(399 days)
The Invader® MTHFR 677 test is an in vitro diagnostic test intended for the detection and genotyping of a single point mutation (C to T at position 677) of the human 5.10-methylenetetrahydrofolate reductase (MTHFR) gene in isolated genomic DNA obtained from whole blood Potassium EDTA samples from patients with suspected thrombophilia.
The Invader MTHFR 677 test consists of the following components:
MTHFR 677 Oligo Mix
Universal Buffer
Universal Enzyme Mix
No DNA Control
MTHFR 677 Wild Type Control
MTHFR 677 Heterozygous Control
MTHFR 677 Mutant Control
Invader Call Reporter™ Software
The Invader® MTHFR 677 test is an in vitro diagnostic test for detecting and genotyping the C to T mutation at position 677 of the MTHFR gene.
1. Table of Acceptance Criteria and Reported Device Performance:
The document doesn't explicitly state "acceptance criteria" but demonstrates performance through several studies of 100% agreement with the ground truth for most tests. The primary performance metric is agreement with bi-directional sequencing.
| Performance Metric | Acceptance Criteria (Implied) | Reported Device Performance |
|---|---|---|
| Inter-laboratory Reproducibility | 100% Agreement | 100% (n=540) |
| Lot-to-Lot Reproducibility | 100% Agreement | 100% (n=108) |
| Real-Time Stability | 100% Agreement | 100% (7 months interim) |
| Reagent Freeze-Thaw Stability | 100% Agreement | 100% (up to 15 cycles) |
| Analytical Sensitivity (5-80 ng/uL) | 100% Concordance | 100% (40/40 replicates) |
| Analytical Specificity (Interfering Substances) | 100% Agreement with Sequencing | 100% (18/18 for all substances) |
| Pre-Analytical Equivalency (Extraction Methods) | 100% Agreement with Sequencing | 100% (n=40 per method) |
| Instrument Equivalency | 100% Agreement with Sequencing | 100% (78/78 per instrument combination) |
| Method Comparison (Bi-directional Sequencing) | 100% Agreement (Final Run) | 100% (359/359) |
| Method Comparison (Bi-directional Sequencing) | >95% (95% LCB) | 99.45% First Run Agreement (359/361) |
2. Sample Sizes and Data Provenance:
- Inter-laboratory Reproducibility (Study #1):
- Test set sample size: 9 whole blood samples (3 wild type, 3 heterozygous, 3 homozygous mutant). Tested in duplicate over 5 non-consecutive days by 6 operators (2 from each of 3 sites), resulting in 90 samples tested per operator, totaling 540 data points.
- Data provenance: Not explicitly stated, but includes 2 external sites and 1 internal site, suggesting geographically diverse testing within a controlled study. Prospective based on the study design.
- Lot-to-Lot Reproducibility (Study #9):
- Test set sample size: 9 genomic DNA samples (3 wild type, 3 heterozygous, 3 mutant). Tested in quadruplicate using 3 different kit lots, totaling 108 data points.
- Data provenance: Not explicitly stated, but part of analytical performance studies, likely within a controlled laboratory setting.
- Real-Time Stability Study (Study #5):
- Test set sample size: Samples representing all 3 genotypes (including 3 controls and 4 gDNA samples) tested in quadruplicate at each time point.
- Data provenance: Not explicitly stated, likely internal laboratory testing.
- Reagent Freeze-Thaw Stability Study (Study #6):
- Test set sample size: Genomic DNA isolated from cell lines, representing all possible genotypes.
- Data provenance: Not explicitly stated, likely internal laboratory testing.
- Analytical Sensitivity (Study #3):
- Test set sample size: 3 genomic DNA samples (WT, HET, MUT). Each sample diluted to 8 different concentrations and tested in replicates of 40, totaling 960 data points.
- Data provenance: Whole blood collected in potassium EDTA, likely laboratory-controlled study.
- Analytical Specificity (Interfering Substances) (Study #4):
- Test set sample size: 9 whole blood samples of different genotypes (3 WT, 3 HET, 3 MUT).
- Data provenance: Not explicitly stated, likely laboratory-controlled study.
- Pre-Analytical Equivalency Study/Genomic DNA Extraction Reproducibility (Study #7):
- Test set sample size: 30 human whole blood samples and 10 leukocyte-depleted whole blood samples spiked with cell lines (total 40 samples). These were analyzed with 4 different extraction methods, leading to 160 extracted DNA samples tested.
- Data provenance: Human whole blood and spiked cell lines, likely from a controlled laboratory setting.
- Instrument Equivalency (Study #8):
- Test set sample size: 29 human whole blood samples and 10 leukocyte-depleted whole blood samples spiked with cell lines (total 39 samples). Tested across 3 thermal cyclers and 3 fluorometers (39*3 = 117 tests per fluorometer in Table 9, but recorded as 78 of 78 = 100% for each cell, implying 78 distinct samples or tests per fluorometer/thermal cycler combination).
- Data provenance: Human whole blood and spiked cell lines, likely from a controlled laboratory setting.
- Method Comparison (Bi-directional Sequencing) (Study #2):
- Test set sample size: 361 human whole blood samples.
- Data provenance: Human whole blood samples. Not specified if retrospective or prospective or country of origin, but generally such studies involve controlled collection.
