VITROS® Solutions

VITROS ® Solutions

The 10th International & 15th National Congress on Quality Improvement in Clinical

Laboratories 2017.

Teheran

© Ortho Clinical Diagnostics 2017

Agenda

2

• Company Overview

• Daily Challenges

• VITROS® Systems

• VITROS® Technologies

• Added Values

• VITROS ® Automation Solutions

Who We AreWe are a global leader of in vitro diagnostics

serving the global clinical laboratory and

immunohematology communities.

What We Do and WhyWorldwide across hospitals, hospital networks, blood

banks and labs, our high-quality products and services

enable health care professionals to make better-informed

treatment decisions to improve and save lives.

© Ortho Clinical Diagnostics 2017 5

At a GlanceA Pioneer of Life-Impacting Advances in Diagnostics – From Our Earliest

Work in Blood Typing to the Latest Developments in Laboratory Systems

120+COUNTRIES

4,200EMPLOYEES

worldwide

$1.7BIN REVENUE

75YEARS

improving and

saving lives withdiagnostics

250PRODUCTS

across instruments and assays

More than Customers in More than More than Approximately


We Reimagine What’s PossibleIt’s Defined Us for More Than 75 Years. It Inspires Us. It Drives Us Forward.

6

Organization

established (1939)

First blood bank product

launched to determine Rh+

or Rh- blood type (1947)

Introduced patented

thin-film, dry-slide

technology (1978)

Introduced the first

test for the detection

of antibodies to

hepatitis C (1989)

Launched the VITROS®

250 System (1992)Introduced the first test

kit to screen blood for

antigens to HIV-1 (1996)

Launched the

ORTHO ProVue®

Analyzer, the first

fully automated

blood banking

system in North

America (2003)

Launched the VITROS®

5600 Integrated System and

the VITROS® 3600 (2008)

Launched the

VITROS®

4600 (2011)

Became an

independent

company (2014)

Launched the

ORTHO VISION®

platform of analyzers

globally (2014-16)

Launched HIV combo

assay in Europe (2016)

Launched VITROS®

Automation Solutions

globally (2015-2016)

© Ortho Clinical Diagnostics 2017 7

Our Journey as an Independent Company

Part of The Carlyle Group portfolio

Since becoming independent

Transitioning into 2017

• One of the world’s largest and most successful investment firms, founded in 1987.

• Forty locations in 23 countries and $169B under management.

• More than 275 companies and 250 active real estate investments in portfolio.

• Renewed emphasis on our core businesses (clinical labs, immunohematology).

• Established Rochester, N.Y., as our global center of excellence for R&D.

• R&D spend is nearly $90M annually, or more than 5 percent of sales.

• Launched our ORTHO VISION® platform of analyzers for immunohematology.

• More than 1,600 new hires.

• A revitalized organization with a new spirit.

• Day 1 countries closed on June 30, 2014.

• Day 2 countries close by end of Q2 2017.

• Transition services agreement in place for key support systems.


Agenda

8





• Summary

• VITROS® Automation Solution


The Clinical Laboratory TodayYour requirements

• Trouble-free

analyzers

• Low annual

maintenance

• Reliability of patient

results

• Influence of

endogenous

Interference

• Less calibrations

• Higher throughput

• No hidden cost

• Staff optimization

Biomedical engineering Laboratory personnel Purchasingg

Quality of Results Cost ControlReliability

9


Agenda

10





• Summary


VITROS® series of Systems

11

Chemistry Immunodiagnostics Integrated

VITROS® 350 System

VITROS® 4600 System

VITROS® ECiQ System

VITROS® 3600 SystemVITROS® 5600 System


Total β-hCG II

Estradiol

Progesterone

Testosterone

LH

FSH

Prolactin

Anemia

Oncology

Thyroids

Metabolic

Diabetes

Infectious DiseasesEndocrineCardiologyProteinsTDMChemistry

DAT & Toxicology

Albumin

Alkp Phos

ALT

Ammonia

Amylase

AST

BuBc-Bilirubin

Total-Bilirubin

Total-Protein

BUN

Bicarbonate

Calcium

Chloride

Cholesterol

CK

CK-MB

Creatinine

CSF Protein

dHDL

dLDL

dTIBC

GGT

Glucose

Iron

Lactate

LDH

Lipase

Magnesium

Phosphor

Potassium

Sodium

Triglyceride

TIBC

Uric Acid

Urine-Protein

Amikacin*

Carbamazepine

Caffeine

Digoxin

Gentamycin

Lithium

Phenobarbital*

Phenytoin

Theophylline

Tobramycin

Valproic Acid

Vancomycin

Acetaminophen

Alcohol

Amphetamine

Barbiturate

Benzodiazepine

Cannabinoids

Cocaine Metabolite

Ecstasy*

Methadone

Opiate

Oxycodone*

Phencyclidine

Salicylate

THC

Alpha-1 Acid

glycoprotein *

Alpha-2

Macroglobulin*

Antithrombin III*

Apo A1

Apo B

ASO

C3

C4

Ceruloplasmin*

CRP

Fibrinogen*

hsCRP

IgA

IgG

IgM

Albumin (turbidimetric)

Micro albumin

Pre Albumin

RF

Transferrin

CK-MB

Troponin I ES

Myoglobin

NT-proBNP

Free T4

FreeT3

T4 total

T3 total

TSH 3rd Gen

T3 uptake

Homocysteine II

% Hemoglobin A1c

Cortisol (Serum & Urine)

NTx

iPTH

VIT D

C-Peptid (D)

Glucose

% Hemoglobin A1c

Insulin (D)

Total PSA II

fPSA

CEA

AFP

CA 125 II™

CA 15-3™

CA 19-9™

Ferritin

B12

Folate

HBsAg ES

HBsAg, Confirmatory

Anti-HBs

Anti-HCV

Anti-HBc

Anti-HBc IgM

HBeAg

HIV Ag/Ab Combo

Anti-Hbe

Anti-HAV Total

Anti-HAV IgM

Anti-HIV 1+2

Toxoplasma IgG

Toxoplasma IgM

Rubella IgG

Rubella IgM

CMV IgG

CMV IgM

Syphilis

Drugs

Cyclosporine *

Everolimus* (D)

Mycophenolisäure*

Tacrolimus* (D)

• (D) In develoment• (*) MPA Assay

Ortho Panel: 125 VITROS® AssaysIncl. MicroTip Partnership Assays (MPA)

New


Agenda

13





• Added Values


The Five Enabling Technologies

14


5 unique VITROS® Technologies

15

3 Assay technologies 2 Quality technologies

VITROS® MicroSlide

(or Dry slide technology) is the foundation of all

VITROS® chemistry systems.

VITROS® MicroTip

For specific proteins, TDMs and DOAs plus open

channels.

Enhanced Chemiluminescence

The critical immunoassay technology for the

VITROS® MicroWell menu, including infectious

disease

Poor sample quality, which could compromise results, is

flagged without impacting workflow by VITROS®

MicroSensor.

VITROS® Intellicheck® Technology verifies sample

integrity and reduces the risk of an incorrect result to near

zero.

