When to Say No: Sample Rejection Criteria for the General Practitioner

As a clinician, you want answers. When a sample looks borderline, the temptation is to say, "Just run it and let's see what we get."

In cfDNA testing, this approach is dangerous. A compromised sample doesn't just give a "blurry" result that you can interpret with caution. It can give a confidence-destroying False Positive (indicating high cell death where there is none, triggering an unnecessary cancer workup) or an equally dangerous False Negative (missing a cancer because the tumor signal was diluted below detection).

Knowing when to reject a sample and redraw is a critical clinical skill. This article covers the absolute deal-breakers that every practice team should recognize.

Why Rejection Matters More for cfDNA

The Trace Analyte Challenge

Unlike creatinine (present at mg/dL concentrations) or even C-reactive protein (µg/mL), cfDNA is measured in nanograms per milliliter. We are looking for tiny signals against a background of normal DNA.

Contamination that would be irrelevant for a chemistry panel can completely invalidate a liquid biopsy. A 5% error in a glucose measurement is clinically insignificant. A 5x contamination of cfDNA from lysed cells can bury a cancer signal completely.

The Asymmetry of Errors

False Positive: The test says cfDNA is high → you suspect cancer → you do a full workup → imaging is negative → owner spent money and experienced stress for nothing → your confidence in the test drops.

False Negative: The test says cfDNA is normal → you reassure the owner → the cancer continues growing → months later, the dog presents with advanced disease → the early detection opportunity was lost.

Both errors are harmful. Both are preventable with proper sample rejection criteria.

The 5 Absolute Deal-Breakers

1. The Clot: When Anticoagulation Failed

The Sign:
- A small jelly-like blob visible in an EDTA (purple top) tube
- Blood that is solid or partially solid
- Fibrin strands visible when the tube is inverted
- Failure to see the classic layering after centrifugation

What Happened:
The clotting cascade activated despite the EDTA anticoagulant. This can occur due to:
- Insufficient anticoagulant (tube underfilled, or expired tube)
- Inadequate mixing after collection
- Prolonged time before mixing
- Tissue factor contamination from traumatic venipuncture

The Reality:
Once clotting initiates, you no longer have plasma—you have serum in a purple tube. The clotting process has:
- Trapped and lysed white blood cells in the fibrin mesh
- Released platelet contents
- Contaminated the sample with massive amounts of genomic DNA

The cfDNA Impact:
The sample is completely unsuitable for liquid biopsy. Any result will be meaningless.

Action: Reject and redraw.
- Check tube expiration dates
- Ensure adequate fill volume
- Mix immediately after collection (8-10 gentle inversions)

2. The Cherry Soda: Severe Hemolysis (2+ or Greater)

The Sign:
- Plasma is red, cherry-colored, or dark pink after centrifugation
- Clear pink tinge extending throughout the sample (not just a streak)
- Cannot see through the plasma (opaque red)

What Happened:
Red blood cells ruptured, releasing hemoglobin into the plasma. More importantly, the conditions that caused RBC hemolysis (mechanical trauma, osmotic stress, temperature extremes) also damaged white blood cells, causing them to lyse and release their genomic DNA.

The Reality:
While mild hemolysis (faint pink) may be tolerable with careful interpretation, moderate to severe hemolysis (2+) implies:
- Massive cellular destruction in the tube
- Genomic DNA contamination from WBC lysis
- Dilution of any tumor signal below detection limits

The cfDNA Impact:
A negative cancer result from a hemolyzed sample cannot be trusted. The tumor DNA may be present but buried in genomic noise.

Action: Reject and redraw.
- Use appropriate needle gauge for vein size
- Avoid excessive vacuum/syringe pressure
- Don't "fish" for the vein with the needle
- Remove needle before transferring to tube

3. The Warm Weekender: Exceeded Stability Window

The Sign:
- An EDTA tube drawn Friday arrives at the lab on Tuesday
- The ice pack in the shipping container has melted
- The sample spent >4 hours at room temperature before processing
- Documentation shows extended time between collection and centrifugation

What Happened:
The 4-hour stability window for EDTA blood was exceeded. White blood cells in the tube, starved of glucose and under metabolic stress, underwent apoptosis and lysis.

The Reality:
After 24-48 hours at room temperature, an EDTA tube contains:
- Plasma heavily contaminated with genomic DNA from dead WBCs
- Degraded cfDNA fragments (nuclease activity resumed)
- Altered fragment size distribution (artifactual fragmentation)

The sample is effectively a soup of necrotic cellular debris.

The cfDNA Impact:
Any result is uninterpretable. Total cfDNA will be falsely elevated. Fragment patterns are artifactually altered. Tumor detection sensitivity approaches zero.

