(464 days)
The eQ-PCR™ LC Warfarin Genotyping kit is an in vitro diagnostic test for the detection and genotyping of two single nucleotide polymorphisms (SNP) in the cytochrome P450 enzyme gene CYP2C9 known as CYP2C92 (C430T) and CYP2C93 (A1075C), and a SNP in the vitamin K epoxide reductase complex 1 gene VKORC1, known as VKORC1 (-1639G>A) obtained from human peripheral blood samples. The eQ-PCR LC Warfarin Genotyping kit is a qualitative assay for use in clinical laboratories upon prescription by the attending physician.
The eO-PCR™ LC Warfarin Genotyping kit is indicated for use as an aid in identifying patients who may be at risk of warfarin sensitivity.
The eQ-PCR 1M LC Warfarin Genotyping Kit assay requires extracted DNA obtained by using any commercially available DNA extraction kit. The extracted DNA sample, within a range of 50-200ng of total DNA, is mixed with a PCR Mix, and an eQ-PCR "M specific Probe Mix reagent containing specific primers and fluorescent labeled probes for the CYP2C9 and/or VKORC1 gene polymorphisms. Amplification and detection are then performed in the Roche Diagnostics LightCycler® Real-Time PCR System instrument model 1.2 using conditions defined in the specific eQ-PCRTM LC Warfarin Genotyping Kit Product Insert. After the PCR reaction is completed, the Roche Diagnostics LightCycler® Real-Time PCR System instrument automatically proceeds to the melting curve-based detection method. This real time PCR test is a closed test system and does not require post PCR operations. It reduces human errors and eliminates post-PCR handling contamination.
The instrument's standard melting curve analysis software algorithm of peak patterns and melting temperatures (Tm) determine the genotype (wild type, mutant, heterozygous) for each of the three specified polymorphisms.
This document describes the TrimGen eQ-PCR™ LC Warfarin Genotyping Kit, an in vitro diagnostic test for genotyping SNPs associated with warfarin sensitivity. The study aims to demonstrate substantial equivalence to a predicate device through clinical performance and reproducibility testing.
1. Table of Acceptance Criteria and Reported Device Performance:
The document doesn't explicitly state pre-defined quantitative acceptance criteria in a dedicated section. However, the performance is reported as "overall 100% clinical sensitivity" for the assay compared to bi-directional sequencing after re-sequencing of discrepant samples. The table below presents the detailed clinical sensitivity for each SNP and genotype, which can be inferred as the performance measures.
| SNP | Genotype | Reported Clinical Sensitivity (# / total) | Reported Clinical Sensitivity (%) | 95% Confidence Interval |
|---|---|---|---|---|
| 2C9*2 | Wild Type | 123/126 | 97.6% | 94.40% |
| Heterozygous | 27/28 | 96.4% | 87.68% | |
| Variant | 5/5 | 100% | 47.98% | |
| 2C9*3 | Wild Type | 138/140 | 98.6% | 96.09% |
| Heterozygous | 12/13 | 92.3% | 75.32% | |
| Variant | 5/6 | 83.3% | 54.28% | |
| VKORC1 | Wild Type | 77/79 | 97.5% | 93.16% |
| Heterozygous | 62/63 | 98.4% | 94.32% | |
| Variant | 17/17 | 100% | 80.52% |
For reproducibility, the criteria were implicitly met by "no discordance among observed results or between observed and expected results across sites, days or operators" and all results matching bi-directional sequencing.
2. Sample Size Used for the Test Set and Data Provenance:
- Sample Size: 159 donors provided whole blood samples for the clinical performance study. For the reproducibility study, a panel of 31 samples (whole blood) was used at each of the three sites across five days.
- Data Provenance: The whole blood samples were collected from adult volunteers under IRB approval and with Informed Consent. The country of origin is not explicitly stated, but the submission is to the US FDA, implying testing within the US or for the US market. The clinical performance study is a prospective collection for testing.
3. Number of Experts Used to Establish the Ground Truth for the Test Set and Qualifications of Those Experts:
- The ground truth for the clinical performance study was established by bi-directional sequencing performed at a "reference laboratory." The number of experts and their specific qualifications (e.g., geneticists, molecular biologists) are not specified within the provided text.
4. Adjudication Method for the Test Set:
- The clinical performance study mentions that "Only 0.4% (2/450) of the results was discordant and resolved in agreement with the eQ-PCR LC Warfarin Genotyping Kit result when bi-directional sequencing was repeated." This indicates an adjudication method where initial discrepancies were re-evaluated by re-sequencing using the ground truth method (bi-directional sequencing). This could be considered a form of "tie-breaker" or confirmatory testing for discordant results.
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, a Multi-Reader Multi-Case (MRMC) comparative effectiveness study was not conducted. This device is an in vitro diagnostic (IVD) kit for genotyping, not an AI-assisted diagnostic imaging or interpretation tool. Therefore, the concept of human readers improving with AI assistance is not applicable here.
6. If a Standalone (i.e., algorithm only without human-in-the-loop performance) was Done:
- Yes, the clinical performance study is essentially a standalone performance evaluation of the device (the eQ-PCR™ LC Warfarin Genotyping Kit) against the established ground truth (bi-directional sequencing). The device's algorithm for determining genotype from melting curve analysis is used independently.
7. The Type of Ground Truth Used:
- The ground truth used was bi-directional sequencing. This is a highly accurate method for determining DNA sequences and is considered a gold standard for genotyping in many contexts.
8. The Sample Size for the Training Set:
- The document describes a post-market clearance study demonstrating assay performance. It does not mention a separate training set for the device's algorithm. The algorithm for interpreting melting curves is likely pre-defined based on known principles of real-time PCR and melting curve analysis, rather than a machine learning model requiring a specific training dataset in the context presented here. The "training" of such a system would typically involve establishing robust assay conditions and expected melting curve profiles for different genotypes.
9. How the Ground Truth for the Training Set Was Established:
- As a dedicated training set is not explicitly mentioned for an algorithm that learns from data, the establishment of ground truth for a training set is not applicable as per the provided text. The device relies on a pre-established methodology (real-time PCR and melting curve analysis) for genotype determination, where the "ground truth" for defining peak patterns and melting temperatures for different genotypes would be based on well-characterized samples and molecular biology principles.
§ 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.