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Chemistry

Special Precautions

Accurate testing for proteins and nitrogen compounds depends on careful technique and proper handling. This section will discuss

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

Specimen Collection

  • Patient Preparation
    • Fasting: Some tests require fasting (e.g., lipid profile, ammonia) to minimize interferences. Follow specific guidelines
    • Medications: Certain medications can affect protein and nitrogen compound levels. The healthcare provider should review the patient’s medication list
    • Recent Procedures: Recent surgeries, injections, or other medical procedures can affect levels of some substances
    • Diet: High-protein diets can affect urea and uric acid levels. Standardize dietary intake prior to testing
    • Exercise: Strenuous exercise can affect muscle enzyme levels (e.g., CK) and creatinine levels. Avoid strenuous exercise prior to blood collection
  • Specimen Type
    • Serum is the most common specimen type for protein and nitrogen compound assays
    • Plasma may be used for some assays, but follow the laboratory’s guidelines for anticoagulant selection
    • Urine is used for protein, creatinine, and porphyrin measurements
    • Cerebrospinal fluid (CSF) is used for protein measurements in neurological disorders
  • 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
    • Urine should be collected in a clean, dry container, with or without preservatives, depending on the test
    • CSF should be collected in sterile tubes
  • 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 degradation or changes in analyte concentrations
    • Analyze unstable analytes (e.g., ammonia) immediately
  • 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 proteins and other analytes
  • Hemolysis
    • Avoid hemolysis, as red blood cells contain high concentrations of many enzymes and proteins, which can falsely elevate results
    • Visually inspect the sample for hemolysis and reject hemolyzed samples
  • Lipemia
    • Lipemia (excess lipids) can interfere with spectrophotometric assays
    • Use lipemia clearing techniques (ultracentrifugation, lipid clearing reagents) or consider methods less affected by turbidity
  • Urine Specimens
    • Preservatives may be required for some urine tests to prevent bacterial growth or analyte degradation
    • Follow specific instructions for urine collection and storage

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
    • High Protein Concentrations: Can interfere with enzymatic assays by affecting the reaction kinetics or detection system
  • Exogenous
    • Medications: Certain drugs can directly affect protein and nitrogen compound levels
    • Anticoagulants: Some anticoagulants can interfere with enzymatic reactions
    • Contaminants: Improperly cleaned glassware or containers can introduce contaminants that affect assay performance
  • 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 Results
    • Pre-Analytical:
      • Patient not fasting
      • Prolonged tourniquet time
      • Hemolysis
      • Improper collection tube
    • Analytical:
      • Incorrect calibration
      • Reagent deterioration
      • Interfering substances
      • Instrument malfunction
  • Unexpectedly Low Results
    • Pre-Analytical:
      • Improper storage
      • Analyte 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

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: Reproducibility of a measurement