GenMark Diagnostics

eSensor® Warfarin Sensitivity Test (IVD)

GNMK-IMC-6013-C

Training Presentation

Training Agenda Warfarin Sensitivity Test

• Test Overview

• Kit Components and Handling

• Assay Setup

• Reporting

• Quality Control

• Ordering and Contact Information

Test Overview

Warfarin Sensitivity Test (IVD) Intended Use

The eSensor® Warfarin Sensitivity Test

is an in vitro diagnostic for the detection

and genotyping of the *2 and *3 alleles

of the cytochrome P450 (CYP450) 2C9

gene locus and the Vitamin K epoxide

reductase C1 (VKORC1) polymorphism

(-1639G>A) as an aid in the

identification of patients at risk for

increased warfarin sensitivity.

*The complete intended use statement may be viewed in the Package Insert, which can be

found in the Customer Training Binder or on the GenMark Customer Resource Center.

Panel Overview Detection and typing of CYP450 2C9 and VKORC1

Mutations Panel

CYP450 2C9 (*2)

Warfarin Metabolism

CYP450 2C9 (*3) Warfarin Metabolism

VKORC1

(-1639G>A)

Warfarin Sensitivity

• 100% accuracy and

reproducibility in clinical trials

• Single tube multiplex test for

all three warfarin-related

mutations

• Efficient, time-saving

alternative to performing

individual tests

• Objective results automatically

generated by GenMark’s

eSensor® XT-8 system

Warfarin Metabolism 2C9 Genotype/Phenotype

Allele Genotype

Result Summary

Result Phenotype Description

430C>T (*2) C/C 2C9 *1/*1

Extensive (normal) warfarin

metabolism anticipated 1075A>C (*3) A/A

430C>T (*2) C/T 2C9 *1/*2

Intermediate warfarin metabolism anticipated 1075A>C (*3) A/A

430C>T (*2) C/C 2C9 *1/*3

Slow warfarin metabolism anticipated

1075A>C (*3) A/C

430C>T (*2) T/T 2C9 *2/*2

1075A>C (*3) A/A

430C>T (*2) C/T 2C9 *2/*3

1075A>C (*3) A/C

430C>T (*2) C/C 2C9 *3/*3

Extremely slow warfarin

metabolism anticipated 1075A>C (*3) C/C

Warfarin Sensitivity VKORC1 Genotype/Phenotype

Allele Genotype

Result Summary

Result Phenotype Description

-1639G>A

G/G -1639 G/G Low warfarin

sensitivity anticipated

G/A -1639 G/A Intermediate warfarin sensitivity anticipated

A/A -1639 A/A High warfarin

sensitivity anticipated

Kit Components and Handling

Kit Pack/Boxes Component Packaging & Quantity Storage

Cartridge Pack Warfarin Sensitivity Test

Cartridge Pouch

3 foil bags with 8

cartridges each 10 – 25 °C

PCR Box

Warfarin PCR Mix 1 vial containing 1100 µL -20 °C

(Pre-PCR Location) Taq Polymerase 1 vial containing 60 µL

Genotyping Box

Exonuclease 1 vial containing 145 µL

-20 °C

(Post-PCR Location)

Warfarin Signal Buffer 1 vial containing 2500 µL

Buffer-1 1 vial containing 350 µL

Buffer-2 1 vial containing 700 µL

Warfarin Sensitivity Test Components Kit contains components and reagents for 24 tests

eSensor Warfarin Sensitivity Test Package Insert can be downloaded from GenMark’s Customer Resource

Center at http://genmarkdx.com/customer_resources.php.

Lot Number and Storage Information Package location

Reagent Storage, Handling, and Stability

• 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.

• Cartridges must be used within 14 days of opening the pouch.

• Do not use a kit after its expiration date.

Assay Set Up

Warfarin Sensitivity Test Workflow Overview

Total assay time of ~4 hours with 40 minutes of hands-on time

* Based on actual workflow studies conducted by GenMark scientists at customer sites. Times used are averages across multiple

sites for calculations.

