The cobas® Factor II and Factor V Test is an in vitro diagnostic device that uses real-time PCR for the detection and genotyping of the Factor II (Prothrombin) G20210A mutation and the Factor V Leiden G1691A mutation in genomic DNA obtained from K2EDTA whole blood specimens as an aid in diagnosis of patients with suspected thrombophilia. The cobas® Factor II and Factor V Test and the cobas z 480 analyzer are used together for automated amplification and detection.

Device Description

The cobas® Factor II and Factor V Test is a real-time polymerase chain reaction (PCR) test for the qualitative detection and genotyping of a single point mutation in the human Factor V gene (G to A at position 1691, referred to as the Factor V Leiden mutation) and a single point mutation in the human Factor II gene (G to A at position 20210), in genomic DNA isolated from peripheral whole blood, as an aid in diagnosis of patients with suspected thrombophilia.

DNA is extracted offline from whole blood specimens collected in K2EDTA tubes. The userselected DNA extraction method must provide DNA of sufficient concentration. Automated realtime PCR is then performed on the cobas z 480 analyzer. The Factor II and Factor V mutations are detected simultaneously in the same real-time PCR reaction. Depending upon the test order, results for one or both mutations are reported for each DNA sample.

The assay consists of one reagent kit and a system platform. The reagent kit provides the necessary reagents and controls to perform automated real-time PCR amplification and detection. The system platform consists of a real-time PCR thermal cycler (cobas z 480 analyzer) and a control unit (cobas® 4800 System CU). The amplification reactions generate a Factor II specific amplicon and a Factor V specific amplicon in all samples, and utilize four fluorescent-dye labeled oligonucleotide probes for detection of the Factor II and Factor V genotypes.

AI/ML Overview

Acceptance Criteria and Study for cobas® Factor II and Factor V Test

This document outlines the acceptance criteria and the studies performed to demonstrate that the cobas® Factor II and Factor V Test meets these criteria.

1. Table of Acceptance Criteria and Reported Device Performance

Factor II (Prothrombin) G20210A Mutation Detection:

Metric	Acceptance Criteria (Study Objective)	Reported Device Performance (95% LCB)
Overall Percent Agreement (OPA)	Compare performance using Sanger sequencing as the reference method.	100% (98.78%)
Positive Percent Agreement (PPA)	Compare performance using Sanger sequencing as the reference method.	100% (97.59%)
Negative Percent Agreement (NPA)	Compare performance using Sanger sequencing as the reference method.	100% (97.55%)
Heterozygous Agreement	Compare performance for heterozygous genotypes using Sanger sequencing.	100% (97.20%)
Homozygous Mutant Agreement	Compare performance for homozygous mutant genotypes using Sanger sequencing.	100% (83.89%)
Reproducibility (WT)	Evaluate reproducibility across sites, lots, and methods for Wild Type (WT).	100% (98.78%)
Reproducibility (HET)	Evaluate reproducibility across sites, lots, and methods for Heterozygous (HET).	100% (97.96%)
Reproducibility (MUT)	Evaluate reproducibility across sites, lots, and methods for Homozygous Mutant (MUT).	100% (94.04%)

Factor V Leiden G1691A Mutation Detection:

Metric	Acceptance Criteria (Study Objective)	Reported Device Performance (95% LCB)
Overall Percent Agreement (OPA)	Compare performance using Sanger sequencing as the reference method.	100% (98.78%)
Positive Percent Agreement (PPA)	Compare performance using Sanger sequencing as the reference method.	100% (97.62%)
Negative Percent Agreement (NPA)	Compare performance using Sanger sequencing as the reference method.	100% (97.52%)
Heterozygous Agreement	Compare performance for heterozygous genotypes using Sanger sequencing.	100% (97.20%)
Homozygous Mutant Agreement	Compare performance for homozygous mutant genotypes using Sanger sequencing.	100% (85.18%)
Reproducibility (WT)	Evaluate reproducibility across sites, lots, and methods for Wild Type (WT).	100% (98.78%)
Reproducibility (HET)	Evaluate reproducibility across sites, lots, and methods for Heterozygous (HET).	100% (97.96%)
Reproducibility (MUT)	Evaluate reproducibility across sites, lots, and methods for Homozygous Mutant (MUT).	100% (94.04%)

Analytical Sensitivity (Lower Limit of Detection):

Metric	Acceptance Criteria (Study Objective)	Reported Device Performance (Correct Call Rate)
Limit of Detection	Determine minimum input DNA necessary to yield correct genotype results.	98% at 0.01 ng/uL (100% at higher concentrations)

Analytical Sensitivity (Upper Limit of Detection):

Metric	Acceptance Criteria (Study Objective)	Reported Device Performance (Correct Call Rate)
Accuracy at High DNA Input	Evaluate performance at higher DNA concentrations (up to 300 ng/uL).	100%

DNA Extraction Method Study:

