Human Platelet Antigen Genotyping Research Articles

PLT antigen discrepancy pattern among couples with recurrent abortion.

Recurrent abortion refers to a condition of two or more consecutive pregnancies without known etiology affected by miscarriage before the completion of the 20th week of gestational age. However, several hypotheses have been proposed, but not much data are available concerning the relationship between human platelet antigens (HPAs) polymorphisms and recurrent abortion. This study was conducted to evaluate the genetic differences between HPA-1, -2, -3, -5, and - 15 in Iranian couples with a history of recurrent abortion. In this cross-sectional study, a total of 74 couples with at least 2 recurrent abortions without any known specified reasons enrolled in the study. HPA polymorphisms genotyping was performed by single-specific primer PCR. Genotype frequency was calculated using the Hardy-Weinberg equation. A total of 39 couples (52.7%) had HPA genotyping partial mismatches. The most common partial mismatch pairs were found concomitantly on both HPA-15a and HPA-15b in three couples (4%), followed by two (2.7%) on HPA-3a and one (1.3%) in each HPA-2b and HPA-5b. There was a deviation from the Hardy-Weinberg equilibrium in the HPA-2 and -5 systems. The present study declared that partial mismatches of HPA-3 and -15 genotypes were common among Iranian couples due to the history of recurrent abortion and approximately half of the couples carried at least one HPA gene that was absent in their partners. Further studies might be helpful to clarify the association between HPA polymorphisms and recurrent abortion, such as an investigation into the alloantibodies against HPAs.

HPA1-29w Genotyping and the Foundation for the Platelet Apheresis Registry in Jiangsu Province of China by MassARRAY Spectrometry

Introduction: This study aimed to investigate the allele frequencies of the human platelet antigens (HPA) HPA-1-29w system in Jiangsu (China) and establish the platelet apheresis registry in blood donors. Methods: HPA genotyping was performed using the MassARRAY iPLEX^® platform. Allele and genotype frequencies were estimated by direct counting and tested for Hardy-Weinberg equilibrium. The transfusion mismatch probability was calculated for every HPA specificity. Results: The HPA allele frequencies in the Jiangsu Han population of HPA-1b, -2b, -3b, -4b, -5b, -6b, -11b, -15b, and -21b were 0.0055, 0.0530, 0.4116, 0.0015, 0.0155, 0.0162, 0.0003, 0.4683, and 0.0070, respectively, in which a heterozygote of HPA-11a/b was first detected in China. Only allele a was detected for HPA-7-10w,-12-14w,-16-20w, and -22-29w quasi-systems. The highest mismatch rate of HPA genes in 1,640 platelet donors was the HPA-15 system, followed by the HPA-3 system with a rate of 37.4% and 36.71%, respectively. Conclusion: China’s largest-scale platelet registry of HPA-1-29w has been explored. The MassARRAY platform may help found the platelet apheresis registry which would be useful to provide matching platelets and lead to a more accurate, effective, and safe transfusion for patients with platelet therapy.

Association of the human platelet antigens polymorphisms with platelet count in patients with COVID-19.

Polymorphism in human platelet antigen (HPA)-1 and HPA-3 (GPIIb/IIIa), HPA-2 (GPIb/IX), HPA-4 (GPIIIa), HPA-5 (GPIa/IIa), & HPA-15 (CD109) was investigated in 86 COVID-19-infected patients with thrombocytopenia (Group A) and 136 COVID-19-infected patients without thrombocytopenia (Group B). HPA genotyping was done by the sequence-specific primers PCR method. Lower HPA-3a and higher HPA-3b (P = 0.028) allele frequencies were seen in Group A than in Group B, and homozygosity for HPA 3b (P = 0.038) alleles was more prevalent in Group A than in Group B. The allele and genotype distributions of the other HPA polymorphic variants were similar between the two groups. Univariate analysis identified the CCGGGC (P = 0.016) combined genotype to be negatively associated & the TCGGGC (P = 0.003) and CCGGGC (P = 0.003) to be positively associated with thrombocytopenia. The frequency of anti-HPA-1a and anti-HPA-3a antibodies was significantly higher in all patients compared to other anti-HPAs antibodies (P < 0.05). These results highlight the role of HPAs in the thrombocytopenia of COVID-19 infected patients. This is the first evidence demonstrating the differential association of the six common HPA gene variants and specific HPA genotype combinations with thrombocytopenia in COVID-19-infected patients.

