Comparison of 5 Flow Cytometric Immunophenotyping Systems for Absolute CD4+ T-Lymphocyte Counts in HIV-1-Infected Patients Living in Resource-Limited Settings

Abstract

Enumeration of CD4+ T lymphocytes is important in management of HIV-infected patients. However, CD4 testing by current gold standard bead-based flow cytometer (FCM) system is expensive for developing countries. This study compared 2 affordable volumetric FCMs with the 3 predicate FCM systems. CD4+ T-lymphocyte counts on blood samples from 150 HIV-1-infected Thai patients were determined in parallel by 5 FCM systems: the 2 single-platform volumetric FCM systems, Guava and CyFlow(green); the 2 standard single-platform bead-based systems (2-color FACSCount and the TriTEST/TruCOUNT tube using a FACSCalibur FCM); and the dual-platform TriTEST system. Correlation and agreement were analyzed using linear regression and Bland-Altman analysis. Results from these 2 volumetric systems gave similar results and excellent correlation: R2 > 0.93; mean biases ranged from +6.3 to +24.1 cells per microliter more for the Guava. In contrast, the CyFlow(green) showed the lowest values with R2 > 0.97; mean biases ranged from -9.8 to -27.6 cells per microliter. This indicates that the absolute CD4+ T-lymphocyte counts determined by CyFlow(green) are < FACSCount < DP TriTEST < TriTEST/TruCOUNT < Guava. Although the use of these 2 volumetric FCMs could make CD4+ T-lymphocyte enumeration more affordable in resource-poor settings, variations among these systems should be considered if these are to be interchanged.

Validation of a Single-Platform, Volumetric, CD45-Assisted PanLeucogating Auto40 Flow Cytometer To Determine the Absolute Number and Percentages of CD4 T Cells in Resource-Constrained Settings Using Cameroonian Patients' Samples

“…Although classical flow cytometry represents the reference method for CD4 T cell enumeration, the feasibility of flow cytometric methods in the field remains controversial in resource-poor settings (13). In the present study, a welltrained technician was able to use the Auto40 flow cytometer with low intra-and interrun variability, less than 10%, which is comparable to results of other published reports using single-platform flow cytometers (7,15,16,25) and is acceptable in routine clinical practice (25). The low observed variability of Auto40 confirmed that single-platform flow cytometric methods are more reproduc- ible than dual-platform methods (27).…”

“…Both latter analyzers have proven their usefulness for validating absolute number-and percentage-based CD4 T cell counting in the field (16,25). These single platforms using only a flow cytometer and volumetric systems allow counting of CD4 T cells in a fixed volume, thus leading to a considerable reduction in the costs of CD4-positive T cell enumeration in comparison to microbead-based systems (2).…”

“…To ensure quality control of the flow cytometric immunophenotyping method with regard to the performance of both the personnel and the instrument, the same lots of reagents were used throughout the study, and all sample preparations and flow cytometric analyses were performed by the same operator for each instrument, as previously reported (25). Adequate training on the use of the reverse pipetting technique and electronic pipette was also provided for each operator.…”

Evaluation of Three Medium Throughput Flow Cytometers for Monitoring People Living with HIV in Resources Limited Laboratories: How to Choose?

“…In addition, absolute bias increases between Guava® and FACSCalibur® from low to higher strata. Our results confirmed previous studies reporting that Guava® is less accurate at CD4 counts above 500 cells/μl [16,20,21,31]. This difference could be caused by the way of separating monocyte and lymphocyte count which share CD4 on cell surface.…”

“…Many flow cytometers have been tested [16][17][18][19][20][21][22][23][24]. However, some of this first generation has a few limitations: (i) it was not possible to get CD4 percentage which is very important in children monitoring or (ii) the use of FCM software requires good computer skills by the operator.…”