INVADER FACTOR V

• K Number: K100980
• Device Name: INVADER FACTOR V
• Manufacturer: HOLOGIC, INC.
• Date Cleared: 2011-06-01 ( 419 days)
• Product Code: NPQ
• Regulation Number: 864.7280

Intended Use

The Invader® Factor V test is an in vitro diagnostic test intended for the detection and genotyping of a single point mutation (G to A at position 1691) of the human Factor V gene in isolated genomic DNA obtained from whole blood potassium EDTA samples from patients with suspected thrombophilia.

Device Description

The Invader Factor V test consists of the following components: Factor V Oligo Mix, Universal Buffer, Universal Enzyme Mix, No DNA Control, Factor V Wild Type Control, Factor V Heterozygous Control, Factor V Mutant Control, Invader Call Reporter™ Software, Invader® Factor V Software.

Predicate Devices

K033607

Reference Devices

Not Found

AI/ML (Artificial Intelligence / Machine Learning)

No
The summary describes a genetic test kit and associated software for detecting 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 mentioned appears to be for data analysis and reporting, not for learning or adaptive tasks.

Therapeutic

No
Explanation: This device is an in vitro diagnostic test designed to detect and genotype a specific mutation in the human Factor V gene from blood samples, which is used to assist in the diagnosis of thrombophilia. It does not directly treat or alleviate a disease, but rather provides diagnostic information.

Diagnostic

Yes

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

SaMD (Software as a Medical Device)

No

The device description explicitly lists multiple hardware components (Oligo Mix, Buffers, Enzyme Mix, Controls) in addition to the software. This indicates it is a kit with both software and physical reagents/controls, not a software-only 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 V test is an in vitro diagnostic test intended for the detection and genotyping of a single point mutation...". 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 V test is an in vitro diagnostic test intended for the detection and genotyping of a single point mutation (G to A at position 1691) of the human Factor V gene in isolated genomic DNA obtained from whole blood potassium EDTA samples from patients with suspected thrombophilia.

Product codes

NPQ

Device Description

The Invader Factor V test consists of the following components: Factor V Oligo Mix, Universal Buffer, Universal Enzyme Mix, No DNA Control, Factor V Wild Type Control, Factor V Heterozygous Control, Factor V Mutant Control, Invader Call Reporter™ Software, Invader® Factor V 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)

Analytical Performance

• Precision/Reproducibility:
  • External Reproducibility (Study #1): Two operators from each of three sites (2 external, 1 internal) tested a panel of nine unique leukocyte-depleted whole blood samples spiked with cell lines specific for each of the three possible genotypes (3 wild type, 3 heterozygous, 3 homozygous mutant) in duplicate over five non-consecutive days for a ten-day period.
    • Total Samples Tested: 540
    • First Pass Correct Calls: 521
    • First Pass No Calls (Invalid, EQ): 19*
    • Miscalls: 0
    • Final Correct Calls: 539
    • Final No Calls (Invalid, EQ): 1†
    • Final % Agreement: 99.81%
    • *Eighteen (18) of these “No Call” results were due to an “Invalid Control” result on a single run. Upon retraining of the Operator, and retesting of the run, all controls reported “Valid” and all 18 samples were found to be in agreement with sequencing.
    • †Upon re-extraction
  • Lot-to-Lot Reproducibility (Study #9): Five genomic DNA samples (three wild type and two heterozygous) were tested in quadruplicate using three different kit lots of the Invader® Factor V test.
    • Total Samples Tested: 60
    • Final Agreement: 100%
• Traceability, Stability, Expected values (controls, calibrators, or methods):
  • Real-Time Stability Study #5: Three lots of product were stored at -30 to -15°C and +2° to +8°C. Functional testing was performed with samples representing all three genotypes in quadruplicate at each time point.
    • Interim results demonstrated 6 months stability for the device with 100% agreement.
  • Reagent Freeze-Thaw Stability Study #6: Product subject to 15 freeze-thaw cycles. Functional testing performed using genomic DNA isolate from cell lines representing all possible genotypes.
    • Percent agreement was 100%, demonstrating stability for up to fifteen (15) freeze/thaw cycles.
• Detection limit/Analytical Sensitivity and Normal Range (Study #3): Two genomic DNA samples (WT, HET) were extracted from whole blood, diluted to eight different concentrations (0.5, 5, 20, 40, 80, 200, 400, 800 ng/µL) and tested in forty replicates.
  • 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.
• Analytical specificity (Interfering Substances (Study #4)): Test performance was evaluated with added substances (Heparin, Cholesterol, Bilirubin, Hemoglobin, Potassium EDTA, Ethanol-based Wash Buffer) to four whole blood samples of different genotypes (3 WT, 1 HET) prior to extraction.
  • 100% agreement with sequencing genotype and 100% agreement with untreated sample Invader® Factor V Genotype for all tested substances. All passed.
• Pre-Analytical Equivalency Study/Genomic DNA Extraction Reproducibility (Study #7): Thirty human whole blood samples and ten leukocyte-depleted whole blood samples spiked with cell lines were divided and extracted using four commercially available DNA extraction methods. The 160 extracted DNAs were analyzed in singlicate with one lot of the device.
  • The percent agreement between the Invader® Factor V test for each extraction method and bi-directional sequencing was 100% (n=40 per method).
• Instrument Equivalency (Study #8): Extracts from 29 human whole blood samples and 10 leukocyte-depleted whole blood samples spiked with cell lines were tested with the Invader® Factor V test using three thermal cyclers and three fluorometers. Readouts were compared to bi-directional sequencing.
  • Concordance by Instrument ranged from 98.7% to 100%.
• Secondary Polymorphism Impact (Study #10): Samples included Factor V (G1691A) homozygous normal, Factor V (G1691A) heterozygous, and three Factor V (G1691A) homozygous normal samples each with a known secondary polymorphism (G1689A, A1692C or A1696G). Forty replicates for each of the five different samples were tested.
  • 100% concordance with expected results.

