Education

Current position

Dr. Miswar Fattah, MSiMakassar, 6th June 1978

1997 : SMAK Depkes Makassar 2002 : Chemistry - UNHAS

2006 : Master of Science in Clinical Chemistry, Biomedicine- UNHAS

2012 : Doctor of Medicine - UNHAS

1. Specialty & Research Laboratory Manager, Prodia Clinical Laboratory 2018- Now2. IACC: Member of Scientific Commette, Indonesian Association for Clinical Chemistry 2013- Now3. PATELKI : Vice President 2017-Now & Member of Collegium PATELKI 2015 - Now4. President of ASEAN Association of Clinical Laboratory Scientist (AACLS) 2018-20205. Member of Board of Directors of Asian Association of Medical Laboratory Science (AAMLS) 2017 - Now 6. Corresponding Member Scientific Committee Asia Pacific Federation for Clinical Chemistry (APFCB)

2010 – Now

How to build a Molecular Diagnostics Lab: Challenges and Tips for starting from Scratch

Dr. Miswar Fattah, MSi

Specialty & Research Laboratory, Prodia Clinical Laboratory

Webinar: Establishing a Molecular Diagnostics Lab for Covid-19 to the detection procedure of Covid-19March 28th, 2020

Topics outline

• Basic Principles of PCR

• Principle Lab Setup• Building Design and Site Selection• Laboratory Configuration (Minimum instrument)

• Workflow Consideration

• Important skill in molecular laboratory

Basic principe of PCR

DNA extraction PCR / RT PCRGel

electrophoresisSequencing

General Step in Molecular Laboratory

Professional Guidelines

MM19A: Molecular Testing in Clinical Lab Environments. Clinical & Laboratory Standards Institute, (available at https://clsi.org/standards/products/molecular-diagnostics/documents/mm19/).

Molecular Guideline

https://clsi.org/

IDEAL DESIGN MOLECULAR LABORATORY

• 4 ROOM (Mandatory) • 3 ROOM • 2 ROOM

Reagen Preparation

Nucleic Acid Extraction

Amplification

Post Amplification/Sequensing

Reagen Preparation

Nucleic Acid Extraction

Amplification & post amplification

Reagen Preparation & Nucleic Acid

Extraction

Amplification & post amplification

Closed system

Minimum Instrument in Molecular Laboratory• Reagen Preparation

Spin down CentrifugePCR Cabinet for mastermix preparation

Minimum Instrument in Molecular Laboratory: Nucleic Acid extraction Room

Biosafety Cabinet

Biohazard bin: layered with biohazard bag,

Refrigerator & Freezer

Centrifuge and heat block

Micropippette & tips

Dedicated lab coats

Biosafety Cabinet for Molecular Diagnostics

Class I Class II Class III

Minimum Instrument in Molecular Laboratory: PCR & Post PCR room

13

14

Additional intruments & equipment

Example position Instrument of a Four-Lab Layout

-20°C

freezer

work

bench

Ice

machine

LAB 1

"No Template"

Lab

LAB 2

"Specimen

Processing" Lab

LAB 4

"Post-

amplification" Lab

LAB 3

"Nested PCR"

Lab/Amplification

Lab

-80°C

freezer

-20°C

freezerBSC

work

bench

BSC: bio-safety cabinet

Ice

machine

Ice

machine

-80°C

freezer

-20°C

freezer

work

bench

Sequencer Gel electrophoresis areaPCR

machinesworkbench PCR

machines

-20°C

freezer

Cen

trif

ug

e

Pass-

through

window

Lab coat rack Lab coat rack

Lab coat rack Lab coat rack

NA extraction

equipment

workbench

sink

sink

sink

sink

Eye

wash

Eye

wash

BSC

Cen

trif

ug

e

WHO | HIV drug resistance laboratory training package. WHO, (available at https://www.who.int/hiv/pub/drugresistance/lab_training/en/).

Pass box

Potential Sources of Contamination How to Controle

Cross contamination

between specimens

Amplification product

contamination

Laboratory surfaces Ventilation ducts

Reagents/suppliesHair, skin, saliva,

and clothes of lab personnel

Laboratory design

Laboratory practices

Chemical and enzymatic controls

R. Lee, Molecular Lab design, 2015

Difference Molecular lab

Avoid DNA Contamination Very low Volume

0,5 uL, 1 uL, 5 uL, 10 uL, total volume 25 -50 uL

CONTAMINATION IN MOLECULAR LABORATORY

• Introduction of unwanted nucleic acids into specimen • the sensitivity of PCR techniques makes them vulnerable to

contamination

• Repeated amplification of the same target sequence leads to accumulation of amplification products in the laboratory environment

