Quality Flashcards
Westgard rules
Levy-Jenning plots
13s refers to a control rule that is commonly used with a Levey-Jennings chart when the control limits are set as the mean plus 3s and the mean minus 3s. A run is rejected when a single control measurement exceeds the mean plus 3s or the mean minus 3s control limit
12srefers to the control rule that is commonly used with a Levey-Jennings chart when the control limits are set as the mean plus/minus 2s. In the original Westgard multirule QC procedure, this rule is used as a warning rule to trigger careful inspection of the control data by the following rejection rules.
22s - reject when 2 consecutive control measurements exceed the same mean plus 2s or the same mean minus 2s control limit.
R4s - reject when 1 control measurement in a group exceeds the mean plus 2s and another exceeds the mean minus 2s. This rule should only be interpreted within-run, not between-run.
41s - reject when 4 consecutive control measurements exceed the same mean plus 1s or the same mean minus 1s control limit.
10x - reject when 10 consecutive control measurements fall on one side of the mean.
accreditation vs certification
Certification is the 3rd party CONFIRMATION via audit of an organisation’s systems or products
Accreditation is independent 3rd party RECOGNITION that the organisation has the competence & impartiality to perform specific technical activities such as certification, testing and inspection.
Certification - audit of whether an organisation, product or individual, conforms to the criteria laid out in a recognised standard or scheme, such as ISO 9001 Quality Management Systems.
Accreditation - Assessment of the competence and impartiality of an organisation and the compliance of their work to nationally and internationally recognised standards or schemes such as ISO 15189 medical lab standard.
Certification bodies are the checkers & accreditation bodies are checkers of the checkers.
Eg- UKAS accredits organisations which can then certify other organisations.
NOTE: UKAS accredits labs under ISO 15188 which is separate.
QA vs IQC vs IQA vs EQA
QA - total process whereby the quality of lab results can be guaranteed. Consists of:
Good lab practice
IQC
Audit
IQA
Accreditation
Evaluation
Validation
Education
EQA
IQC - IQC consists of statistical and non-statistical techniques for the verification of the quality of results. Quality control materials are developed or obtained from a source independent of the manufacturer of the test kit. These are used to simulate patient samples in the clinically significant range, and results
are periodically examined.
Monitor’s reproducibility but not necessarily accuracy - compares control material against levels determined during validation of the test
IQA - clinical specimens are anonymously reintroduced back through the lab process ensuring processes are operating at an acceptable level - checking for consistency
EQA - comparison of a lab’s performance to an external source - objectively checking using an external provider. Inter lab comparability.
Detects analytical & Post analytical errors. DOES NOT HELP WITH PREANALYTICAL ERRORS.
Spills
Minor vs major based on volume, pathogen, and location
Major - needs evacuation and hazmat team. If in doubt, treat as major.
RIDDOR reporting - reporting of injuries, diseases and dangerous occurrences regulations.
Investigate once spill it dealt with to prevent reoccurrence.
Lab cleaning
Decontamination -
heat
autoclave
chemical (disinfectant, guanidine, detergents)
Gaseous - formaldehyde fumigation
Disinfection - destruction of microorganisms but not of spores. Reduce the burden to safe levels.
Sterilisation - absence of living microorganisms.
Disinfectants:
1. Halogen releasing - hypochlorite
2. Aldehydes - formaldehyde
3. Peroxygens - Hydrogen peroxide
4. Alcohol
5. Quaternary ammonium compounds - benzalkonium chloride
Disinfection effectiveness - measured by log reduction by basic suspension test.
Cat 3 vs Cat 4 lab
Cat 3:
- Must be sealable for fumigation with an independent air system.
- Must have an ante-room.
- PPE, including goggles and gloves; respirators may also be required
- The use of solid-front wraparound gowns, scrub suits, and/or coveralls is often required
- Access to a hands-free sink and eyewash station available near the exit
- Negative pressure airflow with sustained directional airflow to draw air into the laboratory from clean areas toward potentially contaminated areas. exhaust air cannot be recirculated and must be HEPA filtered.
- Self-closing set of locking doors with access away from general building corridors. Electro-magnetic interlocks so that both lobby doors cannot be opened at the same time; secure card control on main access.
- Walls, floor and ceiling are clad in a continuous vinyl layer to facilitate cleaning and fumigation.
- Access to a BSL-3 laboratory is restricted and controlled at all times.
- MSC I cabinetry
Cat 4:
In addition to biosafety level 3 considerations, biosafety level 4 laboratories must follow these safety protocols:
- Personnel must change clothing before entering the facility and shower upon exiting
- All materials must be decontaminated before leaving the facility
- Personnel must wear the PPE from lower BSL levels, as well as a full-body, air-supplied, positive pressure suit (in the US)
- Access to a MSC III biological safety cabinet
MSC I vs II vs III hoods
I - open fronted, onward airflow, exhaust through HEPA (no protection for sample). For ACDP 2&3.
II - open fronted, downward flow, inward and outward through HEPA. For ACDP 2 & 3.
III/ FFI - closed system with gauntlets, HEPA filtered. Exhaust double HEPA filtered. For ACDP 3&4.
Preventing lab contamination
DNA Contamination can not be reduced or removed once it has occurred.
Identifying contamination
Use “no template controls” (NTCs). In a standard 96-well plate qPCR setup, NTC wells contain all the qPCR reaction components components such as primers, reagents etc., with the exception of the DNA template [2]. If the NTC wells are contamination-free, you should not observe any amplification in these wells following the thermocycling steps. If amplification is observed in the NTC wells, then it may be possible to discern the cause of the contamination, which could help you decide on an appropriate response.