Simultaneous genotyping for human platelet antigen systems and HLA-A and HLA-B loci by targeted next-generation sequencing.

In order to treat the alloimmunization platelet transfusion refractoriness (PTR), human leukocyte antigen (HLA)-type and/or human platelet antigen (HPA)-type matched platelets between donors and patients are usually used. Therefore, genotyping of HLA-A and HLA-B loci, as well as HPA systems, for donors and patients, is of great significance. However, there is a rare report of genotyping for HLA-A and HLA-B loci as well as HPA systems at the same time. In this study, a high-throughput method for simultaneous genotyping of HLA-A and HLA-B loci, as well as HPA genotyping, was developed. A RNA capture probe panel was designed covering all exon sequences of the GP1BA, GP1BB, ITGA2, CD109, ITGB3, and ITGA2B genes and HLA-A and HLA-B loci. The HLA-A, HLA-B, and 34 HPA systems were genotyped using a targeted next-generation sequencing (NGS) method. The genotypes of the HLA-A and HLA-B loci, as well as the HPA, were assigned based on the nucleotides in the polymorphism sites. Using the NGS method, 204 unrelated blood specimens were successfully genotyped for all 34 HPA systems as well as HLA-A and HLA-B loci. The accuracy of the NGS method was 100%. Only HPA-2, HPA-3, HPA-5, HPA-6w, HPA-15, and HPA-21w showed polymorphism with frequencies of 0.9412, 0.6863, 0.9853, 0.9779, 0.4314, and 0.9951 for a allele, respectively. Thirty-two single nucleotide variants (SNVs) were detected. Of them, 12 SNVs can lead to amino acid change. HLA-A*11:01 and HLA-B*46:01 are the most common alleles for HLA-A and HLA-B loci. A targeted next-generation sequencing method for simultaneously genotyping HPA systems and HLA-A and HLA-B loci was first established, which could be used to create a database of HLA-typed and/or HPA-typed unrelated donors.

Next Generation Sequencing of Human Platelet Antigens for Routine Clinical Investigations and Donor Screening.

Human platelet antigen (HPA) genotyping is performed in a number of clinical scenarios, including characterization of immune-mediated thrombocytopenia and provision of HPA-matched platelets. Current gold-standard methods for HPA genotyping utilize single nucleotide variant (SNV) based approaches. This review aims to ascertain if next generation sequencing (NGS) has reasonable grounds to replace SNV-based genotyping for HPA systems. A systematic review was conducted following a comprehensive literature search in accordance with the Preferred Reporting Items for Systematic Review and Meta-Analysis guidelines. Studies were subjected to screening based on a defined set of inclusion/exclusion criteria. Study quality, characteristics and results were extracted and a meta-analysis was performed to assess the concordance of HPA genotyping results between NGS and the SNV-based comparators for HPA-1,-2,-3,-4,-5,-15. In total, 3374 potentially eligible articles were identified, only 6 of which were included in the meta-analysis. The pooled proportion agreement for the overall concordance of the 6 included studies was shown to be 0.998, 95%CI [0.995, 0.999], P < .001. The discrepancies between HPA genotypes obtained by the two platforms were due to allele dropout in real-time PCR, thus discordant results were in favor of NGS over SNV-based comparators. Currently available platforms for NGS are not without their limitations, including high upfront and ongoing costs, data management and storage, accurate variant calling and availability of appropriately trained staff. Despite the high level of concordance between NGS and current gold-standard methods, these significant challenges mean that NGS is currently not viable as a stand-alone technique for HPA typing.

