Investigation of plasma cell-free DNA as a potential biomarker in horses

Abstract

Plasma cell-free DNA (cfDNA) is a biomarker of ischemia, systemic inflammation, and mortality in humans with gastrointestinal disease. Cell-free DNA has not been investigated as a biomarker for equine colic, to our knowledge. We hypothesized that cfDNA could be measured accurately in neat equine plasma using a benchtop fluorometer and that plasma cfDNA would be elevated in emergency patients compared to healthy horses. Plasma was obtained from blood collected in Roche DNA stabilizing tubes. We used the Qubit 4 fluorometer and 1× dsDNA HS assay kit to measure cfDNA concentration in neat patient plasma and following DNA extraction of plasma with a commercial kit. Assay precision and linearity of dilution were satisfactory for neat plasma cfDNA, but DNA spike and recovery results were variable. Further, cfDNA concentrations in paired neat plasma and extracted-plasma samples ( n = 66) were not correlated. Median extracted-plasma cfDNA was higher in emergency patients ( n = 50) and a subgroup of colic patients ( n = 36), compared to healthy horses ( n = 19). Our results with extracted-plasma samples provide proof of concept for further investigation of plasma cfDNA as a biomarker in horses.

Plasma and Synovial Fluid Cell-Free DNA Concentrations Following Induction of Osteoarthritis in Horses

“…The small CV found for the cfDNA assay in horses is consistent with the mean CV (2%) using the same analytical method reported in dogs [ 75 ] and a report that used the assay in horses admitted as emergency patients with colic and a control group [ 70 ]. The precision of the assay meets the USA Food and Drug Administration’s requirements for bioanalytical method validation (Food and Drug Administration, 2018).…”

“…Even though, a similar lack of diagnostic sensitivity was demonstrated in a recent study in foals that showed no significant differences in cfDNA concentrations between critically ill and healthy foals [ 39 ], suggesting that even with significant systemic illness, plasma cfDNA concentration may not be a useful marker in young horses. In contrast, cfDNA is significantly higher in emergency patients > 2 years old with colic than in control horses and ponies [ 70 ]. In the current OA model, plasma cfDNA measurement was not sufficiently sensitive for diagnostic purposes.…”

Visualizing neutrophil extracellular traps in septic equine synovial and peritoneal fluid samples using immunofluorescence microscopy

“… 3 Similarly, plasma cell–free DNA, a marker that can also be increased as a result of NET release, was elevated in equine colic patients. 4 However, the presence of infectious processes was not confirmed in these studies. 3 , 4 Interestingly, plasma cell–free DNA concentrations were not elevated in septic foals.…”

Clinical insights: Clinicopathological parameters for diagnosing and predicting outcome of horses with colic

“…A recent study evaluated the utility of this marker in equine patients presented to a referral centre, in which the majority of cases were admitted for colic. 45 Results showed that the levels of cfDNA were higher in sick compared with healthy horses, including in the colic subgroup. The relatively small number of cases (50 horses), particularly colic cases (36 horses) which was the main goal of the study, somewhat limits the conclusions that can be made.…”

“…Plasma cell‐free DNA (cfDNA; generated from cellular apoptosis and necrosis) is a biomarker used in humans for the diagnosis and prognosis of several conditions, including acute abdominal pain. A recent study evaluated the utility of this marker in equine patients presented to a referral centre, in which the majority of cases were admitted for colic 45 . Results showed that the levels of cfDNA were higher in sick compared with healthy horses, including in the colic subgroup.…”