VITROS® MicroWell Assay Technology

16


MicroWell Technology

17

• Ready to use, Stable reagents

Sable calibration curves (28 days)

• Use of small reaction vessels (MicroWells) with consistent

and reinforced binding capacity

Small sample volume(20 - 80 µl)

Less waist

Immune Assay Testing


Excellent sensitivity and precisionEnhanced Chemiluminescence

18

0

1

10

100

1000

0 Time (log)

Lo

g R

ela

tive

Lig

ht In

ten

sity

Direct Chemiluminescence

VITROS® MicroWell With Enhance Chemiluminescence

Direct Chemiluminescence

Increased and sustained light production compared to Direct and other Indirect methods

Excellent sensitivity & precision

Streptavidin coating

Light

Solid Phase

HRP Label


Proprietary Enhanced Chemiluminescence Detection Technology

19

• Improved Signal light output:

• Use of patented enhancer produces light output at

extremely low-analyte concentrations

• Better detection of low analyte concentrations levels

• Better performance to support medical decisions

• Excellent Sensitivity & Precision:

• More accurate results

• Ability to capture clinically significant low analyte

concentration levels

• Broad Dynamic Range:

• Less Dilutions & Repeats - Faster TAT

• Decreased Costs


Examples of current installationsin Blood Screening Centers

Site name

and City

N°Donations/YR*

Markers N° VITROS

analyzers

Automation

IT

AZ. Santaria

Provinciale Di

Ragusa, Ragusa

80,000aHCV, HbsAg, HIV,

Syphilis3 X VITROS 5600 YES

SP

Instituto Canario

Hemodonacion y

Hemote, Gran

Canaria

80,000

aHCV, HbsAg, HIV,

Syphilis2 X VITROS 3600 YES

SP

Generalitat

Valenciana

Conselsanitat,

Valencia

360,000 aHCV, HbsAg, HIV 5 XVITROS 3600YES

DKRigshospitalet -

Copenhagen150,000

aHCV, HbsAg, HIV

(Syphilis 30%)

3 X VITROS 3600

+

1 X VITROS 5600

YES

SLund University

Hospital, Lund120,000

aHCV, HbsAg, HIV

Syphilis3 X VITROS 3600

YES

IBloodbank,

Reykjavik17,000

aHCV, HbsAg, HIV1 XVITROS 3600

+

1 X VITROS ECi

NO

*Based on internal Ortho data

HIV Combo

21


VITROS® HIV Combo – Earlier Detection. Improved Diagnosis and Prevention.*

Thirty four commercially available seroconversion panels were tested on both the VITROS HIV

Combo Test and a leading commercially available CE-marked 4th generation Ag/Ab combo†

test:

•The VITROS HIV Combo Test became reactive earlier for five of the 34 panels.

•The commercially available CE-marked 4th generation Ag/Ab combo test became reactive

earlier for only one of the 34 panels‡

22

VITROS® HIV Combo detected acute HIV-1 infection earlierthan a leading commercially available test, in seroconversion panels.

*VITROS® Immunodiagnostic Products HIV Combo Assay IFU†Abbott ARCHITECT HIV Ag/Ab Combo‡Agreement for 28 of the 34 panels.

§Acute HIV-1 Infection

Seroconversion panels are a group of serial bleeds

from plasma donors during early stages of infection.

Seroconversion SensitivityEarlier reactivity on bleeds of

seroconversion panels.

Provides early detection of

acute HIV-1 infection§


VITROS® HIV Combo –Class-Leading Analytical Sensitivity – p24 Detection*

23

VITROS® HIV Combo detected p24 at lower concentrationsthan a leading commercially available test

Detection of HIV-1 viral nucleic acid with

Nucleic Acid Test (NAT) remains the most

sensitive method in identifying acute HIV-

1 infection but its use is not widespread

due to associated cost, time and labor.

Analytical SensitivityNIBSC/WHO : 0.48IU/mL

AFSSAPS : 13.1pg/mL

Provides assurance in

detecting infection

*VITROS® Immunodiagnostic Products HIV Combo Assay IFU


VITROS® HIV Combo –Excellent Clinical Sensitivity*

• 500 samples previously determined as positive became reactive (400 for HIV-1

and 100 for HIV-2)

• 83 samples known to be infected with HIV-1 group M tested – all found to be

reactive.

24

VITROS® HIV Combo has a clinical sensitivity of 100%.


Clinical Sensitivity100% (CI 99.26% - 100%)


detecting infection


VITROS® HIV Combo –Detection of HIV Subtypes*

HIV-1 Group M subtypes:

Three HIV-1 group M subtype

specimens tested reactive by

both VITROS® HIV Combo and

Commercially Available 4th

Generation HIV Ag/Ab Test.

49 viral isolates were tested with

both these tests:

25

In detecting a wide panel of subtypes, VITROS® HIV Combo was similar or better compared to a leading commercially available 4th Generation HIV Ag/Ab Test

*VITROS® Immunodiagnostic Products HIV Combo Assay IFU†Countries of Origin specified in Instructions for Use.

Testing performed on VITROS 3600 Immunodiagnostic System.

HIV Subtypes DetectionWide range of subtype detection


detecting infection

Number ofSpecimens Tested†

VITROS® HIV Combo Test

Leading Commercially Available 4th Generation HIV Ag/Ab Test

Viral Isolates*

Subtype A 4 4 4

Subtype B 8 8 7

Subtype C 7 5 5

Subtype D 3 3 3

Subtype F 5 5 5

Subtype G 2 2 1

Subtype H 1 1 1

Subtype CRF01_AE 10 10 10

Subtype CRF01_AE 3 3 3

Subtype Group O 4 4 4

Subtype IIIB 1 1 0

Subtype HIV-2 1 1 1


VITROS® HIV Combo – Competitive Performance Uncompromised Specificity*

Testing performed at two external independent hospital laboratories.

Assay specificity assessed using 5077 blood donor samples and 607 samples from

hospitalized patients.

26

High sensitivity while maintaining specificity.


Clinical Specificity99.84% (Donor Population, CI 99.69-99.93%)

100% (Clinical Population, CI 99.39-100%)

Fewer false positives to

save time, cost and labor

in repeat and

confirmatory tests


VITROS® HIV Combo –Excellent Precision*

Two replicates each of 14 negative or diluted reactive patient sample pools and 5

control samples were tested on 2 separate occasions per day on at least 20

different days.

The experiment was performed using 2 reagent lots on 2 different systems on the

VITROS ECi/ECiQ, 3600 and 5600 Immunodiagnostic Systems.

27

Reliable results due to excellent precision.

*VITROS® Immunodiagnostic Products HIV Combo Assay IFU†Clinical and Laboratory Standards Institute

PrecisionEvaluated consistent with CLSI†

document EP5

Excellent precision ensures

consistency

of test results


VITROS® HIV Combo –Excellent Precision*

Performed using two

reagent lots on two

different VITROS®

3600 systems.

Data on VITROS® 5600

and ECi/ECiQ is found

in IFU

28

Reliable results due to excellent precision.


Panel Member Mean S/CWithin-run*

CV (%)

Within-calibration**

CV (%)

Within-lab***

CV (%)

Anti-HIV-1 0.56 4.4 9.3 9.3

Anti-HIV-1 0.98 3.2 7.8 7.6

Anti-HIV-1 2.17 2.7 5.7 5.4

Anti -HIV-1 Reactive Control 1.90 3.8 6.1 6.0

Anti-HIV-2 0.72 5.1 10.1 10.0

Anti-HIV-2 1.04 3.7 7.3 7.0

Anti-HIV-2 2.44 3.1 5.4 5.1

Anti -HIV-2 Reactive Control 4.20 3.0 4.6 4.5

Anti-HIV-1 Group O 0.86 5.2 8.7 8.7

Anti-HIV-1 Group O 1.10 4.2 7.3 7.3

Anti-HIV-1 Group O 2.38 4.0 5.6 5.6

Anti -HIV-1 Group O Reactive Control 3.30 3.3 5.3 5.0

HIV p24 Ag 0.78 2.3 7.4 7.5

HIV p24 Ag 1.40 2.0 5.3 5.4

HIV p24 Ag 3.33 1.5 3.3 3.4

HIV p24 Ag Reactive Control 1.92 1.7 4.7 4.7

NT-pro BNPVideo

29

../../Documents/Reference documents/Meetings/Turkey Distributor meeting 2017/NT-proBNP Full Video.mp4

