Evaluation of four alternative methodologies for determination of absolute CD4+ lymphocyte counts. The National Heart, Lung, and Blood Institute Retrovirus Epidemiology Donor Study

Abstract

Standard methods for determining absolute CD3+CD4+ T-lymphocyte concentrations using combined results from flow cytometry and hematology analyzers are expensive and require fresh (< 18 h old) blood. We evaluated four new "alternative" methods for determining absolute CD4+ T-lymphocyte counts; (a) the FACSCount System (Becton Dickinson Immunocytometry Systems), (b) VCS Technology/Coulter Cyto-Spheres (Coulter Corporation), (c) Zymmune CD4/CD8 Cell Monitoring Kit (Zynaxis, Inc.), and (d) TRAx CD4 Test Kit (T Cell Diagnostics). EDTA-anticoagulated whole-blood specimens from HIV-seropositive patients and anti-HIV-negative adult blood donors and staff were tested by the standard flow cytometry method on fresh blood (0-6 postphlebotomy) and by the four alternative methods at 0 to 6 h and 24 to 30 h postphlebotomy. Representative specimens (< 6 h old) were tested five times with each system to evaluate reproducibility. Correlation coefficients for absolute CD4+ counts between standard flow cytometry and the alternative methods ranged from 0.84 to 0.92 for both fresh and 24- to 30-h-old specimens. Average coefficient of variation for reproducibility ranged from 4.5 to 7.1%. The four alternative CD4+ T-lymphocyte counting methods performed well relative to standard methods. Each alternative method offers advantages over standard flow cytometry with respect to sample throughout, required technical expertise, and cost per result. These methods should facilitate wider availability of low-cost CD4 counts.