Cell‐Free DNA and DNase Activity in Dogs with Immune‐Mediated Hemolytic Anemia

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 14 days 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: 45 ng/mL, range: 10–2334 ng/mL) than controls (26 ng/mL, range 1–151 ng/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.