Multiplex-PCR technique to predict polymorphic antigens - M, N, S and s - and associations between their alleles and Mi-associated hybrid glycophorins.

Serological typing of MNS polymorphic antigens - M, N, S and s - remains a fundamental technique in transfusion medicine and prenatal care, providing essential information for matching blood donors and recipients and managing haemolytic disease. Although this method is well proven and routinely used, it is not a comprehensive solution, as it has several weaknesses. Alternatively, multiplex polymerase chain reaction (PCR) is a commonly used genotyping tool due to its potency and ability to amplify several DNA targets simultaneously in a single reaction. In this work, we aimed to develop multiplex PCR and evaluate its performance for GYPAM, GYPAN, GYPBS, and GYPBs allele identification using serological and DNA sequencing methods. We also aimed to investigate the correlation between these alleles and Mi-associated hybrid glycophorins (GPs). Remarkably, multiplex PCR was well optimised, and the results aligned with serological phenotyping and DNA sequencing data with maximum accuracy and reliability; this confirmed our findings on its validity in predicting MNSs phenotypes. In addition, this work strongly demonstrates, for the first time, a moderate correlation between the GYPAM/M and GYPBs/s genotypes and Mi-associated hybrid GPs among Thai donors. Individuals with the GYPAM/M and GYPBs/s genotypes, predicted M + N - S- s + phenotypes, will thus most likely to express the Mi(a+) antigen. Nevertheless, further studies are required to validate these results and elucidate the underlying correlations.