(422 days)
The Invader® Factor II test is an in vitro diagnostic test intended for the detection and genotyping of a single point mutation (G to A at position 20210) of the human Factor II gene in isolated genomic DNA obtained from whole blood potassium EDTA samples from patients with suspected thrombophilia.
The Invader Factor II test consists of the following components: Factor II Oligo Mix, Universal Buffer, Universal Enzyme Mix, No DNA Control, Factor II Wild Type Control, Factor II Heterozygous Control, Factor II Mutant Control, Invader Call Reporter™ Software, Invader® Factor II Software.
This document describes the Invader® Factor II test, an in vitro diagnostic test for detecting a specific mutation (G20210A) in the human Factor II gene, associated with thrombophilia.
Here's an analysis of the acceptance criteria and supporting studies:
1. Table of Acceptance Criteria and Reported Device Performance
| Acceptance Criteria / Performance Metric | Reported Device Performance (Invader® Factor II) | Supporting Study |
|---|---|---|
| Inter-laboratory Reproducibility | Overall 99.53% final agreement (538/540 correct calls after retesting 2 initial "No Calls"). All individual sites (5/6 operators) achieved 100% agreement. | External Reproducibility (Study #1) |
| Lot-to-Lot Reproducibility | 100% agreement (48/48) between Invader® Factor II test and sequencing across 3 different kit lots. | Lot-to-Lot Reproducibility (Study #9) |
| Real-Time Stability | 100% agreement between Invader® Factor II test and sequencing/expected genotypes for 7 months (all 3 lots tested at T0, T4, T7). | Real-Time Stability Study (Study #5) |
| Reagent Freeze-Thaw Stability | 100% agreement between Invader® Factor II test and sequencing for up to 15 freeze-thaw cycles. | Reagent Freeze-Thaw Stability Study (Study #6) |
| Analytical Sensitivity / Normal Range (gDNA concentration) | 100% concordance for genomic DNA input concentrations between 5-800 ng/µL. (0.5 ng/µL showed 12.5% concordance for one heterozygous sample.) | Detection limit/Analytical Sensitivity and Normal Range (Study #3) |
| Analytical Specificity (Interfering Substances) | 100% agreement with sequencing and untreated samples in the presence of various interfering substances (Heparin, Cholesterol, Bilirubin, Hemoglobin, K2EDTA, Ethanol-based Wash Buffer). | Analytical specificity (Interfering Substances) (Study #4) |
| Pre-Analytical Equivalency (DNA Extraction Methods) | 100% agreement between Invader® Factor II test and bi-directional sequencing across 4 different commercial DNA extraction methods. | Pre-Analytical Equivalency Study/Genomic DNA Extraction Reproducibility (Study #7) |
| Instrument Equivalency (Thermal Cycler & Fluorometer) | 100% concordance with bi-directional sequencing across 3 different thermal cyclers and 3 different fluorometers. | Instrument Equivalency (Study #8) |
| Secondary Polymorphism Impact | 100% agreement for the targeted G20210A genotype in the presence of various secondary polymorphisms. | Secondary Polymorphism Impact (Study #10) |
| Method Comparison with Bi-directional Sequencing | 100% overall agreement (336/336) between the Invader® Factor II test and bi-directional DNA sequencing for all genotypes (WT, HET, MUT). | Bi-directional Sequencing (Study #2) |
2. Sample Sizes Used for the Test Set and Data Provenance
- External Reproducibility (Study #1):
- Sample Size: 9 unique leukocyte-depleted whole blood samples (3 WT, 3 HET, 3 MUT), tested in duplicate over 5 days by 2 operators at 3 sites. Total tests: 9 samples * 2 duplicates * 5 days * 2 operators * 3 sites = 540 initial tests.
- Data Provenance: Not explicitly stated, but implied to be from internal and external validation sites. Likely retrospective as they were "spiked cell lines."
- Lot-to-Lot Reproducibility (Study #9):
- Sample Size: 4 genomic DNA samples (3 WT, 1 HET), tested in quadruplicate using 3 different kit lots. Total tests: 4 samples * 4 replicates * 3 lots = 48 tests.
- Data Provenance: Not explicitly stated. Likely retrospective as they are "genomic DNA samples."