VITROS® MicroSlide Assay Technology


MicroSlide: The ongoing revolution

Colorimetric Potentiometric Immuno-Rate

Colorimetric MicroSlides


Colorimetric MicroSlide Layers

Spreading Layer

Reagent Layer

Registration/Indicator Layer

Support Layer

Scavenger & Masking Layers


Registration/Indicator Layer

Color formation occurs here

- Color intensity is proportional to analyte

concentrationkkkkkkkkkkkkkkkkkkkkkkkkkkkkkkkkkkkkk

kkkkkkkkkkkkkkkkkkkkkkkkkkkkkkk


Transmission Spectrophotometry: Traditional Liquid Chemistry

• Refracted light in which

light passes through a

liquid

• Must also pass through

interfering substances


Reflectance Spectrophotometry: MicroSlide

• Reflected light to measure

amount of reaction product

• Interfering substances are much

less likely to affect test results

because they are captured by

other layers

Potentiometric MicroSlidesDirect ISE


Potentiometric MicroSlides™ Layers

Patient’s Sample Reference fluid

Ion-Selective Membrane

Internal Reference Layer

Ion-Selective Membrane

AgCl

Ag

Paper Bridge


Potentiometric MicroSlides™

S1 – unknown solution

S2 – reference solution (known)

S3 and S4 – internal reference solutions (known)

S1 – sample

S2 – ERF (known)

S3 and S4 – internal reference layers (known)


Electrometer

Technologies overview - MicroSlide™

DEISE = SAMPLE - ERF


Benefits of MicroSlide™ Technology

No reagent preparation

Extended reagent shelf expiration

On board stability 2 days-5 weeks

Infrequent calibration

Use of disposable VersaTips

No sample cross contamination

Small sample size

Low sensitivity to visual interference

No water, plumbing, or drains

Minimal waste


Direct vs Indirect ISE’s

• All ISE’s measure the activity of an ion

• Direct ISE’s do not dilute the sample prior to analysis

• Indirect ISE’s dilute the sample prior to analysis

The difference between direct and indirect ISE’s

is the impact of sample dilution on ion activity


Electrolyte Exclusion Effect

Indirect ISE compensate for the presence of solids (proteins/lipids) in a sample

• Assumption: all samples contain 7% solids

• If a sample contains > or < than 7% solids, the result calculated for

an indirect ISE method will be inaccurate

• This inaccuracy is particularly important for sodium, which has a very

narrow normal range (140 mmol/L +/- 5 mmol/L)

Source: Sulaiman et al. Clinica Chimica Acta (2011); 412: 1-6.


• At LOW TP levels:

the VITROS SODIUM will be LOWER THAN INDIRECT methods.

e.g. at TP of 4 g/dL (40 g/L) VITROS Na will be

LOWER by approximately 3-5 mmol/L

NOTE: The VITROS result is considered

the MORE PHYSIOLOGICALLY CORRECT RESULT.

• At NORMAL TP levels:

the VITROS SODIUM should AGREE with Indirect methods.

• At HIGH TP levels :

the VITROS SODIUM will be HIGHER THAN INDIRECT methods.

e.g. at TP of 10 g/dL (100 g/L) VITROS Na will be

HIGHER by approximately 3-5 mmol/L

NOTE: The VITROS result is considered

the MORE PHYSIOLOGICALLY CORRECT RESULT.

MicroTip Assay Technology

53


VITROS® MicroTip Reagents

54

• Liquid, ready-to-use — no reagent preparation

• Integrated reagent pack design accommodates single and

dual reagents

• 36 MicroTip reagent positions = 36 MicroTip assays on board

• Refrigerated storage maximizes on system stability

• Reagent on-board stability of 7-28 days

• Conical design and level sensing minimizes dead volume

and waste

• Convenient packaging size minimizes waste


VITROS® MicroTip Reagents Packs

55

• Automatic capping/decapping of reagents extends reagent and calibration

stability

• Eliminates contamination, evaporation, and reagent cross-talk

• Enhances reagent and calibration stability

• Calibration stability up to lot change


VITROS® FS Cuvettes

56

• Single-use reaction cuvettes

• Optimize Labor

• No maintenance

• Minimize Costs

• No expensive water system required

• Six reaction cells per cuvette strip

=

• Six results (no waste)

air pocket insulates cuvettes


VITROS® FS Cuvettes

57

• Ensure quality results

• No sample and reagent carryover

• No reagent evaporation or cross contamination

• 3-Point cuvette read

• Detects bubbles and other imperfections in read

window if present

• Notifies operator and prevents erroneous result

reporting

Read windows

Increases confidence in the quality of the test result


VITROS® Single-Use Tips

58

VITROS® VersaTip

• Pipettes patient sample and dilutions

• When sealed as a CuveTip, it enables

MicroSensor™ sample quality assessment

• Minimizes dead volume requirements

VITROS® MicroTip

• Aspirates the sample from the CuveTip

• Mixes the sample/reagent in the cuvette

• Pipettes patient sample and MicroTip

reagents into cuvettes, minimizing dead

volume

No sample or reagent carryover or extensive maintenance


VITROS® MicroTip Technology Keeps it SIMPLE

59

Feature MicroTip Technology Traditional Wet Chemistry

Single-use tips

No priming or purging

No sample/reagent carryover

Single-use cuvettes

Capped reagents

Water-free

No drains, plumbing, mixing

assemblies

Air-controlled incubator

Intellicheck® Technology

MicroSensor Technology

Intelligent Parallel Processing

Minimal calibration frequency

Minimal maintenance

MPA-UDA MicroTip Assay

62


UDA/ MPA Definitions

UDA= User Defined Assay

An assay that is run on an open channel using reagents supplied by a third party (in some

cases, reagents can also be supplied by Ortho).

The validation of the assay is the customer’s responsibility.

Ortho can suggest these solutions to customers in response to tenders, but not as a validated

solution.

MPA = Micro Tip Partnership Assay (*)

An assay that is run on an open channel using reagents validated by a third party in

collaboration with Ortho for use on the VITROS® 5,1 FS, 4600 and 5600 Integrated System

with the User Defined Assay (UDA) software using MicroTip Technology.

The reagents can be supplied by Ortho for markets where this has been agreed with the third

party.

(*) This was previously called Manufacturer Validated Assay (MVA)

MicroSensor® Technology


VITROS®

MicroSensor Technology

65

Semi Quantification and flagging of

samples for:

• Hemolysis

• Icterus

• Turbidity


MicroSensor ® - How it Works

Intellicheck ® Technology

67


VITROS® SMART Metering

68

Precise sample volume plus detection of air bubbles, clots, fibrin and viscosity

Patented pressure-level-sensing technology

VITROS® disposable sample and

reagents tips


Analytical control process for each test

High Quality–Intellicheck® Technology

• Proprietary technology provides unique results integrity

– Designed to significantly reduce

analytical errors

• SMART Metering

• MicroSensor

• IntelliReport

– Integrated Process Control

– Traceability with real-time

documentation for exceptions

– Prevents reporting of results that

may be affected by exceptions


6

7

9

8

IntelliReport


Reports the quality of each result

Lumino-meter

9

AddReagent 2

AddReagent 1

Well Shuttle to Inner Ring

Incubation

4

Discard to waste

1

3

Microwell Dispense

1

Sample Metering

2

Reagent Metering

3

SignalReagent

7

Incubate4.5 mins

8

MicrowellWash

6

AddSample

105

10

4

2



IntelliReport

• Verifies the quality of

each result reported

•Traceability of all results

•Real-time and retrospective quality verification for each result

• Displays Intellicheck® Technology

Verifications performed

through system processing

Provides real-time status report on every result


Agenda

72





• Added Values



Minimum maintenance and calibration timeWith VITROS® Analyzers

73

Significantly fewer calibrations allow more cost transparency

Non- VITROS®

Systems

3,000

1,000

0

5,000

VITROS®

Systems

Nu

mb

er

of C

alib

ratio

ns p

er

Ye

ar

4,000

2,000

Expected

Actual

Data from on-site studies commissioned by Ortho Clinical Diagnostics.

