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Discrimination of Seven Immune Cell Subsets by Two-fluorochrome Flow Cytometry
Published on: March 5, 2019
Multicentre Harmonisation of a Six-Colour Flow Cytometry Panel for Naïve/Memory T Cell Immunomonitoring
Iole Macchia1, Valentina La Sorsa2, Irene Ruspantini3
1Department of Oncology and Molecular Medicine, Istituto Superiore di Sanità (ISS), Rome 00161, Italy.
Insights
Standardising flow cytometry (FCM) assays is vital for personalised oncology. This study developed a harmonisation workflow reducing variability in multicentre clinical trials for immunological biomarker analysis.
Area of Science:
- Immunology
- Oncology
- Clinical Trials
Background:
- Personalised medicine in oncology requires standardised immunological assays.
- Flow cytometry (FCM) is essential for immunomonitoring in clinical trials.
- Harmonisation of FCM methods is crucial for comparable multicentre data.
Purpose of the Study:
- To design a harmonisation workflow for FCM assays.
- To address intra- and interoperator variabilities in multicentre projects.
- To improve data comparability for immunological biomarkers in oncology.
Main Methods:
- Multicentre harmonisation of a multiparametric FCM panel involving thirteen operators.
- Use of a dried antibody mixture targeting naïve/memory T cells.
- Distribution of samples, reagents, and Standard Operating Procedures (SOPs) to participants.
- Centralised data analysis by the coordinating centre (Istituto Superiore di Sanità, ISS).
Main Results:
- Harmonised and reproducible FCM results were achieved.
- Sharing experimental set-up and procedures reduced cross-centre variability.
- Centralised data analysis significantly reduced variability, especially for naïve/memory T cell subsets in whole blood samples.
Conclusions:
- The developed workflow is suitable for harmonising FCM assays in multicentre settings.
- High-quality data can be generated for evaluating immunological markers.
- This approach supports the selection of better therapeutic options in cancer immunotherapy.
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.
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