Human Platelet Antigen Systems Research Articles

Simultaneous genotyping of human platelet antigen‐1 to 17w by polymerase chain reaction sequence‐based typing

Genotyping for human platelet antigen (HPA) systems is required in the investigation of patients with suspected HPA antibodies and for the provision of compatible blood products from HPA-typed donors. Here, we set up a polymerase chain reaction sequence-based typing method for simultaneous genotyping of HPA-1 to 17w (PCR-SBT). The specific primers for HPA-1 to 17w were designed by primer premier 5.0 software. The PCR amplification conditions were optimized. Amplification products were purified by enzyme digestion and sequenced by a BigDye Terminator Cycle Sequencing kit. We used two standard DNA samples and 16 reference samples, including six with HPA gene mutations, for the HPA-1 to 17w analysis by the PCR-SBT and obtained 100% correct genotypes in these samples. The genotype and allele frequencies of HPA-1 to 17w were analysed from 112 Han Chinese persons. Thirteen new single nucleotide polymorphisms and one microsatellite were found in the sequenced regions, in which two nucleotide polymorphisms of ITGB3 gene could cause amino acid alteration of Val419Met and Glu628Lys on the mature glycoprotein, respectively. The PCR-SBT method is reliable and accurate for HPA genotyping, suitable for routine HPA analysis, and can screen large numbers of donors. We identified 14 new polymorphisms, two of which led to amino acid changes.

Alloantibodies against low‐frequency human platelet antigens do not account for a significant proportion of cases of fetomaternal alloimmune thrombocytopenia: evidence from 1054 cases

Maternal alloantibodies against the five common human platelet antigen (HPA) systems (HPA-1 to -3, -5, and -15) are found in only 20% of cases referred for fetal and neonatal thrombocytopenia (FMAIT) investigations. The question asked was whether mismatches for the remaining 11 low-frequency HPAs (HPA-4 and -6bw to -17bw) might in part explain the remaining 80% of cases. A total of 1054 paternal DNA samples from referred FMAIT cases (among which 223 cases where antibodies against a common HPA were found) were genotyped for 11 low-frequency HPAs as well as a recently discovered polymorphism (ITGA2B-C2320T). The initial genotyping was carried out by TaqMan and potential heterozygotes were confirmed by DNA sequencing. Clinical and serologic data were collected for each case with a heterozygote father. In total, eight heterozygous fathers were identified: four for HPA-6w, one each for HPA-10w and -11w, and two for HPA-12w. Maternal antibodies against the corresponding antigen were identified in four of the eight cases. In two of these cases, antibodies against HPA-1a and HPA-1b were also found. It was concluded that the minor alleles of HPA-4 and -6bw to -17bw are exceptionally rare in the Caucasian population and therefore do not explain the large number of FMAIT referrals which test negative for the common HPA antibodies.

Platelet‐specific alloantigens and antibodies in Tunisian women after three or more pregnancies

Pregnancy may allow alloimmunization against human platelet antigens (HPA), which can lead to neonatal alloimmune thrombocytopenia (NAIT). The specificities of alloantibodies are closely related to the distribution of the HPA systems. A total of 281 Tunisian multiparous women (mean number of pregnancies: 4.5) were phenotyped for the HPA-1, -3 and -5 systems, by monoclonal antibody immobilization of platelet antigens (MAIPA). We searched for antibodies against HPA-1a, HPA-3a, HPA-5b and HPA-5a in HPA-1b1b, HPA-3b3b, HPA-5a5a and HPA-5b5b individuals, respectively. The gene frequencies were: 0.83 for HPA-1a, 0.17 for HPA-1b, 0.78 for HPA-3a, 0.22 for HPA-3b, 0.82 for HPA-5a and 0.18 for HPA-5b. Anti-HPA-5b antibodies were present in eight sera and anti-HPA-3a antibodies were present in one serum. The anti-HPA-5b system is the most frequently involved in platelet alloimmunization in Tunisian multiparous women. However, prospective trials are required to confirm this result and to determine the exact frequencies and clinical relevance of platelet alloantibodies in pregnant Tunisian women.

