(427 days)
The Invader® MTHFR 1298 test is an in vitro diagnostic test intended for the detection and genotyping of a single point mutation (A to C at position 1298) 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 1298 test consists of the following components:
MTHFR 1298 Oligo Mix
Universal Buffer
Universal Enzyme Mix
No DNA Control
MTHFR 1298 Wild Type Control
MTHFR 1298 Heterozygous Control
MTHFR 1298 Mutant Control
Invader Call Reporter™ Software
Invader® MTHFR 1298 Software
Here's an analysis of the Invader® MTHFR 1298 device based on the provided text, addressing your specific questions.
1. Table of Acceptance Criteria and Reported Device Performance
(Note: The document does not explicitly state "acceptance criteria" in a separate section with pass/fail thresholds for each study. Instead, it presents study results and concludes that the information "supports a substantial equivalence decision." The table below infers the implicit acceptance criterion as 100% agreement/concordance where achieved, and notes the precise percentage reported for other metrics.)
| Performance Characteristic | Acceptance Criteria (Inferred) | Reported Device Performance |
|---|---|---|
| Reproducibility (Inter-laboratory) | 100% Correct Calls, 0% No Calls, 0% Miscalls | 100% Final Agreement (540/540 samples across 3 sites, 2 operators each) |
| Reproducibility (Lot-to-Lot) | 100% Agreement with sequencing | 100% Agreement with sequencing (108/108 samples across 3 lots) |
| Real-time Stability | 100% Agreement with sequencing/expected genotype | 100% Agreement (all controls and gDNA samples across 3 lots and 3 timepoints up to 7 months) |
| Freeze-Thaw Stability | 100% Agreement with sequencing | 100% Agreement (all controls and gDNA samples through 15 freeze-thaw cycles) |
| Analytical Sensitivity (Concentration Range) | 100% Concordance with bi-directional sequencing | 100% Concordance for gDNA concentrations between 5-80 ng/µL (3 samples, 240 replicates) |
| Analytical Specificity (Interfering Substances) | 100% Agreement with sequencing and untreated samples | 100% Agreement (all tested interfering substances) |
| Pre-Analytical Equivalency (Extraction Reproducibility) | 100% Agreement with bi-directional sequencing | 100% Agreement (159/160 samples, 4 extraction methods) |
| Instrument Equivalency (Thermal Cycler & Fluorometer) | 100% Concordance with bi-directional sequencing | 100% Concordance (all combinations of 3 thermal cyclers and 3 fluorometers) |
| Method Comparison (Bi-directional Sequencing) | High agreement with bi-directional sequencing (explicit criteria not specified, but 100% is typical for substantial equivalence) | 99.71% First Run Agreement (347/348 samples); 100% (347/347) for valid results |
2. Sample Size Used for the Test Set and Data Provenance
- Inter-laboratory Reproducibility (Study #1):
- Test Set Sample Size: 9 (panel of genomic DNA samples representing 3 genotypes: wild type, heterozygous, homozygous mutant).
- Total Tests Performed: 540 (9 samples x 2 operators/site x 3 sites x 2 duplicates/day x 5 days of testing).
- Data Provenance: Not specified, but involved 2 external sites and 1 internal site. The samples were "whole blood samples specific for each of the three possible genotypes." Implied retrospective as specific genotypes were selected.
- Lot-to-Lot Reproducibility (Study #9):
- Test Set Sample Size: 9 (genomic DNA samples: 3 wild type, 3 heterozygous, 3 mutants).
- Total Tests Performed: 108 (9 samples x 4 replicates x 3 lots).
- Data Provenance: Not specified, but samples were "genomic DNA samples." Implied retrospective.
- Real-time Stability (Study #5):
- Test Set Sample Size: 3 genomic DNA samples (WT, HET, MUT) + 3 controls.
- Total Tests Performed: Performed in quadruplicate at each time point (initial, 4 months, 7 months) for 3 lots. Total: (3 gDNA + 3 controls) x 4 replicates x 3 lots x 3 time points = 216 tests.
- Data Provenance: Not specified.
- Freeze-Thaw Stability (Study #6):
- Test Set Sample Size: 3 genomic DNA samples (WT, HET, MUT) + 3 controls.
- Total Tests Performed: The table indicates varying numbers of tests per sample per freeze/thaw cycle. Total for gDNA: 36 (WT), 48 (HET), 36 (MUT). Total for controls: 45 each. Total overall is 255.
