(419 days)
The Invader® Factor V test is an in vitro diagnostic test intended for the detection and genotyping of a single point mutation (G to A at position 1691) of the human Factor V gene in isolated genomic DNA obtained from whole blood potassium EDTA samples from patients with suspected thrombophilia.
The Invader Factor V test consists of the following components: Factor V Oligo Mix, Universal Buffer, Universal Enzyme Mix, No DNA Control, Factor V Wild Type Control, Factor V Heterozygous Control, Factor V Mutant Control, Invader Call Reporter™ Software, Invader® Factor V Software.
Here's an analysis of the acceptance criteria and supporting study details for the Invader® Factor V test, based on the provided text:
Acceptance Criteria and Device Performance for Invader® Factor V Test
1. Table of Acceptance Criteria and Reported Device Performance
The submission primarily focuses on analytical performance criteria, demonstrating the assay's reliability and accuracy in detecting the Factor V G1691A mutation.
| Acceptance Criteria Category | Specific Criteria/Study Goal | Reported Device Performance |
|---|---|---|
| Precision/Reproducibility | External Reproducibility: Consistent results across sites and operators. | External Reproducibility (Study #1): Overall Final % Agreement: 99.81% (539 correct calls out of 540 samples). Five out of six operators achieved 100% agreement. One operator's initial invalid control issue was resolved, resulting in 98.89% agreement for that operator. |
| Lot-to-Lot Reproducibility: Consistent performance across different kit lots. | Lot-to-Lot Reproducibility (Study #9): 100% agreement between Invader Factor V test and sequencing across three different kit lots (n=60 samples). | |
| Stability | Real-Time Stability: Maintain performance over time under storage conditions. | Real-Time Stability (Study #5): 100% agreement for all tested genotypes (WT, HET, MUT) across three product lots at T₀, T₃, and T₆ months, when stored at -30 to -15°C and +2°C to +8°C. Interim results demonstrated 6 months stability. |
| Freeze-Thaw Stability: Maintain performance after multiple freeze/thaw cycles. | Reagent Freeze-Thaw Stability (Study #6): 100% agreement after up to 15 freeze-thaw cycles (n=255 total tests across controls and gDNA samples). | |
| Analytical Sensitivity | Detection Limit/Reportable Range: Accurate genotyping within a specified DNA concentration range. | Detection limit/Analytical Sensitivity and Normal Range (Study #3): Recommended assay range: 5-80 ng/µL of input gDNA, based on 100% concordance with bi-directional sequencing across all tested replicates (80 replicates total for WT and HET samples at various dilutions). |
| Analytical Specificity | Interfering Substances: Performance unaffected by common interfering substances. | Analytical specificity (Study #4): 100% agreement with sequencing genotype and untreated sample Invader® Factor V genotype for all tested interfering substances (Heparin, Cholesterol, Bilirubin, Hemoglobin, Potassium EDTA, Ethanol-based Wash Buffer). |
| Pre-Analytical Workflow | Genomic DNA Extraction Reproducibility: Consistent performance with various DNA extraction methods. | Pre-Analytical Equivalency Study (Study #7): 100% agreement between Invader® Factor V test and bi-directional sequencing for four commercially available DNA extraction methods (n=40 samples per method). One sample was removed due to traceability loss. |
| Instrument Compatibility | Instrument Equivalency: Consistent performance across different thermal cyclers and fluorometers. | Instrument Equivalency (Study #8): 100% concordance for 7 out of 9 combinations of thermal cyclers and fluorometers. Two combinations showed 98.7% concordance (77 of 78 samples). |
| Secondary Polymorphism | Impact of other polymorphisms on assay accuracy. | Secondary Polymorphism Impact (Study #10): 100% concordance (200/200) for control samples and samples with known secondary polymorphisms (G1689A, A1692C, A1696G). |
| Method Comparison | Comparison to a gold standard method. | Bi-directional Sequencing (Study #2): 100% agreement between the Invader® Factor V test and bi-directional DNA sequencing (352/352 human whole blood samples). |
2. Sample Size for Test Set and Data Provenance
- External Reproducibility (Study #1):
- Sample Size: 9 unique leukocyte-depleted whole blood samples (3 wild type, 3 heterozygous, 3 homozygous mutant) spiked cell lines.
- Provenance: "Spiked cell lines" implies controlled, laboratory-prepared samples. The text does not specify the country of origin, but the study was conducted at "3 different sites (2 external sites and 1 internal site)", suggesting multi-center testing, likely within the US given the FDA submission. This was a prospective study where samples were tested newly.
- Lot-to-Lot Reproducibility (Study #9):
- Sample Size: 5 genomic DNA samples (3 wild type, 2 heterozygous).
- Provenance: Not explicitly stated, likely laboratory-prepared or acquired genomic DNA.
- Real-Time Stability (Study #5):
- Sample Size: Samples representing all three genotypes (WT, HET, MUT) (number not explicitly stated, but tested in quadruplicate at each time point). Also mentions three product lots.
- Provenance: Likely laboratory-prepared/controlled samples.
- Reagent Freeze-Thaw Stability (Study #6):
- Sample Size: Controls (WT, HET, MUT) and gDNA (WT, HET, MUT). Total of 255 tests.
