Defining "Normal": Reference Intervals and Biological Variation in Dogs

A common misconception among veterinarians new to liquid biopsy is that a healthy patient should have zero cell-free DNA (cfDNA).

In reality, cfDNA is a normal physiological byproduct. Every day, billions of cells in a dog's body reach the end of their lifespan and undergo apoptosis (programmed cell death). The liver and kidneys work constantly to clear this debris. The cfDNA level we measure in the blood is the net balance between this production (cell death) and clearance (organ function).

To interpret a test result, we must first define what "normal" looks like—and understand all the factors that can shift that baseline.

Why Zero Is Not the Goal

Some clinicians, upon first encountering cfDNA testing, assume that the ideal result is "undetectable." This misunderstanding can lead to unnecessary concern when a healthy patient shows measurable cfDNA.

Think of cfDNA like bilirubin or creatinine. Every healthy animal has some measurable level. The presence of cfDNA simply reflects the ongoing cellular maintenance happening throughout the body. Red blood cells are recycled, gut epithelial cells are shed, immune cells complete their patrol and are retired. All of this releases small amounts of DNA into circulation.

The question is never "Is there cfDNA?" but rather "How much, and is that amount changing?"

The Healthy Baseline: What the Numbers Mean

While reference intervals vary slightly by methodology (e.g., fluorometry vs. qPCR), multiple studies have converged on a consistent range for healthy, adult dogs.

* Typical Range: 0.5 – 1.2 ng/mL (or 500 – 1,200 µg/L).
* The Cutoff: Most studies consider values consistently above 1.5 – 2.0 ng/mL to be abnormal and worthy of investigation.

For example, in a study by Kim et al. (2021), healthy dogs had a mean cfDNA concentration of approximately 0.88 ng/mL. In contrast, dogs with lymphoma averaged over 2.0 ng/mL, with some advanced cases reaching levels 10x or 100x higher.

Understanding the Range

Why such a wide "normal" range (0.5 to 1.2)? Because individual dogs vary. Just as some healthy dogs run hotter or have higher resting heart rates, some dogs simply have higher baseline cellular turnover. This is why establishing an individual baseline during wellness visits is so valuable—you learn what "normal" looks like for that patient.

Factors Influencing Baseline: The Biological Variables

Before flagging a result as "pathological," consider these biological variables that can shift cfDNA levels in healthy animals:

1. Age: The "Inflammaging" Effect

Older dogs tend to have slightly higher baseline cfDNA than puppies or young adults. This phenomenon is attributed to:

* Inflammaging: The low-grade, chronic inflammation associated with aging. As the immune system ages, it becomes less efficient at resolving inflammation, leading to persistent low-level cellular stress.
* Cellular Senescence: Older cells are more likely to enter senescence (a state where they stop dividing but don't die), and eventually undergo apoptosis.
* Organ Efficiency: The liver and kidneys clear cfDNA from circulation. Subtle age-related decline in organ function can slow clearance.

Practical Implication: A 12-year-old Golden Retriever might run at 1.0 ng/mL, whereas a 2-year-old might run at 0.4 ng/mL. Both can be considered healthy, but the "buffer" before hitting the abnormal threshold is smaller for the older dog.

2. Exercise: The Muscle Micro-Trauma Effect

If a client brings in a working dog (e.g., a Border Collie or hunting dog) immediately after a field trial or intense exercise session, hold off on the draw.

* The Mechanism: Strenuous exercise causes minor muscle micro-trauma and oxidative stress. This leads to a burst of cellular turnover as damaged muscle fibers are repaired.
* The Result: cfDNA levels can elevate transiently by approximately 20% or more following intense activity.
* Recovery Time: Levels typically normalize within 24-48 hours of rest.

Recommendation: For baseline screening or trend monitoring, rest the dog for 24 hours before collecting a sample. For emergency diagnostics, this obviously doesn't apply—just document the circumstances.

