Analytical validation of a quantitative reverse transcriptase polymerase chain reaction assay for evaluation of T-cell targeted immunosuppressive therapy in the dog

Abstract

Cyclosporine is an immunosuppressive agent that inhibits T-cell function by decreasing production of cytokines such as interleukin-2 (IL-2) and interferon-γ (IFN-γ). In dogs, there is currently no reliable analytical method for determining effective cyclosporine dosages in individual patients. Our laboratory has developed a quantitative reverse transcriptase-polymerase chain reaction (RT-qPCR) assay that measures IL-2 and IFN-γ gene expression, with the goal of quantifying immunosuppression in dogs treated with cyclosporine. This study focuses on analytical validation of our assay, and on the effects of sample storage conditions on cyclosporine-exposed samples. Heparinized whole blood collected from healthy adult dogs was exposed to a typical post-treatment blood concentration for cyclosporine (500ng/mL) for 1h, and then stored for 0, 24, and 48h at both room temperature and 4°C. The study was then repeated using a cyclosporine concentration of 75ng/mL, with sample storage for 0, 24, and 48h at 4°C. Cytokine gene expression was measured using RT-qPCR, and assay efficiency and inter- and intra-assay variability were determined. Storage for up to 24h at room temperature, and up to 48h at 4°C, did not significantly alter results compared to samples that were processed immediately. Validation studies showed our assay to be highly efficient and reproducible and robust enough to be feasible under standard practice submission conditions.