Genetic landscape of human platelet antigen variants in the Indian population analysed from 1029 whole genomes.

Genetic variants in human platelet antigens (HPAs) considered allo- or auto antigens are associated with various disorders, including neonatal alloimmune thrombocytopenia, platelet transfusion refractoriness and post-transfusion purpura. Although global differences in genotype frequencies were observed, the distributions of HPA variants in the Indian population are largely unknown. This study aims to explore the landscape of HPA variants in India to provide a basis for risk assessment and management of related complications. Population-specific frequencies of genetic variants associated with the 35 classes of HPAs (HPA-1 to HPA-35) were estimated by systematically analysing genomic variations of 1029 healthy Indian individuals as well as from global population genome datasets. Allele frequencies of the most clinically relevant HPA systems in the Indian population were found as follows, HPA-1a - 0.884, HPA-1b - 0.117, HPA-2a - 0.941, HPA-2b - 0.059, HPA-3a - 0.653, HPA-3b - 0.347, HPA-4a - 0.999, HPA-4b - 0.0010, HPA-5a - 0.923, HPA-5b - 0.077, HPA-6a - 0.998, HPA-6b - 0.002, HPA-15a - 0.582 and HPA-15b - 0.418. This study provides the first comprehensive analysis of HPA allele and genotype frequencies using large scale representative whole genome sequencing data of the Indian population.

Subamniotic Hemorrhage, a Possible New Presentation of Fetal and Neonatal Alloimmune Thrombocytopenia.

BACKGROUND: Fetal and neonatal alloimmune thrombocytopenia (FNAIT) is a rare fetal disease in which maternal antibodies directed toward fetal human platelet antigens (HPA) are formed during pregnancy and cause fetal thrombocytopenia. The diagnosis FNAIT is suspected when a fetus or neonate presents with signs of bleeding. CASE: We describe a pregnancy complicated by a placental hematoma in the 20th week of gestation as the first manifestation of FNAIT. Further evaluation showed signs of germinal matrix hemorrhage and HPA-5b allo-antibodies. After the diagnosis, intravenous immunoglobulin was administered weekly and a healthy daughter was born at 37 weeks. Histopathological analysis revealed that the hematoma was caused by a subamniotic hemorrhage of fetal origin. CONCLUSION: A subamniotic hematoma appears to be the first manifestation of FNAIT.

Analysis of Integrin αIIb Subunit Dynamics Reveals Long-Range Effects of Missense Mutations on Calf Domains.

Integrin αIIbß3, a glycoprotein complex expressed at the platelet surface, is involved in platelet aggregation and contributes to primary haemostasis. Several integrin αIIbß3 polymorphisms prevent the aggregation that causes haemorrhagic syndromes, such as Glanzmann thrombasthenia (GT). Access to 3D structure allows understanding the structural effects of polymorphisms related to GT. In a previous analysis using Molecular Dynamics (MD) simulations of αIIbCalf-1 domain structure, it was observed that GT associated with single amino acid variation affects distant loops, but not the mutated position. In this study, experiments are extended to Calf-1, Thigh, and Calf-2 domains. Two loops in Calf-2 are unstructured and therefore are modelled expertly using biophysical restraints. Surprisingly, MD revealed the presence of rigid zones in these loops. Detailed analysis with structural alphabet, the Proteins Blocks (PBs), allowed observing local changes in highly flexible regions. The variant P741R located at C-terminal of Calf-1 revealed that the Calf-2 presence did not affect the results obtained with isolated Calf-1 domain. Simulations for Calf-1 + Calf-2, and Thigh + Calf-1 variant systems are designed to comprehend the impact of five single amino acid variations in these domains. Distant conformational changes are observed, thus highlighting the potential role of allostery in the structural basis of GT.

A nait-associated and previously unreported mutation in the ITGB3 gene with a low frequency in the local population.