Assessment of Gene Frequencies of Human Platelet Alloantigens in Rivers-State, Nigeria Based on ABO/Rhesus Blood Groups Distribution

Aim: The aim of this study was to assess the Gene Frequencies of Human Platelet Alloantigens in Rivers-State, Nigeria based on ABO/Rhesus blood groups distribution&#x0D; Study Design: A randomized controlled trial.&#x0D; Place and Duration of Study: Rivers State University Medical Centre, Port Harcourt, Safety Molecular Pathology Laboratory, Enugu State, Justcare clinical laboratory Port Harcourt Rivers State and University of Port Harcourt Teaching Hospital, between October 2019 and March 2020.&#x0D; Methodology: The subjects consisted of apparently healthy individuals who were of Rivers State origin totaling 104 persons aged 17 to 42 years. They were under-graduate and post graduate students of Rivers State University of Port Harcourt. Five major ethnic groups were considered which included Ikwerre, Ogoni, Ijaw, Etche and Ogba. Their demographic information was collected using a sample register and a questionnaire. Samples were collected from the antecubital vein. 10ml of blood was collected, 5ml was transferred into EDTA sample bottle (Ethylene diamine tetracetic acid) while 2ml was dispensed into plain bottle and labeled accordingly. Serological testing including HIV (RVS) screening, HBsag, HCV and VDRL were all as part of the inclusion criteria immediately after samples were collected. The remaining sample was analyzed using genotyping of Human Platelet Antigens by High Resolution Melting Curve Analysis Polymerase Chain Reaction (HRM-PCR), while tile method also known as forward/cell grouping method which is based on haem-agglutination reaction was used for ABO/Rh blood grouping. The melt curve analysis was done using the MicPCR software while the frequency analysis was done using Number Cruncher Statistical Software (NCSS) Version 13. GraphPad Prism Version 8.0.2 was used to determine the statistical significance between the various HPA genotypes and the ethnic groups and p-values of &lt; 0.05 were considered to be statistically significant. Results were presented in percentages, mean+/- standard deviation and in tables&#x0D; Results: The results showed that the A+ blood group had highest frequencies of 19.2% and 17.7% for HPA-5 b/b and HPA-4 a/a, while the least was 0.8% each for HPA-3 a/a, HPA-4 b/b and HPA-5. For blood group B+, the highest were 20.0% (HPA-5 b/b) and 16.7% (HPA-3 b/b), and the least were 5.0% each for HPA-1 b/b and HPA-4 a/b, while blood group B- had highest frequencies for HPA-1 a/a, HPA-2 b/b, HPA-3 b/b, HPA-4 a/b and HPA-5 b/b (20.0% each). The blood group O+ HPA gene patterns had their highest values at 19.7% (HPA-5 b/b), 16.5% (HPA-4 a/a) and 13.7% (HPA-3 b/b) and the least was 7.9% (HPA-1 a/b), while for the blood group O-, the highest was observed for HPA-3 b/b and HPA-5 b/b (20.0% each) and the least for HPA-1 a/a and a/b, HPA-2 a/b and b/b, and HPA-4 a/b and b/b (10.0% each).&#x0D; Conclusion: Based on the results, we conclude that A+ blood group had highest HPA frequencies. Whilst, the highest for blood group B+ were (HPA-5 b/b) and (HPA-3 b/b), and blood group B- had highest frequencies for HPA-1 a/a, HPA-2 b/b, HPA-3 b/b, HPA-4 a/b and HPA-5 b/b. The blood group O+ HPA gene patterns had their highest values (HPA-5 b/b), (HPA-4 a/a) and (HPA-3 b/b) and the least was (HPA-1 a/b), while for the blood group O-, the highest was observed for HPA-3 b/b and HPA-5 b/b and the least for HPA-1 a/a and a/b, HPA-2 a/b and b/b, and HPA-4 a/b and b/b.

Genotyping of Eight Human Platelet Antigen Systems in Serbian Blood Donors: Foundation for Platelet Apheresis Registry

