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Three-colour flow cytometric immunophenotyping in HIV-patients; comparison to dual-colour protocols
S T Lillevang1, U Sprogøe-Jakobsen, B Simonsen
1Department of Clinical Immunology, Odense University Hospital, Denmark.
Insights
A new three-color flow cytometry protocol improves CD4+ T-lymphocyte counting for HIV patients. This method accurately quanties lymphocytes, overcoming issues with cell clumping found in older dual-color methods.
Area of Science:
- Immunology
- Biotechnology
- Clinical Diagnostics
Background:
- Accurate enumeration of circulating CD4+ lymphocytes is crucial for monitoring HIV disease progression.
- Advancements in fluorescent dyes enable multi-color flow cytometry protocols, enhancing diagnostic capabilities.
- Conventional dual-color flow cytometry protocols have limitations in precise lymphocyte subset analysis.
Purpose of the Study:
- To compare a novel two-tube, three-color flow cytometry protocol with the conventional dual-color method for CD4+ T-lymphocyte quantification.
- To identify and address potential sources of error in lymphocyte subset analysis, such as cell aggregation.
- To evaluate the applicability of the three-color protocol's gating strategy for other lymphocyte populations, like CD5+ B lymphocytes.
Main Methods:
- Comparison of a three-color protocol (CD45/CD4/CD3 and CD8/CD4/CD3) against a dual-color protocol.
- Utilized flow cytometry with specific antibody combinations and fluorescent dyes (FITC, PE, far-red emitting dyes).
- Employed a lymphocyte gating strategy based on side scatter and CD45 expression, with rigorous vortexing to minimize cell aggregates.
Main Results:
- No significant difference in the percentage of CD3+ lymphocytes was observed between the tested antibody combinations.
- A systematic overestimation of CD3+ CD4+% cells was noted with the CD8/CD4/CD3 combination due to 'CD8-escapees' (aggregated CD8+ cells).
- Rigorous vortexing effectively mitigated the issue of cell clumping, improving gating accuracy. The CD45-based gating strategy proved effective for analyzing CD5+ B lymphocytes.
Conclusions:
- The developed three-color flow cytometry protocol offers significant advantages over conventional dual-color methods for CD4+ T-lymphocyte determination in HIV patients.
- Incorporating anti-CD45 antibody in dual-color combinations can enhance lymphocyte gating and improve accuracy.
- The protocol's gating strategy is adaptable for analyzing other lymphocyte subsets, aiding in the study of HIV-induced immune deviations.
Abstract:
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.