3. Number of Experts and Qualifications for Ground Truth:
The document does not explicitly mention the number or qualifications of experts used to establish the ground truth. However, the ground truth for all performance studies is established by bi-directional DNA sequencing, which is a widely accepted gold standard for determining genetic variants. This method is inherently objective and does not rely on expert interpretation in the same way as, for example, image-based diagnostics.
4. Adjudication Method:
Given that the ground truth is established by bi-directional DNA sequencing, which is a definitive molecular method, there is no mention of an adjudication method in the context of human interpretation (e.g., 2+1, 3+1). The testing process involves comparing the device's output to the objective sequencing results. In cases of discrepancies or invalid results (e.g., in Study #2), these are noted and factored into the agreement calculations.
5. Multi-Reader Multi-Case (MRMC) Comparative Effectiveness Study:
No, a multi-reader multi-case (MRMC) comparative effectiveness study was not done. The device is an in vitro diagnostic test for genetic analysis, not an imaging device or one requiring human interpretation of complex visual data. Therefore, the concept of "human readers improve with AI vs without AI assistance" is not applicable here.
6. Standalone Performance:
Yes, a standalone performance study (algorithm only without human-in-the-loop performance) was done. All the analytical performance studies (precision, stability, sensitivity, specificity, equivalency, and method comparison against sequencing) represent the standalone performance of the Invader® MTHFR 677 test, as it directly outputs genotype calls from fluorescence data without human interpretation influencing the result. The Invader Call Reporter™ software performs the data analysis and determines results autonomously following the import of fluorescence data.
7. Type of Ground Truth Used:
The primary type of ground truth used across all studies is bi-directional DNA sequencing. This is explicitly stated for the method comparison study and implied as the reference method for verifying all genotype calls throughout the analytical performance sections (e.g., "percent agreement between Invader® MTHFR 677 test and sequencing").
8. Sample Size for the Training Set:
The document does not specify the sample size used for a "training set." This device is based on a well-established molecular biology technique (Invader Plus® chemistry, PCR, FRET) and software for data interpretation, rather than a machine learning or AI model that typically requires a distinct training phase with large datasets. The controls included with the kit (Wild Type, Heterozygous, Mutant, No DNA Control) serve to ensure proper functioning and calibration for each run, effectively acting as internal references for the test.
9. How the Ground Truth for the Training Set Was Established:
Since there is no explicit mention of a "training set" in the context of machine learning, the question of how its ground truth was established is not directly applicable. The performance is validated against the gold standard of bi-directional DNA sequencing, which inherently provides the "ground truth" for the MTHFR 677 genotype. The development and optimization of the assay itself would have involved molecular biology techniques to ensure accurate detection of the specific SNP.
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Invader® MTHFR 677 510(k) SUMMARY
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A. 510(k) Number: K100987
: -
B. Purpose for Submission: New Device
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C. Measurand: MTHFR 677
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D. Type of Test:
Qualitative genotyping test for single nucleotide polymorphism detection.
E. Applicant:
Hologic Inc. Third Wave Technologies 250 Campus Drive Marlborough, MA 01752 508-263-8912 Contact Person: Randall J. Covill, Manager, Regulatory Affairs Date of Submission: April 2010
- F. Proprietary and Established Names: Invader® MTHFR 677
G. Regulatory Information:
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- Regulation Sections: 21 CFR 864.7280
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- Classification: Class II
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- Product Code: OMM: Test 5,10-Methylenetetrahydrofolate Reductase Mutations, Genomic DNA PCR
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- Panel:
Hematology (81)
H. Intended Use:
-
- Intended Use(s):
The Invader® MTHFR 677 test is an in vitro diagnostic test intended for the detection and genotyping of a single point mutation (C to T at position 677) of the human 5.10-methylenetetrahydrofolate reductase (MTHFR) gene in isolated genomic DNA obtained from whole blood Potassium EDTA samples from patients with suspected thrombophilia.
- Intended Use(s):
-
Indication(s) for use: 2.
Same as Intended Use -
Special Conditions for use statements(s): ల For prescription use only
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Special instrument requirements: 4. None
I. Device Description:
The Invader MTHFR 677 test consists of the following components:
MTHFR 677 Oligo Mix
- Universal Buffer
Universal Enzyme Mix
No DNA Control
MTHFR 677 Wild Type Control
MTHFR 677 Heterozygous Control
MTHFR 677 Mutant Control
Invader Call Reporter™ Software
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· Invader® MTHFR 677 Software
J. Substantial Equivalence Information:
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- Predicate device name(s):
... .
