cfDNA (cell-free DNA) & cfDNA Extraction: Basics & Development

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What is Cell-free DNA (cfDNA)?

Cell-free DNA (cfDNA) are degraded DNA molecules/fragments that are released into the bloodstream by cells. These fragments, which can vary in length between 50 and 300 base pairs, are no longer confined or encapsulated inside a cell but circulating freely in the bloodstream. Hence it’s called cell-free DNA.

cfDNA in plasma

Figure 1. cfDNA in plasma illustrated in sizes relative to other elements in plasma. cfDNA fragments are released into plasma through cell necrosis. Their sizes are between 17~200nm. The other elements are exosome 40~160nm, miRNA about 15nm, Circulating tumor cells (CTC) about 30um, white blood cell (WBC) about 9um, red blood cell (RBC) 7~8um, platelets 2~3um, and MagVigen magnetic nanoparticles that can be tailor made from 5nm to 1um.

Name Size
CTC (shown in necrosis) 8~30um CTC (in necrosis)
White blood cell 9um white blood cellwhite blood cell
Red blood cell 7~8um red blood cellred blood cell
Platelet 2~3um platelet
Exosome 40~160nm Exosome
cfDNA 17~200nm cfDNA
mRNA 15nm mRNA
MagVigen engineered nanoparticles tailorable size 5nm~1um MagVigen

Table 1. Size comparison of elements in blood plasma vs. Nvigen MagVigen engineered nanoparticles.

cfDNA circulates in both healthy and pathological conditions, i.e. cfDNA comes from both cancerous and noncancerous cells. In addition to presenting in bloodstream, i.e. in plasma and serum, it also presents in cerebral spinal fluid and saliva. Scientists believe cfDNA is not just passive waste products of cell death but have a physiological and pathological function in inflammation and autoimmunity.

As researchers Diana Han etc. summarized, nucleases act both in cfDNA generation via cell death pathways and in cfDNA clearance at the plasma level. Nuclease-associated cfDNA markers, such as its size, genome coverage, end positions, and end motifs, differ in different tissues and pathologies and can be harnessed for molecular diagnostics.

Applications of cfDNA

cfDNA has been successfully exploited as noninvasive biomarkers for biopsies. For example:

  • In pregnancy, the presence of fetal-specific cfDNA has led to the development of noninvasive prenatal testing (NIPT) for fetal sex, genetic inheritance, and chromosomal abnormalities, such as Down syndrome or trisomy 18.
  • Tumor-specific cfDNA can be detected in the plasma of individuals with cancer (case study).
  • Donor-specific cfDNA can be used to monitor organ transplants in the recipient
  • Viral- or bacterial-specific cfDNA can be used to monitor infection-related pathologies or detect causative agents in sepsis.

What is Circulating tumor cells (CTC)?

CTCs are tumor cells shed into the bloodstream from primary and metastatic tumor deposits. CTCs occur at very low concentrations of one tumor cell in the background of millions of blood cells. What mechanisms causing CTCs to break off from the tumor are elusive, thus it is unclear whether CTCs represent the entire makeup of cancer cells in the tumor or only a subpopulation. Nonetheless, analysis of CTCs in peripheral blood of cancer patients holds great promise for the early detection of invasive cancer and the management of advanced disease.

Circulating tumor cells in the blood vessel
Figure 2. Cancerous tumor, metastases and how are circulating tumor cells (CTC) are released into the blood vessel. WBC, RBC and platelets are illustrated in relative sizes.

What is ctDNA?

When cfDNA is released by cancer cells, it is specifically called circulating tumor DNA (ctDNA). ctDNA carries genomic and epigenomic alterations concordant to the tumor mutational spectrum. It’s a specific biomarker that can provide personalized information to detect residual disease or monitor tumor progression during therapy.

Why is CTC and ctDNA so important?

Normally, the cell-free DNA fragments are cleaned up by macrophages but in cases of cancer it is believed that the overproduction of cells leaves more ctDNA behind. For example, studies have reported that the concentration of cfDNA in serum or plasma of hepatocellular carcinoma patients to be 3-4 times higher than patients with chronic hepatitis and nearly 20 times that of healthy controls. ctDNA has the potential to accurately genotype the tumor and identify personalized genetic and epigenetic alterations of the entire tumor. Studies have suggested that the variability of ctDNA levels in cancer patients can reflect tumor burden, stage, vascularity, cellular turnover, and response to therapy. Thus ctDNA can guide potential therapeutic targets, while also being able to monitor minimal residual disease, reducing the need for harmful adjuvant chemotherapy and allowing more rapid detection of relapse.