- Data Provenance: Genomic DNA isolated from cell lines.
- Analytical Sensitivity (Detection Limit) (Study #3):
- Test Set Sample Size: 3 genomic DNA samples (1 MUT, 1 HET, 1 WT).
- Total Tests Performed: 960 (3 samples x 8 concentrations x 40 replicates).
- Data Provenance: "Genomic DNA samples with different genotypes (i.e. WT, HET, MUT) were extracted from whole blood collected in potassium EDTA." Implied retrospective.
- Analytical Specificity (Interfering Substances) (Study #4):
- Test Set Sample Size: 9 whole blood samples (3 WT, 3 HET, 3 MUT).
- Total Tests Performed: 18 tests per substance (9 samples x 2 replicates). 7 substances + 1 control = 8 conditions. Total: 18 x 8 = 144 tests.
- Data Provenance: "human whole blood samples." Implied retrospective.
- Pre-Analytical Equivalency (Genomic DNA Extraction Reproducibility) (Study #7):
- Test Set Sample Size: 30 human whole blood samples + 10 leukocyte depleted whole blood spiked with cell lines = 40 unique samples.
- Total Tests Performed: 160 (40 samples x 4 extraction methods, analyzed in singlicate).
- Data Provenance: "human whole blood samples and leukocyte depleted whole blood spiked with cell lines." Implied retrospective.
- Instrument Equivalency (Study #8):
- Test Set Sample Size: 29 human whole blood samples + 10 leukocyte depleted whole blood samples = 39 unique samples.
- Total Tests Performed: 78 tests were reported per combination of fluorometer/thermal cycler. Overall: 39 samples x 2 extraction methods x 3 thermal cyclers x 3 fluorometers (but results are combined for each fluorometer/cycler pairing). The table shows 78 total tests for each of the 9 combinations.
- Data Provenance: "human whole blood samples and leukocyte depleted whole blood samples spiked with cell lines." Implied retrospective.
- Method Comparison (Bi-directional Sequencing) (Study #2):
- Test Set Sample Size: 348 human whole blood samples.
- Data Provenance: "Human whole blood samples (n = 348)." Implied retrospective.
3. Number of Experts Used to Establish the Ground Truth for the Test Set and Qualifications of Those Experts
The document does not explicitly state the number or qualifications of experts used to establish ground truth.
For ground truth established by bi-directional sequencing, it's a laboratory method that typically involves trained molecular biologists or laboratory technicians. The results are considered highly accurate.
4. Adjudication Method for the Test Set
The document does not describe an adjudication method for the test sets. The ground truth was primarily established by bi-directional sequencing, which is considered a definitive method, minimizing the need for adjudication in the traditional sense (e.g., of discrepant expert reads).
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
This section is not applicable. The Invader® MTHFR 1298 is an in vitro diagnostic molecular test for genotyping a specific gene mutation. It is not an imaging or interpretive AI device that involves human "readers" or assesses "AI assistance." The device outputs a direct genotype call (WT, HET, MUT).
6. If a Standalone (i.e., algorithm only without human-in-the-loop performance) Was Done
Yes, the device's performance studies were conducted in a standalone manner. The Invader® MTHFR 1298 test is an automated system where the Invader Call Reporter™ Software processes raw fluorescence data to determine genotypes. Human input is in sample preparation and loading, and reviewing the generated report, but the genotyping call itself is algorithm-driven without real-time human interpretation embedded in the performance measurement. The various studies (reproducibility, sensitivity, specificity, method comparison) directly evaluate this standalone algorithmic performance against a known ground truth (sequencing).
7. The Type of Ground Truth Used
The primary ground truth used for all performance studies was bi-directional DNA sequencing. This is considered the gold standard for confirming specific genetic variations. For some studies, expected genotypes based on controls or known cell lines were also used, which would have initially been characterized by sequencing.
8. The Sample Size for the Training Set
The document primarily describes validation studies and does not explicitly mention a "training set" in the context of device development. For IVD devices, a training set generally refers to the data used to initially develop and optimize the assay parameters and interpretation algorithms. Such details are typically proprietary and not extensively disclosed in 510(k) summaries, which focus on performance validation. The studies described are validation and verification studies using independent "test sets."
