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.
Publication Date: 2022-02-15 PubMed ID: 35168428PubMed Central: PMC9254060DOI: 10.1177/10406387221078047Google Scholar: Lookup
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- Journal Article
Summary
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The researchers studied the levels of cell-free DNA in horse plasma, to investigate its potential as a biomarker for health issues such as colic, and established a method for its accurate measurement.
Research Objective
- The research aimed to investigate the potential of plasma cell-free DNA (cfDNA) as a biomarker in horses, focusing on its uses related to equine colic conditions.
- The research also aimed to develop accurate means of measuring cfDNA in equine plasma using a benchtop fluorometer.
Methods Used
- To gather the necessary samples for the study, blood was collected using Roche DNA stabilizing tubes.
- The researchers used the Qubit 4 fluorometer and 1× dsDNA HS assay kit to measure the cfDNA concentration in the collected plasma samples.
- A commercial kit was used to extract DNA from the plasma.
Data Analysis
- Assay precision and linearity of dilution were satisfactory enough for the measurement of neat plasma cfDNA, although DNA spike and recovery results were variable.
- The researchers found that cfDNA concentrations in paired neat plasma and extracted-plasma samples were not correlated.
Research Findings
- The researchers found that median extracted-plasma cfDNA was higher in emergency patients and a subgroup of colic patients, compared to healthy horses.
- These results provide a proof of concept for the potential use of cfDNA as a possible biomarker in horses, though further investigation is needed.
Cite This Article
APA
Bayless RL, Cooper BL, Sheats MK.
(2022).
Investigation of plasma cell-free DNA as a potential biomarker in horses.
J Vet Diagn Invest, 34(3), 402-406.
https://doi.org/10.1177/10406387221078047 Publication
Researcher Affiliations
- College of Veterinary Medicine, North Carolina State University, Raleigh, NC, USA.
- College of Veterinary Medicine, North Carolina State University, Raleigh, NC, USA.
- College of Veterinary Medicine, North Carolina State University, Raleigh, NC, USA.
MeSH Terms
- Animals
- Biomarkers
- Cell-Free Nucleic Acids
- Colic / diagnosis
- Colic / veterinary
- DNA
- Horse Diseases / diagnosis
- Horses
- Humans
- Plasma
Grant Funding
- T32 OD011130 / NIH HHS
Conflict of Interest Statement
Declaration of conflicting interests: The authors declared no potential conflicts of interest with respect to the research,authorship, and/or publication of this article.
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Citations
This article has been cited 7 times.- Cooper BL, Hobbs KJ, Bayless R, Stinson-Miller A, Gruber E, Hepworth-Warren K, Lavoie JP, Sheats MK. A Portable Fluorometer Detects Significantly Elevated Cell-Free DNA in Tracheal Wash and Bronchoalveolar Lavage Fluid in Horses with Severe Asthma. Animals (Basel) 2025 Dec 3;15(23).
- Sheahan BJ, Schubert AG, Schubert W, Sheats MK, Schnabel LV, Gilbertie JM. Equine neutrophils selectively release neutrophil extracellular traps in response to chemical and bacterial agonists. Front Vet Sci 2025;12:1512343.
- Bayless RL, Cooper BL, Sheats MK. Extracted Plasma Cell-Free DNA Concentrations Are Elevated in Colic Patients with Systemic Inflammation. Vet Sci 2024 Sep 12;11(9).
- Hobbs KJ, Cooper BL, Dembek K, Sheats MK. Investigation of Extracted Plasma Cell-Free DNA as a Biomarker in Foals with Sepsis. Vet Sci 2024 Aug 1;11(8).
- Birckhead EM, Das S, Tidd N, Raidal SL, Raidal SR. Visualizing neutrophil extracellular traps in septic equine synovial and peritoneal fluid samples using immunofluorescence microscopy. J Vet Diagn Invest 2023 Nov;35(6):751-760.
- Panizzi L, Dittmer KE, Vignes M, Doucet JS, Gedye K, Waterland MR, Rogers CW, Sano H, McIlwraith CW, Riley CB. Plasma and Synovial Fluid Cell-Free DNA Concentrations Following Induction of Osteoarthritis in Horses. Animals (Basel) 2023 Mar 14;13(6).
- Ludwig EK, Hobbs KJ, McKinney-Aguirre CA, Gonzalez LM. Biomarkers of Intestinal Injury in Colic. Animals (Basel) 2023 Jan 7;13(2).
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