Metrological traceability for Molecular Diagnostics Jim Huggett · 2015-12-07 · – Next...
Transcript of Metrological traceability for Molecular Diagnostics Jim Huggett · 2015-12-07 · – Next...
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Metrological traceability for Molecular Diagnostics
Jim Huggett Principal Scientist, Nucleic Acid Research LGC, Queens Road, United Kingdom
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• 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)
INFECTMET partners
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 8th edn.
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WHY NOW?
• Limiting dilution – Some reaction contain 0
templates
• 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)
Partition the reaction
dPCR 20 × 1 µl reac,ons
Digital PCR (dPCR)
qPCR 1 × 20 µl reac,ons
Method B 2ndary reference material
3ary reference material
reference material
Higher order method
Procedures
Calibration material Defined (high) purity
Method A Primary calibrator
Method in clinical lab
Method C
Routine sample
Result
Materials
Unc
erta
inty
Met
rolo
gica
l tra
ceab
ility
qPCR 2ndary reference material
3ary reference material
reference material
Procedures
IU/ml plasma
Method in clinical lab
qPCR
Routine sample
Result
Materials
Met
rolo
gica
l tra
ceab
ility
Molecular quantification
Unc
erta
inty
dPCR 2ndary reference
material
reference material
Weight
Procedures
Copies
Calibration material (E.g. dNMPs) Defined
(high) purity
Physicochemical method
Primary calibrator
Method in clinical lab
Routine sample
Result
Materials
Met
rolo
gica
l tra
ceab
ility
Molecular quantification
Unc
erta
inty
dPCR/qPCR 2ndary reference
material
Reference material
dPCR
Procedures
Copies
Method in clinical lab
Routine sample
Result
Materials
Met
rolo
gica
l tra
ceab
ility
Sources of bias
Calibration material Defined (high) purity
Sources of impurity
Molecular quantification
SI traceable via counting
Unc
erta
inty
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Molecular Analysis
Extraction
Sampling Storage Quality
Quality Yield Storage
Inhibitors
Sample
Nucleic Acid
dPCR
Sta
ndar
disa
tion
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Materials for full analytical workflow
Quantity: Bacilli (cfu)
Quantity: Genomic copies
Quantity: Gene copies
Genomic DNA/RNA within genome
Bacteria/Virus Gene target
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Materials for full analytical workflow
Lysis Analysis (PCR)
Biological matrix
Or Or Purification
Or
1
10
100
1000
10000
100000
1000000
1.00E-07 1.00E-06 1.00E-05 1.00E-04 1.00E-03 1.00E-02
Dilution factor
Cop
ies
/mL
Median values +/- 0.8 log10 3 dPCR results
Human Cytomegalovirus
INSTAND ICBS
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Tuberculosis
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Xpert RIF/MTB
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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
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dPCR platforms/laboratories
5.00E+06
5.00E+07
BioMark QX100 BioMark BioMark QX100
JRC LGC NIB
Cop
ies/
unit
dPCR platform/Laboratory
1 2 3 4
Units
1 1 2 3
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Extraction methods
Extraction method
log 1
0 (G
E/ex
trac
tion)
Method 1 Method 2 Method 3
2
4
6
8
10
qPCR BioMarkQubit QX100
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0%
5%
10%
15%
20%
25%
30%
35%
40%
BioMark QX100 BioMark QX100
CTAB Prepman
Who
le p
roce
ss p
reci
sion
(%C
V)
dPCR platform / Extraction method
Between day Within day Measurement (dPCR)
Precision
1 2
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Quantification Xpert RIF/MTB
0
0.5
1
1.5
2
2.5
3
3.5
1 3 5 7 9 11 13 15 17 19 21 23 25 27 29 31 33 35 37 39 0
0.5
1
1.5
2
2.5
3
3.5
1 3 5 7 9 11 13 15 17 19 21 23 25 27 29 31 33 35 37 39 0
0.5
1
1.5
2
2.5
3
3.5
1 3 5 7 9 11 13 15 17 19 21 23 25 27 29 31 33 35 37 39
Fluo
resc
ence
Cycle
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4,000 copies/vial (2,500-10,000 copies/vial)
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Inter-laboratory comparison
• Materials sent to eight clinical laboratories (three vials of each material per analysis) – Three perform qPCR – Six perform Xpert RIF/MTB
0
2
4
6
8
10
12
14
16
ND Very low Low Medium High
Res
ult f
requ
ency
Gene Xpert result MTB detection level
BCG/ASM
0
2
4
6
8
10
12
14
16
ND Very low Low Medium High
Res
ult f
requ
ency
Gene Xpert result MTB detection level
Total MTB Control
Xpert RIF/MTB
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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
Acknowledgements LGC • Alison Devonshire • Simon Cowen • Denise O’Sullivan • Alexandra Whale • Alice GuDeridge • Gerwyn Jones • Carole Foy • Helen Parkes
JRC • Heinz Schimmel • Maria Karczmarczyk
Bio-‐Rad • Svilen Tzonev/Viresh Patel/DBC
University College London • Tim Mchugh, Isobella Honeybourne • Jeremy Garson & Kathryn Harris
Vircell • Pablo Mendoza
NIB • Mojca Milavec • Jernej Pavšič
Charite/GBD • Heinz Zeichhardt, Hans-‐Peter Grunert
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Acknowledgements
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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