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

Abstract

BACKGROUND: 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. STUDY DESIGN AND METHODS: 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. RESULTS: There was full concordance between the two genotyping methods, and genotype frequencies were comparable with previous studies in caucasians. CONCLUSION: 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.