Three-colour flow cytometric immunophenotyping in HIV-patients; comparison to dual-colour protocols

Flow cytometric measurement of circulating CD4+ lymphocytes is important in the evaluation of disease progression in HIV-infected patients. Development of dyes that can be exited at 488 nm and have emission maximum in the far red area has made three-colour protocols, together with fluorescein isothiocyanate (FITC) and R-phycoerythrin (PE), possible in most clinical flow cytometers. We report here the comparison of a two-tube, three-colour protocol (including CD45/CD4/ CD3 and CD8/CD4/CD3) with our conventional dual-colour protocol. No significant differences were found between percentage of CD3+ lymphocytic cells determined with three different antibody combinations. When the CD8/CD4/CD3 combination was used a systematic overestimation of CD3+ CD4+% cells was found. This turned out to be caused by the formation of 'CD8-escapees'. These are clumps of CD8+ cells that fall outside the lymphocyte gating region, principally because of high side scatter. The problem can be overcome by rigorous vortexing to loosen aggregates. The lymphocyte gating principle used in this protocol (gating on a side scatter/CD45 dot plot) is readily applicable to other antibody combinations. This was demonstrated by measuring CD5+ B lymphocytes, a subset receiving increasing attention in the study of HIV-induced immune deviations. We conclude that our three-colour protocol for CD4+ T-lymphocyte determinations offers significant advantages to the conventional dual-colour method, and we suggest that when possible anti-CD45 be added to dual-colour combinations in order to improve lymphocyte gating.