Multisite Comparison of CD4 and CD8 T-Lymphocyte Counting by Single- versus Multiple-Platform Methodologies: Evaluation of Beckman Coulter Flow-Count Fluorospheres and the tetraONE System

The progressive loss of CD4+ T lymphocytes (CD4 T cells) through virally mediated cell destruction is the predominant pathophysiological manifestation of human immunodeficiency virus type 1 (HIV-1) infection (15). Enumeration of this cell subset provides an estimate of HIV disease progression (17). A major component of the immune response to HIV-1 infection is mediated by CD8+ T lymphocytes (CD8 T cells) (6). CD8 T cells capable of suppressing HIV-1 replication adopt an activation phenotype and appear in increased numbers in the blood and other body compartments following infection (7, 19). Therefore, the CD4 and CD8 T cells in blood are frequently quantified to assess immune competence and disease stage in HIV-1-infected patients. Furthermore, changes in CD4 T-cell numbers are an important estimate of the response to antiretroviral therapy. The CD4 T-cell count remains the most important immunologic surrogate marker of the efficacy of new antiretroviral regimens used in clinical trial evaluations (9). The accurate quantitation of these cells in blood is crucial for providing clinical care to HIV-1-infected patients and for the systematic evaluation of new therapeutic modalities. However, previous studies have identified substantial variability in results between laboratories performing these assays (1, 5). The currently recommended method for CD4 T-cell determination (2, 3) utilizes three independently derived values from two different instruments: a white blood cell (WBC) count and percent lymphocytes derived from a hematology instrument, and percentage of CD4 or CD8 T cells derived from a flow cytometer. A major disadvantage of this multiple-platform assay method is that error in each independent measurement is multiplied at each subsequent step in the calculation. Recently, new analytic methods have emerged that permit absolute CD4 and CD8 T-cell determinations to be performed either using a single-determination, non-flow cytometry-based assay (8, 10, 11) or entirely on the flow cytometer (14). In the single-platform flow cytometry-based techniques, fluorospheres are added to the blood at a known concentration. Absolute cell counts in each specimen can be calculated ratiometrically by simultaneously counting both fluorospheres and the cells of interest (18). Other technological improvements, including automated instrument setup, lymphocyte gating, and cursor placement, could also improve assay performance. In the present study, we have assessed the within-laboratory and between-laboratory precision of two alternative assay methods: a two-color method that uses Beckman Coulter Flow-Count fluorospheres to determine absolute counts, and a four-color method that uses the fully automated tetraONE System. The precision obtained with these alternative methods was compared with the precision obtained with a conventional multiple-platform assay method. We also assessed the accuracy of these two new assay methods by comparing the absolute counts obtained with the single-platform methods and with the conventional multiple-platform method.