Multiplex fluorescent immunocytochemistry for the diagnosis of feline infectious peritonitis: Determining optimal storage conditions.

Abstract

BACKGROUND: Feline Infectious Peritonitis (FIP) is a fatal disease of cats that can be very difficult to definitively diagnose antemortem. Multiplex fluorescent immunocytochemical (MF-ICC) assays are emerging as useful diagnostic tests in veterinary medicine, particularly for fluid samples. OBJECTIVE: We aimed to develop and optimize an MF-ICC assay to detect feline coronavirus within macrophages, with the primary goal of determining the allowable/recommended sample storage conditions for clinical use of this assay. METHODS: A feline macrophage cell line was infected with the FIP virus. Following harvest into EDTA tubes (simulating typical clinical collection of effusion), cells were stored at 4℃, 22℃, and 37℃. For each temperature condition, slides for MF-ICC were made at 0, 1, 2, 3, and 5 days post-collection. To assess the stability of immunoreactivity following fixation, freshly harvested infected cells were fixed onto slides and maintained at 4℃ for 1, 2, 4, and 12 weeks. All slides were analyzed by MF-ICC for the presence of mononuclear cells with co-expression of vimentin and coronaviral antigen. RESULTS: MF-ICC confirmed that cells tested positive for coronavirus at 4℃ through 3 days post-harvest, 22℃ through 48 hours post-harvest, and 37℃ through 24 hours post-harvest. The MF-ICC assay was successfully performed on fixed slides through the 12-week time point. This assay also demonstrated positive results on a clinical sample of abdominal fluid from a cat later confirmed to have FIP. CONCLUSIONS: The MF-ICC assay described here offers a potentially specific and relatively stable antemortem diagnostic test for feline infectious peritonitis. Evaluation of this assay in clinical samples is ongoing.