Comprehensive quality control for multiplex testing: tools

Comprehensive quality
control for multiplex testing:
tools, choices and solutions
Todd Christensen
Research and Development
Sacred Heart Medical Center and Children’s Hospital
Providence Associates Medical Laboratories
Controls should…
„ Confirm
every reagent in the assay is
performing appropriately.
„ Allow for a QC methodology that is
efficient and cheap.
„ Be in ample supply for every mutation
to be tested.
„ Fit the assay in question
Tools and Choices
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Isolation
PCR Amplification
Mutation Detection
Contamination
Control
Troubleshooting
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Source material
Patient Sample
Wild-type Genomic
DNA control
Mutant Genomic DNA
control
No template
Sample specific
measurements
Synthetic DNA controls
Why are synthetic controls
needed?
„ Limited
supply of genomic mutant
controls.
„ Genomic controls do not allow for
multiplex testing.
„ Controlling for all mutations in an assay
would be very expensive.
Multiplex Qualitative Assay
Choices
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Isolation:
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PCR:
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1 WT gDNA, 1-5 Mutant gDNA
Contamination control:
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1 WT gDNA control, patient sample
Detection:
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Quantitation, 260/280, patient sample
No template DNA
Troubleshooting aids:
„
all of the above, validation studies, comparison to other inhouse assays, paper trails, persistence and luck
What is not being checked, what
are we missing and is that OK?
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Mutation specific primers and probes
Making assumptions based on initial
validation results.
The range of yes and no.
Absolute vs. detailed QC
„ Borderline results provide clues
„ Trending can identify tests soon to be in
trouble.
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Reality check / value judgement
Our Approach
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Integrate synthetic controls into a standard
genomic DNA QC system.
A methodology that is easily modified,
efficient, and totally synthetic.
Provide speed and flexibility to be adopted
for many distinct mutations.
Minimize time and reagent costs by testing
for every mutation in a single reaction tube.
Synthetic Control Design
Genomic DNA
Wild-type Genomic DNA
Mutation
Genomic DNA with a CF mutation
Synthetic Oligonuclotide
5’ tag
Sequence of Primer B (yellow)
Complement sequence to Primer A (dark orange)
The DNA sequence flanking the mutation site (blue)
Primer B
3’ tag
Primer A
The Full Reaction
Control Mix
CF32
CF
PCR
ASPE
OLA
QC Comparison
genomic vs. genomic/synthetic strategy
Component
G G/S
Component
Buffer
I148 wt
Taq
I148T
Ligase
621+1 wt
Primers
621+1G-T
F508 wt
711+1 wt
ΔF508
711+1G-T
I507 wt
1078 wt
ΔI507
1078delT
G542 wt
R334 wt
G542X
R334W
G85 wt
R347 wt
G85E
R347P
R117 wt
A455 wt
R117H
A455E
G G/S
Component
G G/S
1717-1 wt
?
?
?
?
?
?
?
1717-1G-A
R560 wt
R560T
R553 wt
R553X
G551 wt
G551D
1898+1 wt
1898+1G-A
2184 wt
2184delA
2789+5 wt
2789+5G-A
Component
G G/S
3120+1 wt
?
?
3120+1G-A
R1162 wt
R1162X
3659 wt
?
3659delC
?
3849+10kbC-T
3849+10kb wt
W1282 wt
?
?
?
?
?
W1282X
?
N1303K
?
G/S:Genomic/Synthetic
N1303 wt
?
?
G:Genomic Strategy
Genomic vs. Genomic/Synthetic
Genomic strategy
Genomic/Synthetic strategy
$=
Mutant genomic DNA control:
Wild-type Genomic DNA Control:
No Template DNA Control:
Synthetic Control:
PCR/OLA Platform CF ASR
Wild-Type gDNA Control
delF508 gDNA Control
What does the data look like?
Variations in Procedure
V520F
PCR/OLA Platform CF IVD
Wild-Type gDNA Control
delI507 gDNA Control
PCR/OLA Platform CF IVD
CF32 Synthetic Control
CF33 Synthetic Control
Switching Gears to another
Platform:
„
Luminex xMAP (Tm Bioscience)
CF Multiplex Assay
„ Ashkenazi Panel multiplex assay
„ Cytochrome p450
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Synthetic Control Results on the
Luminex CF39 IVD Panel
Synthetic Control Results on the
Tm Bioscience CF70 Extended Panel
Synthetic Control Results on the
Tm Bioscience AJ Panel
Cytochrome p450 2D6
Synthetic Mutant
Control
Cytochrome p450 2D6
Genomic DNA
Control
Cytochrome p450 2D6
Synthetic Mutant
Genomic DNA
Mixed Control
Closing Thoughts
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Think critically about QC
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Identify your needs and use the best tool for the job
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Assumptions are dangerous- V520F
Global vs. Platform specific controls
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Genomics
Troubleshooting aids
Cover your blind spots and develop a comprehensive QC
plan
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Heterozygotes vs. Homozygotes
Tradeoffs and needs will determine direction
Variability in assay design and mutations tested
Keep an eye on the details
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Yes/No results are not enough
Monitor for trends
Acknowledgments
„
Sacred Heart Medical Center and
Children’s Hospital
Bassem Bejjani
„ Jill Brandon
„ Marcy Hoffmann
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Eurogentest
Mayo Clinic
ARUP
Specialty Laboratories Inc.
Questions?
Directions
„
Running the Gauntlet to show the necessity of
comprehensive QC strategies.
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Continue evaluation studies with multiple
laboratories.
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Collaborate with other working groups to
develop additional solutions for reference
materials.
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Apply this methodology to address other
multiplex testing needs.
The Gauntlet
Standard Conditions
WT
MUT
Substandard Conditions
Reproducibility
CF40+4 Performance
CF32 Performance
10000
8000
MFI
6000
4000
2000
0
Mutation
AJ Panel Performance
10000
8000
MFI
6000
4000
2000
0
Mutation
Current and Future Projects
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Application and evaluation of CF controls
on additional testing platforms.
Additional multiplex control designs.
CYP450 2C9
„ CYP450 2C19
„ CYP450 2D6
„ VKORC1
„
Asuragen
Roche Gold
WT
CF32
WT
CF32
Platforms Tested
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Methodology
Works
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ABI OLA
Luminex
Asuragen
Innogenetics
Roche Gold
Autogenomics*
TWT Inplex
MassArray
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Doesn’t work
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Osmetech
Elucigene*
Orchid*
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Haven’t tried
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Nanosphere
Elucigene
Orchid
Synthetic Control Results on the
PCR/OLA Platform CFv3.0 ASR
CF32 Synthetic
ΔF508
Heterozygote
2C19
Cytochromes
2C9/VKORC1