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Biological variability of cell-free DNA in healthy females at rest within a short time course

International Journal of Legal Medicine volume 134, pages 911–919 (2020)Cite this article

Abstract

Introduction

Alterations in cell-free DNA concentration (cfDNA) over time have been studied in diseased or injured patients or analyzed in athletes during exhaustive exercise. However, no fluctuations have been examined over a short time course in healthy humans at rest so far, wherefore the aim of this study was to examine individual variations at different time points within 75 min.

Methods

Serial blood drawing was performed in 14 healthy female volunteers at rest within 75 min. Plasma DNA was quantified by real-time qPCR, and absolute levels were analyzed together with relative variations. cfDNA alterations were moreover analyzed in consideration of potential volunteer-related impact factors (e.g., pulse) and were compared to alterations of plasma CK and AST.

Results

Absolute cfDNA concentration ranged from 0.6 to 3.4 ng/ml. Regarding alterations over time, positive and negative variations were identified, whereby the interdecile range of fold changes was from 0.5 to 1.4. The maximum fold change was determined at 10 min. No relations were found between cfDNA levels and the analyzed individual factors.

Conclusion

We evidenced the variability of cfDNA in healthy humans at rest within a short time course. The determined variations should serve in future studies to distinguish small cfDNA increases after minor trauma from natural fluctuations. Without such reference of intra-individual variation at rest, it would not be feasible to distinguish an injury from a fluctuation with certainty. Thus, a basis was established for the application of cfDNA as biomarker for the detection of mild injuries in forensic biomechanics.

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References

  1. Sharma SK, Naidu G (2016) The role of danger-associated molecular patterns ( DAMPs ) in trauma and infections. J Thorac Dis 8(7):1406–1409. https://doi.org/10.21037/jtd.2016.05.22

    Article  PubMed  PubMed Central  Google Scholar 

  2. Ren B, Liu F, Xu F et al (2013) Is plasma cell-free DNA really a useful marker for diagnosis and treatment of trauma patients? Clin Chim Acta 424:109–113. https://doi.org/10.1016/j.cca.2013.05.015

    Article  CAS  PubMed  Google Scholar 

  3. McIlroy DJ, Bigland M, White AE, Hardy BM, Lott N, Smith DW, Balogh ZJ (2015) Cell necrosis-independent sustained mitochondrial and nuclear DNA release following trauma surgery. J Trauma Acute Care Surg 78(2):282–288. https://doi.org/10.1097/TA.0000000000000519

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  4. Brodbeck K, Kern S, Schick S, Steinbrück A, Schwerer M, Bayer B, Anslinger K, Peldschus S (2018) Quantitative analysis of individual cell-free DNA concentration before and after penetrating trauma. Int J Legal Med 133(2):385–393. https://doi.org/10.1007/s00414-018-1945-y

    Article  PubMed  Google Scholar 

  5. Fox A, Gal S, Fisher N, Smythe J, Wainscoat J, Tyler MP, Watt SM, Harris AL (2008) Quantification of circulating cell-free plasma DNA and endothelial gene RNA in patients with burns and relation to acute thermal injury. Burns 34(6):809–816. https://doi.org/10.1016/j.burns.2007.10.003

    Article  CAS  PubMed  Google Scholar 

  6. Shaked G, Douvdevani A, Yair S et al (2014) The role of cell-free DNA measured by a fluorescent test in the management of isolated traumatic head injuries. Scand J Trauma Resusc Emerg Med 22(1):21. https://doi.org/10.1186/1757-7241-22-21

    Article  PubMed  PubMed Central  Google Scholar 

  7. Timmermans K, Kox M, Vaneker M, van den Berg M, John A, van Laarhoven A, van der Hoeven H, Scheffer GJ, Pickkers P (2016) Plasma levels of danger-associated molecular patterns are associated with immune suppression in trauma patients. Intensive Care Med 42(4):1–11. https://doi.org/10.1007/s00134-015-4205-3

    Article  CAS  Google Scholar 

  8. Lam NY, Rainer TH, Chan LY et al (2003) Time course of early and late changes in plasma DNA in trauma patients. Clin Chem 49(8):1286–1291. https://doi.org/10.1373/49.8.1286

    Article  CAS  PubMed  Google Scholar 

  9. Muggenthaler H, von Merten K, Peldschus S, Holley S, Adamec J, Praxl N, Graw M (2008) Experimental tests for the validation of active numerical human models. Forensic Sci Int 177(2–3):184–191. https://doi.org/10.1016/j.forsciint.2007.12.005

    Article  PubMed  Google Scholar 

  10. Bohnert M, Baumgartner R, Pollak S (2000) Spectrophotometric evaluation of the colour of intra- and subcutaneous bruises. Int J Legal Med 113(6):343–348. https://doi.org/10.1007/s004149900107

