esensor thrombophilia risk test package insert · the esensor® thrombophilia risk test is an in...

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eSensor® Thrombophilia Risk Test Package Insert

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Contents Intended Use ..............................................................................................................................................................3 Background .................................................................................................................................................................3 Principle of eSensor

® Technology ..............................................................................................................................3

Warnings and Precautions .........................................................................................................................................4 Safety .....................................................................................................................................................................4 General ...................................................................................................................................................................4

Components and Reagents provided .........................................................................................................................5 Required/Recommended Materials and Equipment Not Provided .............................................................................5 Storage, Stability, and Handling Requirements ..........................................................................................................5 eSensor

® Thrombophilia Risk Test Procedure ...........................................................................................................6

Procedural Notes ........................................................................................................................................................6 General ...................................................................................................................................................................6 PCR Amplification ..................................................................................................................................................6 Exonuclease Digestion ...........................................................................................................................................6 Cartridge Assay ......................................................................................................................................................7

Detailed Procedure: Assay Set-up ............................................................................................................................8 PCR Set-Up ............................................................................................................................................................8 Single-Stranded DNA Generation/Exonuclease Digestion ....................................................................................8 Reagent Preparation and Final Specimen Processing ..........................................................................................9

Assay Calling Parameters ..........................................................................................................................................9 Test Results ............................................................................................................................................................. 10 Results Section ........................................................................................................................................................ 10 Quality Control ......................................................................................................................................................... 11 Controls ................................................................................................................................................................... 11

Positive Control ................................................................................................................................................... 11 Negative Control .................................................................................................................................................. 11 Hardware and Software Controls ........................................................................................................................ 11 PCR Blank ........................................................................................................................................................... 11

Instrument Maintenance and Calibration ................................................................................................................. 12 Expected Values / Reference Range ...................................................................................................................... 12 Performance Characteristics ................................................................................................................................... 12

Polymorphisms Detected .................................................................................................................................... 12 Limit of Detection ................................................................................................................................................. 12 Method Comparison ............................................................................................................................................ 14 Reproducibility ..................................................................................................................................................... 15 Genomic DNA Extraction Reproducibility ............................................................................................................ 15 Lot to Lot Reproducibility ..................................................................................................................................... 16 Interfering Substances ........................................................................................................................................ 16 Interfering Mutations ............................................................................................................................................ 16

Troubleshooting Table ............................................................................................................................................. 16 GenMark Technical Support .................................................................................................................................... 18 References .............................................................................................................................................................. 18 Thrombophilia Risk Test Worksheet ........................................................................................................................ 20

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INTENDED USE

The eSensor® Thrombophilia Risk Test is an in vitro diagnostic for the detection and genotyping of Factor II

(Prothrombin) G20210A, Factor V (Factor V Leiden) G1691A and MTHFR (human 5, 10 methylenetetrahydrofolate reductase gene) C677T and A1298C mutations in patients with suspected Thrombophilia from isolated genomic DNA obtained from whole blood samples. The test is intended to be used on the eSensor

® XT-8™ System.

BACKGROUND

Thrombosis is one of the most common types of blood coagulation disorders, affecting 1 in 1000 individuals with a fatality rate of 1-2%. Thrombosis can be categorized as inherited or acquired. Inherited Thrombosis is associated with congenital predisposing risk factors such as Factor II (Prothrombin, FII)

1 and Factor V (Leiden,

FV)2 proteins involved in the blood coagulation enzyme activity cascade and the Methylenetetrahydrofolate

Reductase (MTHFR) that converts homocysteine to methionine as part of the pathway that coverts 5,10-methylenetetrahydrofolate to 5-methyltetrahydrofolate

3. The FII and FV mutations are present in ~2% and 5% of

individuals with N. European ancestry respectively, but at much lower levels in other populations4, 5

. The MTHFR C677T and A1298C mutations are present in ~40-50% of N.European ancestry, but the frequency varies considerably in other ethnic groups

5. Inherited Thrombosis is characterized by increased risk of deep vein

thrombosis, ectopic pregnancy, pulmonary embolism, myocardial infarction, cardiovascular disease and other complications related to abnormal blood coagulation. In 2001, The American College of Medical Genetics (ACMG) recommended that FV DNA testing should be performed in patients with any type of venous thrombosis (hepatic, mesenteric, cerebral and recurrent), with a strong family history of thrombotic disease, pregnant women with venous thrombosis, women with history of pregnancy difficulties such as miscarriages, placental abruption, intrauterine fetal growth retardation or still birth, or women taking oral contraceptives and female smokers (under age 50) with myocardial infarction. Additional recommendations also include molecular genetic testing for the other most common risk factors such as FII and MTHFR for patients tested positive for FV

6.

The eSensor Thrombophilia Risk Test detects the 4 mutations summarized in Table 1.

Table 1: Mutations Detected by the eSensor® Thrombophilia Risk Test

Gene Panel Mutation

FV Leiden 1691G>A

FII Prothrombin 20210G>A

MTHFR 677C>T

1298A>C

PRINCIPLE OF ESENSOR® TECHNOLOGY

The eSensor Technology uses a solid-phase electrochemical method for determining the genotyping status of a defined panel of mutations. Purified genomic DNA is isolated from the patient specimen according to defined laboratory procedures and is amplified in a multiplex PCR reaction using specific primers and enzyme. The amplified DNA is converted to single stranded DNA via exonuclease digestion and is then combined with a signal buffer containing a pair of ferrocene-labeled, allele-specific oligonucleotide signal probes for each polymorphism. The mixture of amplified sample and signal buffer is loaded onto the eSensor

® cartridge, which contains single-

stranded oligonucleotide capture probes bound to gold-plated electrodes. The cartridge is inserted into the XT-8™ instrument where the single-stranded targets first hybridize to the matched signal probes and then hybridize to the complementary sequences of the capture probes. The final target/signal probe and capture probe structure

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is shown in figure 1. The genotype of each polymorphism is determined by voltammetry, which generates specific electrical signals from the ferrocene-labeled, allele-specific signal probes.

