Lab Sample Collection and Handling

Options: In-house (quick results, quality depends on upkeep/staff skills) or referred out (turnaround time based on transport, clinical pathologist review available).
Decision Factors: Urgency, complexity, clinic resources, specific tests required, client finances.

Whole Blood: Comprises Red Blood Cells (RBCs), White Blood Cells (WBCs), Platelets, and Plasma. Transports oxygen, carbon dioxide, nutrients, waste, hormones; vital for the immune system. Requires an appropriate anticoagulant for examination.
Plasma: Fluid portion of blood containing anticoagulant, after formed elements are removed by centrifuging. Contains fibrinogen, clotting factors, and proteins. Clear and straw-colored.
Serum: Clear, straw-colored liquid portion of blood remaining after clotting. Does not contain fibrinogen, formed elements, or anticoagulant.

Preparation: Know requested tests to determine vein, volume, and required tubes. Collect samples before starting treatments; note if treatments initiated.
Blood Source: Preferred is venous blood. Common sites vary by animal (e.g., Dog: Cephalic, jugular; Cat: Jugular, cephalic).
Site Preparation: Clean site with alcohol, allow to dry. Minimize manual restraint to reduce stress and sample compromise.
Reliability: Proper collection and handling are paramount for reliable results. Develop a consistent routine.

Needle and Syringe (Small Animals/Exotics): Use largest practical gauge needle; syringe size close to required volume. When transferring to vacutainer, remove needle from syringe and cap from vacutainer, then inject gently down the side of the tube (do NOT stick needle into stopper).
Vacutainer System: Composed of needle, needle holder, and collection tubes. Use correct tube size to prevent artifacts and vein collapse.
General Procedure: Gather equipment, label tube before collection (patient name, date, time, sample type). Perform venipuncture with minimal tissue injury. Apply pressure post-withdrawal. Gently mix anticoagulant tubes (minimum $10$ inversions or figure-eight motion).
Filling Tips: Select a site providing sufficient volume and tolerated by animal. Use correct vacutainer size (various sizes from $250 \mu L$ to $15 \text{ mL}$ ). Check tube expiration. Use minimum negative force, gentle/pulsing force on syringe. Use a large bore needle to reduce hemolysis. Fill tubes to required amount for proper anticoagulant mixing.

Anticoagulants: Prevent/delay clotting; use specific anticoagulant for ordered test to avoid result interference.
Red-Top Tube (Serum): No anticoagulant. Used for biochemical tests, storage (histology, hair, urine). NEVER for hematology.
Green-Top Tube (Heparin): Heparin (sodium, potassium, lithium, or ammonium salt) anticoagulant. Used for biochemistry, particularly when whole blood is required. NEVER for hematology.
Lavender-Top Tube (EDTA): Ethylenediaminetetraacetic acid (sodium or potassium salt). Anticoagulant of choice for hematology tests; preserves cell volume and morphology. NEVER for chemistry analysis. Mix gently after blood addition. Excess EDTA can nullify automated analysis.
Blue-Top Tube (Oxalate and Citrate): Interfere with clot formation by binding calcium. Used for coagulation tests. Interfere with chemistry results; some require refrigeration.
Grey-Top Tube (Sodium Fluoride): Preserves blood glucose (effectiveness questionable). Interferes with many enzyme tests.
Tiger-Top Tube (Sure-Sep/Serum Separator): Red-top variation, no anticoagulant. Contains gel to separate cells from serum after centrifugation; prevents cell metabolism of analytes.

Serum: Collect into red-top tube. Allow to sit at room temperature for $20-30$ minutes for clot formation. Loosen clot. Centrifuge $2000-3000$ rpm for $10$ minutes. Pipette serum to new labeled tube. Process immediately or refrigerate/freeze.
Plasma: Collect into appropriate anticoagulant tube. Mix gently. Centrifuge within $1$ hour of collection at $2000-3000$ rpm for $10$ minutes. Pipette plasma to new labeled tube. Process immediately or refrigerate/freeze.

Volume depends on tests and patient hydration status.
Well-hydrated patients yield approximately $1/2$ of whole blood volume as serum or plasma. Dehydrated patients yield about $1/2$ of that.
Ideal to collect enough for $3$ runs to account for technical error, instrument failure, dilution, or forwarding to a reference lab.

Timeliness: Blood for CBC should be analyzed within $1$ hour. If not possible, prepare a blood smear and refrigerate the blood tube. DO NOT refrigerate smears.
Temperature: Analyze refrigerated blood at room temperature; remove about $30$ minutes prior. Mix gently before testing.
Freezing: NEVER freeze whole blood samples for hematology. If freezing for chemistry, harvest serum/plasma first; DO NOT freeze whole blood. Thawed samples cannot be re-frozen.
Tube Filling: Fill tubes completely to prevent artificial reduction in Packed Cell Volume (PCV) due to anticoagulant dilution.
Difficult Veins: For small veins, use heat therapy or liberal alcohol; avoid large volumes of alcohol if blood alcohol testing is required.

Purpose: Minimizes cross-contamination of anticoagulants, which can lead to inaccurate results. Higher cross-contamination risk with needle and syringe.
Needle/Syringe Technique: Ensure syringe tip does not touch the inside of vacutainers.
Recommended Order: Culture tubes/bottles $ ightarrow$ Sodium citrate tubes $ ightarrow$ Serum tube $ ightarrow$ Serum separator tube $ ightarrow$ Heparin tube $ ightarrow$ EDTA tube $ ightarrow$ Sodium fluoride/Potassium oxalate.

Importance: Vital for continuity of care and avoiding human error.
Accuracy: Double-check math; report correct units (conventional vs. SI).
Lab Forms: Should include patient/owner name, signalment, clinical history, clinician info, requested tests, results, RVT initials, remarks, and case identification number.

Abnormal results can arise from pathological conditions, hemolysis, icterus, lipemia, medications, diet, and improper sample handling.

Causes: Aspiration of alcohol, damp syringe, small diameter needle, excessive suction, transferring blood via needle through vacutainer stopper, transferring too quickly, rough sample agitation, over-centrifuging, freezing whole blood.
Impacts: Artificially dilutes sample, decreasing some analyte concentrations. Increases concentrations of some metabolites due to cell rupture. Interferes with lipase activity and alters bilirubin determination.

Contamination: Detergents/disinfectants or cross-contamination from anticoagulants affect chemistry results. Avoid reusing tubes/syringes; follow order of draw; avoid syringe tip touching tubes.
Labelling: Essential and easily avoidable error. Tubes must be labelled with patient ID, date, time of collection, and sample type (e.g., urine, serum, plasma can look similar).

Patient Status: Ideally, patients are fasted for $12$ hours before blood collection. Non-fasted samples can show decreased inorganic phosphorus or increased glucose, cholesterol, and lipids (lipemia increases sample turbidity).
Reference Ranges: