Clinical Biomarker Innovation & Development, Takeda Pharmaceuticals Inc. Co, Cambridge, Massachusetts, USA.
Primity Bio, Inc., Fremont, California, USA.
Cytometry B Clin Cytom. 2021 Jan;100(1):92-102. doi: 10.1002/cyto.b.21986. Epub 2021 Jan 16.
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.
时间飞行质谱流式细胞术（CyTOF）的出现使人们能够对免疫系统进行高维且无偏倚的检测，从而同时检测多个参数，并更好地理解临床试验样本中的免疫数据。在这里，我们描述了一种用于测量乳糜泻（CeD）患者胃肠道（GI）迁移外周血单核细胞（PBMC）的 33 标志物质谱流式细胞术工作流程的开发和验证。该面板基于对特征明确的免疫细胞的鉴定，并扩展到包括在 CeD 患者中对麸质挑战有反应的标志物。该 CeD 面板根据流式细胞术检测验证的公认行业惯例进行了优化和验证，并建立在用于质谱流式细胞术研究的既定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，包括优化样本处理步骤、抗体特异性，并确保整个面板按预期运行。然后，使用专门针对临床试验中数据预期用途的特定用途方法对该面板进行了验证。验证包括评估分析参数，这些参数对于理解 CeD 面板的可靠性和稳健性至关重要，例如：室内精密度、室间精密度、操作员间精密度和样本处理稳定性。总之，经过验证的质谱流式细胞术工作流程为临床研究中的患者样本提供了可靠且可重复的人类外周血免疫细胞的高维分析。
