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Cell-free mitochondrial DNA (cf-mtDNA) has recently been in the spotlight as an endogenously produced danger molecule that can potentially elicit inflammation. However, its clinical and prognostic implications are uncertain in patients undergoing hemodialysis. We examined the association of baseline cf-mtDNA categorized as tertiles with health-related quality of life (HRQOL), inflammatory cytokines, and mortality in a multicenter prospective cohort of 334 patients on hemodialysis. To better understand cf-mtDNA-mediated inflammation, we