Evaluation of a Simplified Dual-Platform Flow Cytometric Method for Measurement of Lymphocyte Subsets and T-Cell Maturation Phenotypes in the Population of Nouna, Burkina Faso

Abstract

In the context of a larger clinical study in Nouna, Burkina Faso, we evaluated a simplified dual-platform (DP) flow cytometric (FCM) method that allows the determination of major lymphocyte subsets in a single test tube. We compared the phenotyping of lymphocytes with DP FCM and simultaneous measurements with standard single-platform (SP) FCM for samples from 177 individuals. Analysis of the comparative measurements revealed that DP FCM systematically underestimates the proportion of NK cells, overestimates the percentage of CD3 ؉ CD8 ؉ lymphocytes, and yields proportions of B cells and CD4 ؉ T cells comparable with the results from SP FCM. Bland-Altman analysis showed a low bias between both methods and an acceptable precision for percent values of CD4 ؉ T cells (bias ؎ precision, ؊1% ؎ 6%) and CD8 ؉ T cells (؊3% ؎ 6%). The absolute cell numbers of all lymphocyte subpopulations, however, were systematically biased towards lower values being obtained by DP FCM. Reference values for the distribution of T-cell maturation phenotypes in 177 healthy adults were calculated using DP FCM. The mean ؎ standard deviation (SD) CD4؉ -to-CD8 ؉ T-cell ratio was 1.61 ؎ 0.61, the mean percentage ؎ SD of CD4 ؉ T cells was 42% ؎ 7%, and that of CD8 ؉ T cells 29% ؎ 7%. Among CD4 ؉ lymphocytes, 28% ؎ 7% were classified as central memory (CD45RA low CCR7 ؉ ), 22% ؎ 10% as naïve (CD45RA high CCR7 ؉ ), 45% ؎ 12% as effector memory (CD45RA low CCR7 ؊ ); and 5% ؎ 3% as terminally differentiated effector memory expressing CD45RA (CD45RA high CCR7 ؊ ). Among CD8 bright lymphocytes, 3% ؎ 2% had a central memory phenotype, 27% ؎ 13% were naïve, 37% ؎ 13% had an effector memory phenotype, and 34% ؎ 12% were terminally differentiated effector memory cells expressing CD45RA.In the years 2004 and 2005, a population-based study was performed in Nouna, Burkina Faso, in order to generate site-and gender-specific reference values for lymphocyte subsets in healthy adults in the context of an expanding program for prevention of mother-to-child transmission of human immunodeficiency virus type 1 (HIV-1) (17). During that study, single-platform (SP) flow cytometry (FCM) was used (9), a method which is not available to most laboratories in developing countries due to its relatively high cost (7).Since lymphocyte differentiation and counting by FCM is needed for immunological monitoring of antiretroviral treatment in resource-limited settings and immunological field studies on cohorts of young infants suffering from diseases other than infection with HIV-1 were planned in our research setting, we wanted to use an FCM test which allows the determination of the complete lymphocyte differential. The test should be reliably performed with low volumes of venous and capillary blood and should be resistant against preanalytic errors. It should be as inexpensive as possible and should be run on a simple flow cytometer equipped with only one laser.In the present study, we evaluated such a simplified dualplatform (DP) FCM method for its clinical use in Nouna. The m...

Phenotypic and Functional Profiling of CD4 T Cell Compartment in Distinct Populations of Healthy Adults with Different Antigenic Exposure

“…Boolean gating analysis identified 16 subpopulations expressing each possible combination of markers within the CD4 T cell population (Figure 1, gating strategy is shown in Figure S1). In all groups, approximately 26% of CD4 T cells could be defined as naïve as they expressed all 4 markers, in agreement with previous studies on healthy individuals in the United States [22] and Burkina Faso [23].…”

“…Collectively, our data suggest that environmentally triggered T cell activation in rural Kenya does not seem to drive the general exhaustion of CD4 T cells, despite significantly higher levels of reported chronic and acute conditions in the rural as compared with the urban setting [26]. Earlier studies, using only CD45RA and CCR7 or CD27 as markers, found more memory and fewer naïve T cells in PBMC of African compared to European donors [23], [25], [27]. We did not observe similar skewed distribution of CD4 T cell phenotypes, when 4 markers were used to distinguish CD4 T cell types.…”

Comparison of interlaboratory variation in absolute T‐cell counts by single‐platform and optimized dual‐platform methods

“…Sites 1 and 4 showed higher CD4+ and CD8+ cell counts for single-platform than for dual-platform, a finding reported in other studies (11,12). …”

Activation and maturation of peripheral blood T cells in HIV‐1‐infected and HIV‐1‐uninfected adults in Burkina Faso: a cross‐sectional study

“…CD4 + and CD8 + naïve and memory T cell subsets were assessed with anti-CD4 or anti-CD8-PerCP, anti-CD45RA-FITC and anti-CCR7-PE conjugated monoclonal antibodies as described [10]. Naïve T cells were identified as CD45RA bright CCR7 + CD4 + or CD8 bright lymphocytes [18][19][20].…”

“…Previously, we had observed significant differences in the distribution of T cell maturation phenotypes between healthy adults in Burkina Faso [10] and published data from populations in Europe [11]. In the few studies that have addressed the immunological consequences of HIV-1-infection in Africans, the focus has been directed towards the expression of CD38 on CD8 + T cells [12][13][14].…”