Establishment of reference panel for human platelet antigen genotyping.
OBJECTIVE
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.


METHODS
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.


RESULTS
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.


CONCLUSIONS
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.
Shanghai Institute of Blood Transfusion, Shanghai Blood Center, Shanghai, China.
R S
RS
Li
Z L
ZL
Qiao
B
B
Ling
P
P
Lu
D016824:Antigens, Human Platelet / Q000235:genetics*; D001483:Base Sequence; D001792:Blood Platelets / Q000276:immunology; D005838:Genotype; D060005:Genotyping Techniques / Q000592:standards*; D006801:Humans; D016297:Mutagenesis, Site-Directed; D010957:Plasmids / Q000235:genetics; D017713:Platelet Transfusion; D012150:Polymorphism, Restriction Fragment Length; D018807:Polymorphism, Single-Stranded Conformational; D012015:Reference Standards; D017422:Sequence Analysis, DNA
D016824:Antigens, Human Platelet
alloantibody; human platelet antigen; reference panel; site-directed mutagenesis; thrombocytopenia