Affordable CD4+ T cell counts by flow cytometry. II. The use of fixed whole blood in resource-poor settings.

We tested the feasibility and precision of affordable CD4+ T cell counting in resource-poor settings using a recently standardised fixative, TransFix in whole blood (WB) by flow cytometry (FCM). The precision of the assays was established under optimal conditions for single-platform FCM such as the volumetric CytoronAbsolute and the bead-based FACSCan. Fresh WB samples from HIV-seropositive and seronegative patients were tested in Tanzania and South Africa, fixed and sent to the UK for reanalysis 7 days later. Correlation, bias and limits of agreements were analysed by linear regression and the Bland-Altman test. Absolute CD4+ T cell counts remained stable for at least 10 days when TransFix was added to WB in 1:10 dilution at 20-25 degrees C, and for 7 days when added in 1:10 or 1:5 dilution to samples stored to mimic 'tropical' conditions at 37 degrees C. Higher temperatures such as 42 degrees C were tolerated for only short periods since the recovery had decreased to 63% by day 3. The reproducibility of lymphocyte subset analysis remained unchanged by TransFix with coefficient of variations <6% for all T cell subsets. Absolute CD4+ T cell counts and CD4+ T cell % values on fixed samples in the UK showed a high correlation with the results using fresh samples in Tanzania (r=0.993 and 0.969, respectively) and with the samples handled in Johannesburg (r=0.991 and 0.981) with minimal bias. Primary CD4 gating using only a single CD4 antibody also remained accurate in TransFixed samples (r=0.999). Thus, TransFix permits optimal fixation and transport of WB samples in the developing world for FCM to local regional laboratories and for quality assurance in international centres. When used together with inexpensive primary CD4 gating, TransFix will allow reliable and affordable CD4+ T cell counting by FCM in resource-poor settings.