusion purpura and platelet refractoriness. The existing minimum potency preparation for the detection of anti-HPA-1a (NIBSC code 93/710) was established by World Health Organization in 1997 and is used by laboratories to validate new assays or to calibrate 'in-house' controls. However, it has been well-used and a replacement is required. This report describes the production and comparative evaluation of a freeze-dried preparation of pooled human plasma, coded 05/106, containing anti-HPA-1a. Plasma containing anti-HPA-1a was obtained and 2974 1-ml aliquots were prepared and freeze-dried in glass ampoules. In order to characterize the material and compare it to the existing reference material, three collaborative studies were organized, involving a total of 50 different laboratories in 23 countries. As expected only anti-HPA-1a could be detected in the plasma and no additional HPA or human leucocyte antigen antibodies were detected. When tested in titration, there was a wide variation in the sensitivity of antibody detection by different laboratories, irrespective of the technique used. However, there was no significant difference between the two materials when compared using a t-test. When diluted 1 in 2, most laboratories were able to detect the presence of anti-HPA-1a in both materials and the participants agreed that this was an appropriate level to set as the minimum sensitivity required. In October 2007, the World Health Organization Expert Committee on Biological Standardization approved the material 05/106 as an International Reference Reagent.

Interlaboratory variation in the detection of HPA‐specific alloantibodies and in molecular HPA typing

Platelet immunology quality assurance exercises have been organized by National Institute for Biological Standards and Control since 1991 and, as of 2006, 35 laboratories participate in the serology section. Molecular human platelet antigen (HPA) typing has been included in the exercises since 1998 and, as of 2006, 29 laboratories participate in this part of the exercise. This report details the performance of laboratories in these two areas. Every 6 months laboratories are sent up to four coded serum/plasma samples for testing in their in-house assays for HPA antibody detection/identification and four coded whole blood samples to be typed for HPA-1, -2, -3 -5 and (since 2003) -15 by their molecular typing assays ('genotyping'). Fifty-two sera containing HPA-specific alloantibodies and 13 sera that were inert, contained only human leucocyte antigen (HLA) class I or high-titre anti-A+B antibodies were distributed; 15 sera were issued in more than one exercise. The percentage of participating laboratories that were able to detect HPA-specific alloantibodies ranged from 15.0 to 100%; the percentage that were able to correctly determine the specificities also ranged from 15.0 to 100%. Over the 20 serology exercises the percentage of laboratories classified as poor performers ranged from 3.1 to 36.7%. A total of 12 780 individual HPA genotyping results were assessed. The overall error rate was 0.7% but there was considerable variation between HPA alleles. Over 11 exercises the percentage of laboratories classified as poor performers varied from 6.3 to 26.3%. The ability to detect and to identify platelet-specific alloantibodies varied widely between laboratories and between various examples of antibodies issued. An increase in the number of laboratories screening for HPA-15 antibodies was seen, although detection and identification of these antibodies was problematic. The majority of examples of HPA-3a antibodies and some examples of HPA-1a and -5b were also difficult to detect and identify. In addition, this scheme has shown that despite the apparent reliability of molecular typing techniques, mistakes do occur, particularly with certain systems. Approximately one in five laboratories participating in the serology exercises and one in seven participating in the genotyping exercises were classified as poor performer at one point or more during the series of exercises.

Post-transfusion alloimmunization to granulocytes and platelets in Japanese patients as determined by the MPHA method

The current occurrence of alloimmunization to granulocytes and platelets after blood transfusion is unclear due to the fact that antibody assays are cumbersome. Using the MPHA method with extracted granulocyte and platelet antigens, a randomized, blinded trial was conducted to investigate three types of alloantibodies in 431 Japanese patients receiving leukocyte-depleted blood transfusions prepared with or without our latest leukocyte-reduction filter. The frequency of granulocyte, platelet and HLA class I alloantibodies was 0.44%, 0.44% and 16.74%, respectively, in patients receiving non-filtered products and 0%, 0% and 0.49%, respectively, in patients receiving filtered products. The granulocyte antibody reacted with an antigen approximately of 51 KDa. The platelet-specific alloantibody was associated with GPIIb/IIIa and GPIa/IIa. The important factors affecting alloimmunization were the transfusion dose and the use of unfiltered platelet products.

