TY - JOUR
T1 - Multicentre Harmonisation of a Six-Colour Flow Cytometry Panel for Naïve/Memory T Cell Immunomonitoring
AU - Macchia, Iole
AU - La Sorsa, Valentina
AU - Ruspantini, Irene
AU - Sanchez, Massimo
AU - Tirelli, Valentina
AU - Carollo, Maria
AU - Fedele, Giorgio
AU - Leone, Pasqualina
AU - Schiavoni, Giovanna
AU - Buccione, Carla
AU - Rizza, Paola
AU - Nisticò, Paola
AU - Palermo, Belinda
AU - Morrone, Stefania
AU - Stabile, Helena
AU - Rughetti, Aurelia
AU - Nuti, Marianna
AU - Zizzari, Ilaria Grazia
AU - Fionda, Cinzia
AU - Maggio, Roberta
AU - Capuano, Cristina
AU - Quintarelli, Concetta
AU - Sinibaldi, Matilde
AU - Agrati, Chiara
AU - Casetti, Rita
AU - Rozo Gonzalez, Andrea
AU - Iacobone, Floriana
AU - Gismondi, Angela
AU - Belardelli, Filippo
AU - Biffoni, Mauro
AU - Urbani, Francesca
N1 - Copyright © 2020 Iole Macchia et al.
PY - 2020
Y1 - 2020
N2 - 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.
AB - 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.
KW - Biomarkers/blood
KW - CD3 Complex/blood
KW - CD4-Positive T-Lymphocytes/immunology
KW - CD8-Positive T-Lymphocytes/immunology
KW - Color/standards
KW - Flow Cytometry/methods
KW - Humans
KW - Immunologic Memory
KW - Immunophenotyping/standards
KW - Italy
KW - Leukocyte Common Antigens/blood
KW - Leukocytes, Mononuclear/immunology
KW - Observer Variation
KW - Receptors, CCR7/blood
KW - T-Lymphocyte Subsets/classification
U2 - 10.1155/2020/1938704
DO - 10.1155/2020/1938704
M3 - Article
C2 - 32322591
VL - 2020
SP - 1938704
JO - Journal of Immunology Research
JF - Journal of Immunology Research
SN - 2314-8861
ER -