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.
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