Introduction: The aim of this study was to investigate the allele and genotype frequencies of 8 human platelet antigen (HPA) systems among blood donors from the Blood Transfusion Institute of Serbia and to compare them with published studies. These data would be useful to establish the basis for a platelet apheresis donor registry. Material and Methods: Seventy-two unrelated male platelet apheresis/blood donors from Serbia were typed for 8 HPA systems (HPA-1 to HPA-6, HPA-9, and HPA-15) via the FluoGene method, based on polymerase chain reaction-sequence-specific amplification (PCR-SSP; PCR using sequence-specific primers) with fluorometric signal detection. Allele and genotype frequencies were estimated by direct counting and compared to the expected genotype frequencies according to the Hardy-Weinberg principle. The transfusion mismatch probability was calculated for every HPA specificity. Results: The allele frequencies were: HPA-1a, 0.868; HPA-1b, 0.132; HPA-2a, 0.917; HPA-2b, 0.083; HPA-3a, 0.611; HPA-3b, 0.389; HPA-5a, 0.903; HPA-5b, 0.097; HPA-9a, 0.993; HPA-9b, 0.007; HPA-15a, 0.472; and HPA-15b, 0.528. For HPA-4 and HPA-6 only allele a was detected. Discussion: The HPA allele frequencies of European populations showed no significant differences in comparison with our results. Statistically significant differences were revealed in comparison with some populations of non-European origin. In the tested donors no HPA-2 bb genotype was detected, but we found 1 donor with the rare HPA-9b allele. The biggest transfusion mismatch probability in the Serbian population is for systems HPA-15 (37.4%) and HPA-3 (36.2%), which means that more than a third of random transfusions could cause mismatch in these systems. This study was enabled by the introduction of molecular HPA typing, and it provides initial results of the HPA allele and genotype frequencies in the population of blood donors in Serbia. They will be used to provide a compatible blood supply on demand for treating patients with alloimmune thrombocytopenic disorders. The successful implementation of PCR-SSP with fluorometric signal detection could be further complemented in the future by the introduction of high-throughput methods, which will largely depend on the available financial resources.

Human platelet antigens polymorphisms: Association to primary immune thrombocytopenia in the Iranian patients

Background: Human platelet antigens (HPAs) are part of platelet GP complexes have the potential to contribute to the autoantibody production. Moreover, these antigens demonstrate different patterns of distribution on different ethnic groups and variation in some types of diseases. This study was objected to determine the incidence of HPA-1 to -5 and -15 polymorphisms in the Iranians suffering from primary Immune thrombocytopenic purpura (ITP). &#x0D; Materials and Methods: In this case-control investigation, 30 patients by definite primary ITP were randomly selected and enrolled in the study. HPA genotyping was performed implicating by the Single Specific Primer PCR (SSP-PCR). For the control group, data of recently published gene polymorphism among Iranian Blood donors were deployed for comparison. &#x0D; Results: The incidence of HPA-1 to -5 and -15 polymorphisms in the Iranian patients with primary ITP was found to be: HPA-1a/1a: 0.933, HPA-1a/1b: 0.067, HPA-2a/2a: 0.133, HPA-2a/2b: 0.867, HPA-3a/3a: 0.2, HPA-3a/3b: 0.533, HPA-3b/3b: 0.267, HPA-4a/4a: 1, HPA-5a/5a: 0.967, HPA-5a/5b: 0.330, HPA-15a/15a: 0.166, HPA-15a/15b: 0.667 &amp; HPA-15b/15b: 0.167. &#x0D; Conclusion: This study provides special new data on the distribution of HPA allele among the Iranians ITP patients.Furthermore, it might useful toccharacterize understanding more presizely about ITP and HPA distribution. However, further studies concerning platelet immunology are needed to do help on best practice on management of immune diseases triggered by platelet antibodies.

Analysis on Polymorphism of Platelet Antigen Gene in Shandong Han Population

To study the Polymorphism of the human platelet antigen(HPA) gene 1-17 and human leukocyte antigen(HLA) gene-A and B locus in Shandong Han population. A total of 962 samples from routine voluntary platelet donors were genotyped for HPA1-17 system and HLA-A site, B by PCR-SSP and PCR-SSOP respectively．Gene frequencies were calculated by counting. HPA1-17 and HLA genotype combinations were analyzed by Arelequin 3.5. The gene frequencies of HPA-la, -1b, HPA-2a, -2b, HPA-3a, -3b, HPA-4a, -4b, HPA-5a, -5b, HPA-6a, -6b, HPA-15a, -15b were 0.9918, 0.0082, 0.9419, 0.0592, 0.5841, 0.4174, 0.9969, 0.0031, 0.9892, 0.0108, 0.9835, 0.0175,0.5488 and 0.4512, respectively． The most common HPA genotype combination was HPA-(1, 2, 4, 5, 6, 7-14, 16, 17) aa-3ab-15ab (0.2048). Moreover, HLA-A*2(0.3094) and HLA-B*13(0.1513) showed the highest frequency in their respective locus. The most common HLA genotype combination was HLA-A*2-B*13(0.1397) . Distributions of HPA and HLA show high polymorphism in Shandong Han population. The ethnic and territorial difference of HPA distribution is also confirmed. It is imperative to establish local genetic database of volunteer platelet donors.

