Reproducibility of estimation of CD3, CD4 and CD8 reference ranges using different monoclonal antibodies

The present study assesses the influence of the immunophenotyping procedure, the type of monoclonal antibody (MAb) and flow cytometer used, upon the reproducibility of the results of estimation of the normal values for CD3, CD4, and CD8 positive lymphocyte subpopulations. A hundred healthy adults were immunophenotyped by direct immunofluorescence with commercially available (OKT3-FITC, OKT4-PE and OKT8-PE), and locally prepared (HL3-FITC, HL4-PE and HL8-PE)MAb. The samples were analysed on FACStar flow cytometer. The maximal values of the analytical (CD3-0.96, CD4-1.05, CD8-1.02), the individual (CD3-2.33, CD4-2.88, CD8-2.86), and the population (CD3-8.7, CD4-8.8, CD8-7.5) variation prove that the precision of immunophenotyping procedure has no effect on the variation of the values of the CD3+, CD4+ and CD8+ subpopulations. The values of all three markers showed Gaussian type of distribution. Regression analysis proved that the values of these subpopulations determined by MAb from two sources were highly correlated (r > 0.9) but the paired t-test gave statistically significant difference in the values of CD8 (1.23%, P > 0.001). Mean, SD and 95% range of the sizes of the positive subsets determined by OKT3, OKT4 and OKT8 showed that the results were very close to the results from a multicenter study of adult Caucasians. The values we find fall within the cited critical range for these markers; so we can conclude that if good care is taken for the precision of the procedure, MAb source and type of flow cytometer do not influence the values of the assayed subsets.