Procedures

Lipid testing plays a critical role in assessing cardiovascular risk and diagnosing lipid disorders. To ensure the accuracy and reliability of these tests, it’s essential to understand the principles behind each method and the precautions necessary for specimen collection, processing, and analysis

Key Principles of Lipid Tests

• Total Cholesterol
  • Enzymatic methods convert cholesterol esters to cholesterol, which is then oxidized to produce hydrogen peroxide. The hydrogen peroxide reacts with a chromogen to form a colored product, which is measured spectrophotometrically
• Triglycerides
  • Enzymatic methods hydrolyze triglycerides to glycerol and fatty acids. Glycerol is then phosphorylated and oxidized to produce hydrogen peroxide. The hydrogen peroxide reacts with a chromogen to form a colored product, which is measured spectrophotometrically
• HDL Cholesterol
  • Direct methods use selective detergents or antibodies to block non-HDL lipoproteins, allowing for the measurement of HDL cholesterol. Indirect methods use precipitation reagents to selectively precipitate non-HDL lipoproteins
• LDL Cholesterol
  • Calculated using the Friedewald equation (LDL-C = Total Cholesterol - HDL-C - Triglycerides/5) when triglycerides are < 400 mg/dL. Direct methods are also available
• Apolipoproteins
  • Immunochemical methods, such as turbidimetry, nephelometry, or ELISA, are used to measure specific apolipoproteins

Special Precautions

• Patient Preparation
  • Fasting: Patients should typically fast for 9-12 hours before lipid testing
  • Medications: Certain medications can affect lipid levels
  • Recent Illness/Surgery: Acute illness, surgery, or trauma can affect lipid levels
  • Posture: The patient should be seated for at least 5 minutes before blood collection
• Specimen Collection
  • Tube Type: Serum separator tubes (SST) or plasma tubes with EDTA are commonly used
  • Order of Draw: Follow the correct order of draw to prevent cross-contamination
  • Tourniquet Time: Minimize tourniquet time to prevent hemoconcentration
• Specimen Processing
  • Timing: Process the specimen as soon as possible after collection
  • Centrifugation: Centrifuge the sample according to the manufacturer’s instructions
  • Storage: If analysis is delayed, store the specimen refrigerated or frozen
• Analytical Considerations
  • Reagent Quality: Use high-quality reagents and store them according to the manufacturer’s instructions
  • Calibration: Calibrate the instrument regularly using appropriate standards
  • Quality Control (QC): Run quality control samples at regular intervals to monitor the accuracy and precision of the assay

Troubleshooting

• High Lipid Results
  • Pre-Analytical: Patient not fasting, prolonged tourniquet time, improper collection tube
  • Analytical: Incorrect calibration, reagent deterioration, interfering substances
• Low Lipid Results
  • Pre-Analytical: Improper storage, lipid degradation
  • Analytical: Incorrect calibration, reagent deterioration
• Inconsistent Results
  • Check patient preparation, review collection and processing procedures, evaluate reagent quality and calibration, consider interfering substances

Interfering Substances

• Endogenous
  • Lipemia: Causes turbidity, affecting spectrophotometric readings
  • Bilirubin: Absorbs light at similar wavelengths, affecting absorbance readings
  • Hemoglobin: Absorbs light and can interfere with enzymatic reactions
• Exogenous
  • Ascorbic Acid (Vitamin C): Can interfere with certain enzymatic reactions
  • Medications: Certain drugs can directly affect lipid levels (e.g., statins, fibrates, beta-blockers, oral contraceptives)