The Double-Spin Protocol: Why One Centrifuge Run is Not Enough

Veterinary technicians are masters of the centrifuge. Every day, they spin down blood to get plasma for chemistry panels or serum for reference lab send-outs. The standard protocol—usually 10 minutes at a moderate speed—works perfectly for measuring creatinine, glucose, or liver enzymes.

However, for cell-free DNA (cfDNA), the standard spin is a recipe for failure.

To get a diagnostic-quality liquid biopsy sample, you must use the Double-Spin Protocol. This extra step takes just 15 additional minutes but makes the difference between a reliable result and a wasted test. Here is the physics of why—and exactly how to do it.

Understanding the Problem: What's Still Floating?

When you spin blood at 2,000 x g (a typical clinic centrifuge speed) for 10 minutes, you successfully pellet the heavy red blood cells and most of the white blood cells. You are left with plasma that looks crystal clear to the naked eye.

But appearances are deceiving.

Suspended in that plasma are millions of microscopic particles that are too small to see but large enough to ruin your test:

1. Platelets: The Mitochondrial Problem

Mammalian platelets do not have nuclei—they are cell fragments derived from megakaryocytes. However, they do contain mitochondria, and those mitochondria contain DNA.

* Mitochondrial DNA: Each platelet contains multiple copies of the 16.5 kb mitochondrial genome.
* The Problem: Total DNA assays (like fluorometry, which is commonly used to quantify cfDNA) cannot distinguish between nuclear DNA and mitochondrial DNA. They measure all DNA present.
* The Result: Platelet-rich plasma will show artificially inflated cfDNA concentrations.

2. Cellular Debris: The Fragile Passengers

After the first spin, the plasma still contains:

* Microvesicles: Tiny membrane-bound particles shed from cells.
* Apoptotic bodies: Fragments of cells undergoing programmed death that haven't fully settled.
* Residual leukocytes: A small number of white blood cells that didn't completely pellet.

These particles may survive the first spin, but they are fragile. The stress of freezing, thawing, or shipping can rupture them, releasing their genomic DNA into the sample at the worst possible time—right before analysis.

3. The Sensitivity Cascade

Liquid biopsy tests are exquisitely sensitive. They're designed to detect tiny amounts of tumor DNA (perhaps 0.1% of total cfDNA) against a background of normal DNA.

* The Signal: Tumor-derived DNA fragments you're trying to detect.
* The Noise: Background DNA from normal cells.

If you run a sensitive test on single-spun plasma, you are analyzing a mixture of true cfDNA plus all the contamination from platelets and debris. This:
- Increases the background noise
- Dilutes the tumor signal
- Decreases sensitivity for detecting cancer
- May cause false-negative results

The Solution: The Two-Step Double-Spin Process

The goal is to create truly cell-free plasma—plasma that contains only the DNA fragments that were already circulating at the time of the blood draw, with no additional contamination.

Step 1: The Separation Spin (Low Speed)

Goal: Separate plasma from the cellular components—red blood cells, white blood cells (the buffy coat), and most platelets.

Settings:
* Speed: 1,600 to 2,000 x g
* Time: 10 minutes
* Temperature: Room temperature (or 4°C if your centrifuge has temperature control)
* Brake: Off or gentle (harsh braking can disturb the layers)

What You'll See:
After this spin, your tube will show three distinct layers:
1. Bottom: Dark red layer of packed red blood cells
2. Middle: Thin, whitish layer called the "buffy coat" (this is where the WBCs live)
3. Top: Straw-colored plasma supernatant

The Harvest:
Using a clean pipette, carefully transfer the plasma to a new, clean tube.

* Critical Rule: Leave a buffer layer of at least 0.5 cm of plasma above the buffy coat. Do NOT try to get every last drop. If you disturb the WBC layer or accidentally aspirate any of the buffy coat, you have contaminated the sample.
* Visual Check: The plasma you transfer should be completely clear with no cloudiness or visible particles.

