BS EN ISO 20184-1:2018 download.Molecular in vitro diagnostic examinations – Specifications for pre-examination processes for frozen tissue.
1 Scope
BS EN ISO 20184-1 gives guidelines on the handling, documentation, storage and processing of frozen tissue specimens intended for RNA examination during the pre-examination phase before a molecular assay is performed.
BS EN ISO 20184-1 is applicable to any molecular in vitro diagnostic examination performed by medical laboratories and molecular pathology laboratories that evaluate RNA extracted from frozen tissue. It is also intended to be used by laboratory customers, in vitro diagnostics developers and manufacturers, biobanks, institutions and commercial organisations performing biomedical research, and regulatory authorities.
Tissues that have undergone chemical stabilization pre-treatment before freezing are not covered in BS EN ISO 20184-1.
NOTE International, national or regional regulations or requirements can also apply to specific topics covered in this document.
2 Normative references
The following documents are referred to in the text in such a way that some or all of their content constitutes requirements of BS EN ISO 20184-1. For dated references, only the edition cited applies. For undated references, the latest edition of the referenced document (including any amendments) applies.
ISO 15189:2012. Medical laboratories — Requirements for quality and competence
3 Terms and definitions
For the purposes of this document, the terms and definitions given in ISO 15189 and the following terms and definitions apply.
3.13
pre-exam I nation processes preanalytical phase preanalytical workilow
processes that start, in chronological order, from the clinician’s request and Include the examination request, preparation and identification of the patient, collection of the primary sample(s), transportation to and within the medical or pathology laboratory, Isolation of analytes. and end when the analytical examination begins
Note 1 to entry: The pre-examination phase indudes preparative processes that influence thc outcome of the intended examination.
(SOURCE: ISO 15199:2012. 3.15, modIfied — pre.analytlcal workflow has been added as a preferred term. Note ito entry has been added and the definition has been extended.)
3.14
primary sample specimen
discrete portion of a body fluid, breath, hair or tissue taken for examination, study or analysis of one or more quantities or properties assumed to apply for the whole
(SOURCE: ISO 15189:2012,3.16, modifIed — Notes to entry ito 3 have been removed,1
3.15
proficiency test
evaluation of participant performance against pre’established criteria by means of inter- laboratory comparisons
(SOURCE: ISO 17043:2010,3.7, modIfied — Notes to entry 1 and 2 have been removed.)
3.16
RNA profile
amounts of the Individual RNA molecules that are present In a sample and that can be measured In the absence ofany losses, inhibition and Interference
3.17
RNA
ribonucleic acId
polymer of ribonucleotides occurring in a double-stranded or single-stranded form
(SOURCE: ISO 22174:2005, 3.13]
3.18
RNase
ribonuclease
enzyme that catalyses the degradation of RNA into smaller components
3.19
room temperature
for the purposes of this document, temperature in the range of 18°C to 25°C
Note I to entry: Local or national regulations can have different definitions.
3.20
sample
one or more parts taken from a primary sample
(SOURCE: ISO 15189:2012,3.24, modified — EXAMPLE has been removed.I
All steps of a diagnostic workilaw can influence the final analytical test result. Thus, the entire workflow including biomolecute stability and sample storage conditions shall be verified and validated. Workflow steps which cannot always be controlled (e.g. warm lschemla) shall be documented. A risk assessment of non-controllable workflow steps including their potential impact on the analytical test performance shall be performed and mitigation measures shall be established to enable the required analytical test performance.
Before or during the design alan analytical test, it should therefore be investigated and assured that the RNA profile(s) intended to be analysed is/are not compromised in a manner impacting the analytical test performance (e.g. by performing a time course experiment or study; see also Annex A).
Before tissues are stabilized by freezing, the RNA profile(s) can change e.g. by gene induction, gene down regulation and RNA degradation. These effects depend on the warm and cold lschemia duration and the ambient temperature before freezing, In addition, the described effects can vary In dIfferent donors/patients’ tissues.
Generally, the longer the duration of warm and cold ischemia and the higher the ambient temperature before freezing the tissue specimen, the higher is the risk that changes in the RNA profile can occur.
NOTE lntraoperatlve warm ischemia can result in more pronounced changes of RNA profiles than during postoperative cold ischemlal7ilUl. RNA profiles can also vary, depending on the orIgin and type of tissue, the underlying disease, the surgical procedure, the drug regimen, and drugs administered for anaesthesia or treatment of concomitant disease and on the dilferent environmental conditions after the tissue removal from the body.