Physically separating qPCR processes
separate, dedicated areas for different processes in the qPCR workflow, e.g. sample preparation, qPCR setup, qPCR amplification, analysis of qPCR products. pre- and post-amplification areas.
Keep pre- and post-amplification areas separate, and ideally in different rooms with completely independent laboratory equipment such as pipettes, centrifuges and vortexers. Ensure each area has its own protective equipment, such as gloves and lab coats, and a dedicated supply of consumables. Ideally, these rooms should not be supplied by the same ventilation/ducting system. You should also maintain a one-way workflow between these different areas, where researchers who have been working in a post-amplification area do not enter a pre-amplification area on the same day. If you need to go from a pre- to a post-amplification area, you should change your gloves and lab coat [2]. De-contaminate anything that is used in post-PCR area before bringing it back to pre-PCR area.
Personal protective equipment, liquid handling and storage
Changing your gloves could prevent you from contaminating the surrounding work surfaces, plasticware and equipment. Open tubes carefully to avoid splashing or spraying their contents. Keep samples and reactions capped/covered as often as possible, and dispose them in a safe, contained place after use. Use a positive-displacement pipette and aerosol-resistant filtered pipette tips, and ensure that your pipetting technique is not causing unnecessary splashing or spraying. This could reduce aerosol formation in your samples or reagents [4].
Finally, store samples separately from kits and reagents in pre-PCR areas, store PCR products in post-PCR areas. If possible, you should aliquot reagents such as primers and probes, into volumes suitable for a single experiment, to prevent repeated opening and freeze-thawing of stock solutions.
Surface and equipment decontamination
Regularly decontaminate surfaces and equipment that are utilized for preparing qPCR reactions. Centrifuges and vortexes are prone to contamination. using 70% ethanol.. Thorough cleaning is particularly important after a spill. Use a 10–15% bleach solution (sodium hypochlorite)
An enzyme called uracil-N-glycosylase (UNG) present in certain qPCR Master Mix formulations removes carryover amplification contamination from your reactions. UNG destroys carryover amplification contamination from previously amplified templates, selectively targeting templates that contain uracil instead of thymine. This technique requires that you use a deoxynucleotide (dNTP) mix that contains uracil instead of thymine when performing your qPCR amplifications; this way, all of your amplification products will contain uracil.
The enzyme is active at room temperature, therefore it can be incubated with your amplification mixture to inactivate any sources of carryover contamination. Once thermocycling is initiated, the high temperatures inactivate the UNG, thus preventing the enzyme from affecting the newly generated amplification products, even if they contain uracil. The UNG method works best with thymine-rich amplification products and is not as effective with guanine/cytosine-rich amplification products. UNG is not effective for sources of DNA contamination, other than uracil-containing amplification products from previous qPCR experiments [1].
Error rectification
Random errors (one-off error):
*Random instrument malfunction
*Power fluctuations
*Temperature fluctuations (environment and incubation chamber)
*Test operator mistake
*Technique (technique-sensitive testing)
* Reagent prep error
* control material contamination
* Software malfunction
* Possible contamination
Systematic errors:
- Calibration - most common
- Reagent integrity/batch - second most common
- instrument maintanence issues
- improper LJ charts
- instrument malfunction
- light source deterioration
- temperature errors
- failure to follow manufacturer specifications
- operator technique in technique dependent procedures
- faulty software updates
- contamination
Lab signs & safety
SEE IMAGE IN ALBUM
Medical lab accreditation
ISO 15189
POCT accreditation
In conjunction with ISO 15189 - ISO 22870
Assay conformity mark
Changed from CE to UKCA marked - CE can be used until 31 December 2024.
Evaluation vs validation vs verification
Evaluation is a generic term used to describe the measurement of the performance capabilities of a system/test method. This is a systematic and extensive process that compares different systems/test methods designed to perform the same or similar functions.
Examples of evaluations within microbiology include comparison of different methods designed to detect the same marker/target, comparison of different culture media to isolate the same organism, or comparison of different equipment with the same function.
According to ISO 15189:2022, validation is defined as “confirmation, through the provision of objective evidence that the requirements for a specific intended use or application have been fulfilled”.
It examines the whole process that is being used to check that results are correct and consistent.
ISO 15189: 2022 defines “verification as the confirmation, through provision of objective evidence that specified requirements have been fulfilled”.
It can also be described as the confirmation of whether or not a product (for example commercial kit system or equipment) complies with a regulation, requirement, specification, or imposed condition.
RT-PCR primer design
- you choose to design your own real-time PCR primers, keep in mind that the amplicon length should be approximately 50–150 bp, since longer products do not amplify as efficiently.
- In general, primers should be 18–24 nucleotides in length. This provides for practical annealing temperatures.
- They should be specific for the target sequence and be free of internal secondary structure. Primers should avoid stretches of homopolymer sequences (e.g., poly (dG)) or repeating motifs, as these can hybridize inappropriately.
- Primer pairs should have compatible melting temperatures (within 5°C) and contain approximately 50% GC content. Primers with high GC content can form stable imperfect hybrids.
- If possible, the 3’ end of the primer should be GC rich (GC clamp) to enhance annealing of the end that will be extended. Analyze primer pair sequences to avoid complementarity and hybridization between primers (primer-dimers).
