Multicentric harmonization of a 6-color flow cytometry panel for immunological monitoring of human whole blood and PBMC samples

Personalized medicine in oncology needs standardized immunological assays. Multiparametric flow-cytometry (MFC) methods represent an essential tool for immunomonitoring, and their harmonization is crucial to obtain comparable data in multicenter clinical trials. Here we report a real-life harmonization protocol of a flow cytometric assay, able to address the most effective issues contributing to intra- and inter-operator variability in the context of a multicenter project. Studies have shown that certain characteristics of the T cell compartment, including the ratio of naïve to memory T cells, the presence of specific memory T cell subsets, and the degree of T cell exhaustion, can impact patient responses to immunotherapy. Naïve/memory status of T lymphocytes is thus recognized as a potential prognostic/predictive immunological biomarker, and various MFC panels are currently applied in both mono- and multi-center clinical studies. Here, we introduce the protocol inspired by a project conducted at our institute, aimed at harmonizing a six-color MFC panel focused on determining the naïve/memory maturation status of CD3+ T lymphocytes, intended for use with both peripheral blood mononuclear cell (PBMC) and whole blood (WB) human samples. To offer a comprehensive overview of the methodological approach, we outline specifics concerning the pre-analytical and experimental analytical phases, emphasizing the final statistical analysis processing. The goal is to promote standardization and reproducibility by addressing variability in sample handling and preparation prior to applying the protocol to patient samples in clinical or translational multicenter trials. At the same time, the protocol design enables proficiency testing for the participating operators, instruments and centers.