**2.1 Necrosis and apoptosis as sources of cell-free DNA**

From the methodological point of view, it is difficult to exactly distinguish between the apoptotic derived fraction of cell-free DNA and the necrotic-derived one in plasma. Veiko et al. (2008) developed a method for in vivo evaluation of cell death in patients with acute and/or chronic heart disease. The main parameters evaluated in the study by Veiko et al. were: total concentrations of cell-free DNA (cfDNA) in the blood (or serum), concentration of serum ribosomal repeat (rDNA), content of rDNA in total cfDNA, but also factors involved in clearance of cfDNA such as nuclease activity and anti-DNA antibodies.

The authors clearly demonstrated significant increase in the concentration of rDNA in the cfDNA pool in patients with acute myocardial infarction. Such an accumulation of rDNA within cfDNA may be caused by the resistance of rDNA to the fragmentation by serum endonucleases, because the nuclease activities in the serum of both acute and chronic coronary disease patients were elevated in comparison to healthy individuals. The titers of anti-DNA antibodies were also higher in the patients group. The anti-DNA antibodies were predominantly bound to cfDNA. It seems that the release of rDNA fragments into the blood may reflect cellular death in the body (Veiko et al., 2008).

Another clinical situation connected with massive cellular death is represented by the multiple-organ dysfunction syndrome (MODS). In MODS, the initial insult damages target organs and leads to tissue necrosis. The necrosis induces a systemic inflammatory response and an alteration of hemodynamics, microcirculation and oxygen metabolism. As a consequence, distant organs may be damaged by necrosis or apoptosis.

The prognostic role of elevated levels of plasma cfDNA in critically ill patients was demonstrated by Wijeratne et al. (2004). Pachl et al. (2005) developed an assay allowing to distinguish between DNA released from apoptotic and necrotic cells. The assay is based on electrophoretic separation of isolated plasma cfDNA fragments on agarose gel. The DNA from apoptotic cells (aDNA) is represented by fragments of typical size resembling the ladder on an electrophoretic gel, but DNA derived from necrotic cells (genomic - gDNA) does not provide this typical pattern when subjected to electrophoretic separation. The authors applied their assay on the samples of plasma cfDNA obtained from intensive care unit patients. They found that the contribution of aDNA to the amount of total plasma DNA in the critically ill patients was 16 fold greater than the contribution of gDNA from necrotic cells. The levels of aDNA were highest on the day of admission and declined thereafter, but the levels of gDNA altered in the opposite manner.

The concentration of apoptotic DNA in samples collected from patients on the day of admission significantly differentiated survivors and non-survivors (Pachl et al., 2005). The study by Pachl confirmed the results of previous research performed on rats (Guan et al., 2002). The most surprising fact in this context is represented by the finding of the highest concentration of cfDNA of apoptotic origin at the time of patient admission to the intensive care unit. The possible explanation for this fact can be found in the induction of apoptosis by the activation of the intrinsic pathway caused by the affection of mitochondria as a consequence of cellular damage caused by primary insult (Crouser et al., 2002).

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

From the methodological point of view, it is difficult to exactly distinguish between the apoptotic derived fraction of cell-free DNA and the necrotic-derived one in plasma. Veiko et al. (2008) developed a method for in vivo evaluation of cell death in patients with acute and/or chronic heart disease. The main parameters evaluated in the study by Veiko et al. were: total concentrations of cell-free DNA (cfDNA) in the blood (or serum), concentration of serum ribosomal repeat (rDNA), content of rDNA in total cfDNA, but also factors involved in clearance of cfDNA such as nuclease activity and anti-DNA

The authors clearly demonstrated significant increase in the concentration of rDNA in the cfDNA pool in patients with acute myocardial infarction. Such an accumulation of rDNA within cfDNA may be caused by the resistance of rDNA to the fragmentation by serum endonucleases, because the nuclease activities in the serum of both acute and chronic coronary disease patients were elevated in comparison to healthy individuals. The titers of anti-DNA antibodies were also higher in the patients group. The anti-DNA antibodies were predominantly bound to cfDNA. It seems that the release of rDNA fragments into the blood

Another clinical situation connected with massive cellular death is represented by the multiple-organ dysfunction syndrome (MODS). In MODS, the initial insult damages target organs and leads to tissue necrosis. The necrosis induces a systemic inflammatory response and an alteration of hemodynamics, microcirculation and oxygen metabolism. As a