Metric	Acceptance Criteria (Study Objective)	Reported Device Performance (Overall Agreement)
Agreement with Sanger Sequencing	Evaluate agreement using different commercial DNA isolation methods.	99.4% (98.6 – 99.8% 95% two-sided CI)

Lot-to-Lot Repeatability:

Metric	Acceptance Criteria (Study Objective)	Reported Device Performance (Overall Agreement)
Agreement with Sanger Sequencing	Evaluate lot-to-lot repeatability across multiple reagent lots.	100% (99.3% one-sided, lower 95% CI)

2. Sample Size Used for the Test Set and Data Provenance

Method Comparison Study:

Sample Size: 300 specimens.
Data Provenance: The specimens were obtained from patients whose routine medical care called for Factor II and/or Factor V measurements, representing an intended use population. The commercial vendors provided these samples. The type of data (retrospective/prospective) is not explicitly stated, but the description of samples being "obtained to represent the intended use population" and collected for "routine medical care" suggests a retrospective collection or at least samples collected for clinical indications. The provenance is likely a mix of clinical labs or commercial biobanks.

Clinical Reproducibility Study:

Sample Size: A 9-member panel was used, consisting of 4 unique K2EDTA blood samples, 3 contrived blood samples, and 2 extracted genomic DNA samples. Each panel member was tested multiple times across different sites, operators, instruments, runs, and days. A total of 540 tests were performed (with 539 valid results and 1 invalid).
Data Provenance: The samples included K2EDTA whole blood samples and extracted genomic DNA samples. The "contrived blood samples" suggest laboratory-prepared samples. The "unique K2EDTA blood samples" were likely sourced from similar vendors as the method comparison study. The study was conducted at three test sites (2 external and 1 internal), implying a multi-center study on potentially diverse datasets.

3. Number of Experts Used to Establish the Ground Truth for the Test Set and the Qualifications of Those Experts

The ground truth for both the Method Comparison Study and the Clinical Reproducibility Study was established by bi-directional Sanger sequencing. The document does not specify the number of experts or their qualifications for performing or interpreting the Sanger sequencing results. However, Sanger sequencing is a widely accepted gold standard for genetic mutation detection, and it's typically performed by trained laboratory professionals.

4. Adjudication Method for the Test Set

The document does not explicitly describe an adjudication method for the test set where discrepancies between results from the cobas® test and Sanger sequencing were adjudicated by experts. The performance metrics are reported as direct agreements or disagreements with the Sanger sequencing results. In the DNA Extraction Method Study, for instance, one inconsistent triplicate result with a method was re-tested, and all results were correct upon re-testing, implying an internal review for outlier data.

5. If a Multi-Reader Multi-Case (MRMC) Comparative Effectiveness Study was Done

No, an MRMC comparative effectiveness study was not done. The cobas® Factor II and Factor V Test is an in vitro diagnostic device for automated amplification and detection of genetic mutations. It is an algorithm-only device; therefore, human readers are not directly involved in its performance or interpretation in a way that would necessitate an MRMC study.

6. If a Standalone (i.e. algorithm only without human-in-the-loop performance) was Done

Yes, the studies presented are standalone performance evaluations. The cobas® Factor II and Factor V Test is described as an "in vitro diagnostic device that uses real-time PCR for the detection and genotyping" and used with the "cobas z 480 analyzer for automated amplification and detection." This indicates an automated system where the algorithm performs the detection and genotyping without direct human intervention in the result generation process. Human interaction is limited to sample preparation and loading, and interpreting the final automated result.

7. The Type of Ground Truth Used

The type of ground truth used for both the Method Comparison Study and the Clinical Reproducibility Study was expert consensus through bi-directional Sanger sequencing. Sanger sequencing is generally considered the "gold standard" for confirming DNA sequences, and thus, genotypes.

8. The Sample Size for the Training Set

The document does not provide information regarding a separate training set or its sample size. The studies described are performance evaluations (method comparison, reproducibility, analytical sensitivity, etc.) which typically use independent test data to assess the device's accuracy and reliability, assuming the algorithm was developed and optimized prior to these evaluation studies. As this is not an AI/ML device in the traditional sense, but a PCR-based diagnostic, the concept of a "training set" for an algorithm's learning phase is not directly applicable in the same way as it would be for an image recognition AI, for example. The "training" for such a system would involve optimizing primers, probes, and reaction conditions during product development.

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

As mentioned above, the document does not discuss a separate training set. For the validation studies, ground truth was established by bi-directional Sanger sequencing.

Summary

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Image /page/0/Picture/0 description: The image contains the logos of the Department of Health & Human Services and the Food and Drug Administration (FDA). The Department of Health & Human Services logo is on the left, and the FDA logo is on the right. The FDA logo includes the text "FDA U.S. FOOD & DRUG ADMINISTRATION" in blue.

