QASI, an international quality management system for CD4 T-cell enumeration focused to make a global difference.

BACKGROUND: A significant worldwide mobilization effort to treat people with HIV disease began in 2003. Most guidelines for initiating antiretroviral therapy require reliable and reproducible CD4 T-cell counting. Therefore, any effort that improves global availability of quality managed assessment schemes for CD4 T-cell enumeration is a positive achievement for the clinical management of AIDS on a worldwide scale. METHODS: The Canadian QASI-Quality Management System (QMS) has been in operation for over a decade. More recently, QMS has fine-tuned its strategy to optimize its global impact in the fight against the HIV/AIDS pandemic. Three modifications were implemented: (1) introduction of skills and knowledge transfer workshops pertaining to the initiation of national quality management programs for CD4 counting, (2) introduction of a road map to establish domestic EQAP for countries that are ready, and (3) introduction of a statistical analysis package which permits continuous monitoring of global impact of the QASI-QMS. RESULTS: Based on QASI-QMS distribution of specimens over four consecutive participation cycles, there was decreased interlaboratory variation for both low and medium CD4 T-cell levels. After three cycles of consecutive participation, there is an average of 38 and 26% error reduction reported for the mid and low CD4 levels, respectively. CONCLUSION: The program improvements mentioned earlier appear to have had a profound effect with regard to enhancing the performance of laboratories participating in the QASI-QMS. Specifically, there is a significant reduction in interlaboratory variability of CD4 T-cell counts resulting from continuous participation in the QASI-QMS.

Development of a quantitative bead capture assay for soluble IL-7 receptor alpha in human plasma.

BACKGROUND: IL-7 is an essential cytokine in T-cell development and homeostasis. It binds to the IL-7R receptor, a complex of the IL-7Ralpha (CD127) and common gamma (CD132) chains. There is significant interest in evaluating the expression of CD127 on human T-cells as it often decreased in medical conditions leading to lymphopenia. Previous reports showed the usefulness of CD127 as a prognostic marker in viral infections such as HIV, CMV, EBV and HCV. A soluble CD127 (sCD127) is released in plasma and may contribute to disease pathogenesis through its control on IL-7 activities. Measuring sCD127 is important to define its role and may complement existing markers used in lymphopenic disease management. We describe a new quantitative assay for the measurement of sCD127 in plasma and report sCD127 concentrations in healthy adults. METHODOLOGY/PRINCIPAL FINDINGS: We developed a quantitative bead-based sCD127 capture assay. Polyclonal CD127-specific antibodies were chosen for capture and a biotinylated monoclonal anti-CD127 antibody was selected for detection. The assay can detect native sCD127 and recombinant sCD127 which served as the calibrator. The analytical performance of the assay was characterized and the concentration and stability of plasma sCD127 in healthy adults was determined. The assay's range was 3.2-1000 ng/mL. The concentration of plasma sCD127 was 164+/-104 ng/mL with over a log variation between subjects. Individual sCD127 concentrations remained stable when measured serially during a period of up to one year. CONCLUSIONS/SIGNIFICANCE: This is the first report on the quantification of plasma sCD127 in a population of healthy adults. Soluble CD127 plasma concentrations remained stable over time in a given individual and sCD127 immunoreactivity was resistant to repeated freeze-thaw cycles. This quantitative sCD127 assay is a valuable tool for defining the potential role of sCD127 in lymphopenic diseases.

A combined HIV-1 protein bead array for serology assay and T-cell subset immunophenotyping with a hybrid flow cytometer: a step in the direction of a comprehensive multitasking instrument platform for infectious disease diagnosis and monitoring.

BACKGROUND: A new generation of bench-top flow cytometers with digital signal processing to perform suspension array technology (SAT) based bead array assays as well as leukocyte immunophenotyping is now available. These hybrid instruments provide an opportunity for the development of a more cost effective multitasking platform to support infectious disease treatment in resource limited countries. METHODS: We report the development and testing of two modules compatible with the hybrid flow cytometers. The first module is an eleven HIV-1 protein bead array (PBA) for the detection of circulating antibodies and the second is a cell based T-cell enumeration assay. RESULTS: The HIV-1 PBA was tested in parallel with two enzyme immunoassays (EIAs) for the detection of plasma antibodies from 4 HIV-1 seroconversion panels and a low antibody titer panel. The PBA as well as the two EIAs performed equally for the detection of antibody positive samples from all seroconversion panels. One antibody positive sample from the low antibody titer panel was missed by the PBA together with one of the two EIAs tested. A parallel analysis of the HIV-1 PBA with Western blot (a confirmatory test for HIV infection) using plasma from nine HIV-1(+) individuals showed that the HIV-1 PBA detected more of the gp41 and gp120 antibody positive samples. Preliminary CD4 T-cell immunophenotyping results from 14 HIV(+) and 10 HIV(-) whole blood specimens with the hybrid flow cytometer platform compared well to conventional flow cytometry data. CONCLUSION: The successful combination of bead and cell based assays on a single hybrid instrument demonstrated the potential utility of a multitasking platform. The results presented are providing groundwork for future development of more cost effective modular architecture for a flexible flow cytometry based platform.