For a typical laboratory with 1 million tests / year


Efficient use of VITROS® systems

74

VITROS® 5600 Integrated analyzer can save

57 (8-hour) working days per year versus the average non-VITROS

systems

0 20 40 60 80 100 120 140

Maintenance Cal/QC Service Downtime

Time the system is not available

VITROS® 5600

Non-VITROS®

System

Data from on-site studies commissioned by Ortho Clinical Diagnostics.

~ 90 min

Min. / System / Day


Efficient reagent use

75

• Any test that does not provide a useful result

will reduce the overall efficiency, which in turn

has an influence on the staff, the TAT and cost

containment

• Inefficient use of the reagents:

• Repeats

• More calibration & QC

94%VITROS® Reagent efficiency

84%Non-VITROS® Reagent

efficiencyData from on-site studies commissioned by Ortho Clinical Diagnostics


VITROS® Systems Efficiency

76

VITROS® 5600

System

Non-VITROS®

System

Less interventions lead to

Reduction of possible errors and delayed results by 12%

96%80

60

20

0

%

40

100

First Pass Yield

(% Of tubes without intervention)

84%

How does this impact your patient results and ongoing costs?


Comparison of non-VITROS® systems with VITROS® systems

With an annual sample volume of 1,000,000 analyzes

VITROS® Systems Efficiency

77

120.000 more Tests / Year

Examination of ~ 15,000 patients

Without errors, delays or additional costs

VITROS® 5600

System

Non-VITROS®

System

96%80

60

20

0

%

40

100

84%


Removing water as a potential source of quality issues

78

Waterless Technology has a huge impact for the clinical laboratory

VITROS® technologies reduce the environmental impact of laboratories

• Removes water as a potential source of poor quality results

• No cost involved for water purification systems

• No resource needed for testing water quality


Agenda

79





• Summary


VITROS ® Automation Solution. VAS

80


HematologyVITROS ®

Immunodiagnostic

Systems

VITROS ®

Chemistry SystemsCoagulation

Provides multidisciplinary connection to extend service to clinicians

Laboratory Information System

INSTRUMENT MANAGER™ for VITROS ®

Automation

TCAutomation

Autoverification Quality controlLaboratory

MaintenanceLaboratory

Intelligence

Open


Start with a workcell

Add analyzerAdd pre-analytical

Connect other disciplines

Aligns hardware and software to actual laboratory activity

Flexible


Grow at your own pace without having to completely redesign your lab

Appropriate automation

“We have relied on Ortho Clinical Diagnostics VITROS® Automation Solutions through a decade of

successful upgrades, reconfiguration and relocation. Their durability and adaptability have

proved to be crucial.” Lab Manager, Belgium.

Volume ROI

Scalable


VITROS® Automation Solutions

WorkcellsIntegrated

AutomationSolutions

Powerful Combinations

LABORATORY ECONOMIC

• Consistent TAT• Reduces errors

• Streamlines operations

• Staged investment• Maximized ROI

• Improved staff utilization

Q & A

Thank You

StripAssay® Technology for inherited diseases, genetic predispositions, pharmacogenomics and oncology diagnosticsDr. Christian OberkaninsIQC Tehran / April 23, 2017

no. of markers format/platform state

low (1-3) RealFastTM currently used

isothermal amplification under evaluation

medium (3-40) StripAssay® currently used

high (>40) array platform under evaluation

next-generation sequencing under evaluation

Technologies

RealFastTM Technology

• dual-labeled, allele-specific probes cleaved by Taq polymerase• physical separation of the fluorophore from the quencher dye

generates a fluorescent signal in real time• signal is proportional to the accumulated PCR product

RealFastTM: fluorogenic 5‘ nuclease (TaqMan®) assays

RealFastTM technology

RealFastTM assays are validated for use on: AB 7500 Fast AB StepOne Roche Lightcycler 480 II Biorad CFX96 Qiagen Rotorgene 6000 Agilent Mx3005P new: MIC (magnetic induction cycler)

RealFastTM features

• small and compact (2 kg)• 2 and 4 optical channels available• very fast thermocycling

(35 PCR cycles in less than 25 min)

• Cardio-Vascular Disease (CVD):FV Leiden, Prothrombin (FII) 20210G>A, MTHFR 677C>T & 1298A>C. PAI-1 4G/5G, Beta-Fibrinogen (FGB) -455G>A, FXII 46C>T, FXIII V34L

• Coumarin anticoagulation:VKORC1 -1639G>A, CYP2C9 *2/*3

• Haemochromatosis:HFE C282Y & H63D

• Lactose intolerance:LCT -13910T>C

• HLA:HLA-B*27 (ankylosing spondylitis)HLA-B*5701 (abacavir hypersensitivity), HLA-B*1502 (carbamazepine hypersensitivity)

RealFastTM applications

• Statin-induced myopathy:SLCO1B1 c.521T>C

• HCV treatment & spontaneous clearance:IL28B

• Gene copy number variation (CNV):CYP2D6 (response to various drugs)CYP21A2 (congenital adrenal hyperplasia - CAH)

• in preparation:alpha-1 antitrypsin (AAT) (COPD risk)Human Papillomavirus (HPV) screening & genotypingMultiplexed versions (4 dyes: FAM, HEX, ROX, Cy5)oncology applications (liquid biopsy!).....

RealFastTM applications

StripAssay® Technology

Human diagnostic assays for genotyping mutations and polymorphisms (SNPs, deletions, insertions) based on polymerase chain reaction (PCR) and reverse-hybridzation to allele-specific oligonucleotide probes immobilized on teststrips

StripAssay®

Technology

or Hybridization and stringent washes at exactly 45ºC

IMPORTANT:no hot-air oven etc. !!!

• simple protocol• inexpensive equipment• less than 6 hours total

assay time• ready-to-use reagents• easy and clear

interpretation of results• only 5-10 ng sample

required

StripAssay® Protocol

for 48 teststrips: Roboblot or ProfiBlot T48 or BeeBlot

Hybridization + Stringent Washes + Color Development fully automated under controlled temperature on one instrument

for 20/30 teststrips: Dynablot Heat or Autoblot 3000H or B20

StripAssay® Protocol

Optional: Automated processing of teststrips

Typical StripAssay® result

EvaluatorTM software

RECORDING, INTERPRETING and electronic ARCHIVING of StripAssay results

Automated StripAssay® Interpretation

Hereditary disorders:germline (i.e. inherited) mutations directly causing a disorder

• alpha- and beta-thalassemia & beta-thal modifier• cystic fibrosis (CF)• familial Mediterranean fever (FMF)• haemochromatosis• Gaucher's Disease• sugar (lactose/fructose) intolerance• congenital adrenal hyperplasia (CAH)

Predispositions:germline mutations confering increased or decresed risk to develop a disease

• cardio-vascular disease (CVD) risk factors• HLA-B27 (risk for ankylosing spondylitis)

Current StripAssay®

applications / 1

Pharmacogenomics:germline mutations determining response to therapy (resistance, side effects, optimal dosing, ...)