- Predicate device name(s):
-
Verigene® MTHFR Nucleic Acid Test (Nanosphere, K070597)
- Predicate 510(k) number(s):
- Nanosphere, K070597
-
- Comparison with predicate:
| Table 1: Comparison with Predicate Device | Use | ||||
|---|---|---|---|---|---|
| Predicate Device | Proposed Device | TargetPopulation | Patients with suspectedthrombophilia | Same as predicate | |
| Product Name(Manufacturer,Submission) | Verigene® MTHFR Nucleic AcidTest(Nanosphere, K070597) | Invader® MTHFR 677(Hologic, Inc. K100987) | Chemistry | SNP discrimination viaoligonucleotide probes; detectionvia evanescent wave light scatterwith nanoparticles. | PCR followed by Invader® achievingSNP discrimination viaoligonucleotide probes. |
| Intended Use | "The Verigene MTHFR NucleicAcid Test is an in vitro diagnosticfor the detection and genotyping ofa single point mutation (C to T atposition 677) of the human 5, 10methylene-tetrahydrofolatereductase gene (MTHFR) inpatients with suspectedthrombophilia, from isolatedgenomic DNA obtained fromwhole blood samples. The test isintended to be used on theVerigene System."The Verigene System is a bench-top molecular diagnosticworkstation that automates the invitro diagnostic analysis anddetection of nucleic acids usinggold nanoparticle probetechnology. The Verigene Systemis intended to be used byexperienced laboratoryprofessionals with training on basiclaboratory techniques and on theuse of the system components. | The Invader® MTHFR 677 test is anin vitro diagnostic test intended forthe detection and genotyping of asingle point mutation (C to T atposition 677) of the human 5,10-methylenetetrahydrofolate reductase(MTHFR) gene in isolated genomicDNA obtained from whole bloodPotassium EDTA samples frompatients with suspectedthrombophilia. | Hardware | The Verigene System consists oftwo instruments, the VerigeneProcessor and the Verigene Reader,and utilizes single-use, disposableTest Cartridges to process andgenotype multiple genes in a DNAsamples in approximately 1.5hours. | Non-specified, third-partyfluorometer and thermal cycler. |
| Specimen Type | Purified DNA isolated from humanwhole peripheral blood | Same as predicate | SoftwareInterface | Embedded software in closedsystem, integrated graphical userinterface. | Java-based software installed on astandalone PC capable of convertingraw fluorescence data into genotypecalls. |
| Indications for | Same as Intended Use | Same as Intended Use | Detection Method | Single-image sensor wherenanoparticles are illuminated usinga fixed-wavelength light source. | PCR and Fluorescence ResonanceEnergy Transfer (FRET) chemistryfor signal reporting. |
| Sample Size | 25µL | 20µl reaction containing 0.25-4ng/ulgDNA extracted from humanperipheral whole blood. | |||
| DetectionProcedure | Single-image sensor wherenanoparticles are illuminated usinga fixed-wavelength light source. | Multi-well fluorometer to detect rawfluorescence. | |||
| DetectionChemistry | Detection via evanescent wavelight scatter with nanoparticles. | PCR and Invader® usingFluorescence Resonance EnergyTransfer (FRET) chemistry for signalreporting. | |||
| Analysis Time | 90 min. processing with 2 minanalysis time. | ~90 min. amplification followed by 1min signal detection. Softwareanalysis post signal detection. |
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.
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K. Standard/Guidance Document Referenced (if applicable):
- Guidance for Industry and FDA Staff Class II Special Controls Guidance Document: Factor V Leiden DNA Mutation Detection Systems issued on March 16, 2004
- Guidance for Industry and FDA Staff Guidance for the Content of . Premarket Submissions for Software Contained in Medical Devices issued May 11, 2005
- Guidance for Industry and FDA Staff Format for Traditional and . Abbreviated 510(k)s issued on August 12, 2005
L. Test Principle:
The Invader® MTHFR 677 test utilizes the Invader Plus® chemistry with DNA isolated from human whole blood, for the detection of the targeted sequence polymorphism. Specifically, the Invader Plus® chemistry utilizes a single-tube, two phase reaction, including target amplification and signal generation (mediated by Invader chemistry). Invader Plus® reaction mixes are assembled by combining the MTHFR 677 Oligo Mix, Universal Enzyme Mix, and Universal Buffer. In a 96-well plate, reaction mix is combined with purified genomic DNA samples, as well as four (4) controls included with the test. The No DNA Control is used by the interpretive software to set the "noise" component of the run for "signal-to-noise" calculations. The genotype-specific controls (WT, HET, MUT) ensure reagents were assembled correctly and perform according to the specifications. The 96-well plate is transferred to an appropriately programmed thermal cycler for target amplification and signal generation. In the target amplification phase of the reaction, amplification is carried out using "two-step" cycling conditions (i.e. denaturation & annealing/extension). Following amplification, Taq polymerase is inactivated by a 10 minute incubation at 99°C, after which the thermal cycler proceeds to 63°C to initiate the signal generation (Invader ) phase of the reaction (see Figure 1).
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Image /page/4/Figure/0 description: The image shows a comparison of two structure formation processes, one for a wildtype specific primary probe and one for a mutation specific primary probe. Both processes involve three steps: structure formation, structure recognition and cleavage, and a secondary reaction. The secondary reaction for the wildtype probe involves a FRET cassette (FAM) and results in fluorescence 1 (FAM), while the secondary reaction for the mutation specific probe involves a FRET cassette (RED) and results in fluorescence 2 (RED).
