Upload
others
View
5
Download
0
Embed Size (px)
Citation preview
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
Prodia.co.id
Thank You
1500-830 e-prodia @prodia_lab Laboratorium Klinik Prodia