Establishment and Application of HPA1-6, 15 Platelet Donor Bank in Beijing Area

To establise the bank of platelet donors with the human platelet antigen (HPA) 1-6, 15 genes so as to provide the HPA-matched platelets for the patients. The HPA genotyping of platelets donors and patients with platelet antibody positive confirmed by sercening was performed by using the SSP-PCR; the efficacy of transfusing the HPA-matched platelets for 37 cases platelet antibody positive was analyzed. The most common genotype in platelet donors were HPA-1a/1a-2a/2a-3a/3b-4a/4a-5a/5a-6a/6a-15a/15b, followed by HPA-1a/1a-2a/2a-3a/3a-4a/4a-5a/5a-6a/6a-15a/15b; the most common genotype in 53 cases of platelet antibody positive confirened by screening were HPA-1a/1a-2a/2a-3a/3b-4a/4a-5a/5a-6a/6a-15a/15b. Among 37 patients with platelet antibody positive confirened by screeming, 28 showed that the transfusion of HPA-matched platelets was effective with statistically significant difference in comparison with random transfusion group. The HPA-3, HPA-15 were the main factors leading to polymorphisms. HPA-3 and HPA-15 are polymorphic, which should be focused on. HPA-matched platelets can improve the efficiency of platelet transfusion, and avoid the waste of blood resources. The genotypes of platelet donors can basically meet the requirements for common genotype transfusion.

Human platelet antigen polymorphisms and the risk of chronic Chagas disease cardiomyopathy

Human platelet antigen (HPA) polymorphisms are considered to be a risk factor for cardiac and vascular diseases, but the role of HPA in chronic Chagas disease cardiomyopathy (CCC) is not available. Therefore, the aim of this study was to investigate the association of HPA polymorphisms, HPA-1, HPA-2, HPA-3, HPA-5 and HPA-15, in the severity of left ventricular systolic dysfunction (LVSD) in CCC patients. For this, 229 CCC patients were separated into three groups: without LVSD, mild/moderate LVSD and severe LVSD. PCR-SSP was performed for HPA genotyping and the risk was assessed using SNPStats software. HPA-1 allele and genotype frequencies were lower in mild/moderate LVSD patients compared to other groups, without statistical significance. After stratified analyzes, the HPA-3a/3b genotype frequency was lower in women with severe LVSD compared to those without LVSD (OR:0.29; 95% CI: 0.10–0.84). In conclusion, HPA-3 variant could be a protection factor for CCC in the female patients.

Neonatal alloimmune thrombocytopenia caused by anti-HPA antibodies in pregnant Chinese women: a study protocol for a multicentre, prospective cohort trial

BackgroundNeonatal alloimmune thrombocytopenia (NAIT), caused by maternal antibodies raised against alloantigens carried on foetal platelets, is a very common haematological abnormality in newborns worldwide. However, baseline data on NAIT in China are lacking. Therefore, this study seeks to explore the incidence of alloantibody against the human platelet antigen (HPA) in pregnant women and its associations with NAIT in China.MethodsA multicentre, prospective cohort study design will be used, and 55,497 pregnant women will be recruited for the first screening of the anti-HPA antibody at 12 to 28 weeks of gestational age. Subjects who are positive in the first screening for the anti-HPA antibody will be included in the exposure group. Re-tests of the antibody titre, antigen-specificity and genotyping of HPA and HLA will be conducted during admission. A ratio of 1:1 paired individuals with the same ethnicity and parity but testing negative for the anti-HPA antibody will be randomly selected to be included in the non-exposure group. NAIT will be diagnosed in the newborns on day one of the birth. The HPA of the neonates in the exposure group will also be genotyped by sequencing. Associations of maternal HLA with the occurrence of the anti-HPA antibody and correlation of the severity of NAIT with the titre of the anti-HPA antibody will be further analysed.DiscussionThe study is expected to provide baseline data on NAIT in China. Besides, we hope to find out a population who expresses particular HLA molecules has significant higher risk of HPA alloimmunization in Chinese individuals. We also hope to find a Chinese-specific cut-off antibody titre for the prediction of the severity of NAIT and to provide a means to evaluate the necessity of antenatal treatment.Trial registrationClinicalTrials.gov: NCT02934906 (date registered: 13.10.2016).