- Real-Time Stability Study (Study #5):
- Sample Size: 3 controls (WT, HET, MUT) and 4 gDNA samples (WT, WT, HET, MUT), tested in quadruplicate at each time point (T0, T4, T7) for 3 lots. Total tests are not explicitly summed, but 7 sample types * 4 replicates * 3 time points * 3 lots = 252 tests.
- Data Provenance: Not explicitly stated. Likely retrospective (genomic DNA and controls).
- Reagent Freeze-Thaw Stability Study (Study #6):
- Sample Size: 3 Controls (WT, HET, MUT) and 3 gDNA samples (WT, HET, MUT) with varying numbers of replicates tested at each of 15 freeze/thaw cycles. Total tests: 255.
- Data Provenance: Not explicitly stated. Likely retrospective (genomic DNA and controls from cell lines).
- Analytical Sensitivity and Normal Range (Study #3):
- Sample Size: 2 genomic DNA samples (1 HET, 1 WT), each diluted to 8 concentrations and tested in 40 replicates. Total tests: 2 samples * 8 concentrations * 40 replicates = 640 tests.
- Data Provenance: Whole blood collected in potassium EDTA, then extracted and diluted. Retrospective for the samples, but the dilution and testing process is controlled.
- Analytical Specificity (Interfering Substances) (Study #4):
- Sample Size: 4 whole blood samples differing genotype (3 WT, 1 HET), each with 9 different interfering substances/conditions. Total tests: 4 samples * 2 (with/without substance) * number of replicates not specified, but results given as "8 of 8" for percent agreement, suggesting at least 8 replicates per condition/sample. Total presented data points: 8 conditions * 8 replicates = 64 tests.
- Data Provenance: Whole blood samples. Retrospective.
- Pre-Analytical Equivalency Study (Study #7):
- Sample Size: 30 human whole blood samples and 10 leukocyte-depleted whole blood samples (total 40 samples), extracted using 4 different methods. Each resulting DNA analyzed in singlicate. Total tests: 40 samples * 4 extraction methods = 160 tests.
- Data Provenance: Human whole blood samples. Implied retrospective for the samples themselves.
- Instrument Equivalency (Study #8):
- Sample Size: 29 human whole blood samples and 10 leukocyte-depleted whole blood samples (total 39 samples), extracted (using 2 methods). These extracts tested with the device on 3 thermal cyclers and raw data acquired on 3 fluorometers. Total presented for agreement: 78 tests per thermal cycler/fluorometer combination (likely 39 samples tested in duplicate, or 39 samples * 2 extraction methods = 78 sample preparations). Total 78 across all combinations * 3 thermal cyclers * 3 fluorometers = 702 measurements.
- Data Provenance: Human whole blood samples. Implied retrospective.
- Secondary Polymorphism Impact (Study #10):
- Sample Size: 6 samples (1 homozygous normal, 1 heterozygous, 4 homozygous normal each with a known secondary polymorphism). 40 replicates for each sample. Total tests: 6 samples * 40 replicates = 240 tests.
- Data Provenance: Samples with known genotypes and secondary polymorphisms. Likely retrospective.
- Method Comparison (Study #2):
- Sample Size: 336 human whole blood samples.
- Data Provenance: Human whole blood samples. Retrospective.
3. Number of Experts and Qualifications for Ground Truth
- General Ground Truth Method: The primary and most frequently cited ground truth method across all studies is bi-directional DNA sequencing.
- Number of Experts: The document does not specify the number of experts used for establishing ground truth via bi-directional DNA sequencing. Sequencing is typically a highly automated process with results interpreted by trained molecular biologists or laboratory staff, rather than a consensus of "experts" in the clinical sense (like radiologists).
- Qualifications of Experts: Not specified. It's assumed that standard molecular biology laboratory practices for sequencing and interpretation were followed.
4. Adjudication Method for the Test Set
- Not Applicable in the traditional sense. For diagnostic tests like this, adjudication by multiple human experts (e.g., radiologists) is not a typical part of ground truth establishment.
- The ground truth is established by a definitive molecular method (bi-directional DNA sequencing).
- In the external reproducibility study (Study #1), two initial "No Call" results were resolved by retesting, which then agreed with the sequencing results. This implies a retesting/re-evaluation process for indeterminate results, rather than expert adjudication of the initial "No Call."
