此前，我们发现血浆细胞外囊泡（EV）中淋巴瘤相关 miRNA miR-155-5p、-127-3p 和 let-7a-5p 水平的定量报告了经典霍奇金淋巴瘤（cHL）患者的治疗反应。在临床实施之前，需要进行质量控制 (QC) 步骤和验证，以满足国际监管标准。大多数已发表的基于 EV 的诊断测定尚未满足这些要求。为了推进检测符合法规（例如 IVDR 2017/746），我们在 ISO-13485 实验性 EV-miRNA 定量实时逆转录 PCR (q-RT-PCR) 检测中纳入了三个 QC 步骤经过认证的质量管理体系（QMS）。脂质体封装有合成的（线虫衍生的）miRNA，通过自动尺寸排阻色谱 (SEC) 控制 EV 分离。额外的 miRNA 掺入可控制 RNA 分离和 cDNA 转化效率。确定质量标准后，46 名患者的 120 个样本中总共有 107 个通过了 QC。采用 bootstrapping 的广义线性混合效应模型确定了质量控制数据的诊断性能，其曲线下面积 (AUC) 为 0.84（置信区间 [CI]：0.76-0.92），而 AUC 为 0.87（CI：0.80） -0.94）的实验测定。纳入 QC 步骤后，该检测方法在预测 cHL 患者治疗期间活动性疾病状态方面的准确度为 78.5%。我们证明，质量控制的血浆 EV-miRNA 测定在技术上是可靠的，将 EV-miRNA 作为液体活检测定向临床评估迈出了重要一步。© 2024 作者。 《细胞外生物学杂志》由 Wiley periodicals LLC 代表国际细胞外囊泡学会出版。

Previously, we showed that quantification of lymphoma-associated miRNAs miR-155-5p, -127-3p and let-7a-5p levels in plasma extracellular vesicles (EVs) report treatment response in patients with classic Hodgkin lymphoma (cHL). Prior to clinical implementation, quality control (QC) steps and validation are required to meet international regulatory standards. Most published EV-based diagnostic assays have yet to meet these requirements. In order to advance the assay towards regulatory compliance (e.g., IVDR 2017/746), we incorporated three QC steps in our experimental EV-miRNA quantitative real-time reverse-transcription PCR (q-RT-PCR) assay in an ISO-13485 certified quality-management system (QMS). Liposomes encapsulated with a synthetic (nematode-derived) miRNA spike-in controlled for EV isolation by automated size-exclusion chromatography (SEC). Additional miRNA spike-ins controlled for RNA isolation and cDNA conversion efficiency. After deciding on quality criteria, in total 107 out of 120 samples from 46 patients passed QC. Generalized linear mixed-effect modelling with bootstrapping determined the diagnostic performance of the quality-controlled data at an area under the curve (AUC) of 0.84 (confidence interval [CI]: 0.76-0.92) compared to an AUC of 0.87 (CI: 0.80-0.94) of the experimental assay. After the inclusion of QC steps, the accuracy of the assay was determined to be 78.5% in predicting active disease status in cHL patients during treatment. We demonstrate that a quality-controlled plasma EV-miRNA assay is technically robust, taking EV-miRNA as liquid biopsy assay an important step closer to clinical evaluation.© 2024 The Author(s). Journal of Extracellular Biology published by Wiley Periodicals LLC on behalf of International Society for Extracellular Vesicles.