Lab Med Online.  2013 Oct;3(4):253-258.

Methods for Flow Cytometric Analysis of T Cell Subsets in HIV-infected Patients: 2-Color versus 4-Color

• Ahn S
• Kang SJ
• Lim YA
• Lee WG
• Cho SR

Abstract

BACKGROUND
Blood CD4+ T-lymphocyte (T4) count is a major clinical marker for the diagnosis and management of AIDS, and flow cytometry is considered the gold standard for T4 enumeration. Our aim was to compare the 2-color and 4-color flow cytometric methods for T-cell subset analysis in HIV-infected patients.
METHODS
T-cell subsets such as T3, T4, T8, and CD3+CD4-CD8- double negative T cells (DN T) were analyzed from the whole blood of 40 HIV-infected patients by using both 2-color and 4-color methods on a Cytomics FC500 analyzer. Statistical analyses using simple linear regression, paired t-tests, and Bland-Altman plots were performed.
RESULTS
The measured T3 (%), T4 (%), T4 (/microL), T8 (%), T8 (/microL), and DN T (%) differed significantly between the 2 methods (P<0.05), whereas the T4/T8 ratio did not. T3 (%), T4 (%), T4 (/microL), T8 (%), T8 (/microL), and T4/T8 measured by the 2 methods showed good correlation, with correlation coefficients above 0.96, whereas DN T (%) did not. The mean differences in T4 (%) and T8 (%) were 0.39% (limit of agreement (LoA), -1.64~2.43) and 1.26% (LoA, -3.37~5.89), respectively.
CONCLUSIONS
Although there were statistically significant differences in the T cell subsets measured between the 2 methods, the differences were minor, and the 2 methods showed good correlation. As confirmed in this study, DN T (%) estimated by the 2-color method is lower than the actual value. We suggest that although the 2 methods can be used interchangeably, the 4-color method is recommended for the analysis of some specific subpopulations such as DN T (%).

Keyword

Flow cytometry; T cell subset; HIV

MeSH Terms

Biomarkers
Flow Cytometry
HIV
Humans
Linear Models
T-Lymphocyte Subsets
T-Lymphocytes

Figure

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  Fig. 1 T lymphocyte subset analyses of a representative patient sample analyzed by the 2-color (A) and 4-color (B) methods. (A) Gating was done with forward scatter and side scatter. CD45 and CD14 were used to measure purity. Absolute counts were calculated from data input from the CBC count and WBC differential count performed before analysis. (B) Gating was automatically set on CD45 and side scatter.

• 

  Fig. 2 Graphic representation of regression analyses (A, C, E, G, I) and Bland-Altman difference plots (B, D, F, H, J) between the 2-color and 4-color methods: percentage of T3 cells (A, B), percentage of T4 cells (C, D), absolute T4 count (E, F), percentage of T8 cells (G, H), and absolute T8 count (I, J). The horizontal dotted lines indicate ±2SD.

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