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.

AI/ML Overview

Here's an analysis of the acceptance criteria and supporting study details for the Invader® Factor V test, based on the provided text:

Acceptance Criteria and Device Performance for Invader® Factor V Test

1. Table of Acceptance Criteria and Reported Device Performance

The submission primarily focuses on analytical performance criteria, demonstrating the assay's reliability and accuracy in detecting the Factor V G1691A mutation.

Acceptance Criteria Category	Specific Criteria/Study Goal	Reported Device Performance
Precision/Reproducibility	External Reproducibility: Consistent results across sites and operators.	External Reproducibility (Study #1): Overall Final % Agreement: 99.81% (539 correct calls out of 540 samples). Five out of six operators achieved 100% agreement. One operator's initial invalid control issue was resolved, resulting in 98.89% agreement for that operator.
	Lot-to-Lot Reproducibility: Consistent performance across different kit lots.	Lot-to-Lot Reproducibility (Study #9): 100% agreement between Invader Factor V test and sequencing across three different kit lots (n=60 samples).
Stability	Real-Time Stability: Maintain performance over time under storage conditions.	Real-Time Stability (Study #5): 100% agreement for all tested genotypes (WT, HET, MUT) across three product lots at T₀, T₃, and T₆ months, when stored at -30 to -15°C and +2°C to +8°C. Interim results demonstrated 6 months stability.
	Freeze-Thaw Stability: Maintain performance after multiple freeze/thaw cycles.	Reagent Freeze-Thaw Stability (Study #6): 100% agreement after up to 15 freeze-thaw cycles (n=255 total tests across controls and gDNA samples).
Analytical Sensitivity	Detection Limit/Reportable Range: Accurate genotyping within a specified DNA concentration range.	Detection limit/Analytical Sensitivity and Normal Range (Study #3): Recommended assay range: 5-80 ng/µL of input gDNA, based on 100% concordance with bi-directional sequencing across all tested replicates (80 replicates total for WT and HET samples at various dilutions).
Analytical Specificity	Interfering Substances: Performance unaffected by common interfering substances.	Analytical specificity (Study #4): 100% agreement with sequencing genotype and untreated sample Invader® Factor V genotype for all tested interfering substances (Heparin, Cholesterol, Bilirubin, Hemoglobin, Potassium EDTA, Ethanol-based Wash Buffer).
Pre-Analytical Workflow	Genomic DNA Extraction Reproducibility: Consistent performance with various DNA extraction methods.	Pre-Analytical Equivalency Study (Study #7): 100% agreement between Invader® Factor V test and bi-directional sequencing for four commercially available DNA extraction methods (n=40 samples per method). One sample was removed due to traceability loss.
Instrument Compatibility	Instrument Equivalency: Consistent performance across different thermal cyclers and fluorometers.	Instrument Equivalency (Study #8): 100% concordance for 7 out of 9 combinations of thermal cyclers and fluorometers. Two combinations showed 98.7% concordance (77 of 78 samples).
Secondary Polymorphism	Impact of other polymorphisms on assay accuracy.	Secondary Polymorphism Impact (Study #10): 100% concordance (200/200) for control samples and samples with known secondary polymorphisms (G1689A, A1692C, A1696G).
Method Comparison	Comparison to a gold standard method.	Bi-directional Sequencing (Study #2): 100% agreement between the Invader® Factor V test and bi-directional DNA sequencing (352/352 human whole blood samples).

