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.

Functional immunologic assays using cryopreserved peripheral blood mononuclear cells (PBMC) are influenced by blood processing, storage and shipment. The objective of this study was to compare the viability, recovery and ELISPOT results of PBMC stored and shipped in liquid nitrogen (LN/LN) or stored in LN and shipped on dry ice (LN/DI) or stored at −70°C for 3 to 12 weeks and shipped on DI (70/DI 3 to 12); and to assess the effect of donor HIV infection status on the interaction between storage/shipment and the outcome measures. PBMC from 12 HIV-infected and 12 uninfected donors showed that LN/LN conferred higher viability and recovery than LN/DI or 70/DI 3, 6, 9 or 12. LN/DI PBMC had higher viability than any 70/DI PBMC. The PBMC viability and recovery linearly decreased with the duration of storage at −70°C from 3 to 12 weeks. This effect was more pronounced in samples from HIV-infected than uninfected donors. Results of ELISPOT assays using CMV pp65, CEF and Candida albicans antigens were qualitatively and quantitatively similar across LN/LN, LN/DI and 70/DI 3. However, ELISPOT values significantly decreased with the duration of storage at −70°C both in HIV-infected and uninfected donors. ELISPOT results also decreased with PBMC viability <70%.

Background: The global HIV/AIDS pandemic and guidelines for initiating anti-retroviral therapy (ART)and opportunistic infection prophylaxis demand affordable, reliable, and accurate CD4 testing. A simple innovative approach applicable to existing technology that has been successfully applied in resourcechallenged settings, PanLeukogated CD4 (PLG), could offer solutions for cost saving and improved precision.Methods: Day-old whole blood from 99 HIV+ donors was simultaneously studied in five North-American laboratories to compare the performance of their predicate methods with the dual-platform PLG method. The predicate technology included varying 4-color CD45/CD3/CD4/CD8 protocols on different flow cytometers. Each laboratory also assayed eight replicate specimens of day-old blood from 10 to 14 local donors. Bias and precision of predicate and PLG methods was studied between-and within-participating laboratories.Results: Significantly (P < 0.0001) improved between-laboratory precision/coefficient of variation (CV%) was noted using the PLG method (overall median 9.3% vs. predicate median CV 13.1%). Within-laboratory precision was also significantly (P < 0.0001) better overall using PLG (median

BACKGROUND The purpose of this study was to assess whether laboratories which do not use CD45 for gating lymphocytes with three‐ (or four‐) color flow cytometry (non‐CD45 laboratories) for CD3+4+% and CD3+8+% do worse on quality assessment (QA) studies than laboratories which do use CD45 (CD45 laboratories). METHODS Data came from blood specimens donated by 62 donors (50 HIV‐positive) assayed over 2 years (November, 1996‐October, 1998) by 35 laboratories in the NIAID DAIDS Flow Cytometry QA Program. RESULTS Non‐CD45 laboratories were significantly more likely to be classified as having unacceptable inter‐laboratory results (far from the group median) than CD45 laboratories (5.6% vs 1.5%, P = 0.005 for CD3+4+%; 10.4% vs 5.0%, P = 0.007 for CD3+8+%). The intra‐laboratory range of results on blinded replicates was significantly more likely to be deemed unacceptable (range >4%) in non‐CD45 laboratories than in CD45 laboratories for CD3+8+% (14.5% vs 3.5%, P = 0.002) but not for CD3+4+% (2.6% vs 1.5%, P = 0.62). These differences in favor of CD45 gating were observed even though the non‐CD45 laboratories had been doing three‐color flow cytometry in the QA program significantly longer (P = 0.05) than the CD45 laboratories, and so would be expected to have fewer problems with the assay. CONCLUSIONS Laboratories which choose to use a single CD3/CD4/CD8 tube for immunophenotyping may be sacrificing both accuracy and reproducibility.Cytometry (Comm. Clin. Cytometry) 42:1–4, 2000 © 2000 Wiley‐Liss, Inc.