Best Practices in Pre-analytical Sample Handling for Free
Circulating DNA Isolation
Dr. Marco Polidori
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Best Practices in Pre-analytical Sample Handling for Free Circulating DNA Isolation
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Legal Disclaimer
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applications. These products are not intended for the diagnosis,
prevention, or treatment of a disease.
For up-to-date licensing information and product-specific
disclaimers, see the respective QIAGEN kit handbook or user
manual. QIAGEN kit handbooks and user manuals are available
at www.QIAGEN.com or can be requested from QIAGEN
Technical Services or your local distributor.
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Best Practices in Pre-analytical Sample Handling for Free Circulating DNA Isolation
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Agenda
Introduction
Sample handling
ccf DNA Isolation
Controls for ccfDNA applications
Summary
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Best Practices in Pre-analytical Sample Handling for Free Circulating DNA Isolation
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The main areas of Liquid Biopsy
Free circulating Nucleic acids
DNA
RNA
miRNA
Circulating Tumor Cells
Enumeration
Genotyping
Exosomes
Total RNA
DNA
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Best Practices in Pre-analytical Sample Handling for Free Circulating DNA Isolation
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Liquid Biopsy: Game changing potential
From sequencing an entire fetal genome from maternal plamsa…
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Best Practices in Pre-analytical Sample Handling for Free Circulating DNA Isolation
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Liquid Biopsy: Game changing potential
…to groundbreaking oncology studies
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Best Practices in Pre-analytical Sample Handling for Free Circulating DNA Isolation
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Liquid Biopsy: Game changing potential
…or even combined both
Eunice Lee took a prenatal test for trisomy
Unusual pattern detected in her ccfDNA profile
Consulted on pattern it might be cancer
Diagnosed with colon cancer after MRI verification
Surgery to remove cancer
No signs of ctDNA in follow up at 7 month pregancy
http://www.buzzfeed.com/virginiahughes/pregnant-women-are-finding-out-they-have-cancer-from-a-genet#.nkZEaBdp9
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Best Practices in Pre-analytical Sample Handling for Free Circulating DNA Isolation
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Application areas
New paradigm: less invasive
bio3400.nicerweb.com
Zeit.de
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Best Practices in Pre-analytical Sample Handling for Free Circulating DNA Isolation
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Circulating nucleic acids: analysis workflow
Pre-analytical Workflow
Sample
Blood draw
(venipuncture)
Separate plasma
Extract circulating nucleic acids:
QIAamp Circulating NA Kit,
QIAsymphony Circulating NA Ki
Analytical
Workflow
Results
Real-time PCR
digital PCR therasceen
assays
Next-generation
sequencing
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Best Practices in Pre-analytical Sample Handling for Free Circulating DNA Isolation
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Critical points along the workflow
Avoid release of cellular
nucleic acids
Blood draw
(venipuncture)
Separate plasma
Highly efficient large-volume
nucleic acid extraction
Extract circulating nucleic acids:
QIAamp Circulating NA Kit,
QIAsymphony Virus/Pathogen Kit
Maximize recovery
improve sensitivity
Optional DNA modification
(e.g., bisulfite treatment)
Real-time PCR
digital PCR
Sequencing library prep
Next-generation
sequencing
Reduce interference of nontarget (“normal”) nucleic acids
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Best Practices in Pre-analytical Sample Handling for Free Circulating DNA Isolation
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Some challenges we will cover
Sample
handling
• Collection tubes
• How to prepare
plasma
• What is the right
approach for me
ccfDNA
isolation
• Isolation
Controls
Example
applications
• Efficiency
• qPCR
• Concentration
• ddPCR
• Sample volume
• Normalization
• NGS
• Size bias
• +/- controls
techniques
• Size distribution
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Best Practices in Pre-analytical Sample Handling for Free Circulating DNA Isolation
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Sample handling: Considerations
Some food for thought
Consider this:
ccfDNA concentration is in the range of 1-50 ng / ml plasma
That includes wild type AND mutant DNA (or fetal and maternal DNA)
The fraction of ctDNA or fetal DNA can range between 1-30%
Mutant allele frequency that you may want to capture can be 1-2%
For an example 60 kg patient the number of ctDNA in the entire system may be in the range of
1-3 x 107 molecules.* That just leaves a few thousand mutant molecules per ml of blood.
