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Chemistry

Special Precautions

Accurate enzyme testing relies on strict attention to detail throughout the entire process, from the patient to the instrument. This section outlines the key considerations for reliable enzyme measurements

  • Specimen Collection
  • Specimen Handling and Processing
  • Analytical Considerations
  • Interfering Substances
  • Troubleshooting

Specimen Collection

  • Patient Preparation
    • Fasting: Some enzyme tests require fasting (e.g., lipase) to minimize the effects of lipemia. Follow specific guidelines for each test
    • Medications: Certain medications can affect enzyme levels. The healthcare provider should review the patient’s medication list and determine if any medications need to be temporarily discontinued before testing
    • Recent Procedures: Recent surgeries, injections, or other medical procedures can affect enzyme levels. Defer testing until the patient has recovered
    • Exercise: Strenuous exercise can elevate muscle enzymes (e.g., CK). Avoid strenuous exercise prior to blood collection
  • Specimen Type
    • Serum is the most common specimen type for enzyme assays
    • Plasma may be used for some enzyme assays, but follow the laboratory’s guidelines for anticoagulant selection
  • Tube Type
    • Serum separator tubes (SST) or red-top tubes are commonly used for serum collection
    • Plasma tubes with heparin or EDTA may be used for some assays, but follow the laboratory’s guidelines
  • Order of Draw
    • Follow the correct order of draw when collecting multiple tubes to prevent cross-contamination
  • Tourniquet Time
    • Minimize tourniquet time (ideally less than 1 minute) to prevent hemoconcentration
  • Collection Technique
    • Use a clean venipuncture technique to minimize hemolysis. Avoid prolonged probing or excessive force

Specimen Handling and Processing

  • Timing
    • Process the specimen as soon as possible after collection to prevent enzyme degradation
    • Analyze unstable enzymes (e.g., CK-MB) within a short time frame
  • Centrifugation
    • Centrifuge the sample according to the manufacturer’s instructions for the specific tube type
    • Proper centrifugation ensures complete separation of serum or plasma from cells
  • Storage
    • If analysis is delayed, store the specimen refrigerated (2-8°C) for short-term storage or frozen (-20°C or -70°C) for longer storage
    • Aliquot the sample into smaller portions to avoid repeated freeze-thaw cycles, which can degrade enzymes
  • Hemolysis
    • Avoid hemolysis, as red blood cells contain high concentrations of many enzymes (e.g., LDH, AST), which can falsely elevate results
    • Visually inspect the sample for hemolysis and reject hemolyzed samples

Analytical Considerations

  • Reagent Quality
    • Use high-quality reagents and store them according to the manufacturer’s instructions
    • Check expiration dates and discard expired reagents
  • Calibration
    • Calibrate the instrument regularly using appropriate standards
    • Use multiple calibrators to ensure linearity over the entire measurement range
  • Quality Control (QC)
    • Run quality control samples at regular intervals to monitor the accuracy and precision of the assay
    • Use both normal and abnormal QC levels to assess performance across the entire range
    • Evaluate QC results before releasing patient results
  • Instrumentation
    • Regularly maintain and service the instrument according to the manufacturer’s recommendations
    • Ensure the instrument is functioning properly and that all components are in good working order
  • Method Validation
    • Validate new methods before implementation to ensure accuracy, precision, linearity, and other performance characteristics

Interfering Substances

  • Endogenous
    • Lipemia: Causes turbidity, affecting spectrophotometric readings
    • Bilirubin: Absorbs light at similar wavelengths, affecting absorbance readings
    • Hemoglobin: Released from hemolyzed red blood cells, interfering with enzymatic reactions
    • Urea: High concentrations can interfere with some enzymatic reactions
    • High Protein Concentrations: Can interfere with enzymatic assays by affecting the reaction kinetics or detection system
  • Exogenous
    • Medications: Certain drugs can directly affect enzyme levels
    • Anticoagulants: Some anticoagulants can interfere with enzymatic reactions
    • Contaminants: Improperly cleaned glassware or containers can introduce contaminants that affect enzyme activity
  • Minimizing Interferences
    • Use methods less susceptible to interference
    • Employ techniques to remove or correct for the interfering substance
    • Dilute the sample to reduce the concentration of the interfering substance
    • Use appropriate blanking or background subtraction techniques

Troubleshooting

  • Unexpectedly High Enzyme Results
    • Pre-Analytical:
      • Patient not fasting
      • Prolonged tourniquet time
      • Hemolysis
      • Improper collection tube
    • Analytical:
      • Incorrect calibration
      • Reagent deterioration
      • Interfering substances
      • Instrument malfunction
  • Unexpectedly Low Enzyme Results
    • Pre-Analytical:
      • Improper storage
      • Enzyme degradation
    • Analytical:
      • Incorrect calibration
      • Reagent deterioration
      • Instrument malfunction
  • Inconsistent Results
    • Check patient preparation
    • Review collection and processing procedures
    • Evaluate reagent quality and calibration
    • Consider interfering substances
    • Repeat the test using a different method or on a new sample
  • Out-of-Control QC Results
    • Check the integrity of the QC materials
    • Prepare fresh QC materials
    • Re-calibrate the instrument
    • Review reagent quality
    • Repeat the QC testing
    • If the problem persists, contact the instrument manufacturer for assistance

Examples

  • Creatine Kinase (CK)
    • Interference from adenylate kinase (AK): AK catalyzes the conversion of ADP to ATP, which can interfere with CK assays
    • Use inhibitors of AK to minimize interference
  • Alkaline Phosphatase (ALP)
    • Inhibition by phosphate: Phosphate is a product of the ALP reaction and can inhibit the enzyme
    • Use optimized substrate concentrations to minimize inhibition
  • Amylase
    • Interference from chloride ions: Chloride ions are required for amylase activity, but high concentrations can inhibit the enzyme
    • Maintain appropriate chloride concentrations in the assay
  • Lipase
    • Requirement for colipase: Colipase is a cofactor required for lipase activity
    • Ensure adequate colipase is present in the assay

Key Terms

  • Pre-analytical: Processes that occur before the sample is analyzed
  • Analytical: Processes involved in analyzing the sample
  • Interfering Substance: A substance that affects the accuracy of a test
  • Hemolysis: The breakdown of red blood cells
  • Lipemia: The presence of excess lipids in the blood
  • Calibration: Adjusting an instrument to ensure accurate readings
  • Quality Control: Samples used to monitor the accuracy and precision of a test
  • SST: Serum Separator Tube
  • EDTA: Ethylenediaminetetraacetic acid, an anticoagulant
  • SOP: Standard Operating Procedure
  • Accuracy: How close a measurement is to the true value
  • Precision: How reproducible a measurement is