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
Article first published online: 8 APR 2003
Volume 43, Issue 4, pages 445–450, April 2003
How to Cite
Randen, I., Sørensen, K., Killie, M. K. and Kjeldsen-Kragh, J. (2003), 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. Transfusion, 43: 445–450. doi: 10.1046/j.1537-2995.2003.00354.x
HPA = human platelet antigen; NAITP = neonatal alloimmune thrombocytopenic purpura; PTP = posttransfusion purpura; SNP = single nucleotide polymorphisms; SSP = sequence-specific primer; Tm = melting temperature.
- Issue published online: 8 APR 2003
- Article first published online: 8 APR 2003
- Received for publication April 3, 2002; revision received November 15, 2002, and accepted November 25, 2002.
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.