Flow cytometric CD4 enumeration of four different HIV-infected blood samples at the cost of one monoclonal antibody reagent

Abstract

Background: The frequency and absolute number of CD4 + T-lymphocytes continue to be one of the major clinical markers for management of HIV/AIDS. The present standard dual-platform (DP) three-color and two-color PanLeucogating flow cytometric (FCM) methods for most developing countries are either expensive if manufacturers' monoclonal antibody reagents are used or limited due to an insufficient supply of generic reagents. Clearly, more affordable FCM methods are needed. Objective: To develop a novel DP FCM method using biotin-streptavidin-fluorochrome labeling in combination with the two standard DP methods for 4 different white blood cells (WBC) using only one monoclonal antibody reagent. Methods: The percentage of CD4 + T-lymphocytes in 116 HIV-infected blood samples was determined using our new method. Results were compared with the two standard methods. Correlation and agreement of the pair method were determined using linear regression, Bland- Altman and percent similarity analysis. Results: Our study showed that percentage of CD4 + T-lymphocyte values obtained from the new method correlated highly with the standard three-color and the two-color methods (r 2 = 0.95 {n = 52} and 0.97 {n = 64}). The mean bias and percent similarity for the new method compared with the two standard methods were -0.53% (limit of agreement {LOA}:-5.22% to +4.16% with percent similarity of 99.28; and -0.22% with LOA of -3.42% to +2.98%, the percent similarity of 98.15, respectively. Conclusions: Our FCM method using biotin to label 4 different WBC samples followed by streptavidin staining is reliable for determination of CD4 + T-lymphocytes. Such an approach will significantly reduce the cost for monitoring HIVinfected patients in resource-limited settings.