Use of Next Generation Sequencing in the Diagnosis of Germline Diseases

What You Need to Know? 👇

What are the key regulatory pathways for NGS-based tests intended to aid in diagnosing suspected germline diseases?

Currently, these tests are automatically classified as Class III devices requiring PMA approval since no predicate devices exist. However, FDA believes they may be suitable for De Novo classification as Class II devices with special controls, potentially enabling future 510(k) submissions.

What performance characteristics must be validated for NGS-based germline diagnostic tests?

Key metrics include accuracy (PPA, NPA, TPPV), precision (reproducibility/repeatability), limit of detection, and analytical specificity. Coverage metrics, test run quality thresholds, and variant calling parameters must also be established and validated for the test’s intended use.

How should test developers handle modifications to FDA-cleared NGS-based tests?

Modifications require risk-based assessment to determine if they could significantly affect safety or effectiveness. Significant changes typically require new 510(k) submissions, while minor modifications may only need internal validation following predetermined protocols and acceptance criteria.

What labeling requirements apply to NGS-based tests for germline disease diagnosis?

Labeling must comply with 21 CFR 809.10, include indications for use, test limitations, performance data in tabular format, coverage metrics, and publicly accessible summary information. Test reports must clearly distinguish pathogenic variants from variants of unknown significance.

What specimen types and quality requirements are recommended for NGS-based germline tests?

Acceptable specimen types (whole blood, saliva, etc.) must be specified and validated. Minimum DNA volume, quantity, and quality thresholds should be established to meet predetermined performance specifications, with documented procedures for specimen handling and storage.

How should bioinformatics pipelines be validated for NGS-based diagnostic tests?

All software components, versions, and modifications must be documented. Validation should include end-to-end testing with well-characterized samples, comparison of pipeline outputs, and procedures for database updates. In silico sequences may supplement biological specimens for pipeline assessment.

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What You Need to Do 👇

Recommended Actions

1. Define and document specific indications for use and user needs for the test
2. Establish and validate test performance characteristics:
   • Accuracy (PPA, NPA, TPPV)
   • Precision (reproducibility and repeatability)
   • Limit of detection
   • Analytical specificity
3. Implement quality control measures:
   • Coverage metrics
   • Base quality thresholds
   • Test run quality metrics
   • Sample acceptance criteria
4. Develop comprehensive documentation:
   • Test design and validation protocols
   • Software components and versions
   • Database versions and updates
   • Modification procedures
   • Test reports format
5. Establish procedures for:
   • Sample handling and processing
   • Test failure management
   • Revalidation after modifications
   • Database updates
6. Create clear labeling and reporting that includes:
   • Indications for use
   • Test limitations
   • Performance characteristics
   • Risk mitigation elements
   • Clear result reporting format
7. Implement a change management system to track and validate modifications
8. Develop procedures for continuous monitoring of test performance during clinical use

Key Considerations

Non-clinical testing

• Establish minimum acceptable thresholds for coverage, base quality, and test run quality metrics
• Evaluate precision (reproducibility and repeatability) for both variant and wild-type calls
• Establish and document the minimum/maximum amount of DNA that will enable expected results
• Establish and document analytical specificity using interference, cross-reactivity and cross-contamination metrics
• Validate test performance using well-characterized reference materials or agreed-upon samples

Software

• Document all software components, versions and modifications
• Validate bioinformatics pipeline end-to-end
• Document software versions and traceability
• Specify whether software will run locally or remotely
• Document which databases will be used and their versions

Labelling

• Include indications for use statement with type of sequence variations detected
• List test limitations and risk mitigation elements
• Specify test performance characteristics and metrics
• Provide information about probability of test failure
• Use clear, consistent language throughout reports
• Report variants using widely accepted nomenclature

Safety

• Document specimen handling, preservation, processing, storage and rejection criteria
• Establish quality control acceptance criteria
• Document procedures for test failures
• Validate sample pooling methods if applicable

Other considerations

• Document all modifications to the test
• Prepare detailed SOPs for revalidation after modifications
• Include procedures to account for database updates
• Document performance after any modifications

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Relevant Guidances 🔗

• Content of Premarket Submissions for Device Software Functions
• Off-The-Shelf Software in Medical Devices: Documentation Requirements for Premarket Submissions
• Software Validation for Medical Device Production, Quality Systems, and Device Components
• Unique Device Identifier (UDI) Form and Content Requirements
• Use of ISO 10993-1 for Biological Evaluation and Testing of Medical Devices

Related references and norms 📂

• CLSI MM09-A2: Nucleic Acid Sequencing Methods in Diagnostic Laboratory Medicine
• CLSI EP12-A2: User Protocol for Evaluation of Qualitative Test Performance

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Original guidance

• Use of Next Generation Sequencing in the Diagnosis of Germline Diseases
• HTML / PDF
• Issue date: 2018-04-13
• Last changed date: 2020-03-02
• Status: FINAL
• Official FDA topics: Medical Devices, Biologics
• ReguVirta ID: 6ddedb8d4e342414e4fcf99f94f6eb56