Abstract
Background 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. Methods 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. Results 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. Conclusion 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.
Highlights
Immunomonitoring has become increasingly relevant in the immunooncology field for the identification of potential prognostic/predictive immune biomarkers and a better understanding of their underlying mechanisms of action, leading to improved personalised treatments.Flow cytometry (FCM) is a powerful technology commonly used to dissect the immune phenotype due to its unique ability to measure quantitatively and simultaneously large numbers of single cells by multiple markers
Hereafter, we will refer intra- and interoperator variability as expression of the contribution of each operator independently from the centre. cryopreserved PBMCs (cPBMCs) replicates were obtained in different experimental rounds, whereas whole blood (WB) replicates were carried out within the same day, as previously stated
We compared local vs. centralised analysis of all T cell subset frequencies by mean of boxes representing triplicate mean values obtained in all observations (n = 12), from one representative donor for cPBMC and one for WB samples
Summary
Immunomonitoring has become increasingly relevant in the immunooncology field for the identification of potential prognostic/predictive immune biomarkers and a better understanding of their underlying mechanisms of action, leading to improved personalised treatments.FCM is a powerful technology commonly used to dissect the immune phenotype due to its unique ability to measure quantitatively and simultaneously large numbers of single cells by multiple markers. In most cases, monitoring of patients’ response is conducted over an extended time lapse, from the pretreatment phase to the follow-up period, and in this context, assay repeatability must be considered mandatory. For this reason, FCM assays need to meet specific analytical method validation especially in settings requiring good laboratory/clinical practice (GLP/GCP) compliance. Flow cytometry (FCM) methods represent an essential tool for immunomonitoring, and their harmonisation is crucial to obtain comparable data in multicentre clinical trials. 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
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