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Veterinary sciences2024; 11(8); 346; doi: 10.3390/vetsci11080346

Investigation of Extracted Plasma Cell-Free DNA as a Biomarker in Foals with 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.
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Publication Date: 2024-08-01 PubMed ID: 39195800PubMed Central: PMC11359113DOI: 10.3390/vetsci11080346Google Scholar: Lookup
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  • Journal Article

Summary

This research summary has been generated with artificial intelligence and may contain errors and omissions. Refer to the original study to confirm details provided. Submit correction.

This research explores the potential for cell-free DNA in foal plasma as a biomarker for sepsis, finding that while a ‘matrix effect’ exists in the plasma, no significant difference was observed in the DNA concentrations between healthy, sick non-septic and septic foals.

Understanding Cell-free DNA as a potential biomarker

  • The study was derived from the understanding that Cell-free DNA (cfDNA) – fragmented extracellular DNA released into various body fluids – has potential as a disease biomarker.
  • Data from adult horses has shown the potential of cfDNA, however, it was understood that direct measurement in plasma could be inaccurate due to matrix effect – an interference affecting the accuracy of measurement.
  • However, it was not known whether a similar matrix effect occurs in foal plasma – thus forming the basis for this research.

Objectives of the study

  • The research aimed to explore any potential ‘Matrix effect’ during the measurement of cfDNA in foal plasma using a Qubit fluorometer.
  • It also sought to identify whether neat and/or extracted plasma cfDNA concentrations are significantly different in healthy, sick non-septic (SNS) or septic foals.

Hypotheses and Methods

  • The researchers hypothesized that the matrix effect would indeed interfere with the accurate fluorescent measurement of cfDNA in foal plasma.
  • Also, they thought that mean extracted cfDNA concentrations, and/or the ratio of extracted cfDNA to neutrophils, would be higher in plasma of septic foals compared to other categories.
  • Cell-free DNA was measured in both neat – meaning unmodified – plasma, and following DNA extraction using a commercial kit, from 60 foals to test these hypotheses.

Key Findings

  • The study confirmed the presence of a matrix effect in foal plasma which showed both autofluorescence and non-specific dye binding.
  • However, contrary to initial theories, following extraction they found no significant difference in cfDNA concentrations, or cfDNA:neutrophil ratios, between healthy, sick non-septic and septic foals.
  • These findings corroborate existing data stating that plasma cfDNA concentrations are not strongly associated with sepsis diagnosis in foals.

Implications and future research

  • While this research ruled out plasma cfDNA as a reliable sepsis biomarker in foals, it pointed out the need for additional studies to better comprehend neutrophil function and dysfunction in the context of foal sepsis.

Cite This Article

APA
Hobbs KJ, Cooper BL, Dembek K, Sheats MK. (2024). Investigation of Extracted Plasma Cell-Free DNA as a Biomarker in Foals with Sepsis. Vet Sci, 11(8), 346. https://doi.org/10.3390/vetsci11080346

Publication

Veterinary sciences
ISSN: 2306-7381
NlmUniqueID: 101680127
Country: Switzerland
Language: English
Volume: 11
Issue: 8
PII: 346

Researcher Affiliations

Hobbs, Kallie J
  • Department of Clinical Sciences, College of Veterinary Medicine, North Carolina State University, Raleigh, NC 27695, USA.
Cooper, Bethanie L
  • Department of Clinical Sciences, College of Veterinary Medicine, North Carolina State University, Raleigh, NC 27695, USA.
Dembek, Katarzyna
  • Department of Clinical Sciences, College of Veterinary Medicine, North Carolina State University, Raleigh, NC 27695, USA.
Sheats, M Katie
  • Department of Clinical Sciences, College of Veterinary Medicine, North Carolina State University, Raleigh, NC 27695, USA.

Grant Funding

  • T-32 Graduate Fellowship Grant / NIH HHS

Conflict of Interest Statement

The authors declare no conflicts of interest.