• response to coumarin anti-coagulants (VKORC1/CYP2C9)• response to clopidogrel/Plavix® anti-coagulant (CYP2C19)• response to HIV highly active anti-retroviral therapy "HAART"• response to thiopurines (TPMT)• response to tamoxifen therapy (CYP2D6)• response to 5-FU chemotherapy (DPYD)• response to IgG therapy in general (FCGR: H133R, F158V)


applications / 2

Oncology:somatic (i.e. aquired) mutations causing tumors or determining response to anti-cancer therapy

• response to anti-EGFR colon cancer therapy:KRAS mutations (sensitivity: 1%): exon 2 (cd 12/13), exon 3 (cd 59/60/61), exon 4 (cd 117/146)NRAS mutations (sensitivity: 1%): exon 2 (cd 12/13), exon 3 (cd 59/80/61), exon 4 (cd 146)

• response to anti-BRAF melanoma therapy:BRAF mutations (sensitivity: 1%): exon 15 (cd 600/601)

• response to anti-EGFR lung cancer therapy:EGFR mutations (sensitivity: 1%): exons 18/19/20/21


applications / 3

RealFastTM plusStripAssay®

For certain applications, RealFastTM Assay and StripAssay® must be used in combination:

1) PGX-CYP2D6 XL StripAssay ® (4-770) to be released Q2/20172) to be released Q2/2017

Point mutations and small InDels (SNPs)

Gene copy number variations (CNVs)

Congenital Adrenal Hyperplasia (CAH)

CAH StripAssay®

(cat.no. 4-380)CAH RealFastTM CNV Assay®

(cat.no. 7-410)

CYP2D6pharmacogenetics

PGX-CYP2D6 StripAssay®

(cat.no. 4-760)1)CYP2D6 RealFastTM CNV Assay

(cat.no. 7-420)2)

StripAssay® Applications for Human Genetics and Pharmacogenomics

Hemoglobin A molecules consist of two alpha- and two beta-globin polypeptide chains plus iron-containing heme groups

Alpha- & Beta-globin StripAssays:

Mutations in globin genes may lead to:• structurally abnormal hemoglobins with altered oxygen-binding and

stability properties (e.g. HbS: "sickle cell anemia")• synthesis disfunctions ("thalassemias") generating hemoglobin molecules

of altered polypeptide chain composition (e.g. HbH: 4 beta-globin chains)

Alpha- and beta-globin mutations are among the most common inherited disorders along the so-called "thalassemia belt"

alpha-globin mutations: mainly large deletions of global prevalencebeta-globin mutations: mainly point-mutations and highly population-specific


Diagnosis of thalassemias and hemoglobin variants usually involves the analysis of hematological parameters and globin genotyping


Genotyping alone is also used for:• carrier screening• prenatal diagnosis (PND) and preimplantation genetic diagnosis (PGD)

ViennaLab offers the globally applicable Alpha-globin StripAssay and three tailored Beta-globin StripAssays for the Mediterranean area (MED), India and Middle East (IME) and Southeast Asia (SEA)


Alpha-globin StripAssay21 mutations detectable(2 teststrips per sample)

Beta-globin StripAssays22 mutations detectable each

Alpha- and Beta-globin StripAssays have more than 90% coverage of known mutations in their respective target areas


beta-globinMED beta-globin

IME

beta-globinSEA

alpha- globin

• beta-thalassemia phenotypes are variable, ranging from the severe transfusion-dependent thalassemia (thal) major to the mild form of thalassemia (thal) intermedia

• thal major patients have severe anemia and usually require medical attention within the first two years of life; without treatment, affected children have severely compromised growth and development and shortened life expectancy

• thal intermedia patients present later in life, have milder anemiaand never or only rarely require blood transfusion; occasionally thal intermedia patients are completely asymptomatic until adult life with only mild anemia

Beta-thal Modifier StripAssay:

• phenotypical variability is mainly associated with the type of beta-globin mutation, the coinheritance of alpha-thalassemia and the ability for persistent production of fetal haemoglobin (HbF) in adult life

• quantitative trait loci (QTL) known to account for HbF variability comprise single nucleotide polymorphisms (SNPs) in the: gamma-globin gene promoter (HBG2): rs7482144

("XmnI"; g.-158 C>T) BCL11A gene: rs1427407 and rs10189857 HBS1L-MYB intergenic region: rs28384513 and rs9399137


Haematologica 2011; 96(11):1712-4



Haematologica 2015; 100(4):452-7


http://tss.unica.it.INPUT:• gender• beta phenotype• alpha genes defect• HBG2• HBS1L-MYB• BCL11A


Red Marker Line (top)

Control

1 HBG2 g.-158 [C>T] T

2 HBG2 g.-158 [C>T] C

3 BCL11A rs1427407 [G>T] T

4 BCL11A rs1427407 [G>T] G

5 BCL11A rs10189857 [A>G] G

6 BCL11A rs10189857 [A>G] A

7 HBS1L-MYB rs28384513 [A>C] C

8 HBS1L-MYB rs28384513 [A>C] A

9 HBS1L-MYB rs9399137 [T>C] C

10 HBS1L-MYB rs9399137 [T>C] T

Green Marker Line (bottom)

Beta-thal Modifier StripAssay

5 SNPs in 3 Genes

CVD StripAssays:

Cardio-Vascular Disease (CVD) is a diverse group of disorders of the heart and blood vessels

Two major manifestations: venous thromboembolismand atherosclerosis

CVD StripAssays:CVD is a leading cause of morbidity and mortality: it causes nearly half of all deaths in Europe (48%) and the USA (41%) and a third of global deaths

Multifactorial origin: environmental & genetic risk factors predispose to CVD[Sources: World Health Organization (WHO), American Heart Association (AHA), European Heart Network (EHN)]

Environmental risk factors:• tobacco smoke• physical inactivity• overweight and obesity• alcohol and drug abuse• stress• .....

CVD StripAssays:Inherited risk factors:• mutated blood coagulation factors• mutated lipoproteins• mutated regulators of blood pressure• mutated regulators of homocysteine• mutated regulators of inflammation• .....

Currently 8 different CVD-related StripAssays covering 17 mutations or polymorphisms in 13 genes

CVD StripAssays:

CVD StripAssay(generic)

12 mutations

CVD StripAssay A(atherosclerosis)

9 mutations

CVD StripAssay T(thrombosis)9 mutations

CVD: Pregnancy complications"Vienna recurrent pregnancy loss study"genetic CVD risk factors were compared between patients with a history of RPL and a matched control group using CVD StripAssays

CVD: Pregnancy complications"Vienna recurrent pregnancy loss study"

PAI-1 4G, FXIII 34L and the angiotensin-converting enzyme (ACE) Del allele act synergistically towards RPL; highly increased risk for RPL in carriers of 2 or more CVD risk mutations

Dossenbach-Glaninger et al. (2008), Br.J.Haematol 141, 269-271

Genotype relative risk (odds ratio)

FXIII V34L 1.9

FXIII V34L + PAI-1 4G/5G 2.4

ACE Ins/Del 3.1

2 or more risk factors 4.4

CVD: Pregnancy complications"Vienna recurrent pregnancy loss study"

Genotype relative risk (odds ratio)

FV Leiden 2.2

EPCR A1 0.8

FV Leiden + EPCR A1 1.0

..... in carriers of FV Leiden the A1 haplotype decreases the relative risk for recurrent pregnancy loss .....

Hopmeier et al. (2008) Clin Biochem 41, 1022-24

Cystic Fibrosis (CF) is the most common life-limiting genetic disorder (incidence approx. 1:3,000 live births in Caucasians)

CF is caused by mutations in the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) gene

Newborn screening programs based on immunoreactive trypsinogen (IRT), sweat cloride and genetic testing are widely in place

CF StripAssay:

• more than 1000 CFTR mutations described to date

• type and distribution vary widely by ethnicity and geographic origin

• F508del accounts for 30-72% of CF chromosomes depending upon ethnicity

CF StripAssay:

Currently 4 different CF StripAssays covering 46 mutations plus IVS8 5T/7T/9T polymorphism

CF StripAssays:

Red Marker Line

Control

1 CFTRdel2,3 (21kb) mutant

2 I507del (-ATC) mutant

3 F508del (-CTT) mutant

4 1717-1G>A mutant

5 G542X mutant

6 G551D mutant

7 R553X mutant

8 R560T mutant

9 2143delT mutant

10 2183AA>G mutant

11 2184delA mutant

12 2184insA mutant

13 2789+5G>A mutant

14 R1162X mutant

15 3659delC mutant

16 3905insT mutant

17 W1282X mutant

18 N1303K mutant

19 CFTRdel2,3 (21kb) wild type

20 codon 507 to 508 wild type

21 1717-1G>A wild type

22 codon 542 wild type

23 codon 551 to 553 wild type


25 2143delT wild type

26 2183AA>G+2184delA/insA wild type

27 2789+5G>A wild type


29 3659delC wild type

30 3905insT wild type



Green Marker Line

MU

TA

NT

WIL

DT

YP

E

Red Marker Line

Control

33 G85E mutant

34 394delTT mutant

35 R117H mutant

36 Y122X mutant

37 621+1G>T mutant

38 711+1G>T mutant

39 1078delT mutant

40 R334W mutant

41 R347H mutant

42 R347P mutant

43 A455E mutant

44 1898+1G>A mutant

45 3120+1G>A mutant

46 3272-26A>G mutant

47 Y1092X mutant

48 3849+10kbC>T mutant

49 codon 85 + 394delTT wild type



52 621+1G>T wild type

53 711+1G>T wild type

54 1078delT wild type






60 3272-26A>G wild type


62 3849+10kbC>T wild type

63 IVS8 5T variant

64 IVS8 7T variant

65 IVS8 9T variant

Blue Marker Line

MU

TA

NT

WIL

DT

YP

E

CF StripAssay34 mutations plus

1 polymorphism detectable(2 teststrips per sample)

