Predicate Devices

Reference Devices

Not Found

AI/ML (Artificial Intelligence / Machine Learning)

No
The summary describes a genetic test kit and associated software for analyzing a specific gene mutation. There is no mention of AI or ML in the device description, intended use, performance studies, or any other section. The software appears to be for reporting and calling results based on the assay's output, not for complex pattern recognition or learning.

Therapeutic

No
This device is an in vitro diagnostic test used to detect and genotype a specific gene mutation, which is a diagnostic purpose, not a therapeutic one. It does not treat or alleviate a medical condition.

Diagnostic

Yes

The "Intended Use / Indications for Use" explicitly states that the device is an "in vitro diagnostic test."

SaMD (Software as a Medical Device)

No

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

IVD (In Vitro Diagnostic)

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

Here's why:

Intended Use: The "Intended Use / Indications for Use" explicitly states that the test is an "in vitro diagnostic test."
Sample Type: It uses "isolated genomic DNA obtained from whole blood potassium EDTA samples," which are biological samples taken from the body and tested in vitro (outside the body).
Purpose: The purpose is for the "detection and genotyping of a single point mutation... of the human 5,10-methylenetetrahydrofolate reductase (MTHFR) gene," which is a diagnostic purpose related to a medical condition (suspected thrombophilia).
Device Description: The components listed are typical for an in vitro diagnostic assay (reagents, controls, software for analysis).

All of these factors align with the definition of an In Vitro Diagnostic device.

PCCP (Predetermined Change Control Plan) Authorized

N/A

Overview

Intended Use / Indications 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 5,10-methylenetetrahydrofolate reductase (MTHFR) gene in isolated genomic DNA obtained from whole blood potassium EDTA samples from patients with suspected thrombophilia.

Product codes

NPR, OMM

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.

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

Not Found

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

1. Analytical performance:
a. Precision/Reproducibility:
* External Reproducibility (Study #1): Two operators from three (3) different sites (2 external, 1 internal) tested in duplicate over five (5) non-consecutive days (10-day period) using a panel of nine (9) whole blood samples covering wild type, heterozygous, and homozygous mutant genotypes.
* Results: 100% Final % Agreement across all sites and operators (540 samples tested, 540 correct calls, 0 no calls, 0 miscalls).
* Lot-to-Lot Reproducibility (Study #9): Nine (9) genomic DNA samples (3 wild type, 3 heterozygous, 3 mutants) were tested in quadruplicate using three (3) different kit lots of the Invader® MTHFR 1298 test.
* Results: The percent agreement between Invader® MTHFR 1298 test and sequencing was 100% (n=108). All 108 samples tested resulted in 100% agreement.

**c. Real-Time Stability Study (#5):** Three (3) lots of product stored at -30° to -15°C and +2° to +8°C were functionally tested with samples representing all 3 genotypes in quadruplicate at each time point.
    *   **Results:** Interim test results demonstrated 7 months stability for the device, with 100% percent agreement for all controls and gDNA samples at T0, T4, and T7.

**c. Reagent Freeze-Thaw Stability Study (#6):** Product in final configuration was subjected to 15 freeze-thaw cycles prior to final thaw and testing. Functional testing performed using genomic DNA isolated from cell lines, representing all possible genotypes.
    *   **Results:** The percent agreement between the sequencing result and the Invader® MTHFR 1298 test was 100% across all 255 samples tested, demonstrating stability for up to fifteen (15) freeze/thaw cycles.

**d. Detection limit/Analytical Sensitivity and Normal Range (Study #3):** Three (3) genomic DNA samples with different genotypes (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/µl) and tested in replicates of forty (40).
    *   **Results:** The recommended range of the assay was determined to be between 5-80 ng/µl of input gDNA, based on 100% concordance of all tested replicates with bi-directional sequencing within this range. At 0.5 ng/µl, the HET sample showed 67.5% agreement.

**e. Analytical specificity (Interfering Substances) (Study #4):** Test performance examined with addition of various substances (Heparin, Cholesterol, Bilirubin, Hemoglobin, Potassium EDTA, Ethanol-based Wash Buffer) to nine (9) whole blood samples (3 WT, 3 HET, 3 MUT) prior to extraction.
    *   **Results:** Test performance was not affected by the addition of listed substances, showing 100% agreement with sequencing genotype and with untreated sample Invader® MTHFR 1298 Genotype (18 of 18 samples each). All substances passed.

