Oral Abstract Presentations|
Clinical Trials in Resource-Limited Settings
Session Day and Time: Thursday 4 - 6:15 pm
Presentation Time: 6:00
Room: Ballroom C
Background: In resource-poor settings, HIV diagnostics are largely unavailable. Technically simple, affordable, point-of-care CD4 counts are urgently needed. We have developed a microchip-based method for measuring CD4:CD8 ratios and CD4 percentages rapidly (<10 minutes) from whole blood, portably and at low cost.
Methods: Microchips were developed containing miniaturized reaction wells with 30 nanoliter volumes. Microporous filters, flow parameters, stain protocols, and imaging systems were tested for the ability to capture and analyze T lymphocytes, and a prototype microchip was optimized for CD4 cell counting. 20 mL of whole blood was then obtained from healthy donors, stained with 2 mL of fluorophore-conjugated anti-CD3, anti-CD4, and anti-CD8 antibodies, introduced to the prototype microchip, and imaged on an interfaced video CCD chip. Optical image analysis algorithms, based on fluorescence intensity, size and geometry, were evaluated for their ability to identify and count labeled lymphocytes. CD4:CD8 ratios and CD4 percentages were calculated in an automated fashion based on optimized algorithms. Automated counts were compared with manual counts.
Results: In a 10
minute, no-lyse, whole blood assay, T cells were
captured in microchips and analyzed. Algorithms for counting CD3+, CD4+, and
CD8+ cells reliably detected and counted >95% of captured cells. In an
initial evaluation in 4 healthy volunteers, automated CD4:CD8 ratios of 1.19±0.22,
were determined in <15 minutes. Corresponding CD4:CD8 ratios calculated
manually were 1.33±0.31,
respectively. Total reagent costs were less than $3 per assay.
Fig 1. Imaging of whole blood in microchips after staining with Alexa488-anti-CD4 and Alexa647-anti-CD8 antibodies in a rapid, small volume assay. A. CD4+ cells. B. Identical field showing CD8+ cells.
Conclusions: Rapid, affordable microchip-based CD4 counts are technically feasible. Standard microchip fabrication methods could lead to further cost savings and large-scale production. Further development of a portable microchip reader would allow for rapid, point-of-care CD4 counts in resource-poor settings. Validation, comparison with flow cytometry, and evaluation of performance characteristics in resource-poor settings should help determine whether microchip CD4 counting could be rapidly scaled up for wide application in areas currently without access to HIV diagnostics.