esensor cystic fibrosis genotyping test package insert · pi0822 rev d page 1 of 22 esensor®...

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eSensor® Cystic Fibrosis Genotyping 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

® CF Genotyping 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 Test Report .............................................................................................................................................................. 10

Summary Section ................................................................................................................................................. 10 Details Section ..................................................................................................................................................... 10

Quality Control ......................................................................................................................................................... 11 Controls ................................................................................................................................................................ 12 Positive Control .................................................................................................................................................... 12 Negative Control .................................................................................................................................................. 12 Hardware and Software Controls ......................................................................................................................... 12 PCR Blank ............................................................................................................................................................ 12

Instrument Maintenance and Calibration ................................................................................................................. 12 Test Limitations ........................................................................................................................................................ 13 Interfering Mutations ................................................................................................................................................ 13 Expected Values / Reference Range ...................................................................................................................... 13 Performance Characteristics ................................................................................................................................... 14

Polymorphisms Detected ..................................................................................................................................... 14 Limit of Detection ................................................................................................................................................. 14 Method Comparison ............................................................................................................................................. 16 Reproducibility ...................................................................................................................................................... 16 Interfering Substances ......................................................................................................................................... 18

Troubleshooting Table ............................................................................................................................................. 18 GenMark Technical Support .................................................................................................................................... 20 References .............................................................................................................................................................. 20 eSensor

® Cystic Fibrosis Genotyping Test Worksheet ........................................................................................... 22

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INTENDED USE

The eSensor® Cystic Fibrosis Genotyping Test is an in vitro diagnostic device used to simultaneously detect and

identify a panel of mutations and variants in the cystic fibrosis transmembrane conductance regulator gene in genomic DNA samples isolated from human peripheral whole blood specimens. The panel includes mutations and variants recommended by the 2004 American College of Medical Genetics. The eSensor Cystic Fibrosis Genotyping Test is a qualitative genotyping test that provides information intended to be used for cystic fibrosis carrier screening as recommended by ACMG and the American College of Obstetricians and Gynecologists for adults of reproductive age, as an aid in newborn screening for cystic fibrosis, and in confirmatory diagnostic testing for cystic fibrosis in newborns and children. The test is not indicated for use in fetal diagnostic or pre-implantation testing. This test is also not indicated for stand-alone diagnostic purposes and results should be used in conjunction with other available laboratory and clinical information. The eSensor Cystic Fibrosis Genotyping Test is intended for use on the eSensor XT-8™ System. BACKGROUND

Cystic Fibrosis (CF) is a genetic disease in which defective chloride transport across cellular membranes causes dehydrated secretions. This leads to tenacious mucous in the lungs, to mucous plugs in the pancreas, and to characteristically high sweat chloride levels. CF is inherited as an autosomal recessive disorder; the responsible gene, the CF Transmembrane Conductance Regulator (CFTR), was mapped to chromosome 7 and identified in 1989.

1, 2, 3 Since that time, over 1,500 CFTR mutations have been identified.

The American College of Obstetricians and Gynecologists (ACOG) and the American College of Medical Genetics (ACMG) have recommended that preconception screening for mutations in the CFTR gene be offered to couples contemplating pregnancy. In 2001, an initial panel of 25 CFTR mutations with an allele frequency >0.1% in the general U.S. population was recommended.

4 Subsequently, additional mutation frequency data have

accumulated, prompting a revision of the panel in 2004 to the following 23 mutations: 1717-1G>A, 1898+1G>A, 2184delA, 2789+5G>A, 3120+1G>A, 3659delC, 3849+10KbC>T, 621+G>T, 711+1G>T, A455E, R117H, ΔI507 (DeltaI507), ΔF508 (DeltaF508), G542X, G551D, G85E, R553X, N1303K, R1162X, R334W, R347P, R560T, and W1282X.

5 Four reflex tests were recommended in the 2001 publication: 5/7/9T (reflex for R117H carrier), I506V,

I507V and F508C (reflex tests for unexpected homozygosity of ΔF508). The CF Genotyping Test includes the ACMG recommended panel of 23 mutations plus a reflex test for the 5/7/9T polymorphism. PRINCIPLE OF ESENSOR

® TECHNOLOGY

The eSensor technology uses a solid-phase electrochemical method for determining the genotyping status of a defined panel of CF 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 structure 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.

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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. do not pipette by mouth, wear appropriate protective clothing and eye protection.

Avoid contact of Signal Buffer on the skin, eyes, or mucous membranes. If contact does occur, immediately flush with water for at least 15 minutes. Seek medical attention if irritation develops. If left untreated, burns may result. Refer to MSDS for more details.

