Logistics: Shipping cfDNA Without Ruining the Sample

The most dangerous part of a liquid biopsy's journey is not the needle stick or the PCR machine—it is the FedEx truck.

Once you have collected and processed your sample, you are fighting against two enemies: Time and Temperature. If either wins, white blood cells lyse, DNA degrades, and the result is invalid. All your careful collection and processing work is wasted.

Shipping seems like a mundane logistical detail, but it is actually a critical extension of the diagnostic process. Here is how to navigate the two main shipping protocols—and the common mistakes that destroy samples in transit.

Understanding the Risks During Transit

Before we dive into protocols, let's understand what can go wrong:

Temperature Extremes

- Freezing: Ice crystal formation ruptures cells and shears DNA - Overheating: Accelerates cellular metabolism, enzymatic degradation, and bacterial growth - Freeze-Thaw Cycling: Repeated transitions damage DNA integrity

Transit Time

- Delays: Every extra hour gives DNA more time to degrade - Weekend Holds: Packages sitting in sorting facilities for 2+ days - Customs: International shipments can be held for days at unpredictable temperatures

Physical Stress

- Rough Handling: Tubes can crack or leak; packages can be thrown - Pressure Changes: Air freight involves pressure variations that can affect sealed containers

Protocol A: The Frozen Plasma Route

This is the standard protocol for labs that require you to process the sample in-house using the double-spin method.

What You're Shipping

- The Sample: Cell-free plasma in a cryovial (typically 1-2 mL) - The State: Frozen solid at -20°C or -80°C - The Container: Leak-proof cryovial, properly labeled

The Primary Threat: Thawing

Once plasma is frozen, it must stay frozen until the lab thaws it for analysis. If the sample thaws during transit:

1. Ice Crystal Damage: As frozen plasma melts, ice crystals shift and can shear long DNA fragments
2. Enzyme Reactivation: DNase enzymes that were inactive while frozen become active again
3. Refreezing Damage: If the sample thaws and then refreezes (e.g., sitting on a tarmac then returned to a cold plane), additional damage occurs
4. Integrity Loss: The fragment size profile is altered, potentially affecting fragmentomics analysis

The Solution: Dry Ice

Blue gel ice packs are NOT adequate for frozen plasma.

Gel packs keep things "cool" (around 4°C) for a few hours. They do not keep things frozen. Your -20°C plasma will thaw within hours if packed with gel packs.

Dry ice is mandatory:
- Dry ice temperature: -78.5°C
- This keeps plasma frozen solid even during transit delays
- Minimum: 2-3 kg for overnight shipment
- For longer transit times: 4-5 kg or more

Dry Ice Shipping Checklist

1. Container: Use a thick-walled Styrofoam cooler, ideally inside a cardboard outer box
2. Sample Placement: Cryovials in a sealed secondary container (zip-lock bag) to prevent dry ice sublimation into the sample
3. Dry Ice Packing: Surround the sample completely with dry ice—above, below, and on all sides
4. Fill Gaps: Newspaper or packing material to fill air spaces
5. Labeling:
- UN1845 Dry Ice label with weight in kg
- "Keep Frozen" label
- Orientation arrows ("This End Up")
- Biological specimen documentation if required
6. Carrier Selection: Choose overnight shipping; FedEx Priority Overnight or equivalent
7. Ship Early Week: Monday-Wednesday only; never ship Thursday or Friday unless Saturday delivery is confirmed

Common Frozen Shipping Mistakes

| Mistake | What Happens | Prevention |
|---------|--------------|------------|
| Using gel packs instead of dry ice | Sample thaws in hours | Always use dry ice for frozen plasma |
| Insufficient dry ice | Sample thaws before arrival | Use at least 2-3 kg; more for longer transits |
| Shipping late week | Weekend delays cause thawing | Ship Mon-Wed only |
| Poor insulation | Dry ice sublimates too quickly | Use thick Styrofoam, fill air gaps |
| Sample not pre-frozen | Slow freezing damages sample | Freeze completely before packing |

Protocol B: The Stabilized Whole Blood Route

This protocol is for labs that accept Streck or PAXgene tubes, allowing you to ship whole blood that has been chemically stabilized.

