An Italian national multicenter study for the definition of reference ranges for normal values of peripheral blood lymphocyte subsets in healthy adults.

Reference ranges are necessary in clinical chemistry and hematology to compare an observed value and to provide meaningful information. The aim of this multicenter study was the definition of reference ranges of the relative and absolute numbers of lymphocyte subsets by evaluating a large cohort of healthy adults and by using a standard protocol to reduce the variability in both sample preparation methodology and flow cytometer operation. Other aims of this study were the evaluation of the influence of sex, age, obesity, smoking, sport and some methodological variables on lymphocyte subsets and the comparison of differential white blood cell values obtained by flow cytometry and those obtained by hematology counters.Blood samples from 1311 healthy adults (blood donors and volunteers chosen according to the Italian law for donor selection) were analyzed to study, by flow cytometry, the immunophenotype of lymphocyte subsets and their distribution in terms of percentages and absolute values. Pre-analytical and analytical phases were performed according to the guidelines of the International Federation of Clinical Chemistry (IFCC) and the Italian Group of Cytometry (GIC). T cells were defined by the expression of CD3; T subpopulations by the coexpression of CD4 or CD8 or HLA-DR; B-lymphocytes were identified by the expression of CD19 while natural killer lymphocytes were identified by positivity of CD16 and/or CD56 without CD3. We calculated, for each laboratory and for all data collected, the frequency distribution percent values and absolute values of each lymphocyte subset. The influence of age, sex, smoking, obesity and sport was calculated by the t-test. The influence of some methodological variables was calculated by the t-test and multiple regression test.Fifty-three flow cytometry laboratories at different institutions in Italy participated in this study. Data was obtained from 1311 healthy adults aged from 18 to 70; 968 phenotype analyses (74%) were considered eligible for statistical analysis. Significant results were found as regards sex, smoking and some methodological variables (quantity of sample, washing procedures, brand of monoclonal antibodies and kind of instruments used). The comparison between hematology counters and cytometers showed no difference for any of the parameters considered.The large number of cases, the different kinds of laboratories and their distribution throughout the country make our sample representative of the Italian adult population. The standardization criteria of pre-analytical and analytical phases (the most important issues in evaluating reference values for an indicator) assured good reproducibility among laboratories so that the obtained reference ranges may be useful for interlaboratory comparison of results. Instruments and the brand of monoclonal antibodies may represent an inevitable cause of variability.