Guidance for molecular clinical laboratory Flashcards
What are the gals of molecular diagnosis procedure
- select the right test for each patient
- generate accurate, clinically relevant results to optimally influence clinical care
- Conserve health care resources
enumerate the three division of all molecular diagnosis workflows
pre analytical, analytical, and post analytical
its standards aim at quality, format, and amount of specimen/sample which should be documented in detail
pre analytical
this considers the proof of these aspects, concerning DNA/RNA extraction, quantification, and purity determination via fluorometry or spectrophotometry
analytical standards
this mainly focuses on the documentation and bioinformatics process
post analytical
accession numbers are to be allocated to samples as per which ISOs?
ISO 15189 and ISO9001
what should SOPs follow?
ISO 15189 and any national guidelines
T or F
use of fixed tissue that was previously frozen is advisable as it yields better results
F (not advised)
what is the best fixing reagent
formaline
The process of removing calcium from a bone to make it softer for processing
decalcification
Why should a molecular biology laboratory staff be trained and highly skilled in the pre analytical phase
because mol bio specimens are highly variable
What should labs validate for specimen acceptability?
all potential specimen types and establish criteria
Laboratories must establish a ________ and ________ workflow for nucleic acid quantitation to guarantee accurate and reproducible results
standardized and cost-effective
The _____________________________ must be well developed to clearly define pre analytical sample quality
quality control methods
This method should be highly avoided because it detects all nucleic acids
however, it is still commonly used in laboratories as it is cheaper
spectrophotometric methods
What is recommended over spectrophotometric methods?
double stranded DNA specific fluorometric quantitation methods
It is important to consider whether the testing would ________ or be ______ in addition to existing diagnostic tests
supplant or be conducted
What tests should you do first before running multiplex PCR to ensure presence of pathogen
bacterial culture and susceptibility testing
factors to be considered in the choice of analytical method (yes, need mo siya kabisaduhin)
- number of samples that require testing
- number of genes being tested
- percentage of mutations tested in each gene
- amount of tissue available
- degree of staff expertise
- equipment, and infrastructure required
- need of separation of stages in different compartments
when does ensuring good DNA/RNA quality start?
isolation and extraction
T or F
DNA/RNA samples must be permanently on ice and avoid repeated freeze-thaw cycles
T
T or F
majority of incubation steps must be kept at higher temperatures to inhibit possible nuclease activity
F (lower temps)
enumerate the special sample requesitions by pacific biosciences RS II
- double stranded format of DNA
- prevention of pH extremes (<6, >9)
- absence of chelating agents, detergents, divalent metal cations, denaturants and RNA)
4 . Quality assessment should yield an OD260/280 ratio of 1.8 to 2.0 and an OD260/230
ratio of 2.0 to 2.2,
what should be the quality assessment ratios be?
OD260/280 ratio of 1.8 to 2.0 and an OD260/230
ratio of 2.0 to 2.2,
what temp should DNA and RNA samples be stored at?
less than 20degrees Celsius or lower
PCR products should be stored at what temp?
less than 20degrees Celsius or lower
Sequencing libraries should be stored at what temp?
less than 20degrees Celsius or lower
T or F
DNA and RNA samples, PCR products, and sequencing libraries may be stored at the same freezer as they share the same storing temperatures
F
T or F
RNA is more stable than DNA or cDA
F (DNA>RNA)
T or F
DNA is more likely to survive more years than cDNA if stored properly
F (same lang sila HAHHAHA)
Device almost exclusively used for quality control in sequencing fragments
capillary gel electrophoresis
can be used to investigate fragment size distribution and final library quality assessment
capillary gel electrophoresis
device that offers fluorometric quantitation and checks sample quality
fluorometer
what does the fluorometer determine?
input DNA/RNA amount
T or F
The validation process assess the pre analytical and analytical phase only
F (including the molecular report)
A molecular diagnostic report should include which ________ or regions of
the ________ are investigated, information about the gene coverage, the
sensitivity of the detection, and the frequency of the detected
mutation.
genes
why is it essential to monitor the performance of all tests in the clinical laboratory?
ensure all measurable parameters remain within acceptable limits?
Pertains to the timeliness of reports that is critical to patient care, and the basis of patient pathways in practice in individual hospitals
turnaround time
Acceptability of longer turnaround time can permit _____________ or _______________to improve the cost effectiveness of
testing.
greater control of testing or batching of samples
A molecular diagnostic report should convey _________the information the clinician needs to treat the patient on whom the test was performed, with sufficient information to allow correct __________of the result.
accurately
interpretation
3 key areas that should be included in reports
- patient identification
- reporting style and content
- Interpretation
How many sample identifiers in total should be in the request form and report?
3 sample identifiers
(2 patient id and 1 on the sample id)
T or F
Long reports are always read in full. One page, or better still, single-screen reports
are preferred, provided that they are legible
F (rarely read in full)
what should the report have if it’s more than 1 page long?
