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.


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.


CONCLUSIONS
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.
Lyou
Jau-Yi
JY
Department of Medicine, Veterans General Hospital, and School of Medicine, National Yang-Ming University, Taipei, Taiwan, ROC.
Chen
Ying-Ju
YJ
Hu
Hui-Yu
HY
Lin
Jeong-Shi
JS
Tzeng
Cheng-Hwai
CH
D000483:Alleles; D016824:Antigens, Human Platelet / Q000097:blood / Q000235:genetics*; D001782:Blood Donors; D001792:Blood Platelets / Q000276:immunology*; D005787:Gene Frequency; D005838:Genotype; D006801:Humans; D007519:Isoantigens / Q000235:genetics*; D015335:Molecular Probes / Q000235:genetics*; D016133:Polymerase Chain Reaction / Q000379:methods*
D016824:Antigens, Human Platelet; D007519:Isoantigens; D015335:Molecular Probes