Molecular QC & QA Flashcards
Pre-analytical error is the consequence of
Erroneous or misleading results caused by events that occur prior to ample analysis
What is done upon receipt of specimen in lab?
Condition of specimen and chain of custody if possible.
No specimen is accepted without
Proper labeling and identification
What is done if specimen is unacceptable?
Disposal or retention is documented/recorded.
All specimens are considered
Infectious
How should specimens be handled?
With standard precautions using proper personal protective equipment (gloves, glasses, coat)
What is absolutely required for handling RNA and why?
Gloves, to protect them from nuclease degradation.
What precautions are used for airborne or contact transmissible agents?
Transmission-based precautions like respirators.
Contact precautions are used for
direct patient care.
What are the requirements for molecular testing specimens?
- Specimen with minimal cellular content accepted
- Cross-contamination must be avoided
- No hemolysis
- NO WBC lysis, or DNA/RNA yield will be reduced
- Solid tissues from fresh or frozen tissues
Solid tissues are best analyzed from
Fresh or frozen tissue
the quality of the nucleic acid from fixed tissue depends on what?
Fixing process and fixative used.
What anticoagulants are used for molecular studies?
Brown: Sodium heparin
Lavender: Tripotassium EDTA
Yellow: Acid citrate dextrose solution
Bench equipment requirement for dealing with RNA
Separate lab area designated RNase free
Wiped with RNase zap to get rid of RNase
Disposables requirement when dealing with RNA
RNase free
Rinsed with 0.1% DEPC
What should be added in RNA reactions?
Rnasin (promega)
What are the reagent requirements when dealing with RNA?
RNase free
0.05-0.1% DEPC added (except Tris)
Tested with RNase alert (Ambion)
What must be taken into consideration with RNA/DNA extraction?
Sample type
Temperature stored
Longevity of tissue sample
What are the holding and storage requirements for DNA?
RT several months
4dC for at least a year
-20 to -70 dC for 10 years or longer
What are the holding and storage requirements for RNA?
RT or 4dC not recommended
- 20dC for ~ 6 months if suspended in ethanol
- 70dC for > 6 months is suspended in ethanol
What Federal structure requires validation of the performance of clinical methods and reagents in accurately identifying, detecting or measuring analytes prior to use in human testing? What is this called?
FDA. Test performance.
What two criteria are used in identifying test performance?
Sensitivity and specificity
How is clinical sensitivity calculated?
TP / TP+FN x 100
How is clinical specificity calculated?
TN / TN+FP x 100
What is analytical sensitivity?
Smallest amount of substance in a sample that can be accurately measured.
What is clinical sensitivity?
Percent that a particular assay identifies true positive patients.
What is analytical specificity?
Ability of an assay to measure/identify a specific target/substance.
What is clinical specificity?
Percent that a particular assay identifies true negative patients.
What type of specimen is test validation performed on?
Specimens of types that will be encountered in routine use of the test.
The number of specimen tested in test validation varies with what?
Procedure and availability of test material.
Results of new test methodology are compared to what?
Results from established procedures or correlated to clinical diagnosis.
How are commercially developed and FDA-approved molecular methods verified?
Using purchased reagent sets to test validation in the lab
When a commercial test is modified, what happens?
Validation is required to show equal or superior performance of the modified procedure.
Once a procedure has been validated, it is documented in the lab according to
CLSI guidelines
What is proficiency testing?
External specimens from a source supplied to independent labs.
What structures supply the specimens for molecular analysis proficiency testing?
The CAP and other organizations
What is the solution when proficiency testing specimens are not commercially available?
Labs exchange blinded split specimens or blinded specimens measured/documented by independent means such as chart review are tested within the lab.
What are controls?
Samples of known type or amount that are treated like and ran with patient samples.
What controls are needed in qualitative tests?
Positive control
Negative control
Sometimes sensitivity control
What controls are needed in quantitative tests?
High positive control
Low positive control
Negative control
What is different with amplification procedures?
Amplification control is needed to avoid false-negative results.
What do quantitative PCR methods that automatically analyze results need?
Standard curve or dilution series of the positive control.
A standard curve or dilution series of the positive control is seen in what procedures?
Quantitative PCR
What do methods requiring detection of a target-specific product or relative amounts of a target?
Internal controls ran simultaneously preferably in the same reaction mix as the test specimen.
What is quality assurance?
Periodic review and documentation of test results
Molecular quantitative methods should have a defined
Dynamic range
Sensitivity level
Accuracy
What are cut-off values?
levels that distinguish positive from negative results in an assay
Who supplies recommendations for routine maintenance?
Manufacturers
What is required for detection systems?
Regular calibration or fitting of instrument/test system with the actual concentration of reference analyze.
What are analyte-specific reagents (ASR)?
Probes, primers, Ab or other test components that detect a specific target.