5. Multi-Reader Multi-Case (MRMC) Comparative Effectiveness Study
- No, an MRMC comparative effectiveness study was not done.
- This device is an in vitro diagnostic test for genotyping, where the output is a molecular result (genotype). It does not involve human readers interpreting images or data where AI assistance would directly improve human performance in the same way as, for example, a radiology AI.
- The "Invader Call Reporter™ Software" mentioned is for data analysis and conversion of raw fluorescence data into genotype calls, not for assisting human interpretation that would typically be evaluated in an MRMC study.
6. Standalone Performance Study
- Yes, a standalone performance study was done.
- The entire set of analytical performance studies (Reproducibility, Stability, Analytical Sensitivity, Analytical Specificity, Extraction Equivalency, Instrument Equivalency, Secondary Polymorphism Impact) and the method comparison study against bi-directional sequencing demonstrate the standalone performance of the algorithm/device system.
- The device takes raw fluorescence data and, through its software ("Invader Call Reporter™ Software" and "Invader® Factor II Software"), outputs a genotype call. The studies rigorously validate that these automated calls are accurate and reproducible compared to the gold standard of sequencing. The system is designed to provide automated calls, not to serve as an aid to a human reader's interpretation in a separate step.
7. Type of Ground Truth Used
- The primary ground truth used across all key performance studies is bi-directional DNA sequencing. This is considered a gold standard molecular method for determining specific genetic mutations.
- For controls, "expected genotype" based on the composition of the control material (e.g., cell lines) is used.
8. Sample Size for the Training Set
- The document does not explicitly describe a separate "training set" for the device's algorithm.
- For molecular diagnostic assays like this, the algorithm (e.g., for converting fluorescence data to genotype calls) is typically developed based on known statistical models and pre-defined thresholds related to the chemistry, rather than being "trained" on a large dataset of classified samples in the machine learning sense.
- The various controls (No DNA, WT, HET, MUT) are used within each run to validate performance and inform signal-to-noise calculations, serving as internal calibration rather than an external training set for an AI model.
9. How Ground Truth for the Training Set Was Established
- As a formal "training set" is not explicitly mentioned or implied in the context of supervised machine learning, the process of establishing ground truth for such a set is not described.
- However, the underlying principles for the chemistry and software's interpretive rules would have been developed using well-characterized samples with ground truth established by methods like DNA sequencing during the research and development phase before formal validation studies.
{0}------------------------------------------------
Invader® Factor II 510(k) SUMMARY
JUN - 2 2011
-
A. 510(k) Number:
K100943 -
B. Purpose for Submission: New Device
-
C. Measurand: Factor II
-
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 508-263-8853 Contact Person: Randall J. Covill, Manager, Regulatory Affairs Date of Submission: April 2010
- F. Proprietary and Established Names: Invader® Factor II
G. Regulatory Information:
-
- Regulation Sections: 21 CFR 864.7280
- Classification: 2.
- Class II 3. Product Code: NPR: Test, Factor II G20210A Mutations, Genomic DNA PCR
-
- Panel:
Hematology (81)
H. Intended Use:
-
- Intended Use(s):
The Invader Factor II test is an in vitro diagnostic test intended for the detection and genotyping of a single point mutation (G to A at position 20210) of the human Factor II 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): For prescription use only
-
Special instrument requirements: 4. None
Device Description: I.
