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.

Keywords: biomarker; cell-free DNA; cfDNA; colic; equine; gastrointestinal.

Figure 1.

Comparison of cell-free DNA (cfDNA) concentrations in neat plasma and extracted-plasma samples. A, B. cfDNA concentrations in paired neat plasma and extracted-plasma samples from all equine emergency patients (A. Spearman ρ: −0.0133, p = 0.9291, n = 47) or from healthy horses (B. Spearman ρ: 0.4552, p = 0.0502, n = 19) were not correlated significantly. C. cfDNA concentrations measured in extracted-plasma samples were significantly higher in the overall population of equine emergency patients (p < 0.0001, n = 50; squares) and in the subgroup of colic patients (p < 0.0001, n = 36; triangles) compared to healthy horses (n = 19; circles). Horizontal line represents median. One-tailed Mann–Whitney test, ****p ≤ 0.0001.