Figure 1. Invader® Signal Generation Phase
During the signal generation phase, a discriminatory Probe transiently hybridizes to the amplified target sequence along with an Invader® oligonucleotide, 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 oligonucleotide overlaps the Primary Probe, and does 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 aligned with the Invader® reaction temperature so that under the isothermal reaction conditions (~63°C) the Primary Probes cycle on and off the target DNA. This allows for multiple rounds of Primary Probe cleavage for each DNA target resulting in an accumulation of the number of released 5'-flaps. The released 5'-flap transiently hybridizes with a corresponding FRET cassette forming an overlapping structure that is recognized and the fluorophore is cleaved from the FRET cassette by the Cleavase enzyme. The 5'-flap is designed to have a melting temperature aligned with the Invader reaction temperature, so that the 5'-flaps cycle on and off of the corresponding FRET cassettes. This allows for multiple rounds of FRET cassette cleavage for each 5'flap, and an accumulation of released fluorophore. When the FRET cassette is cleaved, a fluorophore and quencher are separated, generating detectable fluorescence signal. The format uses two different discriminatory Primary Probes, one for the mutant allele and one for the wild type allele (Figure 1). Each Primary Probe is assigned a unique 5-flap, and distinct FRET cassette, with a spectrally distinct fluorophore. By design, the released 5flaps will bind only to their respective FRET cassettes to generate a target-specific signal, linking the wild type allele with one fluorophore (Fluorescence 1: FAM) and the mutant allele with the second fluorophore (Fluorescence 2: RED).
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The Invader® MTHFR 677 software, in combination with Invader Call Reporter™ software, is a data analysis software package developed by Hologic for use with the Invader® MTHFR 677 test. The software package provides a working template for the setup of reaction mixes and sample placement, and following the import of fluorescence data, it determines results and validity for controls and samples. A summary of the Invader Call Reporter™- Invader® MTHFR 677 package workflow is shown in Figure 2.
Image /page/5/Figure/1 description: This image is a flowchart that describes an assay selection process. The process starts with assay selection, where the operator enters the run ID, selects MTHFR 677, and enters the number of samples. The next step is mix preparation, where the master kit lot number and expiration date are entered, as well as the component lot numbers and expiration dates, and the reaction mix amounts are calculated. The next step is sample placement, where the sample IDs and comments are entered, followed by the results, where fluorescence data is imported, Invader MTHFR 677 results and data for controls and samples are displayed for valid plates, results are saved as PDF, and results are exported to spreadsheet and CSV, and finally the summary, where Invader MTHFR 677 results are displayed for controls and samples, the summary is saved as PDF, and the assay is finished.
Figure 2. Invader Call Reporter™. Invader® MTHFR 677 Package Workflow
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M. Performance Characteristics (if/when applicable):
-
- Analytical performance:
- a. Precision/Reproducibility:
External Reproducibility (Study #1): Two operators each from three (3) different sites (2 external sites and 1 internal site) performed the testing, in duplicate, over five (5) non-consecutive days for a ten (10) day period using the same testing materials including a panel of nine (9) whole blood samples specific for each of the three (3) possible genotypes (i.e. wild type, heterozygous, homozygous mutant).
| Table 2: Inter-laboratory Reproducibility of Invader® 677 Test | ||||||||
|---|---|---|---|---|---|---|---|---|
| Site | Operator | Samplestested | CorrectCalls | First PassNo Calls(Invalid, EQ) | Miscalls | CorrectCalls | FinalNo Calls(Invalid, EQ) | Final %AgreementFinal Correct CallsSamples Tested |
| Site001 | 1 | 90 | 90 | 0 | 0 | 90 | 0 | 100% |
| Site001 | 2 | 90 | 90 | 0 | 0 | 90 | 0 | 100% |
| Site002 | 1 | 90 | 90 | 0 | 0 | 90 | 0 | 100% |
| Site002 | 2 | 90 | 90 | 0 | 0 | 90 | 0 | 100% |
| Site003 | 1 | 90 | 90 | 0 | 0 | 90 | 0 | 100% |
| Site003 | 2 | 90 | 90 | 0 | 0 | 90 | 0 | 100% |
| All | All | 540 | 540 | 0 | 0 | 540 | 0 | 100% |
Lot-to-Lot Reproducibility (Study #9): A total of nine (9) genomic DNA samples (three (3) wild type , three (3) heterozygous and three (3) mutants) were tested in quadruplicate using three (3) different kit lots of the Invader® MTHFR 677 test. The percent agreement between Invader® MTHFR 677 test and sequencing was 100% (n=108).
| Table 3: Lot to Lot Reproducibility | ||||||
|---|---|---|---|---|---|---|
| Lot | #SamplesTested | FirstPassCorrectCalls | FirstPass NoCalls | Miscalls | FinalCorrectCalls | FinalAgreement% |
| 1 | 36 | 36 | 0 | 0 | 36 | 100 |
| 2 | 36 | 36 | 0 | 0 | 36 | 100 |
| 3 | 36 | 36 | 0 | 0 | 36 | 100 |
| Total | 108 | 108 | 0 | 0 | 108 | 100 |
- b. Linearity/assay reportable range: Refer to paragraph D below.
- Traceability, Stability, Expected values (controls, calibrators, or methods): C. Real-Time Stability Study (Study #5): Three (3) lots of product in the final configuration are being stored under recommended conditions: (1) -30° to -15°C (Standard Storage of intermediate components) as well as (2) +2° to +8°C (Standard Storage of Genotype-Specific Controls). Functional testing is performed with samples representing all 3 genotypes in quadruplicate at each time point. The interim test results have demonstrated 7 months stability for the device.
