DataSheet_2_Standardization of Workflow and Flow Cytometry Panels for Quantitative Expression Profiling of Surface Antigens on Blood Leukocyte Subsets: An HCDM CDMaps Initiative.pdf

BackgroundThe Human Cell Differentiation Molecules (HCDM) organizes Human Leukocyte Differentiation Antigen (HLDA) workshops to test and name clusters of antibodies that react with a specific antigen. These cluster of differentiation (CD) markers have provided the scientific community with validated antibody clones, consistent naming of targets and reproducible identification of leukocyte subsets. Still, quantitative CD marker expression profiles and benchmarking of reagents at the single-cell level are currently lacking.ObjectiveTo develop a flow cytometric procedure for quantitative expression profiling of surface antigens on blood leukocyte subsets that is standardized across multiple research laboratories.MethodsA high content framework to evaluate the titration and reactivity of Phycoerythrin (PE)-conjugated monoclonal antibodies (mAbs) was created. Two flow cytometry panels were designed: an innate cell tube for granulocytes, dendritic cells, monocytes, NK cells and innate lymphoid cells (12-color) and an adaptive lymphocyte tube for naive and memory B and T cells, including TCRγδ+, regulatory-T and follicular helper T cells (11-color). The potential of these 2 panels was demonstrated via expression profiling of selected CD markers detected by PE-conjugated antibodies and evaluated using 561 nm excitation.ResultsUsing automated data annotation and dried backbone reagents, we reached a robust workflow amenable to processing hundreds of measurements in each experiment in a 96-well plate format. The immunophenotyping panels enabled discrimination of 27 leukocyte subsets and quantitative detection of the expression of PE-conjugated CD markers of interest that could quantify protein expression above 400 units of antibody binding capacity. Expression profiling of 4 selected CD markers (CD11b, CD31, CD38, CD40) showed high reproducibility across centers, as well as the capacity to benchmark unique clones directed toward the same CD3 antigen.ConclusionWe optimized a procedure for quantitative expression profiling of surface antigens on blood leukocyte subsets. The workflow, bioinformatics pipeline and optimized flow panels enable the following: 1) mapping the expression patterns of HLDA-approved mAb clones to CD markers; 2) benchmarking new antibody clones to established CD markers; 3) defining new clusters of differentiation in future HLDA workshops.

背景：人类细胞分化分子（HCDM）组织人类白细胞分化抗原（HLDA）研讨会，以测试和命名与特定抗原发生反应的抗体簇。这些分化簇（CD）标记为科学界提供了经过验证的抗体克隆、目标名称的一致性以及白细胞亚群的重复性识别。然而，目前尚未实现CD标记表达谱的定量分析和试剂在单细胞水平的基准测试。目标：开发一种流式细胞术程序，以对血液白细胞亚群表面的抗原进行定量表达谱分析，并在多个研究实验室中得到标准化。方法：创建了一个用于评估藻红蛋白（PE）标记的单克隆抗体（mAbs）滴度和反应性的高内涵框架。设计了两个流式细胞术面板：一个用于粒细胞、树突状细胞、单核细胞、自然杀伤细胞和先天淋巴细胞（12色）的天然细胞管，以及一个用于原始和记忆B细胞和T细胞（包括TCRγδ+、调节性T细胞和滤泡辅助T细胞，11色）的适应性淋巴细胞管。通过使用PE标记的抗体检测到的选定CD标记的表达谱分析，并使用561 nm激发光进行评估，展示了这两个面板的潜力。结果：通过自动数据标注和干燥的骨架试剂，我们实现了一个稳健的工作流程，适用于在96孔板格式中处理每个实验中的数百次测量。免疫表型分析面板能够区分27个白细胞亚群，并定量检测感兴趣的PE标记CD标记的表达，能够量化超过400单位抗体结合能力的蛋白质表达。4个选定CD标记（CD11b、CD31、CD38、CD40）的表达谱分析显示了跨中心的较高可重复性，以及基准测试针对相同CD3抗原的独特克隆的能力。结论：我们优化了血液白细胞亚群表面抗原的定量表达谱分析程序。该工作流程、生物信息学管道和优化的流式细胞术面板使得以下成为可能：1）将HLDA批准的mAb克隆的表达模式映射到CD标记；2）将新抗体克隆与已建立的CD标记进行基准测试；3）在未来的HLDA研讨会上定义新的分化簇。