CD4 and CD8 enumeration for HIV monitoring in resource‐constrained settings

Abstract

Background: We developed a volumetric single platform image cytometer (SP ICM) that is dedicated to count CD4 1 and CD8 1 T lymphocytes for HIV monitoring in resource-constrained settings. The instrument was designed to be low-cost, yet reliable, easy-to-use, and robust.Methods: Whole blood is incubated with CD3-magnetic nanoparticles, CD4-phycoerythrin (PE), and CD8-peridinin-chlorophyll-protein complex (PerCP). The CD3 cells are immunomagnetically attracted to an analysis surface, where fluorescence images of CD4 1 and CD8 1 T lymphocytes are recorded and analyzed, respectively. We compared CD4, CD8 counts, and CD4/CD8 ratio obtained by the SP ICM with those from a SP flow cytometer (FCM) tetraCXP method on blood samples from 145 patients.Results: Good correlations were obtained (R: 0.96-0.99) between the SP ICM and the SP FCM. There was 10% CD8 undercount in the SP ICM, which could be partly caused by CD8 1dim T lymphocytes that were not detected by the instrument or not counted by the image analysis due to the cross-talk from the CD4-PE signal in the CD8-PerCP image.Conclusions: The SP ICM is a good candidate for HIV monitoring in point-of-care settings of resourceconstrained countries. q

A microflow cytometer exploited for the immunological differentiation of leukocytes

“…This kit is designed for enumerating percentages of various human leukocyte subsets by double or triple staining: T (CD3 (32). The kit also allows the direct measurement of the CD4/CD8 concentration ratio, which is considered to be a more robust marker for HIV disease progression than the concentration of T h/i lymphocytes (33). The amount of blood (100 lL), antibody reagents (between 5 lL and 20 lL), and the incubation time of 900 s at room temperature were chosen as recommended by the manufacturer.…”

Clinical evaluation of a simple image cytometer for CD4 enumeration on HIV‐infected patients

“…Images were taken at three different positions by manually moving the chamber arriving at a total blood volume analyzed of 3.48 μl. This approach was chosen to obtain a theoretical statistical Poisson variation of ∼5.4% at 100 cells/μl and ∼7.6% at 50 cells/μl (10, 12). At each position, the PE and PerCP filters were manually changed resulting in three CD4 and three CD8 captured and stored images per sample.…”

“…Affordable, easy‐to‐use, and reliable CD4 + T lymphocyte enumeration systems are urgently needed and various groups are attempting to achieve this goal (8–16). Earlier, we reported the development of an image cytometer for enumeration of CD4 + T and CD8 + T lymphocytes (10–12). In this system, immunomagnetic selection of CD3 + T lymphocytes is combined with immunofluorescent labeling of CD4‐phycoerythrin (PE) and CD8‐peridinin‐chlorophyll‐protein (PerCP).…”

Centrifugo-Magnetophoretic Purification of CD4+ Cells from Whole Blood Toward Future HIV/AIDS Point-of-Care Applications

“…Many of these devices have made use of paramagnetic beads to bind cells in a mobile phase and subsequently relocate them to detectors or areas on a chip where they can be manually enumerated. [10][11][12][13] Often, the passage of sample in these devices has been driven by capillary flow. In the work presented here, we build on previous work carried out in this group in which the LOC strategy is integrated into a spinning platform, allowing rapid movement of sample through microfluidic structures and an increase in throughput by placing a number of such structures on a single, DVD-sized disc.…”