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

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 (%).