Blood Center of Zhejiang Province, Hangzhou, China.
HLA. 2024 Oct;104(4):e15716. doi: 10.1111/tan.15716.
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-A11:451, HLA-A30:01:26, HLA-B39:201 and HLA-B40:538, 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.
基于基因分型选择相容的 HLA Ⅰ类和/或 HPA 血小板可能会减轻免疫性血小板输注无效（PTR）。为了构建具有已知 HLA Ⅰ类和 HPA 基因型的血小板供体库，需要一种快速可靠的 HLA Ⅰ类和 HPA 基因分型方法。设计了 10 对针对 HLA-A、HLA-B、HLA-C、HPA-1 到 HPA-6w、HPA-15 和 HPA-21w 的特异性引物。优化了合适的片段，以便在单个多重反应中进行扩增。在用磁性珠进行清洗步骤后，制备并测序扩增子文库。为了验证所开发方法的准确性，使用商业 NGS 试剂盒对 HLA-A、HLA-B 和 HLA-C 进行基因分型，以及 TaqMan 实时 PCR 方法对 HPA-1 到 HPA-6w、HPA-15 和 HPA-21w 进行基因分型，同时平行检测所有标本。共检测了 386 个标本，同时获得了 HLA-A、HLA-B、HLA-C 和 HPA-1 到 HPA-6w、HPA-15 和 HPA-21w 的基因分型结果，两种方法之间的结果完全一致。还重新确认了 4 个新的 HLA 等位基因，HLA-A11:451、HLA-A30:01:26、HLA-B39:201 和 HLA-B40:538。还检测到 ITGA2B 编码区中的 2 个新的 SNV，c.2671C > T 和 c.2681T > G，均为个体中的杂合子。建立了一种基于多重 PCR 的新型 NGS 方法来同时检测 HLA Ⅰ类和 HPA，该方法具有高通量、快速和准确的特点，可用于建立血小板供体库。