Handle all specimens and waste materials as if they were capable of transmitting infectious agents in accordance with Universal Precautions. Observe safety guidelines such as those outlined in CDC/NIH Biosafety in Microbiological and Biomedical Laboratories and the CLSI Document M29 Protection of Laboratory Workers from Occupationally Acquired Infections.

Follow your institution’s safety procedures for handling biological samples.

Wash hands thoroughly with soap and water after handling reagents. Launder contaminated clothing prior to re-use.

Reagents are not considered to be a fire hazard. However, 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 to be an explosion hazard.

Dispose of materials used in this test, including reagents, specimens, and used vials in accordance with all Federal, State, and Local regulations.

General

For In Vitro Diagnostic Use.

Do not substitute eSensor CF Genotyping Test reagents with alternate reagents.

Do not reuse cartridges.

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

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Do not pool reagents or mix and match kit components from different lots.

Follow the procedure as described in this package insert.

After test completion, remove cartridges from the XT-8 Instrument. COMPONENTS AND REAGENTS PROVIDED

Table 1: 24 Test Kit

Box Component Packaging & Quantity Storage

eSensor® CF

Genotyping Test Cartridges

eSensor® CF Cartridges

3 foil bags with 8 cartridges in each bag

10-25 ºC

eSensor® CF

Genotyping Test Amplification

Reagents

CF PCR Mix 1 vial with 1100 µL -20 ºC (in a designated pre-PCR location) Taq Polymerase 1 vial with 60 µL

eSensor® CF

Genotyping Test Detection Reagents

Exonuclease 1 vial with 145 µL

-20 ºC (in a designated post-PCR location)

CF Signal Buffer 1 vial with 2500 µL

Buffer-1 1 vial with 350 µL

Buffer-2 1 vial with 700 µL

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

CF Genotyping 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 Table 1. Store frozen reagents in a non-frost-free freezer. Place thawed reagents back into non-frost-free freezer immediately after use. Reagents must be used within 30 days of opening a vial or after 3 thaws. Cartridges must be used within 14 days of opening the bag.

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ESENSOR® CF GENOTYPING 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.

Reagents must be used within 30 days of opening a vial or after 3 thaws.

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 the 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 the 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. Decontaminate laboratory areas and affected equipment before each assay run.

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 proper storage conditions. NOTE: IMPORTANT! Do not thaw exonuclease enzyme.

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

To ensure complete mixing of the enzyme solution with the amplicon solution, it is IMPORTANT that each tube 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 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 14 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® CF Genotyping Test Workflow

Figure 2. Overall schematic of the CF Genotyping Test procedure. The assay consists of 3 steps for each sample: 1) multiplex PCR to generate double-stranded amplicon, 2) exonuclease digestion to yield single-stranded 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 CF Genotyping Test Amplification Reagents, except the Taq polymerase.

3. Use the “PCR Master Mix Set-Up” table from the CF Genotyping 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 freezer and centrifuge. NOTE: IMPORTANT! Do not vortex the Taq polymerase; place on cold block immediately and return to -20 °C promptly after use.

6. Combine all reagents in a tube of sufficient volume. IMPORTANT! Invert the PCR master mix tube three (3) times, and then vortex and centrifuge the tube.

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

8. If extracted samples are frozen, 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 in a calibrated thermal cycler and amplify according to the protocol below, using 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 (37 Cycles)

Denaturation 94 °C 30 seconds

Annealing 56 °C 30 seconds

Extension 72 °C 30 seconds

Cool Down

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 the samples.

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. NOTE: IMPORTANT! Thorough vortexing of the exonuclease enzyme is necessary to generate single-stranded DNA.

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NOTE: Do not use a pipette to mix as this may aerosolize amplicon and is a potential source of contamination.

5. Place PCR tubes 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.

Thermal Cycling Protocol – Exonuclease Digestion


Digestion 37 °C 20 minutes

Inactivation 95 °C 2 minutes


6. After completion of incubation, remove the tubes 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, vortex and centrifuge the Signal Buffer, Buffer-1 and Buffer-2.

2. Use the “Hybridization Solution Set-Up” table from the CF Genotyping 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 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 precipitate clears. If vortexing does not dissolve the precipitate, heat the solution to 37 ºC for 5 minutes before vortexing again. Centrifuge or lightly tap the tube on a bench to minimize the amount of liquid left on the sides 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. Vortex and centrifuge each tube.

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 IDs can be entered by bar code, work list or manually (see XT-8 User Manual for more instructions).

