Background

Extracellular DNA often called cell-free DNA is a term for all DNA found outside of cells especially in diagnostic biofluids. Blood plasma DNA was first discovered by Mandel and Metais (1948). The interest in extracellular DNA research was stimulated much later by the research of the so-called liquid biopsy as a source of DNA-based biomarkers for non-invasive screening and diagnostics (Poon and Lo, 2001). The same extracellular DNA is, however, also involved in the pathophysiology of inflammatory diseases such as sepsis (Lauková et al., 2017), acute kidney injury (Jansen et al., 2017) and acute liver failure (Vokálová et al., 2017). The proinflammatory properties of extracellular DNA are in some of the sequences, especially mitochondrial DNA, resembling the sequences of bacterial DNA such as unmethylated CpG islands. In this case those proinflammatory properties are facilitated via detection with toll-like receptors. The nuclear DNA is more effective to trigger inflammation when associated with proteins such as histones or as a part of neutrophil extracellular traps. Extracellular DNA can be released from dying cells during apoptosis or necrosis. Most of the extracellular DNA in healthy individuals originates from blood elements.

The quantitative assessment of extracellular DNA is, thus, crucial for a wide range of research topics. There are many limitations when it comes to extracellular DNA measurement. In animal experiments, the available volume of blood, urine or saliva greatly limits subsequent analyses. Studies differ in the procedures used for sample collection, processing, isolation and quantification. This makes it impossible to use standard normal ranges for the concentration of extracellular DNA in plasma, urine or saliva. Generally, EDTA-containing tubes that inhibit the activity of deoxyribonucleases are mostly used for isolation of extracellular DNA from blood plasma (Lee et al., 2001). Sampling of urine or saliva is not standardized at all. Sample storage by freezing is common and often cannot be avoided. However, freezing and thawing can lead to unwanted degradation of extracellular DNA (Lahiri and Schnabel, 1993). Our unpublished observations suggest that isolation of DNA from fresh and frozen plasma leads to a completely divergent size profile of the extracellular DNA. Usually, two consecutive centrifugations are carried out to remove cells and smaller cellular debris from body fluids before DNA isolation. However, not all studies follow this procedure (Sun et al., 2018). Results from studies using different isolation and quantification methods cannot be compared. Due to high technical variability of the measurement of extracellular DNA concentrations the use of duplicates or triplicates could help achieve reproducible results. The standardisation of protocols for processing of body fluids intended for extracellular DNA isolation would be valuable for the research of extracellular DNA as a biomarker, but also as a damage-associated molecular pattern.