cobas Factor II and Factor V Test
Device Facts
| Record ID | K172913 |
|---|---|
| Device Name | cobas Factor II and Factor V Test |
| Applicant | Roche Molecular Systems, Inc. |
| Product Code | NPR · Hematology |
| Decision Date | Jan 12, 2018 |
| Decision | SESE |
| Submission Type | Traditional |
| Regulation | 21 CFR 864.7280 |
| Device Class | Class 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 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 cobas z 480 analyzer are used together for automated amplification and detection.
Device Story
The cobas® Factor II and Factor V Test is an in vitro diagnostic assay for qualitative detection and genotyping of Factor II (G20210A) and Factor V Leiden (G1691A) mutations. Input consists of genomic DNA extracted offline from K2EDTA whole blood specimens. The device utilizes real-time PCR on the cobas z 480 analyzer for automated amplification and detection. The assay employs four fluorescent-dye labeled oligonucleotide probes to detect specific genotypes simultaneously in a single reaction. The system produces genotype results for one or both mutations. Used in clinical laboratories by healthcare professionals, the output aids in the diagnosis of patients with suspected thrombophilia. By identifying these genetic risk factors, the test assists clinicians in the management of patients with thrombotic conditions.
Clinical Evidence
Clinical performance was evaluated via a method comparison study of 300 patient samples using bi-directional Sanger sequencing as the reference method. The study demonstrated 100% Overall Percentage Agreement (OPA) for both Factor II and Factor V genotypes, with 95% lower confidence bounds of 98.78%. A clinical reproducibility study across three sites, using three reagent lots and three DNA extraction methods, showed 100% agreement for valid results across all genotypes (Wild Type, Heterozygous, Homozygous Mutant).
Technological Characteristics
Real-time PCR multiplex system. Uses cobas z 480 analyzer. Detection via fluorogenic TaqMan probes. Requires offline DNA extraction. Internal control included. Shelf life 15 months at 2-8°C. Software-controlled automated amplification and detection.
Indications for Use
Indicated for patients with suspected thrombophilia to detect and genotype Factor II (Prothrombin) G20210A and Factor V Leiden G1691A mutations in genomic DNA from K2EDTA whole blood.
Regulatory Classification
Identification
Factor V Leiden mutation detection system is a device that allows the detection and genotyping of a single point mutation of the human Factor V gene, referred to as Factor V Leiden mutation, from DNA isolated from human whole peripheral blood. The system consists of different reagents and instruments which includes polymerase chain reaction (PCR) primers, hybridization matrices, thermal cyclers, imagers, and software packages. The detection system is an aid to diagnosis in the evaluation of patients with suspected thrombophilia.
Special Controls
*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.)
Predicate Devices
- Roche LightCycler® Factor II (Prothrombin) Test (K033612)
- Roche LightCycler® Factor V Leiden Test (K033607)
Related Devices
- K082118 — XPERT HEMOSIL FACTOR II & FACTOR V ASSAY · Cepheid · Sep 18, 2009
- K223046 — Xpert® FII & FV · Cepheid® · Feb 15, 2023
Submission Summary (Full Text)
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# 510(k) SUBSTANTIAL EQUIVALENCE DETERMINATION DECISION MEMORANDUM
A. 510(k) Number:
K172913
B. Purpose for Submission:
To obtain clearance for a new device
C. Measurand:
Factor II and Factor V
D. Type of Test:
Genotyping test
E. Applicant:
Roche Molecular Systems, Inc.
F. Proprietary and Established Names:
cobas® Factor II and Factor V Test
G. Regulatory Information:
1. Regulation section:
21 CFR 84.7280; Factor V Leiden DNA Mutation Detection Systems
2. Classification:
Class II
3. Product code:
NPR; Test, Factor II G20210A Mutations, Genomic DNA PCR, Factor V Leiden DNA Mutation Detection Systems
NPQ; Test, Factor V Leiden Mutations, Genomics DNA PCR, Factor V Leiden DNA Mutation Detection Systems
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4. Panel:
Hematology (81)
H. Intended Use:
1. Intended use(s):
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 cobas z 480 analyzer are used together for automated amplification and detection.
