A blood test tube with the label ‘cfDNA Screening–Test’, held in a hand wearing blue gloves.
Cell-free DNA is found circulating in the peripheral blood and can be tested for disease-related genetic abnormalities.
© iStock, Md Babul Hosen

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What Is Cell-Free DNA and Where Does it Come From?

Cell-free DNA (cfDNA) refers to DNA fragments that circulate within the peripheral blood.1 Synonymous with circulating cell-free DNA (ccfDNA), these fragments are small, with an average size of around 166 base pairs depending on their origin.2 Although not the only source, cfDNA is primarily thought to be released into the bloodstream as a result of cell death,3 which suggests that the quantification and genetic analysis of cfDNA provides information about cell death in the body.

The concentration of cfDNA in the blood plasma of healthy individuals is typically between one and 10 ng/mL.4 However, in diseased individuals, the concentration can be significantly higher. For example, in colorectal cancer (CRC) patients, the concentration can range from 10 to 100 ng/mL.4 The size of cfDNA segments also varies between healthy and diseased individuals, with CRC patients having significantly smaller fragments of cfDNA in their blood compared to the former.4 cfDNA derived from tumors is referred to as circulating tumor DNA (ctDNA).5 

During pregnancy, between 10 and 20 percent of the cfDNA in maternal peripheral blood is derived from the apoptotic trophoblast cells of the placenta.6 Although a misnomer, this is frequently referred to as fetal cfDNA, and is detectable from around seven weeks of gestation.6 cfDNA can also be derived from microorganisms, that cause infections in humans, such as viruses.7 

What Is Cell-Free DNA Testing and How Does It Work?

Scientists can use cfDNA to conduct minimally invasive screens for disease-related genetic abnormalities in a process known as cfDNA testing. Placental cfDNA can reveal genetic abnormalities, such as aneuploidy, in the fetus during pregnancy.

cfDNA Down syndrome test

 One of the most common and well-established applications of cfDNA testing is in screening pregnancies for trisomy 21, commonly called Down syndrome, in which there is an extra copy of chromosome 21.8 To perform a fetal cfDNA Down syndrome test, clinicians take samples of maternal peripheral blood, separate the plasma component, and isolate the cfDNA from the plasma.8 They can then assess the relative abundance of chromosome 21 within the cfDNA sample. 

The most common method, widely considered the gold standard in prenatal cfDNA Down syndrome testing, is chromosome microarray analysis (CMA).9 CMA can analyze thousands of cfDNA fragments simultaneously, typically using a microchip containing probes specific to target regions of chromosomes.9 This approach can also be used to screen pregnancies for other aneuploidies, such as trisomy 18, trisomy 13, and monosomy X. 

Graphic showing the different locations where cell-free DNA (cfDNA) found in the blood can originate from. Lungs are an example of a source of cfDNA from donated organs, a liver with a tumor is an example of a source of cfDNA from a tumor, and a placenta housing a fetus is an example of fetal cfDNA. A test tube holding blood with cfDNA is then depicted being analysis via sequencing or microarray.
cfDNA from various sources can be identified in blood samples via sequencing or microarray analysis.
The Scientist

Additional Applications of cfDNA Testing 

Assessment of allograft injury using cfDNA testing

Scientists also use cfDNA testing to determine if organ transplants have been successful. Donor-derived cfDNA (dd-cfDNA) can be detected circulating in the organ recipient’s peripheral blood if there has been allograft injury or rejection.1 In these cases, early detection can prevent severe adverse outcomes in high-risk patients.10 

To eliminate the need for more invasive tissue biopsy approaches, clinicians have adapted sequencing-based cfDNA testing to diagnose organ rejection in heart transplant recipients.10 They can also use cfDNA to test lung transplant recipients for infections, such as viral infections, which are a common posttransplant complication.7 Compared to traditional tests, the use of dd-cfDNA allows scientists to test transplant recipients for allograft injury, rejection, and infection more frequently in a quantitative and non-invasive way.11 

cfDNA testing in oncology 

The presence of cfDNA in peripheral blood means that scientists can also non-invasively diagnose and monitor cancer through liquid biopsy.3 Scientists can use quantitative methods to determine disease burden and monitor this over time, including in response to treatment.3 Once treatment has concluded, they can also detect residual disease or identify mutations in the cancer DNA that allow it to become resistant to treatment.3 

In recent years, oncologists have also applied the analysis of DNA methylation to cfDNA. Because aberrant methylation patterns are a hallmark of cancer12 and cfDNA bears the same epigenetic marks as its tissue of origin, scientists can determine which organ or tissue the cfDNA is derived from.4 This is particularly relevant in metastatic tumor patients who have a cancer of unknown primary (CUP) because diagnosing the primary cancer can help determine the most effective treatment regimen.1 

Cardiovascular disease diagnosis with cfDNA

cfDNA has also been implicated in cardiovascular disease (CVD). Scientists developed cfDNA-based biomarkers for diagnosing acute coronary syndrome, as well as predicting the severity of coronary artery lesions.13 Additionally, researchers revealed that levels of cfDNA derived specifically from the mitochondria (cf-mtDNA) are elevated in CVD patients and those with associated comorbidities, such as hypercholesterolemia, arterial hypertension, and diabetes mellitus.14 

  1. Moss J, et al. Comprehensive human cell-type methylation atlas reveals origins of circulating cell-free DNA in health and disease. Nat Commun. 2018;9(1):5068. 
  2. Shi J, et al. Size profile of cell-free DNA: A beacon guiding the practice and innovation of clinical testing. Theranostics. 2020;10(11):4737-4748. 
  3. Wan JCM, et al. Liquid biopsies come of age: towards implementation of circulating tumour DNA. Nat Rev Cancer. 2017;17(4):223-238. 
  4. Mouliere F, et al. Multi-marker analysis of circulating cell-free DNA toward personalized medicine for colorectal cancer. Mol Oncol. 2014;8(5):927-941. 
  5. Cisneros-Villanueva M, et al. Cell-free DNA analysis in current cancer clinical trials: a review. Br J Cancer. 2022;126(3):391-400. 
  6. Grace MR, et al. Cell-free DNA screening: complexities and challenges of clinical implementation. Obstet Gynecol Surv. 2016;71(8):477-487. 
  7. De Vlaminck I, et al. Noninvasive monitoring of infection and rejection after lung transplantation. Proc Natl Acad Sci. 2015;112(43):13336-13341. 
  8. Norton ME, et al. Cell-free DNA analysis for noninvasive examination of trisomy. N Engl J Med. 2015;372(17):1589-1597. 
  9. Liu X, et al. Potentials and challenges of chromosomal microarray analysis in prenatal diagnosis. Front Genet. 2022;13. 
  10. De Vlaminck I, et al. Circulating cell-free DNA enables noninvasive diagnosis of heart transplant rejection. Sci Transl Med. 2014;6(241). 
  11. Bloom RD, et al. Cell-free DNA and active rejection in kidney allografts. J Am Soc Nephrol. 2017;28(7). 
  12. Luo H, et al. Liquid biopsy of methylation biomarkers in cell-free DNA. Trends Mol Med. 2021;27(5):482-500. 
  13. Cui M, et al. Cell-free circulating DNA: a new biomarker for the acute coronary syndrome. Cardiology. 2013;124(2):76-84. 
  14. Nie S, et al. Pro-inflammatory role of cell-free mitochondrial DNA in cardiovascular diseases. IUBMB Life. 2020;72(9):1879-1890. 
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