Freeze-Thaw Cycles: How to Shatter a Molecule

In veterinary research and diagnostics, we often freeze serum or plasma to batch samples for later testing. For chemistry (like cortisol or insulin), this is usually fine.

For cfDNA, specifically for Fragmentomics, it is a disaster.

The Physics of Freezing

When plasma freezes, the water content crystallizes. As water turns to ice, it expands. These ice crystals act like microscopic blades.

DNA is a long, strand-like molecule. While cfDNA is already fragmented (mostly ~167 bp), there are longer fragments present (dinucleosomes ~300 bp, or longer necrotic strands).

Each freeze-thaw cycle shears these strands.

The Impact on Diagnostics

1. Fragmentation Analysis: Advanced cancer tests use the "DNA Integrity Index" (DII)—the ratio of long to short DNA. Cancer often creates short DNA.
* If you freeze-thaw a healthy sample 3 times, you artificially shear the long DNA into short pieces.
Result:* The healthy sample now "looks" like cancer (False Positive for fragmentation).

2. Cell Lysis (The bigger problem): Even in "cell-free" plasma, there are often a few residual platelets or micro-debris left (especially if you didn't do a double-spin).
* Freezing bursts these residual structures instantly.
Result:* A spike in total DNA concentration upon thawing.

The "One Thaw" Rule

Best practice for cfDNA is "One Freeze, One Thaw."

1. Process plasma.
2. Freeze at -80°C.
3. Thaw only when ready to run the assay.

What if you need to run it twice?
Aliquot! Before freezing the first time, split the plasma into three small 0.5 mL cryovials. Freeze them all. If you need to re-test, grab a fresh vial. Never put a thawed tube back in the freezer.