• Familial Mediterranean Fever (FMF) is a hereditary inflammatory disorder caused by mutations in the MEFV (marenostrin) gene

• short recurrent attacks of fever accompanied by pain in the abdomen, chest or joints

FMF StripAssay:

• predominantly affects Turks, Arabs, Armenians and Sephardic Jews, and is observed throughout the entire Mediterranean and Middle Eastern area

• most severe complication: progressive amyloidosis leading to end-stage renal failure (risk factor: SAA1 genotype)

• upon correct diagnosis treatable with colchicine

FMF StripAssay:

Ozen & Bilginer(Natue Reviews Rheumatology 2014)

FMF and SAA1 StripAssays:

FMF StripAssay12 MEFV mutations


Control

1 E148Q mutant c.442G>C

2 P369S mutant c.1105C>T

3 F479L mutant c.1437C>G

4 M680I (G/C) mutant c.2040G>C

5 M680I (G/A) mutant c.2040G>A

6 I692del mutant c.2076_2078del

7 M694V mutant c.2080A>G

8 M694I mutant c.2082G>A

9 K695R mutant c.2084A>G

10 V726A mutant c.2177T>C

11 A744S mutant c.2230G>T

12 R761H mutant c.2282G>A





17 codon 692-695 wild type





MU

TA

NT

WIL

DT

YP

E


Control

1 E148Q mutant c.442G>C

2 P369S mutant c.1105C>T

3 F479L mutant c.1437C>G

4 M680I (G/C) mutant c.2040G>C

5 M680I (G/A) mutant c.2040G>A

6 I692del mutant c.2076_2078del

7 M694V mutant c.2080A>G

8 M694I mutant c.2082G>A

9 K695R mutant c.2084A>G

10 V726A mutant c.2177T>C

11 A744S mutant c.2230G>T

12 R761H mutant c.2282G>A





17 codon 692-695 wild type




21 SAA1 c.209C>T T

22 SAA1 c.224T>C C

23 SAA1 c.209C>T C

24 SAA1 c.224T>C T


ME

FV

MU

TA

NT

ME

FV

WIL

DT

YP

ES

AA

1

FMF-SAA1 StripAssay12 MEFV mutations

SAA1 isoforms 1.1/1.3/1.5

• Congenital adrenal hyperplasia (CAH) comprises inborn errors in the synthesis of adrenal corticoid hormones

• average incidence approx. 1:15,000 births worldwide

• more than 90% of cases arise from mutations in the CYP21A2 gene

• severe forms cause life-threatening salt-wasting crisis - treatable!

• newborn screening programs based on 17-hydroxyprogesterone (17-OHP) levels have been introduced, but have a high false positive recall rate

• concurrent genetic testing saves costs and efforts for repeated testing and minimizes emotional stress for parents

CAH StripAssay:

CAH StripAssay:

CAH StripAssay:


Control

1 P30L c.89C>T

2 I2 splice c.290-13A/C>G

3 Del 8 bp E3 c.329_336del GAGACTAC

4 I172N c.515T>A

5 Cluster E6 c.707T>A,c.710T>A,c.716T>A

6 V281L c.841G>T

7 L307 frameshift c.920_921insT

8 Q318X c.952C>T

9 R356W c.1066C>T

10 P453S c.1357C>T

11 R483P c.1448G>C

12 codon 30

13 I2

14 E3

15 codon 172

16 E6

17 codon 281

18 codon 307

19 codon 318

20 codon 356

21 codon 453

22 codon 483


MU

TA

NT

WIL

DT

YP

E

CAH StripAssay11 mutations

CAH RealFast CNV Assaycopy nuber variations

CYP21A2 gene alterations found in CAH patients:• 60-70% point mutations and small InDels• 30-40% large conversions, deletions, duplications

• Coumarin derivatives (e.g. warfarin, phenprocoumon) are the most widespread oral anticoagulant drugs for the prevention and treatment of thromboembolic disorders.

• Coumarins have a narrow therapeutic range and a wide inter-individual variability in dose requirement.

• delays in achieving a stable maintenance dose and adverse eventsare frequently encountered during the initial phase of therapy(overdosing -> bleeding; underdosing -> insufficient anticoagulation).

• known parameters influencing coumarin doserequirement include age, sex, body size andweight, co-medication and genetic factors.

PGX-Thrombo StripAssay:

• the VKORC1 -1639 G>A polymorphism and the functionally defective cytochromes CYP2C9 *2 and *3 account for the majority of variations in the therapeutic response to warfarin.

• based on these data refined warfarin dosing algorithms were developed.• free web-based calculator: www.warfarindosing.org


Sconce et al. (Blood 2005)

PGX-Thrombo StripAssay

3 SNPs in 2 Genes



Control

1 VKORC1 -1639 G>A mutant (A)

2 VKORC1 -1639 G>A wild type (G)

3 CYP2C9 1075 A>C mutant

4 CYP2C9 430 C>T mutant

5 CYP2C9 1075 A>C wild type

6 CYP2C9 430 C>T wild type


Genotyping workflow from blood

StripAssays

DNA extraction

RealFast assays

DNA extraction

PCR PCR & Detection

Hybridization & Detection

6h

2.5h

5h

1.5h

Direct-to-PCR

Direct-To-PCR workflow

Blood

collection(EDTA or citrate tubes)

Saliva

collection(designated devices)

various approachesunder evaluation

(dilution, heating, ..)

various approachesunder evaluation

(dilution, heating, ..)

PCR PCR

Direct-to-PCR

Multi marker testingCVD StripAssay T

blood-derivedDNA DNA

Direct-to-PCRDNA

Single marker testingMTHFR 677C>T RealFast AssayTM

MUT-Ctrl

WT-Ctrl

HET-Ctrl

Direct-to-PCR

purified DNA

• heat denaturation of blood sample (10 min at 98°C)• 5 µl of clear supernantant added to PCR

MTHFR 677C>T

Direct-to-PCR

Sample type: anticoagulated blood

Multi marker testingCVD StripAssay T

→ B

→ A

→ C→ D

blood-derivedDNA DNA

salivaryDNA

salivaDirect-to-PCR

Single marker testingFV Leiden

RealFast AssayTMPTH 20210G>A

RealFast AssayTM

MTHFR 677C>TRealFast AssayTM

MTHFR 1298A>CRealFast AssayTM

A B

C D

Direct-to-PCR

Sample type: saliva• DNA•SALTM Saliva DNA Collection Device• sample dilution buffer incl. detergents

StripAssay® Applications for Oncology

Prognostic biomarker:

Indicates the likely course of the diseasein an untreated individual.

Predictive biomarker:

Identifies subpopulations of patients whoare most likely to respond to a given therapy.