Load PCR

reactions into

thermal cycler

Add 5 µL of

exonuclease

to each sample

PCR

Dispense sample

and hybridization

solution into

cartridges

Load cartridges onto

the eSensor® XT-8

system(s)

Generate

reports

Warfarin Sensitivity Test Workflow Overview

Capable of high-

throughput testing

using a 96-well

plate format OPTIONAL

HOLD POINT

OPTIONAL

HOLD POINT

dsDNA

Amplicon

40 µL

EXONUCLEASE

DIGESTION

Signal Buffer

70 µL

Buffer 1

10 µL

Buffer 2

20 µL

Hybridization

Solution

100 µL

ssDNA

Amplicon

140 µL

GENOTYPING

SETUP

Genomic DNA

5 µL

PCR Reaction Mix

35 µL

PCR SETUP PCR Master Mix

30 µL

PCR Mix

28 µL Taq Polymerase

2 µL

125 µL

eSensor® XT-8

Cartridge

HYBRIDIZATION / DETECTION

EXONUCLEASE SETUP Exonuclease

5 µL

PCR

Warfarin Sensitivity Test Workflow Overview

Exonuclease Technique

X

Exo addition Post-vortexing Post-centrifuging

NOTE: Dye was added to improve visualization of exonuclease and is not added to the

exonuclease supplied with the kit

Proper Cartridge Insertion Technique

X

Entering Lot Information Scan the barcode into the XT-8 system

• Frozen reagents are shipped in a

ziplock bag labeled “eSensor Warfarin

Sensitivity Test Reagents”

• 2D barcode on label is for GenMark

Internal Use Only and can’t be used

for scanning lot information into the

XT-8

• Use the barcode found on the side of

the Detection Reagents box to enter

lot information

Procedural Notes • General

– Dedicated pipettes, tips and equipment are recommended for pre- and post-amplification activities

– Unidirectional laboratory workflow is recommended

• Reagents – Frozen reagents and samples are thawed at 37 °C for 10-15

minutes or at room temperature

– Mix using a vortexer at maximum speed for 3 - 5 seconds

– Consolidate liquids by brief centrifugation (5-10 seconds) in a centrifuge or mini-centrifuge

– Do not thaw or vortex Taq polymerase or exonuclease enzymes!

• Sample Type

– Whole blood collected with EDTA as anticoagulant

– Do not freeze the blood prior to DNA extraction

– Frozen whole blood is not recommended for use with this product

– Genomic DNA Concentration: 2 ng/µL (10 ng DNA/PCR) - 100 ng/µL (500 ng DNA/PCR)

Reporting

Sample Report

• Single-page report

• Summary Section gives genotype and phenotype result

• Results Section lists results for all genotypes tested

• Information can be easily entered into Warfarin dosing algorithm

Reporting Viewing reports

• Reports can be filtered according

to different parameters: − Accession Number

− Cartridge Lot

− Date (see screen shot)

− Protocol

− User

− Batch

− Printed Reports

Warfarin Sensitivity Test Report Using GenMark reports to aid in dosing

*Source: http://www.warfarindosing.org

• Warfarin dosing algorithms can aid in estimating therapeutic doses

• Results from GenMark reports can be entered into dosing algorithms

• Free resources available to doctors and clinicians at WarfarinDosing.org*

Quality Control

Quality Control

• Training Samples – QC samples are provided for use during training, and can be used

as control material during validation

• QC Vendors

– Coriell Institute

• www.Coriell.org

• Phone: 1-856-966-7377

– Maine Molecular Quality Control Institute (MMQCI)

• www.MMQCI.com

• Phone: 1-207-885-1072

• On-Board Positive Control

– Hybridization Failure

– Detection Failure

• On-Board Negative Control

– Assay System Failure

• Metric – Shows distance of the genotyping score from the nearest

relevant boundary (indeterminate zone)

– Acceptable range 0.9 – 1.0

Quality Controls On-Board

Quality Controls No Template Control

• PCR Blank or DCM (DNA Contamination Monitor)

– Method to monitor for amplicon contamination during

setup of the reaction

– Use Nuclease-free water (Blank)

– Summary Result:

• Metric Value: 0.0 – 1.0

VALUE FOLLOW UP ACTION

0.0 No Action

0.1 – 0.8 Monitor for increasing trend

0.9 – 1.0 Decontaminate work areas and repeat entire run

Ordering and Contact Information

Warfarin Sensitivity Test Ordering Information

• Item # MT002002

• Kit Configuration 24 tests per kit

• Item # MT002402

• Kit Configuration 48 tests per kit

• Regulatory Status IVD

• Customer Service

Telephone 1-800-eSensor (373-6767), Option 1

Email CustomerService@genmarkdx.com

Fax Number 1-866-831-2001

• Technical Support

Telephone 1-800-eSensor (373-6767), Option 2

Email TechnicalSupport@genmarkdx.com