validating your real time pcr results/qc issues -...
Post on 09-Jan-2020
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Validating your real time PCR
results/QC issues
Dr RN Gunson
On Behalf of the West of Scotland Specialist Virology Centre
R+D team
Gartnavel General Hospital
Glasgow
Scotland
Overview
• How do you control a PCR reaction?
• How do you know your test has worked?
• What to do if a control fails?
• Ongoing QC methods to consider?
How do you control a PCR
reaction?
• You control a reaction via the inclusion of:
– Positive controls
– Negative controls
– Internal controls
– Other• NTCs/RT controls/Standards
What are the PCR controls for?
• What is the positive control for?– included to ensure that the entire testing protocol
(including the extraction, reverse transcriptase and PCR steps) is optimally sensitive.
• Things to consider….– a positive control will detect general errors/failures
that are assumed to be related to all samples at each stage rather than to the individual sample
– It will not detect sample inhibition or individual sample error.
– If the controls are plasmids they may not control extraction/RT step.
What are the PCR controls for?
• How does it work?
– Should reflect the target (eg virus)
– A positive control contains a pre determined amount of target and therefore should become positive at a known Ct value.
– Based on repeat testing an allowable range will be determined between which the control must always fall.
• Westgard rules
• Levey-Jennings plot
What are the PCR controls for?
• What is the negative control for?– Negative controls are included to detect test contamination,
which can result in a false positive result.
• How does it work?– It should be a “fake” sample known to contain no target (e.g.
VTM, lysis buffer, water)
– It should be processed exactly the same way as your samples.
• Things to consider:• Occasionally contamination may be present but will not be picked
up by the control.
– Depends on number of negative controls incorporated in the run.
What are the PCR controls for?
• What is the internal control for?– An internal control is present in each PCR reaction to
ensure it is working optimally.
– It is the ideal positive control as it can detect extraction failures, PCR inhibition, and technical errors relating to each individual sample
• What are PCR inhibitors?– direct interaction with DNA/RNA
– interference with DNA polymerases.
– Interference with the extraction procedure (e.g. PBS)
What are the PCR controls for?
• How does the internal control work?– The IC template will be added to samples prior to extraction. The same volume
of IC is added to each sample. As a result it should be detected at the same Ct in each sample
• pre-extraction, in the eluate or in the mastermix itself.
• Rule of interpretation differs (e.g. 2SD, 3CT, 3SD)
– The PCR reagents contain an IC PCR multiplexed with the pathogen PCR tests.• Can also be a single
• Things to consider:– >1 targets can compete and result in strange IC traces or even negative IC
traces
– Increase the complexity of interpreation (e.g. What do you do should a test fail its IC?)
• Repeat extract on a different machine
• Dilute
• Freeze thaw
• Report as inhibited.
What are the PCR controls for?
• What is an NTC for?
– It is an alternative negative control. Instead of template/extracted material water is added. It is included in a reaction to show the reagents are not contaminated.
• What is the RT control for?
– An RT control is a DNA control that is added to a rtRT-PCR reaction. Its tells the user whether the PCR step has worked.
How do you know your PCR
has worked?
Commercial kits have strict rules
• All negative controls should be below the threshold. If there is a potential contamination (appearance of a curve), results obtained are not interpretable and the whole run (including extraction) has to be repeated.
• All the positive controls must show a positive (i.e. exponential) amplification trace. The positive controls must fall under a ct of 33. The spreading is based on the variance of the instrument.
• Check the “component” trace before accepting the exponential trace as real. Contact the equipment manufacturer or Fast-track Diagnostics for advice (technical@fast-trackdiagnostics.com).
• All internal controls must show a positive (i.e. exponential) amplification trace. The internal control must fall within a range of CT=30 +/-3. The stated spreading is based on the variance of the instrument and the purification. If the internal control falls out of this range, this points to a purification problem.
Changing the rules = loss in CE marking
Commercial kits have strict rules
• Pro’s (vs in-house methods)– QC/troubleshooting is done for you
– No need for specialised staff
– Responsibility is with the company
• Con’s (vs in-house methods)– Reduced opportunity to learn from mistakes/issues
– Cannot partially pass runs
– Users are often instructed to repeat from extraction• Increases cost and turn around time
• ?sample volume issues.
