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

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(500 ng/mL) for 1 h, and then stored for 0, 24, and 48 h at both room temperature and 4 ◦C.The study was then repeated using a cyclosporine concentration of 75 ng/mL, with sample storage for 0, 24, and 48 h at 4 ◦C. Cytokine gene expression was measured using RT-qPCR,and assay efficiency and inter- and intra-assay variability were determined. Storage for upto 24 h at room temperature, and up to 48 h 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.

Congenital posterior angulation of tibia and fibula.

Congenital posterior angulation of the tibia occurs less frequently than congenital anterior bowing. Concomitant deformities of the foot and contractures of the soft tissue usually exist. A review of the literature indicates that nonoperative treatment is usually successful in correcting the deformity. Tibial growth is frequently less than normal and epiphysiodesis of the opposite extermity may be necessary. Three patients are presented who have been followed up for 17 years, two years, and over one year. Improvement is occurring with nonoperative treatment.