2. Indication(s) for use:
Same as Intended Use
3. Special conditions for use statement(s):
Prescription Use only
4. Special instrument requirements:
cobas z 480 analyzer
I. 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.
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 real-time PCR thermal cycler (cobas z 480 analyzer) and a control unit (cobas 4800 System CU).
J. Substantial Equivalence Information:
1. Predicate device name(s):
Roche Factor II (Prothrombin) G20210A Kit
Roche Factor V Leiden Kit
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2. Predicate 510(k) number(s):
K033612
K033607 (DEN030005)
3. Comparison with predicate:
| | Similarities | | |
| --- | --- | --- | --- |
| Item | Device: cobas Factor II and Factor V Test (K172913) | Predicate: Roche Factor II (Prothrombin) G20210A Kit K033612 | Predicate: Roche Factor V Leiden Kit 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 G1691A 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 except the predicate device was cleared on the LightCycler 1.2 analyzer. | Same except the predicate device was cleared on the LightCycler 1.2 analyzer. |
| Type of Test | Genotyping test | Same | Same |
| Target of Detection | Single nucleotide mutation | Same | Same |
| Indication for Use | Aid in the diagnosis of patients with suspected thrombophilia | Same | Same |
| Intended User | Healthcare professionals | Same | Same |
| Specimen Type | Purified DNA from | Same | Same |
| | cobas Z 480 analyzer | | |
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| | Similarities | | |
| --- | --- | --- | --- |
| Item | Device: cobas Factor II and Factor V Test (K172913) | Predicate: Roche Factor II (Prothrombin) G20210A Kit K033612 | Predicate: Roche Factor V Leiden Kit K033607 |
| | human blood samples | | |
| | Differences | | |
| Item | Device: cobas Factor II and Factor V Test (K172913) | Predicate: Roche Factor II (Prothrombin) G20210A Kit K033612 | Predicate: Roche Factor V Leiden Kit K033607 |
| 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 |
| 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 | <50 allele copies (0.01 ng input DNA/reaction) | Approximately 50 allele copies per reaction | Approximately 50 allele copies per reaction |
| Instrument | cobas z 480 | Roche LightCycler version 1.2 | Roche LightCycler version 1.2 |
| Controls | Endogenous internal control (each gene is internal control for other gene), plus external positive and negative controls required in each run | External positive and negative controls required in each run | External positive and negative controls required in each run |
| Reference method | Bi-directional Sanger sequencing | Sanger sequencing | Sanger sequencing |
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K. Standard/Guidance Document Referenced (if applicable):
CLSI EP7-A2, Interference Testing in Clinical Chemistry; Approved Guideline – 2nd Edition
L. Test Principle:
DNA is extracted offline from whole blood specimens collected in K₂EDTA tubes. The user-selected 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. The amplification reactions generate a Factor II specific amplicon and 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. Depending upon the test order, results for one or both mutations are reported for each DNA sample.
M. Performance Characteristics (if/when applicable):
1. Analytical performance:
a. Precision/Reproducibility:
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. 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%). The table below summarizes the study results by individual lots and combined lots.
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| Sample ID | Factor II Genotype | Factor V Genotype | Number of Tests per Lot | Number (%) Correct Genotype Results | | | |
| --- | --- | --- | --- | --- | --- | --- | --- |
| | | | | Lot 1 | Lot 2 | Lot 3 | Lot 4 |
| 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%) |
Clinical Reproducibility: A panel of nine specimens was tested at three sites, two operators per site, with one instrument per site and three lots of reagent lots with one lot per site. Each site had one operator which performed one run per day using one sample preparation method over five non-consecutive days. Reproducibility was assessed using a nine member panel as demonstrated in the table below: four $\mathrm{K}_2\mathrm{EDTA}$ whole blood samples, three contrived whole blood samples and two extracted genomic (gDNA) samples diluted to $0.2\mathrm{ng} / \mu \mathrm{L}$ . Below are tables which summarize the overall agreement results of the reproducibility study for Factor II and Factor V by overall genotype and testing site/sample preparation method.