The prognostic role of elevated levels of plasma cfDNA in critically ill patients was demonstrated by Wijeratne et al. (2004). Pachl et al. (2005) developed an assay allowing to distinguish between DNA released from apoptotic and necrotic cells. The assay is based on electrophoretic separation of isolated plasma cfDNA fragments on agarose gel. The DNA from apoptotic cells (aDNA) is represented by fragments of typical size resembling the ladder on an electrophoretic gel, but DNA derived from necrotic cells (genomic - gDNA) does not provide this typical pattern when subjected to electrophoretic separation. The authors applied their assay on the samples of plasma cfDNA obtained from intensive care unit patients. They found that the contribution of aDNA to the amount of total plasma DNA in the critically ill patients was 16 fold greater than the contribution of gDNA from necrotic cells. The levels of aDNA were highest on the day of admission and declined thereafter, but

The concentration of apoptotic DNA in samples collected from patients on the day of admission significantly differentiated survivors and non-survivors (Pachl et al., 2005). The study by Pachl confirmed the results of previous research performed on rats (Guan et al., 2002). The most surprising fact in this context is represented by the finding of the highest concentration of cfDNA of apoptotic origin at the time of patient admission to the intensive care unit. The possible explanation for this fact can be found in the induction of apoptosis by the activation of the intrinsic pathway caused by the affection of mitochondria as a

consequence of cellular damage caused by primary insult (Crouser et al., 2002).

the development of effective extraction methods for clinical laboratories.

**2.1 Necrosis and apoptosis as sources of cell-free DNA** 

may reflect cellular death in the body (Veiko et al., 2008).

the levels of gDNA altered in the opposite manner.

consequence, distant organs may be damaged by necrosis or apoptosis.

antibodies.

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

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 the following sections.

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

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

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

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 source of cell-free nucleic acids in cancer patients.

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 here in comparison with genomic DNA.

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

Cell-Free Nucleic Acids as Biomarkers of Biocompatibility in Dialytic Process 203

Recent progress in immunology suggests that nucleic acids are active modulators of the immune system. Both RNA and DNA molecules can be detected by specific receptors - the so called Toll-like receptors, RIG-I-like receptors and NOD-like receptors (Koyama S. et al., 2010). All above mentioned facts contribute to the understanding of cell-free nucleic acids in human plasma as important tools in complex regulatory mechanisms involved in homeostasis and

Cell-free nucleic acids circulate in plasma in different forms as described in previous sections. Their clearance and turnover represent very probably complex mechanism which is known only superficially. The equilibrium between nucleome and genome forms probably the base of genetic homeostasis (Peters & Pretorius, 2011). The individual cells from different organs can not only contribute to the pool of nucleic acids in plasma but also take part in their turnover using them for regulation of intracellular events in variable extent depending on the cell type and physiological state. The live span of the individual molecules and complexes determines the area of their effect in which the group of cells can benefit

Our study (Horinek et al., 2008) brought an indirect evidence for the existence of homeostatic mechanisms between nucleic acids released into circulation and taken up by cells or digested by nucleases. We measured the concentrations of total cfDNA and fetal cfDNA in plasma of healthy pregnant women during pregnancy. Although the concentrations of total cfDNA were kept at the same level through the entire pregnancy, the fractions of cfDNA of fetal origin in the pool of total maternal cfDNA elevated from the first trimestr toward the labor. Lo et al. (1999) reported that fetal DNA is undetectable 2 hours

 Under pathological conditions, the clearance of cfDNA can be affected as demonstrated in the study by Lau et al. (2002) where higher half-life of fetal DNA in preeclamptic (median 114 min) women was observed compared to healthy controls (median 28 min). Impaired clearance of cfDNA can be expected under other pathological conditions, for example in cancer patients. It has been actually documented that the activities of nucleases in cancer

It seems that cfDNA in human circulation is present predominantly in the form of nucleosomes (Holdenrieder et al., 2001). Liver, macrophages and immune system are mainly involved in the clearance of DNA bounded in nucleosomes as has been documented in animal model (Burlingame et al., 1996). Kupfer cells are able to degrade the naked DNA on their surfaces in a saturable process (Gauthier et al., 1996; Kobayashi et al., 2001). Botezatu et al. (2000) demonstrated that kidney is not the main route of elimination of cfDNA from the body, only 0,5 % – 2% of the cfDNA passes from bloodstream through kidney into urine. This group showed the presence of Y- chromosome sequences in the urine of pregnant women carrying male fetuses and detected KRAS mutations in the urine of patients with

The study by Chan et al (2010) confirmed that transrenal excretion of cfDNA is very low. Sequences of the Epstein- Barr virus they are present in plasma of patients suffering with nasopharyngeal carcinoma in high concentrations were used as model system to make the detection of cfDNA in urine more robust. Only minor fraction of DNA representing EBV sequences was found in the urine of examined patients, this fraction represented 0,0028% -

immune response under frequently changing endo- and exogenous conditions.