What You're Shipping

- The Sample: Whole blood in a cell-stabilization tube (Streck BCT, PAXgene, etc.) - The State: Ambient/room temperature (NOT frozen, NOT refrigerated) - The Container: The original collection tube, properly labeled

The Primary Threat: Temperature Extremes

Freezing (The #1 Error):

This is the most common and most devastating mistake with stabilization tubes. If you put a Streck tube in the freezer:
- Water inside the blood cells expands as it freezes
- Ice crystals rupture cell membranes
- The preservative cannot stop physics
- Upon thawing, the sample is 100% hemolyzed
- Result: Complete sample destruction

A frozen whole blood tube is unsalvageable. There is no recovery.

Overheating:

Stabilization tubes are validated for temperatures up to 37°C (body temperature) for their stated stability period. Exceeding this:
- A package sitting in a mail truck in summer heat (potentially 60°C+) can exceed the preservative's capacity
- Cells may begin metabolizing again or lysing
- Degradation accelerates

The Solution: Insulated Shipping at Room Temperature

Do NOT use ice packs. Even regular gel packs can freeze in a cold warehouse or plane cargo hold, potentially freezing your sample.

The Room Temperature Buffer Approach:

1. Insulated Container: Use a Styrofoam-lined box to buffer against external temperature swings
2. Room Temperature Packs: If desired, include gel packs that have been equilibrated at room temperature (not frozen, not refrigerated). These act as a thermal mass to slow temperature changes.
3. Temperature Indicator: Consider including a temperature indicator strip that changes color if extreme temperatures are reached
4. Avoid Heat Sources: Don't leave the package in a hot car or sunny location before shipping
5. Avoid Cold Sources: Don't refrigerate or add any cold packs

Stabilized Blood Shipping Checklist

1. ☐ Sample is in appropriate stabilization tube (Streck, PAXgene, etc.)
2. ☐ Tube is filled to proper volume (check fill line)
3. ☐ Sample is at room temperature (NOT refrigerated or frozen)
4. ☐ Package is insulated but contains NO ice
5. ☐ Ship Monday-Wednesday to avoid weekend delays
6. ☐ Choose priority/express shipping (2-day maximum transit time)
7. ☐ Label as biological specimen per carrier requirements

The "Friday Draw" Dilemma

It's 4 PM on Friday. The courier has left. The lab is closed over the weekend. What do you do?

Option 1: EDTA Tube → Process and Freeze

If you drew into a standard EDTA tube:
1. Do NOT wait until Monday. EDTA blood cannot sit for 72+ hours.
2. Process now: Perform the double-spin protocol immediately.
3. Freeze the plasma: Store at -20°C or -80°C over the weekend.
4. Ship Monday: Send on dry ice at the start of the week.

Option 2: Stabilization Tube → Hold at Room Temperature

If you drew into a Streck or PAXgene tube:
1. Keep at room temperature: Store in a climate-controlled area (not a window, not outside)
2. Do NOT refrigerate or freeze
3. Ship Monday: Send using the room-temperature protocol at the start of the week
4. Check stability window: Streck tubes are stable for 7-14 days; you have time

Option 3: Delay the Draw

If the Friday draw is for routine screening (not urgent), consider:
- Rescheduling for Monday morning
- Avoids weekend logistics entirely
- Fresh sample ships same-day

Geographic Considerations

Extreme Hot Climates

If you're shipping from (or to) locations where summer temperatures exceed 40°C:
- Ship stabilized blood only in climate-controlled aircraft cargo
- Add extra insulation
- Consider early-morning drop-off to minimize time in hot mail trucks
- Temperature indicators are especially valuable

Extreme Cold Climates

If shipping in winter when ambient temperatures drop below freezing:
- Frozen plasma: Continue with dry ice protocol (no change needed)
- Stabilized whole blood: Add insulation to prevent freezing
- Room temperature gel packs (thermal mass) can help
- Extra Styrofoam layers
- Hand warmers (chemical) in outer box layer (not touching sample)

International Shipping

Cross-border shipments introduce additional risks:
- Customs delays (unpredictable duration)
- Storage conditions at customs facilities
- Regulatory requirements vary by country
- Consider: Is there a domestic lab option?

Summary: Quick Reference

| Sample Type | Shipping Temperature | Refrigerant | Biggest Danger |
|-------------|---------------------|-------------|----------------|
| Frozen Plasma | Frozen (-78°C) | Dry Ice (mandatory) | Thawing |
| Stabilized Blood | Room Temperature | None (insulation only) | Freezing |

Universal Rules:
- Ship early in the week (Mon-Wed)
- Use overnight/priority shipping
- Track the package
- Communicate with the receiving lab

Treat the shipment as part of the medical procedure. A bad shipment = no diagnosis. Your patient deserves the same care in logistics as they received in collection.