Specimen Collection, Preparation, Handling, Storage, and Shipping for In-House and Reference Labs

Blood Collection Tubes: Additives and Uses

Choosing the correct tube is the first and most critical step in pre-analytical specimen handling. Each tube contains a specific additive that preserves or anticoagulates the sample in a way matched to the intended test. Using the wrong tube invalidates results and wastes the patient's blood draw.

Order of Draw and Filling Technique

Although veterinary medicine has less rigid order-of-draw consensus than human phlebotomy, a practical recommended sequence minimizes cross-contamination of additives:

1. Blue (citrate) first — citrate tubes are most sensitive to underfilling and additive carryover from other tubes.
2. Red or SST — plain serum tubes for chemistry and serology.
3. Green (heparin) — plasma chemistry.
4. Lavender (EDTA) — hematology.
5. Gray (fluoride) — glucose and lactate preservation last, because potassium oxalate additive would contaminate earlier tubes.

Fill additive tubes to the marked fill line. Underfilling a citrate (blue) tube changes the 1:9 citrate-to-blood ratio and falsely prolongs coagulation times. Underfilling EDTA tubes concentrates the additive and can cause RBC shrinkage, affecting hematocrit and MCV.

Handling After Collection

• Invert immediately. Gently invert additive tubes 8 to 10 times immediately after drawing. Never shake or vortex — vigorous mixing lyses RBCs, causing hemolysis.
• Label at the cage side. Every tube must carry patient name or ID, date, time of collection, and initials of the collector. Mislabeling is a leading cause of rejected specimens at reference laboratories.
• Inspect for hemolysis and lipemia. Note gross appearance; hemolyzed samples may require recollection for tests like potassium, AST, CK, and LDH.
• Maintain temperature. Most whole blood samples for hematology are held refrigerated (2–6°C) if not run within 1–2 hours. Blood smears should be made fresh within minutes of collection, before leukocyte and platelet morphology degenerates.

Time-Sensitive Pre-Analytical Pitfalls

Blood is a living tissue that continues to metabolize after collection. Failing to account for these changes produces falsely low or falsely high results.

• Glucose consumption. RBCs and WBCs continue glycolysis in the tube, consuming glucose at roughly 7 to 10 percent per hour in unspun whole blood. To preserve glucose: use a gray (fluoride) tube, or centrifuge a red/SST tube within 30 minutes and separate the serum. Never leave whole blood at room temperature for hours before running glucose.
• Potassium falsely elevated. Hemolysis releases intracellular potassium from RBCs. In species with high-K RBCs (horses, cattle, some dogs like Akitas and Shiba Inus), potassium rises rapidly if serum sits on the clot. Heparin (green) plasma avoids this problem for potassium measurement.
• Calcium and magnesium falsely low. EDTA chelates divalent cations. If a lavender tube is accidentally submitted for a chemistry panel, calcium and magnesium read falsely low and potassium reads falsely high (with K2-EDTA). This is a classic and dangerous pre-analytical error.
• Sodium and chloride altered by citrate. Blue tubes contain sodium citrate; never submit blue-tube plasma for sodium or chloride measurement.
• Enzyme degradation. LDH and acid phosphatase are heat-labile; AST and ALT are relatively stable. Refrigeration slows but does not stop degradation of sensitive analytes.
• Platelet clumping. EDTA can induce platelet clumping in some patients, causing automated counters to undercount platelets. If a platelet count looks implausibly low, examine a blood smear to confirm.

In-House vs Reference Laboratory Submission

In-house analyzers deliver rapid turnaround for urgent cases but require daily quality control, calibration verification, and operator competency. Reference laboratories offer broader test menus, boarded-pathologist review, and stricter QC, but require correct shipping.

Shipping guidelines for reference labs:

• Serum or plasma (already separated): ship frozen on dry ice or refrigerated on cold packs per lab instructions.
• Whole blood for hematology: ship refrigerated on cold packs, never frozen — freezing lyses all cells and renders the sample useless.
• Coagulation samples: citrate plasma is stable refrigerated for about 4 hours; for longer transport, double-spin and freeze the platelet-poor plasma.
• Blood smears: make and air-dry before shipping; do not stain if the lab prefers to stain in-house. Ship at room temperature in slide mailers.
• Urine and body fluids: follow specific lab instructions; most urine for culture must be refrigerated and shipped on cold packs within 24 hours.

Common Rejection Criteria

Reference labs reject specimens for: incorrect tube, unlabeled or mismatched tube, hemolysis interfering with requested analyte, insufficient volume (QNS — quantity not sufficient), clot in an anticoagulant tube, expired or wrong temperature on arrival, and leaky or broken container. Avoiding these errors ensures the patient's blood draw yields actionable diagnostic results.