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

Background: Thefrequency 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 were 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 (r2= 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 HIV-infected patients in resource-limited settings.

Discordant CD38 measurement of CD8+ T lymphocytes using fluorescein conjugates in comparison with phycoerythrin conjugates.

Background: We have previously shown that monitoring of CD38 expression can be used as a marker for antiretroviral drug efficacy in HIV infected patients. However, the detection of CD38 expression may be affected by the sensitivity of the fluorochrome conjugated reagent. Objective: In this study, we determined the level of CD38 expression using PE and FITC conjugated anti-CD38 monoclonal antibodies in different groups of HIV infected patients. Methods: The frequency and mean fluorescence intensity of CD38 expression using PE and FITC conjugated anti-CD38 monoclonal antibodies were detected by flow cytometry either alone or in combination with HLA-DR. A correlation between CD38 expression and CD4 count, the percentage of CD4 or viral load in antiretroviral drug naïve HIV infected patients was performed. The results were compared with those for antiretroviral treated HIV infected patients who responsed to therapy and patients with virological failure. Results: We found that while both reagents had the ability to detect a high frequency of CD38 expressing cells in untreated patients, only PE conjugated reagent provided correlation with markers for disease progression. More importantly, FITC conjugated reagent cannot monitor the increase in CD38 expression in patients who showed virological failure. Conclusions: The results from this study suggest that a cautious selection of fluorochrome conjugated reagents and a method for utilizing the data are extremely critical in the use of CD38 expression as a monitoring tool for ART efficacy.