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

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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.

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