BACKGROUND: Neonatal alloimmune thrombocytopenia is a rare but potentially severe postnatal complication caused by maternal allo-antibodies against platelet antigens of the newborn. In relatively few cases, immunisation against low-frequency antigens has been reported. METHODS: Platelet antigens of a newborn with severe thrombocytopenia and his family members were investigated by serological and molecular biological methods. A real-time PCR assay was developed to reliably detect this mutation in pools of DNA from up to seven individuals. RESULTS: Serological testing showed positive reactions of maternal plasma with paternal platelets but not with conventional platelet donor panels. Sequencing of the ITGB3 gene revealed a G > A polymorphism in position c.1915 of exon 12 for the father, the newborn and three of four paternal relatives. Screening of samples from a local population of 1575 Caucasian blood donors identified only a single individual with this mutation. CONCLUSION: This finding of a previously unreported private platelet antigen demonstrates that the identification of the target glycoprotein by MAIPA assay followed by sequencing of the affected gene can be combined with an efficient population screening by real-time PCR with pooling of DNA samples.

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.

A new efficient tool for non-invasive diagnosis of fetomaternal platelet antigen incompatibility.

Fetal and neonatal alloimmune thrombocytopenia (FNAIT) is the consequence of platelet destruction by maternal alloantibodies against fetal human platelet antigens (HPA). This may result in intracranial haemorrhages (ICH) or even fetal death. Currently, fetal HPA genotyping is performed using invasive procedures. Here, we carried out a proof-of-concept study for non-invasive prenatal diagnosis of fetal platelet genotyping in four HPA systems (HPA-1, -3, -5 and-15) by droplet digital polymerase chain reaction (ddPCR) using cell-free DNA extracts from the plasma of 47 pregnant women with suspected, or history of, FNAIT. Results showed that 74% (35/47) of pregnant women presented incompatibility in at least one HPA system, and 38% (18/47) of cases presented HPA-1 incompatibility, including nine women with multiple incompatibilities. ICH occurred in one case of profound fetal thrombocytopenia with HPA-15 incompatibility, confirming the need for non-invasive prenatal genotyping in systems other than HPA-1. Fetal HPA genotypes predicted by ddPCR were confirmed in all FNAIT cases after amniocentesis or delivery. Fetal HPA genotyping on maternal plasma based on ddPCR is a fast, safe and reliable non-invasive method. This technique will be useful for the early identification of pregnancies at high risk of FNAIT requiring antenatal management to minimize the risk of fetal/neonatal haemorrhage.

Progress and development of platelet antibody detection.

Human platelet antibody (HPA) detection is necessary for the diagnosis and therapeutic decisions for refractoriness to platelet transfusions, post transfusion purpura and fetal and neonatal alloimmune thrombocytopenia. In the last four to five decades many new developments, both in knowledge and methods, have increased the quality of platelet serology. However, the quest for the optimal antibody detection method(s) encountered, sometimes unexpected, difficulties. In this review the various aspects concerning platelet antibody test methods and detection of platelet antibodies both for the diagnostic and screening setting are discussed.

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.

High-Throughput Screening of Blood Donors for Twelve Human Platelet Antigen Systems Using Next-Generation Sequencing Reveals Detection of Rare Polymorphisms and Two Novel Protein-Changing Variants.

BACKGROUND: Exposure to non-matching human platelet alloantigens (HPA) may result in alloimmunization. Antibodies to HPA can be responsible for post-transfusion purpura, refractoriness to donor platelets, and fetal and neonatal alloimmune thrombocytopenia. For the supply of compatible apheresis platelet concentrates, the HPA genotypes are determined in a routine manner. METHODS: Here, we describe a novel method for genotyping twelve different HPA systems simultaneously, including HPA-1 to HPA-5, HPA-9w, HPA-10w, HPA-16w, HPA-19w, HPA-27w, and the novel HPA-34w by means of amplicon-based next-generation sequencing (NGS). Blood donor samples of 757 individuals with a migration background and 547 of Western European ancestry were genotyped in a mass-screening setup. An in-house software was developed for fast and automatic analysis. TaqMan assay and Sanger sequencing results served for validation of the NGS workflow. Finally, blood donors were divided in several groups based on their country of origin and the allele frequencies were compared. RESULTS: For 1,299 of 1,304 samples (99.6%) NGS was successfully performed. The concordance with TaqMan assay and Sanger sequencing results was 99.8%. Allele-calling dropouts that were observed for two samples with the TaqMan assay caused by rare single nucleotide polymorphisms were resolved by NGS. Additionally, twenty rare and two novel variants in the coding regions of the genes ITGB3, GPB1A, ITGBA2, and CD109 were detected. The determined allele frequencies were similar to those published in the gnomAD database. CONCLUSIONS: No significant differences were observed in the distribution of allele frequencies of HPA-1 through HPA-5 and HPA-15 throughout the analyzed groups except for a lower allele frequency for the HPA-1b allele in the group of donors with Southern Asian ancestry. In contrast, other nucleotide variants that have not yet been phenotypically characterized occurred three times more often in blood donors with a migration background. High-throughput amplicon-based NGS is a reliable method for screening HPA genotypes in a large sample cohort simultaneously. It is easily upgradeable for genotyping additional targets without changing the setup or the analysis pipeline. Mass-screening methods will help building up blood donor registries to provide matched blood products.

