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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
Author(s) -
Randen Ingrid,
Sørensen Kirsten,
Killie Mette K.,
KjeldsenKragh Jens
Publication year - 2003
Publication title -
transfusion
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.045
H-Index - 132
eISSN - 1537-2995
pISSN - 0041-1132
DOI - 10.1046/j.1537-2995.2003.00354.x
Subject(s) - genotyping , melting curve analysis , neonatal alloimmune thrombocytopenia , concordance , high resolution melt , genotype , antigen , allele , biology , immunology , allele frequency , thrombocytopenic purpura , population , polymerase chain reaction , microbiology and biotechnology , platelet , genetics , medicine , gene , pregnancy , fetus , environmental health
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.