Laboratory

Overview of the Testing Process

In clinical laboratories, the testing process is generally divided into three phases:

• Pre-Analytical Phase: Includes all steps that occur before the actual testing of the sample. This is where most errors happen, so it is super important to know these steps!
• Analytical Phase: Includes the actual performance of the tests and quality control procedures
• Post-Analytical Phase: Includes the steps that occur after the tests have been performed, such as result review, interpretation, and reporting

Pre-Analytical Phase

This phase encompasses all the steps that occur before the sample is analyzed, from test ordering to sample collection and transport. Errors in the pre-analytical phase are the most common source of errors in the laboratory and can significantly impact the accuracy and reliability of coagulation results

• Test Ordering:
  • Appropriate Test Selection: Ensure that the correct coagulation tests are ordered based on the patient’s clinical presentation and the physician’s request
    • Common Coagulation Tests:
      • Prothrombin Time (PT) and International Normalized Ratio (INR)
      • Activated Partial Thromboplastin Time (aPTT)
      • Fibrinogen Level
      • D-dimer
      • Mixing Studies
      • Factor Assays (e.g., Factor VIII, Factor IX)
      • Lupus Anticoagulant Testing
  • Review Patient History: Check the patient’s medication list (especially anticoagulants) and history of bleeding or thrombotic disorders
  • Follow Laboratory Guidelines: Adhere to the laboratory’s test ordering policies and procedures
• Patient Preparation:
  • Fasting: Some coagulation tests may require the patient to fast for a certain period of time
  • Medications: Certain medications (e.g., warfarin, heparin, direct oral anticoagulants) can affect coagulation test results and should be noted on the test requisition
  • Patient Identification: Properly identify the patient using two unique identifiers to prevent errors
• Sample Collection:
  • Collection Tubes:
    • Use the correct type of blood collection tube:
      • Sodium Citrate (light blue top): The anticoagulant of choice for most coagulation tests
      • EDTA (purple top): Not appropriate for coagulation testing
    • Check Expiration Date: Ensure that the collection tube is not expired
  • Blood-to-Anticoagulant Ratio:
    • The correct blood-to-anticoagulant ratio is critical for accurate coagulation testing:
      • Typically 9:1 (9 parts blood to 1 part anticoagulant)
    • Underfilling the tube can lead to falsely prolonged clotting times due to excess anticoagulant
    • Overfilling the tube can lead to falsely shortened clotting times due to insufficient anticoagulant
    • Adjust Blood-to-Anticoagulant Ratio in Patients with High Hematocrit: In patients with hematocrits >55%, the amount of anticoagulant should be reduced to maintain the correct ratio
    • Use Vacutainer to fill to the line on the collection tube
  • Order of Draw:
    • Follow the recommended order of draw to prevent contamination of coagulation samples:
      1. Blood culture tubes (if applicable)
      2. Coagulation tube (light blue top)
      3. Serum tubes (red top)
      4. Heparin tubes (green top)
      5. EDTA tubes (purple top)
      6. Fluoride tubes (gray top)
  • Venipuncture Technique:
    • Perform a clean venipuncture to avoid tissue thromboplastin contamination
    • Avoid prolonged tourniquet time, as this can activate coagulation factors
    • Use a 21-gauge needle or larger to minimize hemolysis
  • Mixing:
    • Gently invert the collection tube 5-10 times immediately after collection to mix the blood with the anticoagulant
    • Avoid vigorous shaking, as this can cause hemolysis
  • Labeling:
    • Properly label the collection tube with the patient’s name, date and time of collection, and other required information
    • Ensure that the information on the tube matches the information on the test requisition
• Sample Transport and Storage:
  • Transport:
    • Transport the sample to the laboratory promptly (within 1 hour) to minimize pre-analytical changes
    • Transport at room temperature (18-24°C)
    • Avoid extreme temperatures (e.g., direct sunlight, freezing)
  • Storage:
    • If testing cannot be performed immediately, store the sample according to the laboratory’s guidelines
    • Platelet Poor Plasma (PPP) may need to be prepared and frozen for some assays:
      • For most coagulation tests, plasma should be tested within 4 hours of collection or frozen for testing later. Specifics will vary from lab to lab.
      • Centrifuge the sample at 1500-2000 g for 15 minutes to obtain platelet-poor plasma (platelet count < 10 x 10^9/L)
      • Aliquot the plasma into labeled plastic tubes
      • Freeze the plasma at -20°C or -70°C if needed to test at later date
        • Thaw samples to room temperature before testing

