Rapid and Robust Multiplex Multicolour Melting Curve Analysis Enables Simultaneous Detection of HLA Cross-Reactive...

Platelet transfusion refractoriness (PTR) is a major clinical challenge that increases bleeding risk and wastes blood resources. Immune-mediated PTR is mainly caused by alloantibodies against HLA class I and human platelet antigens (HPA). To overcome this, transfusion of compatible platelets based on HLA cross-reactive groups (CREGs) and HPA genotypes is considered effective; however, current detection methods remain costly, complex and time-consuming, and no simple approach is available for direct CREGs detection. In this study, we developed a novel multiplex multicolour melting curve analysis (MMCA) assay that simultaneously detected HLA CREGs and the genotypes of 14 HPA systems (HPA-1-6, 9, 12, 13, 15, 21, 27, 30 and 31) within just six reaction tubes. Analytical validation demonstrated that the MMCA assay achieved an analytical sensitivity of 0.222-4.725 ng/μL, with excellent reproducibility, as indicated by intra-assay coefficients of variation (CVs) of 0.07%-0.3% and inter-assay CVs of 0.1%-0.6%, depending on the reaction. Clinical validation using 200 blood donor samples showed complete concordance with next-generation sequencing (NGS) for CREGs detection and 95.5%-100% concordance with a commercial kit for HPA genotyping. Sequencing further confirmed the superior accuracy of the MMCA assay, especially for the HPA-2, HPA-3 and HPA-15 systems, thereby effectively preventing misclassification. The newly developed MMCA assay provides rapid turnaround, simplicity and cost-effectiveness. With high sensitivity, reproducibility and clinical accuracy, it offers a valuable tool for high-throughput selection of HLA- and HPA-matched platelets in immune-mediated PTR and establishing platelet donor registries.