Validation of primary CD4 gating as an affordable strategy for absolute CD4 counting in Cambodia

OBJECTIVE: To validate primary CD4 gating in lysed whole blood for absolute CD4 counts in fresh and aged blood using an affordable compact volumetric commercial flow cytometer. DESIGN: Comparison of CD4 counts between the FACSCount and the 2-parameter CyFlow SL Green. METHODS: One hundred twenty fresh blood samples from patients likely to be infected with HIV were simultaneously run on a FACSCount at the Pasteur Institute of Cambodia and on a CyFlow SL Green at the Sihanouk Hospital Center of Hope (SHCH), Phnom Penh, Cambodia. Intra- and interrun precision was assessed using 2 blood samples. Stability of CD4 counting in blood stored up to 96 hours at room temperature was assessed using 27 blood samples. RESULTS: CD4 counts on the CyFlow SL Green and on the FACSCount correlated well apart from a relative bias (R = 0.993, bias of -9.5%, 95% confidence interval [CI]: -11.8% to -7.1%, limits of agreement: -32.5% to 13.6%). Intra- and interrun variability ranged from 3% to 5% and from 5% to 6%, respectively. CD4 counts on aged blood using the CyFlow SL Green showed an interassay variability of <10%. CONCLUSIONS: Primary CD4 gating in lysed whole blood using the CyFlow SL Green is an affordable and precise method for CD4 counting. Because the fluorescence (FL) and light scatter signals have to be analyzed manually, however, intensive training of the technician and/or operator is imperative.