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

Abstract

<jats:title>ABSTRACT</jats:title><jats:p>The study evaluated the single-platform, volumetric, CD45-assisted PanLeucogating Auto40 flow cytometer (Apogee Flow Systems Ltd., Hemel Hempstead, United Kingdom) for CD4 T cell numeration, compared to the reference FACSCalibur flow cytometer. Results of absolute counts and percentages of CD4 T cells by Auto40 and FACSCalibur of 234 tripotassium EDTA (K3-EDTA)-blood samples from 146 adults and 88 children (aged from 18 months to 5 years), living in Yaoundé, Cameroon, were highly correlated (<jats:italic>r</jats:italic><jats:sup>2</jats:sup>= 0.97 and<jats:italic>r</jats:italic><jats:sup>2</jats:sup>= 0.98, respectively). The mean absolute bias and relative bias between Apogee Auto40 and FACSCalibur absolute CD4 T cell counts were +9.6 cells/μl, with limits of agreement from −251 to 270 cells/μl, and +4.1%, with limits of agreement from −16.1 to 24.4%, respectively. The mean absolute bias and relative bias between Apogee Auto40 and FACSCalibur CD4 T cell results expressed as percentages were +0.05% CD4 (95% confidence interval [CI], −0.03 to 0.41), with limits of agreement from −6.0 to 5.9% CD4, and +1.0%, with limits of agreement from −32.3 to 34.4%, respectively. The Auto40 counting allowed identification of the majority of adults with CD4 T cell counts below 200 cells/μl (sensitivity, 87%; specificity, 98%) or below 350 cells/μl (sensitivity, 92%; specificity, 98%) and of children with CD4 T cell counts below 750 cells/μl (sensitivity, 82%; specificity, 98%) or below 25% CD4<jats:sup>+</jats:sup>(sensitivity, 96%; specificity, 99%). The Auto40 analyzer is a reliable alternative flow cytometer for CD4 T lymphocyte enumeration to be used in routine immunological monitoring according to the WHO recommendations for HIV-infected adults as well as children living in resource-constrained settings.</jats:p>

Single-platform, volumetric, CD45-assisted pan-leucogating flow cytometry for CD4 T lymphocytes monitoring of HIV infection according to the WHO recommendations for resource-constrained settings

“…Higher precisions for high than for low CD4 T cell count have been previously reported with single-platform volumetric flow cytometric method [40]. Finally, the Auto40 system showed a high capacity of accurately discriminate CD4 T cell values around the relevant CD4 T cells thresholds recommended by the 2010-revised WHO guidelines to initiate and follow ART in HIV-infected adults or children, as previously reported [15,19,46]. …”

“…In the present study, we carried out a new evaluation of the biological performances of the Auto40 flow cytometer in Chad focusing on the HIV-infected adult and children populations, independently of the manufacturer, and in complement to those previously reported in Senegal [15] and Cameroon [19]. Indeed, several evaluations by at least 3 independent laboratories in the field are strongly recommended for effective validation of the newly introduced affordable CD4 T cell measurement technologies to be used in resources-constrained settings [6,21].…”

“…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 [8]. In the Cameroonese experience, well-trained technicians were able to use the Auto40 flow cytometer with low intra- and inter- run precisions ranging from 4.8% to 5.5% and from 5.3% to 7.9%, respectively, thus less than 10% [19,20], considered as acceptable for routine clinical practice [15], and comparable to other published reports using single-platform flow cytometers [10,24,40,44]. The low observed variability of the Auto40 flow cytometer confirmed that single-platform flow cytometric methods are more reproducible than dual-platform [45].…”

“…The Auto40 analyzer was initially intended for CD4 T cell enumeration based on primary CD4 gating, and has been validated for measurement of absolute CD4 T cell count by reference to the FACSCount system [15]. The analyzer has been recently updated for CD4 T cells measurement within 30 minutes, using pan-leucogating protocol with primary CD45 gating followed by secondary CD4 gating, that allows results to be obtained in a single measurement of the CD4 T cell count expressed both in absolute number and in percentage [19,20], and therefore to address the current WHO recommendations for CD4 T cell measurements in children less than 5 years [2]. The initial version of Auto40 analyzer based on primary CD4 gating for CD4 T cell enumeration in absolute number has been evaluated positively in [15]; and the current version is based on CD45-assisted panleucogating for CD4 T cell enumeration in absolute number as well as in percentage in Cameroon [19,20].…”

Laboratory and Field Evaluation of the Partec CyFlow MiniPOC for Absolute and Relative CD4 T-Cell Enumeration

“…Moreover, they require highly trained personnel, an air conditioned lab and a cold chain to ship and store reagents, facilities which are not always available in resource-limited settings. To ensure decentralization of the HIV-monitoring services, more affordable CD4 dedicated flow cytometers have been introduced on the market, including FACSCount (BD Biosciences), CyFlow Counter (Partec), Guava EasyCD4 (Millipore), Apogee Auto40 (Apogee), PointCare NOW (PointCare) [ 5 – 13 ]. At the end of the previous decade, low-cost Point of Care (POC) technologies were being developed, mainly based on digital imaging [ 14 – 18 ].…”