2. Sample Size for Test Set and Data Provenance

External Reproducibility (Study #1):
- Sample Size: 9 unique leukocyte-depleted whole blood samples (3 wild type, 3 heterozygous, 3 homozygous mutant) spiked cell lines.
- Provenance: "Spiked cell lines" implies controlled, laboratory-prepared samples. The text does not specify the country of origin, but the study was conducted at "3 different sites (2 external sites and 1 internal site)", suggesting multi-center testing, likely within the US given the FDA submission. This was a prospective study where samples were tested newly.
Lot-to-Lot Reproducibility (Study #9):
- Sample Size: 5 genomic DNA samples (3 wild type, 2 heterozygous).
- Provenance: Not explicitly stated, likely laboratory-prepared or acquired genomic DNA.
Real-Time Stability (Study #5):
- Sample Size: Samples representing all three genotypes (WT, HET, MUT) (number not explicitly stated, but tested in quadruplicate at each time point). Also mentions three product lots.
- Provenance: Likely laboratory-prepared/controlled samples.
Reagent Freeze-Thaw Stability (Study #6):
- Sample Size: Controls (WT, HET, MUT) and gDNA (WT, HET, MUT). Total of 255 tests.
- Provenance: Genomic DNA isolate from cell lines.
Detection limit/Analytical Sensitivity and Normal Range (Study #3):
- Sample Size: 2 genomic DNA samples (1 WT, 1 HET), each diluted to 8 concentrations and tested in 40 replicates (total 2 * 8 * 40 = 640 tests).
- Provenance: Genomic DNA extracted from whole blood collected in potassium EDTA.
Analytical specificity (Study #4):
- Sample Size: 4 whole blood samples (3 WT, 1 HET). Each substance tested on 8 of these samples.
- Provenance: Human whole blood samples.
Pre-Analytical Equivalency Study (Study #7):
- Sample Size: 30 human whole blood samples and 10 leukocyte-depleted whole blood spike cell lines (total 40 unique samples). These were processed by 4 different extraction methods, leading to 160 extracted DNAs.
- Provenance: Human whole blood samples and cell lines.
Instrument Equivalency (Study #8):
- Sample Size: 29 human whole blood samples and 10 leukocyte-depleted whole blood samples spiked with cell lines (total 39 unique samples). These were tested across various instrument combinations, leading to 78 samples per combination (likely some samples were tested multiple times or a subset of the 39 samples were used to create 78 data points.
- Provenance: Human whole blood samples and cell lines.
Secondary Polymorphism Impact (Study #10):
- Sample Size: 5 samples (one homozygous normal, one heterozygous, and three homozygous normal with different secondary polymorphisms). Tested in 40 replicates each (total 5 * 40 = 200 tests).
- Provenance: Known Factor V genotypes with or without secondary polymorphisms.
Method comparison (Bi-directional Sequencing) (Study #2):
- Sample Size: 352 human whole blood samples (289 Homozygous Wild Type (GG), 45 Heterozygous (GA), 18 Homozygous Mutant (AA)).
- Provenance: Human whole blood samples. The text does not specify the country of origin, but it is implied to be a retrospective collection based on the phrasing "underwent DNA extraction and subsequent bi-directional DNA sequence analysis" then "The same DNA samples were then analyzed using the Invader® Factor V test."

3. Number of Experts and Qualifications for Ground Truth

The primary method for establishing ground truth across most studies is bi-directional DNA sequencing. The text does not specify the number of experts or their specific qualifications (e.g., molecular geneticists) used to interpret these sequencing results. However, bi-directional sequencing is a standard and highly accurate method for genotyping, and its interpretation would typically be performed by trained molecular diagnosticians or geneticists.

4. Adjudication Method for the Test Set

The text does not explicitly describe an adjudication method (e.g., 2+1, 3+1). For discrepancies, the sequencing results are considered the gold standard to which the device's results are compared. In cases like the "No Call" results in Study #1, troubleshooting and retraining of the operator were performed, and retesting was done against sequencing as the reference.

5. Multi-Reader Multi-Case (MRMC) Comparative Effectiveness Study

No, an MRMC comparative effectiveness study involving human readers with and without AI assistance was not done.

This device (Invader® Factor V test) is an in vitro diagnostic assay for genotyping, not an AI-assisted diagnostic imaging or clinical decision support system that would typically involve human readers. Its output is a genotype call (WT, HET, MUT), which is then interpreted by a healthcare professional. Therefore, the concept of "how much human readers improve with AI vs without AI assistance" does not apply to this type of device.

6. Standalone (Algorithm Only) Performance

Yes, the standalone performance (algorithm only without human-in-the-loop performance) was done and is the primary focus of the performance studies.

The Invader® Factor V test is an automated in vitro diagnostic system. The "Invader® Factor V Software, in combination with Invader Call Reporter™ software," processes raw fluorescence data to determine genotype calls (WT, HET, MUT). All the performance studies described (Precision, Stability, Sensitivity, Specificity, Method Comparison, etc.) evaluate the accuracy of these software-generated genotype calls against a recognized gold standard (bi-directional sequencing). There is no "human-in-the-loop" step described for data interpretation that would alter the genotype call generated by the software. The human operator's role is primarily experimental execution and loading/reviewing results from the software.

7. Type of Ground Truth Used

The primary type of ground truth used across all studies is bi-directional DNA sequencing. Where applicable, expected genotypes from known control materials (cell lines) also served as ground truth. This is a highly accurate and widely accepted method for determining genetic mutations.

8. Sample Size for the Training Set

The document does not explicitly mention a separate "training set" for the device's algorithm. For in vitro diagnostic kits like the Invader® Factor V test, the "training" (development and optimization) phase would typically involve a proprietary set of samples used by the manufacturer to establish the assay parameters, cutoff values for fluorescence, and the logic within the software for genotype calling. These samples are not typically disclosed or enumerated as a formal "training set" in regulatory submissions but are part of the overall development process. The studies described are validation studies (test sets) demonstrating the finalized device's performance.