**2.3 Clearance of circulating nucleic acids in human plasma** 

from horizontal gene transfer (Peters & Pretorius, 2011).

postpartum, with a mean half-life of 16.3 min.

patient are lowered (Cherepanova et al., 2008).

pancreatic and colorectal cancers.

0,00018% of the clearance of creatinine.

cancer. The higher representation of certain repetetive elements in the nucleomes of breast cancer patients was found and the finding was validated with regard to the diagnostic use for staging and outcome prediction (Beck et al., 2010).

During apoptosis DNA and RNA are packed in separate apoptotic bodies which are rapidly ingested by adjacent cells and professional phagocytes. The contribution of apoptotic DNA under physiological conditions to the pool of cfDNA is probably insignificant because DNA from apoptotic bodies is completely digested by DNAseII in lysosomes of phagocyting cells (Peters & Pretorius, 2011).

Cell-free DNAs are mostly in complex with histones in the form of nucleosomes. The association with histones can play the key role in the translocation of cfDNA across cell membrane. The binding of cfDNA with histone H3K27me2b was described, this histone could be crucial for externalization and stabilization of cfDNA in plasma (Beck et al., 2009).

It is known for longer time that regulated release of newly synthetized DNA/RNA – lipoprotein complexes requires energy. The newly synthetized particles contain DNA, RNA DNA-dependent DNA and RNA polymerases, lipoproteins and are referred as virtosomes. Synthesis of DNA for virtosomes probably takes place mainly in the G0 or G1 phase - it seems not to be limited to the mitosis (Peters & Pretorius, 2011).

Not only cell-free DNA can be detected in human plasma, but there is also broad spectrum of mRNA and microRNA. Surprisingly, these RNA molecules due their relative stability can be also isolated from patient plasma and analyzed in clinical laboratories. Their relative stability within the plasma samples is linked to the form in which they are released into circulation. These RNA molecules are enclosed in small vesicles referred as exosomes. Exosomes are 40 - 100 nm membrane bound vesicles of endocytic origin secreted by most cell types *in vivo*. More than 2,300 proteins and 270 microRNAs have been linked with exosomes derived from different biological fluid (Taylor et al., 2011). Exosomes were found *in vivo* in body fluid such as blood, urine, amniotic fluid, malignant ascites, bronchoalveolar lavage fluid, synovial fluid and breast milk *(*Simpson et al., 2008).

Recently it is not exactly known, if exosomes contains DNA or not. According to the research done by García-Olmo et al. (2000) it seems that exosomes can also carry DNA containing complexes.

The advent of genomic and proteomic technologies contributed to the understanding of the molecular composition of exosomes, but their biological functions remain still unclear. It is becoming apparent that they may be involved in the transfer of both mRNA and microRNA to the distant target cells to modulate their expression and behavior. The secretion of exosomes by tumor cells and their implication in the transport and propagation of infectious cargo such as prions and retroviruses provide the evidence that exosomes are important participants of different pathological processes (Simpson at al., 2008).

The group of García-Olmo demonstrated in an animal study that plasma from tumorbearing rats was able to stably transfect cultured cells. The authors proposed a hypothesis that the metastasis might occur via transfection of susceptible cells located in distant target organ with dominant oncogenes circulating in plasma (García- Olmo et al., 1999) and called such a putative phenomenon "genometastasis."

One of the most common alterations of tumor related cfDNA is its hypermethylation. The methylated DNA fragment have been shown to be taken up by HeLa and human umbilical vein endothelial cells twice as efficiently as unmethylated fragments (Skvortsova, Vlassov & Laktionov, 2008).

cancer. The higher representation of certain repetetive elements in the nucleomes of breast cancer patients was found and the finding was validated with regard to the diagnostic use

During apoptosis DNA and RNA are packed in separate apoptotic bodies which are rapidly ingested by adjacent cells and professional phagocytes. The contribution of apoptotic DNA under physiological conditions to the pool of cfDNA is probably insignificant because DNA from apoptotic bodies is completely digested by DNAseII in lysosomes of phagocyting cells

Cell-free DNAs are mostly in complex with histones in the form of nucleosomes. The association with histones can play the key role in the translocation of cfDNA across cell membrane. The binding of cfDNA with histone H3K27me2b was described, this histone could be crucial for externalization and stabilization of cfDNA in plasma (Beck et al., 2009). It is known for longer time that regulated release of newly synthetized DNA/RNA – lipoprotein complexes requires energy. The newly synthetized particles contain DNA, RNA DNA-dependent DNA and RNA polymerases, lipoproteins and are referred as virtosomes. Synthesis of DNA for virtosomes probably takes place mainly in the G0 or G1 phase - it