References

This article includes 38 references
  1. Hytychová T, Bezděková B. Retrospective evaluation of blood culture isolates and sepsis survival rate in foals in the Czech Republic: 50 cases (2011–2013). J. Vet. Emerg. Crit. Care 2015;25:660–666.
    doi: 10.1111/vec.12348pubmed: 26220509google scholar: lookup
  2. Colon D.F, Wanderley C.W, Franchin M, Silva C.M, Hiroki C.H, Castanheira F.V.S, Donate P.B, Lopes A.H, Volpon L.C, Kavaguti S.K. Neutrophil extracellular traps (NETs) exacerbate severity of infant sepsis. Crit. Care 2019;23:113.
    doi: 10.1186/s13054-019-2407-8pmc: PMC6454713pubmed: 30961634google scholar: lookup
  3. Clementi A, Virzì G.M, Brocca A, Pastori S, de Cal M, Marcante S, Granata A, Ronco C. The Role of Cell-Free Plasma DNA in Critically Ill Patients with Sepsis. Blood Purif 2016;41:34–40.
    doi: 10.1159/000440975pubmed: 26960212google scholar: lookup
  4. Jackson Chornenki N.L, Coke R, Kwong A.C, Dwivedi D.J, Xu M.K, McDonald E, Marshall J.C, Fox-Robichaud A.E, Charbonney E, Liaw P.C. Comparison of the source and prognostic utility of cfDNA in trauma and sepsis. Intensive Care Med. Exp. 2019;7:29.
    doi: 10.1186/s40635-019-0251-4pmc: PMC6531595pubmed: 31119471google scholar: lookup
  5. Bayless R.L, Cooper B.L, Sheats M.K. Investigation of plasma cell-free DNA as a potential biomarker in horses. J. Vet. Diagn. Investig. 2022;34:402–406.
    doi: 10.1177/10406387221078047pmc: PMC9254060pubmed: 35168428google scholar: lookup
  6. Li R.H, Tablin F. A comparative review of neutrophil extracellular traps in sepsis. Front. Vet. Sci. 2018;5:291.
    doi: 10.3389/fvets.2018.00291pmc: PMC6280561pubmed: 30547040google scholar: lookup
  7. Sun M, Chen P, Xiao K, Zhu X, Zhao Z, Guo C, He X, Shi T, Zhong Q, Jia Y. Circulating Cell-Free DNAs as a Biomarker and Therapeutic Target for Acetaminophen-Induced Liver Injury. Adv. Sci. 2023;10:e2206789.
    doi: 10.1002/advs.202206789pmc: PMC10238175pubmed: 37035952google scholar: lookup
  8. Fingerhut L, Ohnesorge B, von Borstel M, Schumski A, Strutzberg-Minder K, Morgelin M, Deeg C.A, Haagsman H.P, Beineke A, von Kockritz-Blickwede M. Neutrophil Extracellular Traps in the Pathogenesis of Equine Recurrent Uveitis (ERU). Cells 2019;8:1528.
    doi: 10.3390/cells8121528pmc: PMC6953072pubmed: 31783639google scholar: lookup
  9. Colmer S.F, Luethy D, Abraham M, Stefanovski D, Hurcombe S.D. Utility of cell-free DNA concentrations and illness severity scores to predict survival in critically ill neonatal foals. PLoS ONE 2021;16:e0242635.
    doi: 10.1371/journal.pone.0242635pmc: PMC8075268pubmed: 33901192google scholar: lookup
  10. Panizzi L, Dittmer K.E, Vignes M, Doucet J.S, Gedye K, Waterland M.R, Rogers C.W, Sano H, McIlwraith C.W, Riley C.B. Plasma and Synovial Fluid Cell-Free DNA Concentrations Following Induction of Osteoarthritis in Horses. Animals 2023;13:1053.
    doi: 10.3390/ani13061053pmc: PMC10044647pubmed: 36978592google scholar: lookup
  11. Brewer B.D, Koterba A.M. Development of a scoring system for the early diagnosis of equine neonatal sepsis. Equine Vet. J. 1988;20:18–22.
    doi: 10.1111/j.2042-3306.1988.tb01445.xpubmed: 3366100google scholar: lookup
  12. Letendre J.A, Goggs R. Measurement of plasma cell-free DNA concentrations in dogs with sepsis, trauma, and neoplasia. J. Vet. Emerg. Crit. Care 2017;27:307–314.
    doi: 10.1111/vec.12592pubmed: 28295988google scholar: lookup
  13. Smith S.A, Lawson C.M, McMichael M.A, Jung K, O’Brien M, Achiel R. Evaluation of assays for quantification of DNA in canine plasma as an indirect marker of NETosis. Vet. Clin. Pathol. 2017;46:278–286.
    doi: 10.1111/vcp.12478pubmed: 28370080google scholar: lookup
  14. Devonshire A.S, Whale A.S, Gutteridge A, Jones G, Cowen S, Foy C.A, Huggett J.F. Towards standardisation of cell-free DNA measurement in plasma: Controls for extraction efficiency, fragment size bias and quantification. Anal. Bioanal. Chem. 2014;406:6499–6512.
