ISO 20184-2:2018 download.Molecular in vitro diagnostic examinations Specifications for pre-examination processes for frozen tissue Part 2: Isolated proteins.
Molecular in vitro diagnostics, including molecular pathology, has enabled a significant progress in medicine. Further progress is expected with new technologies analysing nucleic acids, proteins, and metabolites in human tissues and body fluids. However, the profiles and/or integrity of these molecules can change drastically during specimen collection, transport, storage, and processing thus making the outcome from diagnostics or research unreliable or even impossible because the subsequent examination assay will not determine the situation in the patient but an artificial molecular pattern generated during the pre-examination process. Therefore, a standardization of the entire process from specimen collection to the protein examination is needed. Studies have been undertaken to determine the important influencing factors. This document draws upon such work to codify and standardize the steps for frozen tissue with regard to protein examination in what is referred to as the pre-examination phase.
Protein profiles and protein—protein interactions in tissues can change drastically before, during (e.g. due to warm ischemia) and after tissue collection (e.g. due to cold ischemia). The changes are caused by e.g. gene induction, gene down regulation, protein degradation. Protein species amounts can change differently in different donors’/patients’ tissues. The expression of genes can be influenced by the given treatment or intervention (surgery, biopsy), or drugs administered for anaesthesia or even treatment of concomitant disease as well as by the different environmental conditions after the tissue removal from the body.
Therefore, it is essential to take special measures to minimize the described protein profile changes and modifications within the tissue for subsequent examination.
ISO 20184-2 gives guidelines on the handling, documentation, storage and processing of frozen tissue specimens intended for the examination of isolated proteins during the pre-exarnination phase before a molecular assay is performed.
ISO 20184-2 is applicable to any molecular in vitro diagnostic examination performed by medical laboratories and molecular pathology laboratories that evaluate proteins isolated 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 a u t ho r it I e s.
NOTE International, national or regional regulations or requirements can also apply to specific topics covered in ISO 20184-2.
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 ISO 20184-2. 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
For general statements on medical laboratory quality management systems and in particular on specimen collection, reception, and handling (including avoidance of cross contaminations) see Iso 15189:2012, 4.2, 5.4.4, 5.4.6, or ISO/IEC 17020:2012, Clause 8 and 7.2. The requirements on laboratory equipment, reagents, and consumables in accordance with ISO 15189:2012, 5.3 shall be followed; ISO 15189:2012, 5.5.1.2 and 5.5.1.3, and ISO/IEC 17020:2012, 6.2 can also apply.
All steps of a diagnostic workflow can influence the final analytical test result. Thus, the entire workflow including biornolecule stability and sample storage conditions shall be verified and validated. Workflow steps which cannot always be controlled (e.g. warm ischernia) shall be documented. A risk assessment of non-controllable workflow steps including their potential impact on the examination test performance shall be performed and mitigation measures shall be established to enable the required examination test performance.
The stability of the specific proteins to be examined and their posttranslational modifications (if important for the assay) should be investigated throughout the complete pre-examination process prior to the development and implementation of an examination test (e.g. by performing a time course experiment or study; see also Annex A and Reference [8]).
Before tissues are stabilized by freezing, protein amounts, conformations and binding status can change e.g. by protein degradation and altered synthesis following gene induction, gene down regulation, RNA degradation, and changes of the biochemical pathway and energy status. These effects depend on the duration of warm and cold ischemia and the ambient temperature before freezing. In addition, the described effects can vary in different donors7patients’ 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 protein profile can occur.
NOTE Prolonged cold ischcmia durations result in changes of protein (e.g. cytokeratin 18) and phosphoprotein (e.g. phospho-p42/44) amountsrrnl9l. Keeping the specimen on wet-icc diminishes this effcct[IQ1. Protein amounts as well as posttranslational modifications can also vary during the pre-exarnination phase, 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 different environmental conditions after the tissue removal from the body.
As warm ischernia cannot be easily standardized, its duration shall be documented. When it is not possible to avoid cold ischeinia, its duration shall be documented and temperatures of the specimen container’s surroundings shall 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.
The protein isolation kit provider’s specific instructions for storing the isolated protein should be followed. Where the examination provider’s instructions are more stringent, these shall be followed.
If there is no information available from the protein isolation kit provider or if the laboratories’ own validated total protein isolation procedures are used, the isolated proteins should be assayed immediately. Where the protein cannot be assayed immediately, the laboratory shall have verified procedures in place on how to store the isolated protein.
NOTE 1 Storage in solution on wet-ice for a short period of time (i.e. 2 h) can be appropriate in certain circu mstances.
Storage for long-term purposes (i.e. for several years) should be at 70 °C.
For long-term storage, aliquots of the isolated protein should be generated to avoid repeated freezing and thawing. The aliquots should not be further diluted to avoid a reduction of the protein stability. Avoid more than two freeze—thaw cycles, use aliquots instead. If lyophilized, proteins can be stored for several years at 4 °C or —20 °C.
NOTE 2 Protein stability is affected by numerous factors, including freeze/thaw cycles, pH, protein concentration, salt conditions and others. Optimal conditions for storing specific proteins can vary from protein to protein.