Hemolysis: The "Red" Flag That Ruins Results

We have all seen it: you spin down a blood tube, and instead of straw-colored yellow plasma, you get pink or red fluid. In a chemistry panel, this might mess up your bilirubin or potassium readings—annoying, but often the lab can still report most values with a caveat.

In cfDNA testing, hemolysis is catastrophic. It doesn't just interfere with the test—it can completely invalidate the results. Understanding why this happens, and how to prevent it, is essential for any practice offering liquid biopsy services.

The Biology of the Pink Tube

Hemolysis is the rupture of Red Blood Cells (RBCs), releasing hemoglobin into the plasma and turning it pink or red. But here's the critical insight that many clinicians miss:

While canine RBCs are anucleated (they don't contain nuclear DNA), hemolysis is rarely an isolated event affecting only red cells.

The conditions that cause RBC hemolysis—mechanical shear forces, osmotic stress, prolonged storage, temperature extremes—affect all cells in the tube. If the draw was traumatic enough to lyse RBCs, you can virtually guarantee that White Blood Cells (WBCs) have also been damaged or lysed.

And WBCs are packed with DNA—approximately 6 picograms per cell of nuclear genomic DNA.

Why Hemolysis Destroys cfDNA Results

The Contamination Problem

When WBCs burst in the tube, they release healthy, genomic DNA that floods the plasma. This creates a massive dilution of any tumor-derived signal you were trying to detect.

The Math of Signal Dilution

Let's walk through a realistic example:

Scenario: Detecting early-stage cancer

Imagine you are trying to detect a faint signal from a small tumor. The tumor is shedding circulating tumor DNA (ctDNA) at a rate that produces 10 mutant copies per milliliter of plasma.

Clean Draw (No Hemolysis):
- Background normal cfDNA: 1,000 copies/mL
- Tumor DNA: 10 copies/mL
- Tumor fraction: 10/1,010 = ~1%
- Assay sensitivity: Can detect down to 0.1%
- Result: DETECTED ✅

Hemolyzed Draw (Moderate Hemolysis):
- Original cfDNA: 1,000 copies/mL
- Genomic DNA from lysed WBCs: 1,000,000 copies/mL (released from just ~0.1% of WBCs lysing)
- Tumor DNA: Still 10 copies/mL (unchanged)
- Tumor fraction: 10/1,001,010 = 0.001%
- Assay sensitivity: Can detect down to 0.1%
- Result: NOT DETECTED ❌ (False Negative)

The tumor DNA is still there. It hasn't gone anywhere. But it has been diluted below the limit of detection by the massive influx of normal DNA from lysed white cells.

The Clinical Consequence

Hemolysis causes False Negatives.

This is the most dangerous type of error for cancer screening. The dog has cancer, the tumor is shedding DNA, but the test says "No Cancer Detected" because the signal was drowned out by contamination.

The owner goes home relieved. The cancer continues to grow. By the time it's caught, it may have progressed significantly.

Visual Grading: The Hemolysis Scale

Most labs use a visual scale or a spectrophotometric "Hemolysis Index" (H-index) to grade samples:

| Grade | Appearance | cfDNA Impact | Action |
|-------|------------|--------------|--------|
| 0 | Clear, straw-yellow | None | Acceptable |
| 1+ | Faint pink tinge | Mild | Caution—may affect sensitivity |
| 2+ | Clearly pink/salmon | Moderate | Reject—risk too high |
| 3+ | Red/cherry color | Severe | Unusable |
| 4+ | Dark red/opaque | Gross | Completely destroyed |

The Rejection Decision

For cfDNA testing, most laboratories will reject samples at 2+ hemolysis or above. Some highly sensitive assays may require even stricter standards.

Do not try to "salvage" a hemolyzed sample. The contamination has already occurred. Running the test will waste money and—worse—may provide a falsely reassuring negative result.

What Causes Hemolysis?

Understanding the causes helps you prevent them:

1. Traumatic Venipuncture

- "Fishing" for the vein: Redirecting the needle through tissue - Vein wall damage: Puncturing through both sides of the vein - Small needle, large vacuum: Creates shear forces that tear cells

2. Excessive Vacuum/Pressure

- Syringe draws: Pulling back too hard or too fast on the plunger - Vacutainer on small veins: The fixed vacuum may be too strong - Forceful transfers: Squirting blood through a needle under pressure

3. Improper Handling

- Vigorous mixing: Shaking instead of gentle inversion - Rough transport: Dropping, throwing, or agitating tubes - Pneumatic tube systems: High-speed capsule transport can cause hemolysis

4. Temperature Extremes

- Freezing whole blood: Ice crystals rupture cells - Excessive heat: Can damage cell membranes - Leaving tubes in hot cars: Summer temperatures accelerate lysis

5. Delayed Processing

- Old blood hemolyzes: Cells become fragile over time - Beyond the 4-hour window: Even without visible hemolysis, cellular integrity is compromised

Prevention: Best Practices

During Collection

1. Choose the right vein: Use the largest accessible vein (usually jugular in dogs)
2. Use appropriate needle gauge: Match needle size to vein size (typically 20-22 gauge)
3. Achieve clean access: Enter the vein smoothly on the first pass when possible
4. Control vacuum pressure: If using a syringe, pull back slowly and steadily
5. Fill tubes properly: Don't over-fill or under-fill

During Transfer

1. Remove the needle: When transferring from syringe to tube, remove the needle first
2. Pop the cap: Open the tube rather than puncturing through the stopper
3. Let blood flow: Allow gentle flow rather than forceful squirting
4. Mix gently: Invert the tube 8-10 times—do not shake

During Storage and Transport

1. Process promptly: The sooner you spin, the less time for cells to become fragile
2. Avoid temperature extremes: Room temperature storage until processing
3. Handle gently: No dropping, throwing, or rough transport
4. Skip the pneumatic tube: Walk cfDNA samples to the lab if possible

What If You See Hemolysis?

If you spin down a sample and see pink or red plasma:

Step 1: Assess the Grade

- Faint pink (1+)? May be acceptable for some tests—check with your lab - Pink/salmon or darker (2+)? Reject the sample

Step 2: Investigate the Cause

- Was the draw difficult? - Was there a delay in processing? - Was the sample exposed to temperature extremes?

Step 3: Redraw if Necessary

- For 2+ or worse, schedule a redraw - Address the underlying cause before the second attempt - Consider a different phlebotomist, vein site, or technique

Step 4: Document

- Note the hemolysis in the medical record - Track patterns—repeated hemolysis may indicate a technique issue

Summary: The Non-Negotiable Standard

| Plasma Appearance | Grade | cfDNA Testing Decision |
|-------------------|-------|------------------------|
| Clear, straw-yellow | 0 | ✅ Proceed |
| Faint pink | 1+ | ⚠️ Proceed with caution |
| Pink/salmon | 2+ | ❌ Reject—redraw |
| Red/cherry | 3+ | ❌ Reject—redraw |
| Dark red | 4+ | ❌ Reject—redraw |

The bottom line: If the plasma is red, the result will be wrong. Don't run it. Don't hope for the best. Redraw.

A rejected sample causes a delay. A false-negative result causes a missed diagnosis. Choose the delay.