Allele frequencies of the human platelet antigen-1 in the Egyptian population

BackgroundThe human platelet alloantigen system HPA-1 in the Egyptian population was examined by polymerase chain reaction using sequence-specific primers (PCR-SSP). The objectives of this study were to evaluate the allele frequency of HPA-1a and -1b in healthy Egyptian individuals and compare these with the international literature. Human platelet antigen (HPA) systems are associated with alloimmunization and organ transplantation rejection as well as the development of cardiovascular disease. Of the various HPA systems, HPA-1 specifically has been considered to be the most important antigenic system implicated in the Caucasian population. No study has yet examined this system in the Egyptian populations, however. We therefore investigated the allele frequency of the HPA-1 system in the Egyptian population.FindingsTo determine the allele frequency of the HPA-1a and -1b, we tested genomic DNAs from 206 healthy, unrelated Egyptian individuals using PCR-SSP. Our results showed that the 1a/1a genotype was the most predominant (59.22%) followed by 1a/1b (34.95%) and 1b/1b (5.83%) with allele frequencies for 1a and 1b of 0.77 and 0.23, respectively, in the population.ConclusionAs compared with other geographic groups, a relatively high allele frequency of the HPA-1b in the Egyptian population may indicate a higher risk of alloimmunization. This study is the first to investigate the allele frequency of the HPA-1 system in the Egyptian population and serves as an outline for future clinical research associated with platelet disorders in this group.

Genotyping for Human Platelet Alloantigen Polymorphisms: Applications in the Diagnosis of Alloimmune Platelet Disorders

Molecular typing for platelet allelic polymorphisms was first made possible by discovery of the HPA-1a/1b single nucleotide polymorphism in 1989. Since then, six other biallelic human platelet antigen (HPA) systems have been determined and can be typed using genomic DNA. The introduction of polymerase chain reaction enabled development of several different assays including polymerase chain reaction-sequence-specific primer, melting curve analysis by LightCycler, and 5'-nuclease assays. More recently, multiplex polymerase chain reaction has allowed for the development of high-throughput assays for genotyping large numbers of patients and blood donors for not only platelet gene polymorphisms but also for those of other blood cell genes. Platelet genotyping is a valuable tool in confirming platelet antigen specificities of alloantibodies detected in patient sera to complement the clinical history in the diagnosis of alloimmune platelet disorders such as fetal and neonatal alloimmune thrombocytopenia (FNAIT), posttransfusion purpura, and multiplatelet transfusion refractoriness. In addition, it has made possible prenatal platelet typing of the fetus in suspected cases of FNAIT and large-scale blood donor typing for provision of antigen-negative platelets to transfuse highly alloimmunized patients. Platelet genotyping may also someday prove important as an aid in determining the relative risk of patients for various thrombotic disorders.

Human platelet‐specific antigens frequencies in the Argentinean population

The frequencies of several human platelet antigens (HPAs) vary between different populations and are a major determinant for the prevalence of HPA alloimmunization and its clinical associated entities. The aim of this study was to characterize the allele frequencies of seven HPA systems in two different ethnic groups from the Argentinean city of Rosario, the major population and a minority Amerindian group recently arrived from the north of the country, the Tobas. A total of 192 healthy unrelated individuals from blood donors and hospital staff from the Hospital Italiano Garibaldi and 27 unrelated Toba Amerindians were genotyped for HPA-1, -2, -3, -4, -5, -6 and -15 systems by polymerase chain reaction with sequence specific primers (PCR-SSP). The present data showed that the distribution of the HPA alleles among Argentineans from Rosario is quite similar to that reported among Europeans. The frequencies seen in Tobas, although limited by the small number of aboriginal samples studied, are similar to those reported for other Amerindians populations. Statistically significant differences were found for the genotype distribution of HPA-1, -3, -5 and -15 between both groups, indicating important differences in the potential risk of HPA alloimmunization associated to transfusion and pregnancy. The study of these polymorphisms represents the first step in the elucidation of pathological conditions that are underdiagnosed in our population. It allowed us to establish a panel of characterized blood donors necessary for the serological work out and as a source for compatible platelets transfusion.

