Absolute CD4 T-Cell Counting in Resource-Poor Settings

Abstract

Flow cytometry is an accurate but expensive method to determine absolute CD4 cell counts. We compared different methods to measure absolute CD4 counts in blood samples from HIV-infected and uninfected subjects using a research/clinical flow cytometer (FACScan); a dedicated clinical instrument (FACSCount); and a volumetric, mobile, open-system flow cytometer equipped with 3 fluorescence and 2 light scatter detectors (Cyflow SL blue). The FACScan and Cyflow were used as single-platform instruments, but they differ in running cost, which is a central factor for resource-poor settings. Direct volumetric and bead-based CD4 measurements on the Cyflow were compared with 2 bead-based single-platform CD4 measurements on the FACSCount and on FACScan (TruCount) in "Le Dantec" Hospital, Dakar, Senegal, using whole blood samples from 102 HIV+ and 28 HIV- subjects. The agreement between the various measurement methods was evaluated by Bland-Altman analysis. Volumetric CD4 measurements on the Cyflow using a no-lyse-no-wash (NLNW) procedure and a lyse-no-wash (LNW) procedure correlated well with each other (R2 = 0.98) and with CD4 measurements on the FACSCount (R2 = 0.97) and FACScan (R2 = 0.97), respectively. Red blood cell lysis had no negative effect on the accuracy of absolute CD4 counting on the Cyflow. An excellent correlation was observed between bead-based CD4 measurements on the Cyflow and CD4 measurements on the FACSCount (R2 = 0.99) and FACScan (R2 = 0.99). Rigid internal and external quality control monitoring and adequate training of technicians were considered essential to generate accurate volumetric CD4 measurements on the Cyflow.

Normal Range of CD4 Cell Counts and Temporal Changes in Two HIV Negative Malawian Populations

“…Several North American studies have demonstrated consistent inter-laboratory variability in measurements despite shared protocols and participation in quality assurance programmes [23-25]. Due to the distance between laboratories we were unable to conduct comparability experiments on shared samples although comparison studies at one of the sites found no systematic variation in results between the two platforms even at low CD4 counts [26] as has been demonstrated in similar settings [27] and UK’s national external quality assurance programme shows no difference in mean CD4 count returns. It is possible that the recent exposure of the rural group to tuberculosis might have had an effect on CD4 count as it is known that active disease may depress the level [28].…”

Validation of a single-platform, volumetric, flow cytometry for CD4 T cell count monitoring in therapeutic mobile unit

“…The Auto40 analyzer uses a volumetric syringe moved by a stepper motor that draws and delivers a known sample volume. Therefore, its absolute volumetric counting allows the direct determination of the number of cells per unit of sample volume without the need for reference material such as micro beads [8,22]. Thus, direct volumetric CD4 T cell measurements were performed on the Auto40 using PE-conjugated anti-CD4 and PE-Dyomics649-conjugated anti-CD45 monoclonal antibodies (Apogee Flow Systems Ltd) for CD4 T cell count measurement.…”

The intersection of flow cytometry with microfluidics and microfabrication

“…Pressure is also used to deliver sample, but the volumetric delivery offered by precision syringe pumps enables the direct counting of cells or particles without the use of external microsphere counting standards that are used to precisely meter volumetric delivery rates. 59 Peristaltic pumps are also effectively used, but care must be taken to dampen fluidic pulses in such systems. 60 Overall, pressure driven flow with external pumps can generate a few μL/min up to several mL/min, as long as the microfluidic system can with stand the high pressure.…”