Neutrophil Extracellular Traps in Plasma from Dogs with Immune-mediated Hemolytic Anemia

BACKGROUND: Neutrophil extracellular traps (NETs) are part of the innate immune response and are essential in local pathogen control, but are associated with pathological inflammation, organ damage, autoimmunity, and thrombosis. Immune-mediated hemolytic anemia (IMHA) is a pro-inflammatory, prothrombotic disease associated with high mortality. HYPOTHESIS/OBJECTIVES: Neutrophil extracellular traps (NETs) are a feature of the inflammatory process in dogs with IMHA. The objective of the study was to evaluate plasma from dogs with IMHA for the presence of 2 indirect markers and 1 direct marker of NETs. ANIMALS: Healthy client-owned dogs (56) and hospitalized dogs with IMHA (n = 35). METHODS: Prospective study. Plasma samples for all dogs were evaluated for cell-free DNA using a fluorescence assay, histone-DNA (hisDNA) complex using an ELISA, and citrullinated histone H3 (specific for NETosis) using Western blot. Reference intervals were generated using plasma from healthy dogs. RESULTS: In dogs with IMHA, cell-free DNA concentration was above the reference interval in 17% of samples with a median (range) of 1.0 μg/mL (0.1-17.3), and hisDNA concentration was above the reference interval in 94% of samples with a median (range) of 30.7 × pooled normal plasma (PNP; 0.6-372.1). Western blot for citrullinated histone H3 identified detectable bands in 84% samples from dogs with IMHA. CONCLUSIONS AND CLINICAL IMPORTANCE: The assay for cell-free DNA detected evidence of NETs in fewer dogs than did the other approaches. Excessive NETs appears to be a feature of IMHA in dogs and contributions to the prothrombotic state deserve further study.