Metrological traceability for Molecular Diagnostics. Jim Huggett Principal Scientist, Nucleic Acid Research LGC, Queens Road, United Kingdom
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1 Metrological traceability for Molecular Diagnostics Jim Huggett Principal Scientist, Nucleic Acid Research LGC, Queens Road, United Kingdom INFECTMET: (
2 INFECTMET partners NMI partners LGC, UK (co-ordinating) NIB, Slovenia JRC-IRMM, EU TUBITAK-UME, Turkey PTB, Germany BAM, Germany Biometrology expertise Biometrology capabilities and knowledge (LGC, NIB, JRC, TUBITAK), clinical reference standard development (JRC, LGC), physical separation (PTB) surface analysis (BAM) metrology expertise Technical expertise World leaders in digital PCR (dpcr), Next Generation Sequencing (NGS) standardisation Cell counting by flow cytometry REG partners: Charité, Germany Leading provider of external quality assessment (EQA) schemes for viral diagnostics. UCL & University College Hospital London University Clinic of Respiratory and Allergic Diseases (UCG) (Golnik), Slovenia Internationally recognised clinical microbiology experts in Tuberculosis (TB) and Chronic obstructive pulmonary disease (COPD)!
3
4 2.2.3 Units for dimensionless quantities, also called quantities of dimension one Another class of dimensionless quantities are numbers that represent a count, such as a number of molecules, degeneracy (number of energy levels), and partition function in statistical thermodynamics (number of thermally accessible states). All of these counting quantities are also described as being dimensionless, or of dimension one, and are taken to have the SI unit one, although the unit of counting quantities cannot be described as a derived unit expressed in terms of the base units of the SI. For such quantities, the unit one may instead be regarded as a further base unit. SI brochure 8 th edn.
5 WHY NOW?
6 Digital PCR (dpcr) qpcr 1 20 µl reac,ons dpcr 20 1 µl reac,ons Limiting dilution Some reaction contain 0 templates Partition the reaction PCR performed as normal using standard real-time PCR chemistry Absolute quantification +ve or ve reactions Poisson statistics to account for multiple targets per partition (> 1)
7 Materials Procedures Calibration material Defined (high) purity Metrological traceability Primary calibrator reference material 2ndary reference material 3ary reference material Routine sample Higher order method Method A Method B Method C Method in clinical lab Uncertainty Result
8 Molecular quantification IU/ml plasma Materials Procedures Metrological traceability reference material 2ndary reference material 3ary reference material Routine sample qpcr qpcr Method in clinical lab Uncertainty Result
9 Molecular quantification Copies Materials Procedures Metrological traceability Calibration material (E.g. dnmps) Defined (high) purity Primary calibrator reference material 2ndary reference material Routine sample Weight Physicochemical method dpcr Method in clinical lab Uncertainty Result
10 Molecular quantification SI traceable Copies via counting Materials Procedures Calibration material Defined (high) purity Metrological traceability Sources of impurity Reference material 2ndary reference material Routine sample dpcr dpcr/qpcr Method in clinical lab Sources of bias Uncertainty Result
11 Sampling Storage Quality Extraction Storage Yield Quality Inhibitors Molecular Analysis Sample Nucleic Acid dpcr Standardisation
12 Materials for full analytical workflow Quantity: Bacilli (cfu) Quantity: Genomic copies Quantity: Gene copies Bacteria/Virus Genomic DNA/RNA within genome Gene target
13 Materials for full analytical workflow Biological matrix Or Lysis Or Purification Or Analysis (PCR)
14 Human Cytomegalovirus Copies /ml Median values +/- 0.8 log 10 3 dpcr results E E E E E E-02 Dilution factor INSTAND ICBS
15 Tuberculosis
16
17 Xpert RIF/MTB
18
19 M. bovis BCG in synthetic sputum CFU data: Mean: 1.09E+07 ± 1.52E+06 (14%) 300 units prepared in synthetic sputum. Molecular homogeneity & Short term stability complete, Long term stability ongoing
20 dpcr platforms/laboratories 5.00E+07 Copies/unit Units E+06 BioMark QX100 BioMark BioMark QX dpcr platform/laboratory JRC LGC NIB
21 Extraction methods 10 log 10 (GE/extraction) Method 1 Method 2 Method 3 Extraction method Qubit qpcr BioMark QX100
22 Precision 40% 35% Whole process precision (%CV) 30% 25% 20% 15% 10% 5% 0% BioMark QX100 BioMark QX CTAB Prepman dpcr platform / Extraction method Between day Within day Measurement (dpcr)
23 Quantification Xpert RIF/MTB
24 Fluorescence Cycle
25 4,000 copies/vial (2,500-10,000 copies/vial)
26 Inter-laboratory comparison Materials sent to eight clinical laboratories (three vials of each material per analysis) Three perform qpcr Six perform Xpert RIF/MTB
27 Xpert RIF/MTB Result frequency BCG/ASM ND Very low Low Medium High Gene Xpert result MTB detection level Result frequency Total MTB Control ND Very low Low Medium High Gene Xpert result MTB detection level
28 Summary Concept of developing SI traceable reference measurement system based on nucleic acid enumeration demonstrated dpcr with optimised extraction has proven to be reproducible and robust in laboratory comparisons Potential for dpcr as reference method DNA copy number enumeration by dpcr can form a reliable and informative basis for range of different measurements in molecular diagnostics Potential for dpcr value assignment of RMs INFECTMET publications available for download: http//infectmet-lgcgroup.com
29 Acknowledgements LGC Alison Devonshire Simon Cowen Denise O Sullivan Alexandra Whale Alice GuDeridge Gerwyn Jones Carole Foy Helen Parkes University College London Tim Mchugh, Isobella Honeybourne Jeremy Garson & Kathryn Harris Charite/GBD Heinz Zeichhardt, Hans- Peter Grunert Vircell Pablo Mendoza Bio- Rad Svilen Tzonev/Viresh Patel/DBC JRC Heinz Schimmel Maria Karczmarczyk NIB Mojca Milavec Jernej Pavšič
30 Acknowledgements Inter!Laboratory!Comparison!! Great!Ormond!Street!Hostpital! NUI!Galway! Forschungszentrum!Borstel! KCMC/KCRI! Lancet!Laboratories!! San!Raffaele!Scientific!Institute! TASK Applied Science University College London!!
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