Establishment and Application of a Multiplex PCR NGS Method for the Genotyping of HLA‐Class I and HPA

ABSTRACT Selecting compatible HLA‐Class I and/or HPA platelets based on genotyping could alleviate immune platelet transfusion refractoriness (PTR). A fast and reliable method of HLA‐Class I and HPA genotyping is necessary to construct a platelet donor bank with known HLA‐Class I and HPA genotypes. Ten pairs of specific primers for HLA‐A, HLA‐B, HLA‐C, HPA‐1 through HPA‐6w, HPA‐15 and HPA‐21w were designed. The appropriate fragments were optimised for amplification in a single multiplex reaction. After a cleanup step using paramagnetic beads, the amplicon library was prepared and sequenced. To validate the accuracy of the developed method, commercial NGS kits for the genotyping of HLA‐A, HLA‐B and HLA‐C and the TaqMan real‐time PCR method in‐house for the genotyping of HPA‐1 through HPA‐6w, HPA‐15 and HPA‐21w were used to detect all the specimens in parallel. A total of 386 specimens were detected and the results of genotyping HLA‐A, HLA‐B, HLA‐C and HPA‐1 through HPA‐6w, HPA‐15 and HPA‐21w were obtained simultaneously, which is 100% consistent between the two methods. Four new HLA alleles, HLA‐A*11:451, HLA‐A*30:01:26, HLA‐B*39:201 and HLA‐B*40:538, were also reconfirmed. Two novel SNVs, c.2671C > T and c.2681T > G, in the coding region of ITGA2B were detected, all of which are heterozygous in individuals. A novel NGS method based on multiplex PCR was established to detect HLA‐Class I and HPA simultaneously, which is high‐throughput, rapid and accurate and could be applied to build a platelet donor bank.