The Invader Factor II test consists of the following components:
- Factor II Oligo Mix
- Universal Buffer
- Universal Enzyme Mix
- No DNA Control
- Factor II Wild Type Control
- Factor II Heterozygous Control
- Factor II Mutant Control
- Invader Call Reporter™ Software
- Invader® Factor II Software
{1}------------------------------------------------
J. Substantial Equivalence Information:
- l. Predicate device name(s): Factor II (Prothrombin) G20210A, Roche
-
- Predicate 510(k) number(s): Roche, K033612
-
- Comparison with predicate:
・
| Table 1: Comparison with Predicate Device | ||
|---|---|---|
| Predicate Device | Proposed Device | |
| Product Name(Manufacturer,Submission) | Factor II (Prothrombin) G20210A Kit (Roche, K033612) | Invader® Factor II (Hologic, Inc., N/A) |
| Intended Use | The Factor II (Prothrombin) G20210A Kit is an in vitro diagnostic test for the detection and genotyping of a single point mutations (G to A at position 20210) of the human Factor II gene, from DNA isolated from human whole peripheral blood. The Factor II (Prothrombin) G20210A Kit is indicated as an aid to diagnosis in the evaluation of patients with suspected thrombophilia. The test is intended to be used on the LightCycler instrument. The sample preparation must be performed according to a workflow procedure described in the package insert. | The Invader® Factor II test is an in vitro diagnostic test intended for the detection and genotyping of a single point mutation (G to A at position 20210) of the human Factor II gene in isolated genomic DNA obtained from whole blood potassium EDTA samples from patients with suspected thrombophilia. |
| Specimen Type | Purified DNA isolated from human whole peripheral blood. | Same as predicate |
| Indications for Use | Same as Intended Use | Same as Intended Use |
| Target Population | Patients with suspected thrombophilia | Same as predicate |
| Chemistry | The amplicon is detected by fluorescence using a specific pair of H probes. The H probes consist of two different oligonucleotides that hybridize to an internal | PCR and Invader® using Fluorescence Resonance Energy Transfer (FRET) chemistry for signal reporting. Both our device and predicate device detect signal from |
| Table 1: Comparison with Predicate Device | ||
| Predicate Device | Proposed Device | |
| Product Name(Manufacturer,Submission) | Factor II (Prothrombin) G20210AKit (Roche, K033612) | Invader® Factor II(Hologic, Inc., N/A) |
| during the annealing phase of thePCR cycle. One probe is labeled atthe 5'-end with LightCycler® Red640-N-hydroxy-succinimide ester(Red 640-NHS ester), and to avoidextension, modified at the 3'-endby phosphorylation. The otherprobe is labeled at the 3'-end withfluorescein.3. Only afterhybridization to the template DNA,do the two probes come in closeproximity, resulting in fluorescenceresonance energy transfer (FRET)between the two fluorophores.During FRET, fluorescein, thedonor fluorophore, is excited by thelight source of the LightCycler® 2.0Instrument, and part of theexcitation energy is transferred toLightCycler® Red 640-NHS ester,the acceptor fluorophore. | Resonance Energy Transfer (FRET). | |
| Hardware | LightCycler® Instrument using SW3.5 | Non-specified, third-partyfluorometer and thermal cycler. |
| SoftwareInterface | LightCycler® Instrument using SW3.5. Expro database and macros. | Java-based software installed on astandalone PC capable of convertingraw fluorescence data into genotypecalls. |
| Detection Method | The LightCyler® uses opticaldetection of stimulatedfluorescence generated by thefollowing chemistry:The H probes are also used todetermine the genotype byperforming a melting curveanalysis after the amplificationcycles are completed and the | PCR and Fluorescence ResonanceEnergy Transfer (FRET) chemistryfor signal reporting. |
| Table 1: Comparison with Predicate Device | ||
| Predicate Device | Proposed Device | |
| Product Name(Manufacturer, Submission) | Factor II (Prothrombin) G20210A Kit (Roche, K033612)amplicon is present at increased concentration.•The Red 640-labeled H probe hybridizes to a part of the target sequence that is not mutated and functions as an anchor probe.•The Fluorescein-labeled H-probe spans the mutation site (mutation probe). During the melting curve analysis, increasing temperature causes the fluorescence to decrease because the shorter of the two probes (mutation probe) dissociates first and the two fluorescent dyes are no longer in close proximity. If the Factor II (Prothrombin) G20210A mutation is present, the mismatch of the mutation probe with the target destabilizes the hybrid so the decrease in fluorescence will occur at a lower temperature. With the wild-type genotype, mismatches will not occur, and therefore, the heteroduplex DNA has a higher melting temperature (Tm). The heterozygous genotype exhibits a distinctive combination of properties. | Invader® Factor II (Hologic, Inc., N/A) |
| Sample Size | 10-20ul in glass capillaries. | 20ul reaction containing 0.25-4ng/ul gDNA extracted from human peripheral whole blood. |
| Detection Procedure | Optical detection of stimulated fluorescence using a specific pair of probes. | Multi-well fluorometer to detect raw fluorescence. |
| Table 1: Comparison with Predicate Device | ||
| Predicate Device | Proposed Device | |
| Product Name(Manufacturer,Submission) | Factor II (Prothrombin) G20210AKit (Roche, K033612) | Invader® Factor II(Hologic, Inc., N/A) |
| DetectionChemistry | Paired hybridization probes usingfluorescence resonance energytransfer (FRET) followed bymelting curve analysis. | PCR and Invader® usingFluorescence Resonance EnergyTransfer (FRET) chemistry for signalreporting. |
| Analysis Time | A multi-step assay with differenttimes required for each step.Detection occurs at definedintervals during PCR cycle and canbe reviewed in real-time. | ~90 min. amplification followed by 1min signal detection. Softwareanalysis post signal detection. |
{2}------------------------------------------------
{3}------------------------------------------------
.
