(3 days)
The Roche AmpliChip CYP450 test is intended to identify a patient's CYP2D6 genotype from genomic DNA extracted from a whole blood sample. Information about CYP2D6 genotype may be used as an aid to clinicians in determining therapeutic strategy and treatment dose for therapeutics that are metabolized by the CYP2D6 gene product.
The Roche AmpliChip CyP4502D6 test is a microarray-based genotyping test. The test is based on several processes: PCR amplification of purified genomic DNA, fragmentation and labeling of the amplified products, hybridization of the amplified products to a microarray, staining of the bound products, scanning of the microarray, and determination of the CYP450 genotype and predicted phenotype. The AmpliChip CYP4502D6 Test is designed to identify specific nucleic acid sequences and query for the presence of certain known sequence polymorphisms through analysis of the pattern of hybridization to a series of probes that are specifically complementary either to wild-type or mutant sequences.
Microarrays of oligonucleotide probes synthesized on a glass substrate are utilized for the analysis. Probe microarrays are manufactured in a series of cycles. The glass substrate is coated with linkers containing photolabile protecting groups. A mask is then applied that exposes selected portions of the probe microarray. Illumination removes the photolabile protecting groups enabling selective nucleoside phosphoramidite addition only at the previously exposed sites. Next, a different mask is applied and the cycle of illumination and chemical coupling is performed again. By repeating this cycle, a specific set of oligonucleotide probes is synthesized, with each probe type in a known location.
The AmpliChip CYP450 microarray consists of a square grid of 15,129 probes, each of which contains approximately 10' copies of the specific oligonucleotide probe. Each probe sequence is 16 to 22 bases in length. A single Probe Set consists of four Probes, or Features, which have a fixed target except for at the substitution position where an A, C, G, and T are included to generate four unique probes. Of these four probes, one is designated the Perfect Match (PM) Probe based on the known genome sequence, and the other three are called Mismatch (MM) Probes. A Probe Set Pair consists of a Wild-type Probe Set and a Mutant Probe Set. Both Probe Sets are designed to hybridize to the same region of the target, but one is designed for the Wildtype allele and the other includes a known polymorphism. The assay is designed to distinguish 29 polymorphisms in the CYP2D6 gene, including gene duplication and gene deletion. Detection of these CYP2D6 polymorphisms is designed to result in the identification of 27 distinct alleles, including 7 CYP2D6 gene duplication alleles.
This document describes the 510(k) submission for the Roche AmpliChip CYP450 microarray, a genotyping system for Cytochrome P450 2D6 (CYP2D6). As this is a premarket notification for a new device without a predicate, the submission focuses on establishing the device's performance through various analytical studies. There are no explicit acceptance criteria stated as numerical thresholds; instead, the studies aim to demonstrate high agreement with reference methods and robust performance.
Here's a breakdown of the requested information based on the provided text:
1. Table of Acceptance Criteria and Reported Device Performance
Since explicit numerical acceptance criteria are not presented in the document as clear pass/fail thresholds, the "Acceptance Criteria" column below interpolates the implied performance target based on the observed "Reported Device Performance." The studies aim to demonstrate high accuracy, reproducibility, and a low failure rate.