• A typical PCR generates as many as 109 copies of target sequence • Aerosols from pipettes will contain as many as 106 amplification

products• Buildup of aerosolized amplification products will contaminate

laboratory reagents, equipment, and ventilation systems

R. Lee, Molecular Lab design, 2015

Basic Principe of Setting Up a Molecular Laboratory

• Mechanical barriers to prevent contamination

• Spatial separation of pre- and post-amplificationwork areas

• Area 1 – Reagent preparation

• Area 2 – Specimen/control preparation, PCR set-up

• Area 3 – Amplification

• Area 4 – Product Amplification detection

• Physically separated and, preferably, at a substantial distance from each other

R. Lee, Molecular Lab design, 2015

sticky mats

Unidirectional workflow

Reagent Preparation

SpecimenPreparation

Amplification Sequencing

Copy #

0 105 1010 1012

WHO | HIV drug resistance laboratory training package. WHO, (available at https://www.who.int/hiv/pub/drugresistance/lab_training/en/).

Unidirectional Flow

Both personnel, including

cleaning personnel, &specimens

Amplification product-free to

product-rich

Remove PPE before leaving

one area

Avoid or limit reversedirection

Reusable supplies in the

reverse direction need to be

bleached

R. Lee, Molecular Lab design, 2015

Example Lab Design for a Molecular Microbiology facility

Mitchel et.al., Molecular Microbiology: Diagnostic Principles and Practice, (American Society of Microbiology), 2004

LAB 3

“1st Round PCR” Lab1st round amplification

2nd round PCR set-up

Ideal Lab Workflow

LAB 1

"No Template" LabReagent storage and

Master mix preparation

LAB 2

"Specimen Processing" LabSpecimen receipt & storage

RNA extraction and 1st round

PCR set-up

LAB 4

"Post-amplification" Lab2nd round amplification

Gel electrophoresis

Sequencing reactions

Capillary electrophoresis

SPECIMENSFrozen plasma

Whole blood

DBS

WHO | HIV drug resistance laboratory training package. WHO, (available at https://www.who.int/hiv/pub/drugresistance/lab_training/en/).

Pressure & Temperature Controller

UV LAMP and Electronic Timer SwitchesElectronic Timer Switches

UV lamp

4 LAB Molecular laboratory Setup

Three-Lab Setup PCR Laboratory

http://www.lifesci.com/xcart/product.php?productid=16&cat=1&page=1

123

Three-Lab Setup (Option 1)

LAB 1

"Specimen Processing" LabSpecimen receipt & storage

RNA extraction and 1st round

PCR set-up

LAB 2

"Amplification" Lab 1st round amplification

2nd round amplification

LAB 3

"Post-amplification" LabGel electrophoresis

Sequencing reactions

Capillary electrophoresis

Master mix preparation

2nd round PCR set-up

In dead-air cabinet with dedicated pipettes

SPECIMENSFrozen plasma

Whole blood

DBS

WHO | HIV drug resistance laboratory training package. WHO, (available at https://www.who.int/hiv/pub/drugresistance/lab_training/en/).

Three-Lab Setup (Option 2)

LAB 1

"No Template" LabReagent storage and

Master mix preparation

LAB 2

"Specimen Processing and

PCR" LabSpecimen receipt & storage

RNA extraction and 1st round

PCR set-up

LAB 3

"Post-amplification" Lab2nd round amplification

Gel electrophoresis

Sequencing reactions

Capillary electrophoresis

1st round amplification

2nd round PCR set-up

In dead-air cabinet with dedicated pipettes

SPECIMENSFrozen plasma

Whole blood

DBS

WHO | HIV drug resistance laboratory training package. WHO, (available at https://www.who.int/hiv/pub/drugresistance/lab_training/en/).

Two-Lab Setup

LAB 1

"Specimen Processing" LabSpecimen receipt & storage

RNA extraction and 1st round

PCR set-up

LAB 2

"Post-Amplification" Lab 1st round amplification

2nd round amplification

Gel electrophoresis

Sequencing reactions

Capillary electrophoresis

Master mix preparation

2nd round PCR set-up

In dead-air cabinet with dedicated pipettes

SPECIMENSFrozen plasma

Whole blood

DBS

WHO | HIV drug resistance laboratory training package. WHO, (available at https://www.who.int/hiv/pub/drugresistance/lab_training/en/).

Close system in Molecular laboratory

Important Skill in Molecular Laboratory• Understood of Consept of Contamination & PCR

• Micropipetting technique

http://www.hkki.org/downloads

Key Trick to Fast Learn PCR

Watch video Basic principle PCR (minimum 5 video)

Simulate master mix calculation (minimum 10 Protocols)

Simulate set up PCR instrument (minimum 5 protocol different protocols)

Simulate pipetation low volume volume 2 uL, 1 uL, 0,5 ul (minimum 20 times before start PCR)

Simulate purchase PCR reagent (minimum 5 protocol)

Optimate minimum 1 protocol

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