Platelet alloantibodies in transfused patients.

Patients receiving cellular blood components may form HLA antibodies and platelet-specific alloantibodies. Serum samples from a cohort of 252 patients with hematologic or oncologic diseases who are receiving cellular blood components were studied for platelet-reactive antibodies. Specificity of platelet alloantibodies was determined with a panel of typed platelets Platelet-reactive antibodies were detected in the sera of 113 patients (44.8% of 252), HLA antibodies in the sera of 108 (42.9%), and platelet-specific antibodies in the sera of 20 (8%). The following platelet-specific antibodies were identified: anti-HPA-5b (n = 10), anti-HPA-1b (n = 4), anti-HPA-5a (n = 2), anti-HPA-1a (n = 1), anti-HPA-2b (n = 1), anti-HPA-1b+5b (n = 1), and anti-HPA-1b+2b (n = 1). Fifteen sera from the 108 patients with anti-HLA (13.9%) contained additional platelet-specific alloantibodies, while in 5 sera, platelet-specific alloantibodies only were detected: anti-HPA-5b (n = 4) and anti-HPA-1a (n = 1). Of the 108 sera with HLA antibodies, 29 (26.9%) showed discordant results when studied with the lymphocytotoxicity test and the glycoprotein-specific immunoassay. Ten sera contained panreactive antibodies against platelet glycoproteins (GP) IIb/IIIa, GPIa/IIa, and/or GPIb/IX. Alloimmunization occurred in 58.3 percent of female patients with previous pregnancies, but in only 23.3 percent of those without previous pregnancies (p = 0.0049). Platelet alloantibody specificities in transfused patients (predominantly anti-HPA-5b and -1b with antigen frequencies <30% among whites) differ significantly from those observed in patients with neonatal alloimmune thrombocytopenia or posttransfusion purpura, in whom anti-HPA-1a (antigen frequency >95%) is the most prevalent specificity. HLA antibody detection yields discordant results when the lymphocytotoxicity assay and a glycoprotein-specific immunoglobulin-binding assay are used.

Platelet antibodies in transfused patients

P atients and M ethods We studied sera of 252 patients with haematological or oncological diseases, who received multiple transfusions of rbc and/or platelet concentrates. The aim of our study was to analyse the specificity of platelet reactive antibodies (platelet specific and HLA-antibodies) in a large series of transfused patients. All sera were analysed by platelet immunofluorescence, platelet agglutination, MAIPA and the lymphocytotoxic test (LCT). R esults Platelet reactive antibodies were detected in 113 patients. Among female patients with previous pregnancies 21/36 were alloimmunized but only 7/30 female patients who had not been pregnant (P = 0·0049). In 108 (42·9%) of all sera, we found HLA-specific antibodies. In a subset of 40 sera, reactions of HLA class I antibodies were compared with an identical cell panel using LCT and MAIPA assay. Twenty-nine of these sera showed discrepant results in both techniques. Sera from 20 patients contained platelet specific alloantibodies with defined specificity: HPA-5b (10), HPA-1b (4), HPA-5a (2) HPA-1a (1), HPA-2b (1), HPA-5b + HPA-1b (1), HPA-1b + HPA-2b (1). All sera with anti-HPA-1b contained additional HLA antibodies, 4 sera with anti-HPA-5b apparently contained no HLA-antibodies. Ten sera contained ‘panreactive’ antibodies with broad specificity: GP IIb/IIIa (4), GP IIb/IIIa + Ia/IIa (2), GPIIb/IIIa + Ib/IX (1), GP IIb/IIIa + Ia/IIa + Ib/IX (3). C onclusion (1) Platelet alloantibody specificities in transfused patients (predominantly anti-HPA-5b and − 1b) differ significantly from those observed in neonatal alloimmune thrombocytopenia and post-transfusion purpura, where anti-HPA-1a is the most prevalent specificity. (2) Results for detection of HLA-antibodies differ considerably in relation to serological technique and target cells.

