PCR with sequence-specific primer-based simultaneous genotyping of human platelet antigen-1 to -13w

BACKGROUND : Accurate human platelet antigen (HPA) typing is important for patients with diagnosis of alloimmune thrombocytopenic syndromes and provision of HPA‐matched blood components for these patients. STUDY DESIGN AND METHODS : Thirteen sequence‐specific primers (SSPs) designed on the basis of known published polymorphisms for HPA‐1 to HPA‐13w, respectively, were employed for simultaneous HPA genotyping. All PCR amplifications were carried out with identical cycling conditions in 96‐well plates containing primer mixtures. A total of 300 blood samples from unrelated volunteer donors in Taiwan were included in the study. RESULTS : All primers had specific amplification products. The typing results were available within 4 hours each time for up to four blood samples tested. Among the 13 HPAs, HPA‐3 had the greatest heterozygosity with a gene frequency of 0.3267, 0.4967, and 0.1767 for HPA‐3a/HPA‐3a, HPA‐3a/HPA‐3b, and HPA‐3b/HPA3‐b, respectively. For the remaining 12 HPAs, the predominance of a/a homozygosity was noted for HPA‐1, ‐2, ‐4, ‐5, and ‐6, with a frequency ranging from 0.9200 to 0.9967. The frequency of a/a homozygosity was 1.0000 for HPA‐7w to ‐13w, except for HPA‐10w, for which one case was observed to be HPA‐10aw/HPA‐10bw heterozygous. Excluding HPA‐3, b/b homozygosity was noted in only one case ( HPA‐6b/HPA‐6b ). The prevalence rates of HPA‐1 to ‐13w in this study were consistent with previous reports using different methods. CONCLUSION : An extended, streamlined PCR‐SSP protocol for simultaneous genotyping of HPA‐1 to HPA‐13w was established. This allows fast and reliable diagnosis of alloimmune thrombocytopenia, and is readily applicable to large‐scale genetic population studies.