Cerebrospinal fluid surrounds the entire CNS and provides a number of vital functions for the nervous tissue, including physical support, protection, nutrition, and transport.1 Biopsy of the CNS is invasive and can be associated with patient morbidity and death, whereas sampling of the CSF is relatively routine and can provide a less invasive sampling that is reflective of changes in the CNS.2 Cerebrospinal fluid contains valuable biochemical and cellular information that aids in the diagnosis and treatment of neurologic disease. In veterinary medicine, routinely evaluated CSF parameters include NCC, RBCC, and protein concentration.3 However, changes in these parameters can be non-specific for different neurologic diseases, and additional diagnostic tools are needed.
Cell-free DNA is extracellular DNA released during cellular apoptosis or necrosis secondary to normal cell turnover or a variety of disease processes, and plasma cfDNA has been measured in human and veterinary patients.4–16 A study16 shows that cfDNA released from tumors in humans is predominantly the result of cellular necrosis, whereas cfDNA released secondary to nonneoplastic processes is predominately the result of apoptosis. Consequently, more malignant tumors result in a higher degree of necrosis and a corresponding greater plasma concentration of circulating cell-free tumor DNA.16 A recent study4 of dogs shows that the plasma concentration of cfDNA increased with disease severity in dogs and could be prognostically useful. Dogs that did not survive to hospital discharge had significantly higher cfDNA concentrations than did those discharged alive.4 These studies4,16 suggest that the plasma concentration of cfDNA may correlate with disease severity and prognosis.
Although plasma cfDNA concentration has been used in determining the diagnosis, treatment, and prognosis of systemic diseases,4–6,8,10,12–14 plasma cfDNA concentration may be of limited use for such purposes with CNS disease.15,17,18 This is presumably because of the presence of the blood-brain barrier, which may prevent cfDNA released secondary to CNS disease from circulating systemically.17 Because CSF surrounds the CNS, analysis of CSF cfDNA should provide a more accurate indication of the CNS environment. Concentrations of cfDNA in the CSF have been evaluated in human medicine, particularly in patients with neoplastic and inflammatory diseases.17–21 Tumor-specific mutations in cfDNA have been identified in CSF samples from humans with various primary and metastatic brain tumors.17 Additionally, a mutation specific for metastatic melanoma of the leptomeninges in a human patient was identified in CSF cfDNA but not plasma cfDNA, and the patient's CSF concentration of mutant alleles coincided with clinical symptoms in that as the patient's symptoms diminished with recovery, so did the CSF cfDNA concentration of mutant alleles.18
Although evaluation of CSF cfDNA has diagnostic and prognostic uses in human medicine, to our knowledge, there have been no investigations into whether CSF cfDNA is detectable or has diagnostic or prognostic use in dogs. Because the CSF volumes (≥ 5 mL) used in human studies19,20 would not be practical in small dogs, it was also unknown whether smaller-volume CSF samples obtained from dogs would yield accurate quantitation of cfDNA.
The main objectives of study reported here were to determine whether cfDNA was detectable in CSF samples from dogs and whether CSF sample volume impacted cfDNA concentration measurement. If cfDNA was measurable in CSF samples from dogs, the second aim was to investigate whether CSF cfDNA concentration was associated with the CNS disease category or with the CSF NCC, RBCC, or protein concentration. We hypothesized that cfDNA would be detectable in CSF samples from healthy dogs and dogs with neurologic disease, the CSF sample volume would not meaningfully affect measurability of the CSF cfDNA concentration, and the CSF cfDNA concentration would be higher in dogs with (vs without) neurologic disease (neoplastic, infectious, or inflammatory).
All CSF samples were processed at the Clinical Pathology Laboratory, College of Veterinary Medicine and Biomedical Sciences, Colorado State University.
Supported by the Young Investigator Grant Program, Center for Companion Animal Studies, Colorado State University.
The authors declare that there were no conflicts of interest.
The authors thank Dr. Steven Dow for technical support and Dr. Sangeeta Rao for statistical analysis.
Nucleated cell count (WBC count)
Cobas c501 chemistry analyzer, Roche Diagnostics, Indianapolis, Ind.
Sorvall ST 16R centrifuge, Thermo Fisher Scientific Inc, Waltham, Mass.
QIAamp DNA mini kit, Qiagen, Hilden, Germany.
Qubit dsDNA HS assay kit, Thermo Fisher Scientific Inc, Waltham, Mass.
Qubit 2.0 Fluorimeter, Thermo Fisher Scientific Inc, Waltham, Mass.
SAS, version 9.4, SAS Institute Inc, Cary, NC.
1. Johanson CE, Duncan JA III, Klinge PM, et al. Multiplicity of cerebrospinal fluid functions: new challenges in health and disease. Cerebrospinal Fluid Res 2008;5:10.
3. Jurado R, Walker HK. Cerebrospinal fluid. In: Walker HK, Hall WD, Hurst JW, eds. Clinical methods: the history, physical, and laboratory examinations. 3rd ed. Boston: Butter-worths, 1990;371–382.
4. Burnett DL, Cave NJ, Gedye KR, et al. Investigation of cell-free DNA in canine plasma and its relation to disease. Vet Q 2016;36:122–129.