Three Cases of Platelet Alloimmunisation Associated with the Presence of a Novel Platelet Specific Antibody

In three cases of platelet alloimmunisation, a platelet-specific alloantibody was detected which could not be classified within the known human platelet alloantigen or HLA systems. The first case was of a family in which two siblings suffered neonatal alloimmune thrombocytopenia at birth. In the second case, the newborn was suffering from phocomelia with hypoplastic thrombocytopenia. The third case was a male who became refractory to transfusions of HLA-matched platelets after a related bone marrow transplantation. The serum samples were investigated by: enzyme-linked immunosorbent assay, platelet suspension immunofluorescence test (PSIFT), monoclonal antibody immobilisation of platelet antigens assay (MAIPA), and by the lymphocytotoxicity test. The antibody gave positive reactions with 26% of normal donor platelets. Surprisingly, no platelet-specific antibody was detected by PSIFT or by MAIPA and there was no evidence found to support classifying the antibody within the HLA system. The reactivity pattern of the antibody detected and the clinical presentation of the three cases described, strongly suggest the presence of an additional platelet-specific alloantigen system.

Three Cases of Platelet Alloimmunisation Associated with the Presence of a Novel Platelet-Specific Antibody

Background and Objectives: In three cases of platelet alloimmunisation, a platelet-specific alloantibody was detected which could not be classified within the known human platelet alloantigen or HLA systems. The first case was of a family in which two siblings suffered neonatal alloimmune thrombocytopenia at birth. In the second case, the newborn was suffering from phocomelia with hypoplastic thrombocytopenia. The third case was a male who became refractory to transfusions of HLA-matched platelets after a related bone marrow transplantation. Materials and Methods: The serum samples were investigated by: enzyme-linked immunosorbent assay, platelet suspension immunofluorescence test (PSIFT), monoclonal antibody immobilisation of platelet antigens assay (MAIPA), and by the lymphocytotoxicity test. Results: The antibody gave positive reactions with 26% of normal donor platelets. Surprisingly, no platelet-specific antibody was detected by PSIFT or by MAIPA and there was no evidence found to support classifying the antibody within the HLA system. Conclusion: The reactivity pattern of the antibody detected and the clinical presentation of the three cases described, strongly suggest the presence of an additional platelet-specific alloantigen system.

Human monoclonal Fab fragments recovered from a combinatorial library bind specifically to the platelet HPA-1a alloantigen on glycoprotein IIb-IIIa.

Certain clinical conditions are related to the presence of platelet-specific alloantibodies in the patient's serum. We studied the molecular diversity of HPA-1a antibodies to analyze some peculiarities of this antibody response. Human antibody Fab fragments that bind to the platelet alloantigen HPA-1a on glycoprotein IIb-IIIa (GPIIbIIIa) were generated by using a recombinant phage display system. We established an immunoglobulin G1, kappa combinatorial library from the peripheral blood lymphocytes of a person undergoing a severe posttransfusion purpura. Characterization of Fab clones selected from the fifth round of antigen-specific panning of this library demonstrates a highly specific reactivity to the HPA-1a alloantigen. The nucleotide sequence analysis of representative HPA-la-specific clones reveals at least 3 distinct V1 and 3VH gene segments that present an extensive degree of mutation as demonstrated by comparison of gene usage and homologies to the nearest germline genes. These human HPA-la-specific Fab reagents should allow us to better understand the molecular mechanism involved in HPA-la alloimmunization.

