1.1 Specimen Collection, Order of Draw & Pre-analytical Interference

Specimen Collection and Pre-analytical Variables

Pre-analytical errors are the most common source of mistakes in the clinical laboratory, accounting for up to 70% of all diagnostic errors. Because clinical chemistry relies on precise measurement of analytes in serum or plasma, the Medical Laboratory Technician (MLT) must possess a deep understanding of proper specimen collection, handling, and the myriad of pre-analytical variables that can compromise test results.

The Order of Draw and Tube Additives

The Clinical and Laboratory Standards Institute (CLSI) defines a strict order of draw during venipuncture. This protocol minimizes the risk of cross-contamination from tube additives, which can drastically alter chemistry results. A classic example of such an error is drawing an EDTA tube (which contains potassium salts and chelates calcium) before a serum chemistry tube. This error leads to a critically elevated potassium level and a critically decreased calcium level, potentially resulting in inappropriate and dangerous patient treatment.

Specimen Types: Serum vs. Plasma

• Serum: The fluid portion of blood that remains after the blood has been allowed to clot. Serum does not contain fibrinogen, as it is consumed during the clotting process. Serum separator tubes (SSTs) must be allowed to clot fully in a vertical position for 20-30 minutes before centrifugation to prevent fibrin strand formation.
• Plasma: The fluid portion of anticoagulated blood. Plasma contains fibrinogen and all other clotting factors. Plasma separator tubes (PSTs), usually containing lithium heparin, can be centrifuged immediately, making them ideal for STAT chemistry testing.
• Whole Blood: Used for specific testing methodologies, including arterial blood gases (ABGs), lead testing, and hemoglobin A1c, where the cellular components or the intact whole blood matrix are required for analysis.

Pre-analytical Interferences (The HIL Index)

Automated chemistry analyzers routinely measure the HIL (Hemolysis, Icterus, Lipemia) indices using spectrophotometric readings at specific wavelengths. These indices help the laboratory flag compromised specimens and append appropriate disclaimers to the patient's report.

Hemolysis

Hemolysis is the rupture of red blood cells, which releases their intracellular components into the surrounding serum or plasma. It is visually characterized by a pink-to-red hue in the fluid portion of the spun sample.

Mechanisms of Interference:

• Release of Intracellular Contents: Hemolysis causes a significant artifactual elevation of analytes that are found in high concentrations inside red blood cells. The most critical of these is Potassium (K+). Other analytes that are falsely elevated include Lactate Dehydrogenase (LD), Aspartate Aminotransferase (AST), Acid Phosphatase (ACP), Magnesium (Mg2+), and Phosphate (PO4-).
• Colorimetric/Spectral Interference: Free hemoglobin absorbs light strongly at approximately 415 nm (the Soret band) and between 540-580 nm. This can interfere with spectrophotometric assays that measure absorbance at these wavelengths, causing falsely elevated or falsely decreased results depending on whether the assay measures an increase or decrease in absorbance.

Icterus (Hyperbilirubinemia)

Icterus refers to an abnormally high concentration of bilirubin in the blood, imparting a deep yellow, amber, or brown color to the serum or plasma. Icterus is commonly seen in patients with liver disease or hemolytic anemia.

Mechanisms of Interference:

• Spectral and Chemical Interference: Bilirubin strongly absorbs light between 400-500 nm (peaking around 460 nm). More importantly, bilirubin acts as a reducing agent and can interfere chemically with peroxidase-catalyzed reactions. For example, in the Trinder reaction (widely used for measuring glucose, cholesterol, and triglycerides), hydrogen peroxide is generated and reacts with a chromogen to produce a color. Bilirubin can scavenge the hydrogen peroxide before it reacts with the chromogen, leading to a falsely decreased measurement of the target analyte.

Lipemia

Lipemia is caused by the presence of large lipoprotein particles, specifically chylomicrons and very-low-density lipoproteins (VLDL), which scatter light and make the sample appear cloudy, milky, or turbid. Lipemia often occurs when blood is drawn shortly after a patient has eaten a fatty meal.

Mechanisms of Interference:

• Light Scattering: The large lipid particles scatter light across a wide range of wavelengths, causing false elevations in absorbance readings for many colorimetric assays.
• The Electrolyte Exclusion Effect (Volume Displacement): This is a critical source of error when measuring sodium using indirect Ion-Selective Electrode (ISE) methods. Indirect ISE methods dilute the sample before measurement, assuming that the sample is composed of roughly 93% water and 7% solids (proteins and lipids). In a lipemic sample, lipids displace a significant portion of the plasma water. Since electrolytes dissolve only in the water phase, the dilution factor is thrown off, leading to a falsely decreased sodium result (pseudohyponatremia). Direct ISE methods, which do not dilute the sample (often used in blood gas analyzers), are unaffected by this phenomenon.
• Resolution: Lipemic interference can be resolved by using ultracentrifugation (which forces the low-density lipids to the top, allowing the clear infranatant to be tested) or by treating the sample with lipid-clearing agents (cyclodextrins or lipases).

Physiological Pre-analytical Variables

Beyond collection errors, the patient's physiological state profoundly impacts chemistry results:

• Prolonged Tourniquet Application: Leaving the tourniquet on for more than one minute causes venous stasis. Fluid leaks from the veins into the tissues, resulting in hemoconcentration. This falsely elevates large, non-filterable molecules such as total protein, albumin, lipids, and protein-bound ions like calcium. Additionally, local tissue hypoxia causes cells to release potassium and switch to anaerobic metabolism, increasing lactic acid and decreasing pH.
• Delayed Centrifugation: If blood cells are not separated from serum/plasma promptly (within 2 hours), cellular metabolism continues. White and red blood cells will consume glucose (glycolysis), causing glucose levels to drop by 5-7% per hour. Simultaneously, potassium, LD, and AST will slowly leak out of the cells, causing false elevations.
• Posture and Position: Moving from a supine (lying down) to an upright position shifts fluid from the intravascular space to the interstitial space. This can increase the concentration of proteins and protein-bound substances (calcium, cholesterol, triglycerides) by up to 10%.
• Diurnal Variation: Some analytes exhibit significant fluctuations based on the time of day. For example, cortisol levels peak in the early morning (around 8:00 AM) and reach their lowest point in the late evening. Iron levels are also typically higher in the morning.
• Special Handling Requirements:
  • Chilling on Ice: Specimens for ammonia, lactic acid, and blood gases must be transported in an ice slurry to slow down cellular metabolism.
  • Protection from Light: Bilirubin and porphyrins are photosensitive and will degrade rapidly if exposed to ambient light; tubes must be wrapped in foil or placed in amber transport bags.
  • Keeping Warm: Cold agglutinins and cryoglobulins must be maintained at 37°C until the serum is separated to prevent precipitation.