What is cfDNA? 

cfDNA refers to short DNA fragments that circulate freely outside of cells in the blood and other body fluids like urine or cerebrospinal fluid.

The origins of cfDNA are not yet entirely understood and continue to be discussed in literature.^1,2 Some scientists argue that apoptosis – a process of programmed cell death responsible for eliminating unneeded, old or damaged cells – is the main mechanism of cfDNA release.^1,2,3 It is believed that apoptosis could play a major role in cfDNA release because most cfDNA fragments are about 167 base pairs long, a fragment length that is characteristic for endonucleases cleaving DNA during apoptosis. 

To understand why the cfDNA length of 167 base pairs is typical for apoptosis, it is essential to know how DNA is packed into chromosomes in our cells. Chromosomes consist of DNA strands wrapped around molecules to form structures called nucleosomes. Between nucleosomes are short linker regions, and endonucleases cut the DNA at these sites during apoptosis. As a nucleosome plus its linker region is roughly 167 base pairs in length, it is hypothesized that the majority of cfDNA is released during apoptosis.^1,4,5

Other scientists argue that processes besides apoptosis – such as alternative cell death mechanisms or active secretion – could also influence cfDNA release. They suggest that the high proportion of cfDNA fragments measuring approximately 167 base pairs might result from the enzymatic degradation of longer cfDNA sequences after their release into body fluids.⁶ 

The clearance mechanisms of cfDNA are not yet entirely understood either, but the fragments seem to be eliminated from body fluids through one of the following pathways.^3,6

• Organ uptake via the liver, spleen, kidneys and lymph nodes
• Enzymatic degradation in body fluids
• Cellular internalization by circulating cells or endothelial cells 

cfDNA clearance happens relatively quickly. There is no consensus on the half-life of cfDNA in general but, for fragments of around 167 base pairs, it is estimated to lie between 16 minutes and 2.5 hours.⁶

The level of cfDNA in the body fluid of an individual is determined by a balance between the release and clearance rates. In blood samples of healthy individuals, the cfDNA concentration is commonly 0-100 ng/ml whereas concentrations of >1000 ng/ml have been observed in patients with cancer.³ It was these high concentrations that led to the discovery of cfDNA in 1948 by Mandel and Metais, who examined blood samples from cancer patients.⁷ Nowadays, oncology is one of the main application fields of cfDNA testing, but before exploring this in more detail, we'd like to briefly introduce the various types of cfDNA. 

The term cfDNA refers to freely circulating DNA fragments originating from the cell nuclei of an individual's own cells, but also includes various subtypes.

• ctDNA: circulating tumor DNA is released by tumor cells
• cffDNA: cell-free fetal DNA can be found in the circulation of pregnant women
• dd-cfDNA: donor-derived cfDNA can be detected in recipients of organ transplants
• cf-mtDNA: cell-free mitochondrial DNA is released by mitochondria. While the cfDNA subtypes listed above can only be found in cancer patients, pregnant women or transplant recipients, cf-mtDNA is present in every individual's body fluids

Applications of cfDNA testing

While the existence of cfDNA has been known since 1948, sufficiently sensitive and specific laboratory techniques for its detection and analysis only became available much later, when methods such as real-time PCR (qPCR), digital droplet PCR (ddPCR) and next generation sequencing (NGS) were developed.^7,8,9 cfDNA testing is minimally invasive, accurate and suitable for the early detection of various health issues, and therefore has become a popular screening method in various fields – primarily oncology, gynecology and transplantology.

