INVADER FACTOR II

• K Number: K100943
• Device Name: INVADER FACTOR II
• Manufacturer: HOLOGIC, INC.
• Date Cleared: 2011-06-02 ( 422 days)
• Product Code: NPR
• Regulation Number: 864.7280

Intended 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.

Device Description

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.

Predicate Devices

K033612

Reference Devices

Not Found

AI/ML (Artificial Intelligence / Machine Learning)

No
The summary does not mention AI, ML, or related terms, and the device description and performance studies focus on standard molecular diagnostic techniques and software for data analysis, not AI/ML algorithms.

Therapeutic

No
The device is an in vitro diagnostic test for detecting and genotyping a genetic mutation associated with thrombophilia, not a device for treating a condition.

Diagnostic

Yes
The "Intended Use / Indications for Use" section explicitly states that "The Invader® Factor II test is an in vitro diagnostic test."

SaMD (Software as a Medical Device)

No

The device description explicitly lists multiple physical components (Oligo Mix, Buffers, Enzyme Mix, Controls) in addition to the software components. This indicates it is not a software-only medical device.

IVD (In Vitro Diagnostic)

Yes, this device is an IVD (In Vitro Diagnostic).

The "Intended Use / Indications for Use" section explicitly states: "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."

This statement directly identifies the device as an in vitro diagnostic test.

PCCP (Predetermined Change Control Plan) Authorized

N/A

Overview

Intended Use / 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.

Product codes (comma separated list FDA assigned to the subject device)

NPR

Device Description

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

Mentions image processing

Not Found

Mentions AI, DNN, or ML

Not Found

Input Imaging Modality

Not Found

Anatomical Site

Not Found

Indicated Patient Age Range

Not Found

Intended User / Care Setting

For prescription use only

Description of the training set, sample size, data source, and annotation protocol

Not Found

Description of the test set, sample size, data source, and annotation protocol

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).

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).

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)

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.

Twenty-nine (29) human whole blood samples and ten (10) leukocyte depleted whole blood samples spiked with cell 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).

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.

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.

Summary of Performance Studies (study type, sample size, AUC, MRMC, standalone performance, key results)

• 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). The agreement was 100% for Site 001 and Site 002. Site 003 had 97.78% agreement. Overall agreement was 99.53%.
• 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).
• Real-Time Stability Study (Study #5): Interim test results have demonstrated 7 months stability for the device with 100% agreement.
• 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.
• Detection limit/Analytical Sensitivity and Normal Range (Study #3): Two (2) genomic डीएनए 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.
• Analytical specificity (Interfering Substances) (Study #4): Test performance was not affected by addition of various substances. % Agreement with Sequencing Genotype was 100% and % Agreement with Untreated Sample Invader® Factor II Genotype was 100% for all tested interfering substances.
• Pre-Analytical Equivalency Study/Genomic DNA Extraction Reproducibility (Study #7): The percent agreement between the Invader® Factor II test for each extraction method and bi-directional sequencing was 100% (n=40).
• Instrument Equivalency (Study #8): Results from the three (3) fluorometers were transferred into the interpretive software and genotype calls compared to bi-directional sequencing. Concordance by Instrument was 100% for all thermal cycler and fluorometer combinations.
• 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. The results show correct genotyping with the expected results.
• 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).

Key Metrics (Sensitivity, Specificity, PPV, NPV, etc.)

Not Found

Predicate Device(s): If the device was cleared using the 510(k) pathway, identify the Predicate Device(s) K/DEN number used to claim substantial equivalence and list them here in a comma separated list exactly as they appear in the text. List the primary predicate first in the list.

K033612

Reference Device(s): Identify the Reference Device(s) K/DEN number and list them here in a comma separated list exactly as they appear in the text.

Not Found

Predetermined Change Control Plan (PCCP) - All Relevant Information for the subject device only (e.g. presence / absence, what scope was granted / cleared under the PCCP, any restrictions, etc).

Not Found

Regulation Number and Section

§ 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.)

Summary

0

K100943

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:

• 1. Regulation Sections: 21 CFR 864.7280
• Classification: 2.
• Class II 3. Product Code: NPR: Test, Factor II G20210A Mutations, Genomic DNA PCR
• 4. Panel:

Hematology (81)

H. Intended Use:

• 1. 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.

