Simultaneous genotyping of human platelet alloantigen‐1 to 28bw systems by multiplex polymerase chain reaction sequence‐based typing

Hong, Xiaozhen; Chen, S.; Ying, Yanling; Liu, Y.; Xu, Xianguo; He, J.; Zhu, Faming

doi:10.1111/vox.12507

Cited by 6 publications

(11 citation statements)

References 26 publications

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“…In this study, gene frequencies of HPA‐1b, HPA‐2b, HPA‐3b, HPA‐4b, HPA‐5b, HPA‐6b, HPA‐15b, and HPA‐21b were demonstrated to be over 0.1%. These frequencies were consistent with those in the Han populations in China 7 . Therefore, at least in Japan and China, these HPA systems show a similar response faced with HPA antibody–associated thrombocytopenia.…”

Section: Discussionsupporting

confidence: 86%

“…These frequencies were consistent with those in the Han populations in China. 7 Therefore, at least in Japan and China, these HPA systems show a similar response faced with HPA antibody-associated thrombocytopenia. In previous studies, HPA-2, HPA-3, HPA-4, HPA-5, HPA-6, HPA-15, and HPA-21 were reported to be major HPA systems in clinical cases associated with PLT transfusion refractoriness and NAIT in Japan, 17,[19][20][21] but HPA-1a has never been reported in Japan.…”

Section: Gene Frequenciesmentioning

confidence: 99%

“…Currently, new HPA genotyping methods are being developed. These include polymerase chain reaction with sequence‐specific primers (PCR‐SSP), 5 PCR–restriction fragment length polymorphism analysis, 6 PCR sequence‐based typing, 7 BeadChip Technology, 8 next‐generation sequencing, 9–11 and matrix‐assisted laser desorption/ionization time‐of‐flight mass spectrometry 12 . We have also reported a PCR‐based high‐resolution melt (PCR‐HRM) assay combined with direct DNA sequencing, by which we identified the HPA‐21bw allele from Japanese infants with severe NAIT, and determined its frequency in Japanese blood donors at 0.3% 13 …”

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confidence: 99%

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Frequency of allotype “b” in human platelet antigen 1 to 29 systems among blood donors in Japan estimated using high‐resolution melt analysis

et al. 2020

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Background: Antibodies against human platelet antigens (HPAs) cause thrombocytopenias. It is thus important to know the frequency of "b" allotypes in each HPA system for the diagnosis and treatment of anti-HPA antibodymediated thrombocytopenia. Study Design and Methods: Genomic DNA was extracted from peripheral blood cells obtained from 2170 blood donors in Japan and was subjected to highresolution melt (HRM) analysis using polymerase chain reaction for each of the HPA genes, using 23 primer pairs. For genotyping, the resulting amplicons were classified based on their HRM curves. In some cases, direct sequence analysis was performed after HRM analysis to determine nucleotide substitutions. In cases where amino acid substitutions were predicted, protein expression levels were examined in a cell line using 293T cells. Results: The frequencies of each of the HPA-b genotypes were as follows: HPA-1b, 0.4%; HPA-2b, 11.8%; HPA-3b, 41.3%; HPA-4b, 0.8%; HPA-5b, 4.3%; HPA-6b, 1.9%; HPA-15b, 48.8%; HPA-21b, 0.6%; and "b" allotype in the other HPA systems, 0.0%. Twenty-eight variants were found; nine of them were predicted to cause amino acid substitution. However, expression analysis revealed that they did not affect protein expression levels on the cell surface. Conclusion: Nine HPA systems are of primary importance in Japan in potentially triggering thrombocytopenia via the HPA antibodies. Similar studies in other countries or races, together with ours, could provide basic information for clinicians in multiethnic societies.

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Section: Discussionsupporting

confidence: 86%

Section: Gene Frequenciesmentioning

confidence: 99%

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confidence: 99%

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Frequency of allotype “b” in human platelet antigen 1 to 29 systems among blood donors in Japan estimated using high‐resolution melt analysis

et al. 2020

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“…[16–20] Thus, HPA-3a would appear to be significantly immunogenic factor only less than HPA-1a and HPA-5b. With the development of technology, numerous new approaches provide highly sensitive and specific, multiplex bead-based assay for detecting human platelet antibodies, [21–23] which can contribute to a high diagnostic rate of the NAIT.…”

Section: Discussionmentioning

confidence: 99%

Timely diagnosis and treatment of neonatal alloimmune thrombocytopenia caused by anti HPA-3a antibody