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| Sample/Control | Sequencing/ExpectedMTHER 677Genotype | T₀ Result | T₁ Result | T₂ Result | ||||||
|---|---|---|---|---|---|---|---|---|---|---|
| Lot 1 | Lot 2 | Lot 3 | Lot 1 | Lot 2 | Lot 3 | Lot 1 | Lot 2 | Lot 3 | ||
| Control 1 | WT | WT | WT | WT | WT | WT | WT | WT | WT | WT |
| Control 2 | HET | HET | HET | HET | HET | HET | HET | HET | HET | HET |
| Control 3 | MUT | MUT | MUT | MUT | MUT | MUT | MUT | MUT | MUT | MUT |
| gDNA 1 | WT | WT | WT | WT | WT | WT | WT | WT | WT | WT |
| gDNA 2 | WT | WT | WT | WT | WT | WT | WT | WT | WT | WT |
| gDNA 3 | HET | HET | HET | HET | HET | HET | HET | HET | HET | HET |
| gDNA 4 | MUT | MUT | MUT | MUT | MUT | MUT | MUT | MUT | MUT | MUT |
| Percent Agreement | 100 | 100 | 100 | 100 | 100 | 100 | 100 | 100 | 100 |
Reagent Freeze-Thaw Stability Study (Study #6): Product in the final configuration was subject to 15 freeze-thaw cycles prior to the final thaw at the time of testing. Functional testing was performed using genomic DNA isolated from cell lines, representing all possible genotypes. The percent agreement between the sequencing result and the Invader® MTHFR 677 test were 100%, therefore demonstrating stability for up to fifteen (15) freeze/thaw cycles.
| Table 5: Freeze/Thaw Stability of Invader® MTHFR 677 | |||||||||||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| 1 | |||||||||||||||||
| Sample 1 1 11 | َ َ َ َ َ َ َ َ | 3 3 8 8 8 9 1 8 8 9 1 10 2 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 | |||||||||||||||
| Control 1(WINE KEEK | ﻢ ﺍﻟﻤﺮﺍﺟﻊ | ﻢ ﺍﻟﻤﺴﺘﻘﻠﺔ ﺍﻟﻤﺘﺤﺪﺓ ﺍﻟﻤﺘﺤﺪﺓ ﺍﻟﻤﺘﺤﺪﺓ ﺍﻟﻤﺘﺤﺪﺓ ﺍﻟﻤﺘﺤﺪﺓ ﺍﻟﻤﺘﺤﺪﺓ ﺍﻟﻤﺘﺤﺪﺓ ﺍﻟﻤﺘﺤﺪﺓ ﺍﻟﻤﺘﺤﺪﺓ ﺍﻟﻤﺘﺤﺪﺓ ﺍﻟﻤﺘﺤﺪﺓ ﺍﻟﻤﺘﺤﺪﺓ ﺍﻟﻤﺘﺤﺪﺓ ﺍﻟﻤﺘﺤﺪﺓ ﺍﻟﻤﺘﺤﺪﺓ ﺍﻟﻤﺘﺤﺪﺓ ﺍﻟﻤﺘﺤﺪﺓ ﺍﻟﻤﺘﺤﺪﺓ ﺍﻟﻤﺘﺤﺪﺓ ﺍﻟﻤﺘﺤﺪﺓ ﺍﻟ | ਪ੍ | 3 | ਤੇ | 3 | 3 | ﺔ | 3 | ﻓ | ﻢ ﺍﻟﻤﺴﺘﻮﻯ ﺍﻟﻤﺴﺘﻮﻯ ﺍﻟﻤﺴﺘﻮﻯ ﺍﻟﻤﺴﺘﻮﻯ ﺍﻟﻤﺴﺘﻮﻯ ﺍﻟﻤﺴﺘﻮﻯ ﺍﻟﻤﺴﺘﻮﻯ ﺍﻟﻤﺴﺘﻮﻯ ﺍﻟﻤﺴﺘﻮﻯ ﺍﻟﻤﺴﺘﻮﻯ ﺍﻟﻤﺴﺘﻮﻯ ﺍﻟﻤﺴﺘﻮﻯ ﺍﻟﻤﺴﺘﻮﻯ ﺍﻟﻤﺴﺘﻮﻯ ﺍﻟﻤﺴﺘﻮﻯ ﺍﻟﻤﺴﺘﻮﻯ ﺍﻟﻤﺴﺘﻮﻯ ﺍﻟﻤﺴﺘﻮﻯ ﺍﻟﻤﺴﺘﻮﻯ ﺍﻟﻤﺴﺘﻮﻯ ﺍﻟﻤﺴﺘﻮﻯ ﺍﻟﻤ | 3 | ব | 3 | વર્તર | 100 | |
| Control 2; 4CHET) THET | 3 | 7 | ﻢ ﺍﻟﻤﺮﺍﺟﻊ ﺍﻟﻤﺮﺍﺟﻊ | 3 | 3 | ತೆ | ನ | 3 | 3 | 3 | ಗೆ | 8 | ತಿ | 3 | 3 | વે રે | 100 |
| Control 3EMUT | ਪ੍ | ਪ੍ | ﻢ | 3 | 3 | 3 | 3 | ಗ | 3 | 3 | ਪੈ | 3 | 3 | 3 | વર્સ | 100 | |
| ISEDNATWT) : | 6 | 6 | 12 | 1 | ત | 6 | 本 | * | 6 | 36 | 100 | ||||||
| gDNA(HET) | ನ್ನ | 8 | 8 | 18 | * | * | 8 | 8 | 本 | * | 8 | 48 | 100 | ||||
| gDNA®: "(MUT) | 6 | 6 | ત | ત્ | 6 | * | * | 6 | 36 | 100 | |||||||
| Total | 29 | 9 | 29 | Q | 29 | ರ | 0 | 9 | ਉ | 29 | 0 | 29 | ರ | ರಿ | 29 | 255 | 100 |
| * Testing with oDNA samples did not occur at this testing point. |
*Testing with gDNA samples did not occur at this testing point.