    Article  CAS  PubMed  Google Scholar 

  11. Mühlbauer JA (2016) In Vivo Kollisionsuntersuchungen an den Oberen Extremitäten zur Ermittlung von Schmerz- und Belastungsgrenzen von Weichgewebe vor dem Hintergrund der Mensch-Roboter-Kollaboration. Master Thesis, Technische Universität München

  12. Rowan P, Hill M, Gresham GA, Goodall E, Moore T (2010) The use of infrared aided photography in identification of sites of bruises after evidence of the bruise is absent to the naked eye. J Forensic Legal Med 17(6):293–297. https://doi.org/10.1016/j.jflm.2010.04.007

    Article  Google Scholar 

  13. Lombardi M, Canter J, Patrick PA, Altman R (2015) Is fluorescence under an alternate light source sufficient to accurately diagnose subclinical bruising? J Forensic Sci 60(2):444–449. https://doi.org/10.1111/1556-4029.12698

    Article  PubMed  Google Scholar 

  14. Helm T, Bir C, Chilstrom M, Claudius I (2016) Ultrasound characteristics of bruises and their correlation to cutaneous appearance. Forensic Sci Int 266:160–163. https://doi.org/10.1016/j.forsciint.2016.05.022

    Article  PubMed  Google Scholar 

  15. Fleischhacker M, Schmidt B (2007) Circulating nucleic acids (CNAs) and cancer—a survey. Biochim Biophys Acta 1775:181–232. https://doi.org/10.1016/j.bbcan.2006.10.001

    Article  CAS  PubMed  Google Scholar 

  16. Lo YM, Rainer TH, Chan LY, Hjelm NM, Cocks RA (2000) Plasma DNA as a prognostic marker in trauma patients. Clin Chem 46:319–323

    Article  CAS  Google Scholar 

  17. Macher H, Egea-Guerrero JJ, Revuelto-Rey J et al (2012) Role of early cell-free DNA levels decrease as a predictive marker of fatal outcome after severe traumatic brain injury. Clin Chim Acta 414:12–17. https://doi.org/10.1016/j.cca.2012.08.001

    Article  CAS  PubMed  Google Scholar 

  18. Tamkovich SN, Bryzgunova OE, Rykova EY, Permyakova VI, Vlassov VV, Laktionov PP (2005) Circulating nucleic acids in blood of healthy male and female donors. Clin Chem 51(7):1317–1319. https://doi.org/10.1373/clinchem.2004.046284

    Article  CAS  PubMed  Google Scholar 

  19. Meddeb R, Pisareva E, Thierry A (2019) Guidelines for the preanalytical conditions for analyzing circulating cell-free DNA. Clin Chem 65(5):000–000. https://doi.org/10.1373/clinchem.2018.298323

    Article  CAS  Google Scholar 

  20. Nishimoto S, Fukuda D, Higashikuni Y et al (2016) Obesity-induced DNA released from adipocytes stimulates chronic adipose tissue inflammation and insulin resistance. Sci Adv 2(3):1–12

    Article  Google Scholar 

  21. Vieira de Sousa M, Madsen K, Fukui R et al (2012) Carbohydrate supplementation delays DNA damage in elite runners during intensive microcycle training. Eur J Appl Physiol 112:493–500. https://doi.org/10.1007/s00421-011-2000-6

    Article  CAS  Google Scholar 

  22. Breiter T, Fragasso A, Hudemann J et al (2011) Short-term treadmill running as a model for studying cell-free DNA kinetics in vivo. Clin Chem 57(4):633–636. https://doi.org/10.1373/clinchem.2010.158030

    Article  Google Scholar 

  23. Pölcher M, Ellinger J, Willems S, el-Maarri O, Höller T, Amann C, Wolfgarten M, Rudlowski C, Kuhn W, Braun M (2010) Impact of the menstrual cycle on circulating cell-free DNA. Anticancer Res 30:2235–2240

    PubMed  Google Scholar 

  24. Korabecna M, Horinek A, Bila N, Opatrna S (2009) Circadian rhythmicity and clearance of cell-free DNA in human plasma. In: Gahan PB (ed), Proceedings of the 6th International Conference on Circulating Nucleic Acids in Plasma and Serum, pp 195–198

  25. Tranberg Madsen A, Hojbjerg JA, Sorensen BS, Winther-larsen A (2019) Day-to-day and within-day biological variation of cell-free DNA. EBioMedicine. https://doi.org/10.1016/j.ebiom.2019.10.008

  26. Vanezis P (2001) Interpreting bruises at necropsy. J Clin Pathol 54(5):348–355. https://doi.org/10.1136/jcp.54.5.348

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  27. Klein G, Berger A, Bertholf R et al (2001) Multicenter evaluation of liquid reagents for CK, CK-MB and LDH with determination of reference intervals on Hitachi systems. Clin Chem 47(6):A30–A30