**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 (Qiagen QlAamp® 96 DNA Blood Kit, Qiagen QIAamp® DNA Blood Mini Kit, Gentra Generation® Capture Column Kit (Qiagen), Roche MagNA Pure LC DNA Isolation Kit I). The 160 extracted DNAs were analyzed in singlicate with one (1) lot of the device.
    *   **Results:** The percent agreement between the Invader® MTHFR 1298 test for each extraction method and bi-directional sequencing was 100% (n=40 for each method). One sample removed from method B due to loss of traceability, but final agreement remained 100% for the 39 valid samples.

**g. Instrument Equivalency (Study #8):** Twenty-nine (29) human whole blood samples and ten (10) leukocyte depleted whole blood samples spiked with cell lines were extracted using two (2) commonly used extraction methods. Extracts were tested with the Invader® MTHFR 1298 test using three (3) commercially available thermal cyclers (ABI GeneAmp® PCR System 9700, ABI Veriti™, MJ Research PTC-100) and raw fluorescent data acquired on three (3) commercially available fluorometers (Tecan Infinite®, Tecan Genios®, BioTek®, FLx800).
    *   **Results:** Results from the three (3) fluorometers were transferred into the interpretive software and genotype calls compared to bi-directional sequencing. All combinations of thermal cyclers and fluorometers showed 100% concordance (78 of 78 samples).

2. Comparison studies:
a. Method comparison: Bi-directional Sequencing (Study #2): 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.
* Results: The observed agreement between the Invader® MIHFR 1298 test and bi-directional DNA sequencing was 100% (347/347). The first run agreement with bi-directional sequencing was 99.71% (347/348). One homozygous wild type sample failed to generate valid results on the first run (reported as invalid (EQ)), but was not a miscall.

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

Not Found. However, following metrics are reported.

Inter-laboratory Reproducibility (Study #1): Final Correct Calls / Samples Tested = 100% across all sites.
Lot-to-Lot Reproducibility (Study #9): Final Agreement % = 100%.
Real-time Stability (Study #5): Percent Agreement = 100% for all time points and lots.
Freeze/Thaw Stability (Study #6): % Agreement = 100%.
Detection limit/Analytical Sensitivity (Study #3): Percent Agreement Between Replicates at 5-80 ng/µl was 100% for all genotypes.
Analytical specificity (Interfering Substances) (Study #4): % Agreement with Sequencing Genotype and % Agreement with Untreated Sample Invader® MTHFR 1298 Genotype consistently 100%.
Pre-Analytical Equivalency Study (Study #7): Final Agreement % = 100% for all extraction methods.
Instrument Equivalency (Study #8): Concordance by Instrument = 100% for all thermal cycler and fluorometer combinations.
Method comparison: Bi-directional Sequencing (Study #2): Overall Agreement between the Invader® MTHFR 1298 Test and Bi-directional DNA Sequencing = 99.71% (347/348) on first run, and 100% (347/347) after excluding invalid result.

Predicate Device(s):

K070597

Reference Device(s):

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

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 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.

Indication(s) for use: 2. Same as Intended Use
Special Conditions for use statements(s): 3. For prescription use only
1. 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

1

J. Substantial Equivalence Information:

1. Predicate device name(s):

,

1. Predicate 510(k) number(s):
Nanosphere, K070597
1. Comparison with predicate:

Table 1: Comparison with Predicate Device
	Predicate Device	Proposed Device
Product Name
(Manufacturer,
Submission)	Verigene® MTHFR Nucleic Acid
Test
( Nanosphere, K070597 )	Invader® MTHFR 1298
( Hologic, Inc., K100496 )
Intended Use	"The Verigene MTHFR Nucleic
Acid Test is an in vitro diagnostic
for the detection and genotyping of
a single point mutation (C to T at
position 677) of the human 5, 10
methylene-tetrahydrofolate
reductase gene (MTHFR) in
patients with suspected
thrombophilia, from isolated
genomic DNA obtained from
whole blood samples. The test is
intended to be used on the
Verigene System."

