Procedures
Enzymes play a crucial role in diagnosing and monitoring various diseases. Understanding the principles of enzyme assays, along with proper pre-analytical and analytical techniques, is essential for generating accurate and reliable results
Principles of Enzyme Assays
- Kinetic Assays: Measure the rate of enzyme-catalyzed reactions continuously
- Fixed-Time Assays: Measure the amount of product formed or substrate consumed at a single point in time
- Enzyme Activity Assays: Measure the catalytic activity of the enzyme
- Enzyme Concentration Assays: Measure the amount of enzyme protein present
Specimen Collection
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Patient Preparation
- Fasting: Some enzymes require fasting
- Medications: Certain medications can affect enzyme levels
- Recent Procedures: Defer testing if affected
- Exercise: Strenuous exercise can elevate some levels
- Specimen Type: Serum is the most common, some use plasma
- Tube Type: Follow lab guidelines
- Order of Draw: Follow the CLSI or other standards
- Tourniquet Time: Keep this short to avoid hemoconcentration
- Collection Technique: Clean venipuncture to avoid hemolysis
Specimen Handling and Processing
- Timing: Process ASAP
- Centrifugation: Follow instructions
- Storage: Refrigerate or freeze, avoid repeated freeze-thaw cycles
- Hemolysis: Avoid and reject hemolyzed samples
Analytical Considerations
- Reagent Quality: Store reagents according to instructions, check expiration dates
- Calibration: Use appropriate standards, calibrate regularly
- Quality Control (QC): Run controls regularly, evaluate results before releasing data
- Instrumentation: Maintain and service instruments per schedule
- Method Validation: Validate new methods
Interfering Substances
- Endogenous: Lipemia, bilirubin, hemoglobin, urea, high protein concentrations
- Exogenous: Medications, anticoagulants, contaminants
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Minimizing Interferences
- Use interference-reducing techniques
- Dilute sample (if appropriate)
- Employ blanking or background subtraction
Troubleshooting
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High Enzyme Results
- Pre-Analytical: Patient factors, collection/processing errors
- Analytical: Calibration, reagents, interferences, instruments
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Low Enzyme Results
- Pre-Analytical: Enzyme degradation, improper storage
- Analytical: Calibration, reagents, instrument issues
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Inconsistent Results
- Check patient factors, collection/processing, reagents/calibration, interferences, and instruments
- Out-of-Control QC: Troubleshoot QC materials, calibration, reagents, and instruments