Antibodies to human platelet antigens form a significant proportion of platelet antibodies detected in Indian patients with refractoriness to platelet transfusions.

BACKGROUND: The transfusion of platelets is an important therapeutic strategy in bleeding patients with thrombocytopenia. However, some chronically transfused patients fail to achieve the appropriate platelet count increment following transfusion due to the presence of platelet alloantibodies. OBJECTIVES: The aims of this research were to study the prevalence of platelet alloimmunisation and to characterise the platelet-reactive (PR) antibodies in haematology patients refractory to platelet transfusions in an Indian setting. PATIENTS AND METHODS: A total of 80 patients with a prior history of multiple transfusions (minimum of five cellular transfusions) were included in the study if they did not achieve an adequate corrected count increment within 24 h of the platelet transfusion. Patients with non-immunological causes of platelet refractoriness were excluded from the study. The test was performed on a blood sample of 4 mL of Ethylenediaminetetraacetic acid (EDTA) blood sample in which plasma was separated and stored at -80 °C and underwent batch testing in PAK-2LE. RESULTS: The overall prevalence of platelet alloimmunisation in our study was 60%. Of the 48 patients who were detected to have platelet antibodies, the combination of anti-human leucocyte antigen (HLA) and platelet-specific (PS) antibodies together constituted the majority of 54·2%. The overall prevalence of anti-HLA antibodies was 51·25% and of PS antibodies was 41·25% in the total study population of 80. CONCLUSION: The overall prevalence of PS antibodies in our study was greater than that reported by other groups in India and other countries. This needs to be considered, particularly in the management of patients refractory to platelet transfusions, where HLA-matched platelets constitute current best practice.

Human Platelet Antigens in Brazilian Multiethnic Populations: Occurrence of Regional Variation and Frequency in a Large Urban Center (Belo Horizonte).

BACKGROUND: The frequency of human platelet antigens (HPA) varies according to ethnicity, which causes differences in the morbidity of alloimmune and autoimmune thrombocytopenic disorders in different populations. Studies on HPA frequencies in Brazil have reported differences among Brazilian populations produced by the diverse degrees of admixture throughout the country. METHODS: In the present study, we investigated the variation of HPA distribution in Brazil, compared with worldwide populations, and describe the frequencies of HPA-1, -2, -3, -5, and -15 in a large urban center in Southern Brazil (Belo Horizonte) based on a sample of blood donors. RESULTS: The principal component analysis and the dendrogram based on genetic distance revealed a clear relationship between Brazilian populations and the groups formed by European and African populations. The coefficients of variation for HPA allele frequencies suggest that Brazilian populations presented variations for HPA alleles comparable with the populations from continental groups. In Belo Horizonte, the allele a frequencies for HPA-1, -2, -3, -5 and -15 were 0.8575, 0.8400, 0.6225, 0.8525 and 0.5825 respectively. The genotypes with higher frequencies were a/a (72-74%), except for HPA-3 and -15, whose heterozygous a/b genotypes were shown to be more prevalent (43.5 and 44.5%, respectively). CONCLUSION: We confirmed the heterogeneity of HPA antigens in Brazilian populations, reinforcing the importance of HPA panels composed of regional blood donors, or a national panel that contemplates the specificities of the different regions of the country, in order to provide support in platelet transfusions and to minimize the risks associated with HPA alloimmunization. The evaluation of HPA data from Belo Horizonte represents the initial step toward the development of a genotyped platelet donor registry in order to treat HPA alloimmunized patients in this region.