- Detection limit/Analytical Sensitivity and Normal Range (Study #3): d. Three (3) genomic DNA samples with different genotypes (i.e. WT, HET, MUT) were extracted from whole blood collected in potassium EDTA. Each sample was diluted to eight different concentrations 0.5, 5, 20, 40, 80, 200, 400, 800 ng/ uL and tested in replicates of forty (40). The recommend range of the assay was
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determined to be between 5-80 ng/uL of input gDNA, based on 100% concordance of all tested replicates with bi-directional sequencing.
| Table 6: Percent Agreement Between Replicates | |||
|---|---|---|---|
| Sample ID (Genotype based on Sequencing) | |||
| Input SampleConcentration | 03-4520 (MUT) | 03-4525 (HET) | 03-4524 (WT) |
| 0.5 ng/µl | 100% (40/40) | 100% (40/40) | 100% (40/40) |
| 5 ng/µl | 100% (40/40) | 100% (40/40) | 100% (40/40) |
| 20 ng/µl | 100% (40/40) | 100% (40/40) | 100% (40/40) |
| 40 ng/µl | 100% (40/40) | 100% (40/40) | 100% (40/40) |
| 80 ng/µl | 100% (40/40) | 100% (40/40) | 100% (40/40) |
| 200 ng/µl | 100% (40/40) | 100% (40/40) | 100% (40/40) |
| 400 ng/µl | 100% (40/40) | 100% (40/40) | 100% (40/40) |
| 800 ng/µl | 100% (40/40) | 100% (40/40) | 100% (40/40) |
-
Analytical specificity (Interfering Substances) (Study #4): e.
Test performance was not affected by addition of the following substances to nine (9) whole blood samples of different genotype (3 WT, 3 HET, 3 MUT) prior to extraction: -
Heparin (1500 U/dL human whole blood) ●
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Cholesterol (300 mg/dL human whole blood) .
-
. Bilirubin (10 mg/dL human whole blood)
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Hemoglobin (up to 0.2% in whole blood) .
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Potassium EDTA (1.8 mg/mL human whole blood) .
-
Ethanol-based Wash Buffer (5% in DNA sample) .
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| Table 7: Summary, Comparison of Invader MTHFR 677 Interfering Substance Results to Sequencing | ||||
|---|---|---|---|---|
| InterferingSubstanceCode | Substance Concentration /(in blood or DNA sample) | % AgreementwithSequencingGenotype | % AgreementwithUntreatedSampleInvader®MTHFR 677Genotype | PASS /FAIL |
| A | No Addition Control(Untreated) | 100% (18 of 18) | N/A | PASS |
| B | Bilirubin 10mg/dl (blood) | 100% (18 of 18) | 100% (18 of 18) | PASS |
| C | Cholesterol 300mg/dl (blood) | 100% (18 of 18) | 100% (18 of 18) | PASS |
| D | K2EDTA 1.8mg/ml (blood) | 100% (18 of 18) | 100% (18 of 18) | PASS |
| E | Heparin 1500 U/dl (blood) | 100% (18 of 18) | 100% (18 of 18) | PASS |
| F1 | Hemoglobin 0.2% (blood) | 100% (18 of 18) | 100% (18 of 18) | PASS |
| F2 | Hemoglobin 0.1% (blood) | 100% (18 of 18) | 100% (18 of 18) | PASS |
| F3 | Hemoglobin 0.05% (blood) | 100% (18 of 18) | 100% (18 of 18) | PASS |
| F4 | Hemoglobin 0.025% (blood) | 100% (18 of 18) | 100% (18 of 18) | PASS |
| G | Buffer AW2 5% (DNA) | 100% (18 of 18) | 100% (18 of 18) | PASS |
61677 Summers Ecomparison of Invodor Max 2010 September 10, 2016 Conservering Substance Count
- Pre-Analytical Equivalency Study/Genomic DNA Extraction Reproducibility f (Study #7): Thirty (30) human whole blood samples and ten (10) leukocyte depleted whole blood spiked with cell lines were divided and extracted using four (4), commercially available DNA extraction methods (A. Qiagen QIAamp® 96 DNA Blood Kit, B. Qiagen QIAamp® DNA Blood Mini Kit, C. Gentra Generation® Capture Column Kit (Oiagen), D. Roche MagNA Pure LC DNA Isolation Kit I). The 160 extracted DNAs were analyzed in singlicate with one (1) lot of the device. The percent agreement between the Invader® MTHFR 677 test for each extraction method and bi-directional sequencing was 100% (n=40).