12. Scan in the Reagent Barcode located on the CF Genotyping 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 go from orange to yellow, meaning that this test is ready to start.

15. Start the hybridization and scanning protocol by touching 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

® CF Genotyping Test uses the principle of redundant electrodes.

Two parameters of the data generated from each electrode are evaluated by the XT-8 Software when making a genotyping call:

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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 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. See the eSensor XT-8™ User Manual for details. TEST REPORT

Summary Section

Valid results: If the assay controls pass and a valid call can be made from the signals generated from all CF mutations in the assay, the genotype will be given in the Summary Section of the report. Qualitative results can have a specific genotype call for the mutation, such as ‘Wild-Type’, ‘Heterozygous’, or ‘Homozygous’. If the CF Genotyping Test Screening Report is selected, the 5/7/9T reflex test result is displayed in the Summary Section only in cases where the R117H genotype is either Heterozygous or Homozygous. If the CF Genotyping Test Diagnostic Report is selected, the 5/7/9T result is included in the Summary Section regardless of the R117H genotype. Invalid results: If an assay control fails, or if any of the panel mutations tests fail, the genotype result for the sample will not be reported in the Summary Section. Instead, ‘Invalid Control(s)’, ‘Invalid Test(s)’, ‘Incomplete Report’, or ‘No Report’ will be reported. Repeat testing is recommended in these cases. Details Section

The Details Section of the report includes qualitative results for each mutation on a separate line and can have a specific genotype call such as ‘WT’, ‘HET’, or ‘MUT’ (or 9T/9T’, ‘9T/7T’, ‘7T/7T’, ‘9T/5T’, ‘5T/5T’ for 5/7/9T reflex polymorphism) or one of the following descriptive messages: ‘Valid Test’, ‘Low Signal’, ‘Indeterminate Score’, ‘Contradictory Score’, ‘Error’, ‘Invalid Control(s)’ or ‘Incomplete Report’. In both the CFTR Diagnostic report and the CFTR Screening report the genotyping status of each of the 23 mutations will be listed on separate lines in the Details Section of the report. In the CFTR screening report, the results of the 5/7/9T reflex test will be absent from the Details Section when R117H is classified as a wild type. The CFTR Diagnostic report will list the results of the 5/7/9T reflex test in the Details Section regardless of the R117H classification. 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.

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Genotyping status and Genotyping Metric value for each polymorphism will only be reported if the assay controls and all panel mutations are valid. For an invalid test, one of the following results will be reported for every mutation in the Details Section:

‘Valid Test’ occurs when data generated for a mutation would have allowed a call to be made, but another mutation(s) or a control failed.

‘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 on the two electrodes for that polymorphism.

‘Error’ occurs when a system error prevents a result from being generated. If a sample reports more than one non-R117H mutation result as ‘Heterozygous’ or ‘Homozygous’ and it is not a multi-mutation control, then the sample should be retested as it is unlikely that a sample from a non-affected individual would carry two mutations on one allele. In this case, a possible system malfunction is indicated. The eSensor CF Genotyping Test does not identify the I506V, I507V, and F508C polymorphisms, thus in the case of ΔF508 homozygosity, reflex testing by bi-directional DNA sequencing is recommended.

When a sample is reported as R117H ‘Heterozygous or Homozygous, 9T/9T or 9T/7T or 7T/7T’, the following information will appear in the Summary Section notes: ‘The phenotypic expression of the R117H mutation is influenced by the presence of the 5T variant. Thus, additional testing was performed, and the results indicated that this individual is negative for the 5T variant. In the absence of the 5T variant, the R117H mutation is not expected to lead to a typical CF clinical phenotype. The presence of R117H, however, has been associated with CBAVD, leading to infertility in males with no known clinical features in females’.

When a sample is reported as R117H ‘Heterozygous or Homozygous, 5T/9T or 5T/7T’ the following information will appear in the Summary Section notes: ‘The R117H mutation produces a partially functional CFTR protein product. The amount of this protein is further reduced when the R117H mutation is on the same allele as the 5T variant. The eSensor

® XT-8 System is unable to determine whether the R117H mutation and the 5T variant are

on the same chromosome. See product insert for recommended additional testing’.

NOTE: It is recommended that parents of such individuals submit blood samples for mutation analysis to resolve this issue. If parents are unavailable, other close family members may be helpful. Genetic counseling is also recommended.

