Molecular Automation in a Diagnostic Laboratory. Cheryl Bletchly PhD Supervising Scientist, Molecular Diagnostic Unit, Pathology Queensland

Transcription

1 Molecular Automation in a Diagnostic Laboratory Cheryl Bletchly PhD Supervising Scientist, Molecular Diagnostic Unit, Pathology Queensland

2 RBWH Campus, Herston Block 7 (Pathology Qld - Central)

3 Monthly Test Numbers 3

4 PCR Reaction Detection DNA Ladder Samples Add mastermix, and controls

5 Automated Extraction of Nucleic Acid MagNAPure (uses magnetic beads) Instrument for batched extraction of nucleic acids (96 per run) At low ph the magnetic beads are positively charged and attract the negatively charged DNA. Washing steps allow the protein and contaminants to be washed away

6 Real time PCR Instruments 6

7 Real-time PCR Software 7

8 Facilities One way workflow shown in red arrows

9 Class II Biological Safety Cabinet

10 Prevention of amplicon contamination Amplicon Contamination Prevention Spatial Physical separation (one way workflow), BSCs, filter tips, gloves, lab coats, sealed amplification tubes Environmental Pressurization Chemical use of UDG

11 Pressurization

12 Regulatory Requirements 12

13 Risk Based Classification IVD classification Class 1 Class 2 Class 3 Class 4 Level of risk No public health risk or low personal risk Low public health risk or moderate personal risk Moderate public health risk or high personal risk High public health risk 13

14 Respiratory Virus PCR Panel Influenza A and B Parainfluenza 1, 2, 3 Respiratory Syncytial Virus and Adenovirus Human metapneumovirus

15 Respiratory Virus Mastermix In-house assays, multiplexed Stockpile of reagents eg. Primers / probes (working batch + spare) + Pandemic preparedness QC of all reagents in advance of batch making and then tested in parallel with working batch Controls for each assay

16 Manual Set Up Respiratory virus PCR

17 MIX Respiratory Virus Panel plate AB Influenza Parainfluenza P123 RSV / Adeno RAD humpv MPV AB Influenza Parainfluenza P123 RSV / Adeno RAD humpv MPV Controls 22 patient samples per plate

18 MIX AB Influenza A & B H1 Swine Influenza H1 AB H1 AB H1 1 2 Flexibility AB H1 Controls 46 patient samples per plate

19 Advantages / Disadvantages Ability to choose inclusions and exclusions in the panel according to local requirements Flexibility to rapidly change the panels in response to outbreaks Ability to modify panels after review of their utility within the local population, and feedback from clients Extensive QC required, maintaining reagents, stocks Compliance with NPAAC guidelines for validation of in-house IVDs 19

20 Window Periods Laboratory Testing for organ and tissue donation Serology Molecular HIV days 5-6 days HCV 70 days 3-5 days HBV days days Testing is regulated by the Therapeutic Goods Administration (TGA) under the Code of Good Manufacturing Practice (GMP). The laboratory is audited and licensed as a production facility. Baleriola et al (2011) Trans Inf Dis 14:

21 Manual Extraction Method Add lysis reagent to specimen and vortex Incubate for 15 minutes at room temperature Add isopropanol, invert, vortex HIV Pos control Neg control Ext control Patient samples Centrifuge g for 20 minutes at 21 degrees Remove supernatant and add 1.0 ml of 70% EtOH, Vortex Centrifuge 1400 g for 10 minutes at 21 degrees HBV Remove supernatant and pulse spin Resuspend pellet in diluent and pulse spin. HCV 21

22 Cobas Amplicor Instrument Combines thermal cycler, automatic pipettor, incubator, washer and reader Turn around time = 7.5 hours Utilizes the AmpErase contamination control system. AmpErase uracil-n-glycosylase) chemically inactivates amplified material and prevents it from contaminating future reactions

23 Lysis and extraction Lysis buffer contains internal control PCR amplification with a biotin labelled primer and dutp dsdna Cobas Amplicor Amplification and Detection Denaturation Convert to ssdna Read absorbance at 660 nm Wash, add TMB substrate with H2O2 Incubate, wash, add avidin-horseradish peroxidase conjugate Magnetic particles coated with specific oligonucleotide probe (HIV, HBV, HCV)