CANCER DRUG TRADE NAME TARGET PREDICTIVE BIOMARKER

ColorectalCetuximab ERBITUX EGFR KRAS, NRASPanitumumab VECTIBIX EGFR KRAS, NRAS

Lung

Afatinib GILOTRIF EGFR, HER2, HER4 EGFRAlectinib ALECENSA ALK, RET ALKCeritinib ZYKADIA ALK, IGFR-1R, InsR, ROS1 ALK Crizotinib XALKORI ALK, ROS1, cMET, RON, HGFR ALK, ROS1Erlotinib TARCEVA EGFR EGFRGefitinib IRESSA EGFR EGFROsimertinib TAGRISSO EGFR EGFR T790M

Melanoma

Dabrafenib TAFINLAR BRAF BRAFCobimetinib COTELLIC MEK1, MEK2 BRAFTrametinib MEKINIST MEK BRAFVemurafenib ZELBORAF BRAF BRAF

Ovarian Olaparib LYNPARZA PARP1,2,3 BRCAPancreas Erlotinib TARCEVA EGFR EGFR

GIST Imatinib GLEEVEC BCR-ABL, PDGFR, c-KIT, SCF Ph+, PDGFR rearr. and. fusion with FIP1L1, KIT

LeukemiaVenetoclax VENCLEXTA BCL-2 del(17p)

Imatinib GLEEVEC BCR-ABL, PDGFR, c-KIT, SCF Ph+, PDGFR rearr. and fusion with FIP1L1, KIT

Various cancers 5-Fluorouracil 5-FU DPD DPYD

ThyroidDabrafenib TAFINLAR BRAF BRAFVemurafenib ZELBORAF BRAF BRAF

Predictive biomarkers and drugs in targeted cancer therapies

Somaticmutations

Hereditarymutations

Germline-mutation(heterozygous or

homozygous)

Acquired mutations(non-germline)

Intratumor heterogeneity

… An often little addressed but important issue in external quality assurance schemes is a low percentage of tumor cells in the test samples, where the analytical sensitivity of most tests becomes critical …

Tumor cells with somatic mutationin a wildtype cell context

… although traditional Sanger sequencing detects all clinically important KRAS mutations, its sensitivity is too low to be used in routine clinical tests (mutant alleles must be present in at least 30-40% of cells for reproducible detection) …

… if a mutation is identified, the affected codon and the specific change should be reported. This is an important aspect of the report because recommendations for treatment eligibility are continuously evolving …

... One can imagine that a perfect approach would be the combination of manual microdissection (under the microscope) with high-sensitivity (1% mutation in a 99% wild-type background limit of detection)methods for KRAS mutation testing.

Three KRAS StripAssay formats are available coveringup to 29 mutations in KRAS exons 2, 3 and 4

KRASStripAssayCat.# 5-590


Control

1 KRAS p.Gly12Ala c.35G>C

2 KRAS p.Gly12Arg c.34G>C

3 KRAS p.Gly12Asp c.35G>A

4 KRAS p.Gly12Cys c.34G>T

5 KRAS p.Gly12Ile c.34_35delGGinsAT

6 KRAS p.Gly12Leu c.34_35delGGinsCT

7 KRAS p.Gly12Ser c.34G>A

8 KRAS p.Gly12Val c.35G>T

9 KRAS p.Gly13Ala c.38G>C

10 KRAS p.Gly13Arg c.37G>C

11 KRAS p.Gly13Asp c.38G>A

12 KRAS p.Gly13Cys c.37G>T

13 KRAS p.Gly13Ser c.37G>A


15 KRAS p.Ala59Glu c.176C>A

16 KRAS p.Ala59Gly c.176C>G

17 KRAS p.Ala59Thr c.175G>A


19 KRAS p.Gln61Arg c.182A>G

20 KRAS p.Gln61His c.183A>C

21 KRAS p.Gln61His c.183A>T

22 KRAS p.Gln61Leu c.182A>T

23 KRAS p.Gln61Lys c.181C>A

24 KRAS p.Lys117Asn c.351A>C

25 KRAS p.Lys117Asn c.351A>T

26 KRAS p.Lys117Glu c.349A>G

27 KRAS p.Ala146Pro c.436G>C

28 KRAS p.Ala146Thr c.436G>A

29 KRAS p.Ala146Val c.437C>T

30 KRAS 12/13 PCR Negative Control

31 KRAS 59/60/61 PCR Negative Control

32 KRAS 117 PCR Negative Control

33 KRAS 146 PCR Negative Control

34 PCR Positive Control



Control

1 KRAS codon 12 Ala c.35G>C

2 KRAS codon 12 Arg c.34G>C

3 KRAS codon 12 Asp c.35G>A

4 KRAS codon 12 Cys c.34G>T

5 KRAS codon 12 Ile c.[34G>A; 35G>T]

6 KRAS codon 12 Leu c.[34G>C; 35G>T]

7 KRAS codon 12 Ser c.34G>A

8 KRAS codon 12 Val c.35G>T



11 PCR Negative Control




Control

1 KRAS codon 12 Ala c.35G>C

2 KRAS codon 12 Arg c.34G>C



5 KRAS codon 12 Ile c.[34G>A; 35G>T]

6 KRAS codon 12 Leu c.[34G>C; 35G>T]

7 KRAS codon 12 Ser c.34G>A

8 KRAS codon 12 Val c.35G>T



11 BRAF V600E

c.1799T>A

12 KRAS PCR Negative Control

13 BRAF PCR Negative Control



KRAS-BRAFStripAssayCat.# 5-580

KRAS XLStripAssayCat.# 5-680

KRAS StripAssays®

KRAS XL StripAssay®

29 mutationsin exons 2, 3 and 4

5-50 ng DNA per PCR(concentration 1-10 ng/µl)

single amplification mix

mutant enrichment by wild-type blocking allows mutation detection at 1% sensitivity

NRAS XL StripAssay®

22 mutations in exons 2, 3 and 4




NRAS StripAssay®Red Marker Line (top)