Description of Specimens
| Panel Number | Genotype of Specimen | | Sample Type |
| --- | --- | --- | --- |
| 1 | Factor II Wild Type | Factor V Wild Type | Whole Blood |
| 2 | Factor II Wild Type | Factor V Wild Type | Whole Blood |
| 3 | Factor II Wild Type | Factor V Heterozygous | Whole Blood |
| 4 | Factor II Heterozygous | Factor V Wild Type | Whole Blood |
| 5 | Factor II Wild Type | Factor V Homozygous | Contrived Whole Blood |
| 6 | Factor II Homozygous Mutant | Factor V Wild Type | Contrived Whole Blood |
| 7 | Factor II Heterozygous | Factor V Heterozygous | Contrived Whole Blood |
| 8 | Factor II Wild Type | Factor V Heterozygous | Genomic DNA |
| 9 | Factor II Heterozygous | Factor V Wild Type | Genomic DNA |
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Factor II – Reproducibility Study Summary by Site
| Genotype by Sequencing^{a} | Correct Results/Number of Samples Tested | | | Incorrect Results | Invalid Results^{c} | Correct Results/Number of Valid Results (%) | 95% LCB^{d} |
| --- | --- | --- | --- | --- | --- | --- | --- |
| Factor II | Site1^{b}/Lot 1/Method A | Site 2^{b}/Lot 2/Method B | Site 3^{b}/Lot 3/Method C | | | | |
| WT^{e} | 100/100 | 100/100 | 100/100 | 0 | 0 | 300/300 (100%) | 98.78 |
| HET^{e} | 60/60 | 59/60 | 60/60 | 0 | 1 | 179/179 (100%) | 97.96 |
| MUT^{f} | 20/20 | 20/20 | 20/20 | 0 | 0 | 60/60 (100%) | 94.04 |
aWT: Wild Type; HET: Heterozygous; MUT: Homozygous Mutant
bEach site used a different DNA isolation method (A, B, C) and different reagent lot
cInvalid result was not retested
dTwo-sided 95% lower confidence bound (LCB) (exact method)
eAt each site, 20 results were from gDNA samples, and 20 were from contrived whole blood samples
fFrom contrived whole blood samples only
Factor V – Reproducibility Study Summary by Site
| Genotype by Sequencing^{a} | Correct Results/Number of Samples Tested | | | Incorrect Results | Invalid Results^{c} | Correct Results/Number of Valid Results (%) | 95% LCB^{d} |
| --- | --- | --- | --- | --- | --- | --- | --- |
| Factor V | Site1^{b}/Lot 1/Method A | Site 2^{b}/Lot 2/Method B | Site 3^{b}/Lot 3/Method C | | | | |
| WT^{e} | 100/100 | 100/100 | 100/100 | 0 | 0 | 300/300 (100%) | 98.78 |
| HET^{e} | 60/60 | 59/60 | 60/60 | 0 | 1 | 179/179 (100%) | 97.96 |
| MUT^{f} | 20/20 | 20/20 | 20/20 | 0 | 0 | 60/60 (100%) | 94.04 |
aWT: Wild Type; HET: Heterozygous; MUT: Homozygous Mutant
bEach site used a different DNA isolation method (A, B, C) and different reagent lot
cInvalid result was not retested
dTwo-sided 95% lower confidence bound (LCB) (exact method)
eAt each site, 20 results were from gDNA samples, and 20 were from contrived whole blood samples
fFrom contrived whole blood samples only
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Factor II – Reproducibility Study Summary by Operator
| Site | Operator | Genotype Panel^{1} | Total Samples Tested | Correct Calls | Missed Calls | No Calls^{2} | Percent Agreement % (Lower Bound of 95% CI^{3}) |
| --- | --- | --- | --- | --- | --- | --- | --- |
| Site 1 | Operator 1 | WT | 50 | 50 | 0 | 0 | 100.0 (92.89) |
| | | HET | 30 | 30 | 0 | 0 | 100.0 (88.43) |
| | | MUT | 10 | 10 | 0 | 0 | 100.0 (69.15) |
| | Operator 2 | WT | 50 | 50 | 0 | 0 | 100.0 (92.89) |
| | | HET | 30 | 30 | 0 | 0 | 100.0 (88.43) |
| | | MUT | 10 | 10 | 0 | 0 | 100.0 (69.15) |
| Site 2 | Operator 1 | WT | 50 | 50 | 0 | 0 | 100.0 (92.89) |
| | | HET | 30 | 30 | 0 | 0 | 100.0 (88.43) |
