What are the recommended vials and techniques for collecting blood samples for different laboratory tests?

Blood Sample Collection: Vial Selection and Techniques

The choice of blood collection tube depends entirely on the intended laboratory test, with EDTA tubes for hematology and plasma biomarkers, citrate tubes for coagulation studies, and plain/SST tubes for serum chemistry—each requiring specific handling protocols to prevent preanalytical errors that compromise test accuracy.

Tube Selection by Test Type

For Plasma Collection (EDTA or Heparin Tubes)

• EDTA tubes are preferred for hematology testing, cell counts, and most plasma biomarker analyses including Alzheimer's disease biomarkers, with K3EDTA at 1.5 ± 0.15 mg/mL blood being the standard anticoagulant 1.
• EDTA is particularly suitable for RNA analysis and genomic studies, whereas heparin interferes with PCR-based assays 1.
• Heparin tubes (sodium or lithium heparin) serve as alternatives when EDTA is contraindicated or for specific chemistry panels 1.
• Fill tubes completely until vacuum is exhausted to maintain proper blood-to-anticoagulant ratio, as underfilling causes clinically significant bias in test results 1, 2.

For Serum Collection (Plain or SST Tubes)

• Plain red-top tubes (glass) or serum separator tubes (SST) are used for chemistry panels, hormone assays, and serology 1.
• Glass plain tubes require no mixing, while all other serum tubes require 5 gentle inversions immediately after blood draw to mix with clot activators 1.
• Serum is preferred over plasma for small RNA studies (e.g., microRNA) despite additional extracellular vesicle release during clotting 1.

For Coagulation Testing (Citrate Tubes)

• Sodium citrate tubes (0.109 mol/L final concentration) are the gold standard for coagulation studies including PT, aPTT, and fibrinogen, as recommended by the International Society on Thrombosis and Haemostasis 1, 3.
• Citrate prevents platelet activation more effectively than other anticoagulants, critical for accurate coagulation testing 1.
• Tubes must be filled to at least 89% capacity for aPTT, 78% for fibrinogen, and 67% for factor VIII to avoid clinically significant bias 2.

For Specialized Applications

• Extracellular vesicle (EV) studies require citrate or acid citrate dextrose (ACD), with citrate-theophylline-adenosine-dipyridamole (CTAD) providing superior platelet activation inhibition 1, 4.
• Buffy coat collection for genomic analysis uses EDTA or heparin tubes with subsequent density gradient centrifugation 1.

Critical Collection Techniques

Order of Draw

Follow the CLSI-recommended order to prevent cross-contamination of additives 1:

1. Blood culture tubes
2. Sodium citrate tubes (coagulation)
3. Serum tubes (plain/SST)
4. Heparin tubes
5. EDTA tubes
6. Sodium fluoride/potassium oxalate tubes

Even microliter-level carryover of heparin into citrate tubes causes severe interference with coagulation testing at volumes beyond 5-100 μL 5.

Venipuncture Technique

• Use a 21-gauge needle to minimize hemolysis and cellular activation 1.
• Discard the first 2-3 mL of blood to eliminate tissue thromboplastin contamination, particularly critical for coagulation studies 1.
• Avoid prolonged tourniquet use (>1 minute) to prevent hemoconcentration and platelet activation 1.
• Collect blood into plastic tubes at room temperature, keeping tubes vertical during transport 1.

Immediate Post-Collection Handling

• Gently invert EDTA tubes 8-10 times immediately after collection to ensure proper anticoagulation and prevent microclot formation 1.
• Serum tubes (except glass plain tubes) require 5 inversions, then must sit upright at room temperature for 30-60 minutes to allow complete clotting 1, 6.
• Never shake tubes vigorously, as this causes hemolysis and cellular activation 1.

Processing Timelines

EDTA/Heparin Plasma

• Centrifuge within 2 hours of collection at room temperature, or refrigerate at 4°C for maximum 24 hours before processing 1, 7.
• Centrifuge at 1500-2000 × g for 10 minutes at 4°C 1.
• For cell-free DNA analysis, the 2-hour limit is critical to prevent genomic DNA contamination from cell lysis 7.

Serum (Plain/SST Tubes)

• Allow 30-60 minutes clotting time at room temperature, then centrifuge within 2 hours total from collection 1, 7, 6.
• If immediate centrifugation is impossible, refrigerate at 4°C for maximum 2 hours after clotting 1, 7, 6.
• Centrifuge at 1500-2000 × g for 10 minutes at 4°C 1, 6.

Citrate (Coagulation)

• Process within 4 hours of collection for most coagulation tests, though specific assays may require faster processing 3.
• Centrifuge at room temperature to avoid cold activation of factor VII 1, 3.

Storage and Aliquoting

Immediate Post-Centrifugation

• Transfer plasma/serum to clean tubes without disturbing the cellular layer or clot 1.
• For plasma, avoid the buffy coat (white layer) between plasma and red cells 1.
• Aliquot 250 μL to 1 mL volumes into O-ring-sealed screw-cap cryovials to minimize freeze-thaw cycles 1.

Long-Term Storage

• Store aliquots at -80°C or colder immediately after processing 1, 7.
• For extracellular vesicle studies, snap-freeze in liquid nitrogen before -80°C storage to prevent ice crystal formation 1.
• Never use -20°C for clinical specimens, as this temperature is insufficient for long-term stability 6.
• Avoid repeated freeze-thaw cycles, which degrade most biomarkers 1.

Common Pitfalls and Quality Control

Hemolysis Prevention

• Inspect all samples for hemolysis using a standardized reference chart (e.g., CDC Hemolysis Reference Palette), as hemolysis is the primary cause of unusable specimens 1.
• Hemolyzed samples should be rejected or results interpreted with extreme caution 1.

Tube Filling

• Underfilled tubes cause spurious results due to incorrect blood-to-anticoagulant ratios, with coagulation tests being most sensitive 1, 2.
• Document tube filling height if not completely filled 1.

Temperature Management

• Room temperature storage accelerates biomarker degradation exponentially compared to refrigeration 7, 6.
• For EV studies, maintain samples at 4°C and process within 6 hours when using EDTA-CTAD anticoagulant 4.

Documentation Requirements

• Record collection time, tube type, storage temperature, and time to processing for all research and specialized clinical samples 1.
• Note any deviations from protocol, including hemolysis grade and delayed processing 1.

Specimen-Specific Considerations

• Sodium citrate as anticoagulant yields falsely lower lactate concentrations compared to heparin or EDTA 8.
• Plasma and serum lactate concentrations are consistently 0.11-0.14 mmol/L higher than whole blood values 8.
• For overnight fasting subjects collecting EV samples, use citrate anticoagulant and discard first 2-3 mL 1.