Additionally, compared with tradtional tissue biopsy, which is costly, sometimes inaccessible, and associated with potential morbidity, the analysis of blood samples for circulating tumor cells (CTC) or cell-free circulating tumor DNA (ctDNA), which is called “liquid biopsy”, provides rapid, cost-effective, and noninvasive surrogates.

The origin of cfDNA and ctDNA

The mechanisms of cfDNA release from cells, and ctDNA from tumor cells, fall into two categories: cell death and active cell secretion. Cell dealth includes processes such as apoptosis, necrosis and NETosis.

cfDNA origins

Figure 3. Where does cfDNA come from? Apoptosis, Necrosis, NETosis and secretion.

  1. Apoptosis

  2. Apoptosis is the process of programmed cell death. This includes:

    • to eliminate unwanted cells during early development in a life cycle.
    • For example, the cells between the fingers of a developing embryo.

    • to rid the body of cells that are old and worn out or have been damaged beyond repair.
    • For example, blood cells will go into apoptosis after 120 days. It makes way for nice, new young cells.

    The average adult human loses between 50 and 70 billion cells each day due to apoptosis. For an average human child between eight and fourteen years old, approximately twenty to thirty billion cells die per day. Since most cfDNA fragments are measured between 180 and 200 base pairs (bp), which falls within the size range of a mononucleosome, apoptosis has been postulated for many years to be the primary source of circulating cfDNA.

    Apoptosis plays an important role in preventing cancer. If for some reason apoptosis can’t happen, it can lead to uncontrolled cell division and the subsequent development of a tumor. On the other hand, too much apoptosis, i.e. when cells die when they’re not supposed to die, causes a number of so-called neurodegenerative diseases, e.g. Parkinson’s disease, Huntington’s disease, Alzheimer’s disease and Lou Gehrig’s disease, with which, in a certain part of the lower part of the brain, the cells get messages from some place, most of which we don’t understand, to tell them to die.

    When apoptosis happens, there’s a whole scripted choreography of pathways and proteins within a cell that get activated in a concerted action to kill the cell without making a mess. The prcoess includes chromatin condensation, membrane blebbing, nucleus collapse. There are at least two stages of DNA fragmentation in apoptosis: an initial cleavage of DNA into high molecular weight 50-300 kb fragments, followed by cleavage of DNA into oligonucleosomally sized DNA that are multiples of 180 bp. Finally, fragmented DNA are packaged along with other degraded cell components into apoptotic bodies (1-5 μm in diameter) and microparticles (0.1-1 μm in diameter) and released.

  3. Necrosis

  4. The second origin is necrosis, which is a spontaneous and unregulated form of cell death in an organ or tissue. Necrosis can have various causes, which can lead to different types of damage to tissues:

    • Hypoxia: Caused by ischemia (insufficient blood flow to an organ), shock, or respiratory failure.
    • Physical agents: External injuries like trauma, extreme temperatures (for example, frostbite), radiation exposure, or electric shock.
    • Chemical agents: Caused by poison, drug toxicities, or recreational drugs.
    • Biological agents: Bacteria, viruses, or fungi.
    • Immunologic reactions: Autoimmune responses (where the immune system attacks healthy tissues).

    Necrosis can be treated, with the dead tissue removed, but the affected tissue can not be healed or returned to good health. It generates larger DNA fragments due to an incomplete and random digestion of genomic DNA.

  5. NETosis

  6. Neutrophils are a type of white blood cell (leukocytes) that act as your immune system’s first line of defense. They constitute 50% to 60% of the total circulating white blood cells. In the blood, neutrophils are inactive but are swept along at high speed. When they receive signals from the sites of inflammation, they slow down and leave the blood to the tissues, where they are activated by cytokines and arrive at the battle scene ready to kill. Activated neutrophils eject neutrophil extracellular traps (NETs), webs of chromatin and mtDNA coated with antimicrobial proteins, to immobilize and neutralize pathogens. In the process of such NETs release, Neutrophils die. This cell death process is called NETosis.

    A proportion of cfDNA may be produced as part of the innate immune response in NETosis. Here is an illustration for Putative stimuli and steps in NETosis.

  7. Cell secretion

  8. Live cells spontaneously release newly synthesized DNA as part of a homeostatically regulated system. Stimulation of lymphocytes also results in the release of large amounts of cell-free DNA in the absence of cell death. Active secretion of cfDNA may serve a physiological role. It has been shown that cfDNA secreted in exosomes may serve to maintain tissue homeostasis by removing damaged DNA.

The clearance mechanisms of cfDNA

cfDNA clearance mechanisms

Figure 4. cfDNA clearance mechanisms.