Step 2: The Purification Spin (High Speed)

Goal: Pellet the remaining platelets, microvesicles, and cellular debris that escaped the first spin.

Settings:
* Speed: >10,000 x g (maximum speed on most microcentrifuges)
* Alternative: If your clinic only has a general centrifuge that maxes out at 3,000-4,000 x g, spin at maximum speed for 15-20 minutes instead
* Time: 10 minutes
* Temperature: Room temperature or 4°C
* Brake: Off or gentle

What You'll See:
Look carefully at the bottom of the tube. You will often see a tiny, pinhead-sized pellet—white, cream-colored, or barely visible. That is the contamination you just removed: platelets, debris, and any residual cells.

If you don't see a visible pellet, that's okay—the debris may be too small to see. It's still there, and it's now at the bottom where you can avoid it.

The Final Harvest:
Transfer the top 80-90% of the plasma to your final storage container (cryovial). Deliberately leave the bottom 10-20% behind to ensure you don't disturb that pellet.

Equipment Considerations

Centrifuge Options

Ideal: A microcentrifuge that reaches 10,000+ x g. These are common in molecular biology labs and are increasingly available in veterinary specialty practices.

Acceptable: A general clinic centrifuge at maximum speed with extended spin time. Most clinic centrifuges max out around 3,000-4,000 x g. This can work if you extend the second spin to 15-20 minutes.

Tip: Check your centrifuge manual for the maximum RCF (relative centrifugal force) in g-force. Speed in RPM is not the same as g-force and depends on the rotor radius.

Tubes and Pipettes

* Use clean, DNA-free tubes for all transfers
* Avoid touching the inside of tubes or pipette tips
* Change pipette tips between steps to avoid cross-contamination

Common Mistakes and How to Avoid Them

| Mistake | Why It Happens | How to Prevent |
|---------|----------------|----------------|
| Disturbing the buffy coat | Trying to get all the plasma | Leave a visible buffer layer—quality over quantity |
| Skipping the second spin | "It looks clear already" | Clear to the eye ≠ cell-free; always double-spin |
| Harsh braking | Default centrifuge settings | Turn brake off or to lowest setting |
| Using the same pipette tip | Rushing | Change tips between every transfer |
| Pipetting from the bottom | Habit from other protocols | Always pipette from the top, leaving the bottom behind |

Validation: Is It Worth the Extra Effort?

Yes. Comparative studies of single-spin versus double-spin plasma consistently show:

* Higher DNA in single-spin: Single-spun samples have significantly higher measured DNA concentrations—not because there's more cfDNA, but because there's more contamination.
* Higher variability in single-spin: Results are less reproducible because contamination varies sample to sample.
* Lower sensitivity in single-spin: For cancer detection, the increased noise floor means subtle signals get missed.

The "extra" DNA in single-spun samples is artifact. It's not the signal—it's noise.

Workflow Integration

The double-spin protocol adds about 15-20 minutes to your processing time. Here's how to integrate it efficiently:

1. Batch processing: If you're processing multiple samples, run them through Spin 1 together, then transfer all plasmas, then run Spin 2 together.
2. Scheduling: If you draw blood at 9 AM, processing can be complete by 10 AM. This easily fits within the 2-4 hour window for EDTA samples.
3. Documentation: Note the exact spin settings and times in your lab records for quality assurance.

Summary: The Double-Spin Checklist

1. ☐ Spin 1: 1,600-2,000 x g for 10 minutes
2. ☐ Transfer plasma: Leave 0.5 cm above buffy coat
3. ☐ Spin 2: >10,000 x g for 10 minutes (or max speed for 15-20 min)
4. ☐ Final transfer: Take top 80-90%, leave pellet behind
5. ☐ Visual check: Plasma should be crystal clear, no visible particles
6. ☐ Freeze or ship: Proceed with storage protocol

In liquid biopsy, we are looking for a needle in a haystack (tumor DNA hiding among normal DNA). The Double-Spin protocol removes the extra hay, making the needle easier to find.