Document 0816
 DOCN  M9550816
 TI    Three-colour flow cytometric immunophenotyping in HIV-patients;
       comparison to dual-colour protocols.
 DT    9505
 AU    Lillevang ST; Sprogoe-Jakobsen U; Simonsen B; Kristensen T; Department
       of Clinical Immunology, Odense University Hospital,; Denmark.
 SO    Scand J Immunol. 1995 Feb;41(2):114-20. Unique Identifier : AIDSLINE
       MED/95167408
 AB    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.
 DE    Antigens, CD/*IMMUNOLOGY  B-Lymphocytes/IMMUNOLOGY  Comparative Study
       Flow Cytometry/*METHODS  Human  HIV Infections/*IMMUNOLOGY
       Immunophenotyping/*METHODS  T-Lymphocytes/IMMUNOLOGY  JOURNAL ARTICLE

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