Human Monoclonal Fab Fragments Recovered from a Combinatorial Library Bind Specifically to the Platelet HPA-1a Alloantigen on Glycoprotein IIb—IIIa

Background and objectives: Certain clinical conditions are related to the presence of platelet-specific alloantibodies in the patient’s serum. We studied the molecular diversity of HPA-1a antibodies to analyze some peculiarities of this antibody response. Materials and methods: Human antibody Fab fragments that bind to the platelet alloantigen HPA-la on glycoprotein Ilb-IIIa (GPIIbllla) were generated by using a recombinant phage display system. We established an immunoglobulin Gl, kappa combinatorial library from the peripheral blood lymphocytes of a person undergoing a severe posttransfusion purpura. Results: Characterization of Fab clones selected from the fifth round of antigen-specific panning of this library demonstrates a highly specific reactivity to the HPA-1a alloantigen. The nucleotide sequence analysis of representative HPA-1a-specific clones reveals at least 3 distinct VL and 3 V(H) gene segments that present an extensive degree of mutation as demonstrated by comparison of gene usage and homologies to the nearest germline genes. Conclusions: These human HPA-laspecific Fab reagents should allow us to better understand the molecular mechanism involved in HPA-1a alloimmunization.

Report on the Sixth International Society of Blood Transfusion Platelet Serology Workshop.

The Sixth International Society of Blood Transfusion Platelet Serology Workshop continued studies to identify methods to detect platelet-specific antigens and antibodies. The study was designed to meet three goals. The first was the establishment of antigen-typed platelet panels and determination of the correlation between serologic and DNA typing of platelet-specific antigens. The second goal was the determination of the proficiency of detecting platelet-specific antibodies by laboratory and by technique. The third goal was the identification of any platelet-specific antibodies present in uncharacterized (unknown) antisera. For platelet-antigen typing, concordance between serologic testing and DNA techniques was 93 percent for oligonucleotide typing and 92 percent for allele-specific restriction site analysis. Agreement between these two was 98 percent. Individual laboratories correctly identified the antibodies contained in coded sera 79 +/- 17 percent of the time. The expected results were obtained from the modified antigen-capture enzyme-linked immunosorbent assay in 75 +/- 46 percent of instances, from the monoclonal antibody-specific immobilization of platelet antigens assay in 72 +/- 24 percent of instances, from the mixed passive hemagglutination assay in 71 +/- 13 percent, from radioimmunoprecipitation procedures in 67 +/- 47 percent, and from Western blot 34 +/- 40 percent. Seven (54%) of 13 antisera of unknown specificity were determined to contain clearly identifiable platelet-specific alloantibodies. Concordant results were achieved by using either serologic or DNA techniques to identify platelet-specific antigens. Except for the significantly lower results found with Western blotting, all other platelet-specific antibody assays were comparable. Established serologic laboratories can identify and characterize plate-specific antibodies.

Diagnostik und Therapie der fetalen Alloimmunthrombozytopenie

In fetal alloimmune thrombocytopenia, maternal IgG antibodies directed against platelets pass into the fetal circulation and lead to the destruction of fetal platelets. Fetal thrombocytopenia is usually first noted postnatally, but the maternal alloantibodies may lead to severe fetal haemorrhage in utero in the second or, more commonly, third trimester. Platelet-specific antibodies appear to play the major role in the pathogenesis of fetal alloimmune thrombocytopenia. Antenatal serological diagnosis is complicated by the fact that no platelet-specific alloantibodies are detectable in untreated maternal serum in about 20% of cases. Even when antibodies are detected, repeated estimation of the titre in the mother gives no indication of the severity of the fetal thrombocytopenia. Therefore, when the diagnosis of fetal alloimmune thrombocytopenia is suspected, it should be confirmed and subsequently monitored by cordocentesis. The intrauterine transfusion of compatible platelets and the administration of high dose IgG infusions to the mother and/or fetus are currently being used as approaches to treatment. Despite advances in the antenatal diagnosis and therapy of fetal alloimmune thrombocytopenia, the clinical effects on the fetus remain unpredictable.

