A Practical and Efficient Multiplex qPCR Assay for Simultaneous Detection of HLA Cross‐Reactive Groups and Human Platelet Antigens

Background Immune‐mediated platelet transfusion refractoriness (IPTR) remains a major clinical challenge, with immune alloimmunization against HLA and HPA as the predominant causes. Although HLA cross‐reactive groups (CREGs) provide a practical alternative to allele‐level matching, methods for simultaneous HLA‐CREG and HPA genotyping remain limited despite their potential to improve platelet matching. Methods Specific U‐anchoring primers (UAPs) and amplification refractory mutation system (ARMS) primers were designed and optimized based on one or more SNPs within HLA‐CREG subgroups and HPA alleles. Reaction conditions were optimized, and analytical performance was evaluated. A total of 200 donor DNA samples were tested and compared with NGS and a commercial HPA sequencing kit, with discrepant results verified by Sanger sequencing. Results We developed and optimized a novel multiplex real‐time quantitative PCR (qPCR) assay integrating UAPs and ARMS primers for simultaneous detection of HLA‐A/B CREGs and major HPA alleles in 12 reaction tubes. The assay demonstrated an overall limit of detection (LOD) of 0.838 ng/μL and the intra‐/inter‐assay coefficients of variation were both below 5%, indicating high reproducibility. HLA‐CREG typing results were 100% concordant with next‐generation sequencing. Sixteen discrepant HPA genotyping results were identified compared with the commercial kit, all of which were confirmed by Sanger sequencing and were consistent with the multiplex qPCR assay. Conclusions This multiplex qPCR assay enables rapid, accurate, and cost‐effective simultaneous detection of HLA‐CREGs and HPA alleles, providing a practical tool for comprehensive platelet compatibility assessment and improved transfusion matching.