(417 days)
The Xpert™ HemosIL® Factor II & Factor V Assay is a qualitative in vitro diagnostic genotyping test for the rapid detection of Factor II and Factor V alleles from sodium citrate or EDTA anticoagulated whole blood. The test is performed on the Cepheid GeneXpert® Dx System. This test is intended to provide rapid results for Factor II (G20210A) and Factor V (Leiden) mutations as an aid in the diagnosis in individuals with suspected thrombophilia.
The Cepheid Xpert® HemosIL® Factor II & Factor V Assay is an automated DNA test for detecting Factor II and Factor V normal and mutant alleles directly from sodium citrate or EDTA anticoagulated whole blood specimens. Blood specimens are drawn into either sodium citrate or EDTA anticoagulant tubes. Following brief mixing of the sample, the blood sample and two single-use reagents (Reagent 1 and Reagent 2) that are provided with the assay are transferred to different, uniquely-labeled chambers of the disposable fluidic cartridge (the Xpert HemosIL Factor V cartridge). The user initiates a test from the system user interface and places the cartridge into the GeneXpert® Dx System instrument platform, which performs hands-off real-time, multiplex polymerase chain reaction (PCR) for detection of DNA. In this platform, sample preparation, amplification, and real-time detection are all fully-automated and completely integrated.
The GeneXpert Dx System consists of a GeneXpert instrument, personal computer, a barcode scanner and the multi-chambered fluidic cartridges that are designed to complete sample preparation and real-time PCR for detection of Factor II and Factor V normal and mutant alleles in approximately 30 minutes. Each system has 1 to 16 randomly accessible modules that are each capable of performing separate sample preparation and real-time PCR tests. Each module contains a syringe drive for dispensing fluids, an ultrasonic horn for lysing nuclei, and a proprietary I-CORE® thermocycler for performing real-time PCR and detection.
The Xpert HemosIL Factor II & Factor V Assay includes reagents for the detection of Factor II and Factor V normal and mutant alleles. The primers and probes in the Xpert HemosIL Factor II & Factor V Assay determine the genotype of the Factor II gene (at position 20210) and the Factor V gene (at position 1691).
The test includes a Probe Check Control (PCC) that verifies reagent rehydration, PCR tube filling in the cartridge, probe integrity, and dye stability and an instrument control verifies the instrument is performing properly. Additionally, the sample functions as its own internal control since both normal and mutant gene sequences are detected, and each person tested is expected to have one of these sequence signatures.
Here's a breakdown of the acceptance criteria and the study that proves the device meets them, based on the provided text:
Cepheid Xpert® HemosIL® FII & FV: Acceptance Criteria and Performance Study
The Cepheid Xpert® HemosIL® Factor II & Factor V Assay is a qualitative in vitro diagnostic genotyping test designed to detect Factor II (G20210A) and Factor V Leiden (G1691A) mutations in whole blood, aiding in the diagnosis of individuals with suspected thrombophilia. The primary study presented demonstrates the device's accuracy compared to bi-directional sequencing, which serves as the gold standard.
1. Table of Acceptance Criteria and Reported Device Performance
The document doesn't explicitly define "acceptance criteria" through numerical thresholds but presents performance metrics that implicitly indicate the successful validation of the device. Based on the clinical comparison study, the implicit acceptance criterion is a high agreement with the gold standard (bi-directional sequencing).
| Acceptance Criteria (Implicit) | Reported Device Performance |
|---|---|
| Overall Accuracy for Factor II (G20210A) relative to bi-directional sequencing | 99.3% |
| Overall Accuracy for Factor V (G1691A) relative to bi-directional sequencing | 99.3% |
| Reproducibility (Inter-site, Inter-operator, Inter-lot) for Factor II and Factor V genotypes | Generally 98.3% - 100% agreement, with a few indeterminate results on first run. Overall reproducibility rates are very high (e.g., 99.7% overall across sites/operators). |
| Analytical Specificity (correct genotyping with SNPs) | Correct genotyping call, or invalid result in case of SNP in probe binding region. |
| Interference with common blood components/treatments | No inhibition from heparin, bilirubin, cholesterol, triglycerides, or one freeze-thaw cycle (hemolyzed blood). |
2. Sample Size Used for the Test Set and Data Provenance
- Sample Size for Clinical Comparison Study:
- Factor II: 1018 samples (plus 6 human genomic DNA samples homozygous for Factor II to supplement homozygous sample size).
- Factor V: 1014 samples (plus 5 human genomic DNA samples homozygous for Factor V to supplement homozygous sample size).
- Data Provenance: The samples were collected in a multi-site investigational study at seven U.S. institutions.
- The study involved prospective collection of aliquots from routine clinical whole blood specimens (intended for Factor II and/or Factor V testing).
- Excess DNA from these samples was sent to a contract laboratory for bi-directional sequencing.
3. Number of Experts Used to Establish the Ground Truth for the Test Set and Qualifications
- The ground truth was established by bi-directional sequencing, which is a laboratory technique rather than expert interpretation. Therefore, the concept of "experts" to establish ground truth in the traditional sense (e.g., radiologists reviewing images) does not directly apply here. The expertise lies in the laboratory personnel performing and interpreting the sequencing results, but specific numbers or qualifications of these individuals are not detailed in the summary.
4. Adjudication Method for the Test Set
- The document primarily describes a direct comparison of the device's results against bi-directional sequencing.
- No explicit adjudication method (e.g., 2+1, 3+1) is mentioned beyond the direct comparison with the gold standard.
- Invalid results are noted. For Factor II, there were 43 invalid results on the first run, reduced to 7 after repeat runs. For Factor V, there were 42 invalid results on the first run, reduced to 7 after repeat runs. These "invalid results" are referred to as "indeterminate" results, meaning they could not be called by the device, but there were no discordant results (where the device definitively called a genotype incorrectly compared to sequencing). This implies that if the device did make a call, it was correct relative to the gold standard.
5. Multi-Reader Multi-Case (MRMC) Comparative Effectiveness Study
- No MRMC comparative effectiveness study was done. This is a diagnostic test where the device directly provides a result, not an assistive AI tool for human readers.
6. Standalone Performance
- Yes, a standalone (algorithm only without human-in-the-loop performance) study was conducted. The clinical comparison study directly assessed the performance of the Xpert HemosIL Factor II & Factor V Assay (the device) against bi-directional sequencing. The device automatically performs sample preparation, amplification, and real-time detection without human interpretation of the final genotype call. The results in Table 5.2 (Agreement after Repeat Run) represent the standalone performance of the device after initial run and any necessary repeat testing for "indeterminate" results.
7. The Type of Ground Truth Used
- The ground truth used was bi-directional sequencing results, which is considered the gold standard for genetic sequencing.
8. The Sample Size for the Training Set
- The document does not report the sample size for a training set. For an in-vitro diagnostic (IVD) PCR-based assay like this, "training set" doesn't typically apply in the same way it would for machine learning algorithms. The device's primers and probes are designed based on known genetic sequences, and its performance is validated through analytical and clinical studies, not typically through a distinct machine learning training phase on patient data.
9. How the Ground Truth for the Training Set Was Established
- As there's no explicitly mentioned "training set" in the context of the device's development as a machine learning model, the method for establishing ground truth for a training set is not applicable. The device's underlying principles are molecular biology and biochemistry (PCR and hybridization probes) rather than learned patterns from a large dataset. The specificity of the probes for Factor II and Factor V mutations defines the "ground truth" the assay is designed to detect.
§ 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.)