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 NIAID DAIDS New Technologies Evaluation Group

Abstract

New analytic methods that permit absolute CD4 and CD8 T-cell determinations to be performed entirely on the flow cytometer have the potential for improving assay precision and accuracy. In a multisite trial, we compared two different single-platform assay methods with a predicate two-color assay in which the absolute lymphocyte count was derived by conventional hematology. A two-color method employing lymphocyte light scatter gating and Beckman Coulter Flow-Count fluorospheres for absolute counting produced within-laboratory precision equivalent to that of the two-color predicate method, as measured by coefficient of variation of replicate measurements. The fully automated Beckman Coulter tetraONE System four-color assay employing CD45 lymphocyte gating, automated analysis, and absolute counting by fluorospheres resulted in a small but significant improvement in the within-laboratory precision of CD4 and CD8 cell counts and percentages suggesting that the CD45 lymphocyte gating and automated analysis might have contributed to the improved performance. Both the two-color method employing Flow-Count fluorospheres and the four-color tetraONE System provided significant and substantial improvements in between-laboratory precision of absolute counts. In some laboratories, absolute counts obtained by the single-platform methods showed small but consistent differences relative to the predicate method. Comparison of each laboratory's absolute counts with the five-laboratory median value suggested that these differences resulted from a bias in the absolute lymphocyte count obtained from the hematology instrument in some laboratories. These results demonstrate the potential for single-platform assay methods to improve within-laboratory and between-laboratory precision of CD4 and CD8 T-cell determinations compared with conventional assay methods.

FIG. 1

Comparison of within-laboratory precision in absolute CD4 and CD8 T-cell counts between predicate method and single-platform methods by laboratory. Median %CVs are illustrated for each method from eight replicates of ≥14 specimens. (A) Flow-Count method versus predicate method; (B) tetraONE method versus predicate method. ∗, significantly different from predicate method, P < 0.025.

FIG. 2

Absolute differences in CD4 and CD8 T-cell counts between single-platform and predicate methods. (A and B) Flow-Count method minus predicate for CD4 (A) and CD8 (B); (C and D) tetraONE method minus predicate for CD4 (C) and CD8 (D). Box indicates median and interquartile range (25th and 75th percentiles); upper and lower lines indicate 90th and 10th percentiles, respectively.

FIG. 3

Source of absolute CD4 T-cell count bias in individual laboratories. The absolute CD4 T-cell count generated in each laboratory by the predicate or tetraONE method was subtracted from the five-laboratory median value by that method to determine whether method biases occurred in individual laboratories. A through E are laboratory designations. The shaded region indicates the interquartile range (25th and 75th percentiles) of these differences.