Simultaneous genotyping of human platelet antigen-1 to 17w by polymerase chain reaction sequence-based typing.

BACKGROUND AND OBJECTIVES: Genotyping for human platelet antigen (HPA) systems is required in the investigation of patients with suspected HPA antibodies and for the provision of compatible blood products from HPA-typed donors. Here, we set up a polymerase chain reaction sequence-based typing method for simultaneous genotyping of HPA-1 to 17w (PCR-SBT).

MATERIALS AND METHODS: The specific primers for HPA-1 to 17w were designed by primer premier 5.0 software. The PCR amplification conditions were optimized. Amplification products were purified by enzyme digestion and sequenced by a BigDye Terminator Cycle Sequencing kit.

RESULTS: We used two standard DNA samples and 16 reference samples, including six with HPA gene mutations, for the HPA-1 to 17w analysis by the PCR-SBT and obtained 100% correct genotypes in these samples. The genotype and allele frequencies of HPA-1 to 17w were analysed from 112 Han Chinese persons. Thirteen new single nucleotide polymorphisms and one microsatellite were found in the sequenced regions, in which two nucleotide polymorphisms of ITGB3 gene could cause amino acid alteration of Val419Met and Glu628Lys on the mature glycoprotein, respectively.

CONCLUSIONS: The PCR-SBT method is reliable and accurate for HPA genotyping, suitable for routine HPA analysis, and can screen large numbers of donors. We identified 14 new polymorphisms, two of which led to amino acid changes.