Multicentre Harmonisation of a Six-Colour Flow Cytometry Panel for Naïve/Memory T Cell Immunomonitoring.
Biomarkers
/ blood
CD3 Complex
/ blood
CD4-Positive T-Lymphocytes
/ immunology
CD8-Positive T-Lymphocytes
/ immunology
Color
/ standards
Flow Cytometry
/ methods
Humans
Immunologic Memory
Immunophenotyping
/ standards
Italy
Leukocyte Common Antigens
/ blood
Leukocytes, Mononuclear
/ immunology
Observer Variation
Receptors, CCR7
/ blood
T-Lymphocyte Subsets
/ classification
Journal
Journal of immunology research
ISSN: 2314-7156
Titre abrégé: J Immunol Res
Pays: Egypt
ID NLM: 101627166
Informations de publication
Date de publication:
2020
2020
Historique:
received:
14
10
2019
revised:
10
02
2020
accepted:
26
02
2020
entrez:
24
4
2020
pubmed:
24
4
2020
medline:
3
2
2021
Statut:
epublish
Résumé
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.
Sections du résumé
BACKGROUND
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
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
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
CONCLUSIONS
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.
Identifiants
pubmed: 32322591
doi: 10.1155/2020/1938704
pmc: PMC7153001
doi:
Substances chimiques
Biomarkers
0
CCR7 protein, human
0
CD3 Complex
0
Receptors, CCR7
0
Leukocyte Common Antigens
EC 3.1.3.48
Types de publication
Journal Article
Multicenter Study
Langues
eng
Sous-ensembles de citation
IM
Pagination
1938704Commentaires et corrections
Type : ErratumIn
Informations de copyright
Copyright © 2020 Iole Macchia et al.
Déclaration de conflit d'intérêts
Each contributing author declares to have no competing interests.
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