Gene frequencies of the HPA‐1 platelet antigen alleles in the Lebanese population

The objective was to study the gene frequencies of HPA-1 in the Lebanese population for the first time. The aims of this study were to assess the prevalence of 1a and 1b HPA-1 alleles in healthy Lebanese individuals and compare with the international literature. Human platelet antigen (HPA) systems are involved in alloimmunization, organ transplantation rejection and the development of cardiovascular disease. Of several classified HPA systems, HPA-1 specifically has been considered to be the most important antigenic system implicated in the Caucasian population. This specific gene has never been investigated in our population. DNA was extracted from specimens collected from 205 healthy unrelated Lebanese individuals and tested, using a reverse hybridization polymerase chain reaction (PCR) assay, for the prevalence of 1a and 1b HPA-1 alleles. Genotypes 1a/1a, 1a/1b, and 1b/1b were assigned accordingly. We observed that the 1a/1a genotype was the most prevalent (65.85%) followed by 1a/1b (30.24%) and 1b/1b (3.91%) with allelic frequencies for 1a and 1b of 0.81 and 0.19, respectively. As compared with other ethnic groups, the Lebanese population was found to have a relatively high prevalence of the HPA-1b, which may predispose to a higher risk of alloimmunization. This report is the first to study the prevalence of the HPA-1 system in the Lebanese population and serves as a template for future clinical research involving platelet disorders and cardiovascular diseases.

Multicolor real‐time polymerase chain reaction genotyping of six human platelet antigens using displacing probes

Several genotyping methods for six clinically relevant human platelet antigens (HPAs) have been reported. A four-color real-time polymerase chain reaction (PCR) method using displacing probes for genotyping of the six HPAs is described. Primers and four differently fluorophor-labeled displacing probes were designed and synthesized to detect single-nucleotide polymorphisms responsible for each of the HPA-1, -2, -3, -4, -5, and -15 genotypes. Two HPA systems were analyzed in a single PCR procedure. After validation with samples of known genotypes, a total of 150 blood samples from healthy donors were genotyped. The results were compared with PCR with sequence-specific primers (SSP), PCR-restriction fragment length polymorphism (RFLP), and/or direct DNA sequencing. The frequencies of each HPA allele were calculated. Unequivocal real-time PCR genotyping results were obtained with minimal manual manipulation and carryover contamination. All 150 blood samples were correctly genotyped as confirmed by PCR-SSP, PCR-RFLP, and/or direct DNA sequencing. The allelic frequencies of HPA-1 through -5 and -15 among the Chinese population in Xiamen were comparable with those previously reported with Chinese living in other territories. For each specimen, genotyping of all six HPA biallelic systems was achieved in three tubes of PCR within 90 minutes and with material cost of no more than $1. Genotyping of HPA with real-time PCR using displacing probes is more rapid and reliable compared with PCR-SSP and PCR-RFLP methods and is more affordable than existing real-time PCR-based HPA genotyping assays. Thus, our approach is more suitable for routine HPA analysis and ideal for both urgent clinical testing and high-throughput screening.

Gene frequencies of human platelet alloantigens in Bahraini Arabs

Human platelet antigens (HPA) are implicated in the pathophysiology of certain hematological disorders, and as varied distribution of HPA-1 alleles and genotypes were reported fordifferent countries and ethnic populations, we determined the distribution of HPA-1, -2, -3, -4, and -5 alleles, genotypes and haplotypes for 194 healthy Bahraini subjects by polymerase chain reaction with sequence specific primers. The distribution of the HPA polymorphisms was in Hardy-Weinberg equilibrium. Allele frequencies of 0.76 and 0.24 (HPA-1a and -1b), 0.77 and 0.23 (HPA-2a and -2b), 0.57 and 0.43 (HPA-3a and -3b), 0.93 and 0.07 (HPA-4a and -4b), and 0.86 and 0.13 (HPA-5a and -5b) were seen. With the exception of HPA-3a/a (30.4%), the frequencies of homozygous HPA-1a/a (56.8%), 2a/a (60.1%), 4a/a (87.2%), and 5a/a (75.7%) were higher than those of heterozygous (a/b) or homozygous (b/b) variants. Our results provide basic information for further studies of the HPA system polymorphism, which in turn will be instrumental in understanding and treating immune-mediated platelet disorders.