    Google Scholar 

  28. Klauke R, Schmidt E, Lorentz K (1993) Recommendations for carrying out standard ECCLS procedures (1988) for the catalytic concentrations of creatine kinase, aspartate aminotransferase, alanine aminotransferase and gamma-glutamyltransferase at 37 degrees C. Eur J Clin Chem Clin Biochem 31(12):901–909

    CAS  PubMed  Google Scholar 

  29. World Health Organization. http://www.euro.who.int/en/health-topics/disease-prevention/nutrition/a-healthy-lifestyle/body-mass-index-bmi. Accessed: January 2019

  30. Liaw PC, Ito T, Iba T, Thachil J, Zeerleder S (2016) DAMP and DIC: the role of extracellular DNA and DNA-binding proteins in the pathogenesis of DIC. Blood Rev 30(4):257–261. https://doi.org/10.1016/j.blre.2015.12.004

    Article  CAS  PubMed  Google Scholar 

  31. Suzuki N, Kamataki A, Yamaki J, Homma Y (2008) Characterization of circulating DNA in healthy human plasma. Clin Chim Acta 387(1–2):55–58. https://doi.org/10.1016/j.cca.2007.09.001

    Article  CAS  PubMed  Google Scholar 

  32. Stawski R, Walczak K, Kosielski P et al (2017) Repeated bouts of exhaustive exercise increase circulating cell free nuclear and mitochondrial DNA without development of tolerance in healthy men. PLoS One 12(5):1–17. https://doi.org/10.1371/journal.pone.0178216

    Article  CAS  Google Scholar 

  33. Breitbach S, Tug S, Helmig S et al (2014) Direct quantification of cell-free, circulating DNA from unpurified plasma. PLoS One. https://doi.org/10.1371/journal.pone.0087838

  34. Breitbach S, Tug S, Simon P (2012) Circulating cell-free DNA: an up-coming molecular marker in exercise physiology. Sports Medi (Auckland, NZ) 42(7):565–586

    Article  Google Scholar 

  35. Tug S, Tross A-K, Hegen P, Neuberger EWI, Helmig S, Schöllhorn W, Simon P (2017) Acute effects of strength exercises and effects of regular strength training on cell free DNA concentrations in blood plasma. PLoS One 12(9):1–12. https://doi.org/10.1371/journal.pone.0184668

    Article  CAS  Google Scholar 

  36. Hummel EM, Hessas E, Müller S, Beiter T, Fisch M, Eibl A, Wolf OT, Giebel B, Platen P, Kumsta R, Moser DA (2018) Cell-free DNA release under psychosocial and physical stress conditions. Transl Psychiatry 8(236):236. https://doi.org/10.1038/s41398-018-0264-x

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  37. Jeong DW, Moon JY, Choi YW, Moon H, Kim K, Lee YH, Kim SY, Kim YG, Jeong KH, Lee SH (2015) Effect of blood pressure and glycemic control on the plasma cell-free DNA in hemodialysis patients. Kidney Res Clin Pract 34(4):201–206. https://doi.org/10.1016/j.krcp.2015.09.002

    Article  PubMed  PubMed Central  Google Scholar 

  38. Lazar L, Rigójr J, Nagy B et al (2009) Relationship of circulating cell-free DNA levels to cell-free fetal DNA levels, clinical characteristics and laboratory parameters in preeclampsia. BMC Med Genet 10(120):1–6. https://doi.org/10.1186/1471-2350-10-120

    Article  CAS  Google Scholar 

  39. Maeo S, Yamamoto M, Kanehisa H, Nosaka K (2017) Prevention of downhill walking-induced muscle damage by non-damaging downhill walking. PLoS One 12(3):1–11. https://doi.org/10.1371/journal.pone.0173909

    Article  CAS  Google Scholar 

  40. Gutenbrunner C (2000) Circadian variations of the serum creatine kinase level – a masking effect? Chronobiol Int 17(4):583–590

    Article  CAS  Google Scholar 

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Authors and Affiliations

  1. Institute of Forensic Medicine, Ludwig Maximilian University of Munich, Nußbaumstr. 26, 80336, Munich, Germany

    Katrin Brodbeck, Sylvia Schick, Birgit Bayer, Katja Anslinger & Steffen Peldschus

  2. Institute of Laboratory Medicine, German Heart Centre Munich, Lazarettstr. 36, 80636, Munich, Germany

    Kimberly Krüger, Zsuzsanna Mayer & Stefan Holdenrieder

Authors
  1. Katrin Brodbeck
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  2. Sylvia Schick
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Correspondence to Katrin Brodbeck.

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Brodbeck, K., Schick, S., Bayer, B. et al. Biological variability of cell-free DNA in healthy females at rest within a short time course. Int J Legal Med 134, 911–919 (2020). https://doi.org/10.1007/s00414-019-02240-9

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  • DOI: https://doi.org/10.1007/s00414-019-02240-9

Keywords

  • Cell-free DNA
  • Individual variation
  • Natural fluctuation
  • Time course
  • Trauma