Molecular diagnosis of infectious disease - Method validation and environmental setting 傳染病的分子診斷 - 方法確認與環境背景 WC Yam 任永昌 Dept of Microbiology Queen Mary Hospital The University of Hong Kong Clinical and Laboratory Standards Institute (CLSI) 1
Area 2 Area 1 Area 3 Laboratory Setup for in-house PCR Area 1 - Reagent preparation ( Clean ) Area 2 - Specimen preparation Area 1 & 2 can be located at 2 different corners of the same lab Area 3 - Amplification (PCR) and Detection Area 3 should be located as far away as possible from Areas 1 & 2 2
Area 1 Reagent preparation ( Dead air-box with UV light ) Dedicated micro-pipettes with plugged (aerosolbarrier) tips Sterile reaction tubes and pipettes Master mix will be prepared and added to clean reaction tubes in area 1 Reagent preparation ( clean room ) - Dead Airbox (UV); UV Cross linker 3
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UV Chamber ( Pre-PCR decontamination ) 6
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Replacement of UV lamp (UV-C) Area 2 Specimen preparation Nucleic Acid Extraction system Dedicated micropipettes with plugged (aerosolbarrier) tips Micro-centrifuge and Vortex Reaction tubes Disposal tank with disinfectant 10
Biosafety Class II Cabinet Area 3 Amplification (PCR) and Detection Temp Cycler / qpcr machine / DNA sequencer Dedicated micropipettes with plugged (aerosol-barrier) tips Disposal reservoir Power pack, electrophoresis gel tank, UV transilluminator & camera Dedicated refrigerator for PCR products 11
PCR / gel electrophoresis / Dot blot hybridization Southern Blot hybridization 12
First and Second Generation PCR, DNA Sequencer 13
Post-amplification ( product detection ) Remote printing Sink Gel doc DNA sequencer Temp cycler qpcr Gel tank Power pack Spin UV box ( for cycle sequencing and nested PCR) 14
Post-amplification ( product detection ) - Chlorox, overnight UV 15
Test Evaluation and Validation As guidelines / NOT as regulatory standards The most important attributes of a laboratory test are its ability to produce accurate, precise, and reproducible results with rapid turnaround time and clinical utility. 1. Analytical sensitivity (Limit of Detection-LOD) 2. Analytical specificity 3. Precision ( linear range esp. for viral load ) 4. Accuracy 5. Cutoff values 6. Detection of inhibitors and interfering substances 7. Diagnostic sensitivity 8. Diagnostic specificity (1-6) are usually provided by for FDA or CE certified kits, minimal verification is needed 16
Validation The documentation that a test which has already been verified is repeatedly giving the expected results ( over a period of time ) Test validation includes personnel competency assessment, quality control, internal and external proficiency testing, and correlation with clinical findings Validation is an integral part of Quality Assurance program 17
Validation (in-house protocol) In-house designed primers should be re-evaluated at least annually. http://blast.ncbi.nlm.nih.gov/blast.cgi http://blast.ncbi.nlm.nih.gov/blast.cgi?program=blastn&blast_programs=megablast&page_type =BlastSearch&SHOW_DEFAULTS=on&LINK_LOC=blasthome 18
Molecular Diagnosis of M. tuberculosis 350 respiratory specimens ( 68 culture + ) AFB smear + ( 56 ) AFB smear negative ( 12 ) PCR inhibitors among 350 samples ( 1.7% ) 520 non-respiratory specimens ( 19 culture + ) AFB smear + ( 1 ) CSF, EMU, abscess, PF, tissue, lymph node asp PCR inhibitors among 320 samples ( 8.8% ) Viral Load monitoring (HBV, HCV, HIV) Real time Nucleic Acid Amplification assays (eg. qpcr) technology Correlation with disease progression and clinical outcome Monitoring of clinical latency or therapeutic phase Copies/mL or IU/mL 19
Viral load monitoring ( Linear range ) CP/CT values Sample A Sample B 10 2 80 copies/ml 10 2 Concentration (Log scale) ( IU or copies/ml) 10 5 10 6 copies/ml 20
Viral load monitoring CP/CT values Sample A Sample B 10 2 Less than 100 copies/ml Concentration (Log scale) ( IU or copies/ml) 10 5 Dilute original plasma, re-extract and qpcr Validation of New HBV viral load assay Initial verification using reference material ( eg. WHO or Accromatrix HBV+ plasma ) Old system (FDA) Select minimal 10-20 recently tested archived plasma ( from -70C ) of low, medium and high viral load ( different runs and operators ) New system (FDA) 21
New system Least Square Linear Regression (HOKLAS SC38-3.5.3) HBV viral load copies/ml Acceptance criteria: Correlation coefficient r 0.975 Or Deming regression Old system http://www.itc.gov.hk/en/quality/hkas/doc/supplementarycriteria/hoklas_sc038.pdf Thank you http://www.microbiology.hku.hk/staff/staff_hp/wcyam/lecture.pdf 22