YS04 Recombinant Mini‐Β3 Integrin Fragments for the Detection of HPA Antibodies

Background The β3 integrin [Glycoprotein (GP) IIIa] gene is highly polymorphic and encodes eight of the Human Platelet Antigen (HPA) systems. HPA immunisation is of clinical relevance in neonatal alloimmune thrombocytopenia (NAIT), post‐transfusion purpura (PTP) and platelet refractoriness. However, the current methods of antibody detection are cumbersome and rely upon the use of platelets from genotyped donors. In the case of the rare HPA antigens, obtaining donor platelets is particularly problematic. We therefore set out to develop an antibody screening assay that uses recombinant, soluble β3‐integrin fragments expressing the HPA‐1, –4, –6, –7, –10, –11 and –16 epitopes.Methods A total of four integrin fragments of different lengths (ΔSDL, ΔβA, PSI‐Hybrid and PSI) were designed using structural data and expressed in Drosophila S2 cells. HPA‐1a (33) and HPA‐1b (P33) forms of all four proteins were expressed as were the fragments ΔSDL‐4b and ΔSDL‐SuperRare, the latter two for the detection of HPA‐4b and the rare HPA antigens (HPA‐6, 7, 10, 11 and 16) respectively. The recombinant proteins were then screened for reactivity with monoclonal antibodies (mAb) and polyclonal antisera containing anti‐HPA in an ELISA based assay.Results The reactivity pattern seen with murine mAbs Y2/51, SZ21 and CRC54 was as expected. Two of three human monoclonal HPA‐1a antibodies (19–7, 23–15, both from a PTP patient) reacted with the four L33 fragments but the third (CamTran007, derived from a NAIT patient) only reacted with the ΔSDL‐L33 and ΔβA‐L33 fragments, defining an epitope split. The reactivity of both potency (NIBSC‐03/152) and sensitivity (NIBSC‐93/710) standards for anti‐HPA‐1a detection were comparable or better than those seen by MAIPA. In addition, samples from 132 NAIT patients with anti‐HPA‐1a [15 with cerebral haemorrhage (CH), 49 with platelet counts <20 × 109 L−1 but no CH, 35 with counts ≥ 20 × 109 L−1 and 33 with antenatal treatment] were tested. Anti‐HPA‐1a was detected in 126 samples including all CH cases. The reactivity patterns against the four different β3‐integrin fragments did not correlate with clinical outcome. Finally, mini‐β3 integrins Δ SDL‐4b and ΔSDL‐SuperRare allowed the detection of 14 of the 15 anti‐HPA samples tested (5 HPA‐4a, 5 HPA‐4b, 1 HPA‐6b, 1 HPA‐7b and 2 of 3 HPA‐11b).Conclusions Recombinant fragments of the β3‐integrin expressing HPA antigens can be used to successfully detect platelet alloantibodies. These fragments with be of use in the screening and diagnosis of suspected cases of NAIT.

Gene frequencies of human platelet antigens in the Macedonian population

Human platelet antigen (HPA) systems consist of more than 12 bi-allelic antigen polymorphisms. Due to these polymorphisms, platelet-membrane glycoproteins can be recognized as alloantigens or autoantigens and can cause conditions such as fetomaternal alloimmune thrombocytopenia, post-transfusion refractoriness to platelets, and post-transfusion throbocytopenic purpura. The purpose of this study was to investigate the distribution of HPA-1, -2, -3, and -5 in Macedonian population by using the polymerase chain reaction and restriction fragment length polymorphism. The allele frequencies were 0.865 for HPA-1a, 0.135 for HPA-1b, 0.852 for HPA-2a, 0.148 for HPA-2b, 0.578 for HPA-3a, 0.422 for HPA-3b, 0.909 for HPA-5a, and 0.091 for HPA-5b. Results of our study were not significantly different from those reported in the other European studies. Our population displayed the highest frequency for HPA-2b allele (0.148) reported among European population.

The influence of human platelet antigen match on the success of allogeneic peripheral blood progenitor cell transplantation following a reduced‐intensity conditioning regimen

