(109 days)
The cobas® Factor II and Factor V Test is an in vitro diagnostic device that uses real-time PCR for the detection and genotyping of the Factor II (Prothrombin) G20210A mutation and the Factor V Leiden G1691A mutation in genomic DNA obtained from K2EDTA whole blood specimens as an aid in diagnosis of patients with suspected thrombophilia. The cobas® Factor II and Factor V Test and the cobas z 480 analyzer are used together for automated amplification and detection.
The cobas® Factor II and Factor V Test is a real-time polymerase chain reaction (PCR) test for the qualitative detection and genotyping of a single point mutation in the human Factor V gene (G to A at position 1691, referred to as the Factor V Leiden mutation) and a single point mutation in the human Factor II gene (G to A at position 20210), in genomic DNA isolated from peripheral whole blood, as an aid in diagnosis of patients with suspected thrombophilia.
DNA is extracted offline from whole blood specimens collected in K2EDTA tubes. The userselected DNA extraction method must provide DNA of sufficient concentration. Automated realtime PCR is then performed on the cobas z 480 analyzer. The Factor II and Factor V mutations are detected simultaneously in the same real-time PCR reaction. Depending upon the test order, results for one or both mutations are reported for each DNA sample.
The assay consists of one reagent kit and a system platform. The reagent kit provides the necessary reagents and controls to perform automated real-time PCR amplification and detection. The system platform consists of a real-time PCR thermal cycler (cobas z 480 analyzer) and a control unit (cobas® 4800 System CU). The amplification reactions generate a Factor II specific amplicon and a Factor V specific amplicon in all samples, and utilize four fluorescent-dye labeled oligonucleotide probes for detection of the Factor II and Factor V genotypes.
Acceptance Criteria and Study for cobas® Factor II and Factor V Test
This document outlines the acceptance criteria and the studies performed to demonstrate that the cobas® Factor II and Factor V Test meets these criteria.
1. Table of Acceptance Criteria and Reported Device Performance
Factor II (Prothrombin) G20210A Mutation Detection:
| Metric | Acceptance Criteria (Study Objective) | Reported Device Performance (95% LCB) |
|---|---|---|
| Overall Percent Agreement (OPA) | Compare performance using Sanger sequencing as the reference method. | 100% (98.78%) |
| Positive Percent Agreement (PPA) | Compare performance using Sanger sequencing as the reference method. | 100% (97.59%) |
| Negative Percent Agreement (NPA) | Compare performance using Sanger sequencing as the reference method. | 100% (97.55%) |
| Heterozygous Agreement | Compare performance for heterozygous genotypes using Sanger sequencing. | 100% (97.20%) |
| Homozygous Mutant Agreement | Compare performance for homozygous mutant genotypes using Sanger sequencing. | 100% (83.89%) |
| Reproducibility (WT) | Evaluate reproducibility across sites, lots, and methods for Wild Type (WT). | 100% (98.78%) |
| Reproducibility (HET) | Evaluate reproducibility across sites, lots, and methods for Heterozygous (HET). | 100% (97.96%) |
| Reproducibility (MUT) | Evaluate reproducibility across sites, lots, and methods for Homozygous Mutant (MUT). | 100% (94.04%) |
Factor V Leiden G1691A Mutation Detection:
| Metric | Acceptance Criteria (Study Objective) | Reported Device Performance (95% LCB) |
|---|---|---|
| Overall Percent Agreement (OPA) | Compare performance using Sanger sequencing as the reference method. | 100% (98.78%) |
| Positive Percent Agreement (PPA) | Compare performance using Sanger sequencing as the reference method. | 100% (97.62%) |
| Negative Percent Agreement (NPA) | Compare performance using Sanger sequencing as the reference method. | 100% (97.52%) |
| Heterozygous Agreement | Compare performance for heterozygous genotypes using Sanger sequencing. | 100% (97.20%) |
| Homozygous Mutant Agreement | Compare performance for homozygous mutant genotypes using Sanger sequencing. | 100% (85.18%) |
| Reproducibility (WT) | Evaluate reproducibility across sites, lots, and methods for Wild Type (WT). | 100% (98.78%) |
| Reproducibility (HET) | Evaluate reproducibility across sites, lots, and methods for Heterozygous (HET). | 100% (97.96%) |
| Reproducibility (MUT) | Evaluate reproducibility across sites, lots, and methods for Homozygous Mutant (MUT). | 100% (94.04%) |
Analytical Sensitivity (Lower Limit of Detection):
| Metric | Acceptance Criteria (Study Objective) | Reported Device Performance (Correct Call Rate) |
|---|---|---|
| Limit of Detection | Determine minimum input DNA necessary to yield correct genotype results. | 98% at 0.01 ng/uL (100% at higher concentrations) |
Analytical Sensitivity (Upper Limit of Detection):
| Metric | Acceptance Criteria (Study Objective) | Reported Device Performance (Correct Call Rate) |
|---|---|---|
| Accuracy at High DNA Input | Evaluate performance at higher DNA concentrations (up to 300 ng/uL). | 100% |
DNA Extraction Method Study:
| Metric | Acceptance Criteria (Study Objective) | Reported Device Performance (Overall Agreement) |
|---|---|---|
| Agreement with Sanger Sequencing | Evaluate agreement using different commercial DNA isolation methods. | 99.4% (98.6 – 99.8% 95% two-sided CI) |
Lot-to-Lot Repeatability:
| Metric | Acceptance Criteria (Study Objective) | Reported Device Performance (Overall Agreement) |
|---|---|---|
| Agreement with Sanger Sequencing | Evaluate lot-to-lot repeatability across multiple reagent lots. | 100% (99.3% one-sided, lower 95% CI) |
2. Sample Size Used for the Test Set and Data Provenance
Method Comparison Study:
- Sample Size: 300 specimens.