Simultaneous human platelet antigen genotyping and detection of novel single nucleotide polymorphisms by targeted next‐generation sequencing

Twenty-nine human platelet antigen systems have been described to date, but the majority of current genotyping methods are restricted to the identification of those most commonly associated with alloantibody production in a clinical context. This can result in a protracted investigation if causative human platelet antigens are rare or novel. A targeted next-generation sequencing approach was designed to detect all known human platelet antigens with the additional capability of identifying novel mutations in the encoding genes. A targeted enrichment, high-sensitivity HaloPlex assay was designed to sequence all exons and flanking regions of the six genes known to encode human platelet antigens. Indexed DNA libraries were prepared from 47 previously human platelet antigen-genotyped samples and subsequently combined into one of three pools for sequencing on an Illumina MiSeq platform. The generated FASTQ files were aligned and scrutinized for each human platelet antigen polymorphism using SureCall data analysis software. Forty-six samples were successfully genotyped for human platelet antigens 1 through 29bw, with an average per base coverage depth of 1144. Concordance with historical human platelet antigen genotypes was 100%. A putative novel mutation in Exon 10 of the integrin β-3 (ITGB3) gene from an unsolved case of fetal neonatal alloimmune thrombocytopenia was also detected. A next-generation sequencing-based method that can accurately define all known human platelet antigen polymorphisms was developed. With the ability to sequence up to 96 samples simultaneously, our HaloPlex design could be used for high-throughput human platelet antigen genotyping. This method is also applicable for investigating fetal neonatal alloimmune thrombocytopenia when rare or novel human platelet antigens are suspected.

Gene frequency of human platelet alloantigens‐1 to ‐6 and ‐15 in Saudi blood donors

Human platelet antigens (HPAs) are involved in the pathogenesis of several clinical conditions, such as platelet transfusion purpura (PTP), refractoriness to platelet transfusion and neonatal alloimmune thrombocytopenia (NAITP). Typing of HPA (1-6 and 15) has not been carried on the Saudi population. This is the first study of all the seven HPA systems on Arabs. The aim of this prospective study was to determine the frequency of HPA (1-6 and 15) in Saudis. A total of 100 randomly selected Saudi blood donor samples were genotyped using the polymerase chain reaction with sequence-specific primers (PCR-SSP). The most common HPA genotypes among Saudis were HPA-1 a + b- (75%), HPA-2 a + b- (62%), HPA-3 a + b- (51·5%), HPA-4 a + b- (99%), HPA-5 a + b- (76·5%), HPA-6 a + b- (100%) and HPA-15 a + b + (50%). The prevalent allele among the HPA systems was (a), except in the HPA-15 system where the (b) allele was found in 52% of the subjects. Comparisons with other ethnic populations uncovered marked differences in the distribution of HPA alleles. Studying the prevalence of HPA antigens in Saudi population will help in the understanding of its role in platelet-related disorders. It will also enable the blood bank to establish an HPA-based donor registry that will be a valuable source of compatible platelet-therapeutic products to alloimmunised patients. This will also enhance the safety and efficacy of platelet transfusion. This data obtained will form an addition to the existing body of literature in transfusion research.

Declined presentation Human platelet antigen genotype : effect on aspirinated platelets in patients with essential thrombocythemia

