Evaluation of Dynabeads and Cytospheres Compared With Flow Cytometry to Enumerate CD4+ T Cells in HIV-Infected Ugandans on Antiretroviral Therapy

Abstract

Background Laboratory-based monitoring of antiretroviral therapy is essential but adds a significant cost to HIV care. The World Health Organization 2006 guidelines support the use of CD4 lymphocyte count (CD4) to define treatment failure in resource-limited settings. Methods We compared CD4 obtained on replicate samples from 497 HIV-positive Ugandans (before and during ART) followed for 18 months by 2 manual bead-based assays, Dynabeads (Dynal Biotech), and Cytospheres (Beckman Coulter) with those generated by flow cytometry at the Infectious Diseases Institute in Kampala, Uganda. Results We tested 1671 samples (123 before ART) with Dynabeads and 1444 samples (91 before ART) with Cytospheres. Mean CD4 was 231 cells/mm3 (SD, 139) and 239 cells/mm3 (SD, 140) by Dynabeads and flow cytometry, respectively. Mean CD4 was 186 cells/mm3 (SD, 101) and 242 cells/mm3 (SD, 136) by Cytospheres and flow cytometry, respectively. The mean difference in CD4 count by flow cytometry versus Dynabeads were 8.8 cells/mm3 (SD, 76.0) and versus Cytospheres were 56.8 cells/mm3 (SD, 85.8). The limits of agreement were -140.9 to 158.4 cells/mm3 for Dynabeads and -112.2 to 225.8 cells/mm3 for Cytospheres. Linear regression analysis showed higher correlation between flow cytometry and Dynabeads (r = 0.85, r2 = 0.73, slope = 0.85, intercept = 28) compared with the correlation between flow cytometry and Cytospheres (r = 0.78, r2 = 0.60, slope = 0.58, intercept = 45). Area under the receiver operating characteristics curve to predict CD4 <200 cells/mm3 was 0.928 for Dynabeads and 0.886 for Cytospheres. Conclusion Although Dynabeads and Cytospheres both underestimated CD4 lymphocyte count compared with flow cytometry, in resource-limited settings with low daily throughput, manual bead-based assays may provide a less expensive alternative to flow cytometry.

Evaluation of the Blood Stabilizers TransFix™ and Cyto-Chex®BCT for Low-Cost CD4 T-Cell Methodologies

“…In this study, our baseline data supported the welldocumented findings that the Dynal low-cost manual CD4 assay provides similar accuracy to flow cytometry (8,11,13). We note that the mean CD4 counts obtained at baseline using the Dynal method were slightly, although not significantly, lower than matched flow cytometry results for the samples tested here (Table 1).…”

“…We note that the mean CD4 counts obtained at baseline using the Dynal method were slightly, although not significantly, lower than matched flow cytometry results for the samples tested here (Table 1). Previous evaluations by both ourselves (19) and others (3,13) found small variations in mean CD4 T-cell counts obtained using the Dynal assay and flow cytometry (which is expected when comparing different methodologies), although these previous studies did not find a trend toward over-or underestimating CD4 counts.…”

“…The Dynal manual assay shows very good correlation with flow cytometry, with r ¼ 0.85-0.93 (5,8,13). While the approximate cost of the reagents used per test (3-5 U.S. dollars [USD]) is similar to that offered by automated systems (e.g., FACSCount 5-20 USD), the initial equipment expenditure is significantly less (2000 USD for Dynal versus 27,000 USD for FACSCount) (13).…”

CD4 immunophenotyping in HIV infection

“…Timely monitoring of the CD4 count in HIV-infected patients in resource-constrained settings demands the development of simple, low-cost, accessible, reliable and accurate CD4 enumeration techniques. These techniques can be categorized in three ways: gating and counting strategies that can be applied to existing bench-top flow cytometers within centralized laboratories that can drastically reduce costs; small and less technically demanding flow cytometers that can be installed in clinics64–66; and non-flow cytometric methods that can provide rapid results without the need for expensive equipment or even electricity67–69. Although unproven as yet, some of these advances might permit point-of-care CD4 testing where the results can be obtained in a rural clinic setting.…”

Electrochemical magneto-actuated biosensor for CD4 count in AIDS diagnosis and monitoring

“…PIMA TM is based on a dualfluorescence image analysis to count CD3 + /CD4 + cells (Diaw et al, 2011). The Coulter ® CD4Count Kit and the Dynal T4 Quant Kit are manual CD4 tests based on the use of beads for lymphocyte capturing, labeling and counting under light microscope (Lutwama et al, 2008).…”