(109 days)
No
The device description and performance studies focus on real-time PCR technology and standard analytical methods for detecting and genotyping specific genetic mutations. There is no mention of AI or ML in the intended use, device description, or performance study summaries.
No
The device is described as an in vitro diagnostic device used as an aid in diagnosis, not for treatment.
Yes
The "Intended Use / Indications for Use" section explicitly states that the device is "an in vitro diagnostic device" and is used "as an aid in diagnosis of patients with suspected thrombophilia."
No
The device is an in vitro diagnostic device that includes a reagent kit and a system platform consisting of a real-time PCR thermal cycler (cobas z 480 analyzer) and a control unit (cobas® 4800 System CU), which are hardware components.
Yes, this device is an IVD (In Vitro Diagnostic).
The "Intended Use / Indications for Use" section explicitly states: "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."
This statement clearly identifies the device as an in vitro diagnostic device.
N/A
Intended Use / 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 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.
Product codes
NPR, NPQ
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 real-time 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.
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
Health Care Professional
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
Method Comparison Study:
Sample size: 300 specimens.
Data source: Specimens from patients whose routine medical care called for Factor II and/or Factor V measurements, obtained from commercial vendors. 284 samples were whole blood, and some rare mutations were acquired as DNA samples without identified extraction methodology.
Annotation protocol: Bi-directional Sanger sequencing was used as the reference method to determine Factor II and Factor V genotypes.
Summary of Performance Studies
-
Analytical Sensitivity (Lower Limit):
- Study type: Analytical sensitivity study.
- Sample size: Genomic DNA 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). Tested with 24 replicates per concentration per genomic DNA sample across two kit lots for 10 concentrations.
- Key results: The rate of correct Factor II and Factor V genotype results was 98% at 0.01 ng/uL and 100% at all higher concentrations. The limit of detection is 0.01 ng/uL.
-
Analytical Sensitivity (Upper Limit):
- Study type: Analytical sensitivity study.
- Sample size: Genomic DNA isolated from four K2EDTA whole blood samples using three different commercial DNA isolation methods, concentrated genomic DNAs from cell lines added to yield 300 ng/uL, 150 ng/uL, and 75 ng/uL. Tested with 24 replicates per concentration per genomic DNA sample across two kit lots.
- Key results: Genomic DNA samples at 300 ng/uL, 150 ng/uL, and 75 ng/uL yielded 100% correct Factor II and Factor V genotype results. The highest recommended DNA input concentration is 150 ng/uL.
-
DNA Extraction Method Study:
- Study type: Analytical performance evaluation.
- Sample size: 15 whole blood samples, each isolated using three commercially available DNA isolation methods by two operators for three days (total of six DNA isolations per sample per method). Each isolated DNA sample was tested in triplicate.
- Key results: 100% agreement with Factor II and Factor V genotype by bi-directional Sanger sequencing. Overall 99.4% correct results, 0.1% incorrect, 0.5% invalid.
-
Analytical Specificity (Potentially Interfering Mutation Study):
- Study type: Analytical specificity study.
- Sample size: Plasmid DNAs containing eight known SNPs (20207A>C, 20209C>T, 20218A>G, 20221C>T, 1689G>A, 1690C>T, 1692A>C, and 1696A>G).
- Key results: None of the SNP plasmids caused false positive results for Factor II or Factor V Leiden mutations, and none interfered with detection of wild type sequences. One Factor V SNP plasmid (1689G>A) was not detected.
-
Potential Interfering Substances Study:
- Study type: Interfering substances study.
- Sample size: Not explicitly stated, but substances like triglycerides, bilirubin, cholesterol, hemoglobin, K2EDTA, heparin, coumadin (Warfarin), rivaroxaban (Xarelto), and dabigatran etexilate (Pradaxa) were tested.
- Key results: Most tested substances did not interfere. A commercial extraction buffer containing guanidine hydrochloride and ethanol interfered, causing invalid results.
-
Lot-to-Lot Repeatability:
- Study type: Repeatability study.
- Sample size: Genomic DNA samples isolated from seven K2EDTA whole blood samples, tested with three lots of the device. 60 replicates of each sample were tested.
- Key results: Overall agreement between cobas® Factor II and Factor V Test results and nucleic acid sequencing was 100% across all samples and reagent lots.
