A Simplified, Scalable Solution for Simultaneous Genotyping of Human Leukocyte Antigen Classical Class I and Human Platelet Antigen Genes Utilising Oxford Nanopore Technologies Long-Read Sequencing.

Human leucocyte antigen (HLA) class I and human platelet antigens (HPAs) can elicit immune responses, leading to platelet transfusion refractoriness (PTRs). To support the clinical management of patients with PTR, transfusion of HLA class I and sometimes HPA-selected apheresis platelets is recommended. To facilitate this service, the HLA class I and HPA genotypes of platelet donors must be defined so that appropriate platelet units may be selected. Currently, National Health Service Blood and Transplant (NHSBT) utilises two separate assays for genotyping HLA class I and HPAs. In this study, a simple, single-well, multiplex PCR assay has been developed to genotype classical HLA class I genes (HLA-A, -B and -C) and 27/35 currently described HPAs, including all clinically relevant HPAs. Genomic DNA from 90 consented English apheresis platelet donors and 17 reference samples provided by the Australian Red Cross were amplified with the custom PCR protocol. Sequencing libraries were prepared using Oxford Nanopore Technologies (ONTs) native barcoding kit v14 (SQK-NBD114.96) and sequenced on MinION R10.4.1 flow cell. Base calling was performed live in MinKNOW v24.02.16, and sequence alignment, variant calling and genotype assignment of HPA were performed by a bespoke bioinformatics pipeline. Analysis of HLA class I genes was carried out using NGSengine v4.0.0. English apheresis platelet donors successfully genotyped (89/90) using the described assay showed 100% concordance with historic results for both HLA and clinically relevant HPAs. Further testing with reference samples from the Australian Red Cross samples showed 100% concordance with externally provided HPA results. This assay offers a simplified workflow for simultaneous HLA class I and HPA genotyping of apheresis platelet donors, permitting automation and significant expansion to include more HPA specificities. This assay provides accurate HLA class I genotyping and HPA genotyping in a single assay, requiring a minimal laboratory footprint and less maintenance than current in-use assays.