It is unknown whether other factors such as circadian rhythms, inflammation, or particular therapies affect ctDNA release and clearance. There are at least three potential contributing mechanisms:

  • active uptake by the macrophages of the reticuloendothelial system in the liver and spleen
  • passive filtration by the renal system (kidneys)
  • direct degradation/digestion by nucleases in the blood

One study of intravenous injection of radiolabeled mononucleosomes into mice showed that the liver was a major organ for removing circulating nucleosomes, with 70-90% of the injected dose recovered in the liver, and ~3% in the spleen and ~4% in the kidneys, with trace amounts in the heart and lungs.

What is cell-free DNA (cfDNA) extraction?

cfDNA extraction is the removal of cfDNA fragments from proteins and other cellular components in a biological sample by using physical and/or chemical methods, e.g. organic extraction, Chelex extraction, and solid phase extraction.

Why is cell-free DNA (cfDNA) extraction important?

cfDNA extraction is important because it is the starting point for the following processes such as PCR or NGS for all the applications above and because it’s challenging to extract due to the following reasons:

  • The concentrations of cfDNA is considerably low in the plasma (average 30ng/mL, range 1.8-44ng/mL). The average concentration of cfDNA in cancer patients tends to increase to 180ng/mL, but this still remains difficult to detect.
  • While ctDNA can provide tumor genetic information, it can constitute as low as 0.1% to 0.01% of total cfDNA. And large amounts of interfering cfDNA can make it difficult to accurately detect rare mutant targets.
  • Cell-free circulating DNA appears to be rapidly cleared by the liver, spleen, liver and kidneys. The half-life of cfDNA is quite short, estimated to be 16 minutes to 2.5 hours.

Precision liquid biopsy requires sensitive assays and accurate, easy and rapid cfDNA extaction methods.

How to extract cell-free DNA?

Isolation, quantification and evaluation of cfDNA is not a straightforward task. It requires a sensitive and reliable workflow.

And cfDNA is an extremely difficult sample type to work with because cfDNA is often limited in yield and highly fragmented. This is where MagVigen™ cfDNA Extraction Kit (Cat #K61003) comes into play. It can provide higher yield and higher quality of cfDNA samples for NGS assays (vs. Qiagen Kit).

nvigen cfdna extration workflow

Figure 5. cfDNA Extraction workflow.

  1. Lyse: Lyse plasma sample.
  2. Bind & Capture: Mix MagVigen™ nanoparticles with sample. Nanoparticles bind to the cfDNA fragments.
  3. Pellet & Re-disperse 1: Magnetic pellet the beads until the solution is clear. The beads respond to a magnetic force (by use of a magnet, e.g. magnetic separation rack), allowing bound material to be rapidly and efficiently separated from the rest of the sample. Remove supernatant (unbound materials) by aspiration, what remains is the nanoparticle-bound targets. Then re-disperse beads pellet in Wash 1 Buffer.
  4. Transfer: Transfer Wash 1 Solution to 1.5 or 2ml tube.
  5. Pellet & Re-disperse 2: Magnetic pelleting, remove supernatant as in step 3. This time re-disperse the beads pellet in 75% Ethanol.
  6. Wash & Pellet 2,3: Wash the beads pellet using 75% Ethanol. Pellet the beads and remove all supernatant. Re-disperse the beads pellet in 75% Ethanol. Do this step twice.
  7. Elute: Final elution of high quality cfDNA. Ready for downstream NGS, PCR or other Applications.
    2 times of elution can help improve cfDNA yield.

Please refer to the K61003 Product Sheet for detailed steps.

How can we help?

The quality of cfDNA sample will strongly influence the downstream applications, which makes it a critical step in library preparation.

MagVigen does high molecular weight cfDNA extraction and DNA sizing and cleanup

The studies discussed above and many other studies strongly support the necessity of continuous improvement and refinement of cfDNA extraction and DNA fragment size selection to enhance the understanding of this important type of circulating biomarkers, and better develop ctDNA clinical testing assays for cancer treatment monitoring and guidance or earlier detection.

At NVIGEN, we are dedicated to developing highly sensitive sample and assay technology to empower personalized medicine. Our cfDNA Extraction Kit and DNA sizing and cleanup nanobeads are popular choices for high molecular weight molecules to achieve high yield and high quality cell free DNA, and discrete clean DNA fragment size ranges for a wide range of downstream genome-wide or targeted sequencing workflow.

Customized product and assay development requests can be addressed at, e.g. to increase the potential applications of cfDNA and ctDNA for discovery research of tumor biology, clinical diagnostics, earlier cancer detection and targeted cancer therapy.

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