Dr. Miswar Fattah, MSi 1997 : SMAK Depkes Makassar th 2002 : … · 2020. 3. 31. · 1500-830...
Transcript of Dr. Miswar Fattah, MSi 1997 : SMAK Depkes Makassar th 2002 : … · 2020. 3. 31. · 1500-830...
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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
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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
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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
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Basic principe of PCR
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DNA extraction PCR / RT PCRGel
electrophoresisSequencing
General Step in Molecular Laboratory
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Professional Guidelines
MM19A: Molecular Testing in Clinical Lab Environments. Clinical & Laboratory Standards Institute, (available at https://clsi.org/standards/products/molecular-diagnostics/documents/mm19/).
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Molecular Guideline
https://clsi.org/
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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
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Minimum Instrument in Molecular Laboratory• Reagen Preparation
Spin down CentrifugePCR Cabinet for mastermix preparation
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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
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Biosafety Cabinet for Molecular Diagnostics
Class I Class II Class III
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Minimum Instrument in Molecular Laboratory: PCR & Post PCR room
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14
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Additional intruments & equipment
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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
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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
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Difference Molecular lab
Avoid DNA Contamination Very low Volume
0,5 uL, 1 uL, 5 uL, 10 uL, total volume 25 -50 uL
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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
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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
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sticky mats
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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/).
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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
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Example Lab Design for a Molecular Microbiology facility
Mitchel et.al., Molecular Microbiology: Diagnostic Principles and Practice, (American Society of Microbiology), 2004
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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/).
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Pressure & Temperature Controller
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UV LAMP and Electronic Timer SwitchesElectronic Timer Switches
UV lamp
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4 LAB Molecular laboratory Setup
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Three-Lab Setup PCR Laboratory
http://www.lifesci.com/xcart/product.php?productid=16&cat=1&page=1
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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/).
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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/).
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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/).
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Close system in Molecular laboratory
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Important Skill in Molecular Laboratory• Understood of Consept of Contamination & PCR
• Micropipetting technique
http://www.hkki.org/downloads
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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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Prodia.co.id
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