3-COLOR FLOW CYTOMETRIC IMMUNOPHENOTYPING IN HIV PATIENTS - COMPARISON TO DUAL-COLOR PROTOCOLS

Flow cytometric measurement of circulating CD4(+) lymphocytes is impor tant in the evaluation of disease progression in HIV-infected patients . Development of dyes that can be exited at 488 nm and have emission m aximum in the far red area has made three-colour protocols, together w ith fluorescein isothiocyanate (FITC) and R-phycoerythrin (PE), possib le 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 differ ences were found between percentage of CD3(+) lymphocytic cells determ ined with three different antibody combinations. When the CD8/CD4/CD3 combination was used a systematic overestimation of CD3(+)CD4(+)% cell s was found. This turned out to be caused by the formation of 'CD8-esc apees'. These are clumps of CD8(+) cells that fall outside the lymphoc yte gating region, principally because of high side scatter. The probl em can be overcome by rigorous vortexing to loosen aggregates. The lym phocyte gating principle used in this protocol (gating on a side scatt er/CD45 dot plot) is readily applicable to other antibody combinations . This was demonstrated by measuring CD5(+) B lymphocytes, a subset re ceiving increasing attention in the study of HIV-induced immune deviat ions. We conclude that our three-colour protocol for CD4(+) T-lymphocy te determinations offers significant advantages to the conventional du al-colour method, and we suggest that when possible anti-CD45 be added to dual-colour combinations in order to improve lymphocyte gating.