Essential thrombocythemia (ET) is a myeloid neoplasm characterized by platelet hyperaggregation. Low-dose aspirin (ASA) is the standard treatment to normal platelet function and to prevent the thrombosis. It is debated if inherited thrombophilia affects the ASA platelet responsiveness. Therefore, we evaluated human platelet antigen-1 (HPA-1) gene polymorphism, as thrombophilic molecular mutation associated with platelet hyperfunction, platelet count, b-thromboglobulin (b-TG) and platelet factor 4 (PF4), as markers of platelet activation, the platelet functional activity (PFA) as indicator of ASA platelet sensitivity, clot formation time (CFT) and the maximum clot firmness (MCF), as indicator of aspirinated platelet contribution to clot firmness. We studied 48 patients (16 men, 32 women; mean age 51 years, range 32-70) with ET according to WHO criteria. The mean duration of disease was 11 years. All patients were on ASA 100 mg once daily. Fifty subjects served as controls. The HPA-1 genotype was determined using a commercialized polymerase chain reaction kit with sequence-specific primers. Platelets were measured by automated analyzer. b-TG and PF4 were determined by ELISA. ASA platelet sensitivity, CFT and MCF were measured by Platelet Function Analyzer (PFA-100) and by ROTEM delta, respectively. Of 48 patients, 36 were HPA-1a/1a and 12 were HPA-1a/1b. The mean platelet count was 462±240x109/L. All patients had high b-TG and PF4 (244±15 IU/ml vs 20±11 IU/ml and 162±56 IU/ml vs 6±2 IU/ml, respectively) (p<.0001 and p<.0001, respectively), prolonged C/EPI closure time (CT, unit: s, n.v. 84-160 s) (252±48 s), normal CFT (CFT, unit: s, n.v. 30-110 s) (50±7s) and MCF (MCF, unit: mm, n.v. 50-72 mm) (71±2 mm). These findings suggest that HPA-1 gene polymorphism does not affect aspirinated platelets responsiveness in patients with ET as assessed by PFA-100 and thromboelastometry.

Human platelet antigen genotyping of platelet donors in southern Brazil.

Human platelet antigens (HPA) are immunogenic structures that result from single nucleotide polymorphisms (SNPs) leading to single amino acid substitutions. This study sought to determine the allele and genotype frequencies of HPA-1, HPA-2, HPA-3, HPA-4, HPA-5 and HPA-15 in platelet donors from the state of Rio Grande do Sul (RS), Brazil, and compare their allele frequencies to those observed in other populations. HPA genotyping was performed by PCR-SSP method. The study sample comprised 201 platelet donors (167 Caucasians and 34 non-Caucasians). Allele 'a' was that most commonly found for HPA-1 to 5 in both groups. The HPA-15ab genotype predominated over homozygous genotypes of this system. Fisher's exact test revealed statistically significant differences for the HPA-5 system, with a greater prevalence of the HPA-5b allele in non-Caucasians. The neighbour-joining method and principal components analysis revealed genetic proximity between our Caucasian group and European populations. We conclude that the allele frequencies of HPA-1 to 5 and HPA-15 found in our Caucasian sample are similar to those reported for European populations. These findings corroborate the ethnic makeup of the population of RS. The higher frequency of the HPA-5b allele found in the non-Caucasian group of our sample suggests the possibility of allosensitization in patients who receive platelet transfusions from genetically incompatible donors.

Report of the First Workshop of the ISBT International Platelet Immunobiology Working Party, Asia Regional

Background and ObjectivesThe aims of the first workshop of the International Platelet Immunology Working Party, Asia Regional (IPIWP‐AR), were to identify antibodies against human platelet antigen (HPA), to compare laboratory performance in the detection of the antibodies and to help laboratories to audit and standardize the own in‐house platelet DNA genotyping methods.Materials and MethodsFourteen laboratories from eight countries participated, which were provided with six DNA samples for genotyping, six serum samples, monoclonal antibodies for monoclonal antibody immobilization of platelet antigens (MAIPA) and the mixed passive hemagglutination (MPHA) kits for antibody detection.ResultsMost of the laboratories could detect the anti‐HPA‐2b, anti‐HPA‐4b, anti‐HPA‐5b and Naka antibodies, by the combination of MAIPA and MPHA. However, some had difficulties with the HPA‐1a antibody, possibly dependent on the rarity of HPA‐1b alloantigen among Asian. Although a good concordance was obtained in genotyping, a significant inter‐laboratory variation was observed, possibly dependent on the methods used.ConclusionsThe IPIWP‐AR and the workshop were successfully established, and most laboratories were able to identify majority of HPA antibodies and genotype HPA correctly.

