CD4⁺ T lymphocytes enumeration by an easy‐to‐use single platform image cytometer for HIV monitoring in resource‐constrained settings

Abstract

Background: HIV monitoring in resource-constrained settings demands affordable and reliable CD4 + T lymphocytes enumeration methods. We developed a simple single platform image cytometer (SP ICM), which is a dedicated volumetric CD4 + T lymphocytes enumeration system that uses immunomagnetic and immunofluorescent technologies. The instrument was designed to be a low-cost, yet reliable and robust one. In this article we test the instrument and the immunochemical procedures used on blood from HIV negative and HIV positive patients.Methods

A single platform image cytometer for resource‐poor settings to monitor disease progression in HIV infection

“…About 320 ll of the sample solution was transferred into an analysis chamber (Immunicon) with inner dimensions of 30 3 2.7 3 4 mm 3 . Separate titration experiments were performed to determine the optimal concentration of both immunomagnetic and fluorescent labels, the incubation time, and the magnetic separation time (12). For CD4 and CD8 tests, we used 3 ll of CD3 Ferrofluid (Immunicon) for magnetic separation, and 5 ll CD4 PE and 2 ll CD8 PerCP (BD Biosciences, San Jose, CA) for fluorescent detection.…”

“…Here, we give the technical details of the cytometer and show that it gives results comparable to a single platform flow cytometer (SP FCM) method. In a different paper, we specify the results from more extensive testing on HIV-positive patients (12).…”

“…These images are analyzed in the SBC using a dedicated image analysis algorithm, from which the number of T lymphocytes per unit volume is determined. The CCD camera, LED units, the SBC, and the touchscreen are connected to a 12 V rechargeable battery [13] via a custom-built electrical circuit [12]. All components are enclosed in a mechanical frame (not shown), with a separate frame for the batteries.…”

“…In addition, it confirms the good agreement between our instrument and gold standard methods such as FCM in this case. More extensive testing with HIV-negative and -positive patients for CD4 count is reported elsewhere (12).…”

Absolute CD4+ cell count using a plastic microchip and a microscopic cell counter

“…However, CD4 counting by flow cytometry is mostly unavailable in developing countries because of the complexity of the method and an associated high cost, whereas manual methods are extremely labor intensive, have a low throughput and are less accurate than the use of traditional flow cytometry methods (5)(6)(7)(8). Therefore, efforts have been made to develop alternative and affordable CD4 1 T cell counting methods for resource-poor settings (9)(10)(11)(12)(13)(14). These recently introduced CD4…”