1.3 Specimen Collection, Handling, Order of Draw, and Phlebotomy Complications

The pre-analytical phase of testing encompasses everything that happens to a specimen before it is placed on an analyzer. It is universally recognized that up to 70% of all laboratory errors occur in the pre-analytical phase, with improper phlebotomy technique, incorrect specimen handling, and misidentification being the primary culprits. Ensuring specimen integrity is paramount; a laboratory can only produce a result as good as the sample it receives.

Phlebotomy Fundamentals

Phlebotomy is typically performed using the evacuated tube system (ETS). The standard needle gauge for routine adult venipuncture is 21-gauge to 22-gauge. Using a needle that is too small (e.g., 25-gauge) creates excessive shear force on erythrocytes, leading to mechanical hemolysis.

A critical step is the application of the tourniquet. The tourniquet should be applied 3-4 inches above the intended puncture site and must never be left on for longer than 1 minute. Prolonged tourniquet application causes hemoconcentration, where plasma water filters into the tissue, leading to artificial, localized increases in non-filterable blood components such as total protein, calcium, lipids, red blood cells, and potassium.

The Order of Draw

To prevent cross-contamination of tube additives, the Clinical and Laboratory Standards Institute (CLSI) mandates a strict order of draw. Exam questions frequently test the order, the additives, and the consequences of drawing tubes out of sequence.

1. Blood Cultures / Yellow Top (SPS): Drawn first to maintain absolute sterility. Sodium Polyanethol Sulfonate (SPS) is an anticoagulant that also inhibits complement and phagocytosis, aiding bacterial recovery.
2. Light Blue Top (Sodium Citrate): Used for coagulation testing (PT/INR, aPTT). Citrate binds calcium reversibly. Crucial Rule: This tube must be filled exactly to the fill line to maintain a precise 9:1 ratio of blood to anticoagulant. An underfilled tube will falsely prolong coagulation times.
3. Red, Gold, or Tiger Top (Serum Tubes): Contains no additive (plain red) or a clot activator and thixotropic gel (Serum Separator Tube, SST). Used for chemistry and serology. Must be allowed to clot completely (typically 30-60 minutes) before centrifugation.
4. Green Top (Heparin): Contains sodium, lithium, or ammonium heparin. Heparin inhibits thrombin formation. Used for STAT chemistry tests because it does not require time to clot. Exam Trap: Lithium heparin is preferred for most chemistries, but cannot be used to measure a lithium level. Sodium heparin cannot be used to measure sodium.
5. Lavender Top (EDTA): Used for hematology (CBC, reticulocyte count, HgbA1C). EDTA binds calcium tightly and preserves cellular morphology better than any other anticoagulant.
6. Gray Top (Sodium Fluoride / Potassium Oxalate): Used for glucose and lactic acid testing. Potassium oxalate is the anticoagulant (binds calcium), while sodium fluoride is an antiglycolytic agent. Fluoride inhibits the enzyme enolase, preventing RBCs from consuming glucose in the tube. Without fluoride, glucose levels in a tube drop by approximately 5-7% per hour.

Clinical Consequence of Wrong Order: If an EDTA tube is drawn before a Serum or Heparin tube, EDTA (potassium salt) can carry over on the needle. This causes a severe, artifactual increase in potassium (hyperkalemia) and a decrease in calcium (hypocalcemia) in the subsequent chemistry tube, mimicking a life-threatening emergency.

Specimen Interferences (HIL Indices)

Laboratories evaluate specimens for three major intrinsic interferences:

• Hemolysis (H): The rupture of RBCs, releasing intracellular contents into the serum/plasma. Visualized as a red/pink tint. Because potassium is highly concentrated inside RBCs, hemolysis causes a dramatic, false elevation in Potassium (K+), Lactate Dehydrogenase (LD), and Aspartate Aminotransferase (AST).
• Icterus (I): Extremely high levels of bilirubin, giving the serum a dark yellow, orange, or brown appearance. Icterus interferes with spectrophotometric assays by absorbing light at certain wavelengths.
• Lipemia (L): High levels of triglycerides/chylomicrons, giving the serum a milky, turbid, or cloudy appearance. Lipemia causes light scattering (turbidity), which falsely elevates absorbance readings in colorimetric assays and can cause false-low sodium results in indirect ion-selective electrode (ISE) methods due to the volume displacement effect.

Phlebotomy Complications

• Hematoma: The most common complication, caused by blood leaking into the surrounding tissue. If a hematoma begins to form, immediately remove the tourniquet, withdraw the needle, and apply firm pressure.
• Syncope (Fainting): If a patient faints or exhibits presyncopal symptoms (pallor, sweating, dizziness), immediately remove the needle and lower the patient's head.
• Petechiae: Small, non-raised red spots on the skin below the tourniquet. This indicates a capillary/platelet defect (e.g., thrombocytopenia) and warns the phlebotomist that the puncture site may bleed excessively after the draw.
• Iatrogenic Anemia: Anemia caused by excessive blood draws, particularly critical in neonatal and pediatric populations.

Special Specimen Handling Requirements

Certain analytes require special temperature or light handling immediately after collection:

• Chilled (Slurry of ice and water): Slows metabolic processes. Required for Ammonia, Lactic Acid, Parathyroid Hormone (PTH), and Arterial Blood Gases (ABGs, if not analyzed immediately).
• Warmed (37°C incubator/block): Prevents precipitation or binding. Required for Cold Agglutinins and Cryofibrinogen.
• Protected from Light (Aluminum foil or amber tube): Prevents photodegradation. Required for Bilirubin, Vitamin B12, Carotene, and Porphyrins.