| Metric | Acceptance Criteria (Implied) | Reported Device Performance |
|---|---|---|
| Reproducibility (Genotype Call Rate) | High percentage of genotype calls, ideally >99% | 99.7% (941/944) of samples provided a genotype call. |
| Reproducibility (Correct Call Rate) | High percentage of correct genotype calls, ideally >99% | 99.9% (940/941) of genotype calls were correct. |
| Whole System Failure Rate | Low failure rate, typically 99%) | For 492 sequenced alleles, there was 99.2% agreement (488 correct calls, 4 no calls). |
| For 806 unique alleles tested against various reference methods: 99.3% agreement. | ||
| Method Comparison (Overall Sample Agreement) | High agreement with reference sequencing/PCR methods (>99%) | For 403 unique samples (genotypes), there was 99.3% agreement (400 correct calls, 3 no calls). |
| Method Comparison (Rare Allele Plasmid Blends) | 100% genotype call rate and accuracy | 100% genotype call rate (100/100) for plasmid clone-genomic DNA blends simulating rare alleles (*7, *8, *11, *19, *20). |
| No Call Rate | Low rate of "No Calls" | Reproducibility study: 0.3% (3/944 samples). |
| Sequencing comparison: 0.8% (2/246 samples). | ||
| Overall allele agreement: 0.7% (6/806 alleles). | ||
| Overall sample agreement: 0.7% (3/403 samples). | ||
| For some specific alleles/genotypes, higher no-call rates or miscalls were observed or noted (e.g., *1XN/*2 sample had a no-call as it could not detect which allele was duplicated). A "No Call" is given if multiple candidate allele pairings are perfect matches. |
2. Sample Size Used for the Test Set and Data Provenance
- Reproducibility Study Test Set: 7 cell line samples (representing 11 known 2D6 alleles), each tested in triplicate over 5 runs by one operator at each of 3 sites, using 3 lots of reagents. This yielded 944 total results (7 samples x 3 replicates x 5 runs x 3 sites x, potentially, 3 lots, but stated as 944 total results).
- Data Provenance: Cell lines.
- Whole System Failure Rate Study Test Set: 100 replicates of genomic DNA from a whole blood specimen (*10/*10 CYP2D6 genotype).
- Data Provenance: Whole blood specimen, likely from a commercially available blood DNA extraction kit.
- Limit of Detection Study Test Set: 144 arrays each for DNA amounts of 50 ng, 25 ng, and 2.5 ng. Total 432 arrays. Two genomic DNA samples (*4DxN/*41 and *4/*5) were used.
- Data Provenance: Genomic DNA samples.
- Analytical Specificity (Interference) Study Test Set: Ten unique patient samples, tested with and without spiking of albumin, bilirubin, and triglycerides.
- Data Provenance: Patient samples (whole blood).
- Analytical Specificity (Carryover) Study Test Set: Five runs of alternating two specimens of distinct genotype.
- Data Provenance: Not specified beyond "specimens."
- Method Comparison Study Test Set:
- Sequencing Comparison: 246 clinical samples previously analyzed by the AmpliChip.
- Data Provenance: Clinical samples.
- Comprehensive Genotype Detection: 403 unique genomic DNA samples for CYP2D6 (including those from the sequencing comparison), with rare alleles tested multiple times. This resulted in 806 unique alleles tested (counted individually for agreement) and 1088 total replicates tested for alleles.
- Data Provenance: Genomic DNA samples, including clinical samples and potentially some internal controls.
- Plasmid Clone-Genomic DNA Blends: 25 blends (simulating rare alleles *7, *8, *11, *19, *20, each blended with 5 genomic DNA backgrounds), with 4 replicates of each blend. Total 100 chip analyses.
- Data Provenance: Genomic DNA from unspecified sources, blended with plasmid clones created by site-directed mutagenesis.
- Sequencing Comparison: 246 clinical samples previously analyzed by the AmpliChip.
3. Number of Experts Used to Establish the Ground Truth for the Test Set and Their Qualifications
The document does not explicitly state the number or qualifications of experts used to establish ground truth.
- For the analytical performance studies (reproducibility, LoD, specificity), the ground truth for samples (e.g., cell line genotypes, genomic DNA genotypes) would have been established through a highly characterized process (e.g., prior sequencing, internal validation). This would typically involve molecular biology experts, but no specific number or qualification is given.
- For the method comparison studies, bidirectional DNA sequencing, allele-specific PCR, and/or PCR-RFLP were used as comparator methods to establish the "reference CYP2D6 genotype" (ground truth). This implies that a molecular biologist or geneticist would interpret these reference standard results. However, there is no mention of "experts" in the sense of clinical specialists or adjudicators for establishing this ground truth. The ground truth essentially relies on the accuracy of these established molecular techniques.