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| Table 8a: Pre-Analytical Equivalency | ||||||
|---|---|---|---|---|---|---|
| ExtractionMethod | #SamplesTested | FirstPassCorrectCalls | FirstPassNo.Calls | Miscalls | FinalCorrectCalls | FinalAgreement% |
| A | 40 | 40 | 0 | 0 | 40 | 100 |
| B | 40 | 39 | 1* | 0 | 39* | 100* |
| C | 40 | 40 | 0 | 0 | 40 | 100 |
| D | 40 | 40 | 0 | 0 | 40 | 100 |
| Total | 160 | 159 | 1 | 0 | 159 | 100 |
| *Sample was removed from study due to loss of traceability of the sampleidentification. |
| Table 8b: Genotype Distribution Across Sample Type | |||||
|---|---|---|---|---|---|
| Sample Type Type WT WT MET MET HET Total | |||||
| Human Whole Blood | 0 * | 10 | |||
| LDWB Spiked with Cell Lines** | 10 | ||||
| Total | 0* | રું તેમ | |||
| *Sample was removed from study due to loss of traceability of the sample identification.**LDWB=Leukocyte Depleted Whole Blood |
- Instrument Equivalency (Study #8): Twenty-nine (29) human whole blood g. samples and ten (10) leukocyte depleted whole blood samples spiked with cell lines were extracted using two (2) commonly used extraction methods. The extracts were tested with the Invader® MTHFR 677 test using three (3) commercially available thermal cyclers (1. ABI GeneAmp® PCR System 9700 with 96-well gold block, 2. ABI Veriti™ and 3. MJ Research PTC-100) and the raw fluorescent data acquired on three (3) commercially available fluorometers (A. Tecan Infinite®, B. Tecan Genios® and C. BioTek®, FLx800). Results from the three (3) fluorometers were transferred into the interpretive software and genotype calls compared to bi-directional sequencing.
| Table 9: Concordance by Instrument | |||
|---|---|---|---|
| Fluorometer | Thermal Cycler1 | 2 | 3 |
| A | 78 of 78 = 100% | 78 of 78 = 100% | 78 of 78 = 100% |
| B | 78 of 78 = 100% | 78 of 78 = 100% | 78 of 78 = 100% |
| C | 78 of 78 = 100% | 78 of 78 = 100% | 78 of 78 = 100% |
2. Comparison studies:
a. Method comparison: Bi-directional Sequencing (Study #2):
- Human whole blood samples (n = 361) underwent DNA extraction and subsequent bi-directional DNA sequence analysis. The same DNA samples were then analyzed using the Invader® MTHFR 677 test. The observed agreement between the Invader® MTHFR 677 test and bidirectional DNA sequencing was 100% (359/359). The first run agreement with bi-directional sequencing was 99.45% (359/361).
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| MTHFR 677Genotype* | Numbertested | Number ofvalid resultson 1st run | Number ofCorrectgenotypecalls on FirstRun | First RunAgreement |
|---|---|---|---|---|
| HomozygousWild Type(GG) | 180 | 178** | 178** | 98.89% |
| Heterozygous(GA) | 104 | 104 | 104 | 100% |
| HomozygousMutant(AA) | 77 | 77 | 77 | 100% |
| Total | 361 | 359** | 359** | 99.45% |
| * Genotype determined through bi-directional DNA sequencing | ||||
| ** Two samples failed to generate valid results. These samples were reported as invalid (EO) and no genotype calls were assigned by the interpretive software. |
The EQ result was used to determine the First Run Agreement.
| Table 10b: Agreement between the Invader® MTHFR 677 Test and | |||||||||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| Bi-directional DNA Sequencing | |||||||||||||||
| Genotype* | First Pass Results | Final Results | |||||||||||||
| WT | HET | MUT | No-Call | Mis-Call | % Agreement | 95% LCB** | WT | HET | MUT | No-Call | Mis-Call | % Agreement | 95% LCB | ||
| WT | 178 | 0 | 0 | 2 | 0 | 99.45 | 96.04 | 178 | 0 | 0 | 2 | 0 | 99.45 | 96.04 | |
| HET | 0 | 104 | 0 | 0 | 0 | 100.00 | 97.16 | 0 | 104 | 0 | 0 | 0 | 100.00 | 97.16 | |
| MUT | 0 | 0 | 77 | 0 | 0 | 100.00 | 96.18 | 0 | 0 | 77 | 0 | 0 | 100.00 | 96.18 | |
| *Genotype determined through bi-directional DNA sequencing. **Lower boundary of the 95% confidence interval for each genotype. †Calculated across all samples. |
- External Reproducibility studies:
Clinical Sensitivity: please refer to section 1d above. a.
Clinical specificity: please refer to section 1e above. b.
-
- Expected values/Reference range: (Prevalence) MTHFR 677: 30-40% Caucasians
N. System Descriptions:
- Expected values/Reference range: (Prevalence) MTHFR 677: 30-40% Caucasians
.
-
- Modes of Operation: · Closed System
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-
- Software:
FDA has reviewed applicant's Hazard Analysis and software development processes for this line of product type. Yes X
- Software:
-
- Specimen Identification: Manual Labeling
-
- Specimen Sampling and Handling:
DNA should be extracted using a validated DNA extraction method that generates DNA concentration range of greater than 5ng/ul.
