Detection of apoptotic T lymphocytes in peripheral blood of human immunodeficiency virus (HIV)-infected subjects by apostain.

Abstract

Apoptosis has been indicated as a mechanism of T cell depletion in HIV-infected subjects and useful in monitoring disease progression. We investigated for the presence of apoptotic T lymphocytes in 130 HIV subjects in various stages of disease by the newly developed cell permeant DNA dye Apostain. Blood was collected in EDTA, lysed in buffered ammonium chloride, fixed in freshly prepared 1% paraformaldehyde and stored in aliquots at -80 degrees C. Samples were thawed and double stained with FITC conjugated-CD3 monoclonal antibody and Apostain. Flow cytometry was then performed and T cells gated on a CD3 versus side scatter dot plot. Normal samples treated in the same manner served to establish the boundary separating non-apoptotic from apoptotic cells. There was no statistically significant association between the proportion of subjects with detectable apoptotic cells and CDC clinical categories A, B and C at the time of admission to the study, although a trend toward a lower apoptotic rate in category A (A= 29%, B=40% and C=41%) was noticed. Conversely, CDC T cell categories 2 and 3 contained significantly higher proportions of Apostain positive patients (1=6%, 2=32% and 3=49%, P=0.072, by chi(2) test). Most importantly, Apostain test identified subjects at risk of disease progression during a 3.5-7 months follow-up in CDC category B and 2 (P=0.008 and P=0.0003, by Fisher's exact test, respectively). A similar, albeit not statistically significant trend was observed also in the other categories. Not requiring extensive manipulation of fresh samples nor cumbersome culture techniques, Apostain test appears suitable for identifying HIV subjects at higher risk of disease progression in clinical settings.