-
Method Comparison Study:
- Study type: Clinical performance evaluation / Method Comparison Study.
- Sample size: 300 specimens.
- Key results: All runs and tests were valid.
- Factor II: Overall Percentage Agreement (OPA) was 100% (95% LCB 98.78%). Positive Percent Agreement (PPA) and Negative Percent Agreement (NPA) were 100% (LCB for PPA 97.59%, for NPA 97.55%).
- Factor V: Overall Percentage Agreement (OPA) was 100% (95% LCB 98.78%). Positive Percent Agreement (PPA) and Negative Percent Agreement (NPA) were 100% (LCB for PPA 97.62%, for NPA 97.52%).
-
Clinical Reproducibility Study:
- Study type: Reproducibility study.
- Sample size: Nine-member panel (four unique K2EDTA blood samples, three contrived blood samples, and two extracted genomic DNA samples) tested across three sites, three reagent lots, and two operators over five non-consecutive days, with 30 valid runs and 540 tests performed.
- Key results:
- Factor II: All results from valid tests were correct calls, leading to 100% agreement for each genotype (Wild Type, Heterozygous, Homozygous Mutant). With one invalid result treated as incorrect, percent agreement was 99.4% for Heterozygous and 100% for Wild Type and Mutant.
- Factor V: All results from valid tests were correct calls, leading to 100% agreement for each genotype (Wild Type, Heterozygous, Homozygous Mutant). With one invalid result treated as incorrect, percent agreement was 99.4% for Heterozygous and 100% for Wild Type and Mutant.
Key Metrics
Method Comparison Study (Factor II):
- Overall Percentage Agreement (OPA): 100% (95% LCB: 98.78%)
- Positive Percent Agreement (PPA): 100% (95% LCB: 97.59%)
- Negative Percent Agreement (NPA): 100% (95% LCB: 97.55%)
- Percent Agreement for Heterozygous: 100% (95% LCB: 97.20%)
- Percent Agreement for Homozygous Mutant: 100% (95% LCB: 83.89%)
Method Comparison Study (Factor V):
- Overall Percentage Agreement (OPA): 100% (95% LCB: 98.78%)
- Positive Percent Agreement (PPA): 100% (95% LCB: 97.62%)
- Negative Percent Agreement (NPA): 100% (95% LCB: 97.52%)
- Percent Agreement for Heterozygous: 100% (95% LCB: 97.20%)
- Percent Agreement for Homozygous Mutant: 100% (95% LCB: 85.18%)
Reproducibility Study (Factor II, based on valid results):
- Percent Agreement for Wild Type: 100% (95% LCB: 98.78%)
- Percent Agreement for Heterozygous: 100% (95% LCB: 97.96%)
- Percent Agreement for Homozygous Mutant: 100% (95% LCB: 94.04%)
Reproducibility Study (Factor V, based on valid results):
- Percent Agreement for Wild Type: 100% (95% LCB: 98.78%)
- Percent Agreement for Heterozygous: 100% (95% LCB: 97.96%)
- Percent Agreement for Homozygous Mutant: 100% (95% LCB: 94.04%)
Predicate Device(s)
Reference Device(s)
Not Found
Predetermined Change Control Plan (PCCP) - All Relevant Information
Not Found
§ 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.)
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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
1
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
2
Indications for Use
510(k) Number (if known) K172913
Device Name cobas® Factor II and Factor V Test
Indications for Use (Describe)
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.
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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3
Attachment 3. 510k Summary
4
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 Shah | |
| Phone: 925-596-8342 | ||
| FAX: 925-225-0207 | ||
| Email: 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 |
5
1. 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.
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.
6
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 V
Test | Predicate Device:
Roche LightCycler®
Factor II (Prothrombin) Test
K033612 | Predicate Device:
Roche LightCycler®
Factor V Leiden Test
K033607 |
|------------------------------------------|--------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------|-----------------------------------------------------------------------------------------------------------------------------|-----------------------------------------------------------------------------------------------------------------------------|
| Intended 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 in
genomic DNA obtained from
K2EDTA whole blood specimens
as an aid in diagnosis of patients
with suspected thrombophilia.
