Metrological traceability for Molecular Diagnostics Jim Huggett · 2015-12-07 · – Next Generation ... Leading provider of external quality assessment (EQA) schemes for viral diagnostics

INFECTMET: (www.infectmet.lgcgroup.com) INFECTMET: (www.INFECTMET.lgcgroup.com)

Metrological traceability for Molecular Diagnostics

Jim Huggett Principal Scientist, Nucleic Acid Research LGC, Queens Road, United Kingdom

!

•  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.

INFECTMET: (www.infectmet.lgcgroup.com)

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


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


Routine sample

Result

Materials

Met

rolo

gica

l tra

ceab

ility


Unc

erta

inty

dPCR/qPCR 2ndary reference

material

Reference material

dPCR

Procedures

Copies


Routine sample

Result

Materials

Met

rolo

gica

l tra

ceab

ility

Sources of bias

Calibration material Defined (high) purity

Sources of impurity


SI traceable via counting

Unc

erta

inty


Molecular Analysis

Extraction

Sampling Storage Quality

Quality Yield Storage

Inhibitors

Sample

Nucleic Acid

dPCR

Sta

ndar

disa

tion


Materials for full analytical workflow

Quantity: Bacilli (cfu)

Quantity: Genomic copies

Quantity: Gene copies

Genomic DNA/RNA within genome

Bacteria/Virus Gene target


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


Tuberculosis



Xpert RIF/MTB



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


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


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


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


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


4,000 copies/vial (2,500-10,000 copies/vial)


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


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


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

Documents

Metrological traceability for Molecular Diagnostics Jim Huggett · 2015-12-07 · – Next Generation ... Leading provider of external quality assessment (EQA) schemes for viral diagnostics