An Inexpensive, Simple, and Manual Method of CD4 T-Cell Quantitation in HIV-Infected Individuals for Use in Developing Countries

Abstract

CD4+ T lymphocytes are currently the most common surrogate marker indicating immune status and disease progression with HIV infection. The cost of monitoring disease progression and response to therapy is still prohibitively expensive. Flow cytometry is the gold standard for the estimation of CD4+, but the high initial investment for this technology and expensive reagents makes it unaffordable for developing countries like India. We evaluated the Coulter cytosphere assay for quantifying CD4+ T lymphocytes in comparison with the standard method, flow cytometry, in 122 HIV-infected individuals. The correlation coefficient of the cytosphere assay compared with that of flow cytometry for CD4+ T lymphocytes was 0.97 (P< 0.0001), with a confidence interval of 0.95 to 0.98. The sensitivity, specificity, positive predictive value, and negative predictive value of the cytosphere assay in enumerating absolute CD4+ T-lymphocyte counts of less than 200/microL were 94.9%, 96.4%, 92.5%, and 97.6%, respectively. This is a simple inexpensive method and has a strong correlation with flow cytometry. Hence, the cytosphere assay can be an alternate to flow cytometry for the estimation of CD4+ T-lymphocyte counts, especially in resource-poor settings of developing countries, for monitoring HIV progression and response to therapy.

A Simple Manual Rosetting Method for Absolute CD4 + Lymphocyte Counting in Resource-Limited Countries

“…In recent years, several such technologies have been developed (4-7, 9-12, 14). Of these alternative methods available, the cytosphere assay (Beckman Coulter) is widely regarded as a method of choice (1,3,5). In the cytosphere method, inert particles coated with a CD4 MAb are used to identify and manually enumerate the absolute CD4 ϩ lymphocytes by visible light microscopy.…”

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

“…As monocytes and macrophages also express the CD4 1 molecule, they must be identified and excluded from the absolute count. Some studies have used a blocking reagent with CD14 monoclonal antibody-labeled beads added to the CD4 samples to achieve the blocking (6).…”

“…The precise determination of CD4 1 T cell counts by flow cytometry using a technique for fluorescence-based measurement of immuno-labeled cells has been designated as the 'gold standard' for CD4 counting by the WHO (3). 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).…”

Clinical applications and availability of CD4+ T cell count testing in sub-Saharan Africa

“…Importantly, the CD4þ T cell counts by both methods were shown to be highly correlated in a United States (n 5 77) and a Ugandan (n 5 141) cohort (50). Other relatively low-cost methods with excellent correlation with flow cytomeric enumeration of CD4þ T cells have been validated (51)(52)(53)(54)(55).…”