Validating the HPA-1 to -5 and -15 Detection by Homemade PCR-SSP, Real-Time PCR, and PCR-RFLP Methods.

ABSTRACT

OBJECTIVE: Human platelet antigens (HPAs) are antigenic determinants on platelet membrane glycoproteins that stimulate the host's immune system and cause platelet destruction. In this study, we share our experience with implementing sequence-specific primer-polymerase chain reaction (PCR-SSP), real-time PCR, and PCR-RFLP (restriction fragment length polymorphism) and the validation process used to evaluate the results. METHODS: At the Ardabil Blood Transfusion Center, 10 samples were obtained from blood donors. Validation using PCR-SSP, real-time PCR, and PCR-RFLP methods for genotyping HPAs was done by sequencing. A commercial DNA sample and a commercial kit were also used for validation. RESULTS: The results of PCR-SSP, TaqMan Real-Time PCR, melting curve analysis (HPA-15), and PCR-RFLP (HPA-3) were 100% consistent with sequencing (gold standard) and commercial kit results. CONCLUSIONS: There was a 100% correlation between repeating the methods and the expected results for repeatability, and no false positives and negatives were observed.