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.
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.
Böhler
T
T
Department of Virology, University of Heidelberg, Im Neuenheimer Feld 324, D-69120 Heidelberg, Germany. thomas.boehler@med.uni-heidelberg.de
von Au
M
M
Klose
N
N
Müller
K
K
Coulibaly
B
B
Nauwelaers
F
F
Spengler
H P
HP
Kynast-Wolf
G
G
Kräusslich
H-G
HG
D000293:Adolescent; D000328:Adult; D000911:Antibodies, Monoclonal / Q000378:metabolism; D002050:Burkina Faso; D016516:CD4-CD8 Ratio; D015496:CD4-Positive T-Lymphocytes / Q000145:classification* / Q000166:cytology / Q000276:immunology; D018414:CD8-Positive T-Lymphocytes / Q000145:classification* / Q000166:cytology / Q000276:immunology; D005260:Female; D005434:Flow Cytometry / Q000295:instrumentation* / Q000379:methods*; D016650:Fluorescein-5-isothiocyanate; D005456:Fluorescent Dyes; D006801:Humans; D016130:Immunophenotyping; D017493:Leukocyte Common Antigens / Q000032:analysis; D018655:Lymphocyte Count; D008297:Male; D012016:Reference Values; D012424:Rural Population; D013601:T-Lymphocytes / Q000145:classification / Q000166:cytology / Q000276:immunology*
D000911:Antibodies, Monoclonal; D005456:Fluorescent Dyes; D017493:Leukocyte Common Antigens; D016650:Fluorescein-5-isothiocyanate