Past Papers 12 Flashcards
What are the causes of false positive PCR results
Pre-test
- Misidentification: wrong sample
- Transcription error
Sample:
- Lack of physical separation between extraction and PCR rooms
(dirty versus clean areas) - unidirectional workflow
- Sharing of equipment between PCR room and other rooms in the
lab
- Shared ventilation systems between PCR room and extraction
- aliquoting error
- Genomic and/or amplicon contamination due to above reasons
Test:
- Misinterpretation of results: e.g. drifters
- Use of uncalibrated equipment (e.g. pipettes, thermocyclers,)
- Poor compliance with universal precautions: gloves, gowns, hand
washing
- Poor staff training
- Lack of standard operating procedures
- Poor assay specificity
What are the causes of false negative PCR results
Pre-test:
-Misidentification: wrong sample
-Transcription errors
Sample:
- Failure to add sample to PCR mix
- repeated freezing/thawing
- Presence of inhibitory substances in the sample: EDTA, heparin,
bile, haem, etc
- Sub- optimal tissue biopsies
- Degradation of nucleic acid (RNA less stable than DNA)
Test:
- Use of uncalibrated equipment (e.g. pipettes, thermocyclers, ect)
- Expired or poorly stored reagents
-Inefficient extraction of target nucleic acid
-
- Poor staff training
- Lack of standard operating procedures
- Poor assay sensitivity
What steps can be taken to prevent false positive/ negative results
Separateinto pre-test, test, and post-test
Proper identification of samples e.g. use bar codes
Adequate IT support: samples and results linked
electronically with the least possible human intervention
Reagents should be sourced from high-
quality suppliers and stored under optimal conditions
Regular maintenance and calibration of equipment as per
the manufacturer’s instructions
Use of exogenous internal positive control at extraction,
reverse transcription (if RNA target), and amplification
steps to monitor the efficiency of the entire reaction
Use endogenous internal positive control to monitor the
efficiency of the reaction as well as the adequacy of tissue
samples
Ensure physical separation between dirty and clean areas
within the lab - unidirectional workflow
Use PCR only tips (fitted with a filter to prevent
aerosolisation) for pipetting reagents
Consider incorporating uracil-N-glycosolase (UNG)
into the PCR master mix in order to reduce carryover
contamination
Avoid unnecessary freezing/thawing of specimens
Use SOPs
Ensure adequate training of staff
QC schemes: IQA; EQA; QCMD
Proper validation of assays prior to their introduction
Use software to detect drifters
Cleaning - use 2% hypochlorite disinfectants to clean work areas
environmental swabbing - monitor environment for contamination
Discuss the implications for discovering a number of incorrect results produced by your in-house CMV DNA PCR assay over a 5
-year period, which only came to light now during the introduction of a new commercial assay. All the samples were EDTA blood samples
taken from allogeneic HSCT recipients.
What measures are you going to take to address this problem?
Conduct a root cause analysis: establish what went wrong, why it went wrong, how it can be prevented from happening again.
Find out more information about the new assay:
- Establish whether the nucleic acid target is same or different from the in-house assay
- Establish whether positive internal controls/negative external controls are used.
- Check if there is any published data to support the utility of the new assay.
- Check whether the new assay has CE marking or FDA approval
Collect and analyse lab data in order to provide explanations for the poor performance of the in-house assay: extraction efficiency, assay
sensitivity/specificity, monitoring of inhibition throughout PCR cycle,
contamination, IT problems, etc
Discuss the situation with relevant specialists e.g. transplant unit, clinical governance team, safety committee, local Caldicott
guardian, legal advisors, press officer
Set up an adverse incident committee and fill in all the necessary
lab/clinical incident forms
Two action options:
do nothing
look back exercise + patient notification
Discuss the implications for discovering a number of incorrect results produced by your in-house CMV DNA PCR assay over a 5
-year period, which only came to light now during the introduction of a new commercial assay. All the samples were EDTA blood samples
taken from allogeneic HSCT recipients.
Two action options:
do nothing
look back exercise + patient notification
What would be the pros/ cons of doing nothing?
The in-house assay was the standard of care at the time of
testing
There is no point in re-testing whenever there is a new assay
being introduced as this is a costly exercise with serious
ethical implications.
Samples might have already been thrown away due to limited
storage capacity.
