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.

Intracellular production of type I and type II cytokines during HIV-1 progression in Thai patients.

A type I to type II cytokine switch on cells of the immune system has been suggested as a critical step in the etiology of HIV infection. In this study, type I and type II cytokine production of both CD4+ and CD8+ T cells activated by superantigen were investigated in 10 healthy donors and 39 HIV-1 infected patients. Patients were divided into 3 groups based on their CD4 count (< 200, 200-500, > 500 cells/microl). Whole blood from each subject was activated by staphylococcal enterotoxin B (SEB) and anti-CD28. Intracellular cytokine stainings for proinflamatory cytokine (TNF-alpha), type I cytokines (IFN-gamma and IL-2) and type II cytokines (IL-4 and IL-5) in CD4+ and CD8+ T lymphocytes were determined by flow cytometer. Type I cytokine (IFN-gamma) expression in CD4+ T cells co-expressing with CD69 were significantly increased in HIV infected patients, particularly in patients with CD4 counts < 200 and 200-500 cells/microl (means +/- S.D. of 20.7 +/- 18.7% and 10.5 +/- 5.9%, respectively) when compared with 4.8 +/- 1.8% in the normal group (p < 0.05). But IL-2 production in both groups of patients was significantly lower than the normal (3.8 +/- 2.6% and 3.2 +/- 1.4% in patients with < 200, 200-500 cells/microl, and 5.9 +/- 1.5% in the normal group) (p < 0.05). For type II cytokines, there was no difference in all groups of subjects when IL-4 was determined. However, IL-5 production was significantly higher in patients with a CD4 count < 200 cells/microl (0.6 +/- 0.5%) than that in the normal group (0.1 +/- 0.1%) (p < 0.005). CD8+ T cells also showed higher IFN-gamma production in patients with a CD4 count < 200 cells/microl (11.9 +/- 4.7%) and 200-500 cells/microl (12.0 +/- 4.3%) than the normal group (5.3 +/- 2.5%) (p < 0.005). In contrast, IL-2 production in CD8+ T cells was low in these HIV infected patients (0.3 +/- 0.2%, 0.3 +/- 0.2%, and 0.3 +/- 0.4% in patients with < 200, 200-500, and > 500 cells/microl, respectively), which was significantly different compared to the control group (1.2 +/- 0.8%) (p < 0.05). For type II cytokines, only IL-4 production in patients with a CD4 count < 200 cells/microl (0.1 +/- 0.1%) was significantly reduced when compared to the other groups (p < 0.05). This study shows that although HIV infection alters the production of both type I and type II cytokines, it does not induce a polarized type I or type II state in the course of HIV-1 progression in Thai patients.