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• Cell-free DNA (cfDNA), a fragmented DNA present in the human bloodstream in a cell-free state, predominantly arises from processes of cellular necrosis and apoptosis. In particular cases, such as cancer patients, pregnant individuals, and those who have undergone organ transplantation, a distinct subset of cfDNA derived from "allogeneic" cells can be harnessed as a biomarker for genetic analysis.
• The main roles of cfDNA include: in prenatal testing, it allows for the screening of genetic diseases by detecting fetal DNA in the blood of pregnant women; in oncology, it enables the discovery of tumor-related mutations and genetic alterations by analyzing cfDNA derived from tumors, facilitating the study of tumor DNA in a non-invasive manner; in organ transplantation, it is used to monitor transplant rejection and organ health; and in cardiovascular diseases, such as myocardial infarction, the levels of cfDNA can serve as an indicator of myocardial damage.
• The significance of cfDNA applications lies in its potential as a non-invasive biomarker, making liquid biopsy techniques possible for early disease diagnosis, prognosis assessment, and therapeutic monitoring. Particularly in cancer treatment, changes in cfDNA levels are closely related to tumor burden, allowing for the monitoring of treatment response and the development of resistance. Moreover, the analysis of cfDNA can reveal tumor heterogeneity and identify multiple concurrent resistance changes in different tumor metastases, which is crucial for guiding personalized treatment and subsequent therapeutic choices.
• Technological advancements in detecting and analyzing cfDNA are swiftly progressing, with promising applications in the fields of oncology, prenatal care, and transplant medicine. As these technologies continue to evolve, cfDNA testing is anticipated to increase the precision of disease detection, refine treatment selection, and deliver more tailored healthcare solutions to patients.

What Is Cell-Free DNA (cfDNA)?

• cfDNA refers to nucleic acid fragments in the bloodstream, originating from both healthy and disease-affected cells. It is released into the circulatory system through various biological processes, including necrosis (abnormal cell death), apoptosis (programmed cell death), and direct secretion.
• cfDNA is typically found in fragments ranging from 120–220 base pairs, with a predominant fragment size of 170 base pairs, which corresponds to the length of DNA that can wrap around a nucleosome plus an additional stretch of DNA linking two nucleosome cores. The half-life of cfDNA in blood ranges from 15 minutes to 2.5 hours.

Where Does cfDNA Come From?

• cfDNA originates from various biological processes and sources within the human body. Under normal physiological conditions, cfDNA primarily comes from the degradation of genomic DNA of senescent and apoptotic cells。 When the body is affected by diseases such as cancer, trauma, organ transplant rejection, organ failure, and severe infections, abnormal necrotic cells can release a large amount of DNA into the bloodstream. Additionally, cfDNA can also be derived from the fetus-maternal unit during pregnancy, with the fetus's DNA finding its way into the maternal circulation。 Furthermore, in the context of organ transplantation, cfDNA can be released into the blood due to cell death associated with immune rejection. The release of cfDNA into the bloodstream can also occur through active secretion by cells, although the exact mechanisms are not fully understood.
• In summary, cfDNA is a complex biomarker that can be traced back to multiple sources, including cellular apoptosis, necrosis, and active secretion, making it a valuable tool in liquid biopsy and non-invasive diagnostics.

Sources of cfDNA in blood samples. (Florent Mouliere al.,2022)

What Does cfDNA Test For?

cfDNA testing is a non-invasive diagnostic technique that targets and analyzes circulating DNA fragments in a patient's bodily fluids, most commonly in the blood. This testing serves several medical applications, including:

Prenatal Screening and Diagnosis

cfDNA tests, such as non-invasive prenatal testing (NIPT), analyze fetal DNA derived from maternal blood to screen for chromosomal abnormalities like Down syndrome (trisomy 21), Edwards syndrome (trisomy 18), and Patau syndrome (trisomy 13). These tests can be performed as early as the 10th week of pregnancy and provide a high rate of accuracy with minimal risk to the mother and fetus.

Oncology

In cancer management, cfDNA testing is used for early diagnosis, therapeutic evaluation, disease progression monitoring, and disease prognosis determination. Circulating tumor DNA (ctDNA), a subset of cfDNA derived from tumor cells, is particularly valuable for monitoring cancer relapse or metastasis and guiding treatment decisions.

Future direction for early detection of cancers based on cfDNA tests. (Qiang Gao al.,2022)

Organ Transplantation

Donor-derived cfDNA (dd-cfDNA) can be detected in the recipient's blood to assess allograft injury or rejection, allowing for early intervention and preventing severe outcomes.

Infectious Diseases

cfDNA can also be derived from microorganisms causing infections in humans, such as viruses, aiding in the diagnosis of infectious diseases.

Cardiovascular Disease

cfDNA has been implicated in the diagnosis of acute coronary syndrome and in predicting the severity of coronary artery lesions, with mitochondrial cfDNA (cf-mtDNA) levels elevated in cardiovascular disease patients.

In summary, cfDNA testing is a versatile tool in medicine, offering non-invasive insights into genetic conditions, cancer management, transplant outcomes, infectious diseases, and cardiovascular health.

References

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4. Buenrostro, Jason D., et al. Transposition of native chromatin for fast and sensitive epigenomic profiling of open chromatin, DNA-binding proteins and nucleosome position. Nature methods 10.12 (2013): 1213-1218.