Figure 1: Hybridization complex formed at the surface of each electrode. Different, target-specific capture probes are covalently attached to the gold electrodes in the eSensor

® microarray. The amplified target DNA hybridizes to

the capture probe and to a complementary ferrocene-labeled signal probe. The ferrocene label is detected at the electrode surface using voltammetry. WARNINGS AND PRECAUTIONS

Safety

CAUTION! REAGENTS CAUSE IRRITATION TO SKIN, EYES, AND RESPIRATORY TRACT. HARMFUL IF SWALLOWED OR INHALED! OXIDIZING LIQUIDS!

Follow routine laboratory safety procedures for handling of reagents e.g. wear appropriate protective clothing, eye protection, and do not pipette by mouth.

Wash hands thoroughly with soap and water after handling reagents.

Launder contaminated clothing prior to re-use.

Reagents are not considered a fire hazard. Liquid dries to leave a residue that may support a fire when combined with combustible materials and reducing agents. Reagents will emit hazardous fumes under fire conditions.

EXTINGUISHING MEDIA: Water, CO2, Dry Chemical, Foam

Not considered an explosion hazard.

Handle all specimens in accordance with Universal Precautions. Waste must be classified and disposed of in accordance with all Federal, State, and Local environmental regulations.

General

Do not substitute eSensor® Thrombophilia Risk Test reagents with alternate reagents.

Do not reuse cartridges.

Do not insert a wet cartridge into the XT-8 Instrument.

Do not combine different reagents lots.

Follow the procedure as described in this package insert.

After test completion, remove cartridges from the XT-8 Instrument. Do not leave cartridges in the Instrument for longer than 7 days.

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COMPONENTS AND REAGENTS PROVIDED

Table 2: 48 Test Kit

Box Component Packaging & Quantity Storage

eSensor®

Thrombophilia Risk Test Cartridges

eSensor® Thrombophilia Risk Test

Cartridges 6 foil bags with 8 cartridges in each cartridge bag 10-25 °C

eSensor®

Thrombophilia Risk Test Amplification

Reagents

TRT* PCR Mix 2 vials with 1100 µL -20 °C (in a designated pre-PCR location) Taq Polymerase 1 vial with 60 µL

eSensor®

Thrombophilia Risk Test Detection

Reagents

Exonuclease 2 vials with 145 µL

-20 °C (in a designated post-PCR location)

TRT* Signal Buffer 2 vials with 2500 µL

Buffer-1 2 vials with 350 µL

Buffer-2 2 vials with 700 µL

*Thrombophilia Risk Test-(TRT)

REQUIRED/RECOMMENDED MATERIALS AND EQUIPMENT NOT PROVIDED

Water, Molecular Biology grade, nuclease free

Vortex Mixer

Disposable Gloves

Disposable thin-wall PCR tubes or strips of 8 tubes, nuclease-free

Microfuge Tubes

Microfuge Tube Racks

Dry Heat block

Cold block or ice

Pipettes

Pipette Tips, Aerosol resistant

Thermal cycler

Micro-Centrifuge

Biosafety Cabinet or Dead Air Hood STORAGE, STABILITY, AND HANDLING REQUIREMENTS

eSensor Thrombophilia Risk Test Kits should be stored with a unidirectional workflow in mind (i.e. maintaining physical separation of the Amplification Reagents from the Detection Reagents and Cartridge Packs).

The unopened kits should be stored as recommended in the table above.

Freeze reagents immediately after use.

Store frozen reagents in a non-frost-free freezer.

Reagents may be frozen and thawed up to 5 times.

Cartridges must be used within 30 days of opening the pouch.

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ESENSOR® THROMBOPHILIA RISK TEST PROCEDURE

PROCEDURAL NOTES

General

Throughout the procedure section of this Package Insert, there are references to specific actions. The details of these actions are defined below:

Thaw: Frozen reagents and samples are thawed at 37°C for 10-15 minutes. It is acceptable to thaw at room temperature, although this may take longer. NOTE: IMPORTANT! Do not thaw the Taq Polymerase or exonuclease enzyme; place on cold block immediately and return to -20 °C promptly after use. Vortex: Thoroughly mix volumes together using a vortexer set at maximum speed for 3-5 seconds. Centrifuge: Consolidate liquids by centrifuging briefly (5-10 seconds) in a centrifuge set at > 6000 rpm.

Do not pool reagents from different lots.

Immediately after use, close all vials to prevent spillage or contamination.

Store all vials in an upright position.

Do not use a kit after its expiration date.

This test is for use with human DNA extracted from fresh whole blood collected using EDTA as anticoagulant. Frozen whole blood is not recommended for use with this product.

Genomic DNA should be extracted from whole blood within 4 weeks of collection.

Laboratory workflow must be in accordance with CLIA laboratory standards for high complexity tests. The flow of testing should proceed in a unidirectional manner (i.e. sample preparation, PCR set-up and PCR amplification / genotyping assay must be physically separated).

All instruments and pipettes should be calibrated.

Specific instructions provided below must be carefully followed. PCR Amplification

The PCR amplification uses genomic DNA that should be extracted by a validated method from whole human blood collected using EDTA as an anticoagulant. The concentration of genomic DNA should be at least 2 ng/µL (10 ng DNA/PCR) and no more than 100 ng/µL (500 ng DNA/PCR), and is recommended to have an A260:A280 ratio between 1.5 and 2.2.

