Evaluation of TruCount Absolute-Count Tubes for Determining CD4 and CD8 Cell Numbers in Human Immunodeficiency Virus-Positive Adults

A single-platform technology that uses an internal bead standard and three-color flow cytometry to determine CD4 and CD8 absolute counts was evaluated for reproducibility and agreement. Values obtained using TruCount absolute-count tubes were compared to those obtained using a two-color predicate methodology. Sixty specimens from human immunodeficiency virus type 1-infected donors were shipped to five laboratories. Each site also analyzed replicates of 14 human immunodeficiency virus type 1-infected local specimens at 6 h and again at 24 h. The interlaboratory variability was significantly less with TruCount (median difference in percent coefficient of variation [%CV] between the two methods was −8% and −3% for CD4 and CD8, respectively) than with the predicate method. Intralaboratory variability was smaller, with a median difference in %CV of −1% for both CD4 and CD8 with 6-h samples and −2% and −3% for CD4 and CD8, respectively, with 24-h samples. Use of TruCount for shipped samples resulted in a median CD4 count change of 7 cells (50th estimated percentile) when all laboratories and CD4 strata were combined. For on-site samples, the median CD4 count change was 10 CD4 cells for 6-h samples and 2 CD4 cells for 24-h samples. Individual site biases occurred in both directions and cancelled each other when the data were combined for all laboratories. Thus, the combined data showed a smaller change in median CD4 count than what may have occurred at an individual site. In summary, the use of TruCount decreased both the inter- and intralaboratory variability in determining absolute CD4 and CD8 counts

Shipment Impairs Lymphocyte Proliferative Responses to Microbial Antigens

Lymphocyte proliferation assays (LPAs) are widely used to assess T-lymphocyte function of patients with human immunodeficiency virus infection and other primary and secondary immunodeficiency disorders. Since these assays require expertise not readily available at all clinical sites, specimens may be shipped to central labs for testing. We conducted a large multicenter study to evaluate the effects of shipping on assay performance and found significant loss of LPA activity. This may lead to erroneous results for individual subjects and introduce bias into multicenter trials

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

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