January 12, 2018

Roche Molecular Systems, Inc. Pooja Shah Sr. Regulatory Affairs Specialist 4300 Hacienda Drive Pleasanton, California 94588

Re: K172913

Trade/Device Name: cobas® Factor II and Factor V Test Regulation Number: 21 CFR 864.7280 Regulation Name: Factor V Leiden DNA mutation detection systems Regulatory Class: Class II Product Codes: NPR, NPQ Dated: December 13, 2017 Received: December 14, 2017

Dear Pooja Shah:

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. Please note: CDRH does not evaluate information related to contract liability warranties. We remind you, however, that device labeling must be truthful and not misleading.

If your device is classified (see above) into either class II (Special Controls) or class III (PMA), it may be subject to additional controls. Existing major regulations affecting your device can be found in the Code of Federal Regulations, Title 21, Parts 800 to 898. 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 Part 801 and Part 809); medical device reporting of medical device-related adverse events) (21 CFR 803); good manufacturing practice requirements as set forth in the quality systems (QS) regulation (21 CFR

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Part 820); and if applicable, the electronic product radiation control provisions (Sections 531-542 of the Act); 21 CFR 1000-1050.

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.

For comprehensive regulatory information about mediation-emitting products, including information about labeling regulations, please see Device Advice (https://www.fda.gov/MedicalDevices/DeviceRegulationandGuidance/) and CDRH Learn (http://www.fda.gov/Training/CDRHLearn). Additionally, you may contact the Division of Industry and Consumer Education (DICE) to ask a question about a specific regulatory topic. See the DICE website (http://www.fda.gov/DICE) for more information or contact DICE by email (DICE@fda.hhs.gov) or phone (1-800-638-2041 or 301-796-7100).

Sincerely,

Leonthena R. Carrington -S

Lea Carrington Director Division of Immunology and Hematology Devices Office of In Vitro Diagnostics and Radiological Health Center for Devices and Radiological Health

Enclosure

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Indications for Use

510(k) Number (if known) K172913

Device Name cobas® Factor II and Factor V Test

Indications for Use (Describe)

Type of Use (Select one or both, as applicable)

X Prescription Use (Part 21 CFR 801 Subpart D)
☐ Over-The-Counter Use (21 CFR 801 Subpart C)

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Attachment 3. 510k Summary

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cobas® Factor II and Factor V Test 510(k) Summary

This summary of 510(k) safety and effectiveness information is being submitted in accordance with the requirements of 21 CFR 807.92.

Submitter Name	Roche Molecular Systems, Inc.
Address	4300 Hacienda Drive, Pleasanton, CA 94588-2722 USA
Contact	Pooja ShahPhone: 925-596-8342FAX: 925-225-0207Email: pooja.shah.ps1@roche.com
Date Prepared	September 13th 2017
Proprietary Name	cobas® Factor II and Factor V Test
Common Name	N/A
Classification Name	Factor V Leiden DNA mutation detection system
Product Codes	NPR,NPQ 21 CFR 864.7280
Predicate Devices	K033612 & K033607
Establishment Registration	2243471 & 3004141078

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1. DEVICE DESCRIPTION

2. INDICATIONS FOR USE

The cobas® Factor II and Factor V Test is an in vitro diagnostic device that uses real-time PCR for the detection and genotyping of the Factor II (Prothrombin) G20210A mutation and the Factor V Leiden mutation G1691A in genomic DNA obtained from K2EDTA whole blood specimens as an aid in diagnosis of patients with suspected thrombophilia. The cobas® Factor II and Factor V Test and cobas z 480 analyzer are used together for automated amplification and detection.

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TECHNOLOGICAL CHARACTERISTICS 3.

The cobas® Factor II and Factor V Test has the same general intended use, technological characteristics and principles of operation as the two predicate devices. A substantial equivalence table comparing the similarities and differences between the cobas® Factor II and Factor V Test and its predicate devices is provided in Table 1

	Submitted Device:cobas® Factor II and Factor VTest	Predicate Device:Roche LightCycler®Factor II (Prothrombin) TestK033612	Predicate Device:Roche LightCycler®Factor V Leiden TestK033607
Intended Use	The cobas® Factor II and FactorV Test is an in vitro diagnosticdevice that uses real-time PCRfor the detection and genotypingof the Factor II (Prothrombin)G20210A mutation and theFactor V Leiden mutation ingenomic DNA obtained fromK2EDTA whole blood specimensas an aid in diagnosis of patientswith suspected thrombophilia.The test is performed on thecobas z 480 analyzer forautomated amplification anddetection.	Same as the Submitted deviceexcept the predicate devicewas cleared on theLightCycler® 1.2 Instrument.	Same as the Submitted deviceexcept the predicate devicewas cleared on theLightCycler® 1.2 Instrument.
Type of Test	Genotyping Test	Same	Same
Target ofDetection	Single nucleotide polymorphism	Same	Same
Indication forUse	Aid in the diagnosis of patientswith suspected thrombophilia	Same	Same
Intended User	Health Care Professional	Same	Same
Specimen Type	Purified DNA from human bloodsamples.	Same	Same
TechnologicalDetectionPrinciples	Real-time PCR test forsimultaneous amplification of twotargets and detection of specificSNPs in the PCR amplified DNAsequences (multiplex system).	Real-time PCR test foramplification of one target anddetection of specific SNP inPCR-amplified DNAsequences	Real-time PCR test foramplification of one targetand detection of specific SNPin PCR-amplified DNAsequences
SamplePreparation	DNA extraction and purificationfrom whole blood, performed off-line	Same	Same