A platelet monoclonal antibody inhibition assay for detection of glycoprotein [formula omitted] platelet alloantibodies

Post-transfusion purpura (PTP) and neonatal alloimmune thrombocytopenia (NAT) result from formation of alloantibodies to platelet membrane glycoprotein-associated antigens. The detection and identification of platelet-specific alloantibodies in patient sera is often complicated by the presence of co-existing HLA antibodies and/or more than one platelet specificity in the same serum. We describe a solid phase assay that specifically detects antibodies to platelet membrane associated alloantigens by measuring the ability of patient antisera to inhibit the binding of glycoprotein GPIIb or GPIIIa monoclonal antibodies to intact platelets. When tested in the GPIIIa assay against a panel of random platelet donors, the reactivities of two known PL A1 antisera that also contained different HLA antibodies were highly correlated ( r = 0.99) and allowed PL A phenotyping of the population. A standard direct binding platelet ELISA, on the other hand, was unable to accurately PL A phenotype the same population. The reactivities of two known Bak a antisera (one containing additional anti-PL A2 and the other anti-Br b specificities) were highly correlated ( r = 0.95) in the GPIIb assay, and Bak phenotype determination was similarly accomplished for a random platelet panel. Furthermore, a comparison of platelet phenotype results (using the monoclonal inhibition assay) and genotype results (using DNA analysis) for the PL A and Bak systems showed a concordance of 98% for 146 alleles tested. In conclusion, the platelet monoclonal antibody inhibition assay: (1) allows determination of platelet-specific alloantibodies in the presence of contaminating HLA antibodies and/or in sera containing multiple platelet alloantibodies; (2) allows accurate platelet phenotyping for the GPIIIa-associated PL A and GPIIb-associated Bak antigen systems; and (3) may be applicable to the detection of other known or even novel platelet glycoprotein-associated antigens.

Simultaneous DNA typing of human platelet antigens 2, 3 and 4 by an allele-specific PCR method.

We developed an allele-specific polymerase chain reaction (ASPCR) method using originally designed primers to determine the genotype of the human platelet antigens (HPAs) 2, 3 and 4 in parallel. The results were compared with those obtained by PCR restriction fragment length polymorphism and the mixed passive hemagglutination test. Seventy-three individuals were tested and the ASPCR results were in good agreement with those determined by the other two methods. This method enables the genotyping of HPA-2, -3 and -4 in parallel without the use of platelets, platelet-specific alloantibodies or restriction enzymes.

Simultaneous DNA Typing of Human Platelet Antigens 2, 3 and 4 by an Allele-Specific PCR Method

We developed an allele-specific polymerase chain reaction (ASPCR) method using originally designed primers to determine the genotype of the human platelet antigens (HPAs) 2, 3 and 4 in parallel. The results were compared with those obtained by PCR restriction fragment length polymorphism and the mixed passive hemagglutination test. Seventy-three individuals were tested and the ASPCR results were in good agreement with those determined by the other two methods. This method enables the genotyping of HPA-2, -3 and -4 in parallel without the use of platelets, platelet-specific alloantibodies or restriction enzymes.

Non-correlation of HLA and platelet-specific alloantibody production

Non-correlation of HLA and platelet-specific alloantibody production

３卵性児に認められた抗ＨＰＡ‐４ｂによる新生児血小板減少症の１症例

We recently came across a case of neonatal alloimmune thrombocytopenic purpura (NAITP) in a set of triplets.The serum obtained from the mother of the child with NAITP contained platelet-specific alloantibody, anti HPA-4b. The antibody was of IgG class detected by anti-IgG indicator cells. The serum of the mother strongly reacted with the platelets of her husband (1:1, 024), but not with her own.After delivery, the platelet count of the triplet with NAITP was 1.2×104/μl. The platelet count of the other two infants were 29.5×104/μl, 29.7×104/μl each.