Not only cell-free DNA can be detected in human plasma, but there is also broad spectrum of mRNA and microRNA. Surprisingly, these RNA molecules due their relative stability can be also isolated from patient plasma and analyzed in clinical laboratories. Their relative stability within the plasma samples is linked to the form in which they are released into circulation. These RNA molecules are enclosed in small vesicles referred as exosomes. Exosomes are 40 - 100 nm membrane bound vesicles of endocytic origin secreted by most cell types *in vivo*. More than 2,300 proteins and 270 microRNAs have been linked with exosomes derived from different biological fluid (Taylor et al., 2011). Exosomes were found *in vivo* in body fluid such as blood, urine, amniotic fluid, malignant ascites, bronchoalveolar

Recently it is not exactly known, if exosomes contains DNA or not. According to the research done by García-Olmo et al. (2000) it seems that exosomes can also carry DNA

The advent of genomic and proteomic technologies contributed to the understanding of the molecular composition of exosomes, but their biological functions remain still unclear. It is becoming apparent that they may be involved in the transfer of both mRNA and microRNA to the distant target cells to modulate their expression and behavior. The secretion of exosomes by tumor cells and their implication in the transport and propagation of infectious cargo such as prions and retroviruses provide the evidence that exosomes are important

The group of García-Olmo demonstrated in an animal study that plasma from tumorbearing rats was able to stably transfect cultured cells. The authors proposed a hypothesis that the metastasis might occur via transfection of susceptible cells located in distant target organ with dominant oncogenes circulating in plasma (García- Olmo et al., 1999) and called

One of the most common alterations of tumor related cfDNA is its hypermethylation. The methylated DNA fragment have been shown to be taken up by HeLa and human umbilical vein endothelial cells twice as efficiently as unmethylated fragments (Skvortsova, Vlassov &

for staging and outcome prediction (Beck et al., 2010).

seems not to be limited to the mitosis (Peters & Pretorius, 2011).

lavage fluid, synovial fluid and breast milk *(*Simpson et al., 2008).

participants of different pathological processes (Simpson at al., 2008).

such a putative phenomenon "genometastasis."

(Peters & Pretorius, 2011).

containing complexes.

Laktionov, 2008).

Recent progress in immunology suggests that nucleic acids are active modulators of the immune system. Both RNA and DNA molecules can be detected by specific receptors - the so called Toll-like receptors, RIG-I-like receptors and NOD-like receptors (Koyama S. et al., 2010). All above mentioned facts contribute to the understanding of cell-free nucleic acids in human plasma as important tools in complex regulatory mechanisms involved in homeostasis and immune response under frequently changing endo- and exogenous conditions.

### **2.3 Clearance of circulating nucleic acids in human plasma**

Cell-free nucleic acids circulate in plasma in different forms as described in previous sections. Their clearance and turnover represent very probably complex mechanism which is known only superficially. The equilibrium between nucleome and genome forms probably the base of genetic homeostasis (Peters & Pretorius, 2011). The individual cells from different organs can not only contribute to the pool of nucleic acids in plasma but also take part in their turnover using them for regulation of intracellular events in variable extent depending on the cell type and physiological state. The live span of the individual molecules and complexes determines the area of their effect in which the group of cells can benefit from horizontal gene transfer (Peters & Pretorius, 2011).

Our study (Horinek et al., 2008) brought an indirect evidence for the existence of homeostatic mechanisms between nucleic acids released into circulation and taken up by cells or digested by nucleases. We measured the concentrations of total cfDNA and fetal cfDNA in plasma of healthy pregnant women during pregnancy. Although the concentrations of total cfDNA were kept at the same level through the entire pregnancy, the fractions of cfDNA of fetal origin in the pool of total maternal cfDNA elevated from the first trimestr toward the labor. Lo et al. (1999) reported that fetal DNA is undetectable 2 hours postpartum, with a mean half-life of 16.3 min.

 Under pathological conditions, the clearance of cfDNA can be affected as demonstrated in the study by Lau et al. (2002) where higher half-life of fetal DNA in preeclamptic (median 114 min) women was observed compared to healthy controls (median 28 min). Impaired clearance of cfDNA can be expected under other pathological conditions, for example in cancer patients. It has been actually documented that the activities of nucleases in cancer patient are lowered (Cherepanova et al., 2008).

