Does Faecal Calprotectin Have to Be on a Frozen Sample?
No, faecal calprotectin does not have to be measured on a frozen sample, but refrigeration at 4°C is strongly recommended if testing cannot be performed immediately, and freezing at -20°C or -80°C is preferred for longer storage periods.
Optimal Sample Handling Strategy
The evidence clearly demonstrates that calprotectin stability depends critically on temperature and time:
Immediate Testing (Ideal Scenario)
- Best practice: Test fresh samples as soon as possible after collection
- Calprotectin is remarkably stable in fresh stool and can even be sent by ordinary mail 1
Short-Term Storage (Up to 3 Days)
- Refrigeration at 4°C is acceptable for samples that will be tested within 3 days 2
- After 3 days at room temperature, calprotectin concentrations remain unchanged, but after 7 days, a significant 28% decrease occurs 2
- Room temperature storage is problematic: One study showed a 35% decrease in calprotectin after 6 days at room temperature, while samples stored at 4°C were significantly better preserved 3
Longer-Term Storage
- For storage beyond 3 days: Freeze samples at -20°C (short-term, up to 3 months) or -80°C (long-term) 4, 5
- Frozen samples are stable: Calprotectin extracted and frozen remains stable for up to 2.5 months 6
- Immediate freezing at -20°C or -80°C is recommended for optimal preservation 5
Critical Preanalytical Considerations
The Refrigeration Imperative
Children with IBD and their caretakers may be falsely reassured by low calprotectin values if samples are not properly refrigerated 3. The best advisable standard for preanalytical calprotectin handling is refrigeration of the stool sample until delivery at the hospital laboratory 3.
Extraction Buffer Stability
- Calprotectin in extraction buffer shows excellent stability at room temperature for 4 days when using specific extraction buffers (Calpro AS buffer) 7
- This finding suggests that patients could perform extraction at home for convenience and hygiene, then mail the extracted sample 7
- However, extraction buffer stability showed a 46% decrease after 6 days at room temperature 3
Freeze-Thaw Cycles
- Avoid multiple freeze-thaw cycles: These have considerable impact on microbial cell integrity and can alter results 5
- Aliquot fresh samples before initial freezing to facilitate subsequent analyses and avoid repeated thawing 5
Practical Clinical Algorithm
- Same-day testing: Use fresh sample without freezing
- Testing within 3 days: Refrigerate at 4°C immediately after collection
- Testing delayed 3-7 days: Refrigerate at 4°C (acceptable but suboptimal)
- Testing beyond 7 days: Freeze at -20°C or -80°C immediately after collection
- Long-term storage: Use -80°C for storage beyond 3 months
Important Caveats
Assay Variability
- Use the same assay type and laboratory for a given patient to compare results over time 8, 9
- Fecal calprotectin assays are not interchangeable - variations of 2.5- to 5-fold differences exist between different assays testing the same sample 9
- Within-stool variation ranges from 8-9% at lower calprotectin levels to 18-33% at higher levels 9
When Results Are Uncertain
If borderline or unexpected results occur, repeat fecal calprotectin testing or endoscopic evaluation for confirmation may be required 8, 9, as there can be substantial within-stool and within-day variation from a single patient.
Temperature Excursions
The key message is that calprotectin is not stable at room temperature beyond 3 days 3. Any delay in refrigeration or freezing risks underestimation of true inflammatory burden, potentially leading to undertreatment.