Assay Development and Validation Requirements for Lamotrigine and Zonisamide Testing in Clinical Laboratories
This guidance addresses lamotrigine and zonisamide assays that: - Employ immunoassay technologies - Are for use in central clinical laboratories - Are for use with serum or plasma samples - Are regulated under 21 CFR 862.3350 as Class II devices
What You Need to Know? 👇
What are the key performance characteristics required for lamotrigine and zonisamide assay validation?
The guidance requires evaluation of precision, trueness/bias, linearity, lower limits, interference testing, matrix comparison, specimen storage, method comparison, and calibrator/control material validation across the entire reportable range using appropriate patient samples.
How many patient samples are needed for method comparison studies?
A minimum of 100 patient samples distributed throughout the measuring range is recommended, following CLSI EP9-A2 guidelines, with sufficient sample size to account for assay precision and inter-individual variations.
What specific interferents must be tested for these antiepileptic drug assays?
Testing should include endogenous compounds (bilirubin, triglycerides, hemoglobin), all antiepileptic drugs, antipsychotics, antidepressants, tranquilizers, antibiotics, and over-the-counter drugs at concentrations three times therapeutic peak levels.
What metabolites require cross-reactivity testing for these assays?
For lamotrigine: N-2 glucuronide, N-5 glucuronide, N-2 methyl metabolite, and N-2 oxide. For zonisamide: N-acetyl zonisamide (NAZ) and 2-sulfamoylacetyl phenol (SMAP) at concentrations three times peak patient levels.
How should therapeutic reference ranges be presented in labeling?
The guidance recommends stating that therapeutic ranges are not well-established, citing balanced literature studies, and emphasizing that considerable overlap exists between responders/non-responders and that results should be interpreted with clinical observation.
What calibrator traceability requirements apply to these assays?
Calibrators should be traceable to the best available reference material (preferably USP material), with clear characterization of assigned value accuracy, uncertainty of measurement, and recovery evaluation relative to the higher-level standard.
What You Need to Do 👇
Recommended Actions
- Design comprehensive validation protocol covering all performance characteristics
- Conduct precision studies over 20 days with multiple concentrations
- Perform recovery/bias studies using reference materials
- Evaluate linearity across entire measuring range
- Validate lower limits of quantitation
- Complete interference testing for endogenous and exogenous substances
- Test cross-reactivity with key metabolites
- Conduct method comparison with minimum 100 patient samples
- Validate stability of calibrators and controls
- Prepare detailed labeling addressing all required elements
- Document traceability of calibrators to reference materials
- Consider need for external site studies based on assay complexity
Key Considerations
Clinical testing
- Method comparison study with minimum 100 patient samples across measuring range
- Samples should be from intended use population with unaltered patient samples
- Multiple specimens from individual patients should be appropriately analyzed statistically
Non-clinical testing
- Precision evaluation over 20 day period for 3+ drug concentrations spanning reportable range
- Recovery/bias studies using reference materials spiked into drug-free clinical samples
- Linearity evaluation across entire reportable range with 7-9 concentration levels
- Lower limits evaluation including precision and bias relative to reference materials
Software
- Follow FDA guidance for software contained in medical devices if device includes software
Labelling
- Must satisfy requirements in 21 CFR 809.10
- Include specimen collection/storage instructions
- Detail quality control and calibration procedures
- List warnings and precautions
- State limitations of procedure
- Provide reference ranges with appropriate context
- Summarize performance characteristics
Safety
- Test blood products for communicable disease agents per 21 CFR 610.40
- Include appropriate warnings for hazardous materials
Other considerations
- Evaluate endogenous and exogenous interference following CLSI EP7-A2
- Test cross-reactivity with drug metabolites
- Validate matrix comparison for all recommended matrices
- Demonstrate calibrator and control materials stability
- Consider external site studies for complex assays
Relevant Guidances 🔗
- Content of Premarket Submissions for Device Software Functions
- Recommended Format and Content for Non-Clinical Bench Performance Testing in Medical Device Premarket Submissions
- Content and Decision-Making Process for 510k Submissions: Determining Substantial Equivalence
- Refuse to Accept Policy for 510k Submissions: Minimum Threshold Requirements for Administrative and Technical Review
- The Abbreviated 510k Program: Using Guidance Documents, Special Controls, and Standards for Substantial Equivalence Demonstration
Related references and norms 📂
- CLSI EP5-A2: Evaluation of Precision Performance of Quantitative Measurement Methods
- CLSI EP7-A2: Interference Testing in Clinical Chemistry
- CLSI EP9-A2: Method Comparison and Bias Estimation Using Patient Samples
- CLSI EP17-A2: Evaluation of Detection Capability for Clinical Laboratory Measurement Procedures
- ISO 17511: Metrological Traceability of Values Assigned to Calibrator and Control Materials