| | | MUT | 10 | 10 | 0 | 0 | 100.0 (69.15) |
| | Operator 2 | WT | 50 | 50 | 0 | 0 | 100.0 (92.89) |
| | | HET | 30 | 29 | 0 | 1 | 100.0 (88.06) |
| | | MUT | 10 | 10 | 0 | 0 | 100.0 (69.15) |
| Site 3 | Operator 1 | WT | 50 | 50 | 0 | 0 | 100.0 (92.89) |
| | | HET | 30 | 30 | 0 | 0 | 100.0 (88.43) |
| | | MUT | 10 | 10 | 0 | 0 | 100.0 (69.15) |
| | Operator 2 | WT | 50 | 50 | 0 | 0 | 100.0 (92.89) |
| | | HET | 30 | 30 | 0 | 0 | 100.0 (88.43) |
| | | MUT | 10 | 10 | 0 | 0 | 100.0 (69.15) |
¹WT: Wild Type; HET: Heterozygous; MUT: Homozygous Mutant
²Invalid results that were not retested
³95% CI = Two-sided 95% Exact Binomial Confidence Interval
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Factor V – Reproducibility Study Summary by Operator
| Site | Operator | Genotype Panel^{1} | Total Samples Tested | Correct Calls | Missed Calls | No Calls^{2} | Percent Agreement % (Lower Bound of 95% CI^{3}) |
| --- | --- | --- | --- | --- | --- | --- | --- |
| Site 1 | Operator 1 | WT | 50 | 50 | 0 | 0 | 100.0 (92.89) |
| | | HET | 30 | 30 | 0 | 0 | 100.0 (88.43) |
| | | MUT | 10 | 10 | 0 | 0 | 100.0 (69.15) |
| | Operator 2 | WT | 50 | 50 | 0 | 0 | 100.0 (92.89) |
| | | HET | 30 | 30 | 0 | 0 | 100.0 (88.43) |
| | | MUT | 10 | 10 | 0 | 0 | 100.0 (69.15) |
| Site 2 | Operator 1 | WT | 50 | 50 | 0 | 0 | 100.0 (92.89) |
| | | HET | 30 | 30 | 0 | 0 | 100.0 (88.43) |
| | | MUT | 10 | 10 | 0 | 0 | 100.0 (69.15) |
| | Operator 2 | WT | 50 | 50 | 0 | 0 | 100.0 (92.89) |
| | | HET | 30 | 29 | 0 | 1 | 100.0 (88.06) |
| | | MUT | 10 | 10 | 0 | 0 | 100.0 (69.15) |
| Site 3 | Operator 1 | WT | 50 | 50 | 0 | 0 | 100.0 (92.89) |
| | | HET | 30 | 30 | 0 | 0 | 100.0 (88.43) |
| | | MUT | 10 | 10 | 0 | 0 | 100.0 (69.15) |
| | Operator 2 | WT | 50 | 50 | 0 | 0 | 100.0 (92.89) |
| | | HET | 30 | 30 | 0 | 0 | 100.0 (88.43) |
| | | MUT | 10 | 10 | 0 | 0 | 100.0 (69.15) |
¹WT: Wild Type; HET: Heterozygous; MUT: Homozygous Mutant
²Invalid results that were not retested
³95% CI = Two-sided 95% Exact Binomial Confidence Interval
b. Linearity/assay reportable range:
Not applicable
c. Traceability, Stability, Expected values (controls, calibrators, or methods):
Whole Blood Stability Study: This study was conducted to determine the stability of K₂EDTA whole blood specimens for isolation of gDNA for testing with the cobas Factor II and Factor V test. The study also compared the performance of the cobas Factor II and Factor V test on DNA isolated from freshly drawn whole blood samples to the performance on DNA isolated from whole blood samples frozen and thawed up to three times. DNA was isolated from K₂EDTA whole blood was collected from six donors and stored at -20°C and ≤ -70°C. In addition, one K₂EDTA blood collection tube from each donor was placed at: 2–8°C, 25°C and 32°C. The cobas Factor II and Factor V test yielded the correct genotype results for all samples, storage conditions and time intervals tested, including up to three freeze/thaw cycles at -20°C and ≤ -70°C. Genomic DNA for
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testing with the cobas Factor II and Factor V test may be isolated from $\mathrm{K}_2\mathrm{EDTA}$ whole blood specimens after storage at $2 - 8^{\circ}\mathrm{C}$ for up to seven days, at $9 - 30^{\circ}\mathrm{C}$ for up to three days. The performance of the cobas Factor II and Factor V test on whole blood samples frozen and thawed up to three times was comparable to the performance on fresh, never frozen samples.