…And that is not even considering error rates of PCR/NGS
* Heidary et al. Breast Cancer Research 2014, 16:421
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Best Practices in Pre-analytical Sample Handling for Free Circulating DNA Isolation
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Sample handling: Amount of blood
So how much sample material do you really need?
Successful studies used as little as 500µl but in all cases, sensitivity can be increased by
increasing the volume of plasma
2-4 ml of plasma is the gold standard for NIPT
We recommend at least 2 ml, 5 ml are better for oncology related topics
In some cases (with poor ctDNA shedding) 10 ml or more might be justified
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Best Practices in Pre-analytical Sample Handling for Free Circulating DNA Isolation
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Sample handling: An example
Mutant allele frequency variation and concentration example
Amount of mutated fragments vary strongly
from <100 to >100,000 per 5 ml.
Amount of ctDNA also depends on cancer
type and tumor burden!
Chetan Bettegowda et al. 2014 doi:10.1126/scitranslmed.3007094
Effectively, between 10 and 20 ml of
blood should be taken for ccfDNA
studies
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Best Practices in Pre-analytical Sample Handling for Free Circulating DNA Isolation
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Blood sample processing
1-2h
Draw EDTA whole
blood
Draw whole blood in CellFree DNA BCT (Streck)
Spin @ 1900g for
plasma separation
Spin @ 300g for plasma
separation
14 days
Carefully save
supernatant
Spin @ 16,000g
Supernatant:
plasma w/o cell
debris and reduced
gDNA background
Store at -80 °C
Thaw plasma
(Spin @ 16,000g
to remove any
precipitate if
needed)
Extraction of
cfDNA
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Best Practices in Pre-analytical Sample Handling for Free Circulating DNA Isolation
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Sample handling
What type of tubes?
Pros and cons for different tubes
EDTA:
cells are less stable increasing risk of background
very good compatibility to molecular analysis
Cheap
Streck:
Very good stabilization of cells reducing background risk
stabilization agents may impair DNA quality
Fragile glass tubes
costly
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Best Practices in Pre-analytical Sample Handling for Free Circulating DNA Isolation
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Sample handling
General considerations
Where is the blood taken and where is it analysed ?
In many cases the blood is taken at one site and analysed at another requiring some sort of
transportation.
Scenarios depend on the Blood collection tubes used
EDTA
Prepare plasma immediately (recommended)
Transport on ice
Streck:
Transportation of whole blood is ok
Transportation at RT is ok
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Best Practices in Pre-analytical Sample Handling for Free Circulating DNA Isolation
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Some challenges we will cover
Sample
handling
• Collection tubes
• How to prepare
plasma
• What is the right
approach for me
ccfDNA
isolation
• Isolation
Controls
Example
applications
• Efficiency
• qPCR
• Concentration
• ddPCR
• Sample volume
• Normalization
• NGS
• Size bias
• +/- controls
techniques
• Size distribution
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Best Practices in Pre-analytical Sample Handling for Free Circulating DNA Isolation
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ccfDNA isolation: What really matters
The most important things to consider
Type of collection tube (compatibility)
Sample volume (the more the merrier)
Fragment size bias (no bias or towards small fragments)
Elution volumes (reasonably small)
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Best Practices in Pre-analytical Sample Handling for Free Circulating DNA Isolation
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ccfDNA isolation: Techniques
Two main isolation techniques
Spin columns / vacuum:
QIAamp cfNA Kit
Magnetic beads: QIAsymphony cfDNA Kit
1-5 ml plasma, serum, urine
2-4 ml plasma, serum
20-100 µL elution volume
60 µL elution volume
24 samples in 3 hours
96 samples in 6 hours (fully
automated)
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Best Practices in Pre-analytical Sample Handling for Free Circulating DNA Isolation
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ccfDNA isolation considerations: Fragmentation
Size bias can influence your downstream results
genome equiv./ sample x1000
Standard kit not intended to deal with fragmented DNA show lower isolation efficiency
The small fragments are the ones your are interested in most
Ultimately results in better sensitivity
2000
QIAamp circulating NA Kit 66 bp
Standard blood kit 66 bp
1500
1000
500
0
sample 1
sample 2
sample 3
Devonshire et al. doi:10.1007/s00216-014-7835-3
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Best Practices in Pre-analytical Sample Handling for Free Circulating DNA Isolation
