The advent of time‐of‐flight mass cytometry (CyTOF) has enabled high dimensional and unbiased examination of the immune system to simultaneous interrogate a multitude of parameters and gain a better understanding of immunologic data from clinical trial samples. Here we describe the development and validation of a 33‐marker mass cytometry workflow for measuring gastrointestinal (GI) trafficking peripheral blood mononuclear cells (PBMCs) in patients with celiac disease (CeD). This panel builds upon identification of well‐characterized immune cells and expands to include markers modulated in response to gluten challenge in patients with CeD. The CeD panel was optimized and validated according to accepted industry practice for validation of flow cytometry assays and builds upon established sample processing workflows for mass cytometry studies. Several critical parameters were evaluated during the assay development phase of this study including optimization of the sample processing steps, antibody specificity, and ensuring the panel as a whole performed to expectation. The panel was then validated using a fit‐for‐purpose approach tailored to the intended use of the data in the clinical trial. Validation included assessment of analytical parameters essential to understanding the reliability and robustness of the CeD panel such as intra‐assay precision, inter‐assay precision, inter‐operator precision and sample processing stability. Together, this validated mass cytometry workstream provides robust and reproducible high‐dimensional analysis of human peripheral blood immune cells to characterize patient samples from clinical trials.

中文翻译：

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开发和验证用于破译乳糜泻免疫调节变化的高参数质谱流式细胞术工作流程

飞行时间质谱流式细胞术 (CyTOF) 的出现使免疫系统的高维度和无偏见检查能够同时询问大量参数并更好地了解来自临床试验样本的免疫学数据。在这里，我们描述了用于测量乳糜泻 (CeD) 患者的胃肠道 (GI) 运输外周血单个核细胞 (PBMC) 的 33 标志物质谱流式细胞仪工作流程的开发和验证。该小组基于对特征良好的免疫细胞的鉴定，并扩展到包括针对 CeD 患者的麸质挑战而调节的标志物。CeD 面板根据公认的流式细胞仪检测验证行业惯例进行了优化和验证，并建立在已建立的用于质谱研究的样本处理工作流程之上。在本研究的检测开发阶段评估了几个关键参数，包括优化样品处理步骤、抗体特异性，以及确保整个面板符合预期。然后使用针对临床试验中数据的预期用途量身定制的适合目的的方法对小组进行验证。验证包括评估对了解 CeD 面板的可靠性和稳健性至关重要的分析参数，例如批内精度、批间精度、操作员间精度和样品处理稳定性。总之，这个经过验证的质谱流式细胞仪工作流程提供了对人类外周血免疫细胞的可靠且可重复的高维分析，以表征来自临床试验的患者样本。