Comparison Studies

• Method comparison: Bi-directional Sequencing (Study #2): Human whole blood samples (n=352) underwent DNA extraction and bi-directional DNA sequence analysis, then analyzed using the Invader® Factor V test.
  • Observed agreement between the Invader® Factor V test and bi-directional DNA sequencing was 100% (352/352).

External Reproducibility Studies

• Clinical Sensitivity: Refer to section 1d above.
• Clinical Specificity: Refer to section 1e above.

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

• Precision/Reproducibility: 99.81%
• Lot-to-Lot Reproducibility: 100%
• Real-Time Stability: 100%
• Freeze/Thaw Stability: 100%
• Analytical Sensitivity: 100% concordance between 5-80 ng/uL
• Analytical Specificity (Interfering Substances): 100% agreement
• Pre-Analytical Equivalency / Genomic DNA Extraction Reproducibility: 100% agreement
• Instrument Equivalency: 98.7% to 100% concordance
• Secondary Polymorphism Impact: 100% concordance
• Agreement with Bi-directional Sequencing: 100%

Predicate Device(s)

K033607

Reference Device(s)

Not Found

Predetermined Change Control Plan (PCCP) - All Relevant Information

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

• A. 510(k) Number: JUN - 1 2011 K100980 B. Purpose for Submission: New Device C. Measurand: Factor V 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 V G. Regulatory Information: Regulation Section: 21 CFR 864.7280 1. Classification: 2. Class II 3. Product Code: NPQ: Test, Factor V Leiden Mutations, Genomic DNA PCR Panel: 4. Hematology (81) H. Intended Use: l. Intended Use(s): The Invader® Factor V test is an in vitro diagnostic test intended for the detection and genotyping of a single point mutation (G to A at position 1691) of the human Factor V 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
  • 4. Special instrument requirements: None

I. Device Description:

The Invader Factor V test consists of the following components:

• Factor V Oligo Mix Universal Buffer Universal Enzyme Mix No DNA Control Factor V Wild Type Control Factor V Heterozygous Control Factor V Mutant Control Invader Call Reporter™ Software Invader® Factor V Software
  Page 1 of 14

1

J. Substantial Equivalence Information:

·

• 1. Predicate device name(s):
  • Factor V Leiden Kit, Roche
• 2. Predicate 510(k) number(s): Roche, K033607
• 3. Comparison with predicate:

Table 1: Comparison with Predicate Device
Characteristic	Predicate Device	Proposed Device
Product Name
(Manufacturer,
Submission)	Factor V Leiden Kit
( Roche, K033607 )	Invader® Factor V
( Hologic, Inc., N/A )
Intended Use	The Factor V Leiden Kit is an in vitro diagnostic test for the detection and genotyping of a single point mutation (G to A at position 1691) of the human Factor V gene, from DNA isolated from human whole peripheral blood. The Factor V Leiden 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 V test is an in vitro diagnostic test intended for the detection and genotyping of a single point mutation (G to A at position 1691) of the human Factor V 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 sequence of the amplified fragment during the annealing phase of the PCR cycle. One probe is labeled at the 5'-end with LightCycler® Red 640-N-hydroxy-	PCR and Invader® using Fluorescence Resonance Energy Transfer (FRET) chemistry for signal reporting. Both our device and predicate device detect signal from amplicons using Fluorescence Resonance Energy Transfer (FRET.)
	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.
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 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 | PCR and Fluorescence Resonance Energy Transfer (FRET) chemistry for signal reporting. |
| 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 V Leiden | | |
| 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. | | |
| Sample Size | 10-20µl 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. |
| 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

:

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
• 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 V 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 V 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, Tag polymerase is inactivated by a 10 minute incubation at 99℃, after which the thermal cycler proceeds to 63°C to initiate the signal generation (Invader®) phase of the reaction (see Figure 1).