Dedicated pipettes, tips and equipment are recommended for all pre-amplification activities.

Precautions must be taken to avoid amplicon contamination throughout the procedure: samples, consumables, and lab areas should be protected from aerosol or direct contamination with amplicon.

Taq Polymerase should be handled with care to minimize the risk of inactivation; it should be kept in a cold block during use and then promptly returned to -20 ºC storage conditions. NOTE: IMPORTANT! Do not vortex the Taq Polymerase. Centrifuge briefly prior to use.

Following set up of the PCR master mix, a large volume of viscous Taq polymerase solution may settle at the bottom of the tube. To ensure complete mixing of this solution, it is IMPORTANT that the tube be inverted at least three times before vortexing.

Exonuclease Digestion

This post-PCR step should be performed only in laboratory areas designated for PCR products.

Dedicated pipettes, tips and equipment are recommended for all post-amplification activities.

Precautions must be taken to avoid amplicon contamination throughout the procedure; samples, consumables, and lab areas should be protected from aerosol or direct contamination with amplicon. Decontaminate laboratory areas and affected equipment before each assay run.

Exonuclease should be handled with care to minimize the risk of inactivation; it should be kept in a cold block during use and then promptly returned to -20 °C storage conditions. NOTE: IMPORTANT! Do not thaw exonuclease enzyme.

Visually verifying the addition of exonuclease directly into the amplicon solution is highly recommended.

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To ensure complete mixing of the enzyme with the amplicon solution, it is IMPORTANT that each tube/plate be vortexed for at least 3 seconds prior to centrifuging.

Cartridge Assay

This post-PCR step should be performed only in laboratory areas designated for PCR products.

Avoid contact of Signal Buffer on the skin, eyes, or mucous membranes. If contact does occur, refer to the MSDS. If left untreated, burns may result.

Add Buffer-1 to the Signal Buffer BEFORE adding Buffer-2.

A cloudy white precipitate may form following addition of Buffer-2. If extended vortexing does not dissolve the precipitate, heat solution to 37°C for 5 minutes before vortexing again.

Once amplicon is added to the hybridization solution, it should be loaded into the cartridge and the scanning protocol started within 8 hours.

Cartridges must be used within 30 days of opening the pouch. If stored, cartridges should be kept in their original foil pouch in a dry place at room temperature with the zip-lock closure sealed.

eSensor®

Thrombophilia Risk Test Workflow

Figure 2: Overall Schematic of the eSensor Thrombophilia Risk Test procedure. The test consists of 3 steps for each sample: 1) multiplex PCR to generate amplicon, 2) exonuclease digestion to yield single-stranded target amplicon, and 3) hybridization and detection. The volumes indicated are per sample/reaction.

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DETAILED PROCEDURE: ASSAY SET-UP

PCR Set-Up 1. Decontaminate the work area used for setting up the PCR.

2. Thaw, vortex, and centrifuge all frozen Thrombophilia Risk Test Amplification Reagents, except the Taq Polymerase.

3. Use the “PCR Master Mix Set-Up” table from the Thrombophilia Risk Test Worksheet to calculate the volumes of reagents required to formulate the PCR master mix. The PCR Blank (DNA contamination monitor) reaction should be included in the number (N) of reactions. The calculations are designed to make 10% excess to ensure that there will be enough solution for aliquot dispensing.

4. Label PCR reaction tubes/plate with sample IDs.

5. Remove Taq Polymerase from the freezer and centrifuge. NOTE: IMPORTANT! This tube should not be vortexed, and should be stored in a cold block while preparing master mix, and returned to -20°C immediately after use.

6. Combine all reagents in a tube of sufficient volume. IMPORTANT! Vortex and centrifuge the PCR Master Mix tube.

7. Dispense 30 µL of PCR Mix into the labeled PCR tubes/plate.

8. If extracted samples are frozen then thaw, vortex, and centrifuge, the samples.

9. Change gloves before adding genomic DNA to PCR reaction tubes.

10. Add 5 L of 2-100 ng/µL genomic DNA sample to each reaction tube containing master mix. For the PCR

Blank reaction, add 5 L water.

11. Vortex and centrifuge all tubes/plates.

12. Place PCR tubes/plate in a calibrated thermal cycler and amplify according to the protocol outlined below. Use the heated lid option if available. If a saved protocol is being used, confirm all settings before use.

Thermal Cycling Protocol – PCR

Step Temperature Duration

Initial PCR Activation Step 95 °C 4 minutes

3-Step Cycling (35 Cycles)

Denaturation 95 °C 25 seconds

Annealing 60 °C 30 seconds

Extension 72 °C 25 seconds

Hold 4 °C Until use in next step

13. After completion of PCR, remove the reaction tubes from the thermal cycler. Proceed immediately with

exonuclease digestion, or store at 4 °C or -20 °C for up to 7 days. Single-Stranded DNA Generation/Exonuclease Digestion

1. If the PCR products are frozen then thaw, vortex, and centrifuge.

2. Remove the exonuclease from the freezer, centrifuge, and place in a cold block. NOTE: IMPORTANT! Do not vortex the exonuclease enzyme; place on cold block immediately and return to -20 °C promptly after use.

3. Add 5 µL of exonuclease to each PCR reaction tube. After dispensing, visually confirm liquid has been expelled from the pipette tip. NOTE: IMPORTANT! Load the exonuclease enzyme directly into each sample.

4. Vortex and centrifuge each tube/plate. NOTE: IMPORTANT! Thorough vortexing of the exonuclease enzyme is necessary to generate single-stranded DNA.

5. Place PCR tubes/plate in a thermal cycler and incubate according to the protocol outlined below. Use the heated lid option if available. If a saved protocol is being used, confirm all settings before use.

