Multicentre Harmonisation of a Six-Colour Flow Cytometry Panel for Naïve/Memory T Cell Immunomonitoring

Iole MacChia, Valentina La Sorsa, Irene Ruspantini, Massimo Sanchez, Valentina Tirelli, Maria Carollo, Giorgio Fedele, Pasqualina Leone, Giovanna Schiavoni, Carla Buccione, Paola Rizza, Paola Nisticò, Belinda Palermo, Stefania Morrone, Helena Stabile, Aurelia Rughetti, Marianna Nuti, Ilaria Grazia Zizzari, Cinzia Fionda, Roberta MaggioCristina Capuano, Concetta Quintarelli, Matilde Sinibaldi, Chiara Agrati, Rita Casetti, Andrea Rozo Gonzalez, Floriana Iacobone, Angela Gismondi, Filippo Belardelli, Mauro Biffoni, Francesca Urbani

Research output: Contribution to journalArticle

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- A nd 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.

Original languageEnglish
Article number1938704
Pages (from-to)1-15
Number of pages15
JournalJournal of Immunology Research
Volume2020
DOIs
Publication statusPublished - Jan 1 2020

ASJC Scopus subject areas

  • Immunology and Allergy
  • Immunology

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