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Size distribution: how it should look like
Agilent High Sensitivity
DNA Kit (5-500 pg/μl)
QIAamp Circulating NA Kit (red)
QIAsymphony Circulating DNA (blue)
www.broadinstitute.org
Agilent DNA 7500 Kit
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Best Practices in Pre-analytical Sample Handling for Free Circulating DNA Isolation
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Some challenges we will cover
Sample
handling
• Collection tubes
• How to prepare
plasma
• What is the right
approach for me
ccfDNA
isolation
• Isolation
Controls
Example
applications
• Efficiency
• qPCR
• Concentration
• ddPCR
• Sample volume
• Normalization
• NGS
• Size bias
• +/- controls
techniques
• Size distribution
Sample to Insight
Best Practices in Pre-analytical Sample Handling for Free Circulating DNA Isolation
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ccfDNA controls
General considerations for controls
Do not rely on standard DNA concentration measurements
Spectrophotometric assessment not feasible due to low concentration of ccfDNA and
absorbance from salts
Fluorometric assessment not feasible due to low concentration
Use qPCR for controls
Easy, “cheap”, fast, reliable
Use different markers with different fragment sizes
Allows easier normalisation
Additional assessment of ctDNA / fetal DNA enrichment
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Best Practices in Pre-analytical Sample Handling for Free Circulating DNA Isolation
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ccfDNA Controls: Normalisation
Different markers can be used to normalise ccfDNA in a sample
Devonshire et al. Anal Bioanal Chem
DOI 10.1007/s00216-014-7835-3
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Best Practices in Pre-analytical Sample Handling for Free Circulating DNA Isolation
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ccfDNA Controls: Combined approach
Combined controls provide most useful insights
2-dimensional control assessment using qPCR:
gDNA background (B-cell allele quantification)
ccfDNA isolation efficiency (artificial spike-in control)
Allows reliable normalisation of ccfDNA fractions plus assesses the isolation efficiency
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Best Practices in Pre-analytical Sample Handling for Free Circulating DNA Isolation
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Controlling your work
So what controls do we really need?
Efficiency of ccfDNA isolation (spike-in)
gDNA background normalization
Negative control (e.g. no template for PCR based methods; healthy donor)
Positive control (spike-in; reference sample
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Best Practices in Pre-analytical Sample Handling for Free Circulating DNA Isolation
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Example: plasma circulating DNA and therascreen
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Best Practices in Pre-analytical Sample Handling for Free Circulating DNA Isolation
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Digital PCR
http://www.sysmex-inostics.com/science-and-technology/beaming-digital-pcr.html
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Best Practices in Pre-analytical Sample Handling for Free Circulating DNA Isolation
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NGS
Forshew et al. Sci Transl Med 4, 136ra68 (2012); DOI:
10.1126/scitranslmed.3003726
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Best Practices in Pre-analytical Sample Handling for Free Circulating DNA Isolation
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QIAGEN Sample to Insight Solutions for ccfDNA
Sample
collection
Sample
Isolation
Any collection
tube (e.g. EDTA,
K3EDTA)
QIAamp
Circulating
Nuecleic Acid Kit
Amplification
GeneRead
DNA seq cancer
panel v2
Library
preparation
GeneRead
Library prep Kits
QIAsymphony
free criculating
DNA Kit
Sequencing
Any sequencer
Data
Analysis &
Interpretation
CLC cancer
workbench
Ingenuity
Variant Analysis
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Best Practices in Pre-analytical Sample Handling for Free Circulating DNA Isolation
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Summary
Some take home points
Proper tube choice and handling can minimize gDNA background
Use at least 2-5 ml plasma (or other biofluids) whenever possible to maximize sensitivity
Use a dedicated isolation technique that also recovers small fragments
Use appropriate controls to validate your experiments
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Title, Location, Date
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Thank you for attending
Questions?
Contact QIAGEN Technical Service
Call: 1-800-426-8157 for US
Call: +49 2103-29-12400 for EU
Email:
[email protected]
[email protected]
Marco Polidori, Ph.D.
[email protected]
Any webinar related questions:
[email protected]
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