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

Abstract

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.

Recovery of viable CD34+ cells from cryopreserved hemopoietic progenitor cell products

“…[19][20][21][22] Various microsphere bead products are currently available for single platform absolute counting, as either beads suspended in liquid medium, or as lyophilized pellets. However, a number of limitations exist when using liquid-phase beads for absolute counting, including the need for thorough mixing before sampling, and the extra pipetting step involved in adding beads to the sample.…”

CD4+ T‐lymphocyte enumeration with a flow‐rate based method in three flow cytometers with different years in service

“…The poor performance of the conventional DP method was shown to be due to the error-prone lymphocyte counts obtained from hematological cell analyzers (7,10,15). It has been demonstrated in multicenter trials that the interlaboratory variation for absolute CD4 1 counts was usually >30% for the DP method, whereas the SP method showed a variation of <17% (16)(17)(18)(19). To avoid inherent variability of the input of total white blood counts and the percentage of lymphocytes from the hematological cell analyzer, the SP method (using a bead-based approach) has thus gained popularity.…”

“…This can be assessed either by counting CD4 1 T-lymphocyte populations by using the known numbers of fluorescent microbeads admixed to a known volume of CD4 1 -stained blood, or by counting CD4 1 Tlymphocytes in a precisely determined blood volume. Unfortunately, the use of bead-based flow cytometry for the SP approach in Thailand is still expensive (US$ [15][16][17][18][19][20] when compared with the present DP approach using a hematological cell analyzer (US$ [10][11][12]. The use of volumetric methods, although simple and inexpensive, requires a new volumetric flow cytometer, thus making this approach impractical in Thailand where large numbers of nonvolumetric flow cytometers already exist.…”

Stem cell source-dependent reconstitution of FOXP3+ T cells after pediatric SCT and the association with allo-reactive disease

“…Total numbers of CD4 þ T lymphocytes were determined by using TruCOUNT technology (Becton Dickinson (BD) Biosciences, Franklin Lakes, NJ, USA) according to the manufacturer's instructions. 26 In addition, after erythrocyte lysis, cells were stained with PerCp-conjugated anti-CD4 monoclonal antibodies (BD Biosciences). After staining of surface markers, cells were fixed and permeabilized using Fixation/Permeabilization buffer (eBiosciences, San Diego, CA, USA) and stained with APC-conjugated anti-FOXP3 Mab (clone PCH 1.01, eBiosciences) to determine the proportion of FOXP3 þ T cells.…”