Investigation of Extracted Plasma Cell-Free DNA as a Biomarker in Foals with Sepsis

Simple Summary Cell-free DNA (cfDNA) are pieces of DNA released from cells into body fluids. Previous studies in adult horses found that plasma cfDNA concentrations differed significantly between healthy horses and those with emergencies like colic. These studies also showed that accurate measurement of plasma cfDNA in adult horses required extraction, due to matrix effects of equine plasma. It's unclear if similar issues exist in foal plasma. In our study, we aimed to determine if foal plasma has similar interference, and if there are differences in cfDNA levels between healthy, sick non-septic (SNS), and septic foals. Cell-free DNA was measured directly in plasma and after extracting it using a kit in 60 foals. Direct measurement of cfDNA in foal plasma was found to be inaccurate due to matrix effect. However, even after cfDNA extraction, there were no significant differences in plasma cfDNA levels or the ratio of cfDNA to neutrophils between healthy foals, SNS foals, and septic foals. Future research should focus on understanding how neutrophils function during foal sepsis.Abstract Cell-free DNA (cfDNA) is fragmented extracellular DNA that is released from cells into various body fluids. Previously published data from adult horses supports cfDNA as a potential disease biomarker, but also shows that direct measurement in plasma is inaccurate due to matrix effect. It is currently unknown whether a similar matrix effect exists in foal plasma. Given this, the objectives of the current study were to investigate foal plasma for potential matrix effect during fluorescence measurement of cfDNA using a Qubit fluorometer, and to determine whether neat and/or extracted plasma cfDNA concentrations are significantly different in healthy, sick non-septic (SNS) or septic foals. We hypothesized that matrix effect would interfere with accurate fluorescent measurement of cfDNA in foal plasma. Further, we hypothesized that mean extracted cfDNA concentrations, and/or extracted cfDNA:neutrophil ratio, would be elevated in plasma of septic foals compared to healthy or SNS foals. Cell-free DNA was measured in neat plasma, and following DNA extraction with a commercial kit, from 60 foals. Foal plasma exhibited both autofluorescence and non-specific dye binding, confirming matrix effect. However, even with extraction, no significant difference was found in cfDNA concentrations, or cfDNA:neutrophil ratios, between healthy (sepsis score <= 5), SNS (sepsis score 6-11 and negative blood culture), or septic (sepsis score >= 12 +/- positive blood culture) foals. Our data show that matrix effect interferes with accurate Qubit measurement of cfDNA in foal plasma and supports previous findings that plasma cfDNA concentrations are not associated with sepsis diagnosis in foals. Further research is needed to better understand neutrophil function and dysfunction in foal sepsis.