    doi: 10.1007/s00216-014-7835-3pmc: PMC4182654pubmed: 24853859google scholar: lookup
  15. Pérez-Barrios C, Nieto-Alcolado I, Torrente M, Jiménez-Sánchez C, Calvo V, Gutierrez-Sanz L, Palka M, Donoso-Navarro E, Provencio M, Romero A. Comparison of methods for circulating cell-free DNA isolation using blood from cancer patients: Impact on biomarker testing. Transl. Lung Cancer Res. 2016;5:665–672.
    doi: 10.21037/tlcr.2016.12.03pmc: PMC5233878pubmed: 28149760google scholar: lookup
  16. Kloten V, Ruchel N, Bruchle N.O, Gasthaus J, Freudenmacher N, Steib F, Mijnes J, Eschenbruch J, Binnebosel M, Knuchel R. Liquid biopsy in colon cancer: Comparison of different circulating DNA extraction systems following absolute quantification of KRAS mutations using Intplex allele-specific PCR. Oncotarget 2017;8:86253–86263.
    doi: 10.18632/oncotarget.21134pmc: PMC5689682pubmed: 29156792google scholar: lookup
  17. Goggs R, Jeffery U, LeVine D.N, Li R.H.L. Neutrophil-Extracellular Traps, Cell-Free DNA, and Immunothrombosis in Companion Animals: A Review. Vet. Pathol. 2020;57:6–23.
    doi: 10.1177/0300985819861721pubmed: 31342866google scholar: lookup
  18. Polatoglou E, Mayer Z, Ungerer V, Bronkhorst A.J, Holdenrieder S. Isolation and Quantification of Plasma Cell-Free DNA Using Different Manual and Automated Methods. Diagnostics 2022;12:2550.
    doi: 10.3390/diagnostics12102550pmc: PMC9601152pubmed: 36292239google scholar: lookup
  19. Bayless R.L. Investigation of plasma cell-free dna (cfdna) as a novel biomarker of inflammation and disease severity in equine colic patients. Equine Vet. Educ. 2021;33:22–23.
    doi: 10.1111/eve.36_13534google scholar: lookup
  20. Ponti G, Maccaferri M, Manfredini M, Kaleci S, Mandrioli M, Pellacani G, Ozben T, Depenni R, Bianchi G, Pirola G.M. The value of fluorimetry (Qubit) and spectrophotometry (NanoDrop) in the quantification of cell-free DNA (cfDNA) in malignant melanoma and prostate cancer patients. Clin. Chim. Acta 2018;479:14–19.
    doi: 10.1016/j.cca.2018.01.007pubmed: 29309771google scholar: lookup
  21. Charoensappakit A, Sae-Khow K, Rattanaliam P, Vutthikraivit N, Pecheenbuvan M, Udomkarnjananun S, Leelahavanichkul A. Cell-free DNA as diagnostic and prognostic biomarkers for adult sepsis: A systematic review and meta-analysis. Sci. Rep. 2023;13:19624.
    doi: 10.1038/s41598-023-46663-2pmc: PMC10638380pubmed: 37949942google scholar: lookup
  22. Nguyen D.N, Stensballe A, Lai J.C, Jiang P, Brunse A, Li Y, Sun J, Mallard C, Skeath T, Embleton N.D. Elevated levels of circulating cell-free DNA and neutrophil proteins are associated with neonatal sepsis and necrotizing enterocolitis in immature mice, pigs and infants. Innate Immun. 2017;23:524–536.
    doi: 10.1177/1753425917719995pubmed: 28714327google scholar: lookup
  23. Perkins G.A, Wagner B. The development of equine immunity: Current knowledge on immunology in the young horse. Equine Vet. J. 2015;47:267–274.
    doi: 10.1111/evj.12387pubmed: 25405920google scholar: lookup
  24. Demmers S, Johannisson A, Grondahl G, Jensen-Waern M. Neutrophil functions and serum IgG in growing foals. Equine Vet. J. 2001;33:676–680.
    doi: 10.2746/042516401776249327pubmed: 11770989google scholar: lookup
  25. Gardner R.B, Nydam D.V, Luna J.A, Bicalho M.L, Matychak M.B, Flaminio M.J. Serum opsonization capacity, phagocytosis, and oxidative burst activity in neonatal foals in the intensive care unit. J. Vet. Intern. Med. 2007;21:797–805.
    doi: 10.1111/j.1939-1676.2007.tb03024.xpubmed: 17708402google scholar: lookup
  26. Marcos V, Nussbaum C, Vitkov L, Hector A, Wiedenbauer E.-M, Roos D, Kuijpers T, Krautgartner W.D, Genzel-Boroviczény O, Sperandio M. Delayed but functional neutrophil extracellular trap formation in neonates. Blood 2009;114:4908–4911.