Similarities and Differences between the cobas® Factor II and Factor V Test Table 1: and the Predicate Device

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	Submitted Device:cobas® Factor II and Factor VTest	Predicate Device:Roche LightCycler®Factor II (Prothrombin) TestK033612	Predicate Device:Roche LightCycler®Factor V Leiden TestK033607
Oligonucleotideprobes andprimers	Specific for Factor V G1691A,Factor II G20210A and the wildtype Factor II and Factor Vsequences	Specific for Factor II G20210Aand Factor II wild typesequences.	Specific for Factor V LeidenG1691A and Factor V wildtype sequences
DetectionChemistry	Fluorogenic detection of SNPs ineach PCR amplification productby allele-specific cleavage ofTaqMan probes	Fluorogenic detection of SNPin PCR amplification productby melting curve analysis	Fluorogenic detection of SNPin PCR amplification productby melting curve analysis
AnalyticalSensitivity	< 50 allele copies (0.01 ng inputDNA/reaction)	Approximately 50 allelecopies per reaction	Approximately 50 allelecopies per reaction
Instrument	cobas® 4800 System	Roche LightCycler® version1.2	Roche LightCycler® version1.2
Controls used	Endogenous Internal control(each gene is internal control forother gene), plus externalpositive and negative controlsrequired in each run.	External positive and negativecontrols required in each run.	External positive and negativecontrols required in each run.
ReferenceMethod	Bi-directional Sanger sequencing	Sanger Sequencing	Sanger Sequencing

NON-CLINICAL PERFORMANCE EVALUATION 4.

Analytical sensitivity (Lower Limit) 4.1.

To determine the minimum input of genomic DNA necessary to yield correct Factor II and Factor V genotype results, genomic DNA was isolated from three K2EDTA whole blood samples (Factor II heterozygous, Factor V heterozygous, Factor V homozygous mutant) and one cell line (Factor II homozygous mutant), using three different commercial DNA isolation methods for the whole blood samples, and one method for the cell line. Each genomic DNA sample was tested with the cobas® Factor II and Factor V Test at 10 concentrations: undiluted (which varied between 6 and 38 ng/uL) and nine serial dilutions from 1.0 to 0.0001 ng/uL. Each dilution was tested with 24 replicates with each of two kit lots, for a total of 48 replicates per concentration per genomic DNA sample. The undiluted genomic DNA samples were tested with six replicates with each kit lot, for a total of 12 replicates per genomic DNA sample.

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The rate of correct Factor II and Factor V genotype results across all four samples, all three DNA isolation methods and both kit lots was 98% at 0.01 ng/uL and 100% at all higher concentrations (Table 2). The cobas® Factor II and Factor V Test is designed to yield Invalid results if the input DNA concentration is too low. There were no incorrect genotype results in the study. The limit of detection is 0.01 ng/uL. which is 10 times lower than the lowest recommended DNA input concentration.

Concentration(ng/μL)	Number	cobas® Factor II and Factor V Test Results
		Number (%) Correct	Number (%) Incorrect	Number (%) Invalid
Undiluted*	120	120 (100%)	0 (0%)	0 (0%)
1	480	480 (100%)	0 (0%)	0 (0%)
0.3	480	480 (100%)	0 (0%)	0 (0%)
0.1	480	480 (100%)	0 (0%)	0 (0%)
0.03	480	480 (100%)	0 (0%)	0 (0%)
0.01	480	473 (98%)	0 (0%)	7 (2%)
0.003	480	86 (18%)	0 (0%)	394 (82%)
0.001	480	0 (0%)	0 (0%)	480 (100%)
0.0003	480	0 (0%)	0 (0%)	480 (100%)
0.0001	480	0 (0%)	0 (0%)	480 (100%)
0	480	0 (0%)	0 (0%)	480 (100%)

Analytical sensitivity of the cobas " Factor II and Factor V Test Table 2:

*6 to 38 nq/µL

4.2. Analytical sensitivity (Upper Limit)

To evaluate higher concentrations of DNA input for the cobas® Factor II and Factor V Test, genomic DNA was isolated from four K2EDTA whole blood samples using three different commercial DNA isolation methods, and concentrated genomic DNAs from cell lines were added to yield total DNA concentrations of 300 ng/uL , 150 ng/uL and 75 ng/uL. Genomic DNAs from Factor II heterozygous, Factor V heterozygous and Factor V homozygous mutant cell lines were added to genomic DNAs from whole blood samples of the same Factor II and Factor V genotypes. Factor II homozygous mutant cell line DNA was added to genomic DNA from a leukocyte depleted whole blood (LDWB) sample. The genomic DNA samples at 300 ng/μL, 150 ng/μL and 75 ng/μL were tested with 24 replicates with each of two kit lots, for