9. How the Ground Truth for the Training Set Was Established
As no specific "training set" details are provided, the method for establishing its ground truth is also not described. However, it can be inferred that any samples used for initial assay development and algorithm optimization would also have had their MTHFR 1298 genotype established using a highly accurate method, most likely bi-directional DNA sequencing, similar to the validation studies.
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1004196
2011
Invader® MTHFR 1298 510(k) SUMMARY
| A. | 510(k) Number: | |||
|---|---|---|---|---|
| K100496 | APR | 2 5 | ||
| B. | Purpose for Submission: | |||
| New Device | ||||
| C. | Measurand: | |||
| MTHFR 1298 | ||||
| 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 1298 | ||||
| G. | Regulatory Information: | |||
| 1. Regulation Sections: 21 CFR 864.7280 | ||||
| 2. Classification: | ||||
| Class II | ||||
| 3. Product Code: | ||||
| NPR: Test, MTHFR A1298C Mutations, Genomic DNA PCR | ||||
| 4. Panel: | ||||
| Hematology (81) | ||||
| H. | Intended Use: | |||
| 1. Intended Use(s): | ||||
| The Invader® MTHFR 1298 test is an in vitro diagnostic test intended for thedetection and genotyping of a single point mutation (A to C at position 1298) 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.
- Indication(s) for use: 2. Same as Intended Use
- Special Conditions for use statements(s): 3. For prescription use only
-
- Special instrument requirements: None
I. Device Description:
The Invader MTHFR 1298 test consists of the following components:
MTHFR 1298 Oligo Mix Universal Buffer Universal Enzyme Mix No DNA Control MTHFR 1298 Wild Type Control MTHFR 1298 Heterozygous Control MTHFR 1298 Mutant Control Invader Call Reporter™ Software Invader® MTHFR 1298 Software
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J. Substantial Equivalence Information:
-
- Predicate device name(s):
,
-
- Predicate 510(k) number(s):
- Nanosphere, K070597
-
- Comparison with predicate:
| Table 1: Comparison with Predicate Device | ||
|---|---|---|
| Predicate Device | Proposed Device | |
| Product Name(Manufacturer,Submission) | Verigene® MTHFR Nucleic AcidTest( Nanosphere, K070597 ) | Invader® MTHFR 1298( Hologic, Inc., K100496 ) |
| 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 1298 test isan in vitro diagnostic test intendedfor the detection and genotyping of asingle point mutation (A to C atposition 1298) of the human 5,10-methylenetetrahydrofolate reductase(MTHFR) gene in isolated genomicDNA obtained from whole bloodpotassium EDTA samples frompatients with suspectedthrombophilia. |
| Specimen Type | Purified DNA isolated from humanwhole peripheral blood | Same as predicate |
| Indications forUse | Same as Intended Use | Same as Intended Use |
| TargetPopulation | Patients with suspectedthrombophilia | Same as predicate |
| Chemistry | SNP discrimination via.oligonucleoptide probes; detectionvia evanescent wave light scatterwith nanoparticles. | PCR and Invader® usingFluorescence Resonance EnergyTransfer (FRET) chemistry for signalreporting. Same as predicate device.Both our device and predicate devicedetect signal from amplicons usingFluorescence Resonance EnergyTransfer (FRET). |
| 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. |
| SoftwareInterface | Embedded software in closedsystem, integrated graphical userinterface. | Java-based software installed on astandalone PC capable of convertingraw fluorescence data into genotypecalls. |
| Detection Method | Single-image sensor wherenanoparticles are illuminated usinga fixed-wavelength light source. | PCR and Fluorescence ResonanceEnergy Transfer (FRET) chemistryfor signal reporting. Same aspredicate device. Both our device andpredicate device detect signal fromamplicons using FluorescenceResonance Energy Transfer (FRET). |
| Sample Size | 25μL | 20ul 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 | SNP discrimination viaoligonucleoptide probes; detectionvia evanescent wave light scatter | PCR and Invader® usingFluorescence Resonance EnergyTransfer (FRET) chemistry for signalreporting. Both our device and |
| with nanoparticles. | predicate device detect signal fromamplicons using FluorescenceResonance Energy Transfer (FRET). | |
| Analysis Time | 90 min. processing with 2 min.analysis 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 t Abbreviated 510(k)s issued on August 12, 2005
L. Test Principle:
The Invader® MTHFR 1298 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 1298 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/5/Figure/0 description: The image shows two diagrams illustrating structure formation, recognition, and cleavage processes. The left diagram, labeled '1a, 1b, 1c,' depicts the process for a wildtype-specific primary probe, while the right diagram, labeled '2a, 2b, 2c,' illustrates the process for a mutation-specific primary probe. Each diagram includes steps for structure formation, structure recognition and cleavage, and a secondary reaction involving FRET cassettes, resulting in fluorescence. The left diagram shows fluorescence 1: FAM, and the right diagram shows 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 51-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 1298 software, in combination with Invader Call Reporter™ software, is a data analysis software package developed by Hologic for use with the Invader® MTHFR 1298 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® MTHER 1298 package workflow is shown in Figure 2.
