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Measurement uncertainties associated to digital PCR

Presentation · October 2016

DOI: 10.13140/RG.2.2.18128.51208

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 The European Commission’s
science and knowledge service
Joint Research Centre

Measurement
uncertainties associated
to digital PCR
Philippe Corbisier

4th qPCR and Digital PCR Congress

20-21 October, London, UK
 ? 3 questions
• Which parameters should be investigated when
performing an in-house validation of a ddPCR
method ?

• Which factors have a significant effect on the

measurement results ?

• How to estimate the contribution to such factors

towards the overall measurement uncertainty ?
2012

value assigned by cdPCR

(BioMark°Fluidigm°12,765 biochips)

LGC
NMIA
IRMM
 2015
QX100 ° Biorad

BioMark vs QX100

Volume of the droplets

BioMark ° Fluidigm
 In-house validation of ddPCR
• Introduce ddPCR in QS

• A formal recognition that a laboratory is

competent to perform specified tests or
measurements

• ISO/IEC 17025:2005 (general requirement)

• ISO 15189: 2012 (medical laboratories)
• Eurachem Guide 2014
• IUPAC technical report 2002
Critical performance characteristics
• Data Analysis
• Selectivity
• working range
• Accuracy
Precision
Trueness
• Measurement uncertainty
• LOD, LOQ
• Robustness
Data analysis
• Fluorescent threshold placed manually by the analyst at the
midpoint between the average fluorescent amplitude of the
+ and – cluster

• Same threshold applied for all the wells of a same plate

• Data rejected for technical reasons if :

• Total number of analysed droplets < 10.000

• Ave fluorescence amplitude positive or negative
droplet are clearly different from those of other
wells
• > 5 % accepted droplets had a fluorescence below
the average of the negative droplet cluster
Selectivity
+
-
Verify the frequency of missed calls
2 types of miscall:

• false positive ← non-specific amplification

→ oligo design

• false negative ←PCR inhibitors

→ quality / purity samples
Selectivity

Blank CTRL ? interference

no false positive
(0/61275)

CTRL
Routine @ high cp/µL ? interference
spiked
false negative
(33/57895) 0.057 %
Clear segregation + and - clusters (no rain)
Working range

n=8
RSD < 5 % between 26 and 5400 cp/µL
RSD 16.9 % @ 2.5 cp/µL

LOQ

, , ,

1 !
,
"!
Accuracy
Precision

estimation within & between variability

Trueness

estimation of the bias

@ medium
@low copy number concentration
Precision
Each of five
ERM-AD623
solutions

Run 1 Run 2 Run 3

n= 12 n = 16 n = 12
within run: between runs:
= analyst ≠ analyst
= cartridge type ≠ instruments
= reagent batches ≠ cartridges types
= instrument ≠ reagent batches
= PCR plate ≠ soLware versions
Precision

within run

between run:
Accuracy - Trueness

Reference value

a) CRM
b) recovery of spiked samples
c) results from another method

Relative bias
 Average (-9.6 %)

Pooled (6.1 %)

Pooled (2.9 %)

Pooled (1.9 %)
U bias,rel = 10.9 % > l -9.6 l %

No significant bias BUT tendency to measure lower cp/µL

Measurement uncertainty – Fish bone diagram
Droplet generation

Viscosity PCR mix

Binominal
distribution Supermix
Distribution target
Droplet Pipet
sequence over generator manipulations Sample matrix
droplets Single stranded
DNA Vd
DNA structures
Cartridge Random Assigned
variation value
Dfsample
Balance Droplet reading
Mdil + Mdil+sample
Sampling Droplet reader
Density Software

Threshold
Density Sampling Supermix Software
Mpremix + Mmix Assay design
Balance
Data analysis
DfPCR Thermocycler
Inhibitors

Accessibility Incomplete mixing reagents

Target sequence Cprimers/probe
Intactness Primers/probe
Quality/purity
PCR amplification
Volume of the droplets (Vd)
0.91 nL 0.85 nL

[186-3026] ddPCR Supermix for Probes

[186-3023] ddPCR Supermix for Probes (No dUTP)

no dUTP with dUTP

[nL] [nL]

NIST* cp/µL
0.780 cp/µL
0.815
NMIA - 90.795
% - 30.833
%
IRMM 0.771 ± 0.012 0.834 ± 0.030§
0.782 nL 0.827 nL

Urel = 3-4 %

* Dagata et al. 2016 ° dilute method

§ Corbisier et al. 2015
Measurement uncertainty
Negligible sources (< 1 %)
• Accuracy weighing (gravimetric dilutions)
• Density sample mix
• Quality HPLC purified primers
• Different assays
• Threshold settings

• ? intactness ds DNA
• ? inhibitors
• ? secondary structures

Uexp = 14.2 %
2 2
*
'%+%&, ,+--(%. ,'%( '0/ ,+--(%. ,'%(
#$%& 2 01. ,'%( 2 023& 2
,'%(
/$%& /'0/ ,'%(
LOD
? Lowest cp/µL ≠ 0 (95 %)

1 sample @ 0.5 cp/µL (n = 64)

repeatability

All positive
0.56 cp/µL RSD 34.4 %

LOD < 0.5 cp/µL

LOQ

? Lowest cp/µL with acceptable Uexp

tested @ 3.5 cp/µL

Runs = 2; replicates = 12

→ Uexp = 28.9 %
 Robustness

? ± 10-20 % primer and probe (n = 10)

? ± 1°C annealing T°C (n = 14)

No significant effects

Not evaluated :

• Biological variability of the target sequence

• Sample source
• Sample preparation
• Sample storage
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Conclusions

Example

Extensiveness f (intended use + acceptable level U)

Factors affecting the measurement of routine samples

Liesbet D. et al. Biomol Dect & Quant 9(2016)29-39