8.4 Specimen Processing: Centrifugation, Aliquoting & Storage
Key Takeaways
- Serum and plasma should be separated from cells within 2 hours of collection to prevent analyte drift.
- Serum tubes must clot completely — about 30 minutes at room temperature — before centrifugation.
- Never re-centrifuge a gel tube that has already been spun, because analytes trapped below the gel are redistributed.
- Potassium rises and glucose falls when whole blood sits unseparated, and refrigerating whole blood accelerates potassium leakage.
- Aliquot tubes must be labeled with the same identifiers as the parent tube before any transfer.
8.4 Specimen Processing: Centrifugation, Aliquoting & Storage
Quick Answer: DTP task 3.16 is "process specimens for testing (e.g., centrifuge, aliquot, storage)." The controlling rule is the 2-hour separation window: serum or plasma must be separated from the cells within about 2 hours of collection, because cells continue to metabolize and exchange with the surrounding fluid until they are physically removed.
Why Separation Is Time-Critical
Blood cells do not stop living when the tube leaves the patient. While they remain in contact with serum or plasma, three processes continue:
| Process | Analyte effect |
|---|---|
| Glycolysis — cells consume glucose | Glucose falls roughly 5 to 10% per hour at room temperature |
| Potassium leakage — potassium moves down its gradient out of cells | Potassium rises, and refrigeration accelerates this by inhibiting the sodium-potassium pump |
| Cellular enzyme release | LDH, AST, and phosphorus rise |
[!WARNING] Never refrigerate an unseparated whole-blood specimen to "preserve" it. Cooling inhibits the ATP-dependent sodium-potassium pump that normally holds potassium inside the cell, so potassium leaks out far faster than at room temperature. A refrigerated unspun tube can produce a falsely critical potassium that triggers an emergency workup. If a glucose or potassium specimen cannot be separated promptly, a gray fluoride/oxalate tube is the correct pre-analytical answer for glucose.
Clotting Before Centrifugation
Serum tubes must clot completely before they are spun. Centrifuging a partially clotted tube produces latent fibrin formation in the serum, which clogs analyzer probes and can cause erroneous results.
| Tube | Clotting time before centrifugation |
|---|---|
| Red (plain, no additive) | 60 minutes at room temperature |
| Gold / tiger SST (clot activator) | Approximately 30 minutes |
| Rapid serum tubes (thrombin) | About 5 minutes per manufacturer |
| Anticoagulated tubes (lavender, green, light blue, gray) | No clotting time — spin immediately if the test requires plasma |
Patients on anticoagulant therapy may require longer clotting times, because the clot activator is working against therapeutic anticoagulation.
Centrifugation Rules
- Keep stoppers on. Centrifuging uncapped tubes creates aerosols — a bloodborne pathogen exposure — and allows evaporation and pH shifts from carbon dioxide loss.
- Balance the rotor. Place tubes of equal size and equal fill directly opposite each other. If you have an odd number, add a water-filled balance tube of matching weight. An unbalanced centrifuge damages the instrument and can shatter tubes.
- Spin at the specified speed and time. Typically about 1,000 to 1,300 relative centrifugal force for 10 minutes, per the tube manufacturer and laboratory procedure. Over-spinning can hemolyze; under-spinning leaves cells suspended.
- Never open the lid while the rotor is moving, and wait for a full stop before removing tubes.
- Do not re-centrifuge a gel tube. Once the gel barrier has formed, a second spin drives analytes trapped beneath it back into the serum layer, altering results. If more serum is needed, recollect.
- A broken tube inside a centrifuge is an aerosol event. Stop, allow aerosols to settle per policy, wear appropriate PPE, and decontaminate the chamber.
[!IMPORTANT] Serum versus plasma. Serum is the fluid remaining after blood has clotted — it contains no fibrinogen. Plasma is the fluid from an anticoagulated tube — it still contains fibrinogen and yields a slightly larger volume. They are not interchangeable, and a test validated for one may give a different result on the other.
Aliquoting
An aliquot is a portion of a specimen transferred into a separate labeled tube, usually so several departments can test from one collection or so a specimen can be frozen.
- Label the aliquot tube before transferring, with the same two patient identifiers and accession number as the parent tube. An unlabeled aliquot on a bench is an unidentifiable specimen.
- Never combine aliquots from different collection times or different tubes, even from the same patient.
- Use a transfer pipette, and use a fresh pipette for each specimen to prevent carryover.
- Do not pour, which risks splashing, aerosols, and transfer of cells or gel.
- Aliquot as soon after centrifugation as possible.
- Cap aliquots promptly to prevent evaporation and pH change.
Storage Conditions
| Condition | Typical use | Notes |
|---|---|---|
| Room temperature (15 to 30 °C) | Short-term holding before processing | The default for most specimens awaiting prompt separation |
| Refrigerated (2 to 8 °C) | Many separated serum and plasma specimens for 24 to 48 hours | Never for unseparated whole blood intended for potassium or glucose |
| Frozen (−20 °C or colder) | Extended storage of separated serum or plasma | Avoid repeated freeze-thaw cycles, which degrade analytes |
| 37 °C (warmed) | Cold agglutinins, cryoglobulins | Maintained from collection through separation |
| Ice-water slurry | Ammonia, lactic acid, blood gases | Slurry, not solid ice — direct contact with ice cubes hemolyzes cells |
| Protected from light | Bilirubin, vitamin B12, folate, carotene | Amber tube or foil wrap |
[!WARNING] Whole blood is never frozen. Freezing ruptures red cells and produces gross hemolysis. Only separated serum or plasma is frozen, and it must be in a plastic tube, since glass may crack.
Delivery and Turnaround
Processing ends with the specimen reaching the right place in the right condition. Deliver promptly, use secondary containment (a sealed biohazard bag with the requisition in the outer pocket, not against the specimen), and keep temperature-controlled specimens in their required condition throughout transport, not just at the ends of it. A chilled ammonia specimen that spent twenty minutes on a warm counter is no longer a chilled specimen.
A green-top heparin tube for a potassium level cannot be delivered to the laboratory for three hours. A technician refrigerates the unspun tube to preserve it. What is the consequence?
A laboratory receives a gold SST tube that yielded insufficient serum after centrifugation. A technician spins it a second time. Why is this incorrect?
Which centrifuge practice is correct?