Image /page/6/Figure/1 description: This image is a flowchart outlining a laboratory assay process. It begins with "Assay Selection," where the operator, run ID, MTHFR 1298, and number of samples are entered. The process continues through "Mix Preparation," "Sample Placement," "Results," and concludes with a "Summary," each step involving data input, result analysis, printing, and saving in various formats like PDF, spreadsheet, and CSV.
Figure 2. Invader Call Reporter™- Invader® MTHFR 1298 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® 1298 Test | |||||||||
|---|---|---|---|---|---|---|---|---|---|
| Site | Operator | Samplestested | First Pass | Final | Final %Agreement | ||||
| CorrectCalls | No Calls(Invalid,EQ) | Miscalls | CorrectCalls | No Calls(Invalid,EQ) | Miscalls | Final Correct CallsSamples Tested | |||
| Site001 | 1 | 90 | 90 | 0 | 0 | 90 | 0 | 0 | 100% |
| Site001 | 2 | 90 | 90 | 0 | 0 | 90 | 0 | 0 | 100% |
| Site002 | 1 | 90 | 90 | 0 | 0 | 90 | 0 | 0 | 100% |
| Site002 | 2 | 90 | 90 | 0 | 0 | 90 | 0 | 0 | 100% |
| Site003 | 1 | 90 | 90 | 0 | 0 | 90 | 0 | 0 | 100% |
| Site003 | 2 | 90 | 90 | 0 | 0 | 90 | 0 | 0 | 100% |
| All | All | 540 | 540 | 0 | 0 | 540 | 0 | 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 1298 test. The percent agreement between Invader® MTHFR 1298 test and sequencing was 100% (n=108).
| 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 #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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| Table 4: MTHFR 1298 Genotype Results; Real-time Stability | ||||||||||
|---|---|---|---|---|---|---|---|---|---|---|
| Sample/Control | Sequencing/ExpectedMTHFR 1298Genotype | T₀ Result | T₄ Result | T₇ 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 1298 test were 100%, therefore demonstrating stability for up to fifteen (15) freeze/thaw cycles.
| Table 5: Freeze/Thaw Stability of Invader MTHFR 1298 | |||||||||||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| Number of Freeze/Thaw Cycles | |||||||||||||||||
| Sample | 1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 | 9 | 10 | 11 | 12 | 13 | 14 | 15 | Total | % Agreement |
| Control 1(WT) | 3 | 3 | 3 | 3 | 3 | 3 | 3 | 3 | 3 | 3 | 3 | 3 | 3 | 3 | 3 | 45 | 100 |
| Control 2(HET) | 3 | 3 | 3 | 3 | 3 | 3 | 3 | 3 | 3 | 3 | 3 | 3 | 3 | 3 | 3 | 45 | 100 |
| Control 3(MUT) | 3 | 3 | 3 | 3 | 3 | 3 | 3 | 3 | 3 | 3 | 3 | 3 | 3 | 3 | 3 | 45 | 100 |
| gDNA(WT) | 6 | * | 6 | * | 6 | * | * | * | * | 6 | * | 6 | * | * | 6 | 36 | 100 |
| gDNA(HET) | 8 | * | 8 | * | 8 | * | * | * | * | 8 | * | 8 | * | * | 8 | 48 | 100 |
| gDNA(MUT) | 6 | * | 6 | * | 6 | * | * | * | * | 6 | * | 6 | * | * | 6 | 36 | 100 |
| Total | 29 | 9 | 29 | 9 | 29 | 9 | 9 | 9 | 9 | 29 | 9 | 29 | 9 | 9 | 29 | 255 | 100 |
| *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 | 03-4720 (MUT) | 03-4525 (HET) | 03-4536 (WT) | ||||||
| 0.5 ng/µl | 100% (40/40) | 67.5% (27/40) | 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) .