Extracted Genomic DNA Stability Study: The stability of gDNA isolated from whole blood for testing with the cobas Factor II and Factor V test was determined by isolating gDNA from six $\mathrm{K}_2\mathrm{EDTA}$ whole blood specimens using three commercially available genomic DNA isolation kits, and testing the gDNA samples with the cobas Factor II and Factor V test after storage at $2 - 8^{\circ}\mathrm{C}$, $-20^{\circ}\mathrm{C}$ and $\leq -70^{\circ}\mathrm{C}$. All tests with the cobas Factor II and Factor V test for all samples, DNA isolation methods, storage conditions and time intervals yielded Factor II and Factor V genotype results in agreement with DNA sequencing. Genomic DNA isolated from $\mathrm{K}_2\mathrm{EDTA}$ whole blood for testing with the cobas Factor II and Factor V test may be stored at $2 - 8^{\circ}\mathrm{C}$ for up to seven days or frozen at $-20^{\circ}\mathrm{C}$ to below $-70^{\circ}\mathrm{C}$ for at least nine weeks. Genomic DNA may be frozen and thawed up to three times.
Real-time and accelerated stability study: Three lots of the cobas Factor II and Factor V test kit were subjected to real-time stability testing at $2 - 8^{\circ}\mathrm{C}$, and accelerated stability testing at $25^{\circ}\mathrm{C}$ and $37^{\circ}\mathrm{C}$. The kit performance was assessed at each time point with the kit function release test and by testing genomic DNA extracted from four whole blood specimens at specified timepoints in the real-time study. The accelerated stability testing at $25^{\circ}\mathrm{C}$ and $37^{\circ}\mathrm{C}$ is complete. The real-time stability testing at $2 - 8^{\circ}\mathrm{C}$ is in progress, however real-time stability testing results through seven months from the date of manufacture is complete. The $25^{\circ}\mathrm{C}$ and $37^{\circ}\mathrm{C}$ accelerated stability studies on all three lots support a shelf life at $2 - 8^{\circ}\mathrm{C}$ of 18 months. The initial shelf life assignment for the cobas Factor II and Factor V test, based on the $25^{\circ}\mathrm{C}$ and $37^{\circ}\mathrm{C}$ accelerated stability studies is 15 months at $2 - 8^{\circ}\mathrm{C}$. The 15-month shelf-life assignment will be confirmed and extended with real-time stability study data.
Open Reagent Stability Study: The stability of the cobas Factor II and Factor V test, after the reagents and controls have been used once, was determined by removing half the volume from multiple vials of the kit reagents and controls, storing and recapped vials at $2 - 8^{\circ}\mathrm{C}$ and testing them after approximately 31, 61 and 91 days of storage. At each time interval, the previously opened reagent and control vials were testing with the kit functional release test and by testing gDNA isolated from four $\mathrm{K}_2\mathrm{EDTA}$ whole blood specimens. The study was conducted with one lot of the cobas Factor II and Factor V test. After opening and recapping, the reagents and controls were stable at $2 - 8^{\circ}\mathrm{C}$ for up to 90 days or the expiration date on the vials.
Activated Master Mix Stability Study: The stability of the activated Factor II and Factor V (F2F5) master mix (MMX) was determined by preparing activated F2F5 MMX, and testing the activated F2F5 MMX after storage at $32^{\circ}\mathrm{C}$ for 0, 1, 2, 2.5, 4 and 4.5 hours. Each time interval was tested with the kit functional test and by testing gDNA isolated from four $\mathrm{K}_2\mathrm{EDTA}$ whole blood samples. The study was conducted with three lots of the cobas Factor II and Factor V test. Activated F2F5 MMX may be stored at room temperature for up to 4 hours.