- Provenance: Genomic DNA isolate from cell lines.
- Detection limit/Analytical Sensitivity and Normal Range (Study #3):
- Sample Size: 2 genomic DNA samples (1 WT, 1 HET), each diluted to 8 concentrations and tested in 40 replicates (total 2 * 8 * 40 = 640 tests).
- Provenance: Genomic DNA extracted from whole blood collected in potassium EDTA.
- Analytical specificity (Study #4):
- Sample Size: 4 whole blood samples (3 WT, 1 HET). Each substance tested on 8 of these samples.
- Provenance: Human whole blood samples.
- Pre-Analytical Equivalency Study (Study #7):
- Sample Size: 30 human whole blood samples and 10 leukocyte-depleted whole blood spike cell lines (total 40 unique samples). These were processed by 4 different extraction methods, leading to 160 extracted DNAs.
- Provenance: Human whole blood samples and cell lines.
- Instrument Equivalency (Study #8):
- Sample Size: 29 human whole blood samples and 10 leukocyte-depleted whole blood samples spiked with cell lines (total 39 unique samples). These were tested across various instrument combinations, leading to 78 samples per combination (likely some samples were tested multiple times or a subset of the 39 samples were used to create 78 data points.
- Provenance: Human whole blood samples and cell lines.
- Secondary Polymorphism Impact (Study #10):
- Sample Size: 5 samples (one homozygous normal, one heterozygous, and three homozygous normal with different secondary polymorphisms). Tested in 40 replicates each (total 5 * 40 = 200 tests).
- Provenance: Known Factor V genotypes with or without secondary polymorphisms.
- Method comparison (Bi-directional Sequencing) (Study #2):
- Sample Size: 352 human whole blood samples (289 Homozygous Wild Type (GG), 45 Heterozygous (GA), 18 Homozygous Mutant (AA)).
- Provenance: Human whole blood samples. The text does not specify the country of origin, but it is implied to be a retrospective collection based on the phrasing "underwent DNA extraction and subsequent bi-directional DNA sequence analysis" then "The same DNA samples were then analyzed using the Invader® Factor V test."
3. Number of Experts and Qualifications for Ground Truth
The primary method for establishing ground truth across most studies is bi-directional DNA sequencing. The text does not specify the number of experts or their specific qualifications (e.g., molecular geneticists) used to interpret these sequencing results. However, bi-directional sequencing is a standard and highly accurate method for genotyping, and its interpretation would typically be performed by trained molecular diagnosticians or geneticists.
4. Adjudication Method for the Test Set
The text does not explicitly describe an adjudication method (e.g., 2+1, 3+1). For discrepancies, the sequencing results are considered the gold standard to which the device's results are compared. In cases like the "No Call" results in Study #1, troubleshooting and retraining of the operator were performed, and retesting was done against sequencing as the reference.
5. Multi-Reader Multi-Case (MRMC) Comparative Effectiveness Study
No, an MRMC comparative effectiveness study involving human readers with and without AI assistance was not done.
This device (Invader® Factor V test) is an in vitro diagnostic assay for genotyping, not an AI-assisted diagnostic imaging or clinical decision support system that would typically involve human readers. Its output is a genotype call (WT, HET, MUT), which is then interpreted by a healthcare professional. Therefore, the concept of "how much human readers improve with AI vs without AI assistance" does not apply to this type of device.
6. Standalone (Algorithm Only) Performance
Yes, the standalone performance (algorithm only without human-in-the-loop performance) was done and is the primary focus of the performance studies.
The Invader® Factor V test is an automated in vitro diagnostic system. The "Invader® Factor V Software, in combination with Invader Call Reporter™ software," processes raw fluorescence data to determine genotype calls (WT, HET, MUT). All the performance studies described (Precision, Stability, Sensitivity, Specificity, Method Comparison, etc.) evaluate the accuracy of these software-generated genotype calls against a recognized gold standard (bi-directional sequencing). There is no "human-in-the-loop" step described for data interpretation that would alter the genotype call generated by the software. The human operator's role is primarily experimental execution and loading/reviewing results from the software.
7. Type of Ground Truth Used
The primary type of ground truth used across all studies is bi-directional DNA sequencing. Where applicable, expected genotypes from known control materials (cell lines) also served as ground truth. This is a highly accurate and widely accepted method for determining genetic mutations.
8. Sample Size for the Training Set
The document does not explicitly mention a separate "training set" for the device's algorithm. For in vitro diagnostic kits like the Invader® Factor V test, the "training" (development and optimization) phase would typically involve a proprietary set of samples used by the manufacturer to establish the assay parameters, cutoff values for fluorescence, and the logic within the software for genotype calling. These samples are not typically disclosed or enumerated as a formal "training set" in regulatory submissions but are part of the overall development process. The studies described are validation studies (test sets) demonstrating the finalized device's performance.
9. How the Ground Truth for the Training Set was Established
Since a formal "training set" is not explicitly defined in the provided document, the method for establishing ground truth for any internal development samples would likely be the same as for the validation studies: bi-directional DNA sequencing, or comparison to previously characterized genomic DNA samples.
§ 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.)