addressing the pre-pcr analytical variability of ffpe samples
TRANSCRIPT
What is the impact of assay failure in your laboratory and how do you monitor for it?
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Addressing the Pre-PCR Analytical Variability of FFPE Samples
Pre-analytical Sample Handling and Processing
Biopsy DNA Quantification
StorageDNA Extraction
DNA Quantity & Quality
FFPE Processing
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Analytical Processing and Reporting
DNA Sample AnalysisActionable
Decision
Quality of Diagnostic Result
Sample preparation
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FFPE HDx™ Reference Standards to monitor your complete workflow
Mutant Wild type
FFPE Processing
FFPE Sections
Digital PCR
Sanger Sequencing
RT-PCR
SNP 6.0
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Characterization of FFPE HDx™ Reference Standards as External Controls
Specific Allelic Frequenciesi.e. 1%, 5%, 50%
Consistent FFPE Sections400ng - 700ng per vial
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EGFR Sample Tested
EGFR Genotyping ErrorsExternal Quality Assessment 2014
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External Quality Assessment Proficiency Testing Scheme - 2014
Using HDx™ Reference Standards the EQA scheme was able to identify and recommend improvements
Pre-analytical FFPE challenges
Pre-analytical main challenges
Efficacy of DNA extraction• Small amount of DNA available –
mixture of tumour and normal DNA• Various extraction methods and yields
Sample collection and handling• Different labs follow different
protocols. Tumour sample
Diagnosis
Therapy
DNA extraction
Genotyping
Accuracy of DNA quantification• Various quantification methods
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0%
10%
20%
30%
40%
50%
60%
70%
PromegaMaxwell(n=12)
PromegaMagnesil (n=6)
PromegaReliaPrep
(n=6)
Qiagen Dneasy(n=6)
Roche Cobas(n=6)
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rce
nta
ge D
NA
Re
cove
red
Extraction Kit
DNA Recovery from Total Theoretical Yield
Comparison of DNA Extraction Methods (Internal data)Quantified using QuantiFluor assay
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Qiagen EZ1(n=3)
COBAS (n=1) LifetechRecover All
(n=2)
QiagenDneasy (n=3)
QiagenQIAmp (n=4)
Co
effi
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of
Var
iati
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(%
)
Extraction Method
Kapp J R et al. J Clin Pathol doi:10.1136/jclinpath-2014-202644
Variation within Extraction Methods (External study)Quantified using Qubit
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N/Q
Participant
Nanodrop:Qubit (N/Q Ratio)
Kapp J R et al. J Clin Pathol doi:10.1136/jclinpath-2014-202644
R2 = 0.4868, p<0.0001
DNA Quantitation
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Summary
Different extraction methods can result in varying DNA yields from the same starting material.
Nanodrop is very efficient at quantifying DNA at high concentrations
At low concentrations spectrophotometry methods overestimate the DNA concentration compared with fluorometry quantification methods
Important implications on diagnostic test – false negatives.
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Range of total DNA (ng) recovered from each sample by participants.
Kapp J R et al. J Clin Pathol doi:10.1136/jclinpath-2014-202644
Clinical Sample
Clinical samples cannot be utilised as external controls due to their huge variability
Cell Line Reference Standards
Validated Cell Line Reference Standards are ideal as External Controls
Validated Cell Line Reference Standards as External Controls
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Mild Formalin Fixation Severe Formalin Fixation
Formalin leads to over-quantitation of DNA
Impact of Formalin Treatment on DNA Quantification
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How to Test the Robustness and Sensitivity of your Workflow and Assay
Sensitivity of your Assay
HD701
Formalin Intensity
HD200
Robustness and Sensitivity of your Workflow
HD-C751
FFPE
DNA
Robustness of your Assay
HD-C750
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What are the Outstanding Questions?
What is the impact of assay failure in your laboratory and how do you monitor for it?
What extraction and quantification methods are you
using?
What is the limit of detection of your
workflow?
Is the impact of formalin treatment interesting to you?
Validated cell Line Reference Standards are Ideal as External Controls
References
Variation in pre-PCR processing of FFPE samples leads to discrepancies in BRAF and EGFR mutation detection: a diagnostic RING trial (2014)Kapp J, Diss T, Spicer J,Gandy M, Schrijver I, Jennings L, Li M, Tsongalis G, et al., Journal of Clinical Pathology, Volume 67
Assessing standardization of molecular testing for non-small-cell lung cancer: results of a worldwide external quality assessment (EQA) scheme for EGFR mutation testing (2014) Patton, S., Normanno, N., Murray, S., Kerr, K., Dietel, M., Filipits, M., et al. British Journal of Cancer, 413-420.
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