• Indication(s) for use: 2. Same as Intended Use

• 3. 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

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J. Substantial Equivalence Information:

• l. Predicate device name(s): Factor II (Prothrombin) G20210A, Roche
• 2. Predicate 510(k) number(s): Roche, K033612
• 3. 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) G20210A
Kit (Roche, K033612)	Invader® Factor II
(Hologic, Inc., N/A)
	during the annealing phase of the
PCR cycle. One probe is labeled at
the 5'-end with LightCycler® Red
640-N-hydroxy-succinimide ester
(Red 640-NHS ester), and to avoid
extension, modified at the 3'-end
by phosphorylation. The other
probe is labeled at the 3'-end with
fluorescein.3. Only after
hybridization to the template DNA,
do the two probes come in close
proximity, resulting in fluorescence
resonance energy transfer (FRET)
between the two fluorophores.
During FRET, fluorescein, the
donor fluorophore, is excited by the
light source of the LightCycler® 2.0
Instrument, and part of the
excitation energy is transferred to
LightCycler® Red 640-NHS ester,
the acceptor fluorophore.	Resonance Energy Transfer (FRET).
Hardware	LightCycler® Instrument using SW
3.5	Non-specified, third-party
fluorometer and thermal cycler.
Software
Interface	LightCycler® Instrument using SW
3.5. Expro database and macros.	Java-based software installed on a
standalone PC capable of converting
raw fluorescence data into genotype
calls.
Detection Method	The LightCyler® uses optical
detection of stimulated
fluorescence generated by the
following chemistry:

The H probes are also used to
determine the genotype by
performing a melting curve
analysis after the amplification
cycles are completed and the | PCR and Fluorescence Resonance
Energy Transfer (FRET) chemistry
for 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) G20210A
Kit (Roche, K033612) | Invader® Factor II
(Hologic, Inc., N/A) |
| Detection
Chemistry | Paired hybridization probes using
fluorescence resonance energy
transfer (FRET) followed by
melting curve analysis. | PCR and Invader® using
Fluorescence Resonance Energy
Transfer (FRET) chemistry for signal
reporting. |
| Analysis Time | A multi-step assay with different
times required for each step.
Detection occurs at defined
intervals during PCR cycle and can
be reviewed in real-time. | ~90 min. amplification followed by 1
min signal detection. Software
analysis 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).

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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.

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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

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M. Performance Characteristics (if/when applicable):

• 1. 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	Samples
tested	Correct
Calls	No Calls
(Invalid,
EQ)	Miscalls	Correct
Calls	No Calls
(Invalid,
EQ)	Miscalls	Agreement
Final Correct Calls
Samples Tested
Site
001	1	90	90	0	0	90	0	0	100%
Site
001	2	90	90	0	0	90	0	0	100%
Site
002	1	90	90	0	0	90	0	0	100%
Site
002	2	90	90	0	0	90	0	0	100%
Site
003	1	90	90	0	0	90	0	0	100%
Site
003	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 | #
Samples
Tested | First
Pass
Correct
Calls | First
Pass No
Calls | Miscalls | Final
Correct
Calls | Final
Agreement
% |
|-------|------------------------|-----------------------------------|---------------------------|----------|---------------------------|-------------------------|
| 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.

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| Sample/
Control | Sequencing/
Expected Factor
II 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.

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Table 6: Analytical Sensitivity and Normal Range
Percent Agreement Between Replicates
Sample ID (Genotype based on Sequencing)
Input Sample
Concentration	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

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Table 7: Summary, Comparison of Invader" Factor II Interfering Substance Results to Sequencing
Interfering
Substance
Code	Substance Concentration / (in
blood or DNA sample)	% Agreement with
Sequencing
Genotype	% Agreement
with Untreated
Sample Invader®
Factor II
Genotype	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
Extraction
Method	#
Samples
Tested	First
Pass
Correct
Calls	First
Pass
No
Calls	Miscalls	Final
Correct
Calls	Final
Agreement
%
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 sample
identification.

• Instrument Equivalency (Study #8): Twenty-nine (29) human whole blood g. samples and ten (10) leukocyte depleted whole blood samples spiked with cell

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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
		Sample
01	Sample
02	Sample
03	Sample
04	Sample
05	Sample
06	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

2. 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).

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| Table 10: Agreement between the Invader® Factor II Test and

Bi-directional DNA Sequencing
Factor II
Genotype*	Number
tested	Number of
Valid
Results on
1st Run	Number of
Correct
genotype
calls on
First Run	Agreement
Homozygous
Wild Type
(GG)	305	305	305	100%
Heterozygous
(GA)	24	24	24	100%
Homozygous
Mutant
(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.
• 4. 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_
• 3. 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.

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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/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

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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 [phone]. 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 [phone] or [phone] 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

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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

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510k K100943