For R117H ‘Heterozygous or Homozygous, 5T/5T’ the following information will appear in the Summary Section notes: ‘The R117H mutation produces a partially functional CFTR protein product. The amount of this protein is further reduced when the R117H mutation is on the same allele as the 5T variant’. See the Troubleshooting Table 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 (no template control) be performed and included with each PCR run of the eSensor CF Genotyping Test, as described in the PCR Blank section. Positive Controls: CF panel mutation controls are commercially available. It is recommended to routinely include positive controls in each run of the eSensor

® CF Genotyping Test. For example, a bank of samples containing

individual polymorphisms can be obtained, and a single positive control from the bank can be selected in rotation and tested with each run.

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All quality control testing and requirements should be performed in accordance with Federal, State and Local regulations and guidelines. Controls

Each test contains on-board positive and negative controls to ensure proper functioning of the system. Failure of either on-board control will be indicated as “Invalid Control(s)” in the Summary Section of the test report. The results for the on-board controls are included in the Details Section of the report with a result of Pass/Fail. The genotyping test results will not be reported for any sample for which an on-board 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 a ferrocene label 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 on-board 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 eSensor

® XT-8™ User Manual for details.

PCR Blank

It is routine laboratory practice to perform a no template reaction (PCR Blank or DNA Contamination Monitor, DCM) 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 processed with the other samples in the run, and the DCM Report is reviewed for this sample. The DCM report provides a Summary Section 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.

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 on-board Positive and Negative Control tests. A failure of either of these tests will prevent interpretation of the PCR Blank test (DCM Report), 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.

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TEST LIMITATIONS

The eSensor CF Genotyping Test does not identify all possible mutations in the CFTR gene, for example, the following mutations or variants are not part of the test panel: E60X, I148T, 1078delT, V520F, 2143delT, 3199del6, D1152H, 3876delA, 2183AA>G, R560K, R117L, R347H, G551S, 711+5G>A, 394delTT,and 3905insT. A negative result for all the mutations in this panel does not necessarily indicate that the individual does not carry mutations in the CFTR gene (carrier or affected status). The mutations included in this test represent >80% of the mutations carried by Caucasian American adults. The CF Genotyping Test does not identify the I506V, I507V, and F508C polymorphisms, thus in the case of ΔF508 homozygosity, reflex testing by bi-directional DNA sequencing is recommended. INTERFERING MUTATIONS

More than 1,500 mutations have been identified in the CFTR gene with varying confidence that they cause Cystic Fibrosis. When present in the same region as a panel mutation, they may interfere with genotyping results. The following mutations have been evaluated and demonstrated not to impact the results of the eSensor CF Genotyping Test:

Non-Panel Mutation or Polymorphism ACOG/ACMG Panel Mutation

2183AA>G 2184delA

R117L R117H

EXPECTED VALUES / REFERENCE RANGE

The mutations being genotyped in the eSensor CF Genotyping Test are present at the following frequencies in the indicated ethnic groups among individuals with clinically diagnosed cystic fibrosis

5:

Mutation Non-Hispanic

Caucasian Hispanic American

African American

Asian American

Ashkenazi Jewish

Pan-Ethnic Population

delF508 72.42% 54.38% 44.07% 38.95% 31.41% 66.31%

G542X 2.28% 5.10% 1.45% 0.00% 7.55% 2.64%

W1282X 1.50% 0.63% 0.24% 0.00% 45.92% 2.20%

G551D 2.25% 0.56% 1.21% 3.15% 0.22% 1.93%

621+1G>T 1.57% 0.26% 1.11% 0.00% 0.00% 1.30%

N1303K 1.27% 1.66% 0.35% 0.76% 2.78% 1.27%

R553X 0.87% 2.81% 2.32% 0.76% 0.00% 1.21%

delI507 0.88% 0.68% 1.87% 0.00% 0.22% 0.90%

3120+1G>A 0.08% 0.16% 9.57% 0.00% 0.10% 0.86%

3849+10kbC>T 0.58% 1.57% 0.17% 5.31% 4.77% 0.85%

R117H 0.70% 0.11% 0.06% 0.00% 0.00% 0.54%

1717–1G>A 0.48% 0.27% 0.37% 0.00% 0.67% 0.44%

2789+5G>A 0.48% 0.16% 0.00% 0.00% 0.10% 0.38%

R334W 0.14% 1.78% 0.49% 0.00% 0.00% 0.37%

R347P 0.45% 0.16% 0.06% 0.00% 0.00% 0.36%

711+1G>T 0.43% 0.23% 0.00% 0.00% 0.10% 0.35%

PI0822 REV D of 22

Mutation Non-Hispanic

Caucasian Hispanic American

African American

Asian American

Ashkenazi Jewish

Pan-Ethnic Population

R560T 0.38% 0.00% 0.17% 0.00% 0.00% 0.30%

R1162X 0.23% 0.58% 0.66% 0.00% 0.00% 0.30%

3659delC 0.34% 0.13% 0.06% 0.00% 0.00% 0.28%

A455E 0.34% 0.05% 0.00% 0.00% 0.00% 0.26%

G85E 0.29% 0.23% 0.12% 0.00% 0.00% 0.26%

2184delA 0.17% 0.16% 0.05% 0.00% 0.10% 0.15%

1898+1G>A 0.16% 0.05% 0.06% 0.00% 0.10% 0.13%

5T/5T NA NA NA NA NA NA

TOTAL 88.29% 71.72% 64.46% 48.93% 94.04% 83.59%

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. Polymorphisms Detected

The eSensor® CF Genotyping Test detects the panel of 23 mutations and the 5/7/9T reflex test recommended by

the American College of Medical Genetics listed in the table below.5

Mutations Genotyped by the eSensor® CF Genotyping Test

delF508 621+1G>T 3120+1G>A 2789+5G>A R560T G85E

G542X N1303K 3849+10kbC>T R334W R1162X 2184delA

W1282X R553X R117H R347P 3659delC 1898+1G>A

G551D delI507 1717–1G>A 711+1G>T A455E 5/7/9T

Limit of Detection

Two genomic DNA samples with different genotypes were serially diluted to 1000, 500, 100, 10, 1.0, 0.1 and 0.01 ng per sample and tested 20 times each with the eSensor

® CF Genotyping Test, as shown in the following table.

All replicates above 0.1 ng gave 100% correct results. Thus, the limit of detection for the CF Genotyping Test was determined to be 0.1 ng of gDNA, with 10 to 500 ng as the recommended input for the test.

Total gDNA input (ng)

Genotypes Replicates

Tested

First Pass

Correct Calls

First Pass Final Final Correct

Calls

% Agreement No

Calls Miscalls

No Calls

Miscalls

0.01

WT 7T/7T

20 5 15 0 12 0 8 40

0.1 20 17 3 0 0 0 20 100

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 g 20 20 0 0 0 0 20 100

1000 20 20 0 0 0 0 20 100

PI0822 REV D of 22

Total gDNA input (ng)

Genotypes Replicates

Tested

First Pass

Correct Calls


Calls

% Agreement No

Calls Miscalls

No Calls

Miscalls

0.01

ΔF508 Heterozygous

9T/7T

20 1 18 1* 15 0 4 20

0.1 20 4 16 0 0 0 20 100

1 20 18 2 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 19 1 0 0 0 20 100

1000 20 6 14 0 0 0 20 100

* One (1) 9T/7T was miscalled as 9T/9T due to inefficient PCR.

PI0822 REV D of 22

METHOD COMPARISON

In a method comparison study, a total of 112 gDNA samples extracted from whole blood with A260:280 ratios of 1.2-2.5 were genotyped using the eSensor

® CF Genotyping Test and DNA sequencing. All samples

demonstrated 100% agreement with DNA sequencing.

