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

Transcription

1 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

2 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

3 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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6 UV Chamber ( Pre-PCR decontamination ) 6

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10 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

11 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

12 PCR / gel electrophoresis / Dot blot hybridization Southern Blot hybridization 12

13 First and Second Generation PCR, DNA Sequencer 13

14 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

15 Post-amplification ( product detection ) - Chlorox, overnight UV 15

16 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

17 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

18 Validation (in-house protocol) In-house designed primers should be re-evaluated at least annually. =BlastSearch&SHOW_DEFAULTS=on&LINK_LOC=blasthome 18

19 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

20 Viral load monitoring ( Linear range ) CP/CT values Sample A Sample B copies/ml 10 2 Concentration (Log scale) ( IU or copies/ml) copies/ml 20

21 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 recently tested archived plasma ( from -70C ) of low, medium and high viral load ( different runs and operators ) New system (FDA) 21

22 New system Least Square Linear Regression (HOKLAS SC ) HBV viral load copies/ml Acceptance criteria: Correlation coefficient r Or Deming regression Old system Thank you 22