0
{4}------------------------------------------------
{5}------------------------------------------------
K. Standard/Guidance Document Referenced (if applicable):
- 0 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
- 0 Guidance for Industry and FDA Staff - Format for Traditional and Abbreviated 510(k)s issued on August 12, 2005
L. Test Principle:
The Invader® Factor II 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 Factor II 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).
Image /page/5/Figure/6 description: The image shows a comparison of wildtype and mutation-specific primary probes in a three-step process. The first step, labeled 1a and 2a, shows the structure formation of the probes. The second step, labeled 1b and 2b, shows the structure recognition and cleavage. The third step, labeled 1c and 2c, shows the secondary reaction with FRET Cassettes, resulting in fluorescence 1 (RED) and fluorescence 2 (FAM).
{6}------------------------------------------------
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: RED) and the mutant allele with the second fluorophore (Fluorescence 2: FAM).
The Invader® Factor II software, in combination with Invader Call Reporter™ software, is a data analysis software package developed by Hologic for-use with the Invader® Factor II 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 Factor II package workflow is shown in Figure 2.
{7}------------------------------------------------
Image /page/7/Figure/0 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 factor II, and enters the number of samples. The next step is mix preparation, where the master kit lot number and expiration date are entered, along with the component lot numbers and expiration dates, and the reaction mix amounts are calculated. The process continues with sample placement, results, and summary, with each step involving data input, analysis, and saving of results in various formats such as PDF, spreadsheet, and CSV.
Figure 2. Invader Call Reporter™ Invader® Factor II Package Workflow
{8}------------------------------------------------
M. Performance Characteristics (if/when applicable):
-
- Analytical performance:
- Precision/Reproducibility: a.
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) unique leukocyte depleted whole blood samples spiked cell lines specific for each of the three (3) possible genotypes (i.e. 3 wild type, 3 heterozygous, 3 homozygous mutant).
| Table 2: Inter-laboratory Reproducibility of Invader® Factor II Test | |||||||||
|---|---|---|---|---|---|---|---|---|---|
| First Pass | Final | Final % | |||||||
| Site | Operator | Samplestested | CorrectCalls | No Calls(Invalid,EQ) | Miscalls | CorrectCalls | No Calls(Invalid,EQ) | Miscalls | AgreementFinal 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 | 54 | 36* | 0 | 88 | 2* | 0 | 97.78%* |
| All | All | 540 | 504 | 36* | 0 | 538 | 2* | 0 | 99.53%* |
| *These "No Call" results were due to an "Invalid Control" result on 2 independent runs. Upon an "Invalid Control" result, the call reporting software |
autonatically prevents the display of all sense of the Call" samles. Upon netraning of the Clerant and reesting of upon retesting both samples were found to be in agreement with sequencing
Lot-to-Lot Reproducibility (Study #9): A total of four (4) genomic DNA samples (three (3) wild type and one (1) heterozygous) were tested in quadruplicate using three (3) different kit lots of the Invader® Factor II test. The percent agreement between Invader® Factor II test and sequencing was 100% (n=48).
| Lot | #SamplesTested | FirstPassCorrectCalls | FirstPass NoCalls | Miscalls | FinalCorrectCalls | FinalAgreement% |
|---|---|---|---|---|---|---|
| 1 | 16 | 16 | 0 | 0 | 16 | 100 |
| 2 | 16 | 16 | 0 | 0 | 16 | 100 |
| 3 | 16 | 16 | 0 | 0 | 16 | 100 |
| Total | 48 | 48 | 0 | 0 | 48 | 100 |
- Linearity/assay reportable range: b. 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.
