cfDNA 101: The Biology of the "Liquid Biopsy"

In the last decade, human oncology has been transformed by the "liquid biopsy"—a non-invasive blood test that can detect cancer, monitor treatment, and predict relapse long before traditional imaging. Now, this technology is accelerating into veterinary medicine. But to use it effectively, we must first understand what we are measuring.

Circulating cell-free DNA (cfDNA) refers to short, fragmented segments of DNA found floating in the bloodstream, outside of any cell. While it is easy to think of DNA as the static instruction manual locked inside the nucleus, cfDNA is a dynamic, rapidly changing signal of cellular health and death.

Where Does cfDNA Come From?

In a healthy dog or cat, cfDNA is present at very low baseline levels (typically 0.5–1.2 ng/mL), resulting from the normal turnover of hematopoietic cells (like white blood cells) and the lining of the gastrointestinal tract. However, when disease strikes, the landscape changes.

There are three primary biological mechanisms that release DNA into circulation, and understanding the difference is key to interpreting clinical results:

1. Apoptosis (Programmed Cell Death)

Apoptosis is the body's tidy, organized way of recycling old or damaged cells. During apoptosis, enzymes systematically cut the genomic DNA into regular, bite-sized pieces. These pieces correspond to the length of DNA wrapped around a nucleosome (the protein spool that packages DNA).

* The Signature: Apoptotic DNA appears as a characteristic "ladder" of fragments, mostly around 160–170 base pairs (bp) in length.
* Clinical Context: This is the dominant source of background cfDNA in healthy animals. It is also the mechanism by which some chemotherapy drugs kill tumor cells, potentially causing a transient spike in "healthy-looking" fragments during treatment.

2. Necrosis (Traumatic Cell Death)

Necrosis is chaotic. It occurs when cells die due to external injury, lack of blood supply (ischemia), or when a rapidly growing tumor outstrips its own vasculature. Unlike the precise cutting of apoptosis, necrosis results in the random rupture of cells.

* The Signature: Necrotic DNA is often larger and more irregular. It does not show the neat 167-bp peak seen in apoptosis.
Clinical Context: We see necrotic cfDNA in aggressive, fast-growing malignancies (like hemangiosarcoma or osteosarcoma), but also in non-cancerous emergencies like Gastric Dilatation-Volvulus (GDV), severe trauma, or splenic torsion. This is why total cfDNA is a marker of injury*, not just cancer.

3. NETosis (Immune Defense)

This is perhaps the most fascinating source. Neutrophils, the first responders of the immune system, can undergo a suicide mission called NETosis. They eject their own DNA into the bloodstream to form Neutrophil Extracellular Traps (NETs)—sticky webs of chromatin decorated with antimicrobial enzymes designed to catch bacteria.

* The Signature: NETs are massive complexes of DNA and histones.
* Clinical Context: NETosis is a massive driver of high cfDNA in sepsis and immune-mediated diseases like IMHA. In these cases, the high DNA load can actually promote thrombosis (clotting), linking inflammation to coagulation risks.

The "Real-Time" Biomarker

Unlike antibodies, which can linger in the blood for months, or proteins like albumin that have long half-lives, cfDNA is incredibly transient. Studies suggest the half-life of cfDNA in circulation is on the order of minutes to hours (typically <2 hours).

It is actively cleared by the liver and kidneys and digested by plasma enzymes called nucleases (DNase). This rapid turnover is its superpower.

If cfDNA is high: It means significant cell death is happening right now*.
* If cfDNA drops: It means the source of the damage has stopped.

For example, if a surgeon removes a splenic hemangiosarcoma, the tumor-specific DNA (ctDNA) should vanish from the blood within 24 to 48 hours. If you test the dog three days later and the levels are still high, it implies the surgery did not get it all—there are likely occult metastases shedding DNA elsewhere.

Conclusion

cfDNA is not just a "cancer test." It is a fundamental measure of cellular integrity. Whether released by a dying tumor, an ischemic stomach, or an exploding neutrophil, elevated cfDNA tells us that the body is under attack. The challenge—and the art—of veterinary liquid biopsy lies in distinguishing these sources to guide clinical decisions.

(References: Letendre & Goggs 2017/2018; Tagawa et al. 2019; Kim et al. 2021)