    doi: 10.1182/blood-2009-09-242388pubmed: 19965699google scholar: lookup
  27. Lipp P, Ruhnau J, Lange A, Vogelgesang A, Dressel A, Heckmann M. Less Neutrophil Extracellular Trap Formation in Term Newborns than in Adults. Neonatology 2017;111:182–188.
    doi: 10.1159/000452615pubmed: 27884010google scholar: lookup
  28. Vorobjeva N.V, Chernyak B.V. NETosis: Molecular Mechanisms, Role in Physiology and Pathology. Biochemistry 2020;85:1178–1190.
    doi: 10.1134/S0006297920100065pmc: PMC7590568pubmed: 33202203google scholar: lookup
  29. McTaggart C, Penhale J, Raidala S.L. Effect of plasma transfusion on neutrophil function in healthy and septic foals. Aust. Vet. J. 2005;83:499–505.
    doi: 10.1111/j.1751-0813.2005.tb13304.xpubmed: 16119424google scholar: lookup
  30. Burton A.G, Harris L.A, Owens S.D, Jandrey K.E. The prognostic utility of degenerative left shifts in dogs. J. Vet. Intern. Med. 2013;27:1517–1522.
    doi: 10.1111/jvim.12208pubmed: 24118349google scholar: lookup
  31. Burton A.G, Harris L.A, Owens S.D, Jandrey K.E. Degenerative left shift as a prognostic tool in cats. J. Vet. Intern. Med. 2014;28:912–917.
    doi: 10.1111/jvim.12338pmc: PMC4895473pubmed: 24597712google scholar: lookup
  32. Gayle J.M, Cohen N.D, Chaffin M.K. Factors associated with survival in septicemic foals: 65 cases (1988–1995). J. Vet. Intern. Med. 1998;12:140–146.
    doi: 10.1111/j.1939-1676.1998.tb02109.xpubmed: 9595374google scholar: lookup
  33. Letendre J.-A, Goggs R. Determining prognosis in canine sepsis by bedside measurement of cell-free DNA and nucleosomes. J. Vet. Emerg. Crit. Care 2018;28:503–511.
    doi: 10.1111/vec.12773pubmed: 30299568google scholar: lookup
  34. Barton M.H, Hart K.A. Clinical Pathology in the Foal. Vet. Clin. N. Am. Equine Pract. 2020;36:73–85.
    doi: 10.1016/j.cveq.2019.11.003pubmed: 32145835google scholar: lookup
  35. Weber E.J, Sanchez L.C, Giguère S. Re-evaluation of the sepsis score in equine neonates. Equine Vet. J. 2015;47:275–278.
    doi: 10.1111/evj.12279pubmed: 24750245google scholar: lookup
  36. Pusterla N, Mapes S, Byrne B.A, Magdesian K.G. Detection of bloodstream infection in neonatal foals with suspected sepsis using real-time PCR. Vet. Rec. 2009;165:114–117.
    doi: 10.1136/vetrec.165.4.114pubmed: 19633325google scholar: lookup
  37. Hackett E.S, Lunn D.P, Ferris R.A, Horohov D.W, Lappin M.R, McCue P.M. Detection of bacteraemia and host response in healthy neonatal foals. Equine Vet. J. 2015;47:405–409.
    doi: 10.1111/evj.12307pubmed: 24917427google scholar: lookup
  38. Wong D.M, Ruby R.E, Dembek K.A, Barr B.S, Reuss S.M, Magdesian K.G, Olsen E, Burns T, Slovis N.M, Wilkins P.A. Evaluation of updated sepsis scoring systems and systemic inflammatory response syndrome criteria and their association with sepsis in equine neonates. J. Vet. Intern. Med. 2018;32:1185–1193.
    doi: 10.1111/jvim.15087pmc: PMC5980351pubmed: 29582480google scholar: lookup

Citations

This article has been cited 3 times.
  1. Chen LT, Wesdorp E, Jager M, Siegers EW, Theelen MJP, Besselink N, Vermeulen C, Zomer AL, Broens EM, Wagenaar JA, de Ridder J. Bacterial cell-free DNA profiling reveals the co-elevation of multiple bacteria in newborn foals with suspected sepsis. iScience 2025 Dec 19;28(12):114005.
    doi: 10.1016/j.isci.2025.114005pubmed: 41476946google scholar: lookup
  2. 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).
    doi: 10.3390/vetsci11090427pubmed: 39330806google scholar: lookup
  3. Ge Q, Zhang ZY, Li SN, Ma JQ, Zhao Z. Liquid biopsy: Comprehensive overview of circulating tumor DNA (Review). Oncol Lett 2024 Nov;28(5):548.
    doi: 10.3892/ol.2024.14681pubmed: 39319213google scholar: lookup
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