Analytical Phase

This phase encompasses the actual testing of the sample and quality control procedures

• Instrumentation:
  • Use Properly Calibrated Instruments: Ensure that coagulation analyzers are properly calibrated and maintained according to the manufacturer’s instructions
  • Use Correct Reagents: Verify that the reagents are appropriate for the test being performed and that they are not expired
• Test Performance:
  • Follow Standard Operating Procedures: Adhere to standardized operating procedures (SOPs) for performing coagulation tests
  • Use Appropriate Controls: Run control materials at regular intervals (e.g., daily, with each batch of samples) to assess the accuracy and precision of the tests
  • Monitor Results: Review control results and evaluate them using statistical methods (e.g., Levey-Jennings charts, Westgard rules)
  • Take Corrective Action: If control results are outside the acceptable range, take corrective action before testing patient samples
• Sample Handling:
  • Ensure that samples are properly identified and stored
  • Thaw frozen samples completely before testing and mix gently
  • Check for hemolysis, lipemia, or other interfering substances
  • If necessary, perform corrective actions to minimize interference (e.g., saline replacement for lipemia)
• Common Test Specific Procedures * Prothrombin Time (PT): * Measures the function of the extrinsic and common pathways * Add thromboplastin reagent to plasma and measure the time to clot formation * Report results in seconds and as an International Normalized Ratio (INR) to standardize PT results * INR = (Patient PT / Mean Normal PT)ISI (ISI = International Sensitivity Index) * INR is used to monitor warfarin therapy * Activated Partial Thromboplastin Time (aPTT): * Measures the function of the intrinsic and common pathways * Add a contact activator (e.g., silica, kaolin), phospholipid, and calcium chloride to plasma and measure the time to clot formation * Used to monitor heparin therapy * Fibrinogen Assay: * Measures the concentration of fibrinogen in the plasma * Clauss method: A functional assay that measures the rate of clot formation after the addition of thrombin * D-Dimer Assay: * Measures the level of D-dimer in the plasma * Uses latex agglutination or immunoturbidimetric methods * Elevated in conditions with increased fibrinolysis (e.g., DVT, PE, DIC)
• Monitoring and Troubleshooting:
  • Monitor Instrument Performance: Regularly monitor the instrument’s performance to detect any malfunctions or errors
  • Troubleshoot Problems: Follow established procedures for troubleshooting instrument errors or unexpected results
  • Consult with a Supervisor or Pathologist: Seek guidance from a supervisor or pathologist if the cause of the problem cannot be determined

Post-Analytical Phase

This phase encompasses the steps that occur after the tests have been performed, including result review, interpretation, and reporting

• Result Review:
  • Review all test results and quality control data
  • Check for any flags, alarms, or abnormal results
  • Verify that the results are consistent with the patient’s clinical information and previous laboratory results
• Result Interpretation:
  • Interpret the results in the context of the patient’s clinical history, medication list, and other laboratory findings
  • Consider potential causes for any abnormal results
  • Recognize patterns of results that are suggestive of specific coagulation disorders
• Critical Value Notification:
  • Immediately notify the physician or other authorized personnel of any critical values (e.g., markedly prolonged PT or aPTT, very low fibrinogen level)
  • Follow the laboratory’s policy for critical value reporting
• Reporting:
  • Report the test results in a clear, concise, and accurate manner
  • Include the reference range for each parameter
  • Note any interfering substances or corrective actions taken
  • Add interpretive comments to the report to guide the clinician’s interpretation of the results
• Documentation:
  • Maintain accurate and complete records of all test results, quality control data, and corrective actions taken
  • Follow all regulatory and accreditation requirements for documentation

Key Terms

• PT (Prothrombin Time): Test of the extrinsic and common pathways
• aPTT (Activated Partial Thromboplastin Time): Test of the intrinsic and common pathways
• INR (International Normalized Ratio): A standardized ratio of the PT used to monitor warfarin therapy
• Fibrinogen: A protein that is converted to fibrin, the main component of a blood clot
• D-dimer: A fibrin degradation product that is elevated in thrombotic conditions
• Pre-Analytical Phase: The steps that occur before the sample is analyzed
• Analytical Phase: The actual performance of the tests
• Post-Analytical Phase: The steps that occur after the tests have been performed
• Quality Control: Procedures used to monitor the accuracy and precision of laboratory testing
• Critical Value: A test result that represents a life-threatening condition and requires immediate notification