Human platelet membrane glycoprotein polymorphisms can be immunogenic in man, and are frequently the cause of clinically important immune reactions responsible for disorders such as neonatal alloimmune thrombocytopenia. Platelets from individuals carrying rare polymorphisms are often difficult to obtain, making diagnostic testing and transfusion of matched platelets challenging. Class I HLA antibodies frequently present in maternal sera additionally interfere with detection of platelet-reactive alloantibodies. Detection of alloantibodies to human platelet antigens (HPA) 3 and 9 is especially challenging, in part due to the presence of cell type-specific glycans situated near the polymorphic amino acid that together form the alloepitope. To overcome these limitations, we generated a series of HLA class I-negative, blood group O, induced pluripotent stem cell lines (iPSCs) that were additionally gene-edited to sequentially convert their endogenous HPA-3a alloantigenic epitope to HPA-3b, and HPA-9a to HPA-9b. Subjecting these cell lines, upon differentiation into CD41+/CD42b+ human megakaryocytes (MKs), to flow cytometric detection of suspected anti-HPA-3 and HPA-9 alloantisera revealed that the HPA-3a-positive MKs specifically reacted with HPA-3a patient sera, while the HPA-3b MKs lost reactivity with HPA-3a patient sera while acquiring reactivity to HPA-3b patient sera. Importantly, HPA-9b-expressing MKs specifically reacted with anti-HPA-9b-suspected patient samples that had been undetectable using conventional techniques. The provision of specialized iPSC-derived human megakaryocytes expressing intact, homozygous glycoprotein alloantigens on the cell surface that additionally carry the appropriate endogenous carbohydrate moieties should greatly enhance detection of clinically-important, rare HPA-specific alloantibodies that have to date resisted detection using current methods.

中文翻译：

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用于检测血小板特异性患者同种抗体的生物工程 iPSC 衍生巨核细胞

人血小板膜糖蛋白多态性在人体内具有免疫原性，并且常常是引起新生儿同种免疫性血小板减少症等疾病的临床重要免疫反应的原因。来自携带罕见多态性的个体的血小板通常很难获得，这使得匹配血小板的诊断测试和输血具有挑战性。母体血清中经常出现的 I 类 HLA 抗体还会干扰血小板反应性同种抗体的检测。检测人血小板抗原 (HPA) 3 和 9 的同种抗体尤其具有挑战性，部分原因是在多态性氨基酸附近存在细胞类型特异性聚糖，它们一起形成同种异体表位。为了克服这些限制，我们生成了一系列 HLA I 类阴性、O 型血、诱导多能干细胞系 (iPSCs) 额外进行基因编辑以依次将其内源性 HPA-3a 同种异体抗原表位转化为 HPA-3b，将 HPA-9a 转化为 HPA-9b。将这些细胞系分化为 CD41+/CD42b+ 人巨核细胞 (MKs) 后，对可疑的抗 HPA-3 和 HPA-9 同种抗血清进行流式细胞术检测，结果表明 HPA-3a 阳性 MKs 与 HPA-3a 患者血清发生特异性反应，而 HPA-3b MKs 失去了与 HPA-3a 患者血清的反应性，同时获得了对 HPA-3b 患者血清的反应性。重要的是，表达 HPA-9b 的 MKs 与使用常规技术无法检测到的抗 HPA-9b 疑似患者样本发生特异性反应。提供特化的 iPSC 衍生的人类巨核细胞，表达完整，