Association of human platelet antigens polymorphisms with susceptibility to hepatitis C virus infection in Chinese population.

Hepatitis C virus (HCV) is a major cause of chronic hepatitis. Previous studies have identified a number of single nucleotide polymorphisms that are associated with HCV infection. Human platelet antigens (HPAs) polymorphisms play an important role in several diseases. Here, we demonstrated the association of the HPA-2, HPA-3, HPA-5 and HPA-15 polymorphisms with susceptibility to HCV infection in Chinese population. Overall, 118 patients with HCV and 167 controls were genotyped for HPAs. There were no significant differences in the allele and genotype frequency distribution for the HPA-3, HPA-5 and HPA-15 systems between the patients with chronic HCV infection and the healthy controls (p > .05). However, the genotype frequency of HPA-2aa was significantly lower, while HPA-2ab/bb was significantly higher in patients than that in the controls (p = .006). The allele frequency of HPA-2a in patients was significantly lower than that in the control group (p = .005). In contrast, HPA-2b in patients was significantly higher than that in the control group (p = .005). We conclude that HPA-2 polymorphism is associated with susceptibility to HCV infection, and individuals carrying the HPA-2b allele may have a higher risk of HCV infection compared with individuals carrying HPA-2a.

Human platelet antigens in Burmese, Karen and north-eastern Thais.

OBJECTIVES: A comparative study of allele frequencies at HPA-1 to -6 and HPA-15 in Burmese and Karen populations as well as at HPA-15 in north-eastern Thais (NET) is presented. BACKGROUND: Human platelet antigens (HPAs) are clinically important in several immune platelet disorders, including foetal and neonatal alloimmune thrombocytopenia (FNAIT), post-transfusion purpura (PTP) and platelet transfusion refractoriness (PTR). The knowledge of antigen frequencies in a population is essential for the evaluation of patients suffering from immune-mediated platelet disorders. METHODS: A total of 285 unrelated, healthy Burmese, 242 Karen and 300 NET were recruited to this study. Genotype and allele frequencies of HPA-1 to -6 and HPA-15 were defined using polymerase chain reaction sequence-specific primers (PCR-SSP) RESULTS: No individuals homozygous for HPA-1bb, -2bb, -4bb, -5bb and -6bb were detected. HPA-1a, -2a, -4a, -5a and -6a were present in all samples of Burmese and Karen origin. HPA-1b, -2b, -4b, -5b and -6b were rare in these populations. The frequencies of HPA-3a/-3b were 60·4/39·6% in Burmese and 55·8/44·2% in Karen, respectively. Frequencies of HPA-15a/-15b were 57·2/42·8% in Burmese, 52·5/47·5% in Karen and 49·8/50·2% in NET. CONCLUSIONS: The frequencies of HPA genotypes in our study indicates that HPA-1a, -2a, -4a, -5a and -6a are unlikely involved in FNAIT, PTP and PTR in Burmese and Karen populations. However, HPA-1b, -2b, -3a, -3b, -4b, -5b, -6b, -15a and -15b may likely stimulate alloantibodies in these populations.

Recent progress in understanding the pathogenesis of fetal and neonatal alloimmune thrombocytopenia.

Fetal and neonatal alloimmune thrombocytopenia (FNAIT) occurs in c. 1 in 1000 births and is caused by maternal antibodies against human platelet alloantigens that bind incompatible fetal platelets and promote their clearance from the circulation. Affected infants can experience bleeding, bruising and, in severe cases, intracranial haemorrhage and even death. As maternal screening is not routinely performed, and first pregnancies can be affected, most cases are diagnosed at delivery of a first affected pregnancy. Unlike its erythrocyte counterpart, Haemolytic Disease of the Fetus and Newborn, there is no prophylactic treatment for FNAIT. This report will review recent advances made in understanding the pathogenesis of FNAIT: the platelet alloantigens involved, maternal exposure and sensitization to fetal platelet antigens, properties of platelet Immunoglobulin G antibodies, maternal-fetal antibody transport mechanisms and efforts to develop an effective FNAIT prophylaxis.

