Affordable CD4+ T-cell counts on 'single-platform' flow cytometers I. Primary CD4 gating

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Abstract
Here, we demonstrate the flow cytometric concept of 'primary CD4 gating' utilizing three different CD4 monoclonal antibodies (mAbs) conjugated with five different fluorochromes. CD4(+) lymphocytes were defined by an autogate in a single histogram of CD4 fluorescence intensity (FI) (y-axis) vs. side light scatter (x-axis). A wide range of absolute counts for > 600 individuals, including HIV(+) patients, were compared with those obtained by 'state-of-the-art' single-platform flow cytometers such as the volumetric Ortho CytoronAbsolute and the Becton Dickinson FACSCalibur using TruCount beads. The correlation between CD4 counts obtained with primary CD4 gating and the full test panel on the Ortho Cytoron was excellent (R(2) = 0.999). Bland-Altman statistics showed a mean difference of -2 cells/mm(3) [confidence interval (CI) 95% = -3 to -1; limits of agreement -27 to +23]. In addition to absolute CD4 counts, CD4% values and CD4/CD8 ratios are also frequently requested. To obtain these, lymphocytes need to be counted using scatter gates, and a second tube stained with a CD8 mAb to count CD8(++) lymphocytes can be incorporated. We conclude that primary CD4 gating on single-platform volumetric flow cytometers is one of the most economical and flexible technologies for routine cost-conscious service work, particularly during the follow-up of patients undergoing anti-HIV therapy and/or vaccination in the developing world.