The Verigene System is a bench-
top molecular diagnostic
workstation that automates the in
vitro diagnostic analysis and
detection of nucleic acids using
gold nanoparticle probe
technology. The Verigene System
is intended to be used by
experienced laboratory
professionals with training on basic
laboratory techniques and on the
use of the system components. | 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. |
| 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 | SNP discrimination via.
oligonucleoptide probes; detection
via evanescent wave light scatter
with nanoparticles. | PCR and Invader® using
Fluorescence Resonance Energy
Transfer (FRET) chemistry for signal
reporting. Same as predicate device.
Both our device and predicate device
detect signal from amplicons using
Fluorescence Resonance Energy
Transfer (FRET). |
| Hardware | The Verigene System consists of
two instruments, the Verigene
Processor and the Verigene Reader,
and utilizes single-use, disposable
Test Cartridges to process and
genotype multiple genes in a DNA
samples in approximately 1.5
hours. | Non-specified, third-party
fluorometer and thermal cycler. |
| Software
Interface | Embedded software in closed
system, integrated graphical user
interface. | Java-based software installed on a
standalone PC capable of converting
raw fluorescence data into genotype
calls. |
| Detection Method | Single-image sensor where
nanoparticles are illuminated using
a fixed-wavelength light source. | PCR and Fluorescence Resonance
Energy Transfer (FRET) chemistry
for signal reporting. Same as
predicate device. Both our device and
predicate device detect signal from
amplicons using Fluorescence
Resonance Energy Transfer (FRET). |
| Sample Size | 25μL | 20ul reaction containing 0.25-4ng/ul
gDNA extracted from human
peripheral whole blood. |
| Detection
Procedure | Single-image sensor where
nanoparticles are illuminated using
a fixed-wavelength light source. | Multi-well fluorometer to detect raw
fluorescence. |
| Detection
Chemistry | SNP discrimination via
oligonucleoptide probes; detection
via evanescent wave light scatter | PCR and Invader® using
Fluorescence Resonance Energy
Transfer (FRET) chemistry for signal
reporting. Both our device and |
| | with nanoparticles. | predicate device detect signal from
amplicons using Fluorescence
Resonance Energy Transfer (FRET). |
| Analysis Time | 90 min. processing with 2 min.
analysis time. | ~90 min. amplification followed by 1
min signal detection. Software
analysis post signal detection. |

2

3

.

:

4

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).

5

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).

6

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

7

M. Performance Characteristics (if/when applicable):

1. 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	Samples
tested	First Pass			Final			Final %
Agreement
			Correct
Calls	No Calls
(Invalid,
EQ)	Miscalls	Correct
Calls	No Calls
(Invalid,
EQ)	Miscalls	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	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 | #
Samples
Tested | First
Pass
Correct
Calls | First
Pass No
Calls | Miscalls | Final
Correct
Calls | Final
Agreement
% |
|-------|------------------------|-----------------------------------|---------------------------|----------|---------------------------|-------------------------|
| 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.

8

Table 4: MTHFR 1298 Genotype Results; Real-time Stability
Sample/
Control	Sequencing/
Expected
MTHFR 1298
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® 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

. . . .

9

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 Sample
Concentration	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) .

10

| Table 7: Summary, Comparison of Invader® MTHFR 1298 Interfering Substance Results

to Sequencing
Interfering
Substance
Code	Substance Concentration /
(in blood or DNA sample)	% Agreement
with
Sequencing
Genotype	% Agreement
with
Untreated
Sample
Invader®
MTHFR 1298
Genotype	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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| 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 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 and

Bi-directional DNA Sequencing
MTHFR
1298
Genotype*	Number
tested	Number of
Valid
Results on
1st Run	Number of
Correct
genotype
calls on
First Run	First Run
Agreement
Homozygous
Wild Type
(GG)	183	182**	182**	99.45%
Heterozygous
(GA)	125	125	125	100%
Homozygous
Mutant
(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.

1. 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:

1. 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

1. Specimen Identification: Manual Labeling
1. Specimen Sampling and Handling:

DNA should be extracted using a validated DNA extraction method that generates DNA concentration range of greater than 5ng/ul.

1. 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:

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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

Office of In Vitro Diagnostic Device Evaluation and Safety

510(k) K100496