May create unnecessary anxiety for patients/ staff, especially if no harm has been done
Discuss the implications for discovering a number of incorrect results produced by your in-house CMV DNA PCR assay over a 5
-year period, which only came to light now during the introduction of a new commercial assay. All the samples were EDTA blood samples
taken from allogeneic HSCT recipients.
Two action options:
do nothing
look back exercise + patient notification
What would be the pros/ cons of doing a look back exercise, and notifying patients?
Pros:
-Can help establish whether this testing contributed to any deaths. This may be difficult to ascertain, as transplant will have high mortality anyway
- looks like department is proactive in improving and learning from mistakes
Cons:
- Can create a lot of anxiety for patients
- expensive
- may not be possible if samples have been discarded
Discuss the implications for discovering a number of incorrect results produced by your in-house CMV DNA PCR assay over a 5
-year period, which only came to light now during the introduction of a new commercial assay. All the samples were EDTA blood samples
taken from allogeneic HSCT recipients.
Two action options:
do nothing
look back exercise + patient notification
What are the steps in performing a look back exercise, and notifying patients?
Establish whether these patients are alive or dead (high
transplant-related mortality) and whether conducting a case
-note review to determine whether the in-house assay results
have seriously compromised their care.
Establish if all EDTA blood samples in question are available
for re-testing in parallel with the new assay (some of these
samples may not be available because of limited storage
capacity and/or local policy on duration of sample storage).
Establish, in relation to the new assay, the sensitivity,
specificity, negative/positive predictive values for the in-house
assay (if re-testing agreed)-Issue amended reports as appropriate.
Explain to patients
What are the causes of false results, both false
positive and false negative, in serum IgG and IgM
tests for antiviral antibodies?
What are the patient-related factors that cause false results?
False negative
Misidentification/ mislabelling of sample
Poor sample storage - delay transport/ fridge
“Window period” concept
Hook effect
Immunosuppressed
Flase positive
pregnancy, rheumatoid factor positive individuals
IVIG therapy
What are the causes of false results, both false
positive and false negative, in serum IgG and IgM
tests for antiviral antibodies?
What are the virus-related factors that cause false results?
High level of genetic diversity (false negative)
What are the causes of false results, both false
positive and false negative, in serum IgG and IgM
tests for antiviral antibodies?
What are the test-related factors that cause false results?
Direct immunofluorescence
Design, development, and validation of assays:
commercial/in-house; manual/automated
Direct immunofluorescence: faulty light bulbs, subjective
(over-reading/under-reading), experience
What are the causes of false results, both false
positive and false negative, in serum IgG and IgM
tests for antiviral antibodies?
What are the test-related factors that cause false results?
ELISA
Design, development, and validation of assays:
commercial/in-house; manual/automated
ELISA: immobilisation of antigen/Ab on solid phase, U versus V shaped wells, monoclonal versus polyclonal Ab
detection, type of plastic used for plates, lack of reactivity
in wells near the edge of the plate (manual ELISA), false neg IgM
if high levels of IgG, hook effect in sandwich assays
What are the causes of false results, both false
positive and false negative, in serum IgG and IgM
tests for antiviral antibodies?
What are the test-related factors that cause false results?
Neutralisation assay
Design, development, and validation of assays:
commercial/in-house; manual/automated
Neutralisation assay: false neg if high or low levels of Ag
What are the causes of false results, both false
positive and false negative, in serum IgG and IgM
tests for antiviral antibodies?
What are the test-related factors that cause false results?
Latex agglutination
Design, development, and validation of assays:
commercial/in-house; manual/automated
Latex agglutination: prozone effect
What are the causes of false results, both false
positive and false negative, in serum IgG and IgM
tests for antiviral antibodies?
What are the test-related factors that cause false results?
Equipment
Degraded reagents: outdated, poor storage conditions
Aliquoting errors: calibration of instruments
Malfunctioning equipment: maintenance, calibration
Staff training - SOPs
Contamination
What are the causes of false results, both false
positive and false negative, in serum IgG and IgM
tests for antiviral antibodies?
What are the test-related factors that cause false results?
Human and virus related causes
Well trained staff
Well-trained virology doctors - avoid unnecessary/ inappropriate testing
Quality control - IQA/ EQA, Audit - decidng whether to accept a run or not
