Standardizing Flow Cytometry Immunophenotyping Analysis from the Human ImmunoPhenotyping Consortium.
Standardizing Flow Cytometry Immunophenotyping Analysis from the Human ImmunoPhenotyping Consortium.
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DOI：
10.1038/srep20686
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发表时间：
2016-02-10
影响因子：
4.6
通讯作者：
McCoy JP
中科院分区：
文献类型：
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作者：
Finak G;Langweiler M;Jaimes M;Malek M;Taghiyar J;Korin Y;Raddassi K;Devine L;Obermoser G;Pekalski ML;Pontikos N;Diaz A;Heck S;Villanova F;Terrazzini N;Kern F;Qian Y;Stanton R;Wang K;Brandes A;Ramey J;Aghaeepour N;Mosmann T;Scheuermann RH;Reed E;Palucka K;Pascual V;Blomberg BB;Nestle F;Nussenblatt RB;Brinkman RR;Gottardo R;Maecker H;McCoy JP
免疫表型的标准化需要仔细注意试剂、样品处理、仪器设置和数据分析，这对于成功的交叉研究和跨中心数据比较至关重要。专家们开发了五种标准化的八色面板，用于识别外周血中的主要免疫细胞亚群。这些是预先配置的，冻干的，试剂在96孔板。我们提出了九个实验室使用这些面板与标准化操作程序（sop）协调分析样品的结果。手动门控由每个站点和一个中心站点执行。自动门控算法由FlowCAP联盟开发和测试。集中的手动门控可以减少跨中心变异性，我们试图确定自动化方法是否可以简化和标准化分析。在所有实验中，站点内变异性都很低，但在进行中心分析时，与特定站点分析相比，跨站点变异性更低。对于明确定义的细胞亚群，它也低于基于模糊标记和罕见种群的细胞亚群。自动门控能够匹配所有测试面板的中央手动分析的性能，几乎没有偏差和可比性。标准化染色，数据收集和自动门控可以增加功率，减少可变性，并简化免疫表型分析。
Standardization of immunophenotyping requires careful attention to reagents, sample handling, instrument setup, and data analysis, and is essential for successful cross-study and cross-center comparison of data. Experts developed five standardized, eight-color panels for identification of major immune cell subsets in peripheral blood. These were produced as pre-configured, lyophilized, reagents in 96-well plates. We present the results of a coordinated analysis of samples across nine laboratories using these panels with standardized operating procedures (SOPs). Manual gating was performed by each site and by a central site. Automated gating algorithms were developed and tested by the FlowCAP consortium. Centralized manual gating can reduce cross-center variability, and we sought to determine whether automated methods could streamline and standardize the analysis. Within-site variability was low in all experiments, but cross-site variability was lower when central analysis was performed in comparison with site-specific analysis. It was also lower for clearly defined cell subsets than those based on dim markers and for rare populations. Automated gating was able to match the performance of central manual analysis for all tested panels, exhibiting little to no bias and comparable variability. Standardized staining, data collection, and automated gating can increase power, reduce variability, and streamline analysis for immunophenotyping.