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d. Detection limit:
Analytical sensitivity - Lower Limit: 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/μL) and nine serial dilutions from 1.0 to 0.0001 ng/μL. 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.
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/μL and 100% at all higher concentrations (see table below). 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/μL, which is 10 times lower than the lowest recommended DNA input concentration. The table below summaries the study results.
Lower Limit Detection
| Concentration (ng/μL) | Number | cobas Factor II and Factor V Test | | |
| --- | --- | --- | --- | --- |
| | | 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%) |
*6 to 38 ng/μL
Analytical Sensitivity - Upper Limit: To evaluate higher concentrations of DNA input for 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 DNA from cell lines were added to yield total DNA concentrations of 300 ng/μL, 150 ng/μL and 75 ng/μL. Genomic DNA from Factor II heterozygous, Factor V heterozygous and Factor V homozygous mutant cell lines were added to genomic DNA from whole blood samples of the same Factor II and
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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 $300\mathrm{ng} / \mu \mathrm{L}$ , $150\mathrm{ng} / \mu \mathrm{L}$ and $75\mathrm{ng} / \mu \mathrm{L}$ were tested with 24 replicates with each of two kit lots, for 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 sample. All samples at $300\mathrm{ng} / \mu \mathrm{L}$ , $150\mathrm{ng} / \mu \mathrm{L}$ and $75\mathrm{ng} / \mu \mathrm{L}$ yielded the correct Factor II and Factor V genotype results in all tests (see table below). The LDWB samples without added cell line DNA yielded Invalid results, as expected; the other genomic DNA samples from whole blood yielded correct Factor II and Factor V results. The highest recommended DNA input concentration is $150\mathrm{ng} / \mu \mathrm{L}$ . The table below summarizes the study results.
Upper Limit Detection
| | | 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 whole blood samples (6 to 38 ng/μL) | 108 | 108 (100%) | 0 (0%) | 0 (0%) |
| Leukocyte depleted whole blood sample* | 36 | 36 (100%) | 0 (0%) | 0 (0%) |
*Eluates from LDWB sample yielded "Invalid" results due to the absence of leukocytes. These expected results are considered "correct" for the purpose of this study.
# e. Analytical specificity:
Substances: Six $\mathrm{K}_2\mathrm{EDTA}$ whole blood samples representing Factor II and Factor V wild type and heterozygous genotypes were tested for potential interference by the following substances: triglycerides, bilirubin, cholesterol and hemoglobin, $\mathrm{K}_2\mathrm{EDTA}$ , heparin, warfarin (Coumadin), rivaroxaban (Xarelto) and dabigatran etexilate (Pradaxa). Triglycerides (37 mM), bilirubin (conjugated and unconjugated, 342 $\mu$ M), 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. Hemoglobin did not interfere with the cobas Factor II and Factor V Test when added to whole blood at a concentration of 20 g/L. The anticoagulant $\mathrm{K}_2\mathrm{EDTA}$ did not interfere when it was tested at 5.7 mg/mL, which is approximately three times the concentration of $\mathrm{K}_2\mathrm{EDTA}$ in whole blood when the blood collection tube was filled to capacity. Heparin (3000 U/L), warfarin (32.5 $\mu$ mol/L), rivaroxaban (1.5 $\mu$ mol/L), and dabigatran etexilate (0.6 $\mu$ mol/L) did not interfere with the cobas Factor II and Factor V Test. Correct genotype results were obtained for all replicates of all the gDNA samples.
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For potential interference with extraction buffer and ethanol, these substances were added to gDNA isolated from K₂EDTA whole blood. 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.
Mutations: 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) in the probe binding regions of the cobas Factor II and Factor V Test 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.
f. Assay cut-off:
Not applicable
2. Comparison studies:
a. Method comparison with predicate device:
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 the genotypes as demonstrated in the table below.
