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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 [email protected]
Fax Number 1-866-831-2001
• Technical Support
Telephone 1-800-eSensor (373-6767), Option 2
Email [email protected]