As warm ischemia cannot be easily standardized, its duration shall be documented. When it is not possible to avoid cold ischemia. duration shall be documented and temperatures of the specimen containers surroundings should be documented. Where the specimen is transported to another facility for freezing, the transport duration shall be documented and the ambient conditions should also be documented.
Safety Instructions on transport and handling shall be considered (see ISO 15189:2012. 5.2.3 and 5.4.5 and 15015190).
During the whole pro-examination proce.ss precautions shall be taken to avoid cross contamination between different specimens/samples, e.g. by using single-use material whenever feasible or appropriate cleaning procedures between processing of different specimens/samples.
If a commercial product is not used In accordance with the manufacturer’s Instructions, responsibility for its use and performance lies with the user.
5 Outside the laboratory
5.1 Specimen collection
5.1.1 General
For the collection of the specimen, the requirements (e.g. disease condition, specimen size) for the intended molecular examination (see also Clauseá) should be considered.
See also ISO 15189:2012. 5.4.4.
5.12 Information about the specimen donor/patient
The documentation shall Include the ID of the specimen donor/patient, which can be In the form ola code.
The documentation should Indude, but Is not limited to:
a) the relevant health status of the specimen donor/patient (e.g. healthy, disease type, concomitant disease, demographics e.g. age and gender));
freezing. Pre-cooling can be done with liquid nitrogen (-196 °C). dry ice (-80 DC), -80 DC freezers or dedicated freezing apphances that keep the isopentane S—80 DC with or without controlled cooling rate. The isopentane shall be cooled in a tube or other container (e.g. glass beaker) resistant to the large and sudden temperature shifts. The volume of pre-cooled isopentane shall be at least 10 times the volume of the specimen or sample. For snap freezing the tissue sample shall be completely submerged into the pre-cooled isopentane
After the tissue is frozen it shall be transferred into its designated pre.cooled labelled cryo-vial. The vial shall be closed according to the manufacturers Instructions. The isopent.ane should be refreshed when tissue sediment is seen at the bottom of the tube.
Isopentane is an extremely volatile and extremely flammable liquid at room temperature and pressure. Therefore, the laboratory should be well ventilated, The isopentane in the tube should be cooled.
bj Fast freezing procedure: Tissues shall be fast frozen on a pre-cooled metal plate, or metal basket placed on the surface of liquid nitrogen, or on dry ice. The metal surface shall be pre-cooled ranging from s—BC DC to >—196 DC. The metal plate or metal basket can be fixed into position with a suitable stand and clamp. Alternatively, the sample can be frozen directly in liquid nitrogen or even in the labelled and closed storage vial in liquid nitrogen or in dry ice. However, a slow freezing process can cause membrane disruption by compartmental rising salt concentrations and crystal formation which can seriously affect the morphology. To avoid cross-contamination, the basket or plate should be cleaned between freezing samples.
NOTE Freezing in liquid nitrogen Is characterIzed by the Leidenfrost elfectli-l) caused by boiling of liquid nitrogen around thr tissue due to its relatively high temperature. This reduces the heat conduct from the sample to the liquid nitrogen; this becomes worse when the sample is placed in the labelled vial,
c) Frozen section procedure: For freezing tissue for fast frozen section diagnosIs: The tissue should be transported freshly to the laboratory without delay. The selected part of the specimen shall be frozen onto the specialized metal grids that lit onto the cryostat in an appropriate freezing medium. The freezing medium used should be documented. The metal grid containing the tissue and freezing medium is frozen by holding the metal in liquid nitrogen or dry ice until the tissue Is frozen. After cutting the frozen sections the remainder should be removed from the metal grid without thawing and stored in a pre-cooled vial for long term storage.
NOTE 1 The use of a freezing medium is known to harm protein mass spectrometry where the column separation can become Impaired.
NOTE 2 Samples treated with freezing medium shouLd be stored at -70 C to avoid dryness and the RNA quality should be evaluated for each freezing medium treated sample before use.
The following steps shall be performed before, during and after the freezing procedure:
a) the documentation of the freezing procedure (e.g. freezing In liquid nitrogen, snap-freezing in isopentane cooled by liquid nitrogen or dry ice, freezing in an appropriate freezing medium, freezing with controlled cooling rate);
b) the documentation of the freezing time point and date (to determine the lag time: time period between removal from the body — until freezing of the specimen or sample);
c) determine if the required tissue size of the sample fits into the chosen cryo-vial before freezing. because the tissue size determines the size of the container; it is therefore recommended, that the specimen/sample does not exceed 1 cm In one dimension;
d) the selection of the specimen/sample container for cryo-storage:
1) the container shall have a sufficient volume for the size of the specimen or sample to be stored in;
2) the container shall be certified for the storage temperature.