Guidance for molecular clinical laboratory Flashcards by Nathalie Grace Castillo

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

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enumerate the three division of all molecular diagnosis workflows

pre analytical, analytical, and post analytical

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its standards aim at quality, format, and amount of specimen/sample which should be documented in detail

pre analytical

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this considers the proof of these aspects, concerning DNA/RNA extraction, quantification, and purity determination via fluorometry or spectrophotometry

analytical standards

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this mainly focuses on the documentation and bioinformatics process

post analytical

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accession numbers are to be allocated to samples as per which ISOs?

ISO 15189 and ISO9001

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what should SOPs follow?

ISO 15189 and any national guidelines

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T or F

use of fixed tissue that was previously frozen is advisable as it yields better results

F (not advised)

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what is the best fixing reagent

formaline

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The process of removing calcium from a bone to make it softer for processing

decalcification

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Why should a molecular biology laboratory staff be trained and highly skilled in the pre analytical phase

because mol bio specimens are highly variable

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What should labs validate for specimen acceptability?

all potential specimen types and establish criteria

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Laboratories must establish a ________ and ________ workflow for nucleic acid quantitation to guarantee accurate and reproducible results

standardized and cost-effective

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The _____________________________ must be well developed to clearly define pre analytical sample quality

quality control methods

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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

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What is recommended over spectrophotometric methods?

double stranded DNA specific fluorometric quantitation methods

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It is important to consider whether the testing would ________ or be ______ in addition to existing diagnostic tests

supplant or be conducted

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What tests should you do first before running multiplex PCR to ensure presence of pathogen

bacterial culture and susceptibility testing

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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

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when does ensuring good DNA/RNA quality start?

isolation and extraction

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T or F

DNA/RNA samples must be permanently on ice and avoid repeated freeze-thaw cycles

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T or F

majority of incubation steps must be kept at higher temperatures to inhibit possible nuclease activity

F (lower temps)

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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,

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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,

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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

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

1. patient identification 2. reporting style and content 3. 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?

1. appropriate patient identifiers in all pages 2. 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

_______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

1. prepare sequencing library 2. Prepare and enrich template 3. sequencing 4. 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?

1. predetermined QC checkpoints for monitoring QA 2. extensive documentation (used devices, reagent lot numbers, reagent expiration date, and any deviation from standard procedures) 3. Methods for contamination identification at several stages within the diagnosis workflow 4. 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 :>

1. staff must keep al equipment clean 2. lab must do the standard daily/weekly / monthly routine maintenance and to record the task 3. Written policy / procedure should be in place 4. 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

1. Opening briefing 2. Examination of records 3. Sample handling 4. Interview with technicians 5. Demonstrations of tests and techniques 6. Examination of equipment calibration and maintenance 7. Review of quality documentation 8. Written report of assessor’s findings 9. 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