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a total of 48 replicates per concentration per genomic DNA sample. The genomic DNA samples from whole blood without added cell line DNA were tested with six replicates with each kit lot, for a total of 12 replicates per genomic DNA samples at 300 ng/μL, 150 ng/μL and 75 ng/μL yielded the correct Factor II and Factor V genotype results in all tests (Table 3). The LDWB samples without added cell line DNA vielded Invalid results, as expected: the other genomic DNA samples from whole blood vielded correct Factor II and Factor V results. The highest recommended DNA input concentration is 150 ng/uL.

		cobas® Factor II and Factor V Test Results Number (%)
Concentration (ng/µL)	Number	Number (%)Correct	Number (%)Incorrect	Number (%)Invalid
300	576	576 (100%)	0 (0%)	0 (0%)
150	576	576 (100%)	0 (0%)	0 (0%)
75	576	576 (100%)	0 (0%)	0 (0%)
Genomic DNA from neat wholeblood samples(6 to 38 ng/µL)	108	108 (100%)	0 (0%)	0 (0%)
Leukocyte depletedwhole blood sample*	36	36 (100%)	0 (0%)	0 (0%)

Testing of higher DNA input for the cobas Factor II and Factor V Test Table 3:

*Eluates from leukocyte-depleted whole blood sample yielded "Invalid" results due to the absence of leukocytes. These expected results are considered "correct" for the purpose of the study.

4.3. DNA Extraction Method Study

Genomic DNA was isolated from fifteen whole blood samples using three commercially available DNA isolation methods according to the manufacturer's instructions, by two operators for three days, for a total of six DNA isolations per sample with each DNA isolation method. Each isolated genomic DNA sample was tested in triplicate with the cobas® Factor II and Factor V Test (Table 4). One hundred percent of the results with the cobas® Factor II and Factor V Test were in agreement with the Factor II and Factor V genotype by bi-directional Sanger sequencing. One genomic DNA sample isolated with method A was excluded from the results. It was rust colored and yielded invalid results in all three tests. Isolated DNA samples should appear clear and colorless. DNA samples with any appearance other than clear and colorless should not be tested as they may yield invalid or incorrect results.

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DNA IsolationMethod	Total Number of		Number (%) of
	DNAIsolations	Tests	Correct Results	IncorrectResults	InvalidResults
A	90	270	267a (98.9%)(96.8 – 99.8)b	0 (0.0%)	3a (1.1%)
B	90	270	268c (99.3%)(97.4 – 99.9)b	1c (0.4%)	1c (0.4%)
C	90	270	270 (100.0%)(98.6 – 100)b	0 (0.0%)	0 (0.0%)
Total	270	810	805 (99.4%)(98.6 – 99.8)b	1 (0.1%)	4 (0.5%)

Table 4: Results by DNA Isolation Methods

ª One of 90 DNA samples isolated with method A was rust-colored and generated 3 invalid results. Only clear and colorless DNA samples should be tested. DNA samples with any appearance other than clear and colorless should not be tested, as they may yield invalid or incorrect results.

b 95% two-sided, confidence interval

C The triplicate results from one sample isolated with method B were inconsistent: 1 incorrect, 1 invalid. The sample eluate was re-tested in triplicate and all results were correct upon re-testing.

Analytical Specificity (Potentially Interfering Mutation Study) 4.4.

To determine the effect of known single nucleotide polymorphisms (SNPs) close to the Factor V Leiden mutation (1691G>A) and the Factor II (prothrombin) mutation (20210G>A), plasmid DNAs containing eight known SNPs (20207A>C. 20209C>T. 20218A>G. 20221C>T. 1689G>A, 1690C>T, 1692A>C and 1696A>G) were tested. The Factor II SNP plasmids and the Factor V SNP plasmids were wild type at positions 20210 and 1691, respectively. Each SNP plasmid DNA was tested alone, and in combination with wild type Factor II plasmid DNA, wild type Factor V plasmid DNA, wild type Factor II and wild type Factor V plasmid DNAs, and with genomic DNA from wild type whole blood.

None of the SNP plasmids caused false positive results for the Factor II (prothrombin) or Factor V Leiden mutations, and none of the SNP plasmids interfered with detection of the wild type Factor II or Factor V sequences. All four Factor II SNP plasmids and three of four Factor V SNP plasmids were detected as wild type Factor II or Factor V DNA, respectively. One Factor V SNP plasmid (1689G>A) was not detected by the cobas® Factor II and Factor V Test. If this SNP is present on both alleles, the test result would be Invalid.

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4.5. Potential Interfering Substances Study

Triglycerides (37 mM), bilirubin (conjugated and unconjugated, 342 uM), and cholesterol (13 mM) did not interfere with the cobas® Factor II and Factor V Test when added to whole blood at the test concentrations recommended in CLSI guideline. EP07-A2.11 Hemoglobin did not interfere with the cobas® Factor II and Factor V Test when spiked into whole blood to vield a total hemoglobin concentration of ~25.8 - 31 g/dL.