{9}------------------------------------------------
| Sample/Control | Sequencing/Expected FactorII Genotype | 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® Factor II test were 100%, therefore demonstrating stability for up to fifteen (15) freeze/thaw cycles.
| 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 |
*Testing with gDNA samples did not occur at this testing point.
- Detection limit/Analytical Sensitivity and Normal Range (Study #3): d. Two (2) genomic DNA samples with different genotypes (i.e. WT, HET) 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 determined to be between 5-80 ng/uL of input gDNA, based on 100% concordance of all tested replicates with bi-directional sequencing.
{10}------------------------------------------------
| Table 6: Analytical Sensitivity and Normal Range | ||
|---|---|---|
| Percent Agreement Between Replicates | ||
| Sample ID (Genotype based on Sequencing) | ||
| Input SampleConcentration | 03-4493 (HET) | 03-4723 (WT) |
| 0.5 ng/µl | 12.5% (5/40) | 100% (40/40) |
| 5 ng/µl | 100% (40/40) | 100% (40/40) |
| 20 ng/µl | 100% (40/40) | 100% (40/40) |
| 40 ng/µl | 100% (40/40) | 100% (40/40) |
| 80 ng/µl | 100% (40/40) | 100% (40/40) |
| 200 ng/µl | 100% (40/40) | 100% (40/40) |
| 400 ng/µl | 100% (40/40) | 100% (40/40) |
| 800 ng/µl | 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 four (4) whole blood samples of different genotype (3 WT, 1 HET) 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 (K2EDTA) (1.8 mg/mL human whole blood) ♥
- . Ethanol-based Wash Buffer (5% in DNA sample)
Page 11 of 15
{11}------------------------------------------------
| Table 7: Summary, Comparison of Invader" Factor II Interfering Substance Results to Sequencing | ||||
|---|---|---|---|---|
| InterferingSubstanceCode | Substance Concentration / (inblood or DNA sample) | % Agreement withSequencingGenotype | % Agreementwith UntreatedSample Invader®Factor IIGenotype | PASS / FAIL |
| A | No Addition Control (Untreated) | 100% (8 of 8) | N/A | PASS |
| B | Bilirubin 10mg/dl (blood) | 100% (8 of 8) | 100% (8 of 8) | PASS |
| C | Cholesterol 300mg/dl (blood) | 100% (8 of 8) | 100% (8 of 8) | PASS |
| D | K2EDTA 1.8mg/ml (blood) | 100% (8 of 8) | 100% (8 of 8) | PASS |
| E | Heparin 1500 U/dl (blood) | 100% (8 of 8) | 100% (8 of 8) | PASS |
| F1 | Hemoglobin 0.2% (blood) | 100% (8 of 8) | 100% (8 of 8) | PASS |
| F2 | Hemoglobin 0.1% (blood) | 100% (8 of 8) | 100% (8 of 8) | PASS |
| F3 | Hemoglobin 0.05% (blood) | 100% (8 of 8) | 100% (8 of 8) | PASS |
| F4 | Hemoglobin 0.025% (blood) | 100% (8 of 8) | 100% (8 of 8) | PASS |
| G | Ethanol-based wash buffer 5%(DNA) | 100% (8 of 8) | 100% (8 of 8) | 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 QIAamp® 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® Factor II test for each extraction method and bi-directional sequencing was 100% (n=40).
| Table 8: Pre-Analytical Equivalency | |||||||
|---|---|---|---|---|---|---|---|
| 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 g. samples and ten (10) leukocyte depleted whole blood samples spiked with cell
{12}------------------------------------------------
lines were extracted using Qiagen QIAamp® DNA Blood Mini Kit and Roche MagNA Pure LC DNA Isolation Kit I. The extracts were tested with the Invader® Factor II 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% |
- h. Secondary Polymorphism Impact (Study #10): Samples tested included one Factor II (G20210A) homozygous normal sample, one Factor II (G20210A) heterozygous sample and four Factor II (G20210A) homozygous normal samples each with a known secondary polymorphism, A20207C, C20209T, A20218G, or C20221T. Forty replicates for each of the 6 different samples were tested.