Allogeneic transplantation in elderly patients requires a dose-reduced conditioning regimen. Owing to reduced-intensity conditioning, host- and donor-type immune responses may affect the early posttransplant period, whereas only later on donor-derived reactions may ensue. Mismatches in the HLA system are known to be detrimental for the outcome of transplantation. Mismatches between donor and recipient for human platelet antigens (HPAs) may also affect the success of transplantation owing to serving as minor histocompatibility antigens and therefore rendering recipients at risk for graft-versus-host disease (GVHD) or graft rejection and inhibition of thrombopoiesis attributed to platelet (PLT) antibodies. Therefore, the occurrence of GVHD, incidence of relapse, need of PLT support, and outcome by analysis of 45 donor-recipient pairs for HPA-1, -2, -3, and -5 allotypes and screening for PLT antibodies were evaluated before transplantation and again 1 year thereafter. Mismatches within the HPA system were not associated with an increased occurrence of transplant-related mortality or GVHD, the onset of thrombopoiesis, the frequency of PLT transfusions, or the incidence of relapse. Neither were settings of homozygous donors versus heterozygous recipients (graft-vs.-host direction) nor homozygous recipients versus heterozygous donors (host-vs.-graft direction) associated with any adverse effects on the outcome of the transplantation. Thus, the HPA match does not affect the outcome of transplantation after reduced-intensity conditioning.

Gene Frequencies of the Human Platelet Antigen-3in Different Populations

Human platelet antigen (HPA) systems consist of more than 12 biallelic antigen polymorphisms in which a base pair substitution leads to change in an amino acid of a glycoprotein expressed on the platelet. HPA-3 is a HPA that is mentioned for possible induction of neonatal alloimmune thrombocytopenia, posttransfusion purpura, and platelet refractoriness. A summary is presented of previous reports on the gene frequencies of HPA-3 among different populations. The frequency of HPA-3a and -3b ranges from 0.50 to 0.61 and 0.38 to 0.50, respectively. A significant correlation between the population ethnicity and the gene frequencies was detected in this study. However, it is quite difficult to use HPA-3 gene as a gene marker to determine the similarity of gene population in different populations. In addition, the comparison of the heterogenicity of HPA-3 frequencies to another well-known HPA gene, HPA-1 gene demonstrates that there is a greater variation in HPA-3 frequencies than in the HPA-1 gene. There was no significant correlation between the incidence of autoimmune thrombocytopenia disorder and the HPA-3 gene polymorphism pattern.

The Influence of Human Plateley Antigen Match on the Success of Stem Cell Transplantation after Myeloablative or Reduced-Intensity Conditioning Regimen.

Background: Mismatches between donor and recipient for human platelet antigens (HPA) may affect the success of transplantation due to: a) serving as minor histocompatibility antigens and therefore render recipients at risk for GvHD, b) inhibition of thrombopoiesis due to platelet antibodies.Patients and Methods: We evaluated in 54 patients receiving hematopoietic stem cell transplantation (HSC T) from HLA matched siblings after a myeloablative conditioning regimen the occurrence of GvHD, transplant related mortality (TRM), and the need of platelet support by prospective analyzes of donor-recipient pairs for HPA -1, -2, -3, and -5 allotypes. Patients were screened for platelet antibodies prior to transplantation and in weekly intervals until day 100 after transplantation. Forty five patients receiving HSCT from related or unrelated donors after a reduced-intensity conditioning regimen (RIC) were enrolled retrospectively after a median observation time of 372 days.Results: Neither the incidence of GvHD and TRM, nor the onset of thrombopoiesis, nor the CCI after platelet transfusions, nor the frequency of platelet transfusions were affected by HPA mismatches. TRM among patients receiving a RIC was higher when grafts were from unrelated donors with 2 or more mismatches. Six of 7 patients who died due to TRM had 2 mismatches in the HPA system, although TRM was not significantly associated with mismatched HPA (p = 0.06). However, within donor-recipient pairs with more than one HPA mismatch TRM occurred when grafts were from unrelated donors (p = 0.03).Conclusion: Thus, the HPA match does not affect the success of transplantation.

Gene frequencies of the HPA-1 to HPA-13, Oe and Gov platelet antigen alleles in Taiwanese, Indonesian, Filipino and Thai populations.

