Department of Oncology and Molecular Medicine, Istituto Superiore di Sanità (ISS), Rome 00161, Italy.
Research Coordination and Support Service, CoRI, ISS, Rome 00161, Italy.
J Immunol Res. 2020 Apr 12;2020:1938704. doi: 10.1155/2020/1938704. eCollection 2020.
Personalised medicine in oncology needs standardised immunological assays. Flow cytometry (FCM) methods represent an essential tool for immunomonitoring, and their harmonisation is crucial to obtain comparable data in multicentre clinical trials. The objective of this study was to design a harmonisation workflow able to address the most effective issues contributing to intra- and interoperator variabilities in a multicentre project.
The Italian National Institute of Health (Istituto Superiore di Sanità, ISS) managed a multiparametric flow cytometric panel harmonisation among thirteen operators belonging to five clinical and research centres of Lazio region (Italy). The panel was based on a backbone mixture of dried antibodies (anti-CD3, anti-CD4, anti-CD8, anti-CD45RA, and anti-CCR7) to detect naïve/memory T cells, recognised as potential prognostic/predictive immunological biomarkers in cancer immunotherapies. The coordinating centre distributed frozen peripheral blood mononuclear cells (PBMCs) and fresh whole blood (WB) samples from healthy donors, reagents, and Standard Operating Procedures (SOPs) to participants who performed experiments by their own equipment, in order to mimic a real-life scenario. Operators returned raw and locally analysed data to ISS for central analysis and statistical elaboration.
Harmonised and reproducible results were obtained by sharing experimental set-up and procedures along with centralising data analysis, leading to a reduction of cross-centre variability for naïve/memory subset frequencies particularly in the whole blood setting.
Our experimental and analytical working process proved to be suitable for the harmonisation of FCM assays in a multicentre setting, where high-quality data are required to evaluate potential immunological markers, which may contribute to select better therapeutic options.
肿瘤学中的个性化医学需要标准化的免疫学检测。流式细胞术（FCM）方法是免疫监测的重要工具，其协调对于在多中心临床试验中获得可比数据至关重要。本研究的目的是设计一种协调工作流程，以解决多中心项目中导致个体间和操作者间变异性的最有效问题。
意大利国家卫生研究院（Istituto Superiore di Sanità，ISS）管理了一个由 13 名操作人员组成的多参数流式细胞术面板协调工作，这些操作人员来自拉齐奥地区（意大利）的五个临床和研究中心。该面板基于一种干燥抗体的骨干混合物（抗-CD3、抗-CD4、抗-CD8、抗-CD45RA 和抗-CCR7），用于检测幼稚/记忆 T 细胞，这些细胞被认为是癌症免疫治疗中潜在的预后/预测性免疫生物标志物。协调中心向参与者分发来自健康供体的冷冻外周血单核细胞（PBMC）和新鲜全血（WB）样本、试剂和标准操作程序（SOP），以便他们在自己的设备上进行实验，从而模拟现实生活场景。操作人员将原始和本地分析的数据返回给 ISS 进行中心分析和统计处理。
通过共享实验设置和程序以及集中数据分析，实现了协调和可重复的结果，特别是在全血设置中，降低了不同中心之间幼稚/记忆亚群频率的变异性。
我们的实验和分析工作流程被证明适用于多中心环境中的 FCM 检测协调，在这种环境中需要高质量的数据来评估潜在的免疫标志物，这可能有助于选择更好的治疗选择。
