Results from the first gating proficiency panel of intracellular cytokine staining (ICS) highlighted the value of using a consensus gating approach to reduce the variability across laboratories in reported %CD8⁺ or %CD4⁺ cytokine‐positive cells. Based on the data analysis from the first proficiency panel, harmonization guidelines for a consensus gating protocol were proposed. To validate the recommendations from the first panel and to examine factors that were not included in the first panel, a second ICS gating proficiency panel was organized. All participants analyzed the same set of Flow Cytometry Standard (FCS) files using their own gating protocol. An optional learning module was provided to demonstrate how to apply the previously established gating recommendations and harmonization guidelines to actual ICS data files. Eighty‐three participants took part in this proficiency panel. The results from this proficiency panel confirmed the harmonization guidelines from the first panel. These recommendations addressed the (1) placement of the cytokine‐positive gate, (2) identification of CD4⁺ CD8⁺ double‐positive T cells, (3) placement of lymphocyte gate, (4) inclusion of dim cells, (5) gate uniformity, and (6) proper adjustment of the biexponential scaling. In addition, based on the results of this proficiency gating panel, two new recommendations were added to expand the harmonization guidelines: (1) inclusion of dump channel marker to gate all live and dump negative cells and (2) backgating to confirm the correct placement of gates across all populations. © 2020 International Society for Advancement of Cytometry

中文翻译：

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细胞内细胞因子染色的门控协调指南在癌症免疫治疗联盟 (CIC/CRI) 进行的第二次国际多联盟熟练小组中得到验证

细胞内细胞因子染色 (ICS) 的第一个门控能力小组的结果强调了使用共识门控方法来减少实验室报告的 %CD8 ⁺或 %CD4 ^{+的变异性的价值}细胞因子阳性细胞。基于第一个能力小组的数据分析，提出了共识门控协议的协调指南。为了验证第一个小组的建议并检查第一个小组中未包含的因素，组织了第二个 ICS 门控能力小组。所有参与者都使用他们自己的门控协议分析了同一组流式细胞术标准 (FCS) 文件。提供了一个可选的学习模块来演示如何将先前建立的门控建议和协调指南应用于实际的 ICS 数据文件。八十三名参与者参加了这个能力评估小组。该熟练度小组的结果证实了第一小组的协调指南。这些建议涉及 (1) 细胞因子阳性门的位置，⁺ CD8 ⁺双阳性 T 细胞，(3) 淋巴细胞门的位置，(4) 暗细胞的包含，(5) 门的均匀性，以及 (6) 双指数缩放的适当调整。此外，根据该能力门控小组的结果，添加了两项新建议以扩展协调指南：(1) 包含转储通道标记以门控所有活细胞和转储阴性细胞，以及 (2) 后门控以确认正确放置所有人群的大门。© 2020 国际细胞术进步协会