Image /page/4/Picture/6 description: The image shows a comparison of wildtype and mutation-specific primary probe structure formation, recognition, and cleavage. On the left side, labeled 1a, 1b, and 1c, the wildtype specific primary probe undergoes structure formation, recognition and cleavage, and a secondary reaction with a FRET cassette, resulting in red fluorescence. On the right side, labeled 2a, 2b, and 2c, the mutation-specific primary probe undergoes the same steps, but the secondary reaction with a FRET cassette results in green fluorescence.

5

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 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 5'-flaps 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 V software, in combination with Invader Call Reporter™ software, is a data analysis software package developed by Hologic for use with the Invader Factor V 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 V package workflow is shown in Figure 2.

6

Image /page/6/Figure/0 description: This image is a flowchart that describes an assay selection process. The process begins with assay selection, where the operator and run ID are entered, factor V is selected, and the number of samples is entered. The next step is mix preparation, where the master kit lot number and expiration date are entered, component lot numbers and expiration dates are entered, and reaction mix amounts are calculated. The process continues with sample placement, results, and summary, with print and save options available at different steps.

Figure 2. Invader Call Reporter™ Invader® Factor V 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) 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).

| Site | Operator | Samples
tested | First Pass | | | | Final | | | Final %
Agreement
Final Correct Calls/
Samples Tested |
|--------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|----------|-------------------|------------------|------------------------------|----------|--|------------------|------------------------------|----------|------------------------------------------------------------------------------------------------------|
| | | | Correct
Calls | No Calls
(Invalid,
EQ) | Miscalls | | Correct
Calls | No Calls
(Invalid,
EQ) | Miscalls | |
| 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 | 71 | 19* | 0 | | 89 | 1† | 0 | 98.89% |
| All | All | 540 | 521 | 19* | 0 | | 539 | 1† | 0 | 99.81% |
| *Eighteen (18) of these “No Call” results were due to an “Invalid Control” result on a single run. Upon an “Invalid Control” result, the call reporting
software automatically prevents the display of all sample genotypes, which resulted in 18 “No Call” samples. Upon retraining of the Operator, and
retesting (see Figure 4) of the run, all controls reported “Valid” and all 18 samples were found to be in agreement with sequencing. †Upon re-extraction | | | | | | | | | | |

Lot-to-Lot Reproducibility (Study #9): A total of five (5) genomic DNA samples (three (3) wild type and two (2) heterozygous) were tested in quadruplicate using three (3) different kit lots of the Invader® Factor V test. The percent agreement between Invader Factor V test and sequencing was 100% (n=60).

Table 3: Lot to Lot Reproducibility
Lot	#
Samples
Tested	First
Pass
Correct
Calls	First
Pass No
Calls	Miscalls	Final
Correct
Calls	Final
Agreement
%
1	20	20	0	0	20	100
2	20	20	0	0	20	100
3	20	20	0	0	20	100
Total	60	60	0	0	60	100

• Linearity/assay reportable range: b. Refer to section 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 three (3) genotypes in quadruplicate at each time point. The interim test results have demonstrated 6 months stability for the device.

8

Table 4: Factor V Genotype Results; Real-Time Stability
Sample/
Control	Sequencing/
Expected Factor V
Genotype	T₀ Result			T₃ Result			T₆ 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	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 isolate from cell lines, representing all possible genotypes. The percent agreement between the sequencing result and the Invader® Factor V test were 100%, therefore demonstrating stability for up to fifteen (15) freeze/thaw cycles.

Table 5: Freeze/Thaw Stability of Invader Factor V
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

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.

9

| Table 6: Analytical Sensitivity and Normal Range

Percent Agreement Between Replicates
Sample ID (Genotype based on Sequencing)
Input Sample Concentration	03-4542 (HET)	03-4420 (WT)
0.5 ng/µl	100% (40/40)	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) .