Oncology

In oncology, cfDNA testing is often referred to as liquid biopsy, and it offers a simple alternative to surgical tumor biopsies. By testing if ctDNA fragments are present in an individual's sample, it is possible to determine whether someone has cancer (early detection),⁷ or whether the disease has returned in a patient previously affected by it (relapse detection).¹⁰ Moreover, ctDNA testing can inform practitioners about the best treatment options, and help to identify mutations or resistance mechanisms that can make patients unresponsive to certain therapies.¹⁰ In some cases, ctDNA tests can even help to predict how likely a successfully treated patient is to experience relapse.¹¹

Gynecology

As cfDNA testing is minimally invasive, it is useful as a prenatal test. cffDNA in the blood of pregnant women can help to determine whether the baby is likely to have aneuploidies such as Down syndrome (trisomy 21), Edwards syndrome (trisomy 18) or Patau syndrome (trisomy 13). cffDNA analysis for genetic anomalies is highly accurate and offers an early, reliable screening option with a sensitivity and specificity typically exceeding 99 percent.¹² However, it is a screening tool and not a definitive diagnostic test. Moreover, the technique can be used to determine a baby's gender, check the mother's Rh blood type, and perform a paternity test by comparing a buccal swab or blood sample from the potential father with the cffDNA fragments in the pregnant woman's blood.^13,14,15

Transplantology

Minimally invasive cfDNA testing is also used to monitor the health of transplanted organs. An increase in dd-cfDNA levels in transplanted patients can indicate cellular damage within the transplanted organ, for example, due to an immune response leading to organ rejection or due to an infection. By monitoring dd-cfDNA levels, graft injury can be detected early before irreversible damage occurs. Moreover, tracking dd-cfDNA levels allows clinicians to tailor immunosuppressive therapy to the needs of the individual patient, reducing the risk of under-medication.^16,17

Other applications

Beyond its roles in oncology, prenatal screening and transplantology, cfDNA has also emerged as a biomarker for a variety of medical conditions in recent years; trauma, sepsis, inflammation, infectious and autoimmune diseases, myocardial infarction and stroke all result in altered cfDNA profiles. Additionally, some chronic disorders like diabetes or sickle cell disease can also lead to higher cfDNA levels.^{3,18,19,20,21,22}

How to collect, purify and analyze cfDNA 

Different body fluids can be used for cfDNA testing depending on the application, however, blood samples are the most common choice. If you're working with blood samples, the first step is to separate out the plasma containing the cfDNA fragments from the blood cells by using a 2-step centrifugation workflow.^23,24 If the plasma is not immediately processed further, it needs to be stored at -80 °C or colder and should undergo no more than 1 freeze-thaw cycle.²⁴ 

After sample collection, you then need to perform a cfDNA purification step. Several purification methods are available, the most frequently used being phenol chloroform, spin column or magnetic bead purification.^25,26 To learn how these methods work, and what their advantages, disadvantages and equipment requirements are, refer to our articles on DNA extraction and purification. If cfDNA is not immediately analyzed after purification, you should store it at -20 °C or less and avoid more than 1 freeze-thaw cycle.²⁴ 

Next, the purified cfDNA sample needs to undergo a quantification step to determine its concentration, yield and purity. Common methods for cfDNA quantification include spectrophotometry and fluorometry. How these methods work, along with their pros and cons, is explained in this article on DNA quantification. 

Subsequent cfDNA analysis comes with 2 main challenges: cfDNA concentrations in a sample are usually low, and fragments of interest – for instance, from the tumor, placenta or donor organ – only make up a small portion of the total amount of cfDNA extracted. Analysis methods that can cope with these challenges are usually PCR or NGS based. The most popular PCR-based methods are qPCR and ddPCR.^27,28,29 PCR-based methods are faster, easier and more affordable than NGS-based methods. However, PCR can only be used for applications with a limited number of cfDNA fragments of interest with known base pair sequences.²⁹ For multiplexing assays with a large number of sequences of interest, NGS-based methods are usually preferred. Moreover, NGS-based methods can be used to discover previously unknown variants, for example, in cancer research.^27,29 

If you want to learn more about the different types of cfDNA analysis methods used in oncology, we recommend reading this article. Illumina also gives a great overview of cfDNA analysis methods used for prenatal testing here.

Conclusion 

cfDNA has revolutionized several areas of medical screening and monitoring, offering a minimally invasive alternative to traditional methods. From early cancer detection and prenatal testing to monitoring transplant health, cfDNA testing provides valuable insights into patient health and disease progression. A better understanding of mechanisms of release and clearance pathways, as well as technological advancements to detect and analyze cfDNA, hold potential to expand the scope of cfDNA applications even further. 

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