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Thermal Cycling Protocol – Exonuclease Digestion


Digestion 37 °C 20 minutes

Inactivation 95 °C 2 minutes


6. After completion of incubation, remove the tubes/plate from the thermal cycler. Proceed immediately with

the next step, or store at -20 °C for up to 7 days.

Reagent Preparation and Final Specimen Processing 1. Thaw and vortex Signal Buffer, Buffer-1 and Buffer-2.

2. Use the “Hybridization Solution Set-Up” table from the Thrombophilia Risk Test Worksheet to calculate the required reagent volumes. The calculations are designed to make 20% excess to ensure that there will be enough solution for aliquot dispensing.

3. Label a tube of sufficient volume as “Hyb”.

4. Combine the appropriate volumes of each reagent into the Hyb tube. It is important that Buffer-1 is added to the Signal Buffer prior to Buffer-2. A cloudy white precipitate may form after the addition of Buffer-2, but the solution will clear with vortexing in the next step.

5. Vortex the tube at maximum speed for 3-5 seconds, or until the precipitate clears. If vortexing does not dissolve the precipitate, heat the solution to 37°C for 5 minutes before vortexing again. Centrifuge or lightly tap the tube on a bench to minimize the amount of liquid on the sides of tube and inside the cap.

6. If the exonuclease-digested samples are frozen, thaw, vortex and centrifuge before proceeding.

7. Transfer 100 µL of “Hyb” solution to each sample tube/well, changing pipette tips between samples to avoid cross contamination.

8. Label cartridges with the sample IDs and place in cartridge trays.

9. Pipette 125 µL of Hyb-sample mix into the appropriately labeled cartridge.

10. Close each cartridge by firmly pressing the attached cap until the top of the cap is level with the top of the loading reservoir to ensure proper sealing.

11. Enter the sample ID for each cartridge into the XT-8™ Software. Sample ID can be entered by barcode, worklist or manually (see XT-8™ User Manual for more instructions).

12. Scan the Reagent Barcode located on the Thrombophilia Risk Test Reagents box. This barcode will apply to all cartridges inserted into the instrument prior to touching the start button.

13. Insert cartridges logo side up into the appropriate slots of the XT-8 Instrument.

14. Slide the lever half-way to the left to engage the cartridge. The LED for this slot will change from orange to yellow, indicating the test is ready to start.

15. Start the hybridization and scanning protocol by clicking the “Start” button.

ASSAY CALLING PARAMETERS

In order to minimize the likelihood of an incorrect result while also minimizing the frequency of “no-call” results that require repeat testing, the eSensor Thrombophilia Risk Test uses the principle of redundant electrodes. Two parameters of the data generated from each electrode are evaluated by the Application Software when making a genotyping call: Signal Strength: The combined major allele and minor allele current (signal) generated for each electrode is evaluated against a pre-established signal threshold. This threshold ensures sufficient signal has been generated

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to discriminate from background levels. If the signal is above this threshold, the second parameter of genotyping score is evaluated. Genotyping Score: The genotyping score, which is derived from the ratio of the different ferrocene label signals for each electrode passing the signal strength threshold parameter, is evaluated and compared to four boundaries. These boundaries are unique for each polymorphism and were determined empirically using test performance data. The boundaries define zones for classification of scores for homozygous major allele, homozygous minor allele and heterozygote genotypes. There are two ‘indeterminate’ zones; one between the homozygous major allele and heterozygote boundaries, and a second between the heterozygote and homozygous minor allele boundaries. If the score from an electrode falls in this zone, it cannot be classified as a specific genotype, and is considered a no-call due to ‘indeterminate score’. TEST RESULTS

Results are provided in a printable format, may be viewed electronically, or may be exported for additional analysis. The user can select up to 5 different types of reports for the following configurations: FV Report, FII Report, FII-FV Report, MTHFR Report or Thrombophilia Risk Test Report, which contains the full mutation panel. See eSensor

® XT-8 User Manual for details.

RESULTS SECTION

Valid results: If the assay controls pass and a valid genotyping call can be made from the signals generated from the mutations in the assay, the appropriate FII, FV and MTHFR allele genotypes will be given in the Results Section of the report, along with a brief description of the genotype status. The genotype status of each of the mutations in the eSensor Thrombophilia Risk Test and the results of the Positive and Negative controls will be listed on separate lines in the Results Section of the report. Qualitative results can have a specific genotype call for the mutations as ‘Homozygous’, ‘Heterozygous’, or ‘Mutation Absent’. A Genotyping Metric is reported for each valid polymorphism. The Genotyping Metric is a value between 0.9 and 1.0 and may be used to monitor and trend performance of each test over time. As the Metric moves towards 0.9 there is a higher likelihood of no-call results. If a trend toward lower values of the Genotyping Metric is observed for a polymorphism, and is combined with occurrences of low signal, error, indeterminate or contradictory scores for additional polymorphisms, contact GenMark Technical Support for assistance. Invalid results: Genotyping status and Genotyping Metric value for each mutation will only be reported if the assay controls and all panel mutations are valid within each allele. If either the on-board positive or negative control does not pass, a result of “Invalid Control” will be reported in the Summary section and a result of Fail will be reported for the control(s) that failed. No genotype results will be reported when either the positive or negative on-board controls fails. If both of the on-board assay controls pass, but any of the polymorphisms fail to meet the signal strength or genotyping score criteria as defined in the Assay Calling Parameters section above, no genotype results for any polymorphism will be reported for that sample. The test report will indicate a result of “Invalid Test” in the Summary Section and one of the following messages will be displayed in the Results Section for the individual polymorphism(s) that failed to indicate the reason for failure:

• ‘Low Signal’ occurs when insufficient signal has been generated to make a reliable call, i.e. when the signal is below threshold. • ‘Indeterminate Score’ occurs when the score lies in the indeterminate region between the genotyping boundaries. • ‘Contradictory Score’ occurs when different valid genotyping calls are made for that polymorphism.