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

Genotype by Sequencing

Calls per

Mutation

Sequencing Calls

1st Pass CF

Genotyping Calls %

Agreement Final CF

Genotyping Calls % Agreement

Pos Neg Pos Neg No

Calls Overall Pos Neg Pos Neg

No Calls

Overall Pos Neg

∆F508 112 47 65 46 65 1 99.1 97.9 100 47 65 0 100 100 100

G542X 112 7 105 6 105 1 99.1 85.7 100 7 105 0 100 100 100

W1282X 112 6 106 6 106 0 100 100 100 6 106 0 100 100 100

G551D 112 8 104 8 104 0 100 100 100 8 104 0 100 100 100

621+1G>T 112 7 105 7 105 0 100 100 100 7 105 0 100 100 100

N1303K 112 8 104 7 104 1 99.1 87.5 100 8 104 0 100 100 100

R553X 112 4 108 4 108 0 100 100 100 4 108 0 100 100 100

∆I507 112 3 109 3 109 0 100 100 100 3 109 0 100 100 100

3120+1G>A 112 2 110 2 110 0 100 100 100 2 110 0 100 100 100

3849+10kbC>T 112 5 107 5 107 0 100 100 100 5 107 0 100 100 100

R117H 112 8 104 8 104 0 100 100 100 8 104 0 100 100 100

1717-1G>A 112 5 107 5 107 0 100 100 100 5 107 0 100 100 100

2789+5G>A 112 5 107 5 107 0 100 100 100 5 107 0 100 100 100

R334W 112 4 108 4 108 0 100 100 100 4 108 0 100 100 100

R347P 112 4 108 4 108 0 100 100 100 4 108 0 100 100 100

711+1G>T 112 4 108 4 108 0 100 100 100 4 108 0 100 100 100

R560T 112 4 108 3 108 1 99.1 75.0 100 4 108 0 100 100 100

R1162X 112 8 104 8 104 0 100 100 100 8 104 0 100 100 100

3659delC 112 4 108 4 108 0 100 100 100 4 108 0 100 100 100

A455E 112 2 110 2 110 0 100 100 100 2 110 0 100 100 100

G85E 112 7 105 7 105 0 100 100 100 7 105 0 100 100 100

2184delA 112 2 110 2 110 0 100 100 100 2 110 0 100 100 100

1898+1G>A 112 2 110 2 110 0 100 100 100 2 110 0 100 100 100

Reflex Polymorphism

IVS8- 5T/7T/9T Variant (§)

112 6 ‡ 106 6 ‡ 106 0 100 100 100 6 106 0 100 100 100

Polymorphisms Not Specifically Genotyped

I507V 112 1 111 1 111 0 100 100 100 1 111 0 100 100 100

F508C 112 1 111 1 111 0 100 100 100 1 111 0 100 100 100

Potentially Interfering Mutations Not Part of Assay Panel

2183AA>G 112 2 110 2 110 0 100 100 100 2 110 0 100 100 100

R117L 112 1 111 1 111 0 100 100 100 1 111 0 100 100 100

Grand Total: 2688 162 2526 158 2526 4 99.9 97.5 100 162 2526 0 100 100 100

§ For the purpose of the IVS8-5T/7T/9T Variant, "Positive" samples are regarded as those that have at least one copy of the 5T allele while "Negative" samples are regarded as having only the 7T and/or 9T allele.

‡1 sample is 5T/5T Mutant.

The number of positive sequencing calls is greater than the number of independent samples due to the inclusion of compound heterozygous samples.

The Grand Total consists of the total number of sequencing calls for mutations, with the 1 5T/5T sample included. I507V, F508C, 2183AA>G, and R117L are polymorphisms not specifically genotyped and are non-panel polymorphisms containing samples correctly called as Wild-Type, which are not included in the grand total for calls. No samples with I506V were tested. REPRODUCIBILITY

Genomic DNA Extraction Reproducibility

A total of 20 whole blood samples of different genotypes were extracted by three commonly used extraction methods and tested using the eSensor

® CF Genotyping Test. The data were evaluated after first pass results and

following a single additional run for sample no-calls. 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.

PI0822 REV D of 22

Extraction Method

# of Samples Tested/Genotype

First Pass Correct

Calls

First Pass No Calls

Miscalls Final

Correct Calls

Final % Agreement

A 20 17 3 0 20 100

B 20 18 2 0 20 100

C 20 19 1 0 20 100

Lot to Lot Reproducibility

A total of 21 genomic DNA samples covering all possible genotypes were tested using three different kit lots of the eSensor

® CF Genotyping 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 due to kit lot was observed in this study. The following table summarizes the results of the lot to lot reproducibility study.

Lot Samples Tested

First Pass

Correct Calls


Calls

% Agreement

No Calls Miscalls No Calls Miscalls

1 21 21 0 0 0 0 21 100

2 21 21 0 0 0 0 21 100

3 21 20 1 0 0 0 21 100

Total 63 62 1 0 0 0 63 100

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). Each site performed the testing twice each day, using two different operators and the same testing materials. Twenty two (22) gDNA samples containing positive results for all ACOG/ACMG panel mutations in addition to the 5/7/9T polymorphism were used. For practical considerations, the 22 samples were divided into 2 sets of 11 samples, so that the sample set was 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 results and following the additional runs for no-calls. All samples gave 100% correct calls when compared with DNA sequencing. There were 36 first pass no-calls; seven due to assay, nine due to cartridge pumping, nine due to XT-8 instrument slot pumping, and remaining ten were due to operator error in set up of the PCR or set-up of the exonuclease reaction. The following tables summarize the percent agreement between results obtained at each of the sites and DNA sequencing, before (first pass) and after a maximum of 2 repeat tests for no-calls (final). Summary of Inter-laboratory, Inter-operator, Reproducibility Results

By Site By

Operator

Number of Sample

Replicates

First Pass

Correct Calls

First Pass No

Calls

First Pass

Miscalls

Final Correct

Calls

Final Incorrect

Calls

% Agreement

Site A (Internal)

