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Quantification of contaminating cellular DNA in plasma samples by chromatography on short monolithic columns and real-time PCR

Although the presence of extracellular, circulating DNA in human plasma was firstly reported in 1948, the standard method for its precise quantification is still lacking. It is estimated that in healthy individuals the concentration of cell-free DNA is approx. 2-20 ng/ml of plasma. Its primary source are apoptotic cells, and therefore only small fragments (smaller than 220 nucleotides) are predominantly detected. Many diagnostic applications based on circulating DNA have been developed because its level is elevated in patients with various types of cancer (especially metastatic), as well as in acute disorders such as trauma and stroke. Fetus-derived DNA is also found in maternal plasma and it is used for non-invasive prenatal diagnosis and monitoring of pregnancy-associated disorders. In transfusion medicine, the cell-free DNA is not considered to be a safety issue. Still, during blood storage and processing the plasma is contaminated with the DNA released form damaged nucleated blood cells, and therefore DNA fragments larger than 220 nucleotides can also be detected. Concerns regarding DNA oncogenicity in such preparations arise due to the potential introduction of a dominant oncogene, possibly present in the donor leukocytes. Another potential safety concern associated with such larger DNA fragments is the risk that the genome of an infectious virus is present in the DNA, either integrated or as an extrachromosomal element. Our study was designed to quantify the level of contaminating (non-apoptotic) cellular DNA in human plasma samples that were previously stored at -20 °C. In order to purify the DNA, chromatography on CIM DEAE column was performed. Prior to application, plasma samples were thawed, centrifuged at 1200 x g to remove cellular debris and filtered through filter of 0.45 &micro ; m pore size. The volume of applied plasma samples ranged from 1 ml to 10 ml. After elution, DNA was precipitated with isopropanol, and its concentration was determined with sybr-green chemistry-based real-time PCR using primers specific for the GAPDH gene. The PCR amplicon size was 257 nucleotides, and therefore just the contaminating cellular DNA was detected. In the samples in which we were able to determine the concentration of contaminating DNA, it was in a range of 0.06-23 ng/ml of plasma.

DNA; plasma; DNA quantification; real-time PCR

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