GenMark Diagnostics
eSensor® Thrombophilia Risk Test (IVD)
GNMK-IMC-6011-D
Training Presentation
Training Agenda Thrombophilia Risk Test
• Test Overview
• Kit Components and Handling
• Assay Setup
• Reporting
• Quality Control
• Ordering and Contact Information
Test Overview
Thrombophilia Risk Test (IVD) Intended Use
The eSensor® Thrombophilia Risk Test
is an in vitro diagnostic for the detection
and genotyping of Factor II
(Prothrombin), Factor V (FV Leiden) and
MTHFR (methylenetetrahydrofolate
reductase gene) C677T and A1298C
mutations in patients with suspected
thrombophilia.
*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.
Thrombophilia Risk Test Get the complete picture of inherited thrombophilia risk
• 100% accuracy and
reproducibility in clinical trials
• Single tube multiplex test for all
four thrombophilia-related
polymorphisms
• Test results in 4 hours including
extraction − ~40 minutes of hands-on time*
• Objective results automatically
generated by the XT-8 system
Gene Polymorphism
FV Leiden 1691G>A
FII Prothrombin 20210G>A
MTHFR 677C>T
1298A>C
* Based on actual workflow studies conducted by GenMark scientists at customer sites. Times used are averages across multiple
sites for calculations.
Kit Components and Handling
Thrombophilia Risk Test Components Each kit contains components and reagents for 48 tests
eSensor Thrombophilia Risk Test Package Insert can be downloaded from GenMark’s Customer Resource
Center at http://genmarkdx.com/customer_resources.php.
Kit Pack/Boxes Component Packaging & Quantity Storage
Cartridge Pack Thrombophilia Risk Test
Cartridge Pouch
6 foil bags with 8 cartridges in
each bag 10 – 25 °C
PCR Box
TRT PCR Mix 2 vials containing 1100 µL each -20 °C
(Pre-PCR Location) Taq Polymerase 1 vial containing 60 µL
Genotyping Box
Exonuclease 2 vials containing 145 µL each
-20 °C
(Post-PCR Location)
TRT Signal Buffer 2 vials containing 2500 µL each
Buffer-1 2 vials containing 350 µL
Buffer-2 2 vials containing 700 µL
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 may be frozen and thawed up to 5 times.
• Cartridges must be used within 30 days of opening the pouch.
• Do not use a kit after its expiration date.
Assay Set Up
Thrombophilia Risk 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
Thrombophilia Risk 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
TRT 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
29 µL Taq Polymerase
1 µL
125 µL
eSensor® XT-8
Cartridge
HYBRIDIZATION / DETECTION
EXONUCLEASE SETUP Exonuclease
5 µL
PCR
100 µL
Thrombophilia Risk Test Detailed Workflow
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
Thrombophilia Risk 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!
• Samples – Whole blood colleted with EDTA as anticoagulant
– 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
• Results Section lists determination made for each of the four polymorphisms
• Customized reporting options
• Reflex report option – No additional testing required
30
• Full Panel (FV, FII, MTHFR 677 & 1298)
• FII
• FV
• FII/FV
• MTHFR 677 & 1298
5 Report Types
Reflex Report Option- No need to run individual tests for each desired result
Customized Report Options
Reporting Viewing Reports
• Reports can be filtered according
to different parameters:
− Accession Number
− Cartridge Lot
− Date (see screen shot)
− Protocol
− User
− Batch
− Printed Reports
Reporting Viewing reports by accession number
• Select the desired
accession number(s) - Multiple accession numbers
can be selected for a
particular report type
• After accession number(s)
is selected, chose the
Report Type by touching
the Report Type button
Reporting Selecting the Report Type
• Users can select from 5 report
types on the Report Type list: − Full Panel:
(FV, FII, MTHFR 677 & 1298)
− FII
− FV
− FII/FV
− MTHFR 677 & 1298
• Once Report Type is chosen,
use the touch screen buttons
to view, print, or export the
report
Reporting Reflex to MTHFR 677_1298
• To reflex to MTHFR results,
select the corresponding
accession number(s)
- Multiple accession numbers
can be selected
• After accession number(s)
are chosen, select the
“FV_FII_677_1298” Report
Reporting Selecting the _677_1298 Report Type
• To view only MTHFR
results, select the
“_677_1298” report type
• Use the touch screen
buttons to view, print, or
export the report
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
• Genotyping Metric – Monitor and trend performance of test
– 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 (DCM Report)
– 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 trend of increasing value
0.9 – 1.0 Decontaminate work areas and repeat entire run
Ordering and Contact Information
Thrombophilia Risk Test Ordering Information
• Item # MT004018
• 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]