| Table 10: Invader® Factor II Concordance | ||||||||
|---|---|---|---|---|---|---|---|---|
| Expected Results - Factor II (G20210A) Genotype | ||||||||
| Sample01 | Sample02 | Sample03 | Sample04 | Sample05 | Sample06 | Total | ||
| Invader™ Results | Normal | 40 | 0 | 40 | 40 | 40 | 40 | 200 |
| HET | 0 | 40 | 0 | 0 | 0 | 0 | 40 | |
| MUT | 0 | 0 | 0 | 0 | 0 | 0 | 0 | |
| Total | 40 | 40 | 40 | 40 | 40 | 40 | 240 |
- Comparison studies:
- a. Method comparison: Bi-directional Sequencing (Study #2):
- Human whole blood samples (n = 336) underwent DNA extraction and subsequent bi-directional DNA sequence analysis. The same DNA samples were then analyzed using the Invader® Factor II test. The observed agreement between the Invader® Factor II test and bidirectional DNA sequencing was 100% (336/336). The overall agreement with bi-directional sequencing was 100% (336/336).
{13}------------------------------------------------
| Table 10: Agreement between the Invader® Factor II Test andBi-directional DNA Sequencing | ||||
|---|---|---|---|---|
| Factor IIGenotype* | Numbertested | Number ofValidResults on1st Run | Number ofCorrectgenotypecalls onFirst Run | Agreement |
| HomozygousWild Type(GG) | 305 | 305 | 305 | 100% |
| Heterozygous(GA) | 24 | 24 | 24 | 100% |
| HomozygousMutant(AA) | 7 | 7 | 7 | 100% |
| Total | 336 | 336 | 336 | 100% |
| * Genotype determined through bi-directional DNA sequencing |
- External Reproducibility studies: 3.
- Clinical Sensitivity: please refer to section 1d above. a.
- Clinical specificity: please refer to section 1e above. b.
-
- Expected values/Reference range: (Prevalence) Factor II: 1-2%
N. System Descriptions:
- l. Modes of Operation:
- Closed System
- Software: 2. FDA has reviewed applicant's Hazard Analysis and software development processes for this line of product type. Yes_______________________________ or No_
-
- Specimen Identification:
- Manual Labeling
Specimen Sampling and Handling: 4. DNA should be extracted using a validated DNA extraction method that generates DNA concentration range of greater than 5ng/ul.
- ડ. 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:
The genotyping test result will not be reported for any sample for which a positive or negative control failure occurs.
{14}------------------------------------------------
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.
{15}------------------------------------------------
Image /page/15/Picture/1 description: The image shows the seal of the Department of Health & Human Services (HHS) of the United States. The seal features a stylized caduceus, a symbol often associated with medicine and healthcare, to the right. Encircling the caduceus are the words "DEPARTMENT OF HEALTH & HUMAN SERVICES • USA" in a circular arrangement.
Food and Drug Administration 10903 New Hampshire Avenue Silver Spring, MD 20993
Hologic Inc. c/o Mr. Randall J. Covill Manager, Regulatory Affairs 250 Campus Drive Marlborough, MA 01752
DIN 0 2 2011
Re: K100943
Trade/Device Name: Invader® Factor II Regulation Number: 21 CFR §864.7280 Regulation Name: Factor V Leiden DNA Mutation Detection Systems Regulatory Class: Class II Product Code: NPR Dated: May 19, 2011 Received: May 26, 2011
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. Iisting 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 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 will allow you to begin marketing your device as described in your Section 510(k) premarket
{16}------------------------------------------------
Page 2 - Mr. Randall J. Covill
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,
Reena Philip.
For 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
{17}------------------------------------------------
Indications for Use Form
510(k) Number (if known): K100943
Device Name: Invader Factor II test
Indications for Use:
The Invader® Factor II test is an in vitro diagnostic test intended for the detection and genotyping of a single point mutation (G to A at position 20210) of the human Factor II 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 IF NEEDED)
Concurrence of CDRH, Office of In Vitro Diagnostic Devices (OIVD)
Reena Philip
Division Sign-Off
Division Sign-Off
Office of In Vitro Diagnostic Device Evailation and Safety
510k K100943
§ 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.)