- . Cholesterol (300 mg/dL human whole blood)
- . Bilirubin (10 mg/dL human whole blood)
- Hemoglobin (up to 0.2% in whole blood) .
- 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 1298 Interfering Substance Resultsto Sequencing | ||||||||
|---|---|---|---|---|---|---|---|---|
| InterferingSubstanceCode | Substance Concentration /(in blood or DNA sample) | % AgreementwithSequencingGenotype | % AgreementwithUntreatedSampleInvader®MTHFR 1298Genotype | 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 |
- f. Pre-Analytical Equivalency Study/Genomic DNA Extraction Reproducibility (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 QlAamp® 96 DNA Blood Kit, B. Qiagen QIAamp® DNA Blood Mini Kit, C. Gentra Generation® Capture Column Kit (Qiagen), 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 1298 test for each extraction method and bi-directional sequencing was 100% (n=40).
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| ExtractionMethod | #SamplesTested | FirstPassCorrectCalls | FirstPassNoCalls | 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. |
- Instrument Equivalency (Study #8): Twenty-nine (29) human whole blood ള. 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 1298 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 | |||
|---|---|---|---|
| Thermal Cycler | |||
| Fluorometer | 1 | 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% |
Comparison studies: 2.
Method comparison: Bi-directional Sequencing (Study #2): a.
Human whole blood samples (n = 348) underwent DNA extraction and subsequent bi-directional DNA sequence analysis. The same DNA samples were then analyzed using the Invader® MTHFR 1298 test. The observed agreement between the Invader® MIHFR 1298 test and bidirectional DNA sequencing was 100% (347/347). The first run agreement with bi-directional sequencing was 99.71% (347/348).
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| Table 10: Agreement between the Invader® MTHFR 1298 Test andBi-directional DNA Sequencing | ||||
|---|---|---|---|---|
| MTHFR1298Genotype* | Numbertested | Number ofValidResults on1st Run | Number ofCorrectgenotypecalls onFirst Run | First RunAgreement |
| HomozygousWild Type(GG) | 183 | 182** | 182** | 99.45% |
| Heterozygous(GA) | 125 | 125 | 125 | 100% |
| HomozygousMutant(AA) | 40 | 40 | 40 | 100% |
| Total | 348 | 347** | 347** | 99.71% |
- Genotype determined through bi-directional DNA sequencing ** One sample failed to generate valid results. This sample was reported as invalid (EQ) and no genotype call was assigned by the interpretive software. The EQ result was used to determine the First Run Agreement.
-
- 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) 4.
MTHFR A1298C: ~33%
N. System Descriptions:
-
- Modes of Operation:
- Closed System 2. Software:
FDA has reviewed applicant's Hazard Analysis and software development processes for this line of product type. Yes X
-
- 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. Hardware and Software Controls:
- Quality Control:
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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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DEPARTMENT OF HEALTH & HUMAN SERVICES
Image /page/14/Picture/1 description: The image shows the seal of the Department of Health & Human Services - USA. The seal features a stylized eagle with its wings spread, facing to the right. The words "DEPARTMENT OF HEALTH & HUMAN SERVICES - USA" are arranged in a circular pattern around the eagle. The seal is black and white.
Food and Drug Administration 10903 New Hampshire Avenue Silver Spring, MD 20993
Hologic Inc. Third Wave Technologies c/o Mr. Randall J. Covill Senior Specialist Regulatory Affairs 502 South Rosa Road Madison, WI 53719
APR 2 5 2011
Re: K100496
Trade/Device Name: Regulation Number: Regulation Name: Regulatory Class: Product Code: Dated: March 31, 2011 Received: April 7, 2011 Invader® MTHFR 1298 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,
ia m Chan
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): K100496 .
Invader MTHFR 1298 test Device Name:
Indication for Use:
The Invader® MTHFR 1298 test is an in vitro diagnostic test intended for the detection and genotyping of a single point mutation (A to C at position 1298) 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)
Mana m Chan
Division Sign-Off
Division Sign-Off
Office of In Vitro Diagnostic Device Evaluation and Safety
510(k) K100496
§ 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.)