5. Letendre JA, Goggs R. Determining prognosis in canine sepsis by bedside measurement of cell-free DNA and nucleosomes. J Vet Emerg Crit Care (San Antonio) 2018;28:503–511.
6. Letendre JA, Goggs R. Measurement of plasma cell-free DNA concentrations in dogs with sepsis, trauma, and neoplasia. J Vet Emerg Crit Care (San Antonio) 2017;27:307–314.
7. Wilson IJ, Burchell RK, Worth AJ, et al. Kinetics of plasma cell-free DNA and creatine kinase in a canine model of tissue i n j u r y. J Vet Intern Med 2018;32:157–164.
8. Jeffery U, Ruterbories L, Hanel R, et al. Cell-free DNA and DNase activity in dogs with immune-mediated hemolytic anemia. J Vet Intern Med 2017;31:1441–1450.
9. Fleischhacker M, Schmidt B, Weichmann S, et al. Methods for isolation of cell-free plasma DNA strongly affect DNA yield. Clin Chim Acta 2011;412:2085–2088.
11. Devonshire AS, Whale AS, Gutteridge A, et al. Towards standardization of cell-free DNA measurement in plasma: controls for extraction efficacy, fragment size bias and quantification. Anal Bioanal Chem 2014;406:6499–6512.
12. Macher H, Egea-Guerrero JJ, Revuelto-Rey J, et al. Role of early cell-free DNA levels decrease as a predictive marker of fatal outcome after severe traumatic brain injury. Clin Chim Acta 2012;414:12–17.
13. Rainer TH, Wong LK, Lam W, et al. Prognostic use of circulating plasma nucleic acid concentration in patients with acute stroke. Clin Chem 2003;49:562–569.
14. Tsai NW, Lin TK, Chen SD, et al. The value of serial plasma nuclear and mitochondrial DNA levels in patients with acute ischemic stroke. Clin Chim Acta 2011;412:476–479.
15. De Mattos-Arruda L, Mayor R, Ng CKY, et al. Cerebrospinal fluid-derived circulating tumour DNA better represents the genomic alterations of brain tumours than plasma. Nat Commun 2015;6:8839.
16. Benesova L, Belsanova B, Suchanek S, et al. Mutation-based detection and monitoring of cell-free DNA in peripheral blood of cancer patients. Anal Biochem 2013;433:227–234.
18. Li Y, Pan W, Connolly ID, et al. Tumor DNA in cerebral spinal fluid reflects clinical course in a patient with melanoma leptomeningeal brain metastases. J Neurooncol 2016;128:93–100.
19. Wang Y, Springer S, Zhang M, et al. Detection of tumor-derived DNA in cerebrospinal fluid of patients with primary tumors of the brain and spinal cord. Proc Natl Acad Sci U S A 2015;112:9704–9709.
20. Shi W, Lv C, Qi J, et al. Prognostic value of free DNA quantification in serum and cerebrospinal fluid in glioma patients. J Mol Neurosci 2012;46:470–475.
21. Mehta SR, Pérez-Santiago J, Hulgan T, et al. Cerebrospinal fluid cell-free mitochondrial DNA is associated with HIV replication, iron transport, and mild HIV-associated cognitive impairment. J Neuroinflammation 2017;14:72.
22. Ponti G, Maccaferri M, Kaleci S, et al. 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.
23. Xue X, Teare MD, Holen I, et al. Optimizing the yield and utility of circulating cell-free DNA from plasma and serum. Clin Chim Acta 2009;404:100–104.
24. Di Terlizzi R, Platt SR. The function, composition and analysis of cerebrospinal fluid in companion animals: part II—analysis. Vet J 2009;180:15–32.
25. de Graaf MT, van den Broek PDM, Kraan J, et al. Addition of serum-containing medium to cerebrospinal fluid prevents cellular loss over time. J Neurol 2011;258:1507–1512.
26. Talarico LR, Schatzberg SJ. Idiopathic granulomatous and necrotizing inflammatory disorders of the canine central nervous system: a review and future perspectives. J Small Anim Pract 2010;51:138–149.
27. Levine GJ, Cook JR, Kerwin SC, et al. Relationships between cerebrospinal fluid characteristics, injury severity, and functional outcome in dogs with and without intervertebral disc herniation. Vet Clin Pathol 2014;43:437–446.
28. de Lahunta A. Cerebrospinal fluid and hydrocephalus. In: de Lahunta A, Glass E, Kent M, eds. Veterinary neuroanatomy and clinical neurology. 4th ed. St Louis: Elsevier Saunders, 2015;78–101.
29. Dewey CW, da Costa RC, Ducoté JM. Neurodiagnostics. In: Dewey CW, da Costa RC, eds. Practical guide to canine and feline neurology. 3rd ed. Ames, Iowa: John Wiley & Sons Inc, 2016;61–65.
30. Newton PL, Fry DR, Best MP. Comparison of direct in-house cerebrospinal fluid cytology with commercial pathology results in dogs. J Small Anim Pract 2017;58:694–702.
31. Pérez-Santiago J, Schrier RD, Oliveira MF, et al. Cell-free mitochondrial DNA in CSF is associated with early viral rebound, inflammation, and severity of neurocognitive deficits in HIV infection. J Neurovirol 2016;22:191–200.