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?