Effects of universal vs bedside leukoreductions on the alloimmunization to platelets and the platelet transfusion refractoriness.

BACKGROUND: Multiple platelet exposure induces anti-HLA and/or anti-HPA antibody production, which may cause platelet transfusion refractoriness (PTR). In Japan, the universal pre-storage leukocyte reduction (ULR) was fully implemented since 2006, but prior to ULR, in our institution, leukocyte reduction filters were routinely used at the bedside (bedside leukoreduction, BSLR) for all onco-hematological patients receiving multiple platelet transfusions. OBJECTIVE: We retrospectively compared patients receiving platelet transfusions in the era of ULR with those of BSLR era. MATERIALS AND METHODS: Patients of the BSLR group (409 cases) and the ULR group (586 cases) were compared in terms of alloimmunization and immunological PTR. The clinico-pathological features, including gender, history of pregnancy, number of exposed transfusion donors, periods of transfusion, and prior stem cell transplantation were compared, and the risk factors of alloimmunization were determined. RESULTS: The antibody detection rate was significantly higher in the ULR compared to BSLR group (8.7% vs. 5.4%), as well as the immunological PTR rate (7.3% vs. 3.2%). By the multivariate analysis, female gender and the number of platelet donor exposure, but not universal leukoreduction or transfusion period, were found to be the risk factors strongly associated with alloantibody formation. CONCLUSION: Although ULR may be superior to BSLR in terms of preventing non-hemolytic transfusion reactions, BSLR was found to be as effective as ULR in terms of preventing platelet alloimmunization and refractoriness. Thus, BSLR should be actively indicated as a realistic alternative in developing countries, before the universal leukoreduction is fully implemented.

Identification of anti-HPA-1a allo-antibodies using IgG platelet antibody detection and crossmatch system assay with Galileo Echo.

Fetal/neonatal allo-immune thrombocytopenia is the most frequent and the most dangerous clinical condition involving anti-human platelet antigens (HPA)-1a allo-antibodies. Anti-HPA-1a allo-immunization requires rapid and accurate diagnosis to determine appropriate treatment. The Capture-P Ready-Screen assay (C-PRS) is a new qualitative immunoassay to detect IgG anti-human leukocyte antigen (HLA) and anti-HPA allo-antibodies. The aim of this study is to assess the identification of anti-HPA-1a allo-antibodies using the C-PRS assay, associated with HLA class I stripping reagents, on the automated benchtop analyzer Galileo Echo. Forty-nine sera were analyzed: without anti-HLA class I or anti-HPA allo-antibodies, with anti-HLA class I allo-antibodies, with anti-HPA-1a allo-antibodies, among which with anti-HLA class I allo-antibodies. None of the samples without allo-antibodies were reactive. Only anti-HLA antibodies, detected by cytotoxicity-dependent complement and not by Luminex, remained positive before and after stripping reagents. Of the 13 samples, anti-HPA-1a allo-antibodies that were correctly identified before and after incubation with HLA assassin reagent were 70% and 85%, respectively. Anti-glycoprotein auto-antibodies and anti-HLA allo-antibodies do not interfere with the detection of anti-HPA-1a antibodies. This preliminary study indicates that further improvement of the test will be helpful in developing a clinically useful assay in the future.

Frequency of human platelet antigens (HPA)-1, -2, -5 and -15 in Brazilian blood donors and establishment of a panel of HPA-typed donors.