9. How the Ground Truth for the Training Set was Established

Since a formal "training set" is not explicitly defined in the provided document, the method for establishing ground truth for any internal development samples would likely be the same as for the validation studies: bi-directional DNA sequencing, or comparison to previously characterized genomic DNA samples.

Summary

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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
- 1. 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
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J. Substantial Equivalence Information:

1. Predicate device name(s):
- Factor V Leiden Kit, Roche
1. Predicate 510(k) number(s): Roche, K033607
1. 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 forUse	Same as Intended Use	Same as Intended Use
TargetPopulation	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.
SoftwareInterface	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.
DetectionMethod	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/ulgDNA extracted from humanperipheral whole blood.
DetectionProcedure	Optical detection of stimulatedfluorescence using a specific pair ofprobes.	Multi-well fluorometer to detect rawfluorescence.
DetectionChemistry	Paired hybridization probes usingfluorescence resonance energy transfer(FRET) followed by melting curveanalysis.	PCR and Invader® usingFluorescence Resonance EnergyTransfer (FRET) chemistry forsignal reporting.
Analysis Time	A multi-step assay with different timesrequired for each step. Detectionoccurs at defined intervals during PCRcycle and can be reviewed in real-time.	~90 min. amplification followed by1 min signal detection. Softwareanalysis post signal detection.

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K. Standard/Guidance Document Referenced (if applicable):

Guidance for Industry and FDA Staff Class II Special Controls Guidance Document: Factor V Leiden DNA Mutation Detection Systems issued on March 16, 2004
Guidance for Industry and FDA Staff Guidance for the Content of Premarket . Submissions for Software Contained in Medical Devices issued May 11, 2005
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.

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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 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.

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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

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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	Samplestested	First Pass			Final			Final %AgreementFinal Correct Calls/Samples Tested
			CorrectCalls	No Calls(Invalid,EQ)	Miscalls	CorrectCalls	No Calls(Invalid,EQ)	Miscalls
Site001	1	90	90	0	0	90	0	0	100%
Site001	2	90	90	0	0	90	0	0	100%
Site002	1	90	90	0	0	90	0	0	100%
Site002	2	90	90	0	0	90	0	0	100%
Site003	1	90	90	0	0	90	0	0	100%
Site003	2	90	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 reportingsoftware automatically prevents the display of all sample genotypes, which resulted in 18 “No Call” samples. Upon retraining of the Operator, andretesting (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	#SamplesTested	FirstPassCorrectCalls	FirstPass NoCalls	Miscalls	FinalCorrectCalls	FinalAgreement%
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.

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Table 4: Factor V Genotype Results; Real-Time Stability
Sample/Control	Sequencing/Expected Factor VGenotype	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.

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Table 6: Analytical Sensitivity and Normal RangePercent 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) .

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Table 7: Summary, Comparison of Invader" Factor V Interfering Substance Results to Sequencing
InterferingSubstanceCode	Substance Concentration / (in bloodor DNA sample)	% Agreement withSequencingGenotype	% Agreement withUntreated SampleInvader® Factor VGenotype	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
ExtractionMethod	#SamplesTested	FirstPassCorrectCalls	FirstPass NoCalls	Miscalls	FinalCorrectCalls	FinalAgreement%
A	40	40	0	0	40	100
B	40	39	1*	0	39*	100*
C	40	40	0	0	40	100
D	40	40	0	0	40	100
Total	160	159	1	0	159	100
*Sample was removed from study due to loss of traceability of the sampleidentification.

Instrument Equivalency (Study #8): Twenty-nine (29) human whole blood ಕ್ಷsamples 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 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
		Sample01	Sample02	Sample03	Sample04	Sample05	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

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).

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Table 10: Agreement between the Invader® Factor V Test and Bi-directionalDNA Sequencing
Factor VGenotype*	Number tested	Number ofValid Resultson 1st Run	Number ofCorrectgenotypecalls on FirstRun	Agreement
HomozygousWild Type(GG)	289	289	289	100%
Heterozygous(GA)	45	45	45	100%
HomozygousMutant(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.
1. 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________________________________________________________________________________________________________________________________________________

1. Specimen Identification: Manual Labeling

Specimen Sampling and Handling: DNA should be extracted using a validated DNA extraction method that generates DNA concentration 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:

The genotyping test result will not be reported for any sample for which a positive or negative control failure occurs.

O. Proposed Labeling:

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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/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

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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 (301) 796-5450. 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 (800) 638-2041 or (301) 796-7100 or at its Internet address http://www.fda.gov/cdrh/industry/support/index.html.

Sincerely yours,

Reena Philip

้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

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Indications for Use Form

510(k) Number (if known): K100980

Device Name: Invader Factor V test

Indications for Use:

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

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.)