Immunophenotyping of T lymphocytes by three-color flow cytometry in healthy newborns, children, and adults

Abstract

Reagents are now available which allow simultaneous assessment of three different fluorescence wavelengths on most commercially available flow cytometers. Such three-color analyses provide more information than single- or dual-color analyses. The present study was undertaken in order to establish age-related differences in lymphocyte subpopulations by simultaneously measuring three surface antigens in newborns, children, and adults. A whole blood method was used to label cells with antibodies conjugated to FITC, PE, and perCP. We found that the percentage of lymphocytes expressing HLA-DR/CD28/CD8, HLA-DR/CD38/CD8, CD95/CD45RO/CD8, CD95/CD45RO/CD4, CD95/CD4, and CD95/CD8 showed relative increases with age. The percentage of lymphocytes expressing CD28/CD8, CD38/CD8, and CD38/CD4 showed relative decreases with age, while the subset HLA-DR/CD38/CD4 did not change. Three-color flow cytometry is a powerful tool to more precisely define lymphocyte subsets than the current two-color methods. We present values using a three-color panel in healthy newborns, children, and adults.