Cell-Free DNA and DNase Activity in Dogs with Immune-Mediated Hemolytic Anemia.
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BACKGROUND: Immune-mediated hemolytic anemia (IMHA) in dogs has a high risk of thrombosis and is associated with marked neutrophilia and necrosis. Cell death and release of neutrophil extracellular traps contribute to increased serum concentrations of cell-free DNA, and in human autoimmune disease reduced DNase activity further increases cell-free DNA. Free DNA in blood has prothrombotic properties and could contribute to hypercoagulability in IMHA. HYPOTHESIS: Cell-free DNA is elevated and DNase activity reduced in dogs with IMHA compared to healthy dogs. ANIMALS: Dogs presenting to two referral hospitals with IMHA (n=28) and healthy controls (n=20). METHODS: Prospective observational study. Blood was collected and death and thrombotic events occurring in the first 14days after hospitalization recorded. DNA was extracted from plasma with a commercial kit and quantified by PicoGreen fluorescence. DNase activity of serum was measured by radial diffusion assay. RESULTS: Cell-free DNA was significantly higher in cases (median: 45ng/mL, range: 10-2334ng/mL) than controls (26ng/mL, range 1-151ng/mL, P=0.0084). DNase activity was not different between cases and controls (P=0.36). Four cases died and there were five suspected or confirmed thrombotic events. Cell-free DNA concentration was associated with death (odds ratio for upper quartile versus lower 3 quartiles: 15; 95% confidence interval 1.62-201; P=0.03) but not thrombosis (P=0.57). CONCLUSIONS AND CLINICAL IMPORTANCE: Cell-free DNA is elevated in dogs with IMHA and likely reflects increased release rather than impaired degradation of DNA. Cell-free DNA concentration is potentially associated with death and might be a prognostic indicator, but this requires confirmation in a larger population.
