Establishment of reference panel for human platelet antigen genotyping

Abstract

Human platelet antigens (HPAs) are platelet-specific alloantigens associated with polymorphisms of platelet surface glycoproteins (GPs), and they can induce alloantibodies when individuals lacking a particular polymorphism are exposed to them via pregnancy or transfusion. Immune responses to HPAs are involved in the pathogenesis of several clinical syndromes. HPA genotyping is therefore important for clinical diagnosis and laboratory research. This study aims to establish a reference panel for HPA genotyping. Genomic DNA extracted from human blood was used as the template for amplifying HPA (1a-5a and 15a) gene fragments using specific primers. The amplified products were cloned into pGM-T vectors, which were transformed into competent TOP10 cells. After clone screening and amplification, the plasmids were extracted and sequenced. Next, the gene fragments HPA-1b-5b and 15b were obtained by site-directed mutagenesis using the corresponding HPA-1a-5a and 15a plasmids as template DNA. We successfully constructed reference plasmids for HPA genotyping with HPA-1a-5a, 15a, HPA-1b-5b and 15b. The DNA sequences were consistent with those published in GenBank. Obtaining reference DNA for low-frequency HPAs is very difficult, and the successful construction of reference plasmids for the six HPA systems may solve this problem. Establishment of this panel has laid the foundation for future research on HPA genotyping.