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

Abstract

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.

Interest of circulating tumor DNA as a biomarker for canine cancers: illustration in histiocytic sarcoma, oral malignant melanoma and multicentric lymphoma

“…cfDNA is an attractive biomarker in human medicine and particularly in oncology. Thus, it has also been evaluated in different canine diseases 16,17,19,20,[50][51][52][53] . As in human, cfDNA concentration in dogs is correlated with the severity of the disease and prognosis 16,17,20,53 .…”

“…Thus, it has also been evaluated in different canine diseases 16,17,19,20,[50][51][52][53] . As in human, cfDNA concentration in dogs is correlated with the severity of the disease and prognosis 16,17,20,53 . Consistently with previous studies, we found that dogs with multicentric lymphoma had significantly higher concentration of total cfDNA than healthy dogs, dogs with non-cancerous diseases and dogs with other cancers 20,51 .…”

“…In veterinary medicine, cfDNA has recently gained attention and more particularly in dogs. As in human medicine, it has been found that cfDNA has a short half-life in canine plasma (around 5 hours) 15 and its concentration is increased in various diseases such as immune-mediated hemolytic anemia 16 , sepsis, severe trauma or inflammation [17][18][19] . In dogs, cfDNA concentration is also correlated with the severity of the disease and prognosis 18 .…”

Kinetics of Plasma Cell‐Free DNA and Creatine Kinase in a Canine Model of Tissue Injury

“…A significant increase in the blood concentration of cfDNA was found in diseased dogs, as compared to healthy dogs, and a positive association between cfDNA concentration and disease severity and survival was observed . Both dogs with sepsis and those with moderate‐to‐severe trauma had significantly increased cfDNA concentrations compared to healthy dogs, and cfDNA concentration was associated with death in dogs with immune‐mediated hemolytic anemia …”

“…21 22 and cfDNA concentration was associated with death in dogs with immune-mediated hemolytic anemia. 23 A short half-life and a good correlation with disease severity would render cfDNA a useful tool to quantify the extent of tissue injury. Based on current knowledge, we hypothesized that, in dogs, cfDNA would have a short biological half-life and its concentration would be able to stratify mild, moderate, and severe tissue injury.…”