3. Breed and Size: The Volume Question

Currently, there is no strong evidence that breed significantly alters cfDNA concentration per milliliter of plasma. While a Great Dane has more total blood volume (and thus more total circulating DNA) than a Chihuahua, the concentration remains relatively constant because we measure nanograms per milliliter, not total body DNA.

However, breed matters for a different reason: cancer risk.

High-risk breeds (Golden Retrievers, Boxers, Bernese Mountain Dogs, Flat-Coated Retrievers) are the primary candidates for proactive cfDNA screening. For these patients, establishing an individual baseline during wellness visits—say, at age 5—is highly valuable. You create a reference point specific to that dog, against which future tests can be compared.

4. Recent Illness or Injury

A dog recovering from any illness—even something as minor as a respiratory infection—may have elevated cfDNA for days to weeks afterward as the body clears damaged tissue. If a patient recently recovered from an illness, consider waiting 2-4 weeks before establishing a "baseline."

5. Time of Day and Feeding Status

While less impactful than exercise or age, there is some evidence that cfDNA levels can fluctuate throughout the day and may be affected by recent feeding (particularly fatty meals that cause lipemia). For consistency, morning draws on fasted patients are ideal for screening purposes.

The "Gray Zone": Navigating Borderline Results

What do you do with a result of 1.6 ng/mL?

This is the diagnostic "gray zone." It is too high to be perfectly normal, but too low to be a definitive cancer signal (like the >5.0 ng/mL spikes seen in septic peritonitis or metastatic hemangiosarcoma).

Differential Diagnoses for Gray Zone Results

1. Mild, Subclinical Inflammation: Gingivitis, dermatitis, early osteoarthritis, or other low-grade inflammatory conditions.
2. Pre-analytical Error: Was the sample delayed before processing? Did minor hemolysis occur? Was the sample exposed to temperature extremes during shipping?
3. Recent Minor Illness or Injury: A healing wound, recent vaccination, or recovering from a mild infection.
4. Early Disease: A small, localized tumor or early-stage organ dysfunction that is not yet causing clinical signs.
5. Normal Variation: Some healthy dogs simply run higher than average.

The Strategy: Trend Over Time

Do not panic over a single borderline result. The most powerful diagnostic approach is to recheck the sample in 2–4 weeks.

* If it returns to <1.0 ng/mL: The elevation was likely transient—a resolved minor injury, cleared infection, or pre-analytical issue.
* If it stays the same (1.5-1.8 ng/mL): This might be that patient's personal baseline. Continue monitoring every 3-6 months.
* If it rises to 2.5 ng/mL or higher: Now you have a trajectory. A rising trend is far more clinically significant than a single borderline value. This warrants a "confirmatory hunt"—detailed imaging, thorough physical exam, and possibly additional diagnostics to find the source.

Building Individual Baselines: The Proactive Approach

For high-risk breeds or patients whose owners want the most proactive care, consider establishing a baseline cfDNA level during a wellness visit when the patient is healthy—typically between ages 4-6 for large breeds, or earlier for known high-risk breeds.

This baseline becomes that patient's personal reference point. Future tests are compared against their own normal, not just population averages. A result of 1.2 ng/mL is very different if the patient's baseline was 0.4 ng/mL (a 3x increase—concerning) versus if their baseline was 1.0 ng/mL (barely changed—reassuring).

Summary: The Reference Framework

| cfDNA Level | Interpretation | Recommended Action |
|-------------|----------------|--------------------|
| < 1.0 ng/mL | Normal | Routine monitoring |
| 1.0 – 2.0 ng/mL | Gray Zone | Recheck in 2-4 weeks; monitor trends |
| > 2.0 ng/mL | Abnormal | Investigate for neoplasia, trauma, infection, or organ damage |
| > 5.0 ng/mL | Markedly Elevated | Urgent investigation; often indicates significant pathology |

Understanding these baselines allows us to use cfDNA not just as a binary "yes/no" test, but as a nuanced physiological monitor that tracks the body's cellular health over time.

(References: Kim et al. 2021; Hunt et al. 2017; Letendre & Goggs 2018)