Action: Reject. Do not send.
- Process EDTA samples within 2-4 hours
- For delayed processing, use stabilization tubes (Streck) from the start
- Ship processed frozen plasma, not whole blood, if courier timing is uncertain

4. The Drop: Insufficient Sample Volume

The Sign:
- The laboratory requests 1.0 mL of plasma
- You send 0.2 mL or 0.3 mL
- The tube is mostly empty after centrifugation
- Only a small amount of whole blood was collected

What Happened:
Not enough blood was drawn, resulting in inadequate plasma after processing. This might occur with:
- Difficult venipuncture (small patient, collapsed vein)
- Tube underfilling due to poor vacuum
- Loss of sample during transfers

The Reality:
cfDNA is a trace analyte. Modern liquid biopsy tests work by:
- Extracting DNA from a specified plasma volume
- Concentrating and amplifying rare signals
- Using statistical methods that assume adequate input material

With insufficient input volume:
- Less total cfDNA enters the assay
- Rare tumor fragments may be missed by chance alone
- Sensitivity drops proportionally to volume reduction

The cfDNA Impact:
A negative result from an insufficient-volume sample might simply mean the tumor DNA wasn't in the small portion tested. It's a sampling error, not a true negative.

Action: Reject or flag as "quantity not sufficient (QNS)."
- Draw adequate whole blood volume (3x the required plasma volume)
- Use appropriately sized tubes
- Communicate with the lab if volume is borderline

5. The Temperature Abuse: Freeze-Thawed or Overheated

The Sign:
- Frozen plasma that thawed during shipping (ice pack liquid, condensation inside tube)
- Evidence of refreezing (frost on previously clear tube)
- Streck/stabilization tube that was accidentally frozen
- Sample left in hot vehicle before transport

What Happened:
The sample was exposed to temperature conditions that damage DNA or cause cell lysis:
- Freeze-thaw cycles shear DNA fragments
- Freezing whole blood lyses all cells
- Heat accelerates enzymatic degradation

The Reality:
- Freeze-thawed plasma: DNA fragments are artificially shortened, altering fragmentomics analysis
- Frozen Streck tube: Complete hemolysis, total sample destruction
- Overheated sample: Accelerated cell death and DNA degradation

The cfDNA Impact:
Results may be falsely high (lysed cell contamination), falsely low (degraded DNA), or have artifactual fragment patterns (freeze-thaw shearing).

Action: Reject.
- Ship frozen plasma on adequate dry ice
- Ship stabilized whole blood at room temperature (NEVER freeze)
- Use insulated containers and temperature monitors when possible

The Decision Framework

When Rejection Is Mandatory

| Finding | Action |
|---------|--------|
| Visible clot in EDTA tube | Reject—redraw |
| 2+ hemolysis or worse | Reject—redraw |
| EDTA blood >4 hours before processing | Reject—do not send |
| Frozen Streck tube | Reject—redraw |
| Sample grossly insufficient | Reject—redraw |

When Caution Is Warranted (Consider Running with Notes)

| Finding | Action |
|---------|--------|
| 1+ hemolysis (faint pink) | May run with notation; interpret cautiously |
| Volume slightly below minimum | Discuss with lab; may run with reduced confidence |
| Mild lipemia | Attempt clarification; may run with notation |
| Processing at 3-4 hours | Acceptable but note delay |

Communicating with Owners

When you must reject and redraw, explain clearly:

"The sample we collected didn't meet the quality standards required for this particular test. Unlike routine blood work, liquid biopsy is extremely sensitive and requires perfect sample handling. To get results we can trust, we need to collect a fresh sample. I know it's inconvenient, but an unreliable result is worse than no result—it could lead us in the wrong direction."

Most owners understand when you explain that a bad sample could cause either a false alarm or a missed diagnosis.

Building a Culture of Quality

Train Your Team

Everyone involved in sample collection and handling should understand:
- Why cfDNA samples are different from routine bloodwork
- The specific handling requirements (time, temperature, tubes)
- What rejection criteria look like
- That it's okay—and expected—to flag problem samples

Track Rejections

If you're consistently rejecting samples, investigate:
- Are certain staff members having more issues? (Training opportunity)
- Are specific collection scenarios problematic? (Workflow adjustment)
- Is shipping logistics causing issues? (Carrier/packaging review)

Summary: The Rejection Hierarchy

| Priority | Issue | Consequence if Run | Decision |
|----------|-------|-------------------|----------|
| CRITICAL | Clot | Complete invalidation | Always reject |
| CRITICAL | Severe hemolysis | False negatives | Always reject |
| CRITICAL | Time exceeded (EDTA) | False positives/negatives | Always reject |
| CRITICAL | Frozen Streck | Complete destruction | Always reject |
| HIGH | Insufficient volume | Reduced sensitivity | Usually reject |
| MODERATE | Mild hemolysis | Interpret with caution | Run with notes |
| MODERATE | Mild lipemia | Variable interference | May run with notes |

The Bottom Line: A rejected sample causes a delay and requires a redraw. A bad result causes a misdiagnosis that may harm the patient and damage trust. Choose the delay.