The anticoagulant K2EDTA did not interfere when it was tested at 5.7 mg/mL, which is approximately 3 times the concentration of K2EDTA in whole blood when the blood collection tube is filled to capacity. Heparin, coumadin (Warfarin), rivaroxaban (Xarelto), and dabigatran etexilate (Pradaxa) did not interfere with the cobas® Factor II and Factor V Test.

A commercial extraction buffer containing guanidine hydrochloride, a common ingredient in commercial DNA extraction buffers, interfered with the cobas® Factor II and Factor V Test, causing invalid results when it was present in genomic DNA samples at a concentration of 2.5% (v/v). Ethanol, a common ingredient in the wash buffers of commercial DNA isolation methods, interfered with the cobas® Factor II and Factor V Test when added to genomic DNA samples to a concentration of 5% (v/v). For both substances, observed interference caused invalid results.

4.6. Lot-to-Lot Repeatability

The lot-to-lot repeatability of the cobas® Factor II and Factor V Test was evaluated by testing genomic DNA samples isolated from seven K2EDTA whole blood samples with three lots of the cobas® Factor II and Factor V Test. The study was conducted over five non-consecutive days with two operators, two cobas z 480 analyzers, one run per day per kit lot, and two replicates per run, for a total of 60 replicates of each sample (Table 5). The cobas® Factor II and Factor V Test results were compared to the Factor II and Factor V genotype by bi-directional Sanger sequencing. The overall agreement between cobas® Factor II and Factor V Test results and nucleic acid sequencing was 100% across all samples and reagent lots (one-sided, lower 95% confidence limit 99.3%)

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SampleID	Factor IIGenotype	Factor VGenotype	Number ofTests perLot	Number (%) Correct Genotype Results
				Lot 1	Lot 2	Lot 3	All Lots
S1	Wild type	Wild type	20	20 (100%)	20 (100%)	20 (100%)	60 (100%)
S2	Wild type	Wild type	20	20 (100%)	20 (100%)	20 (100%)	60 (100%)
S3	Wild type	Heterozygous	20	20 (100%)	20 (100%)	20 (100%)	60 (100%)
S4	Heterozygous	Wild type	20	20 (100%)	20 (100%)	20 (100%)	60 (100%)
S5	Wild type	Homozygousmutant	20	20 (100%)	20 (100%)	20 (100%)	60 (100%)
S6	Homozygousmutant	Wild type	20	20 (100%)	20 (100%)	20 (100%)	60 (100%)
S7	Heterozygous	Heterozygous	20	20 (100%)	20 (100%)	20 (100%)	60 (100%)
Total			140	140 (100%)	140 (100%)	140 (100%)	420 (100%)

Table 5: Lot-to-lot Repeatability

CLINICAL PERFORMANCE EVALUATION 5.

Method Comparison Study 5.1.

Study Objective 5.1.1. -

The objective of this investigational protocol was to compare the performance of the cobas® Factor II and Factor V Test to bi-directional Sanger sequencing for the identification of the Factor II and Factor V mutations in DNA isolated from whole blood specimens from patients with suspected thrombophilia.

Methodology 5.1.2.

5.1.2.1. Study Design

A total of 300 specimens from patients whose routine medical care called for Factor II and/or Factor V measurements were obtained to represent the intended use population. The commercial vendors that provided these samples indicated that the collection included: individual Factor II and Factor V heterozygous mutations, individual homozygous Factor II and Factor V mutations, compound heterozygous mutations, and samples that were wild type for Factor V Table 6.

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There were 284 samples that were whole blood and represented frequently found genotypes for Factor II and Factor V. The majority of rare mutations such as Factor II homozygous/ Factor V Wild Type were acquired as DNA samples without identified extraction methodology.

Factor II (F2)	Factor V (F5)	Specimen Type
Wild Type (WT F2)	Wild Type (WT F5)	Whole Blood
Wild Type (WT F2)	Heterozygous (HET F5)	Whole Blood
Heterozygous (HET F2)	Wild Type (WT F5)	Whole Blood
Heterozygous (HET F2)	Heterozygous (HET F5)	Whole Blood
Wild Type (WT F2)	Homozygous Mutant (MUT F5)	Whole Blood
Homozygous Mutant (MUT F2)	Wild Type (WT F5)	Whole Blood and DNA

Factor II and Factor V genotypes included in the study Table 6:

Statistical Methods 5.1.2.2.

The performance of the cobas® Factor II and Factor V Test was evaluated separately for Factor II and Factor V genotypes by using bi-directional Sanger sequencing as the reference method. Positive Percent Agreement (PPA), Negative Percent Agreement (NPA) and Overall Percentage Agreement (OPA) were estimated at the genotype level (Wild Type, Heterozygous, and Homozygous Mutant) separately for Factor II and Factor V.

Results 5.1.3.

