Mass cytometry (CyTOF) represents one of the most powerful tools in immune phenotyping, allowing high throughput quantification of over 40 single parameters at single-cell resolution. However, wide deployment of CyTOF-based immune phenotyping studies are limited by complex experimental workflows and the need for specialized CyTOF equipment and technical expertise. Furthermore, differences in cell isolation and enrichment protocols, antibody reagent preparation, sample staining and data acquisition protocols can all introduce technical variation that can potentially confound integrative analyses of large data-sets of samples processed across multiple labs. Here, we present a streamlined whole blood CyTOF workflow which addresses many of these sources of experimental variation and facilitates wider adoption of CyTOF immune monitoring across sites with limited technical expertise or sample-processing resources or equipment. Our workflow utilizes commercially available reagents including the Fluidigm MaxPar Direct Immune Profiling Assay (MDIPA), a dry tube 30-marker immunophenotyping panel, and SmartTube Proteomic Stabilizer, which allows for simple and reliable fixation and cryopreservation of whole blood samples. We validate a workflow that allows for streamlined staining of whole blood samples with minimal processing requirements or expertise at the site of sample collection, followed by shipment to a central CyTOF core facility for batched downstream processing and data acquisition. We further demonstrate the application of this workflow to characterize immune responses in a cohort of hospitalized COVID-19 patients, highlighting key disease-associated changes in immune cell frequency and phenotype.

中文翻译：

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简化的CyTOF工作流程，以简化COVID-19患者的标准化多站点免疫分析。

质谱分析法（CyTOF）代表了免疫表型分析中最强大的工具之一，可以在单细胞分辨率下对40多个单个参数进行高通量定量分析。但是，基于CyTOF的免疫表型研究的广泛应用受到复杂的实验工作流程以及对CyTOF专业设备和技术专长的需求的限制。此外，细胞分离和富集方案，抗体试剂制备，样品染色和数据采集方案中的差异都可能引入技术差异，从而可能混淆跨多个实验室处理的大型数据集的综合分析。这里，我们提出了一种简化的全血CyTOF工作流程，该流程解决了许多此类实验变异来源，并在技术专业知识或样本处理资源或设备有限的情况下，促进了跨站点的CyTOF免疫监测的广泛采用。我们的工作流程利用了市场上可买到的试剂，包括Fluidigm MaxPar直接免疫分析法（MDIPA），干管30标记免疫表型检测板和SmartTube蛋白质组稳定剂，可对全血样品进行简单可靠的固定和冷冻保存。我们验证了一种工作流程，该流程可在样品采集现场以最少的处理要求或专业知识来简化全血样品的染色，然后将其运送到中央CyTOF核心设施，以进行批量下游处理和数据采集。