- Data Provenance: The specimens were obtained from patients whose routine medical care called for Factor II and/or Factor V measurements, representing an intended use population. The commercial vendors provided these samples. The type of data (retrospective/prospective) is not explicitly stated, but the description of samples being "obtained to represent the intended use population" and collected for "routine medical care" suggests a retrospective collection or at least samples collected for clinical indications. The provenance is likely a mix of clinical labs or commercial biobanks.
Clinical Reproducibility Study:
- Sample Size: A 9-member panel was used, consisting of 4 unique K2EDTA blood samples, 3 contrived blood samples, and 2 extracted genomic DNA samples. Each panel member was tested multiple times across different sites, operators, instruments, runs, and days. A total of 540 tests were performed (with 539 valid results and 1 invalid).
- Data Provenance: The samples included K2EDTA whole blood samples and extracted genomic DNA samples. The "contrived blood samples" suggest laboratory-prepared samples. The "unique K2EDTA blood samples" were likely sourced from similar vendors as the method comparison study. The study was conducted at three test sites (2 external and 1 internal), implying a multi-center study on potentially diverse datasets.
3. Number of Experts Used to Establish the Ground Truth for the Test Set and the Qualifications of Those Experts
The ground truth for both the Method Comparison Study and the Clinical Reproducibility Study was established by bi-directional Sanger sequencing. The document does not specify the number of experts or their qualifications for performing or interpreting the Sanger sequencing results. However, Sanger sequencing is a widely accepted gold standard for genetic mutation detection, and it's typically performed by trained laboratory professionals.
4. Adjudication Method for the Test Set
The document does not explicitly describe an adjudication method for the test set where discrepancies between results from the cobas® test and Sanger sequencing were adjudicated by experts. The performance metrics are reported as direct agreements or disagreements with the Sanger sequencing results. In the DNA Extraction Method Study, for instance, one inconsistent triplicate result with a method was re-tested, and all results were correct upon re-testing, implying an internal review for outlier data.
5. If a Multi-Reader Multi-Case (MRMC) Comparative Effectiveness Study was Done
No, an MRMC comparative effectiveness study was not done. The cobas® Factor II and Factor V Test is an in vitro diagnostic device for automated amplification and detection of genetic mutations. It is an algorithm-only device; therefore, human readers are not directly involved in its performance or interpretation in a way that would necessitate an MRMC study.
6. If a Standalone (i.e. algorithm only without human-in-the-loop performance) was Done
Yes, the studies presented are standalone performance evaluations. The cobas® Factor II and Factor V Test is described as an "in vitro diagnostic device that uses real-time PCR for the detection and genotyping" and used with the "cobas z 480 analyzer for automated amplification and detection." This indicates an automated system where the algorithm performs the detection and genotyping without direct human intervention in the result generation process. Human interaction is limited to sample preparation and loading, and interpreting the final automated result.
7. The Type of Ground Truth Used
The type of ground truth used for both the Method Comparison Study and the Clinical Reproducibility Study was expert consensus through bi-directional Sanger sequencing. Sanger sequencing is generally considered the "gold standard" for confirming DNA sequences, and thus, genotypes.
8. The Sample Size for the Training Set
The document does not provide information regarding a separate training set or its sample size. The studies described are performance evaluations (method comparison, reproducibility, analytical sensitivity, etc.) which typically use independent test data to assess the device's accuracy and reliability, assuming the algorithm was developed and optimized prior to these evaluation studies. As this is not an AI/ML device in the traditional sense, but a PCR-based diagnostic, the concept of a "training set" for an algorithm's learning phase is not directly applicable in the same way as it would be for an image recognition AI, for example. The "training" for such a system would involve optimizing primers, probes, and reaction conditions during product development.
9. How the Ground Truth for the Training Set Was Established
As mentioned above, the document does not discuss a separate training set. For the validation studies, ground truth was established by bi-directional Sanger sequencing.
§ 864.7280 Factor V Leiden DNA mutation detection systems.
(a)
Identification. Factor V Leiden deoxyribonucleic acid (DNA) mutation detection systems are devices that consist of different reagents and instruments which include polymerase chain reaction (PCR) primers, hybridization matrices, thermal cyclers, imagers, and software packages. The detection of the Factor V Leiden mutation aids in the diagnosis of patients with suspected thrombophilia.(b)
Classification. Class II (special controls). The special control is FDA's guidance entitled “Class II Special Controls Guidance Document: Factor V Leiden DNA Mutation Detection Systems.” (See § 864.1(d) for the availability of this guidance document.)