The test is performed on the
cobas z 480 analyzer for
automated amplification and
detection. | Same as the Submitted device
except the predicate device
was cleared on the
LightCycler® 1.2 Instrument. | Same as the Submitted device
except the predicate device
was cleared on the
LightCycler® 1.2 Instrument. |
| Type of Test | Genotyping Test | Same | Same |
| Target of
Detection | Single nucleotide polymorphism | Same | Same |
| Indication for
Use | Aid in the diagnosis of patients
with suspected thrombophilia | Same | Same |
| Intended User | Health Care Professional | Same | Same |
| Specimen Type | Purified DNA from human blood
samples. | Same | Same |
| Technological
Detection
Principles | Real-time PCR test for
simultaneous amplification of two
targets and detection of specific
SNPs in the PCR amplified DNA
sequences (multiplex system). | Real-time PCR test for
amplification of one target and
detection of specific SNP in
PCR-amplified DNA
sequences | Real-time PCR test for
amplification of one target
and detection of specific SNP
in PCR-amplified DNA
sequences |
| Sample
Preparation | DNA extraction and purification
from 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
7
| | Submitted Device:
cobas® Factor II and Factor V
Test | Predicate Device:
Roche LightCycler®
Factor II (Prothrombin) Test
K033612 | Predicate Device:
Roche LightCycler®
Factor V Leiden Test
K033607 |
|------------------------------------------|------------------------------------------------------------------------------------------------------------------------------------------------------------|-------------------------------------------------------------------------------------------|-------------------------------------------------------------------------------------------|
| Oligonucleotide
probes and
primers | Specific for Factor V G1691A,
Factor II G20210A and the wild
type Factor II and Factor V
sequences | Specific for Factor II G20210A
and Factor II wild type
sequences. | Specific for Factor V Leiden
G1691A and Factor V wild
type sequences |
| Detection
Chemistry | Fluorogenic detection of SNPs in
each PCR amplification product
by allele-specific cleavage of
TaqMan probes | Fluorogenic detection of SNP
in PCR amplification product
by melting curve analysis | Fluorogenic detection of SNP
in PCR amplification product
by melting curve analysis |
| Analytical
Sensitivity | 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.
11
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%)
12
| Sample
ID | Factor II
Genotype | Factor V
Genotype | Number of
Tests per
Lot | 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 | Homozygous
mutant | 20 | 20 (100%) | 20 (100%) | 20 (100%) | 60 (100%) |
| S6 | Homozygous
mutant | 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.
13
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
14
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 by
Sequencing | Total
Samples
Tested | Correct
Calls1 | No calls or
Invalid
Results2 | Missed or
Incorrect
Calls3 | Percent
Agreement | 95%
LCB4 |
|---------------------------|----------------------------|-------------------|------------------------------------|----------------------------------|----------------------|-------------|
| Wild Type | 149 | 149 | 0 | 0 | 100%
(NPA) | 97.55% |
| Positive | 151 | 151 | 0 | 0 | 100%
(PPA) | 97.59% |
| Heterozygous | 130 | 130 | 0 | 0 | 100% | 97.20% |
| Mutant5 | 21 | 21 | 0 | 0 | 100% | 83.89% |
| Total | 300 | 300 | 0 | 0 | 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 by
Sequencing | Total
Samples
Tested | Correct
Calls1 | No calls or
Invalid
Results2 | Missed or
Incorrect
Calls3 | Percent
Agreement | 95% LCB4 |
|---------------------------|----------------------------|-------------------|------------------------------------|----------------------------------|----------------------|----------|
| Wild Type | 147 | 147 | 0 | 0 | 100%
(NPA) | 97.52% |
| Positive | 153 | 153 | 0 | 0 | 100%
(PPA) | 97.62% |
| Heterozygous | 130 | 130 | 0 | 0 | 100% | 97.20% |
| Mutant5 | 23 | 23 | 0 | 0 | 100% | 85.18% |
| Total | 300 | 300 | 0 | 0 | 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-Directional
Sanger
sequencing
Result | 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.
| Genotype by
Sequencinga | Factor II | Correct Results/
Number of Samples Tested | | | Incorrect
Results | Invalid
Resultsc | Correct Results/
Number of Valid
Results (%) | 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 by
Sequencinga | Correct Results/
Number of Samples Tested | | | Incorrect
Results | Invalid
Resultsc | Correct Results/
Number of Valid
Results (%) | 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.