(44 days)
The Invader® UGTIA1 Molecular Assay is an in vitro diagnostic test for the detection and genotyping of the *1 (TA6) and *28 (TA7) alleles of the UDP glucuronosyltransferase 1A1 (UGTIA1) gene in genomic DNA from whole peripheral blood as an aid in the identification of patients with greater risk for decreased UDP-glucunorosyltransferase activity.
The Invader® UGTIA1 Molecular Assay is an in vitro diagnostic test which utilizes sequence specific Invader DNA probes, a structure-specific cleavage enzyme and a fluorescent resonance energy transfer (FRET) system combined with universal interpretative software and third party microtiter plate reader instrumentation. Invader® is the term used to generically refer to the patented chemistry on which the Invader® UGT A 1 Molecular Assay is based. The assay is designed to identify specific nucleic acid sequences and query for the presence of known sequence polymorphisms through the structurespecific cleavage of a series of probes that are specifically complementary to TA repeat sequences in in the "TATA Box" of of the UGT1A1 promoter region.
In the Invader® UGT1A1 Molecular Assay, two oligonucleotides (a discriminatory Primary Probe and an Invader® Oligo) hybridize in tandem to the target DNA to form an overlapping structure. The 5'-end of the Primary Probe includes a 5'-flap that does not hybridize to the target DNA. The 3'-nucleotide of the bound Invader® Oligo overlaps the Primary Probe, but need not hybridize to the target DNA. The Cleavase® enzyme recognizes this overlapping structure and cleaves off the unpaired 5'-flap of the Primary Probe, releasing it as a target-specific product. The Primary Probe is designed to have a melting temperature close to the reaction temperature. Therefore, under the isothermal assay conditions, Primary Probes, which are provided in excess, cycle on the target DNA. This allows for multiple rounds of Primary Probe cleavage for each target DNA, and amplification of the number of released 5'-flaps.
In the secondary reaction, each released 5'-flap can serve as an Invader® Oligo on a fluorescence resonance energy transfer (FRET) Cassette to create another overlapping structure that is recognized and cleaved by the Cleavase® enzyme. When the FRET Cassette is cleaved, the fluorophore and quencher are separated, generating detectable fluorescence signal. Similar to the initial reaction, the released 5'-flap and the FRET Cassette cycle, resulting in amplified fluorescence signal. The initial and secondary reactions run concurrently in the same well.
The biplex format of the Invader® UGT1A1 Molecular Assay enables simultaneous detection of two DNA sequences, a non-varying segment of the human alpha actin (ACTAI) gene and the TA repeat in the TATA box of the human UGTIA1 gene, in a single well. The biplex format uses two different discriminatory Primary Probes, each with a unique 5'-flap, and two different FRET Cassettes, each with a spectrally distinct fluorophore. By design, the released 5'-flaps will bind only to their respective FRET Cassettes to generate a target-specific signal.
The Invader® UGT1A1 Molecular Assay utilizes four independent wells per sample (one well for each of the TA Oligo mix reactions), to make a single genotype call. Each well contains a TATA box specific probe and an alpha actin probe. The alpha actin probe serves as an internal control to confirm the validity of a given result when a particular TATA box polymorphism is absent.
Here's a summary of the acceptance criteria and study details for the Invader® UGT1A1 Molecular Assay, based on the provided text:
Acceptance Criteria and Reported Device Performance
| Acceptance Criteria Category | Specific Metric | Acceptance Criteria (Implied) | Reported Device Performance |
|---|---|---|---|
| Limit of Detection | Lower Limit (Correct Detection) | 100% correct detection | 100% correct detection at 50 ng/reaction (95% CI: 92.8%) |
| Upper Limit (Genotype Call Agreement) | 100% agreement | 100% agreement at 80 ng/µL (95% CI: 97.5%) | |
| Genotype Detection | Agreement with Bi-directional DNA Sequencing | 100% agreement | 100% agreement for all genotypes (95% CI: 90.5% - 98.95%) |
| Repeat Rate | On first attempt | Low (quantitative not specified) | 0% (in initial study); 5.4% (in external reproducibility study) |
| Stability (Freeze-Thaw) | Agreement with known genotype | 100% agreement | 100% agreement for all samples after 1, 3, 5, 10 freeze-thaw cycles |
| Stability (Storage) | Agreement with bi-directional DNA sequencing | 100% agreement | 100% agreement at -20°C and simulated shipping stress (ongoing for 18 months) |
| Lot-to-Lot Reproducibility | Agreement with bi-directional DNA sequencing | 100% agreement | 100% agreement across three lots (95% CI: 92.8%) |
| Sample Preparation Equivalency | Agreement in genotype calls across different kits | 100% agreement | 100% agreement with two different Qiagen DNA purification kits |
| Reproducibility | Total Correct Genotype Call Rate (after 1st run) | High (quantitative not specified) | 93.3% (840/900) - 1st run (95% CI: 91.8%) |
| Total Correct Genotype Call Rate (combined 1st & 2nd run) | High (quantitative not specified) | 98.1% (883/900) - combined (95% CI: 97.2%) | |
| Interference Studies | Genotype Call Agreement (spiked vs. non-spiked samples) | 100% agreement | 100% agreement for bilirubin, lipids, EDTA, hemoglobin, 1% AW2; 1 incorrect call and 1 low signal for 5% AW2. |
Study Details:
-
Sample sizes used for the test set and the data provenance:
- Limit of Detection (Lower): 3 genomic DNA samples (TA6/6, TA6/7, TA7/7) concentrated at 50, 100, 150 ng DNA/µL. Total genotype calls for calculation: 120 (40 at each concentration for 3 genotypes).
