**2. Origin and clearance of free nucleic acids in human circulation**

The phenomenon of cell-free DNA circulating in human plasma was discovered in 1948 (Mandel &, Metais, 1948,). The authors detected DNA and RNA in blood plasma of healthy control and patients. With regard to the lack of biological understanding of such a finding their work remained unnoticed. During next thirty years only two studies focusing on cellfree DNA appeared. The patients with lupus erythematosus (Tan et al., 1966, in Swarup & Rajeswari, 2007) and the patients with rheumatoid arthritis (Ayala et al., 1951) were studied and elevated levels DNA in circulation were reported. The medical importance of cell-free nucleic acids was recognized when the decreasing levels of these nucleic acids were reported in cancer patients after successful chemotherapy (Leon et al., 1977).

In 1994, mutated oncogene K-ras was discovered in the pool of circulating DNA in pancreatic cancer patients (Sorenson at al., 1994). In 1997 the circulating DNA of fetal origin was found in plasmas of pregnant women (Lo et al., 1997). Till today numerous studies were performed not only to understand the nature and biological meaning of cell-free DNAs and RNAs but mainly to establish their diagnostic use under different clinical conditions such as cancer, autoimmune disorders, pregnancy related disorders and trauma (for review Tong & Lo, 2006).

Although recent molecular biology and genetics employ very broad spectrum of sophisticated methods, the complete understanding of biology of cell-free nucleic acids was so far not achieved. As there are speculations concerning the regulatory function of circulating nucleic acids in plasma, the clinical laboratory research and practice stream to develop approaches allowing the analytic use of these molecules. The main sources of nucleic acids in plasma are necrosis, apoptosis and active release by living, non-apoptotic cells. All mentioned processes will be discussed in details in following sections. Cell-free

Cell-Free Nucleic Acids as Biomarkers of Biocompatibility in Dialytic Process 201

According to the results obtained in an animal study (Guan et al., 2002) and the first human study (Pachl et al., 2005) it seems that primary insult induces apoptosis in the relationship to its severity and necrotic DNA originates from secondary organ damage therefore it can be

Muscle injury caused by athletic overtraining leads also to elevation of plasma cfDNA concentrations as reported by Fatouros et al. (2006). The study demonstrated increase of cfDNA levels in proportion to training load. Overtraining causes an acute breakdown with subsequent repair of skeletal muscle and it is characterized by changes in the functionality of immune system resulting in increased susceptibility to infections. The correlation of plasma cfDNA with creatinine kinase but not with C-reactive protein was reported in this study. The results achieved by Fatouros and coworkers are in good agreement with the results of an earlier study (Atamaniuk et al., 2004) in which long-distance runners were examined and 9- to 17.5 fold increase in concentrations of cfDNA was found immediately after the run. With regard to these reports, the extent of physical activities of examined subjects before the sampling procedure must be taken in account when evaluating the results of cfDNA based studies. The problems concerning the clearance of plasma cfDNA will be discussed in one of

Necrosis and apoptosis were originally sought to be the only sources of cell-free nucleic acids in the circulation. The pioneer study by Stroun (2001a) clearly demonstrated the active release of fragmented DNA from cells in culture where apoptosis has been inhibited. Anker with coworkers (Anker et al., 1976) provided evidence about the release of newly synthesized DNA from human leucocytes. They reported that the release process is unrelated to cell death and is regulated by a homeostatic mechanism. Precursor incorporation into the DNA was inhibited

The original hypothesis that the tumor specific cfDNA originates from lysis of tumor cells on the interface between the tumor and circulation is not more viable with regard to the high concentration of cancer specific cfDNA in circulation. Sorenson (1997, in Stroun et al., 2001a) calculated that 1000 cancer cell per milliliter would be necessary to provide the amount of DNA found in the plasma of pancreatic cancer patients. Later, the extraction methods for cfDNA were improved, and it has been demonstrated that the concentrations of

The tumors are not able to supply the circulation with 10 000 cells per each milliliter therefore the active release of nucleic acids by tumor cells is recently accepted as the main

Cell-free DNA can occur in different forms in circulation. It may be transported by vesicle based particles, nucleosomes and virtosomes. The extracellular DNA bound in all mentioned structures forms the so called nucleome (Peters & Pretorius, 2011). The sequences contained in the nucleome were compared with genomic DNA sequences (Stroun et al., 2001; van der Vaart & Pretorius, 2008; Beck et al., 2009; Puszyk et al., 2009). Beck et al. studied the nucleome of 50 healthy individuals and concluded that practically no gene sequence is highly overrepresented

The overrepresentation of non-coding sequences – *Alu* repeats – in the nucleome was repeatedly reported in studies of this type (Stroun et al., 2001; van der Vaart & Pretorius, 2008; Beck et al., 2009). Next generation sequencing (high-througput sequencing) has been used to analyze and compare the nucleome of healthy controls and patients with breast

cfDNA in plasma of cancer patients are even ten times higher than originally sought.

detected with later onset during the course of the illness (Pachl et al., 2005).

the following sections.

**2.2 Active release of nucleic acids by cells** 

source of cell-free nucleic acids in cancer patients.

here in comparison with genomic DNA.

by DNase, RNase, Pronase, and actinomycin D in their experiments.

nucleic acids in human plasma present very heterogeneous material with heterogeneous function – there are for example fragments of genomic DNA, mitochondrial DNA, but also mRNAs and microRNA. The forms in which they circulate are studied also with regard to the development of effective extraction methods for clinical laboratories.