Yang¹,

Lv²,

Kong³

et al. 2019

Medicine

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Rationale: Neonatal alloimmune thrombocytopenia (NAIT) caused by anti HPA-3a antibody is rare, and the clinical features of the syndrome are not specific. Patient concerns: A male infant was noted to be irritable and physical examination revealed the presence of petechiae and bruising on the right arm and thigh after born. Diagnoses: Platelet antibodies were investigated using the monoclonal antibody-specific immobilization of platelet antigens (MAIPA) assay, platelet genotyping (HPA 1–17) was performed by polymerase chain reaction technique with sequence-specific primers (PCR-SSP). The HPA genotype of the newborn was HPA-3a/b, while that of his mother and his father were HPA-3b/b and HPA-3a/a, respectively. The sera of newborn contained antibody against the platelet of newborn's father. The HPA antibody of the newborn was identified as anti HPA-3a. The newborn was confirmed as a patient of NAIT caused by anti HPA-3a antibody. Interventions: A single dose of intravenous immunoglobulin (IVIG) 1 g/kg was administered from day 3 to day 7. Outcomes: At follow-up 3 months after discharge from the hospital, the baby was developing normally and had a normal platelet count (361 × 109/L). Lessons: NAIT caused by anti HPA-3a antibody is rare, and we believe this study can provide insights for diagnosing prospective cases. Prognosis of NAIT caused by HPA3a seems to be favorable if diagnosed and treated in a timely manner.

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“…Fresh apheresis platelets were collected from three voluntary blood donors (P2, P3 and P4) and genotyped for HPA as previously described [11]. The platelets were centrifuged at 1400 g for 5 min at 4 °C, washed 2 times with TBS-EDTA buffer, and resuspended with TBS-EDTA buffer.…”

Section: Methodsmentioning

confidence: 99%

Novel method for simultaneously detecting HPA and HLA antibodies using Luminex microbeads

et al. 2019

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Background Alloantibodies against human platelet antigens (HPAs) and human leukocyte antigen (HLA) are implicated in several immune-mediated platelet disorders. Detection of these antibodies is crucial in the diagnosis and management of these disorders. The aim of this study was to establish a novel method to simultaneously detect HPA-1, HPA-2, HPA-3, HPA-5 and HLA antibodies with Luminex microbeads technology. Methods Monoclonal antibodies specific for platelet glycoproteins and HLA class I molecules were separately coupled to the Luminex microbeads. We validated specificity of the Luminex platform using the following antibodies: anti-HPA-1a, anti-HPA-2b, anti-HPA-3a, anti-HPA-5a, and anti-HLA positive samples. Sensitivity was evaluated by a serial dilution (from neat to 1/1024) using the following antibodies: anti-HPA-1a, anti-HPA-3a standard sera, and anti-HPA-5a positive serum. Serum samples were collected from 36 neonatal alloimmune thrombocytopenia (NAIT) patients suspected of having HPA or HLA antibodies and 8 samples from ISBT platelet workshop were tested using the Luminex assay. Results The Luminex assay detected all antibodies tested from the known samples. The sensitivities of the Luminex assay detecting anti-HPA-1a, anti-HPA-3a, and anti-HPA-5a were 1:512, 1:64, and 1:128, respectively. The sensitivity of Luminex assay was higher than monoclonal antibody immobilization of platelet antigen method (MAIPA). No cross-reactivity was observed in the samples containing multi-platelet antibodies or mixture antibodies against HPA and HLA. The results of 44 samples with platelet disorders were consistent with those of the same samples processed with the MAIPA assay. Conclusion Luminex microbeads coupled with monoclonal antibodies could be successfully used to detect HPA and HLA antibodies simultaneously, especially with high sensitivity in detecting HPA antibodies.

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Simultaneous genotyping of human platelet alloantigen‐1 to 28bw systems by multiplex polymerase chain reaction sequence‐based typing

Abstract: One multiplex PCR-SBT method for HPAs was established and the data of the study could help to prevent and treat for alloimmune thrombocytopenia.

Cited by 6 publications

References 26 publications

Frequency of allotype “b” in human platelet antigen 1 to 29 systems among blood donors in Japan estimated using high‐resolution melt analysis

Frequency of allotype “b” in human platelet antigen 1 to 29 systems among blood donors in Japan estimated using high‐resolution melt analysis

Timely diagnosis and treatment of neonatal alloimmune thrombocytopenia caused by anti HPA-3a antibody

Novel method for simultaneously detecting HPA and HLA antibodies using Luminex microbeads

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