Human platelet antigen (HPA) systems consist of more than twelve bi-allelic antigen polymorphisms in which a base pair substitution leads to change in an amino acid of a glycoprotein expressed on the platelet. The neonatal alloimmune thrombocytopenia (NAIT), post transfusion purpura, and refractoriness to platelet transfusion can be induced by antibodies against human platelet antigens: e.g. HPA-1a, 3a, 4a, 5a, and Gova. HPA typing is essential for the diagnosis and treatment of a variety of diseases. We developed a PCR-based method to detect HPA-1 to HPA-13, Oe and Gov platelet alloantigens. In this method, the amplified PCR products were used to recognize the polymorphism after restriction enzyme digestions. Among 566 Taiwanese, 107 Indonesian, 100 Filipino and 137 Thai subjects studied, HPA-1a, 2a, 4a, 5a, 6a, 7aW, 8aW, 9a, 10a, 11a, 12a, 13a, Oea genes were present in every sample; while HPA-1b, 2b, 4b, 5b and 6b were rarely found. HPA-7aW, 8aW, 9, 10, 11, 12, 13, and Oea alleles were noted to be monomorphic only. HPA-3a/3b alleles had frequencies of 0.595/0.405, 0.505/0.495, 0.507/0.493, 0.530/0.470, while Gova/Govb of 0.462/0.538, 0.450/0.550, 0.463/0.537, 0.520/0.480 among Taiwanese, Indonesians, Thais and Filipinos respectively. The prevalence rates of HPA-1 to 13 in this study were also consistent with other previous reports using different methods. The alloimmunization due to Gov and HPA-3 antigens need to be emphasized in these populations.

Rapid and reliable genotyping of human platelet antigen (HPA)-1, -2, -3, -4, and -5 a/b and Gov a/b by melting curve analysis.

The probability for occurrence of neonatal alloimmune thrombocytopenic purpura (NAITP) depends largely on the frequency of each individual phenotype in various populations. In caucasians, antibodies to human platelet antigen (HPA)-1a are the major cause of neonatal alloimmune thrombocytopenic purpura, whereas in the Japanese population, antibodies to HPA-4b is most frequently involved in NAITP. Conventional PCR techniques for platelet antigen genotyping rely on sequence-specific primers (SSPs) and detection by gel electrophoresis, a method which is laborious and time consuming. New PCR technology, measuring the match of a hybridization probe with its target and thereby allowing simultaneous detection of both alleles, provides an efficient tool for genotyping of the HPA systems. A total of 105 healthy blood donors were genotyped for HPA-1, -2, -3, -4, and -5 a/b and Gov a/b with new primers and probes designed for mutation detection by melting curve analysis (using LightCycler technology). Donor DNA was independently genotyped by an allele-specific assay, using SSPs, in a reference laboratory. There was full concordance between the two genotyping methods, and genotype frequencies were comparable with previous studies in caucasians. We present rapid and reliable detection systems for HPA-1, -2, -3, -4, and -5 a/b and Gov a/b based on mutation detection of both alleles simultaneously by melting curve analysis. As the Gov system has been reported to have similar frequency of involvement in alloimmune thrombocytopenia as HPA-5, the opportunity for genotyping should aid the diagnosis of such patients.

Genotyping for platelet‐specific antigens: techniques for the detection of single nucleotide polymorphisms

Accurate typing of patients for platelet-specific (human platelet) antigens (HPA) is required in several different clinical situations, and blood services need to maintain panels of HPA-typed apheresis platelet donors and whole-blood donors to support HPA alloimmunized patients. Six clinically relevant HPA alloantigen systems have been described and, in addition, a significant number of HPA alloantigens with a highly skewed allele frequency or of very low immunogenicity have been reported. Certain well-characterized biallelic systems such as Gov have not as yet been included in the HPA nomenclature but are included in this review. Biochemical studies have identified the platelet membrane proteins on which the HPA antigens are localized. Cloning of the genes encoding these proteins and the realization that there is adequate mRNA in fresh platelets has led to identification of the molecular basis of HPA antigens over the last decade. All but one of the biallelic platelet-specific alloantigen systems are based on a single nucleotide polymorphism in the DNA sequence, corresponding to a single amino acid substitution in the encoded primary protein sequence. The discovery of the genetic basis of the alloantigens has allowed the development of polymerase chain reaction-based techniques for HPA genotyping using genomic DNA. The genetic basis of the HPA alloantigens, the most commonly used genome typing techniques and their pitfalls, and future developments, are discussed in this review.

Gene frequencies of the HPA-1 to HPA-8w platelet antigen alleles in Taiwanese, Indonesian, and Thai.