- appropriate patient identifiers in all pages
- number the pages
T or F
For all tests, the percentage of the sample occupied by the target of the test
(e.g., neoplastic cells) is important.
F (some lang, not all tests)
Ideally, the report should contain basic information about ______ for quality control.
fixation
The __________of the individual taking responsibility for the test and ________
details is important, and some form of authorization with date
name and contact
The type and extent of molecular analysis used should be described clearly to allow ________to request further testing if warranted by the clinical situation
oncologists
these are also known as proficiency testing and are inevitable for monitoring a lab’s performance
External Quality assurance (EQA)
One of the requirements to become accredited according to ISO _______
is to participate in EQA programs
15189
It is necessary to periodically assess the analytical performance of molecular tests by __________, which will assist laboratories in monitoring their assays and improve assay performance and evaluation of results.
Inter-laboratory comparison
this is necessary to ensure high assay
reproducibility and performance and enable detection and correction
of errors in daily practice.
internal quality assurance (IQA)
Assays should be performed according to SOPs using appropriate _______ and _______ _________
positive and negative controls
Who performs the control every shift change?
RMT
______ is essential to take charge of
participation and performance in quality assurance schemes and to
organize internal and external audits.
laboratory quality managerR
T or F
the lab quality manager does the control also
f
_______is the critical guidelines to ensure comparability and exchange of experimental data to accelerate the innovation process and aid improvement of transferability, transparency, and
reproducibility of results.
standards
Standardization can ________________, therefore enabling improved financial planning and scheduling and a possible expansion of services
potentially decrease cost
The results gained from successful standardization of NGS might _______ to other fields in life sciences
transfer
Enumerate the general NGS workflow
- prepare sequencing library
- Prepare and enrich template
- sequencing
- data analysis
- control input
- Fragmentation and end-repair
- size selection
- library normalization / quantification
Prepare sequencing library
- Clonally amplified DNA template prepared
- enriched
prepare and enrich template
-create a run
- clean and initialize sequencer
- begin sequencing
sequencing
Data quality check and analysis
data analysis
T or F
Quality assurance is under quality control
F (baliktad)
What should the quality assurance program contain?
- predetermined QC checkpoints for monitoring QA
- extensive documentation (used devices, reagent lot numbers, reagent expiration date, and any deviation from standard procedures)
- Methods for contamination identification at several stages within the diagnosis workflow
- documentation of all methods, reagents, instruments, and controls
Laboratories must define and document _________or ________criteria for each step of the wet bench process, such as DNA/RNA
extraction, library preparation, and sequencing
acceptance or rejection
_____and ____________, such as maintenance logs, must exist to demonstrate and provide evidence that all instrumentation and equipment are validated, operated, inspected, maintained, tested, and standardized to ensure optimal quality of assay results.
SOPs and supporting documentation
All preventive maintenance and calibrations must be scheduled and performed at least as frequently as suggested by the ____________ to ensure continued accuracy, precision, and extended usable life of the equipment in EQA programs
equipment manufacturers
______ must conduct all preventative maintenance and service per manufacturer specifications
staff
Guidelines of preventive maintenance (summarized) go read the whole thing also :>
- staff must keep al equipment clean
- lab must do the standard daily/weekly / monthly routine maintenance and to record the task
- Written policy / procedure should be in place
- primary and backup instruments should be compared to each other to determine result consistency
Calibration / maintenance of automatic pipettors every ____
6 months
Calibration / maintenance of thermometers every ____
1 year
Calibration / maintenance of centrifuge every ____
6 months
We should record the temperature / freezer’s temperature every _____
every day
we should establish tolerance limits for temperatures, carbon dioxide level, and humidity as applicable for which equipment?
incubators and water bath
What do we use to measure centrifuge speeds?
tachometer
When should the autoclave’s pressure and temperature be checked?
every year
When is the autoclave’s sterilization efficiency measured?
weekly
How is content processing verified with each autoclave batch?
heat sensitive tape
where should the analytical balance be placed?
stable location
what can be used to clean the BSC’s UV lamp
70% alcohol
Calibration / maintenance of BSC every ____
1 year
Calibration / maintenance of generators every ____
1 month
Accreditation means that medical laboratories have been assessed against ___________ to demonstrate their competence, impartiality and performance capacity.
internationally recognized standards
What is the international standard for accreditation?
ISO 15189
What is the title of ISO 15189
“Medical laboratories – particular requirements for quality and competence”
what is ISO 15189 based on?
ISO/IEC 17025 and ISO 9001
All laboratories providing molecular diagnostic services should have laboratory accreditation according to_______ or their _______
ISO 15189 or their national equivalent
steps of accreditation
- Opening briefing
- Examination of records
- Sample handling
- Interview with technicians
- Demonstrations of tests and techniques
- Examination of equipment calibration and maintenance
- Review of quality documentation
- Written report of assessor’s findings
- Closure meeting
Accreditation can be granted for ______ years, but the NABs will perform surveillance visits to ensure that the laboratory continues to meet requirements, which is typically every ______
3-5 years
1 to 2 years