Establishment of reference panel for human platelet antigen genotyping

Human platelet antigens (HPAs) are platelet-specific alloantigens associated with polymorphisms of platelet surface glycoproteins (GPs), and they can induce alloantibodies when individuals lacking a particular polymorphism are exposed to them via pregnancy or transfusion. Immune responses to HPAs are involved in the pathogenesis of several clinical syndromes. HPA genotyping is therefore important for clinical diagnosis and laboratory research. This study aims to establish a reference panel for HPA genotyping. Genomic DNA extracted from human blood was used as the template for amplifying HPA (1a-5a and 15a) gene fragments using specific primers. The amplified products were cloned into pGM-T vectors, which were transformed into competent TOP10 cells. After clone screening and amplification, the plasmids were extracted and sequenced. Next, the gene fragments HPA-1b-5b and 15b were obtained by site-directed mutagenesis using the corresponding HPA-1a-5a and 15a plasmids as template DNA. We successfully constructed reference plasmids for HPA genotyping with HPA-1a-5a, 15a, HPA-1b-5b and 15b. The DNA sequences were consistent with those published in GenBank. Obtaining reference DNA for low-frequency HPAs is very difficult, and the successful construction of reference plasmids for the six HPA systems may solve this problem. Establishment of this panel has laid the foundation for future research on HPA genotyping.

Comparison of a Simple-Probe Real-Time PCR and Multiplex PCR Techniques for HPA-1 to HPA-6 and HPA-15 Genotyping

We aimed to compare HPA-1 to HPA-6 and HPA-15 genotyping results obtained by a simple-probe real-time polymerase chain reaction (PCR) technique with the multiplex PCR technique. Five hundred DNA samples from the Thai National Stem Cell Donor Registry (TSCDR) of the National Blood Centre, Thai Red Cross Society were included. Human platelet antigen (HPA) genotyping was performed by simple-probe real-time PCR and multiplex PCR techniques. HPA-1, HPA-2, HPA-3, and HPA-4 genotyping results obtained by both techniques were in agreement. The misinterpretation of HPA-5, HPA-6, and HPA-15 genotypes was found in eight samples by simple-probe real-time PCR and HPA genotypes were confirmed by DNA sequencing. Two samples of HPA-5 were misinterpreted as HPA-5a5a instead of HPA-5a5b due to an NM_002203.3:c.1594A>C mutation (rs199808499) near the HPA-5 polymorphism (5' side). Five samples of HPA-6a6b were misinterpreted as HPA-6b6b because of an NM_000212.2:c.1545G>A mutation (rs4634) adjacent to the HPA-6 polymorphism (3' side). Interestingly, one sample of HPA-15a15b was misinterpreted as HPA-15b15b due to an NM_133493.1:c.2118C>A mutation near the HPA-15 polymorphism (3' side). HPA genotyping results by two PCR techniques were compared. Incorrect assignments were found due to genetic variations near each HPA single nucleotide polymorphism. Therefore, to avoid false assignation, the use of two genotyping techniques is recommended.

Antibody formation in pregnant women with maternal-neonatal human platelet antigen mismatch from a hospital in northern Taiwan

Neonatal alloimmune thrombocytopenia (NAIT) is a clinical syndrome that resembles hemolytic disease of the newborn, affecting the platelets only. The thrombocytopenia results from the maternal alloantibodies reacting with specific human platelet antigens (HPAs) on the fetal platelets. Forty-four maternal plasma samples were screened for platelet alloantibodies using qualitative solid phase enzyme-linked immunosorbent assay (ELISA) commercial kit (LIFECODES Pakplus, Hologic Gen-Probe GTI Diagnostics, Waukesha, WI, USA), and both the maternal and the corresponding cord blood samples were genotyped (LIFECODES ThromboType, Hologic Gen-Probe GTI Diagnostics, Waukesha, WI, USA). HPA genotyping results correlated with the genetic frequencies in the Taiwan population. A total of 34 newborns (77.3%) had partial HPA genotyping mismatches with the corresponding mothers. The most common partial mismatches between mothers and neonates in HPA genotypes were 13 (29.5%) in both HPA-3b and HPA-15a, followed by 12 (27.3%) in HPA-15b, and 8 (18.2%) in HPA-3a. The frequencies of homozygotic mother with heterozygotic neonate were 15.9% in both HPA-3a and HPA-15b, 9.1% in HPA-15a, 6.8% in HPA-3b, and 2.3% in both HPA-2a and HPA-6a. In this study, maternal HPA antibodies were found in five samples, whereas HLA class I antibodies were found in seven maternal plasma samples from the antibody screen. The results from this study have demonstrated that HPA mismatch is not the main cause for the production of HPA alloantibodies.