Human platelet antigen (HPA) systems consist of more than eight biallelic antigen polymorphisms in which a base pair substitution leads to change in an amino acid of a glycoprotein expressed on the platelet. HPA typing is essential in the diagnosis and treatment for a variety of diseases. We developed a polymerase chain reaction (PCR)-based method to detect HPA-1 through HPA-8w. In this method, the amplified PCR products were used to recognize the polymorphism after restriction enzyme digestions. Among 295 Taiwanese, 107 Indonesian, and 137 Thai subjects studied, HPA-1a, 2a, 4a, 5a, 6a, 7aw, and 8aw genes were present in every sample tested. HPA-1b, 2b, 4b, 5b, and 6b were rarely found among subjects. Only monomorphic HPA-7aw and 8aw alleles were noted in the samples. HPA-3a and 3b alleles showed frequencies of 0.595/0.405, 0.504/0.496, and 0.507/0.493 in Taiwanese, Indonesian, and Thai subjects, respectively. Our report is the first PCR-based method to detect most of the HPA antigen variants in Taiwanese, Indonesian, and Thai. The genomic typing results were also confirmed by direct sequencing for uncertain and some representative cases. The prevalence rates of HPA-1, 2, 3, 4, and 5 in this study were also consistent with other previous reports using different methods.

Neonatal Alloimmune Thrombocytopenia

Neonatal alloimmune thrombocytopenia (NAIT) can occur when a mother is immunized against fetal platelet antigens inherited from the father. Early diagnosis and appropriate platelet transfusion therapy are essential to prevent life-threatening intracranial hemorrhage in the thrombocytopenic fetus or neonate. Five major human platelet antigen (HPA) systems are capable of causing this disorder with HPA-1a indicated most frequently. This article reviews the pathophysiology, clinical aspects, and management of NAIT. We also present our experience with treatment of neonates affected with this disorder.

Fetomaternal alloimmune thrombocytopenia

Thrombocytopenia is the second commonest haematological abnormality in the neonatal period after anaemia due to iatrogenic blood letting. One to four percent of all newborn babies have a platelet count <150×10 9/l at birth and approximately 20–40% of neonates in intensive care units are affected by neonatal thrombocytopenia. The most common cause of severe neonatal thrombocytopenia is fetomaternal platelet incompatibility and subsequent alloimmunisation. During the last decade recent advances in molecular techniques have led to rapid and efficient methods for diagnosis. Progress in fetal medicine has enabled accurate determination of fetal status, allowing improvements in fetal diagnosis and therapy. Human platelet antigen (HPA)-1a is by far the most frequently involved platelet antigen system in Caucasians accounting for 90% of cases, followed at a much lower frequency by HPA-5b (5–15%) and HPA-3a. The incidence is estimated to be 1 per 2000 to 1 per 5000 live births, but this is low in comparison to the incidence of fetomaternal platelet antigen incompatibility especially for the HPA-1 alloantigen system in the Caucasian population in whom the estimated frequency of HPA-1b1b individuals is 2%. Retrospective and prospective studies have reported that the immunogenetic background is important, and the chance of HPA-1a alloimmunisation is strongly associated with maternal HLA class II DRB3 ∗ 0101 (DR52a) type. A significant association ( p=0.004) between severe thrombocytopenia and a third trimester antiHPA titre >1:32 has been observed. It is now possible to genotype the fetus or neonate and the parents, which provides confirmation as to which HPA systems are incompatible between the mother and father. Simultaneous genotyping of HPA-1, 2, 3 and 5 can be carried out using the polymerase chain reaction–sequence specific primers (PCR–SSP) protocol, which has been widely used for HLA class II determination. The platelet count may continue to fall during the first 48 h after birth and the risk of intracranial bleeding is highest during this period. The best option is transfusion of specially selected antigen negative compatible donor platelets or if unavailable, maternal washed platelets. Antenatal screening for the most common form of fetomaternal alloimmune thrombocytopenia (FMAIT), due to antiHPA-1a is under consideration, but there is no established method at present. The Scottish National Blood Transfusion Service started a study in August 1999 on 25,000 pregnancies to carry out a cost benefit analysis of routine antenatal screening. The aims of the study are to determine the frequency of HPA-1b homozygosity; monitor antibody titres during pregnancy and confirm correlation of antibody emergence with HLA-DRB3 ∗ 0101, and finally to access cost effectiveness of routine screening across Scotland. Of 26,509 women screened in three Scottish regions 501 (1.9%) are HPA-1b homozygous and about 9% of the consented women are antibody positive.