-
- Quality Control:
Each test contains positive and negative controls to assure proper functioning of the system: Failure of any controls will be indicated as "Invalid" in the test results section of the report. The genotyping test result will not be reported for any sample for which a positive or negative control failure occurs. Positive Control: The genotype controls (WT, HET, MUT) ensure reagents were assembled correctly and perform according to the specifications. Negative Control: The No DNA Control is used by the interpretive software to set the "noise" component of the run for "signal-to-noise" calculations.
- Quality Control:
Hardware and Software Controls:
The genotyping test result will not be reported for any sample for which a positive or negative control failure occurs.
O. Proposed Labeling:
The labeling is sufficient and satisfies the requirements of 21 CFR Part 809.10.
- P. Conclusion:
The submitted information in this 510 (k) notification is complete and supports a substantial equivalence decision.
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Image /page/13/Picture/1 description: The image shows the logo for the U.S. Department of Health and Human Services. The logo features a stylized caduceus, which is a symbol of medicine, with three wavy lines representing the serpent entwined around the staff. The logo is encircled by the text "DEPARTMENT OF HEALTH & HUMAN SERVICES - USA" in a circular arrangement.
Food and Drug Administration 10903 New Hampshire Avenue Document Mail Center - WO66-0609 Silver Spring, MD 20993-0002
Hologic Inc. Third Wave Technologies c/o Mr. Randall J. Covill Senior Specialist Regulatory Affairs 502 South Rosa Road Madison, WI 53719
MAY 1 3 2011
Re: K100987
Trade/Device Name: Regulation Number: Regulation Name: Regulatory Class: Product Code: Dated: March 31, 2011 Received: April 7, 2011 Invader® MTHFR 677 21 CFR §864.7280 Factor V Leiden DNA Mutation Detection Systems Class II OMM
Dear Mr. Covill:
We have reviewed your Section 510(k) premarket notification of intent to market the device referenced above and have determined the device is substantially equivalent (for the indications for use stated in the enclosure) to legally marketed predicate devices marketed in interstate commerce prior to May 28, 1976, the enactment date of the Medical Device Amendments, or to devices that have been reclassified in accordance with the provisions of the Federal Food, Drug, and Cosmetic Act (Act) that do not require approval of a premarket approval application (PMA). You may, therefore, market the device, subject to the general controls provisions of the Act. The general controls provisions of the Act include requirements for annual registration, listing of devices, good manufacturing practice, labeling, and prohibitions against misbranding and adulteration.
If your device is classified (see above) into class II (Special Controls), it may be subject to such additional controls. Existing major regulations affecting your device can be found in Title 21, Code of Federal Regulations (CFR), Parts 800 to 895. In addition, FDA may publish further announcements concerning your device in the Federal Register.
Please be advised that FDA's issuance of a substantial equivalence determination does not mean that FDA has made a determination that your device complies with other requirements of the Act or any Federal statutes and regulations administered by other Federal agencies. You must comply with all the Act's requirements, including, but not limited to: registration and listing (21 CFR Part 807); labeling (21 CFR Parts 801 and 809); medical device reporting (reporting of medical device-related adverse events) (21 CFR 803); and good manufacturing practice requirements as set forth in the quality systems (QS) regulation (21 CFR Part 820). This letter
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Page 2 - Mr. Randall J. Covill
will allow you to begin marketing your device as described in your Section 510(k) premarket notification. The FDA finding of substantial equivalence of your device to a legally marketed predicate device results in a classification for your device and thus, permits your device to proceed to the market.
If you desire specific advice for your device on our labeling regulation (21 CFR Parts 801 and 809), please contact the Office of In Vitro Diagnostic Device Evaluation and Safety at (301) 796-5450. Also, please note the regulation entitled, "Misbranding by reference to premarket notification" (21 CFR Part 807.97). For questions regarding the reporting of adverse events under the MDR regulation (21 CFR Part 803), please go to
http://www.fda.gov/MedicalDevices/Safety/ReportaProblem/default.htm for the CDRH's Office of Surveillance and Biometrics/Division of Postmarket Surveillance.
You may obtain other general information on your responsibilities under the Act from the Division of Small Manufacturers, International and Consumer Assistance at its toll-free number (800) 638-2041 or (301) 796-7100 or at its Internet address http://www.fda.gov/cdrh/industry/support/index.html.
Sincerely yours,
Thava In Chen
Maria M. Chan, Ph.D Director Division of Immunology and Hematology Devices Office of In Vitro Diagnostic Device Evaluation and Safety Center for Devices and Radiological Health
Enclosure
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Indication for Use Statement
510(k) Number (if known): K100987
Invader MTHFR 677 test Device Name:
Indication for Use:
The Invader® MTHFR 677 test is an in vitro diagnostic test intended for the detection and genotyping of a single point mutation (C to T at position 677) of the human 5,10methylenetetrahydrofolate reductase (MTHFR) gene in isolated genomic DNA obtained from whole blood potassium EDTA samples from patients with suspected thrombophilia.
Prescription Use x (Part 21 CFR 801 Subpart D) AND/OR
Over-The-Counter Use (21 CFR 801 Subpart C)
(PLEASE DO NOT WRITE BELOW THIS LINE-CONTINUE ON ANOTHER PAGE OF NEEDED)
Concurrence of CDRH, Office of In Vitro Diagnostic Devices (OIVD)
Division Sign-Off
Office of In Vitro Diagnostic Device Evaluation and Safety
510(k) K100987
§ 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.)