**3.1 Cancer patients**

Numerous studies provided evidence for the value of cfDNA quantification in cancer diagnostics and the therapy monitoring. Healthy individuals have in most cases lower cfDNA concentrations than cancer patients.

Today, the study of cell-free DNAs and RNAs in cancer patients is the most fertile area of free-nucleic aids research (8180 articles for key words "cell free nucleic acids and cancer" on PubMed till April 2011)

The qualitative alterations reported in cfDNA in cancer patients include mutations of oncogenes and tumor suppressor genes, microsatellite alterations, promoter hypermethylation of different cancer related genes and mitochondrial variation. In plasma of cancer patients, the existence of tumor specific RNAs detectable with recent methods was reported.

Detection of circulating RNA in tumor biology has the advantage over DNA-based approaches as transcripts can be both tumor and tissue-specific and therefore the origin and location of the tumor can be followed. For comprehensive review concerning different aspects of the use of circulating nucleic acids as biomarkers in cancer see Vlassov, Laktionov & Rykova (2010).

#### **3.2 Pregnancy related disorders**

Qualitative analysis of fetal cfDNA in maternal circulation has been used for fetal gender determination using detection of Y- chromosomal specific sequences, for the detection of single gene mutations in fetuses with achondroplasia, myotonic dystrophy, congenital adrenal hyperplasia, thalassemia, cystic fibrosis and Huntington disease. Recently, the fetal cfDNA is routinely examined to detect the RhD positive fetuses in RhD negative mothers.

Quantitative aberrations in the concentrations of total levels of cfDNA were found to be associated with different pregnancy related disorders such as preeclampsia, various placental pathologies, preterm labor, intrauterine growth retardation, polyhydramnion, and ectopic pregnancy. Some studies described the elevated values of total cfDNA in maternal circulation in pregnancies with aneuploid fetuses, but such results were not satisfactory confirmed by others (for review Hořínek et al., 2008).

#### **3.3 Other diseases**

Elevated plasma levels of cfDNA have been reported to correlate with the severity of injury in patients with polytrauma (Lo et al., 2000; Rainer & Lam, 2006) and with the severity of stroke (Rainer & Lam, 2006). Another study proposed to use the concentrations of cfDNA in plasma for outcome prognosis in acute myocardial infarction (Antonatos et al., 2006). Strong correlations were also found between increase of cfDNA in plasma and the outcome of intensive care unit patients (Rhodes et al., 2006; Saukkonen et al., 2007).

The elevated plasma levels of cfDNA were described to be an excellent marker for graft rejection in patients after renal transplantation (García-Moreira et al., 2010).

#### **3.4 Inflammatory response**

204 Technical Problems in Patients on Hemodialysis

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.

**3. Concentrations of cell-free nucleic acids in human plasma in health and** 

Numerous studies provided evidence for the value of cfDNA quantification in cancer diagnostics and the therapy monitoring. Healthy individuals have in most cases lower

Today, the study of cell-free DNAs and RNAs in cancer patients is the most fertile area of free-nucleic aids research (8180 articles for key words "cell free nucleic acids and cancer" on

The qualitative alterations reported in cfDNA in cancer patients include mutations of oncogenes and tumor suppressor genes, microsatellite alterations, promoter hypermethylation of different cancer related genes and mitochondrial variation. In plasma of cancer patients, the

Detection of circulating RNA in tumor biology has the advantage over DNA-based approaches as transcripts can be both tumor and tissue-specific and therefore the origin and location of the tumor can be followed. For comprehensive review concerning different aspects of the use of circulating nucleic acids as biomarkers in cancer see Vlassov, Laktionov

Qualitative analysis of fetal cfDNA in maternal circulation has been used for fetal gender determination using detection of Y- chromosomal specific sequences, for the detection of single gene mutations in fetuses with achondroplasia, myotonic dystrophy, congenital adrenal hyperplasia, thalassemia, cystic fibrosis and Huntington disease. Recently, the fetal cfDNA is routinely examined to detect the RhD positive fetuses in RhD negative mothers. Quantitative aberrations in the concentrations of total levels of cfDNA were found to be associated with different pregnancy related disorders such as preeclampsia, various placental pathologies, preterm labor, intrauterine growth retardation, polyhydramnion, and ectopic pregnancy. Some studies described the elevated values of total cfDNA in maternal circulation in pregnancies with aneuploid fetuses, but such results were not satisfactory

Elevated plasma levels of cfDNA have been reported to correlate with the severity of injury in patients with polytrauma (Lo et al., 2000; Rainer & Lam, 2006) and with the severity of stroke (Rainer & Lam, 2006). Another study proposed to use the concentrations of cfDNA in plasma for outcome prognosis in acute myocardial infarction (Antonatos et al., 2006). Strong correlations were also found between increase of cfDNA in plasma and the outcome of

intensive care unit patients (Rhodes et al., 2006; Saukkonen et al., 2007).

existence of tumor specific RNAs detectable with recent methods was reported.

**disease** 

**3.1 Cancer patients** 

PubMed till April 2011)

& Rykova (2010).

**3.3 Other diseases** 

**3.2 Pregnancy related disorders** 

confirmed by others (for review Hořínek et al., 2008).

cfDNA concentrations than cancer patients.

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

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

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 of autoimmunity (Lindau et al., 2011).

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

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 dialytic procedure will be discussed in the following sections.

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 used as exclusion criteria.

For total plasma concentrations of different types of circulating ribonucleic acids, similar clinical data are urgently needed.
