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CD45-assisted PanLeucogating for accurate, cost-effective dual-platform CD4+ T-cell enumeration
Deborah Glencross1, Lesley E Scott, Ilesh V Jani
1Department of Molecular Medicine and Hematology, Faculty of Health Sciences, University of the Witwatersrand, Johannesburg, PO Box 2551 Houghton, 2041, South Africa. glencross@mighty.co.za
Cytometry
|July 13, 2002
Summary
Dual-platform flow cytometry for CD4 T-cell counts is more accurate using total white cell counts as the denominator, not lymphocyte counts. This PanLeucogating method improves precision for CD4 T-cell enumeration.
Area of Science:
- Immunology
- Clinical Diagnostics
- Flow Cytometry
Background:
- Current dual-platform (DP) flow cytometry guidelines for CD4 T-cell counts rely on absolute lymphocyte counts, which can be inaccurate.
- Inaccurate lymphocyte identification between hematology analyzers and flow cytometry systems is a major source of error.
- Total white cell counts (WCC) and CD4 percentages among leukocytes offer greater accuracy.
Purpose of the Study:
- To introduce and evaluate a novel dual-platform (DP) flow cytometry method using total leucocytes as the common denominator for improved absolute CD4 T-cell counting.
- To compare the accuracy and precision of this new method, termed PanLeucogating, against traditional lymphocyte gating and single-platform techniques.
Main Methods:
- Implemented "PanLeucogating," utilizing total leucocyte counts (WCC) as the common denominator for dual-platform (DP) absolute CD4 T-cell counting.
- Employed correlation and Bland-Altman analyses to statistically assess accuracy and precision.
- Validated the method using stabilized blood products and fresh/fixed whole blood samples, comparing against single-platform (SP) volumetric and bead-based methods.
Main Results:
- PanLeucogating demonstrated higher accuracy and precision compared to lymphocyte gating in stabilized blood samples.
- Excellent correlation (R² = 0.990) and comparable precision (bias 6 ± SD 17%) were observed between SP volumetric method and DP PanLeucogating.
- DP lymphocyte gating showed significantly lower agreement with the SP volumetric method (R² = 0.758).
Conclusions:
- Dual-platform (DP) leucocyte counts (WCC) should supersede lymphocyte counts as the "common denominator" for CD4 T-cell enumeration.
- The DP method with CD45 PanLeucogating, when combined with hematology analyzer WCC quality control, offers a robust and accurate CD4 T-cell assay.
- This DP approach requires only CD45 and CD4 antibody reagents and is compatible with standard hematology analyzers and flow cytometers.