Operator 1 220 208 12 0 220 0 100

Operator 2 220 216 4 0 220 0 100

Site B: (External)

Operator 1 220 215 5 0 220 0 100

Operator 2 220 211 9 0 220 0 100

Site C (External):

Operator 1 220 216 4 0 220 0 100

Operator 2 220 218 2 0 220 0 100

Total 1320 1284 36 0 1320 0 100

PI0822 REV D of 22

Summary of Reproducibility Results by Sample Genotype and Site

Sample Genotype by Sequencing

5/7/9T

Number of Sample

Replicates tested by eSensor

® CF

Genotyping Test

Number of CF Sample Calls Before Repeat Testing

Number of CF Sample Calls After Repeat Testing

Site A

Site B

Site C

Site A Correct

calls

Site B Correct

Calls

Site C Correct

calls

No Calls

Mis- calls

Site A Correct

calls

Site B Correct

Calls

Site C Correct

calls

No Calls

1717-1G>A 7T/7T 60 60 60 59 59 59 3 0 60 60 60 0

1898+1G>A/ΔF508 7T/9T 60 60 60 59 60 58 3 0 60 60 60 0

2184delA/ΔF508 7T/9T 60 60 60 58 60 58 4 0 60 60 60 0

3120+1G>A/621+1G>T

7T/9T 60 60 60 59 60 59 2 0 60 60 60 0

2789+5G>A/2789+5G>A

7T/7T 60 60 60 59 60 59 2 0 60 60 60 0

3659delC/ΔF508 7T/9T 60 60 60 59 59 60 2 0 60 60 60 0

3849+10KbC>T/ΔF508

7T/9T 60 60 60 60 59 58 3 0 60 60 60 0

621+1G>T/G85E 7T/9T 60 60 60 58 60 60 2 0 60 60 60 0

711+1G>T/621+1G>T

7T/9T 60 60 60 59 60 60 1 0 60 60 60 0

A455E/621+1G>T 9T/9T 60 60 60 59 60 59 2 0 60 60 60 0

ΔI507 7T/7T 60 60 60 60 60 60 0 0 60 60 60 0

G542X 7T/9T 60 60 60 59 59 60 2 0 60 60 60 0

G551D/R347P 7T/7T 60 60 60 59 59 60 2 0 60 60 60 0

N1303K 7T/9T 60 60 60 60 60 60 0 0 60 60 60 0

R1162X 7T/7T 60 60 60 60 60 59 1 0 60 60 60 0

R117H/ΔF508 5T/9T 60 60 60 60 60 59 1 0 60 60 60 0

R334W 7T/7T 60 60 60 60 59 59 2 0 60 60 60 0

R553X/G551D 7T/7T 60 60 60 59 60 60 1 0 60 60 60 0

R560T/ΔF508 7T/9T 60 60 60 59 60 60 1 0 60 60 60 0

W1282X 5T/7T 60 60 60 59 60 59 2 0 60 60 60 0

WT 7T/7T 60 60 60 60 60 60 0 0 60 60 60 0

R117H/ΔF508 5T/9T 60 60 60 60 60 60 0 0 60 60 60 0

INTERFERING SUBSTANCES

The following interfering substances were added separately to wild type, ΔF508, R117H and G551D heterozygous whole blood samples at the concentration indicated below, with no effect observed on the yield of extracted DNA, multiplex amplification of target gene sequences, or genotyping of CF panel mutations by the eSensor

® CF Genotyping Test: Conjugated Bilirubin (0.3 mg added/mL whole blood), Triglycerides (500 mg

added/dL whole blood), and Hemoglobin (~20 g/dL whole blood). In addition, the effect of elevated EDTA concentration due to a reduced volume blood draw was evaluated by adding a total of 5 times the normal amount of EDTA anticoagulant to a whole blood sample; no effect was observed on the yield of extracted DNA, multiplex amplification of target gene sequences, or genotyping of CF panel mutations by the eSensor

® CF Genotyping

Test. 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

® XT-8 User Manual. If there are any additional questions

regarding the eSensor® CF Genotyping Test, please call Technical Support at 1-800-eSensor (1-800-373-6767),

option 2.

PI0822 REV D of 22

Observation Cause/Remedy

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 reagents were used. Check that all reagents are 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.


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. Summary Section reports ‘Invalid Test(s)’.

If the sample is a no template control (PCR Blank), 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 gDNA or Taq polymerase, faulty thermal cycler operation, or reagent evaporation.


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. Summary Section reports ‘Invalid Controls’.

This indicates a failure of the on-board 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. Summary 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.