24 Cobas 6800 Automates the extraction and real-time PCR in one instrument. Turn around time = 3.5 hours 2.92M (W) x 2.16 (H) x 1.29 (D)

25 Automation Considerations for Automation Staff training (change in focus from technique to troubleshooting) Assays still require laboratory verification LIS interfacing and verification (200 page document) o Verify each scenario eg. Positive result, negative result, invalid result, correct specimen type, validation tables, accepting/rejecting results, repeat specimens. Breakdown and redundancy (service contract, after hours support for 24 hour service) Monitoring of external controls by inter and intra laboratory comparisons of data

26 HPV High Throughput 26

27 HPV Primary Screening Requirements NPAAC Requirements for Laboratories Reporting Tests for the National Cervical Screening Program (First Ed 2017) S4.1 Laboratories must only use commercially supplied HPV NAT that are validated for primary population based screening. S4.2 The HPV NAT method must test for HPV genotype 16,18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59 and 68 and separately identify HPV16 and HPV18 S4.3 The test must be included on the Australian Register of Therapeutic Goods. 27

28 Batch Number and Expiry Data

29 Hydridization Assays Eg. Human Papillomavirus DNA Assay HPV DNA + HPV RNA probe cocktail RNA:DNA hybrid Chemiluminescence Alkaline phosphatase conjugated antibody specific for RNA:DNA hybrids RNA:DNA hybrid RNA:DNA specific hybrid antibody

30 PCR for Gastrointestinal Pathogens 3 separate panels of in-house assays, requested independently Bacterial Panel Viral Panel Protozoan Panel

31 Gene targets from literature Gene Reference Campylobacter cadf Cunningham SA et al (2010) Salmonella spp. inva Antikainen J et al (2013) Shigella spp. ipah Liu J et al (2013) V. cholerae hlya Park JY et al (2013) V. parahaemolyticus tlh Park JY et al (2013) Y. enterocolitica ail Lambertz ST et al (2008) Adenovirus 40/41 Hexon Feeney SA (2011) Norovirus G1 and G11 Kageyama T et al (2003) Rotavirus P3 Pang XL et al (2004 and 2011) Cryptosporidium spp. 18S Liu et al (2013) E. histolytica Pxr Stark et al (2011) G. intestinalis Β giardin Haque et al (2007)

32 96 well plate Processing Janus 8-tip loading instrument Extracts, mastermix, controls 384 well plate Salmonella FAM/ Shigella Cy5 Campylobacter FAM / Yersinia Cy5 Adenovirus HEX Adeno 40/41 FAM EHV (Cy5) Controls Norovirus Noro 1 FAM, Noro 2 Cy5 Controls Rotavirus FAM Controls V. cholerae FAM / V. parahaemolyticus VIC / EHV Cy5 Crypto FAM / Giardia Cy5 E. histolytica FAM Controls Controls Controls 2μL extract + 8μL mastermix provides economy of scale

33 Gastrointestinal pathogen PCR Gastrointestinal Pathogens Specimens are batched daily and therefore limited flexibility to accommodate urgent requests Outbreaks 33

34 Going Viral - The Rise of the world s most contagious bug New Scientist 23 March 2013 pp.43-45

35 Influenza virus GeneXpert

36 BioFire Other Rapid Multiplex assays BioFire FilmArray Respiratory panel on nasopharyngeal swab (20 targets) Blood Culture ID on positive blood culture (27 targets) Gastrointestinal panel on stool in Cary Blair (27 targets) Meningitis/Encephalitis Panel on CSF (14 Targets) 36

37 Source Bacteriology Automation

38 Comparison In house Assays Advantages Choice of target / Specimen type Flexibility of extraction/detection Allows testing for small volume requests Disadvantages Regulatory compliance Dedicated facilities Resources required for mastermix making and QC of reagents, controls, stocks, storage Rigorous staff training for technical laboratory skills and result interpretation and reporting Automation Advantages Pre-validated assays Manufacturer QC Ease of use (limited training) Direct transfer of results with interfacing Capacity for high throughput Disadvantages Creation/Verification of LIS interfacing Limited validated specimen types Restricted specimen requirements eg. time from collection 38

39 Questions 39