BackgroundFeline 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.ObjectiveWe 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.MethodsA 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.ResultsMF‐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.ConclusionsThe 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.