Glasgows guide to the
investigation of in house PCR
control failures……
(some of which may prove useful
to those using commercial kits).
When a control fails..
Important to investigate why:
• To prevent it happening again– Improves training/understanding
– Change protocols
• To determine whether the whole run needsrepeating from extraction.– Expensive (cost and time)
– ?some samples may still be valid despite a failed control
What to do if your PCR fails these
rules?
• Failed positive control:
– No trace/weak trace
• What does this mean?
– The assay is less sensitive than it should be!
• Negative results may not be true negatives.
• Positive results may have stronger Ct values than
those given
What to do if your PCR fails these
rules?
• Reasons for failed positive control:
– Set up error
– Extraction failure
– Reagent degradation
– Control degradation
What to do if your positive control fails?
– Confirm that the PCR set up is correct.• ?control added elsewhere
– Check PCR plate volumes• ?control not added
• ?Check IC to see if this has also failed/less sensitive.
• ?check spectra
– Extraction failure• Check sample was actually extracted.
– Check IC to see if this has also failed/less sensitive– Can be used as an alternative marker of extraction efficiency.
– Check other positive controls from this extraction• This will tell you the extraction process is ok.
– Are there other positive controls for this test on the run (i.e. from previous dates)
• Useful as it will show you the PCR reaction is ok.
What to do if your PCR fails these
rules?• Can you pass a run that has a failed positive
control?– Yes…………partially
– Positive results will still be positive
• But Ct/quant will be wrong.
– ?Repeat negative samples as these may contain positive samples
• From extraction plate or from extraction depending on what troubleshoot has shown.
– NOTE: ongoing issues will lead to an extensive troubleshoot.
Example 1: Flu A control failed.
So would you pass this run?
• The set up is ok.
• The volumes are ok except for the failed control
• The IC is ok in all samples except the failed positive control
• All other RNA positive controls have worked
– Therefore the extraction is ok
• All other Flu A controls worked– PCR reagents ok
• So I would pass the run– Especially if reason can be found for
failed control (sample volume, IC failure)
Flu A
NEG
Sample
Flu A
pos
Sample Sample
Rhino
30
Flu B
30
Sample Sample Sample
Rhino
pos
Rhino
30
Sample Sample Sample NEG
Sample Sample Sample Sample NEG
Sample Sample Sample NEG
Sample Sample NEG
Sample
Rhino
pos
NEG Flu A
30
NEG Sample
Flu A
pos
Flu B
30
Example 2: Flu A control failed.
So would you pass this run?
• The set up is ok.
• The volumes are ok except for the failed control
• The IC is ok in all samples except the failed positive control
• Other RNA positive controls worked– Therefore the extraction ok
• But no proof that flu A PCR reagents are ok.
– Report flu A positive results
– Repeat Flu A negative samples from extraction plate.
Flu A
NEG
Sample
Flu A
pos
Sample
Flu B
30
Sample Sample
Rhino
30
Sample Sample NEG
Sample Sample Sample NEG
Sample Sample Sample NEG
Sample Sample NEG
Sample
Rhino
pos
NEG
NEG Sample
Flu A
pos
What to do if your PCR fails these
rules?
• Failed negative control:
– Positive trace in negative control
• What does this mean?
– The assay may be contaminated!
• positive results may not be true positives.
– The set up is wrong
What to do if your PCR fails these
rules?
• Reasons for failed negative controls– Set up error (wrong sample into wrong well)
• Ct value can be a guide since contamination is often (not always at a low Ct value)
• Strong ct is suggestive of sample mix up
– Contamination• Pre extraction
• At extraction
• PCR set up
• What to do?– Check sample set up
– Check volume of negative control
What to do if your PCR fails these
rules?
• Can I pass a run with a positive negative
control?
– Yes…..partially
– All negative results will remain negative
– If all contamination is at a low level, consider reporting out
positive samples that are much stronger.
– Consider reporting all results if 100% sure why negative is
positive….
• Note: ongoing contamination will not be ignored.
Example 3:
So how would you interpret this run?