7. Summary Section reports ‘Heterozygous or Homozygous’ for more than 1 mutation (not including R117H).

It is unlikely that a sample from a non-affected individual will be heterozygous or homozygous for more than one CF mutation. Check to determine if sample is a known multi-mutation control; otherwise, this may indicate a problem with the test. Repeat test. If result is obtained repeatedly, contact Technical Support.

PI0822 REV D of 22

Observation Cause/Remedy

8. 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 remedies 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 storageand handling of Taq polymerase or exonuclease enzyme.

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

Exonuclease failure. Make sure that 5 µL of exonuclease is delivered into sample tubes, and that tubes are thoroughly mixed after exonuclease addition.

9. One or more polymorphisms report “Error”

This generally indicates that Buffer-1 and Buffer-2 volumes were incorrectly added. Add reagents in the order and volumes as 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. Rommens JM, Iannuzzi MC, Kerem B, Drumm ML, Melmer G, Dean M, Rozmahel R, Cole JL, Kennedy D, Hidaka N, et al. (1989) Identification of the cystic fibrosis gene: chromosome walking and jumping. Science 245(4922):1059-1065.

2. Riordan JR, Rommens JM, Kerem B, Alon N, Rozmahel R, Grzelczak Z, Zielenski J, Lok S, Plavsic N, Chou JL, et al. (1989) Identification of the cystic fibrosis gene: cloning and characterization of complementary DNA. Science 245(4922):1066-1073.

3. Kerem B, Rommens JM, Buchanan JA, Markiewicz D, Cox TK, Chakravarti A, Buchwald M, Tsui LC. (1989) Identification of the cystic fibrosis gene: genetic analysis. Science 245(4922):1073-1080.

4. Grody WW, Cutting GR, Klinger KW, Richards CS, Watson MS, Desnick RJ; Subcommittee on Cystic Fibrosis Screening, Accreditation of Genetic Services Committee, American College of Medical Genetics (ACMG). (2001) Laboratory standards and guidelines for population-based cystic fibrosis carrier screening. Genet Med. 3(2):149-154.

5. Watson MS, Cutting GR, Desnick RJ, Driscoll DA, Klinger K, Mennuti M, Palomaki GE, Popovich BW, Pratt VM, Rohlfs EM, Strom CM, Richards CS, Witt DR, Grody WW. (2004) Cystic fibrosis population carrier screening: 2004 revision of American College of Medical Genetics mutation panel. Genet Med. 6(5):387-391.

PI0822 REV D of 22

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’s eSensor® Cystic Fibrosis Genotyping Test features technology claimed in one or more of the following

United States and European patents owned or licensed by GenMark Diagnostics Inc. or its subsidiaries, with multiple additional foreign and domestic patents pending: US7,655,129, US7,601,507, US7,595,153, US7,582,419, US7,579,145, US7,560,237, US7,514,228, US7,393,645, US7,384,749, US7,381,533, US7,381,525, US7,312,087, US7,267,939, US7,172,897, US7,160,678, US7,125,668, US7,056,669, US7,049,068, US7,090,804, US7,018,523, US6,977,151, US6,960,467, US6,984,487, US6,942,771, US6,761,816, US6,740,518, US6,600,026, US6,495,323, US6,479,240, US6,472,148, US6,361,958, 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, US6,132,971, US6,087,100, US6,071,699, US6,054,270, US6,001,588, US5,981,178, US5,952,172, US5,891,630, US5,846,708. US5,824,473, US5,776,677, 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, US5,407,796, EP1233830, EP1075541, EP0871773, EP0733058, and EP0373203. The purchase of this product grants the purchaser rights under certain Roche patents noted above to use it solely for providing human in vitro diagnostic services. No general patent or other license of any kind other than this specific right of use from purchase is granted hereby. The purchase of this product also conveys rights under patents owned by Harvard University, Johns Hopkins University, University of Michigan, and the Research Institute of the Hospital for Sick Children. Effective Date: December 2013 ©

2013 GenMark Diagnostics, Inc. All rights reserved.

PI0822 REV D of 22

ESENSOR® CYSTIC FIBROSIS GENOTYPING TEST WORKSHEET

OPERATOR: RUN: DATE:

PCR Master Mix Set-Up

Component Volume/Reaction Calculation† Volume (µL)

CF PCR Mix 28 µL 28 * N * 1.1 =

Taq 2 µL 2 * N * 1.1=

Total PCR Master Mix Volume=

† 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 (37 Cycles)

Denaturation 94 °C 30 seconds

Annealing 56 °C 30 seconds

Extension 72 °C 30 seconds

Final Hold


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)

CF 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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