An Integrated Workflow To Assess Technical and Biological Variability of Cell Population Frequencies in Human Peripheral Blood by Flow Cytometry.
An Integrated Workflow To Assess Technical and Biological Variability of Cell Population Frequencies in Human Peripheral Blood by Flow Cytometry.
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DOI：
10.4049/jimmunol.1601750
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发表时间：
2017-02-15
期刊：
影响因子：
--
通讯作者：
Peters B
中科院分区：
文献类型：
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作者：
Burel JG;Qian Y;Lindestam Arlehamn C;Weiskopf D;Zapardiel-Gonzalo J;Taplitz R;Gilman RH;Saito M;de Silva AD;Vijayanand P;Scheuermann RH;Sette A;Peters B
在大规模人体系统免疫学研究的背景下，控制技术和生物变异性对于确保实验数据支持研究结论至关重要。在这里，我们报告了一个通用的工作流程，以评估多参数流式细胞术的技术和生物学变化，应用于开发一个10色面板，以确定冻存PBMC中的所有主要细胞群和T细胞亚群。对照供体的重复运行和不同门控策略的比较评估了与每个细胞群相关的技术变异性，并允许计算质控评分。将我们的小组应用于大量PBMC样本，我们发现大多数细胞群随时间推移显示出较低的个体内变异性。相反，某些亚群如CD56 T细胞和Temra CD4 T细胞与高个体间变异性相关。年龄而不是性别对几个群体的频率有显著影响，在老年供体中观察到幼稚T细胞急剧减少。种族也影响了免疫细胞群体频率的显着比例，强调需要在免疫分析研究中考虑这些协变量。最后，我们通过识别潜伏性结核感染的新细胞亚群特征来验证我们的工作流程的有用性。因此，我们的研究提供了一个通用的工作流程，以建立和评估系统免疫学研究中的任何流式细胞术面板。
In the context of large-scale human system immunology studies, controlling for technical and biological variability is crucial to ensure that experimental data support research conclusions. Here, we report on a universal workflow to evaluate both technical and biological variation in multiparameter flow cytometry, applied to the development of a 10-color panel to identify all major cell populations and T cell subsets in cryopreserved PBMC. Replicate runs from a control donation and comparison of different gating strategies assessed technical variability associated with each cell population and permitted the calculation of a quality control score. Applying our panel to a large collection of PBMC samples, we found that most cell populations showed low intra-individual variability over time. In contrast, certain subpopulations such as CD56 T cells and Temra CD4 T cells were associated with high inter-individual variability. Age but not gender had a significant effect on the frequency of several populations, with a drastic decrease in naïve T cells observed in older donors. Ethnicity also influenced a significant proportion of immune cell population frequencies, emphasizing the need to account for these co-variates in immune profiling studies. Finally, we exemplify the usefulness of our workflow by identifying a novel cell-subset signature of latent tuberculosis infection. Thus, our study provides a universal workflow to establish and evaluate any flow cytometry panel in systems immunology studies.