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Published on: April 23, 2010
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.
Scandinavian Journal of Immunology
|February 1, 1995
Summary
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.