It seems that cfDNA in human circulation is present predominantly in the form of nucleosomes (Holdenrieder et al., 2001). Liver, macrophages and immune system are mainly involved in the clearance of DNA bounded in nucleosomes as has been documented in animal model (Burlingame et al., 1996). Kupfer cells are able to degrade the naked DNA on their surfaces in a saturable process (Gauthier et al., 1996; Kobayashi et al., 2001). Botezatu et al. (2000) demonstrated that kidney is not the main route of elimination of cfDNA from the body, only 0,5 % – 2% of the cfDNA passes from bloodstream through kidney into urine. This group showed the presence of Y- chromosome sequences in the urine of pregnant women carrying male fetuses and detected KRAS mutations in the urine of patients with pancreatic and colorectal cancers.

The study by Chan et al (2010) confirmed that transrenal excretion of cfDNA is very low. Sequences of the Epstein- Barr virus they are present in plasma of patients suffering with nasopharyngeal carcinoma in high concentrations were used as model system to make the detection of cfDNA in urine more robust. Only minor fraction of DNA representing EBV sequences was found in the urine of examined patients, this fraction represented 0,0028% - 0,00018% of the clearance of creatinine.

Cell-Free Nucleic Acids as Biomarkers of Biocompatibility in Dialytic Process 205

The elevated plasma levels of cfDNA were described to be an excellent marker for graft

Due to well documented increase in cfDNA levels during inflammatory process (Fatouros et al., 2006) it is necessary to take in account the actual health status of control subjects and patients with complex clinical diagnosis. The elevated levels of cfDNA interpreted without the clinical context can not indicate pathological situations. The actual physical activity of examined subjects may also contribute to the elevations of total cfDNA in plasma and serum

The mechanism of immune response to increased amount of nucleosomes in circulation was examined in systemic lupus erythematosus (SLE). In this rheumatic autoimmune disease of unknown etiology numerous autoantibodies against circulating nucleosomes are produced.

Polymorphonuclear cells, dendritic cells and monocytes strongly bind nucleosomes on their surfaces, the binding of nuckeosomes to lymphocytes is weaker. Circulating nucleosomes are endocyted in polymorphonuclears, dendritic cells and monocytes. It has been shown in polymorphonuclear cells that the nucleosomes are not translocated into nucleus. Polymorphonuclear cells represent the link between innate and adaptive immunity therefore the perturbations in the regulation of their function could lead to the development

The role of the receptor TLR9 in the binding of circulating DNA and development of SLE is not fully understood. It is known that the TLR9 plays an important role in recognition of dsDNA in extracellular space and in its internalization (Lindau et al., 2011) but the internalized DNA molecules may stimulate the production of IFN I in dendritic cells also in

After a hemodialytic procedure, the concentrations of cfDNA in patients are dramatically elevated as has been demonstrated in numerous studies (Atamaniuk et al., 2006; Garcia-Moreira et al., 2006; Korabecna et al., 2008; Horinek et al. 2011). Garcia-Moreira et al. reported quite rapid normalization to normal values during 30 min after the end of the procedure. The problems associated with the well documented increase of cfDNA during

The interpretations of each cfDNA based study must be performed carefully with the respect to clinical data concerning the presence of autoimmune disease, malignancy, renal transplantation or acute infection in examined subjects. All mentioned conditions must be

For total plasma concentrations of different types of circulating ribonucleic acids, similar

During hemodialysis, the interaction of dialyzer membrane with patient´s blood leads to the activation of alternate pathway of complement. The biologically active complement component C5A is generated and activates the aggregation of neutrophils and their adherence to the endothelial surfaces. Neutrophils harvested during hemodialysis exhibit altered oxidative response, chemotaxis, aggregation, and adherence (Lewis & Van Epps,

rejection in patients after renal transplantation (García-Moreira et al., 2010).

The production of anti-dsDNA autobodies serves as disease marker for SLE.

**3.4 Inflammatory response** 

(Atamaniuk et al., 2004; Fatouros et al., 2006).

of autoimmunity (Lindau et al., 2011).

used as exclusion criteria.

clinical data are urgently needed.

the TLR9–independent pathway (Martin & Elkon, 2006).

the dialytic procedure will be discussed in the following sections.

**4. Cell-free nucleic acids as biomarkers in dialytic process** 

The finding that the cfDNA is stable for at least 4 hours in urine (Su et al., 2005) is important for management of clinical samples and translation medicine based on examination of cfDNA quantity and quality. From the results of our pilot study (Korabecna et al., 2011) it would appear that plasma DNAse II (which is not inhibited in blood samples stabilized by EDTA) makes only minor contribution to the degradation of circulating DNA *in vitro.* If heparin is used for sample stabilization nucleases are not inhibited and cfDNA is degraded very quickly.