• a negative control is positive for Flu A– Ct of ~36
• Report out all Flu A negative results
• Consider reporting out strong positive Flu A results
• Repeat all weak Flu A positive samples with Ct values similar to the false positive neg.– From extraction
Flu A
30
Sample
Flu A
pos (23)
Sample
Flu B
30
Sample Sample
Rhino
30
Sample Sample
Flu A
pos (38)
NEG
Sample Sample Sample
Flu A
pos (24)
NEG
Sample Sample Sample NEG
Sample Sample
Flu A
pos (27)
NEG
Sample
Rhino
pos
NEG
Flu A
pos (36)
NEG Sample
Flu A
pos (34)
Example 4:
How would you interpret
this run?
• Negative control is strongly
false positive.
• Suggestive of set up error.
• Repeat all samples from
extraction plate.
• If problem persists repeat run
from extraction
Flu A
NEG
Sample
Flu A
pos (23)
Sample
Flu B
30
Sample Sample
Rhino
30
Sample Sample
Flu A
pos (38)
NEG
Sample Sample Sample
Flu A
pos (24)
NEG
Sample Sample Sample NEG
Sample Sample NEG
Sample
Rhino
pos
NEG
Flu A
pos (27)
NEG Sample
Flu A
pos (34)
What to do if your PCR fails these
rules?
• Failed IC control
– Negative IC or low Ct
• What does this mean?
– The assay in this sample is less sensitive than
it should be:
• Negative result may not be true negatives.
• Positive result may have stronger Ct values than
those given
What to do if your PCR fails these
rules?• Reasons for the failed control?
– The sample may contain PCR inhibitors
– The extraction process may have failed
– PCR set up errors
• What to do?– Check the volumes of individual sample.
• Repeat from extraction plate if volume in well is wrong.
– Did all ICs fail or is the Ct value less than usual (?IC added wrongly/gone off)
• If all IC range is down it is possible to pass
• If positive control ok, it possible to use alternative rule…..
– If IC failure limited to few samples then repeat sample• From extraction plate if volume low
• Different extraction
• Post dilutions
• Post freeze thaw.
Re-extract samples on the
easyMag
MDx EAV IC easyMag EAV IC
Next best platform - easyMag
• Inhibition was assessed on the easyMag &
compared to MDx
Limits % inhibition
MDx easyMag
+/-3STDEV 94.6 0
+/-2STDEV 95.4 3.8
+/-1.5ct 95.4 3.8
What to do if your PCR fails these
rules?• Reasons for the failed control?
– The sample may contain PCR inhibitors
– The extraction process may have failed
– PCR set up errors
• What to do?– Check the volumes of individual sample.
• Repeat from extraction plate if volume in well is wrong.
– Did all ICs fail or is the Ct value less than usual (?IC added wrongly/gone off)• If all IC range is down it is possible to pass
• If positive control ok, it possible to use alternative rule…..
– If IC failure limited to few samples then repeat sample• From extraction plate if volume low
• Different extraction
• Post dilutions
• Post freeze thaw.
– What if the IC fails but a pathogen is detected?• Complicataed.
• ?depends on the test
Additional aspects that aid QC
Consider a checklist• Tests get more complex (as does
the QC)
• Each test user has to complete a test checklist
– Acts as a test SOP for set-up and test validation
– Ensures consistent test interpretation between users
– Contains useful data for troubleshooting (e.g. lot changes, user, volumes used etc)
– Improved audit trail
– Can be adapted rapidly• Volume countersign
Consider long term monitoring
Benefits
• Loss in sensitivity over time
• Link it to particular lots of control, reagent or primer/probe
• More accurate than a general level.
• Maintain lot to lot
Together = better test stability
202122232425262728293031323334353637383940
1 12 23 34 45 56 67 78 89 100 111 122 133 144 155 166 177 188 199 210 221 232
Test run
CT
va
lue
Control value Warning rule Warning rule
Action rule Action rule
Conclusions
• QC of real time PCR is very important
• Commercial assays come with there own QC program– Often results in repeat extractions…..
• In house tests have to use in house QC– Complicated
– But can aid understanding of the test
– Partially pass failed runs with confidence.
• Consider long term monitoring tools to aid QC.
Any questions?
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