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.
Publication Date: 2024-08-01 PubMed ID: 39195800PubMed Central: PMC11359113DOI: 10.3390/vetsci11080346Google Scholar: Lookup
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- Journal Article
Summary
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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
Researcher Affiliations
- Department of Clinical Sciences, College of Veterinary Medicine, North Carolina State University, Raleigh, NC 27695, USA.
- Department of Clinical Sciences, College of Veterinary Medicine, North Carolina State University, Raleigh, NC 27695, USA.
- Department of Clinical Sciences, College of Veterinary Medicine, North Carolina State University, Raleigh, NC 27695, USA.
- 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.
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