Control

1 NRAS p.Gly12Ala c.35G>C

2 NRAS p.Gly12Arg c.34G>C

3 NRAS p.Gly12Asp c.35G>A

4 NRAS p.Gly12Cys c.34G>T

5 NRAS p.Gly12Ser c.34G>A

6 NRAS p.Gly12Val c.35G>T


8 NRAS p.Gly13Asp c.38G>A

9 NRAS p.Gly13Cys c.37G>T

10 NRAS p.Gly13Val c.38G>T

11 NRAS p.Ala59Asp c.176C>A

12 NRAS p.Ala59Thr c.175G>A


14 NRAS p.Gly60Glu c.179G>A

15 NRAS p.Gln61Arg c.182A>G

16 NRAS p.Gln61Glu c.181C>G

17 NRAS p.Gln61His c.183A>C

18 NRAS p.Gln61His c.183A>T

19 NRAS p.Gln61Leu c.182A>T

20 NRAS p.Gln61Lys c.181C>A

21 NRAS p.Gln61Pro c.182A>C

22 NRAS p.Ala146Thr c.436G>A

23 NRAS 12/13 PCR Negative Control

24 NRAS 59/60/61 PCR Negative Control

25 NRAS 146 PCR Negative Control



NRAS XLStripAssayCat.# 5-620


Control

1 BRAF V600A c.1799T>C

2 BRAF V600D c.1799_1800TG>AT

3 BRAF V600E c.1799T>A

4 BRAF V600E c.1799_1800TG>AA

5 BRAF V600G c.1799T>G

6 BRAF V600K c.1798_1799GT>AA

7 BRAF V600M c.1798G>A

8 BRAF V600R c.1798_1799GT>AG

9 BRAF K601E c.1801A>G




BRAF StripAssay®

V600E mutation in exon 15

BRAF 600/601 StripAssay®

9 mutations in exon 15





Control

1 BRAF V600E c.1799T>A




BRAFStripAssayCat.# 5-570

BRAF 600/601StripAssayCat.# 5-560

BRAF StripAssays®

EGFR StripAssay®

EGFR XL StripAssay®

30 mutations in exons 18, 19, 20 and 21


two amplification mixes



Control

1 EGFR ex18 p.G719A c.2156G>C

2 EGFR ex18 p.G719C c.2155G>T

3 EGFR ex18 p.G719S c.2155G>A

4 EGFR ex19 p.K745_E749del c.2233_2247del

5 EGFR ex19 p.E746_A750del c.2235_2249del

6 EGFR ex19 p.E746_A750delinsIP c.2235_2248delinsAATTC

7 EGFR ex19 p.E746_A750del c.2236_2250del

8 EGFR ex19 p.E746_T751delinsIP c.2235_2251delinsAATTC

9 EGFR ex19 p.E746_T751del c.2236_2253del

10 EGFR ex19 p.E746_T751delinsA c.2237_2251del

11 EGFR ex19 p.E746_T751delinsV c.2237_2252delinsT

12 EGFR ex19 p.E746_T751delinsVA c.2237_2253delinsTTGCT

13 EGFR ex19 p.E746_S752delinsI c.2235_2255delinsAAT

14 EGFR ex19 p.E746_S752delinsA c.2237_2254del

15 EGFR ex19 p.E746_S752delinsV c.2237_2255delinsT

16 EGFR ex19 p.E746_S752delinsD c.2238_2255del

17 EGFR ex19 p.E746_P753delinsVS c.2237_2257delinsTCT

18 EGFR ex19 p.L747_E749del c.2239_2247del

19 EGFR ex19 p.L747_A750delinsP c.2238_2248delinsGC

20 EGFR ex19 p.L747_A750delinsP c.2239_2248delinsC

21 EGFR ex19 p.L747_T751delinsP c.2239_2251delinsC

22 EGFR ex19 p.L747_T751delinsS c.2240_2251del

23 EGFR ex19 p.L747_T751del c.2240_2254del

24 EGFR ex19 p.L747_S752del c.2239_2256del

25 EGFR ex19 p.L747_S752delinsQ c.2239_2256delinsCAA

26 EGFR ex19 p.L747_P753delinsQ c.2239_2258delinsCA

27 EGFR ex19 p.L747_P753delinsS c.2240_2257del

28 EGFR ex20 p.T790M c.2369C>T

29 EGFR ex21 p.L858R c.2573T>G

30 EGFR ex21 p.L861Q c.2582T>A

31 EGFR ex18 PCR Negative Control




35 PCR Positive Control A

36 PCR Positive Control B


EGFR XLStripAssayCat.# 5-630

o Quality of neutral-buffered formalin solution:formalin degradation products impair nucleic acid quality

o Fix tissue as quickly as possible after surgical removal

o Fixation time between 14-24 hours:penetration approx. 1 mm / hour

o To improve DNA quality avoid tissue staining:high pH and heavy metal ions may affect DNA integrity

o Use 5-10 µm thick sections for DNA extraction:discard the first 2-3 sections if the surface was exposed to air

References: Qiagen “Unlocking your FFPE archive” and “QIAamp DNA FFPE Tissue Handbook”.

Mutation detection in DNA from formalin-fixed paraffin-embedded (FFPE) tissue

For DNA extracted from formalin-fixed paraffin-embedded (FFPE) tissue either qPCR or fluorometry (e.g. Invitrogen Qubit or Promega QuantiFluor system) need to be applied. FFPE DNA concentration is up to 100-fold overestimated if determined by OD 260 nm.

Results of five representative FFPE DNA samples measured at ViennaLab by using UV-VIS 260/280, qPCR, Qubit HS ("high sensitivity") and BR ("broad range").

Reference: Simbolo et al., 2013

DNA Qualification Workflow for Next Generation Sequencing of HistopathologicalSamples. PLoSOne 8(6):e62692.

FFPE DNA quantification

BRAF1 2

3 4

(A) (B)

Sample I

1 2 3 4

- + BSA

- + BSASample II

PCR inhibition by melanin

(A) Agarose gel (4%), 50 bp ladder and amplification products of the melanoma FFPE DNA samples I and II (204 bp PCR Positive Control, 154 bp mutated BRAF).

(B) BRAF 600/601 StripAssay of the respective PCR products.

Nucleic acids extracted from melanoma samples may contain melanin that affects PCR. The addition of bovine serum albumin (BSA; 15 µg per 25 µl PCR) reversed melanin-dependent PCR inhibition.

A liquid biopsy, also known as fluid biopsy or fluid phase biopsy, is the sampling and analysis of non-solid biological tissue, primarily blood. (Wikipedia)

A liquid biopsy is a test done on a sample of blood to look for cancer cells from a tumor that are circulating in the blood or for pieces of DNA from tumor cells that are in the blood. (NCI Dictionary of Cancer Terms)

Liquid biopsies

Sources for liquid biopsy samples: primarily blood, but also saliva, urine and other body fluids.

Plasma

fragments of genomic DNA (e.g. from leukocytes)

circulating cell free DNA (cfDNA) from various tissues

circulating tumor DNA (ctDNA)

ctDNA with somatic mutation

miRNA / cfRNA / exosomal RNA

Liquid biopsies

Technical Problems associated with liquid biopsy assays:• low concentrations, short half life (~2 hours) and heavy

fragmentation of cfDNA and ctDNA• contaminating leukocyte DNA• availability of sufficient amounts of well-characterized

samples for test development

Set up of PCRAll Steps have to be done on ice using cold reagents !!

Frequent errors & major mistakes !!!!

cool block

Fresh working dilution of Taq DNA polymerase in Taq

Dilution Buffer(never use

undiluted Taq !!)

Final PCR mix prepared on ice for each sample:• 15 μl Amplification Mix• 5 μl diluted Taq DNA• 5 μl DNA template

Thermocycler preheated and paused !!

Reaction tubes tightly capped and inserted to run the appropriate program.

Instruments for 45ºC incubationsManual: shaking waterbath or suitable thermoshaker plus preheating device

Automated: established teststrip processors

IMPORTANT:no hot-air

oven etc. !!!

+

ProfiBlot T48(for 48 teststrips)

AutoBlot 3000H(for 20 teststrips)

Dynablot Heat(for 30 teststrips

or


Hybridization & stringent washes at 45°CTemperature must be strictly kept at 45±0.5°C !!


Hybridization Buffer and Wash Solution A must be prewarmed to 45°C (approx. 30 min)

Precipitates formed at 2-8°C must be completely re-dissolved

• Water level must be at 1/2 the height of the tray• Moderate shaking frequency must be chosen to

avoid spilling• Cover of the waterbath/thermoshaker must be

closed to avoid variations in temperature

Selected StripAssay® Reports

Alpha-globin StripAssay

Beta-globin StripAssay


Beta-thal Modifier StripAssay

Haemochromatosis StripAssay

Haemochromatosis StripAssay

CAH StripAssay

CVD StripAssay

CVD StripAssay

FMF StripAssay

FMF StripAssay

FMF StripAssay

FMF StripAssay

FMF StripAssay

Gaucher Disease StripAssay

Sugar Intolerance StripAssay

PGX-Thrombo StripAssay

KRAS StripAssay

BRAF StripAssay

KRAS-BRAF StripAssay

EGFR StripAssay

Selected Iranian StripAssay® Reports










"... samples were screened for a panel of 22 relatively common β-globin mutations using an assay based on polymerase chain reaction (PCR) and reverse-hybridization to oligonucleotide arrays immobilized on test strips ... a single amplification and hybridization step, allowing even very small amounts of DNA (e.g. prenatal samples) to be rapidly and comprehensively typed."


FMF StripAssay

"In this regard, the FMF StripAssay demonstrated to be a reliable and cost-effective first-line screening tool ... a very suitable strategy for population-based FMF genotyping in Iran.."

J Genet 2016; 95(3):667-74

FMF StripAssay

FMF StripAssay

CF StripAssay

"In Iran, complete genetic information is currently lacking to implement solid population-based CFTR screening programs that could enable adequate carrier detection of either typical or atypical CF patients or their family members. For national policies of CF prevention, it is acceptable to include only the most frequent mutations present in the population, which allows a 90% detection rate."

Thank you !Dr. Christian [email protected]: +43 1 8120156 40