10

Table 7: Summary, Comparison of Invader" Factor V 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 V
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

• Pre-Analytical Equivalency Study/Genomic DNA Extraction Reproducibility f. (Study #7): Thirty (30) human whole blood samples and ten (10) leukocyte depleted whole blood spiked with cell lines were divided and extracted using four (4), commercially available DNA extraction methods (A. Qiagen QIAamp® 96 DNA Blood Kit, B. Qiagen QIAamp® DNA Blood Mini Kit, C. Gentra Generation® Capture Column Kit (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 V 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 ಕ್ಷsamples and ten (10) leukocyte depleted whole blood samples spiked with cell

11

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 V test using three (3) commercially available thermal cyclers (1. ABI GeneAmp® PCR System 9700 with 96-well gold block, 2. ABI Veriti™ and 3. MJ Research PTC-100) and the raw fluorescent data acquired on three (3) commercially available fluorometers (A. Tecan Infinite®, B. Tecan Genios® and C. BioTek®, FLx800). Results from the three (3) fluorometers were transferred into the interpretive software and genotype calls compared to bi-directional sequencing.

Table 9: Concordance by Instrument
Fluorometer	Thermal Cycler
	1	2	3
A	78 of 78 = 100%	77 of 78 = 98.7%	78 of 78 = 100%
B	78 of 78 = 100%	77 of 78 = 98.7%	78 of 78 = 100%
C	78 of 78 = 100%	77 of 78 = 98.7%	78 of 78 = 100%

• Secondary Polymorphism Impact (Study #10): Samples tested included one h. Factor V (G1691A) homozygous normal sample, one Factor V (G1691A) heterozygous sample and three Factor V (G1691A) homozygous normal samples each with a known secondary polymorphism, G1689A, A1692C or A1696G. Forty replicates for each of the five different samples were tested.

Table 10: Invader® Factor V Concordance
		Expected Results - Factor V (G1691A) Genotype
		Sample
01	Sample
02	Sample
03	Sample
04	Sample
05	Total
Invader® Results	Normal	40	0	40	40	40	160
	HET	0	40	0	0	0	40
	MUT	0	0	0	0	0	0
	Total	40	40	40	40	40	200

2. Comparison studies:

• Method comparison: Bi-directional Sequencing (Study #2) a.
  • Human whole blood samples (n = 352) underwent DNA extraction and subsequent bi-directional DNA sequence analysis. The same DNA samples were then analyzed using the Invader® Factor V test. The observed agreement between the Invader® Factor V test and bi-directional DNA sequencing was 100% (352/352). The overall agreement with bi-directional sequencing was 100% (352/352).

12

| Table 10: Agreement between the Invader® Factor V Test and Bi-directional

DNA Sequencing
Factor V
Genotype*	Number tested	Number of
Valid Results
on 1st Run	Number of
Correct
genotype
calls on First
Run	Agreement
Homozygous
Wild Type
(GG)	289	289	289	100%
Heterozygous
(GA)	45	45	45	100%
Homozygous
Mutant
(AA)	18	18	18	100%
Total	352	352	352	100%
* Genotype determined through bi-directional DNA sequencing

3. External Reproducibility studies:

• a. Clinical Sensitivity: Refer to section 1d above.
• Clinical specificity: Refer to section 1e above. b.
• 4. Expected values/Reference range: (Prevalence) Factor V: 5%

N. System Descriptions:

• Modes of Operation: 1. Closed System
• Software: 2.

FDA has reviewed applicant's Hazard Analysis and software development processes for this line of product type. Yes________________________________________________________________________________________________________________________________________________

• 3. Specimen Identification: Manual Labeling

4. Specimen Sampling and Handling: DNA should be extracted using a validated DNA extraction method that generates DNA concentration greater than 5ng/ul.

• 5. 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.

• O. Proposed Labeling:

13

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.

14

Image /page/14/Picture/1 description: The image shows the seal of the Department of Health & Human Services (HHS) of the United States. The seal features a circular border with the text "DEPARTMENT OF HEALTH & HUMAN SERVICES - USA" arranged around the perimeter. Inside the circle is an emblem of a stylized eagle with its wings spread, symbolizing the department's mission to protect the health of all Americans and provide essential human services.

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

JUN 0 1 2011

Re: K100980

Trade/Device Name: Invader® Factor V Regulation Number: 21 CFR 8864.7280 Regulation Name: Factor V Leiden DNA Mutation Detection Systems Regulatory Class: Class II Product Code: NPQ 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, 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

15

Page 2 - Mr. Randall J. Covill

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 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" (2) 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

้งท

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

16

Indications for Use Form

510(k) Number (if known): K100980

Device Name: Invader Factor V test

Indications for Use:

The Invader® Factor V test is an in vitro diagnostic test intended for the detection and genotyping of a single point mutation (G to A at position 1691) of the human Factor V gene in isolated genomic DNA obtained from whole blood potassium EDTA samples from patients with suspected thrombophilia.

Prescription Use × (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

Office of In Vitro Diagn Device Evaluatio

510K K100980