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• ‘Error’ occurs when a system error prevents a result from being generated. See Troubleshooting Section for additional recommended actions.

QUALITY CONTROL

Results will not be reported unless all on-board positive and negative controls pass and all mutations give a valid result. Negative Controls: It is recommended that a PCR Blank reaction be included with each run of the eSensor Thrombophilia Risk Test, as described in the Package Insert under “PCR Blank”. Positive Controls: Controls for FII, FV and MTHFR are available commercially. All quality control testing should be performed in accordance with Federal, State and Local regulations and guidelines. CONTROLS

Each test contains on-board positive and negative controls to assure proper functioning of the system: Failure of either control will be indicated as “Invalid Control(s)” in the test results section of the report. Test results will not be reported for any sample for which a positive or negative control failure occurs. Positive Control Each cartridge contains a capture probe that is complementary to a synthetic target DNA present in the hybridization mixture. The target also contains ferrocene labels and thus generates an appropriate signal in the assay. The positive control is designed to detect a systematic failure of the hybridization and/or detection processes. A lack of signal for the positive control indicates a genotyping assay failure. If a correct signal is observed for the positive control, but one or more genotyping assays are invalid due to low signals, then a failure of DNA isolation or PCR amplification or Exonuclease digestion is indicated. Negative Control A negative control is present on each cartridge, consisting of a capture probe that does not hybridize to any sequence within the target DNA or signal probes. Signals on the negative control indicate an assay system failure. Hardware and Software Controls The system contains additional controls in the hardware and software to enable proper performance. See the XT-8™ User Manual for details. PCR BLANK

It is routine industry practice to perform a no template reaction (PCR Blank) as part of a PCR run to determine if amplicon contamination from prior reactions has been introduced during reaction set-up. This PCR Blank reaction is then processed with the other samples in the run, and a DNA Contamination Monitor Report is reviewed for this sample. The severity of contamination is determined by the System, and reported as a Metric with a value between 0.0 and 1.0. Results of the PCR Blank are found on the DCM report which provides a summary result of Pass/Fail followed by a value for the Metric (between 0.0 and 1.0) in parentheses. The description indicates what action, if any, is required based on the results of the PCR Blank test.

A Metric value of 0.0 indicates no DNA contamination was present during PCR set-up, and no further action is recommended.

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A Metric value between 0.1 and 0.8 indicates that a minor amount of DNA contamination was present during PCR set-up. The value of the Metric should be monitored for any trend of increasing value, and decontamination of the PCR set up area is recommended before the Metric reaches a value of 0.9.

A Metric value of 0.9 or 1.0 indicates a failed DCM test, and decontamination of the PCR set up area and a repeat of the entire run is indicated.

The Details section of the DCM report describes the results of the Negative and Positive Control tests. A failure of either of these tests will prevent interpretation of the DCM test, and a repeat of the run is indicated. INSTRUMENT MAINTENANCE AND CALIBRATION

No routine calibration or user maintenance is required. Please refer to the eSensor XT-8 User Manual for system diagnostic testing recommendations. Annual preventive maintenance is recommended for all XT-8 systems. Please contact GenMark Technical Support for information on annual preventive maintenance or to schedule preventive maintenance. EXPECTED VALUES / REFERENCE RANGE

The mutations being genotyped in the eSensor Thrombophilia Risk Test are present at the following population frequencies in the general population:

Panel Mutation Population frequency

FV 5%

FII 2%

MTHFR-C677T 40-50% MTHFR-A1298C

PERFORMANCE CHARACTERISTICS

The results obtained during internal and clinical studies of product performance support the following claims. A maximum of two repeat tests were allowed for no-call or invalid results. The no-call rate observed was 1.1%. POLYMORPHISMS DETECTED

The eSensor® Thrombophilia Risk Test detects and genotypes the panel of 4 mutations as listed below:

Gene Panel Mutation

FV Leiden 1691G>A

FII Prothrombin 20210G>A

MTHFR 677C>T

1298A>C

LIMIT OF DETECTION

Two (2) genomic DNA samples with different genotypes were serially diluted to 500, 100, 10, 1.0 and 0.1 ng per sample input and tested 20 times each with the eSensor

® Thrombophilia Risk Test, as shown in the following

tables. All replicates at ≥1.0 ng gave 100% final correct results. Thus, the limit of detection for the eSensor®

Thrombophilia Risk Test was determined to be 1.0 ng of gDNA, with 10 to 500ng gDNA as the recommended input for the test.

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Total gDNA input (ng)

FV Genotype

Replicates Tested

First pass correct calls

First pass Final Final correct calls

% Agreement with Sequencing No

Calls Mis-Calls

No calls

Mis-Calls

0.1

HET (gDNA 1)

20 0 20 0 20 0 0 0

1 20 2 18 0 1 0 19 95

10 20 20 0 0 0 0 20 100

100 20 20 0 0 0 0 20 100

500 20 20 0 0 0 0 20 100

0.1

WT (gDNA 2)

20 4 16 0 16 0 4 20

1 20 20 0 0 0 0 20 100

10 20 20 0 0 0 0 20 100

100 20 19 1 0 0 0 20 100

500 20 18 2 0 0 0 20 100


FII Genotype Replicates Tested




Calls Mis-Calls

No calls

Mis-Calls

0.1

WT (gDNA 1)

20 19 1 0 1 0 19 95

1 20 20 0 0 0 0 20 100

10 20 20 0 0 0 0 20 100

100 20 20 0 0 0 0 20 100

500 20 20 0 0 0 0 20 100

0.1

HET (gDNA 2)