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Factor II and Factor V genotypes included in the method comparison study
| Factor II (F2) | Factor V (F5) | Specimen Type |
| --- | --- | --- |
| Wild Type (WT F2) | Wild Type (WT F5) | K_{2}EDTA whole blood |
| Wild Type (WT F2) | Heterozygous (HET F5) | K_{2}EDTA whole blood |
| Heterozygous (HET F2) | Wild Type (WT F5) | K_{2}EDTA whole blood |
| Heterozygous (HET F2) | Heterozygous (HET F5) | K_{2}EDTA whole blood |
| Wild Type (WT F2) | Homozygous (MUT F5) | K_{2}EDTA whole blood |
| Homozygous (MUT F2) | Wild Type (WT F5) | K_{2}EDTA whole blood and gDNA |
One external site conducted testing with the cobas Factor II and Factor V test. One commercial laboratory performed bi-directional Sanger sequencing. The 300 samples were randomized and tested by two operators. All 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 the cobas test and no repeat tests were performed. A test result was classified as correct for Factor II or Factor V if both the cobas test and Sanger sequencing detected the same genotype.
The table below presents agreement between the cobas test and Sanger sequencing for Factor II results. The Overall Percentage Agreement (OPA) between the two tests was 100% with a two-sided 95% lower confidence bound (exact method) of 98.78%. Both Positive Percent Agreement (PPA) and Negative Percent Agreement (NPA) were 100% with lower confidence bounds of 97.59% for PPA and 97.55% for NPA. The percent agreement of correct results for heterozygous and homozygous mutant genotypes were both 100%.
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 Calls^{1} | No calls or Invalid Results^{2} | Missed or Incorrect Calls^{3} | Percent Agreement | 95% LCB^{4} |
| --- | --- | --- | --- | --- | --- | --- |
| 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% |
| Mutant^{5} | 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 Sanger 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
For Factor V, the table below 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 Sanger Sequencing as a Reference for the Identification of Factor V Genotype
| Genotype by Sequencing | Total Samples Tested | Correct Calls^{1} | No calls or Invalid Results^{2} | Missed or Incorrect Calls^{3} | Percent Agreement | 95% LCB^{4} |
| --- | --- | --- | --- | --- | --- | --- |
| 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% |
| Mutant^{5} | 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 Sanger 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
The table below presents method comparison study results for the combined Factor II and Factor V results.
| Bi-Directional Sanger Sequencing Result | cobas Factor II and Factor V Test Result | | | | | | Total |
| --- | --- | --- | --- | --- | --- | --- | --- |
| | HET F2/HET F5 | HET F2/WT F5 | MUT F2/WT F5 | WT F2/HET F5 | WT F2/MUT F5 | WT F2/WT F5 | |
| 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 | 25 | 105 | 21 | 105 | 23 | 21 | 300 |
b. Matrix comparison:
Matched frozen and fresh K₂EDTA whole blood samples were compared and resulted in 100% correct genotype results.
3. Clinical studies:
a. Clinical Sensitivity:
Not applicable
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b. Clinical specificity:
Not applicable
c. Other clinical supportive data (when a. and b. are not applicable):
DNA Extraction Method Study: Genomic DNA was isolated from 15 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 gDNA sample was tested in triplicate with the cobas Factor II and Factor V Test. 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 gDNA sample was excluded from the results as it was rust colored and yielded invalid results in all three tests. The table below summarizes the results from the DNA extraction method study.
| DNA Isolation Method | Total number of | | Number (%) of | | | |
| --- | --- | --- | --- | --- | --- | --- |
| | DNA Isolations | Tests | Correct Results | | Incorrect Results | Invalid Results |
| 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%) | 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%) |
aOne of 90 DNA samples isolated with method A was rust-colored and generated three invalid results. DNA samples with any appearance other than clear and colorless should not be tested, as they may yield invalid or incorrect results.
b95% two-sided, confidence interval
cThe triplicate results from one sample isolated with method B were inconsistent: one correct, one incorrect, one invalid. The sample eluate was re-tested in triplicate and all results were correct upon testing.
4. Clinical cut-off:
Not applicable
5. Expected values/Reference range:
Not applicable
N. Proposed Labeling:
The labeling is sufficient and it satisfies the requirements of 21 CFR Parts 801 and 809, as applicable.
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O. Conclusion:
The submitted information in this premarket notification is complete and supports a substantial equivalence decision.
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