Molecular techniques, including polymerase chain reaction (PCR), gel electrophoresis, and sequencing approaches like Sanger sequencing and next generation sequencing (NGS), are laboratory methods used to examine and modify molecules at the cellular level, focusing on DNA and proteins.
Next-generation sequencing (NGS) has transformed in vitro diagnostics (IVDs), enhancing disease detection, monitoring, and understanding. NGS-based IVDs analyse genetic information accurately, allowing for the simultaneous examination of millions of DNA or RNA fragments.
They are vital for diagnosing genetic disorders, identifying cancer mutations, and guiding targeted therapies. However, these devices must adhere to strict regulatory standards to ensure safety, accuracy, and clinical reliability. The FDA in the United States supervises their validation and approval, balancing innovation with patient safety.
NGS-based IVDs have become essential tools in modern healthcare, providing high-throughput and cost-effective genetic insights that advance disease diagnostics, treatment planning, and prognostic evaluation.
Submission Strategies for NGS-Based IVDs: Balancing Risk and Intended Use
NGS-based IVDs can follow several FDA regulatory pathways depending on their intended use, risk profile, and predicate availability. For tests intended to aid in the diagnosis of suspected germline diseases, a FDA 510(k)-submission premarket notification may be appropriate if substantial equivalence to a legally marketed predicate device can be established. In cases where no suitable predicate exists, the De Novo submission process may be pursued.
This pathway allows the classification of novel, low- to moderate-risk devices as Class II, provided that general and special controls are sufficient to ensure safety and effectiveness. For higher-risk tests, such as those that directly inform life-altering clinical decisions, the Premarket Approval (PMA) pathway is required.
Device Code and Regulation Number for different type of NGS Based IVDs
HIV-1 Genotyping Assay Using NGS Technology (QIC):
Device description
The HIV drug resistance genotyping assay, using next-generation sequencing technology, is an automated diagnostic device used to detect HIV-1 genomic mutations, aiding in monitoring and treating HIV-1 infection, utilizing techniques such as nucleic acid extraction, amplification, sequencing, bioinformatics, alignment, and results interpretation.
Intended Use: intended for use in detecting HIV genomic mutations that confer resistance to specific anti-retroviral drugs. The device is intended to be used as an aid in monitoring and treating HIV infection. Class II special control
Next Generation Sequencing Based Tumour Profiling Test (PZM):
Device description
The device comprises various components, including equipment, software, and reagents. The assay system encompasses a sequencing instrument, reagents for DNA extraction, library preparation, and sequencing, as well as software for operating the sequencing instrument and performing variant calling.
Intended use: intended to detect mutations in a broad panel of targeted genes that are somatically altered in malignant neoplasms from tumour specimens obtained from patients diagnosed with malignant solid neoplasms using targeted next-generation sequencing in a broad panel of targeted genes to aid in the management of previously diagnosed cancer patients by qualified health care professionals. Class II special control.
Principle:
The assay uses custom DNA primers to identify variant alterations in oncogenes, tumour suppressor genes, drug metabolism genes, and immune-related genes. It uses an overlapping amplicon approach to generate multiple overlapping amplicons. Sequence libraries are prepared through PCR amplification, and target sequences are tagged with index and adaptor oligonucleotides. Multiple barcoded sequence libraries are pooled and sequenced, aligned with the reference human genome. Variant alterations are identified by comparing the tumour DNA and reference human genome.
Analytical Performance Validation of NGS- IVDs
Analytical performance validation serves as a fundamental requirement for FDA compliance concerning NGS-based in vitro diagnostics (IVDs). A key metric in this process is accuracy, which indicates the test’s proficiency in correctly identifying genetic variants. This is evaluated through positive percent agreement (PPA), negative percent agreement (NPA), and technical positive predictive value (TPPV). Specifically, PPA quantifies the percentage of true positive variants identified by the test, whereas NPA measures the test’s capability to accurately recognize wild-type sequences.
Another essential parameter is the limit of detection (LOD), which denotes the minimum quantity of DNA or the lowest variant allele frequency that the test can reliably detect. It is imperative for developers to validate the LOD for all variant types encompassed within the test’s parameters, taking into account potential complications such as mosaicism or low-frequency alleles.
Precision, which includes both repeatability and reproducibility, assesses the reliability of test outcomes across different conditions. This assessment involves evaluating multiple operators, instruments, and reagent lots over various days and locations. Additionally, the bioinformatics pipeline responsible for converting raw sequencing data into clinically relevant results must undergo thorough validation. This process includes confirming the accuracy of variant calling, annotation, and filtering methods to ensure dependable results.
Regulatory Vision and Future Considerations
The FDA aims to foster innovation in NGS-based diagnostics by promoting the development of consensus standards for analytical validation and clinical databases. These standards could simplify the regulatory process, enabling test developers to certify compliance without undergoing extensive premarket review. The FDA also encourages collaboration among stakeholders, including academic institutions, industry leaders, and professional organizations, to advance the field of genomic diagnostics.
Conclusion
NGS-based in vitro diagnostic (IVD) devices have transformed clinical diagnostics by enabling accurate genetic analysis for various applications, including the diagnosis of germline diseases, cancer profiling, and treatment planning. However, maintaining their safety, accuracy, and reliability necessitates strict compliance with FDA regulations. The FDA 510(k) submission process is essential for demonstrating substantial equivalence to a predicate device, while the De Novo and PMA pathways cater to novel or higher-risk devices. By adhering to these regulatory frameworks and emphasizing robust analytical validation, developers can ensure that NGS-based IVDs meet clinical needs while prioritizing patient safety, ultimately fostering innovation in molecular diagnostics.
Author:
Ms Suman Mishra (M. Pharm)
Regulatory Consultant, FDA Compliance | Medical Device
I3CGlobal