- Limit of Detection (Upper): 3 genomic DNA samples (TA6/6, TA6/7, TA7/7) concentrated at 80 ng DNA/µL. Total genotype calls: 120.
- Genotype Detection: 285 blood samples.
- Repeat Rate (Initial): 285 blood samples.
- Repeat Rate (External Study): 20 samples, tested in triplicate at 3 sites on 5 separate days (900 possible genotype calls).
- Stability (Freeze-Thaw): 20 genomic DNA samples.
- Stability (Storage): 3 genomic DNA samples.
- Lot-to-Lot Reproducibility: 40 whole blood samples, analyzed with 3 different lots of reagents (120 data points).
- Sample Preparation Equivalency: 60 human genomic DNA samples.
- Reproducibility (Multi-site): 20 blood samples, tested across 3 sites (900 sample points generated).
- Interference Studies: 16 whole blood samples (for bilirubin, lipids, EDTA); 16 whole blood samples (for hemoglobin, AW2 buffer).
The data provenance is not explicitly stated as retrospective or prospective, nor is the country of origin mentioned for the samples. It is implied to be clinical samples, likely from a patient population relevant to UGT1A1 genotyping.
-
Number of experts used to establish the ground truth for the test set and the qualifications of those experts:
- The ground truth for all performance studies (Limit of Detection, Genotype Detection, Stability, Lot-to-Lot Reproducibility, Sample Preparation Equivalency, Interference, Reproducibility) was established using bi-directional DNA sequence analysis.
- The document does not specify the number or qualifications of experts who performed or interpreted the bi-directional DNA sequencing. It is presented as a gold standard laboratory method.
-
Adjudication method for the test set:
- The document does not describe an adjudication method involving multiple human readers for discrepancies. The genotype calls from the Invader assay were directly compared to the results of bi-directional DNA sequencing.
- For the reproducibility study, where "misidentified" samples occurred at one site, it states "Discrepancy resolution by resequencing of the template in the original Invader assay confirmed that the genotypes present in the assay wells were consistent with the reported genotypes of the assay." This suggests a re-analysis by the measurement method itself rather than a formal expert adjudication panel.
-
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, an MRMC comparative effectiveness study was not done. This device is an in vitro diagnostic molecular assay, not an AI-powered diagnostic imaging tool that would typically involve human readers. The performance is evaluated against a gold standard molecular method (bi-directional DNA sequencing), not against human interpretation of images.
-
If a standalone (i.e. algorithm only without human-in-the-loop performance) was done:
- Yes, this entire submission focuses on the standalone performance of the Invader® UGT1A1 Molecular Assay, which is an algorithm-driven automated test. The "algorithm" here refers to the sequence-specific Invader DNA probes, cleavage enzyme, FRET system, and universal interpretative software that automates genotype calling. There is no human-in-the-loop performance described beyond standard laboratory handling and setup. The assay signal results are interpreted by a software program and assigned a genotype.
-
The type of ground truth used:
- The primary ground truth used for all performance evaluations was bi-directional DNA sequence analysis. This is a highly accurate molecular method for determining DNA sequences.
-
The sample size for the training set:
- The document does not mention a separate training set or training data used to develop the assay. The focus is on the performance data from non-clinical studies (validation studies) comparing the Invader assay results to bi-directional DNA sequencing. This suggests the assay's underlying chemistry and interpretive software were already developed and are being validated, rather than being developed using a specific training dataset in the context of machine learning.
-
How the ground truth for the training set was established:
- As no training set is explicitly described, the method for establishing its ground truth is not applicable or detailed in this document. The assay's fundamental design relies on established molecular biology principles and specific probe-target recognition, rather than learning from a large, pre-labeled training dataset in the way a machine learning algorithm would.
§ 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.