OBJECTIVES: The aim of this study is to keep a record of regular human platelet antigens (HPA)-typed blood donors and to compare their allele frequencies with those reported in other populations. BACKGROUND: HPA are polymorphisms expressed on platelet membrane glycoproteins. They can generate an immune response leading to platelet alloimmunisation that may show clinical manifestations, such as neonatal alloimmune thrombocytopenia, post-transfusion purpura and platelet refractoriness. Platelet alloimmunisation is not uncommon, therefore, for an optimum management, it is advantageous to establish a panel of typed platelets to help matched platelets selection in HPA-alloimmunised patients transfusion. METHODS: The polymerase chain reaction (PCR)-allele-specific primers and PCR-restriction fragment length polymorphism methods were used to determine the genotypes of HPA-1, -2, -5 and -15 systems of 337 blood donors. RESULTS: The three genotypes (AA, AB and BB) were found in all HPA systems analysed, and the most frequent genotypes were AA for HPA-1, -2 and -5 systems (mean: 0·732) and AB for HPA-15 system (mean: 0·523). Allele frequencies were 0·148, 0·155, 0·140 and 0·430 for HPA-1b, -2b, -5b and -15b, respectively, and they were similar to those found in Caucasian populations, especially for HPA-1. However, the B allele was more frequent in all HPA systems when compared with Amazon Indians, and the frequency of the B allele in our study was higher in HPA-1 and -15 systems and lower in HPA-2 and -5 systems in comparison with sub-Saharan African populations. CONCLUSIONS: A record of HPA-typed donors would enable rapid identification and selection of donors when HPA-compatible platelets are required for transfusion.

Platelet antibody detection assays: a single-laboratory comparison of MAIPA, PIFT, and microsphere-based multiplex assays Pak-Lx.

BACKGROUND AND OBJECTIVES: The identification of platelet antibodies is essential for diagnosing and managing conditions such as fetal and neonatal alloimmune thrombocytopenic purpura, post-transfusion purpura, and immune platelet refractoriness. Monoclonal antibody immobilization of platelet antigens (MAIPA) is the standard method for detecting anti-human platelet antigen (HPA) antibodies, while the detection of anti-HLA antibodies once relied on the complement-dependent cytotoxicity method, however advanced technologies such as enzyme-linked immunosorbent assay and Luminex have significantly improved sensitivity and accuracy in identifying these antibodies. Flow cytometry-based techniques (platelet immunofluorescence test - PIFT) and Luminex platform-driven microsphere-based multiplex assays (Pak-Lx) are widely employed in platelet immunology laboratories owing to their remarkable flexibility and versatility. The present study compared the sensitivity, specificity, and concordance of these different serological techniques used in platelet antibody identification. MATERIAL AND METHODS: One hundred serum samples from patients suspected of immune-mediated platelet disorders were examined. Initially, the samples underwent testing using the MAIPA method. Subsequently, the results were compared with three alternative methods: PIFT and microsphere-based multiplex assays for both HLA and HPA antibodies. RESULTS: Pak-Lx demonstrated a 94 % agreement with MAIPA, while PIFT had 88 % agreement for HPA antibodies. For HLA antibody detection, Pak-Lx versus DLX had 75 % concordance, MAIPA versus DLX showed 77 %, and PIFT versus DLX displayed an 81 % concordance rate. Remarkably, there were no significant differences in concordance levels between Pak-Lx and PIFT compared to MAIPA and DLX for anti-HPA and HLA antibodies, respectively. CONCLUSION: This study found no significant differences in concordance among the tested assays for detecting anti-HPA and anti-HLA antibodies. These data suggest that no single method can detect all clinically important antibodies. Therefore, it is advisable that each laboratory develops customized protocols based on their expertise and employs complementary methods for comprehensive patient assessments.

Validating the HPA-1 to -5 and -15 Detection by Homemade PCR-SSP, Real-Time PCR, and PCR-RFLP Methods.

OBJECTIVE: Human platelet antigens (HPAs) are antigenic determinants on platelet membrane glycoproteins that stimulate the host's immune system and cause platelet destruction. In this study, we share our experience with implementing sequence-specific primer-polymerase chain reaction (PCR-SSP), real-time PCR, and PCR-RFLP (restriction fragment length polymorphism) and the validation process used to evaluate the results. METHODS: At the Ardabil Blood Transfusion Center, 10 samples were obtained from blood donors. Validation using PCR-SSP, real-time PCR, and PCR-RFLP methods for genotyping HPAs was done by sequencing. A commercial DNA sample and a commercial kit were also used for validation. RESULTS: The results of PCR-SSP, TaqMan Real-Time PCR, melting curve analysis (HPA-15), and PCR-RFLP (HPA-3) were 100% consistent with sequencing (gold standard) and commercial kit results. CONCLUSIONS: There was a 100% correlation between repeating the methods and the expected results for repeatability, and no false positives and negatives were observed.