Bio-Reference Standards/Materials. Carole Foy LGC Presentation to EuroGentest 15 th May 2007

Transcription

1 Bio-Reference Standards/Materials Carole Foy LGC Presentation to EuroGentest 15 th May 2007

2 Overview of Presentation Role of Horizontal Standards in International Bio- Metrology Bio-reference standards developed at LGC Lessons learnt in production/use of materials

3 Vertical cf. Horizontal Standardisation Vertical reference standards product/gene/matrix specific e.g. Cystic Fibrosis reference panels Horizontal reference standards - generic, link to method performance criteria Technique Product A B C D Real-time PCR Microarray analysis Sequencing PCR-RFLP Calibrate/validate method, operator performance, comparability of different technology platforms.

4 Bio-Reference Materials at LGC Microarray QC Material Real-time PCR QC Material (collaboration with NIST, plasmid based) Real-time PCR QC Kit Material Genotyping/haplotyping QC material Biomarker panel multiple RNA transcripts ( )

5 Additional Bio-reference material activities UNIQ PT scheme launched, with 2 rounds being carried out Reference material production and distribution: ATCC products Cell lines and DNA Gentris GentriSure Human Genomic Reference Controls (Pharmacogenetics)

6 Specificity Standards and Performance Indicators for microarrays Assist in assay optimisation, QC, validation and comparability Multiple sets of standards (high, med, low GC content) Four standards within each set differing by 1,3 and 5 mismatches Fluorescently labelled reverse complements (Cy3 and Cy5) spiked-in Sub-array profiles compared to average profile for slide QC Discriminatory power of each panel assessed Hybridisation efficiency (MFI) % (PM) 60% (1MM) 60% (3MM) 60% (5MM) 50% (PM) 50% (1MM) 50% (3MM) 50% (5MM) Probe Standard 40% (PM) 40% (1MM) 40% (3MM) 40% (5MM)

7 Plasmid Based Performance Standard for qpcr Designed as a independent monitor of equipment and technical staff/lab performance Ct values by real-time PCR within a defined range A plasmid with a synthesised segment that is not homologous to any known gene Sequence suitable for most probe types Ct value Log target amount

8 Real-time PCR Kit QC Material Designed for laboratories wishing to check the consistency of their real-time PCR measurements over time Kit consists of: Forward Primer (21mer oligonucleotide) Reverse Primer (20mer oligonucleotide) Target (86mer oligonucleotide) Probe (24mer)

9 Genotyping/haplotyping QC material Genotype/haplotype-validated reference material platform evaluation analyst training assay QC trace DNA detection (e.g. circulating cancer and fetal markers) rapid DNA detection (e.g. Point Of Care Diagnostics) Non-human (Arabidopsis) Two strains selected which differ by 12 SNPs in ~1500bp fragment Fragments cloned and amplified Poly A tail and T7 promoter added to allow IVT for gene expression

10 Biomarker Reference Panel Increased volume of healthcare information available Many potential new biomarkers Increased complexity of healthcare information Panels of biomarkers rather than a single analyte Multi-analyte analysis Overall trends and patterns Video camera vs single click camera analogy

11 Biomarker material under development (in collaboration with NIST) Calibrator sample for qrt-pcr multi-analyte measurements covering a range of concentrations Used to normalise qrt-pcr measurements Used to evaluate dynamic range and precision of assays Based on ERCC standards Panel of 144 In-vitro transcripts Adopted by the array community as reference standards

12 UNIQ PT scheme Complementary activity to development of ref materials Designed to test laboratories ability to carry out real-time PCR measurements at a generic level Test samples are synthetic oligonucleotides cloned into a plasmid with no direct diagnostic relevance No chance of contaminating in house processes No barriers to acceptance of materials Targets constructed at a range of GC contents Artificial matrix developed and tested Steering committee composed of experts from range of sectors

13 UNIQ PT Scheme 1 round successfully completed December labs (all UK/EU) returned results Results were generally good, but at a few labs had some problems with some measurements Feedback and technical help provided after the report to help labs identify/address potential issues 2 nd round samples with 15 participants nearing completion Some participants outside Europe

14 Lessons learnt.

15 qpcr Plasmid Standard - Round 1 Pilot Study Graphs of equivalence - Round 1- Sample B Estimated concentration (pg/ul) a 3b 4 5a 5b 5c 5d 6a 6b 7a 7b c 17a Group Assigned mean = Consensus mean =

16 qpcr Plasmid Standard - Round 2 Pilot study Graphs of equivalence - Round 2 - Sample B Estimated concentration (pg/ul) a 1b 1c 2a 2b a 6b 7a 7b 8a 8b 9a 9b a 13b 14a 14b 15 Group Assigned mean = 0.18 Consensus mean = 0.16

17 Improvement in performance between rounds Changes between rounds: Experience of participants (potential large effect) Form of target analyte (solution vs. dried-down DNA) Prescriptive protocol Submission of all raw data - negate potential bias Set number of replicates Data handling Verification of data collation Comparisons between rounds - values based on averages Round 2 Round Round 1 0 Round 2 Relative error CV

18 Round 1 of UNIQ PT Participants requested to quantify a high concentration DNA solution in house 12 unknown samples also distributed, with lower concentrations of the target 9 of the 12 unknowns required DNA extraction 2 kits recommended for use All 12 samples then analysed - quantification of target DNA by Q-PCR Primer and probe for 5 -nuclease assay supplied DNA extraction High concentration DNA quantification Q-PCR quantification and reporting

19 Round 1 results Participants mainly used 2 methods for quantification of the high concentration DNA UV spectrophotometry (A 260 measurement) PicoGreen kit (fluorescent intercalating dye) DNA concentration ng/ul Laboratory number Measurements using Pico Green Participants were close to the expected ~20ng/ul Pico Green underestimated the concentration of DNA present by ~50%

20 Variability of DNA quantification methods Common DNA quantification methods can give different results on the same sample May be exacerbated by inappropriate standards and poorly calibrated equipment C o n c ( u g /m l) English et al Use of elemental analysis to determine comparative performance of established DNA quantification methods. Anal Chem. 78(13): U-2000 ND-1000 PicoGreen Quant-iT Platform

21 DNA quantification results Each unknown supplied in triplicate qpcr amplification only Median

22 Extraction and quantification More overall variability with the more complex analytical process Results coming in now for Round 2

23 Useful information on current practice Use of method blanks Separate pre and post PCR No No Yes Yes Separate DNA extraction area Calibrated pipettes No No Yes Yes Feedback on performance and possible problems also provided to participants if requested

24 Summary Need for inter lab comparisons of measurements and associated materials for use Horizontal reference materials can provide high level traceability of measurements for:- calibration/validation of technique operator/lab performance comparability of different technology platforms Homogeneity and stability issues key for production of appropriate materials

25 Acknowledgements Nick Boley Lyndsey Birch Malcolm Burns Carol Donald Steve Ellison Claire English Jacquie Keer Helen Parkes Tim Wilkes Alison Woolford Marcia Holden (NIST) Marc Salit (NIST)