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[Circulating free DNA (cfDNA), also known as cell-free DNA''', are degraded DNA fragments released to the blood plasma. cfDNA can be used to describe various forms of DNA freely circulating the bloodstream, including circulating tumor DNA (ctDNA) and cell-free fetal DNA (cffDNA). Elevated levels of cfDNA are observed in cancer, especially in advanced disease. There is evidence that cfDNA becomes increasingly frequent in circulation with the onset of age.]''' cfDNA has been shown to be a useful biomarker for a multitude of ailments other than cancer and fetal medicine. This includes but is not limited to trauma, sepsis, aseptic inflammation, myocardial infarction, stroke, transplantation, diabetes, and sickle cell disease.

History
Circulated cell-free DNA was first discovered by Mandel and Metais in 1948. It was later discovered that the level of cfDNA is significantly increased in the plasma of diseased patients. This discovery was first made in Lupus patients and later it was determined that the levels of cfDNA are elevated in over half of cancer patients. This increase in cfDNA in cancer patients has been shown to be due to circulating tumor cells (CTC) traveling in the peripheral blood. The ability to extract circulating tumor DNA (ctDNA) from the human plasma has led to huge advancements in noninvasive cancer detection. Most notably, it has led to what is now known as Liquid Biopsy. In short, liquid biopsy is using biomarkers and cancer cells in the blood as a means of diagnosing cancer type and stage. This type of biopsy is noninvasive and allows for the routine clinical screening that is important in determining cancer relapse after initial treatment.

Collection and purification
cfDNA purification is prone to contamination due to ruptured blood cells during the purification process. Because of this, different purification methods can lead to significantly different cfDNA extraction yields. At the moment, typical purification methods involve collection of blood via venipuncture, centrifugation to pellet the cells, and extraction of cfDNA from the plasma. The specific method for extraction of cfDNA from the plasma depends on the protocol desired.

PCR
In general, the detection of specific DNA sequences in cfDNA can be done by two means; sequence specific detection (PCR based) and general genomic analysis of all cfDNA present in the blood (DNA sequencing). The presence of cfDNA containing DNA from tumor cells was originally characterized using PCR amplification of mutated genes from extracted cfDNA. PCR based analysis of cfDNA typically rely on the analytical nature of qPCR and digital PCR. Both of these techniques can detect down to a single targeted molecule present in a sample. For this reason the PCR based method of detection is still very prominent tool in cfDNA detection. This method has the limitation of not being able to detect larger structural variant present in ctDNA and for this reason massively parallel next generation sequencing is also used to determine ctDNA content in cfDNA

Massively Parallel Sequencing
Massively parallel sequencing (MPS) has allowed the deep sequencing of cfDNA. this deep sequencing is required to detect ctDNA present in low concentrations in the plasma. Two main sequencing techniques are typically used for analysis of cfDNA; PCR amplicon sequencing and hybrid capture sequencing.

Cancer
The majority of cfDNA research is focused on DNA originating from circulating tumor cells (ctDNA). In short, the DNA from circulating tumor cells gets released by means that are not fully understood.

Trauma
Elevated cfDNA has been detected with acute blunt trauma and burn victimizes. In both of these cases cfDNA concentration in the plasma were correlated to the severity of the injury, as well as outcome of the patient.

Sepsis
It has been shown that an increase cfDNA in the plasma of ICU patients is an indicator of the onset of sepsis. Due to the severity of sepsis in ICU patients, further testing in order to determine the scope of cfDNA efficacy as a biomarker for septic risk is likely.

Myocardial Infraction
Patients showing signs of myocardial infraction have been shown to have elevated cfDNA levels. This elevation correlates to patient outcome in terms of additional cardiac issues and even mortality within two years.

Transplant Graft Rejection
Foreign cfDNA has been shown to be present in the plasma of solid organ transplant patients. This cfDNA is derived from the grafted organ and is termed GcfDNA. GcfDNA values spike initially after a transplant procedure (>5%) and typically drop down (<0.5%) within one week. If the host body rejects the grafted organ the GcfDNA concentration in the blood will rise to a level greater than 5-fold higher than those without complications. This increase in GcfDNA can be detected prior to any other clinical or biochemical signs of complication.