20 16 4 0 4 0 16 80

1 20 20 0 0 0 0 20 100

10 20 20 0 0 0 0 20 100

100 20 19 1 0 0 0 20 100

500 20 18 2 0 0 0 20 100


MTHFR C677T Genotype

Replicates Tested




Calls Mis-Calls

No calls

Mis-Calls

0.1

WT (gDNA 1)

20 1 19 0 19 0 1 5

1 20 20 0 0 0 0 20 100

10 20 20 0 0 0 0 20 100

100 20 20 0 0 0 0 20 100

500 20 20 0 0 0 0 20 100

0.1

HET (gDNA 2)

20 1 19 0 19 0 1 5

1 20 20 0 0 0 0 20 100

10 20 20 0 0 0 0 20 100

100 20 19 1 0 0 0 20 100

500 20 18 2 0 0 0 20 100

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MTHFR A1298C Genotype

Replicates Tested




Calls Mis-Calls

No calls

Mis-Calls

0.1 ng

HET (gDNA1)

20 8 12 0 11 0 9 45

1 ng 20 20 0 0 0 0 20 100

10 ng 20 20 0 0 0 0 20 100

100 ng 20 20 0 0 0 0 20 100

500 ng 20 20 0 0 0 0 20 100

0.1 ng

WT (gDNA2)

20 20 0 0 0 0 20 100

1 ng 20 20 0 0 0 0 20 100

10 ng 20 20 0 0 0 0 20 100

100 ng 20 19 1 0 0 0 20 100

500 ng 20 18 2 0 0 0 20 100

METHOD COMPARISON

In a method comparison study, a total of 219 gDNA samples extracted from whole blood were genotyped using the eSensor

® Thrombophilia Risk Test and DNA sequencing. All samples gave 100% agreement with DNA

sequencing. The following table summarizes the results of the method comparison study.

FV Mutation

Results by DNA Sequencing

First Pass Results Final Results

eSensor® Results

1st Pass No-calls Miscalls

% Agree-ment

95% LCB

eSensor®

Results Final No-calls Miscalls

% Agree-ment

95% LCB WT HET MUT WT HET MUT

WT 181 0 0 1 0 99.45% 97.42% 182 0 0 0 0 100.00% 98.37%

HET 0 27 0 1 0 96.43% 84.15% 0 28 0 0 0 100.00% 89.85%

MUT 0 0 9 0 0 100.00% 71.69% 0 0 9 0 0 100.00% 71.69%

FII Mutation



eSensor® Results


% Agree-ment

95% LCB

eSensor® Results

Final No-calls Miscalls

% Agree-ment


WT 183 0 0 1 0 99.46% 97.45% 184 0 0 0 0 100.00% 98.39%

HET 0 27 0 0 0 100.00% 89.50% 0 27 0 0 0 100.00% 89.50%

MUT 0 0 6 2 0 75.00% 40.03% 0 0 8 0 0 100.00% 68.77%

MTHFR (C677T) Mutation



eSensor® Results


% Agree-ment

95% LCB

eSensor® Results


% Agree-ment


WT 68 0 0 0 0 100.00% 95.69% 68 0 0 0 0 100.00% 95.69%

HET 0 118 0 2 0 98.33% 94.85% 0 120 0 0 0 100.00% 97.53%

MUT 0 0 31 0 0 100.00% 90.79% 0 0 31 0 0 100.00% 90.79%

PI0971 REV C of 20

MTHFR (A1298C) Mutation



eSensor® Results


% Agree-ment

95% LCB

eSensor® Results


% Agree-ment


WT 69 0 0 0 0 100.00% 95.75% 69 0 0 0 0 100.00% 95.75%

HET 0 117 0 3 0 97.50% 93.67% 0 120 0 0 0 100.00% 97.53%

MUT 0 0 30 0 0 100.00% 90.50% 0 0 30 0 0 100.00% 90.50%

REPRODUCIBILITY

Site to Site, Operator to Operator, Day to Day, Run to Run and Sample to Sample Reproducibility A reproducibility study was performed over 5 non consecutive days at 3 different sites (2 external sites and 1 internal site) expanding 20 calendar days. Each site performed the testing twice each day, using two different operators and the same testing materials. Five (5) gDNA samples containing positive calls for Thrombophilia Risk Test mutations were used. The sample sets were run in duplicate to evaluate intra-assay reproducibility using a single kit. Only one lot of materials was used for this study. The data were evaluated after first-pass result and following the additional runs for no-calls. There were two (2) first-pass no-calls resulting in a first-pass call rate of 99.33%. All samples gave 100% final correct calls when compared with DNA sequencing. The following table summarizes the percent agreement between results obtained at each of the sites and DNA sequencing, before (first-pass) and after one additional run for no-calls (final).

Site Operator Samples Tested

First pass Correct calls

First pass no- calls

Miscalls Final

Correct calls

% Agreement

Site A 1 50 50 0 0 50 100%

2 50 50 0 0 50 100%

Site B 1 50 50 0 0 50 100%

2 50 49 1 0 50 100%

Site C 1 50 49 1* 0 50 100%

2 50 50 0 0 50 100%

All All 300 298 2 0 300 100%

*This no-call was due to MTHFR-A1298C low signal. FII, FV and MTHFR-C677T were correctly called.

Genomic DNA Extraction Reproducibility A total of 6 whole blood samples of different genotypes were extracted by three commonly used extraction methods and tested using the eSensor

® Thrombophilia Risk Test. The data were evaluated after first-pass

results. There were zero no-call or miscalls. All samples gave 100% correct calls when compared with DNA sequencing. There was no impact of extraction method observed in this study. The following table summarizes the results of extraction reproducibility study.

