Should the 24‑hour urine collection be refrigerated during the collection period?

Should 24-Hour Urine Collection Be Refrigerated?

Yes, refrigerate the 24-hour urine collection container at 4°C throughout the entire collection period to prevent bacterial overgrowth, cell lysis, and molecular degradation—this is the standard recommendation for most clinical applications. 1

Standard Refrigeration Protocol

• Keep the collection container refrigerated at 0-4°C continuously during the 24-hour collection period to avoid bacterial growth, cell lysis, molecular degradation of RNA and protein, and formation of sediments 1
• Maximum storage temperature of 4°C is recommended as an obligatory parameter with high evidence level for preventing microbial growth and biomolecule degradation 1
• Process samples within 8 hours maximum after collection completion when kept refrigerated 1

Analyte-Specific Considerations

When Refrigeration Is Essential:

• Catecholamines require continuous refrigeration throughout the 24-hour collection period without preservative, or room temperature storage only if hydrochloric acid (HCl) is added 2
• Routine urinalysis and microscopy require refrigeration if processing will be delayed beyond 1 hour 3, 4
• Subjects collecting for catecholamines should urinate every 3 hours during the collection day, as catecholamines can degrade in the bladder at body temperature 2

When Refrigeration May Be Optional:

• Glucocorticoids can be stored at room temperature during the 24-hour collection period without compromising preservation 2
• Fractionated metanephrines do not require refrigeration or preservatives if samples are frozen within 1 week, showing no clinically relevant degradation (<5%) under any storage conditions 5
• Stone risk metabolic parameters (calcium, oxalate, citrate, uric acid, magnesium, phosphorus, creatinine) show no significant deviation with storage at room temperature or 4°C for up to 72 hours 6, 7

Important Caveats and Pitfalls

Cold-Induced Artifacts:

• Refrigeration causes uromodulin polymer formation when urine is kept below 4°C, which can trap extracellular vesicles and cellular elements, potentially affecting certain specialized analyses 1, 3
• Some RNA content may decline more at 2-4°C compared to room temperature, though this primarily affects molecular studies rather than routine clinical chemistry 1

Preservative Considerations:

• For longer collection times (24 hours), consider adding preservatives such as azide to prevent microbial overgrowth, especially when the preservative is compatible with downstream processing 1
• Acidification is NOT necessary during collection for most metabolic stone parameters—acid can be added after collection is complete without altering results 6, 7
• Post-delivery acidification or alkalinization does not modify measured levels of urinary oxalate, calcium, magnesium, creatinine, and uric acid, allowing all parameters to be determined in a single sample 7

Practical Clinical Algorithm

1. Determine the specific analyte(s) being measured:

   • Catecholamines → Mandatory refrigeration throughout collection 2
   • Glucocorticoids → Room temperature acceptable 2
   • Metanephrines → Refrigeration optional if freezing within 1 week 5
   • Stone risk parameters → Refrigeration preferred but not critical if processed within 72 hours 6
   • Routine urinalysis/microscopy → Refrigeration mandatory if >1 hour delay 3, 4

2. Default to refrigeration at 4°C when in doubt or when multiple analytes are being measured simultaneously 1

3. Instruct patients to:

   • Store the collection container in the refrigerator between voids 1
   • Urinate every 3 hours if collecting for catecholamines 2
   • Deliver the sample to the laboratory promptly after the 24-hour period ends 1

4. Laboratory processing:

   • Process within 8 hours of collection completion 1
   • If acidification is needed, it can be performed after collection without compromising most analytes 6, 7