A total of 300 samples were tested for Factor II and Factor V by both cobas Factor II and Factor V Test and Sanger sequencing. All runs and tests were valid for cobas® test and no repeat tests were performed. A test result was classified as correct for Factor V, if both the cobas test and Sanger sequencing detect the same genotype. A test result was classified as an incorrect for Factor II or Factor V if the cobas® test and Sanger sequencing detect a different genotype for Factor II or Factor V.

Table 7 presents agreement between the cobas® test and Sanger sequencing for Factor II results. The OPA between the two tests was 100% with a two-sided 95% lower confidence bound (exact method) of 98.78%. Both PPA and NPA were 100% with lower confidence bounds of

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97.59% for PPA and 97.55 % for NPA. The percent agreement of correct results for

Heterozygous and Homozygous Mutant genotypes were both 100%.

Table 7: Performance of the cobas Factor II and Factor V Test using Bi-Directional
Sanger Sequencing as a Reference for the Identification of Factor II Genotype

Genotype bySequencing	TotalSamplesTested	CorrectCalls1	PercentAgreement	95%LCB4
Wild Type	149	149	100%(NPA)	97.55%
Positive	151	151	100%(PPA)	97.59%
Heterozygous	130	130	100%	97.20%
Mutant5	21	21	100%	83.89%
Total	300	300	100%(OPA)	98.78%

1 cobas test Factor II genotype results in agreement with the Factor II genotype by sequencing

2 Invalid or failed cobas® result (no Factor II genotype call)

3 cobas test Factor II genotype results discordant with the Factor II genotype by sequencing

4 Two-sided 95% lower confidence boundary is calculated using Exact method

5 Homozygous mutant

Table 8 presents agreement between the cobas® test and Sanger sequencing for Factor V results. The OPA between the two tests was 100% with a two-sided 95% lower confidence bound (exact method) of 98.78 %. Both PPA and NPA were 100% with lower confidence bounds of 97.62% for PPA and 97.52 % for NPA. The percent agreement of correct results for Heterozygous and Homozygous Mutant genotypes were both 100%.

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Performance of the cobas Factor II and Factor V Test using Bi-Directional Table 8: Sanger Sequencing as a Reference for the Identification of Factor V Genotype

Genotype bySequencing	TotalSamplesTested	CorrectCalls1	PercentAgreement	95% LCB4
Wild Type	147	147	100%(NPA)	97.52%
Positive	153	153	100%(PPA)	97.62%
Heterozygous	130	130	100%	97.20%
Mutant5	23	23	100%	85.18%
Total	300	300	100%(OPA)	98.78%

1 cobas test Factor V genotype results in agreement with the Factor V genotype by sequencing

2 Invalid or failed cobas® result (no Factor V genotype call)

3 cobas® test Factor V genotype results discordant with the Factor V genotype by sequencing

4 Two-sided 95% lower confidence boundary is calculated using Exact method

5 Homozygous mutant

Table 9: Performance of the cobas® Factor II and Factor V Test Using Bi-Directional Sanger Sequencing as a Reference for the Identification of Combined Factor II and Factor V Result

Bi-DirectionalSangersequencingResult	HET F2/HET F5	HET F2/WT F5	MUT F2/WT F5	WT F2/HET F5	WT F2/MUT F5	WT F2/WT F5	Total
HET F2/ HET F5	25	0	0	0	0	0	25
HET F2/ WT F5	0	105	0	0	0	0	105
MUT F2/ WT F5	0	0	21	0	0	0	21
WT F2/ HET F5	0	0	0	105	0	0	105
WT F2/ MUT F5	0	0	0	0	23	0	23
WT F2/ WT F5	0	0	0	0	0	21	21
Total	25	105	21	105	23	21	300

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Conclusion 5.1.4.

The results of this Method Comparison Study support the intended use of the cobas "Factor II and Factor V Test for diagnostic purposes as an aid in the clinical management of patients with suspected thrombophilia.

Clinical Reproducibility Study 5.2.

5.2.1. Study Objective

The objective of this study was to evaluate the reproducibility of the cobas® Factor II and Factor V Test for the detection and genotyping of the Factor II (prothrombin) G20210A and Factor V Leiden (G1691A) mutations.

Methodology 5.2.2.

5.2.2.1. Study Design

The reproducibility study included three manual whole blood sample preparation methods. These three whole blood DNA sample preparation kits were intended to be representative of commercially available sample preparation kits for DNA extraction from whole blood.

Reproducibility of the cobas Factor II and Factor V Test was evaluated with K2EDTA whole blood samples and genomic DNA samples across the following factors:

Sample Preparation Method: One (1) sample preparation method per site .
Reagent Lot: Three (3) reagent lots, 1 lot per site .
Test Site: Three (3) sites (2 external and 1 internal) .
Operator: Two (2) operators per site .
Instrument: One (1) instrument per site .
Run: One (1) run per operator per day .
Days: Five (5) non-consecutive days per reagent lot .

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Reproducibility was assessed using a nine-member panel: four unique K2EDTA blood samples, three contrived blood samples and two extracted genomic DNA (gDNA) samples diluted to 0.2 ng/μL.