Extraction Method

# Samples Tested


First pass No Calls

Miscalls Final correct

calls

Final Agreement

%

A 6 6 0 0 6 100

B 6 6 0 0 6 100

C 6 6 0 0 6 100

Total 18 18 0 0 18 100

PI0971 REV C of 20

Lot to Lot Reproducibility A total of 5 genomic DNA samples containing positive calls for Thrombophilia Risk Test panel mutations were tested in duplicate using three different kit lots of the eSensor

® Thrombophilia Risk Test. The data were evaluated

after first-pass results and following an additional run for a single no-call. All samples gave 100% correct calls when compared with DNA sequencing. There was a single first-pass no-call, but no impact of kit lot observed in this study. The following table summarizes the results of lot to lot reproducibility study.

LOT Samples Tested



Final % Agreement No

Calls Mis-Calls

No Calls

Mis-Calls

1 20 20 0 0 0 0 20 100%

2 20 19 1 0 0 0 20 100%

3 20 20 0 0 0 0 20 100%

Total 60 59 1* 0 0 0 60 100%

*The single no-call was due to FII low signal. FV, MTHFR-C677T and MTHFR-A1298C were correctly called.

INTERFERING SUBSTANCES

The following interfering substances were added separately to two whole blood samples at the concentration indicated below, with no effect observed on yield of extracted DNA, multiplex amplification of target gene sequences, or genotyping of mutations by the eSensor

® Thrombophilia Risk Test: Heparin (3,000 U/L),

Cholesterol (250 mg/dL), Bilirubin (30 mg/dL) and Hemoglobin (~20g /dL). In addition, no effect of elevated EDTA concentration due to a reduced volume blood draw was observed by adding a total of 5 times the normal amount of EDTA anticoagulant to a whole blood sample. INTERFERING MUTATIONS

The following interfering mutations were tested in 40 replicates alongside a Wild-Type control gDNA sample, with no effect observed on multiplex amplification of target gene sequence, or genotyping by the eSensor

®

Thrombophilia Risk Test.

Non-Panel Mutation or Polymorphism Panel Mutation

1692A>C FV-1691G>A

1689G>A

1696A>G

20207A>C FII-20210G>A

20209C>T

TROUBLESHOOTING TABLE

The following table may be helpful in resolving issues that may arise when using this product. For more troubleshooting information, please refer to the eSensor

® X-T 8™ System User Manual. If there are any

additional questions regarding the eSensor Thrombophilia Risk Test, please call Technical Support at 1-800-eSensor (1-800-373-6767), option 2.

PI0971 REV C of 20

Observation Remedy/Action

1. Insufficient volume of PCR or hybridization solutions after preparation.

Some of the possible causes for this are:

Calculation errors. Double-check all calculations.

Pipetting error. Check that pipettes are properly calibrated and set at the appropriate volume settings when in use.

Accidental omission of a required reagent.

PCR tubes or plates are not properly sealed. Verify secure closure prior to loading into the thermal cycler. Heated lid option on thermal cycler not used.

Heated lid of thermal cycler does not come into contact with tube caps. Verify that the tube type is compatible with thermal cycler, and that the heated lid properly contacts the tube cap.

Spill.

2. Hybridization buffer precipitates upon formulation.

Some of the possible causes for this are:

Insufficient mixing. Vortex all reagents as directed. Expired components were used. Check that all components are still within their

expiration dates. Reagents were added in the wrong order. Add reagents in the order specified in

the protocol. Improper storage and handling of reagents. Check that all reagents have been

stored as specified in the protocol. Pipetting error. Check that pipettes are properly calibrated and set at the

appropriate volume settings when in use.

3. One mutation reports ‘Heterozygous’ for many samples of a single run

Possible amplicon contamination has occurred. Decontaminate work surfaces and instruments, and then test a no template control sample (PCR Blank) to confirm efficacy of decontamination.

4. Any of the mutations report ‘Invalid Test(s)’.

If the sample is a no template control (PCR Blank) reaction, this is the expected result; print DCM Report.

If the sample is a patient, possible causes of this failure are:

Amplification failure due to the absence of genomic DNA or Taq polymerase, faulty thermal cycler operation, or reagent evaporation.

Pipetting error. Check that pipettes are properly calibrated and set at the appropriate volume settings when in use.

Insufficient mixing. Mix and vortex all reagents as directed. Improper storage and handling of reagents. Check that all reagents have been

stored as specified in the labeling.

If issues persist after the identification and correction of the error, contact Technical Support.

5. Any of the controls report ‘Invalid Controls’.

This indicates a failure of the positive or negative controls. Some of the possible causes of this failure are:

Incorrect preparation of hybridization solution (For invalid positive control, signal buffer may not have been added).

Improper storage and handling of reagents. Check that all reagents have been stored as specified in the labeling.

If issues persist after the identification and correction of the error, contact Technical Support.

6. Results section reports ‘No Report’.

No data has been collected. This could be due to:

Incomplete cartridge scanning.

The protocol was aborted before it was able to read the cartridges.

If necessary, repeat test. If problem persists, contact Technical Support.

PI0971 REV C of 20

Observation Remedy/Action

7. Multiple polymorphisms report ‘Low Signal’ with a passing positive control

This generally indicates a failure in sample preparation, PCR or exonuclease steps. Possible causes and actions include:

Poor quality sample. Verify that the DNA concentration is within specified criteria, and that the sample is not degraded (e.g. by agarose gel electrophoresis) or contaminated (e.g. by A260:A280 ratio).

Genomic DNA template was not added.

Improper storage and handling of Taq Polymerase/ exonuclease enzyme.

