In the world of healthcare, accuracy is more than a goal - it’s a necessity. Interference testing helps labs confirm that no external substance is skewing test results. The main goal of an Interference Experiment is to evaluate whether any other substances influence the accuracy of an analyte’s measurement.
Interference occurs when substances other than the intended analyte disrupt a test’s measurement, leading to patient results that are inaccurate. From hemolysis and lipemia to medications and sample contaminants, these unexpected intruders can affect nearly any assay if not properly understood and controlled.
What is an Interference?
Interference can be any substance that affects the experimental result by producing a response unrelated to the target analyte leading to erroneous results. Interferences can arise from various sources, including the sample matrix, the analytical procedure, or the instrument used for analysis.
These interferences are described as constant systemic errors because the amount of error is proportional to the concentration of the interferer.
During Method Verification, it is essential to identify and eliminate any interferences present to ensure that the analytical method is specific to the analyte of interest only.
Specificity in an MV experiment that refers to the method’s ability to assess the analyte of interest even in the presence of other components expected to be present in the sample matrix. This determines the method’s capability to identify the analyte separate from other substances present in the matrix.
Interference Experiment Design
Following recommendations from Clinical and Laboratory Standards Institute EP07, the minimal experiment design for Interference Verification is as follows:
- 3 replicates
Interference Experiment Procedure
Step 1 - Define the Experiment Design
Start off by establishing the experiment design including but not limited to the following:
- Acceptance Criteria - this is often based on the Total Allowable Error (TEa) for each analyte describing the maximum allowable bias due to interference
- Interferents to Test - for verification typically 1-2 levels of interferents are sufficient
- Hemolysis
- Icterus
- Lipemia
- Medications (e.g. acetaminophen, antibiotics)
- Anticoagulants
Common Interferences include but are not limited to the following:
Step 2 - Prepare Samples to be Used
Prepare the following pool samples for testing:
- Control Sample - this sample contains only the analyte of interest ideally at clinically relevant levels
- Test Sample - also known as a spiked sample containing both the analyte and the interferent to be tested
Step 3 - Test Replicate Measurements
Analyze both control and test samples using the same run and method to minimize variability. Ensure all test measurements following routine conditions and procedures to mirror real-life laboratory settings.
Step 4 - Perform Experiment Calculations
- Mean Values - Start off by computing the mean values for both test and control test results.
- Bias (Difference) - Obtain the difference between the two mean values using the formula:
Step 5 - Determine Acceptability
If calculated bias from Step 4 is equal to or less than the pre-established allowable bias, then the experiment is considered as passed.
Designing an Interference Experiment?
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References
Westgard, James. (2020). Basic Method Validation, 4th Edition. Wisconsin, Westgard QC, Inc.
Westgard, James. (2008). Interference and Recovery Experiments. Basic Method Validation. Retrieved from: https://www.westgard.com/lesson27.htm
Jensen, A.L., Kjelgaard-Hansen, M. (2008). Veterinary Clinical Pathology, 3rd edition, Vol. 35. The American Society for Veterinary Clinical Pathology. https://doi.org/10.1111/j.1939-165X.2006.tb00131.x
Naidis, I. , Turpeinen, S. (2009). Guidance for the Validation of Analytical Methodology and Calibration of Equipment used for Testing of Illicit Drugs in Seized Materials and Biological Specimens. New York, United Nations Publications.
European Medicines Agency. (1995). Note for Guidance on Validation of Analytical Procedures: Text and Methodology. Validation of Analytical Procedures. Retrieved from: https://www.ema.europa.eu/en/documents/scientific-guideline/ich-q-2-r1-validation-analytical-procedures-text-methodology-step-5_en.pdf