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 CustomerService@genmarkdx.com

Fax Number 1-866-831-2001

• Technical Support

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

Email TechnicalSupport@genmarkdx.com