PCR failure. Make sure that PCR Master Mix was prepared and mixed thoroughly.

Exonuclease failure. Make sure that 5 µL of exonuclease was delivered directly into the solution of each sample and that tubes are thoroughly vortexed after exonuclease addition.

8. One or more polymorphisms report “Error”

This generally indicates that Buffer-1 and Buffer-2 reagent volumes were incorrectly added. Add reagents in the order and volumes specified in the protocol.

This may also result from poor fluid pumping, contact Technical Support.

GENMARK TECHNICAL SUPPORT

GenMark Diagnostics, Technical Support 5964 La Place Court, Carlsbad, CA 92008 Phone: 1-800-eSensor (1-800-373-6767), Option 2 Email: [email protected] 7 am to 6 pm (Pacific Time), Monday- Friday REFERENCES

1 Poort, SR, Rosendaal FR, Reitsma PH, et al (1996). A common genetic variation in the 3'-untranslated region of

the prothrombin gene is associated with elevated plasma prothrombin levels and an increase in venous thrombosis. Blood 88, 3698-3703. 2 Bertina RM, Koeleman BP, Koster T, et al (1994). Mutation in blood coagulation factor V associated with

resistance to activated protein C. Nature 369, 64–67. 3 Botto LD and Yang Q (2000). 5, 10-Methylenetetrahydrofolate reductase (MTHFR) Gene Variants and

Congenital Anomalies: A HuGE Review. Am. J. Epidemiol. 151, 862-877. 4 Franco RF, Santos SEB, Elion J, et al (1998). Prevalence of the G20210A Polymorphism in the 3'-Untranslated

Region of the Prothrombin Gene in Different Human Populations. Acta Haematol. 100, 9-12. 5 Gregg JP, Yamane AJ, and Grody WW (1997). Prevalence of the factor V-Leiden mutation in four distinct

American ethnic populations. Am. J. Med. Genet. 73, 334–336. 6 Grody WW, Griffin JH, Taylor AK, Korf BR and Heit JA (2001). American College of Medical Genetics

Consensus Statement on Factor V Leiden Mutation Testing. http://www.acmg.net/resources/policies/pol-009.asp

http://www.acmg.net/resources/policies/pol-009.asp

PI0971 REV C of 20

MEDICAL DEVICE SYMBOL CHART

Symbol Description Symbol Description

Batch Code

Use by YYYY-MM-DD

Caution, Consult Accompanying documents

Serial number

Contains sufficient for <n> tests

Catalog number

In vitro diagnostic medical device

Upper limit of temperature

Consult instructions for use

Temperature limitation

Manufacturer

TRADEMARKS

eSensor®, GenMark™, GenMark Dx™

and XT-8™ are trademarks of GenMark Diagnostics.

PATENT INFORMATION

GenMark Diagnostics eSensor®-based assays all feature technology claimed in one or more of the following

United States and foreign patents owned or licensed by GenMark Diagnostics Inc. or its subsidiaries, with multiple additional foreign and domestic patents pending: US7,601,507, US7,595,153, US7,582,419, US7,579,145, US7,560,237, US7,514,228, US7,393,645, US7,384,749, 525US7,381,533, US7,381,525, US7,312,087, US7,267,939, US7,172,897, US7,160,678, US7,125,668, US7,056,669, US7,090,804, US7,018,523, US6,977,151, US6,960,467, US6,942,771, US6,761,816, US6,740,518, US6,600,026, US6,495,323, US6,479,240, US6,472,148, US6,322,979, US6,306,584, US6,291,188, US6,277,576, US6,268,149, US6,265,155, US6,264,825, US6,258,545, US6,232,062, US6,087,100, US6,071,699, US6,054,270, US5,952,172, US5,891,630, US5,846,708, US5,824,473, US5,773,258, US5,770,369, US5,705,348, US5,677,152, US5,670,322, US5,653,939, US5,620,850, US5,532,128, US5,591,578, EPO1350568, EPO1254372, EPO1246699, EPO1233830, EPO1218541, EPO1075549, EPO871,642, EPO871773, EPO733058, EPO1183102, EPO373203, JP4124830, JP4072206, JP3926625, and JP3548159. THIS PRODUCT IS MANUFACTURED AND SOLD UNDER LICENSE OF US PATENTS 6,074,821, 6,218,120, 6,528,259, 6,566,065, 6,833,243 and foreign equivalents thereof from Siemens Healthcare Diagnostics, Inc. Effective Date: December 2013 © 2013 GenMark Diagnostics, Inc. All rights reserved.

PI0971 REV C of 20

THROMBOPHILIA RISK TEST WORKSHEET

OPERATOR: RUN: DATE:

PCR Master Mix Set-Up

Component Volume/Reaction Calculation† Volume (µL)

PCR Mix 29 µL 29 * N * 1.1 =

Taq polymerase 1 µL 1 * N * 1.1=

Total Volume 30 µL 30 * N * 1.1 =

†N = Total number of reactions in run, including PCR Blank and any controls

Thermal Cycling Protocol – PCR


Initial PCR Activation Step 95 °C 4 minutes

3-Step Cycling (35 Cycles)

Denaturation 95 °C 25 seconds

Annealing 60 °C 30 seconds

Extension 72 °C 25 seconds


Thermal Cycling Protocol – Exonuclease Digestion


Digestion 37 °C 20 minutes

Inactivation 95 °C 2 minutes


Hybridization Solution Set-Up

Component Volume/ Reaction Calculation† Volume (µL)

Signal Buffer 70 L 70 * N * 1.2 =

Buffer-1 10 L 10 * N * 1.2 =

Buffer-2 20 L 20 * N * 1.2 =

Total Hybridization Solution Volume =

†N = Total number of reactions in run, including PCR Blank and any controls

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