4. Adjudication Method (e.g., 2+1, 3+1, none) for the Test Set
No explicit adjudication method (like 2+1, 3+1) is mentioned. The ground truth was established by comparator methods (DNA sequencing, allele-specific PCR, PCR-RFLP). Any discrepancies would likely be investigated by molecular biology specialists comparing the device's output to the reference method.
5. If a Multi-Reader Multi-Case (MRMC) Comparative Effectiveness Study Was Done, If So, What Was the Effect Size of How Much Human Readers Improve with AI vs. Without AI Assistance
No MRMC comparative effectiveness study was done. This device is a genotyping microarray, an automated test that provides a direct genetic result, not an AI-assisted diagnostic tool that human readers would interpret. Therefore, the concept of "human readers improving with AI vs. without AI assistance" is not applicable.
6. If a Standalone (i.e. Algorithm Only Without Human-in-the-Loop Performance) Was Done
Yes, a standalone performance evaluation was done. The entire analytical and method comparison section describes the performance of the device and its associated software (AmpliChip CYP450 Data Analysis Software) in determining genotypes. This is the "algorithm only" performance. The device automates PCR amplification, fragmentation, labeling, hybridization, staining, scanning, and subsequent data analysis (genotyping algorithm) to produce a genotype report. While manual steps are involved (e.g., DNA purification, loading samples), the interpretation and ultimate genotype call is generated by the instrument and its software, making it a standalone system from the perspective of genotype determination.
7. The Type of Ground Truth Used (Expert Consensus, Pathology, Outcomes Data, etc.)
The ground truth for the test sets was primarily established using molecular reference methods:
- Bidirectional DNA sequencing
- Allele-specific PCR
- PCR-RFLP
- For rare alleles, plasmid DNA clones created by site-directed mutagenesis were blended with homozygous genomic DNA to simulate genotypes.
The document also mentions that "the genotypes of the Reproducibility Panel samples are as follows," implying these genotypes were externally validated or known standards.
8. The Sample Size for the Training Set
The document does not explicitly describe a separate "training set" or its size. The AmpliChip CYP450 microarray determines genotypes by analyzing hybridization patterns to a series of pre-synthesized oligonucleotide probes using CYP450-specific algorithms. This is a rule-based or model-based system established during the device's design and manufacturing, rather than a machine learning model that would typically go through a distinct training phase on a large dataset. The "training" in this context would be the comprehensive design and validation of the probe sequences and algorithms.
9. How the Ground Truth for the Training Set Was Established
As there is no distinct "training set" in the context of typical machine learning, the establishment of "ground truth" for the device's fundamental design relies on established genetic knowledge:
- The design of the oligonucleotide probes and the underlying algorithms are based on known CYP2D6 gene sequences and identified polymorphisms (wild-type and mutant sequences).
- The identification of 27 distinct alleles (including duplication/deletion) is based on established genetic literature and previous research.
- The predicted phenotypes associated with each allele are derived from published scientific studies and literature references, which are explicitly listed in the submission (e.g., Marez et al, 1997; Sachse et al, 1997; etc.). This constitutes the "ground truth" for the phenotypic predictions.
§ 862.3360 Drug metabolizing enzyme genotyping system.
(a)
Identification. A drug metabolizing enzyme genotyping system is a device intended for use in testing deoxyribonucleic acid (DNA) extracted from clinical samples to identify the presence or absence of human genotypic markers encoding a drug metabolizing enzyme. This device is used as an aid in determining treatment choice and individualizing treatment dose for therapeutics that are metabolized primarily by the specific enzyme about which the system provides genotypic information.(b)
Classification. Class II (special controls). The special control is FDA's guidance document entitled “Class II Special Controls Guidance Document: Drug Metabolizing Enzyme Genotyping Test System.” See § 862.1(d) for the availability of this guidance document.