Panel Member	Genotype of Panel Member	Sample Type
01	Factor II Wild Type	Factor V Wild Type	Whole Blood
02	Factor II Wild Type	Factor V Wild Type	Whole Blood
03	Factor II Wild Type	Factor V Heterozygous	Whole Blood
04	Factor II Heterozygous	Factor V Wild Type	Whole Blood
05	Factor II Wild Type	Factor V Homozygous mutant	Contrived Whole Blood
06	Factor II Homozygous mutant	Factor V Wild Type	Contrived Whole Blood
07	Factor II Heterozygous	Factor V Heterozygous	Contrived Whole Blood
08	Factor II Wild Type	Factor V Heterozygous	Genomic DNA
09	Factor II Heterozygous	Factor V Wild Type	Genomic DNA

Table 10: Description of Panel Members

Statistical Analyses 5.2.2.2.

Data were summarized by the percentage of correct results, and the associated one-sided 95% exact lower confidence bounds (LCBs) or two-sided 95% exact confidence interval (CIS) and data were presented by panel member, genotype, site/sample preparation method and sample type (gDNA or whole blood).

For a valid test, a correct result was defined as agreement between the cobas test result and the panel member genotype as determined by Sanger sequencing; an incorrect result was defined as disagreement between the cobas test result and the panel member genotype as determined by Sanger sequencing. Test results are for both Factor II and Factor V and are either valid for both results or invalid for both results. A 'No Call' indicated that the test result was not available due to failed or invalid test result. Percent agreement of correct results between Sanger sequencing and cobas® Factor II and Factor V Test was determined from valid results.

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5.2.3. Results

A total of 38 runs were performed in this study. A subset of 8 runs performed at site 1 with an expired sample preparation lot were determined as not acceptable and excluded from the statistical analysis. These runs were repeated with in-date reagents. The rest of 30 runs are all valid and meet all protocol requirements. A total of 540 tests were performed on the 9 panel members in the 30 valid runs, with 539 valid results and one invalid result. The following results are based on the dataset from these 30 runs.

5.2.3.1. Results for Factor II

Table 11 summarizes the overall agreement results of the reproducibility study for Factor II by overall genotype and testing site/sample preparation method. All results from valid tests were correct calls, leading to 100% agreement for each genotype. With the invalid result treated as an incorrect result, the percent agreement was 99.4% for HET and remained 100% for WT and MUT.

Factor II	Correct Results/Number of Samples Tested			IncorrectResults	InvalidResultsc	Correct Results/Number of ValidResults (%)	95% LCBd
	Site1b/Lot1/Method A	Site2b/Lot2/Method B	Site 3b/Lot3/Method C
WTe	100/100	100/100	100/100	0	0	300/300 (100%)	98.78
HETe	60/60	59/60	60/60	0	1	179/179 (100%)	97.96
MUTf	20/20	20/20	20/20	0	0	60/60 (100%)	94.04

Table 11: Summary of Reproducibility Study -- Factor II

ª WT: Wild Type; HET: Heterozygous; MUT: Homozyqous Mutant

b Each site used different DNA isolation method (A, B, C) and different reagent lot

° Invalid result that was not retested

d Two-sided 95% Lower Confidence Bound (LCB) (exact method)

e At each site, 20 results were from gDNA samples, and 20 from contrived whole blood samples

f From contrived whole blood samples only

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5.2.3.2. Results for Factor V

Table 12 summarizes the overall agreement results of the reproducibility study for Factor V by overall genotype and testing site/reagent lot/sample preparation method. All results from valid tests were correct calls, leading to 100% agreement for each genotype. With the invalid result treated as an incorrect result, the percent agreement was 99.4% for HET and remained 100% for WT and MUT.

Genotype bySequencinga	Correct Results/Number of Samples Tested			IncorrectResults	InvalidResultsc	Correct Results/Number of ValidResults (%)	95% LCBd
Factor V	Site1b/Lot1/Method A	Site 2b/Lot2/Method B	Site 3b/Lot3/Method C
WTe	100/100	100/100	100/100	0	0	300/300 (100%)	98.78
HETe	60/60	59/60	60/60	0	1	179/179 (100%)	97.96
MUTf	20/20	20/20	20/20	0	0	60/60 (100%)	94.04

Table 12: Summary of Reproducibility Study -- Factor V

ª WT: Wild Type; HET: Heterozygous; MUT: Homozygous Mutant

b Each site used different DNA isolation method (A, B, C) and different reagent lot

° Invalid result that was not retested

d Two-sided 95% Lower Confidence Bound (LCB) (exact method)

° At each site, 20 results were from gDNA samples, and 20 from contrived whole blood samples

f From contrived whole blood samples only

Conclusions 5.3.

The submitted information in this 510(k) notification demonstrates that the cobas "Factor II and

Factor V Test is as safe and effective as the predicate devices and therefore supports a Substantial Equivalence decision.

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.)