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Updated: May 11, 2026
Isolation of CD4+ T-cells and Analysis of Circulating T-follicular Helper (cTfh) Cell Subsets from Peripheral Blood Using 6-color Flow Cytometry
Published on: January 7, 2019
An eight-colour flow cytometric method for the detection of reference values of lymphocyte subsets in selected
Bianca Rovati1, Sara Mariucci, Rossella Poma
1SC Oncologia, Laboratorio di Citofluorimetria e Terapie Cellulari, Fondazione IRCCS Policlinico San Matteo, 27100, Pavia, Italy .
Insights
This study developed a standard flow cytometry method for counting lymphocyte subsets, establishing reference values in healthy individuals. Age and sex influence T, B, and natural killer cell distributions, crucial for clinical applications.
Area of Science:
- Immunology
- Clinical Flow Cytometry
Background:
- Accurate enumeration of immunoregulatory cells, particularly lymphocyte subpopulations, is vital for disease management and therapeutic strategies.
- Variability in sample preparation and flow cytometry operations can impact the reliability of lymphocyte subset analysis.
Purpose of the Study:
- To establish a standardized multiparametric flow cytometry method for precise detection and enumeration of lymphocyte subsets.
- To determine reference values for lymphocyte populations in a healthy donor cohort.
- To investigate the impact of age and sex on the distribution of lymphocyte subsets expressing specific surface markers.
Main Methods:
- Analysis of eighty healthy donors using a ten-parameter, eight-colour flow cytometry analytical procedure.
- Development of a comprehensive panel for detecting and enumerating lymphocyte subpopulations.
- Statistical analysis to assess age and sex influences on lymphocyte distribution.
Main Results:
- Statistically significant, though not clinically significant, differences were observed in T lymphocyte subsets and natural killer cells.
- Significant age-related correlations were identified in T lymphocyte and natural killer cell populations.
- Distinct age-related trends in T and B lymphocyte subsets were noted between males and females.
Conclusions:
- The study provides a rapid, accurate, and standardized flow cytometry method for lymphocyte subset quantification suitable for clinical settings.
- Established reference values in a healthy population aid in defining disease status and evaluating treatment efficacy in clinical trials.
Abstract:
Determination of immunoregulatory cells in peripheral blood is important in the management of disease or in the therapeutic approaches that involve alterations in lymphocyte subpopulations. The aims of the present study were (1) to develop a standard multiparametric flow cytometric method for phenotypic detection and enumeration of lymphocyte subsets so as to reduce the variability in both sample preparation methodology and flow cytometric operations; (2) to furnish reference values of lymphocytes by using a selected healthy population; and (3) to examine the influence of age and sex on the distribution of lymphocytes expressing surface markers. Eighty healthy donors were analysed, and ten-parameter, eight-colour analytical procedure was performed. We furnished a panel to detect and to enumerate lymphocyte subpopulations by a multiparametric flow cytometric method to set the reference values to a selected healthy population. These values showed statistically but not clinically significant differences in T lymphocyte subsets and natural killer cells. Furthermore, significant age-related correlations in T lymphocyte and natural killer cells were observed. Lastly, males and females in relation to age showed a significant different trend in T and B lymphocyte subsets. We confirmed that this study provides a rapid and accurate method for the detection and quantification of lymphocyte subsets that could be utilized in the clinical settings. The definition of reference values in the healthy selected population could be helpful also to better define the disease status and to evaluate the treatment efficacy during clinical trials.
