Comparison of a Simple-Probe Real-Time PCR and Multiplex PCR Techniques for HPA-1 to HPA-6 and HPA-15 Genotyping

Abstract

We aimed to compare HPA-1 to HPA-6 and HPA-15 genotyping results obtained by a simple-probe real-time polymerase chain reaction (PCR) technique with the multiplex PCR technique. Five hundred DNA samples from the Thai National Stem Cell Donor Registry (TSCDR) of the National Blood Centre, Thai Red Cross Society were included. Human platelet antigen (HPA) genotyping was performed by simple-probe real-time PCR and multiplex PCR techniques. HPA-1, HPA-2, HPA-3, and HPA-4 genotyping results obtained by both techniques were in agreement. The misinterpretation of HPA-5, HPA-6, and HPA-15 genotypes was found in eight samples by simple-probe real-time PCR and HPA genotypes were confirmed by DNA sequencing. Two samples of HPA-5 were misinterpreted as HPA-5a5a instead of HPA-5a5b due to an NM_002203.3:c.1594A>C mutation (rs199808499) near the HPA-5 polymorphism (5' side). Five samples of HPA-6a6b were misinterpreted as HPA-6b6b because of an NM_000212.2:c.1545G>A mutation (rs4634) adjacent to the HPA-6 polymorphism (3' side). Interestingly, one sample of HPA-15a15b was misinterpreted as HPA-15b15b due to an NM_133493.1:c.2118C>A mutation near the HPA-15 polymorphism (3' side). HPA genotyping results by two PCR techniques were compared. Incorrect assignments were found due to genetic variations near each HPA single nucleotide polymorphism. Therefore, to avoid false assignation, the use of two genotyping techniques is recommended.