1、BSI Standards PublicationPD CEN/TS 16826-2:2015Molecular in vitro diagnosticexaminations Specificationsfor pre-examination processesfor snap frozen tissuePart 2: Isolated proteinsPD CEN/TS 16826-2:2015 PUBLISHED DOCUMENTNational forewordThis Published Document is the UK implementation of CEN/TS16826
2、-2:2015.The UK participation in its preparation was entrusted to TechnicalCommittee CH/212, IVDs.A list of organizations represented on this committee can beobtained on request to its secretary.This publication does not purport to include all the necessaryprovisions of a contract. Users are responsi
3、ble for its correctapplication. The British Standards Institution 2015. Published by BSI StandardsLimited 2015ISBN 978 0 580 85035 6ICS 11.100.10Compliance with a British Standard cannot confer immunity fromlegal obligations.This Published Document was published under the authority of theStandards P
4、olicy and Strategy Committee on 31 August 2015.Amendments issued since publicationDate Text affectedPD CEN/TS 16826-2:2015TECHNICAL SPECIFICATION SPCIFICATION TECHNIQUE TECHNISCHE SPEZIFIKATION CEN/TS 16826-2 August 2015 ICS 11.100.10 English Version Molecular in vitro diagnostic examinations - Spec
5、ifications for pre-examination processes for snap frozen tissue - Part 2: Isolated proteins Tests de diagnostic molculaire in vitro - Spcifications relatives aux processus pranalytiques pour les tissus conglation rapide - Partie 2: Protines extraites Molekularanalytische in-vitro-diagnostische Verfa
6、hren - Spezifikationen fr pranalytische Prozesse fr schockgefrorene Gewebeproben - Teil 2: Isolierte ProteineThis Technical Specification (CEN/TS) was approved by CEN on 6 July 2015 for provisional application. The period of validity of this CEN/TS is limited initially to three years. After two year
7、s the members of CEN will be requested to submit their comments, particularly on the question whether the CEN/TS can be converted into a European Standard. CEN members are required to announce the existence of this CEN/TS in the same way as for an EN and to make the CEN/TS available promptly at nati
8、onal level in an appropriate form. It is permissible to keep conflicting national standards in force (in parallel to the CEN/TS) until the final decision about the possible conversion of the CEN/TS into an EN is reached. CEN members are the national standards bodies of Austria, Belgium, Bulgaria, Cr
9、oatia, Cyprus, Czech Republic, Denmark, Estonia, Finland, Former Yugoslav Republic of Macedonia, France, Germany, Greece, Hungary, Iceland, Ireland, Italy, Latvia, Lithuania, Luxembourg, Malta, Netherlands, Norway, Poland, Portugal, Romania, Slovakia, Slovenia, Spain, Sweden, Switzerland, Turkey and
10、 United Kingdom. EUROPEAN COMMITTEE FOR STANDARDIZATION COMIT EUROPEN DE NORMALISATION EUROPISCHES KOMITEE FR NORMUNG CEN-CENELEC Management Centre: Avenue Marnix 17, B-1000 Brussels 2015 CEN All rights of exploitation in any form and by any means reserved worldwide for CEN national Members. Ref. No
11、. CEN/TS 16826-2:2015 EPD CEN/TS 16826-2:2015CEN/TS 16826-2:2015 (E) 2 Contents Page European foreword .3 Introduction .4 1 Scope 5 2 Normative references 5 3 Terms and definitions .5 4 General considerations .7 5 Outside the laboratory 8 5.1 Primary tissue collection manual.8 5.1.1 Information abou
12、t the primary sample donor .8 5.1.2 Information on the primary tissue sample 8 5.1.3 Information on the primary tissue sample processing 8 5.2 Transport requirements 9 6 Inside the laboratory .9 6.1 Information on the primary tissue sample receipt .9 6.2 Evaluation of the pathology of the specimen a
13、nd selection of the sample.9 6.3 Cryo-storage of the specimen . 10 6.4 Storage requirements . 11 6.5 Isolation of total protein . 11 6.5.1 General . 11 6.5.2 Using commercial kits 11 6.5.3 Using the laboratories own protocols . 11 6.6 Quality assessment of isolated proteins 12 6.7 Storage of isolate
14、d total protein 12 Annex A (informative) Quantitative protein analysis demonstrates changes of protein amounts during cold ischemia 13 A.1 Introduction . 13 A.2 Example . 13 A.2.1 General . 13 A.2.2 Experimental procedures . 13 A.2.2.1 General . 13 A.2.2.2 Tissues . 14 A.2.2.3 Protein analysis 14 A.
15、2.3 Results . 15 A.2.4 Further reading . 16 Bibliography . 17 PD CEN/TS 16826-2:2015CEN/TS 16826-2:2015 (E) 3 European foreword This document (CEN/TS 16826-2:2015) has been prepared by Technical Committee CEN/TC 140 “In vitro diagnostic medical devices”, the secretariat of which is held by DIN. Atte
16、ntion is drawn to the possibility that some of the elements of this document may be the subject of patent rights. CEN and/or CENELEC shall not be held responsible for identifying any or all such patent rights. According to the CEN-CENELEC Internal Regulations, the national standards organizations of
17、 the following countries are bound to announce this Technical Specification: Austria, Belgium, Bulgaria, Croatia, Cyprus, Czech Republic, Denmark, Estonia, Finland, Former Yugoslav Republic of Macedonia, France, Germany, Greece, Hungary, Iceland, Ireland, Italy, Latvia, Lithuania, Luxembourg, Malta,
18、 Netherlands, Norway, Poland, Portugal, Romania, Slovakia, Slovenia, Spain, Sweden, Switzerland, Turkey and the United Kingdom. PD CEN/TS 16826-2:2015CEN/TS 16826-2:2015 (E) 4 Introduction Molecular in vitro diagnostics has enabled a significant progress in medicine. Further progress is expected by
19、new technologies analysing signatures of nucleic acids, proteins, and metabolites in human tissues and body fluids. However, the profiles and/or integrity of these molecules can change drastically during primary sample collection, transport, storage, and processing thus making the outcome from diagn
20、ostics or research unreliable or even impossible because the subsequent analytical 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 primary sample collection
21、 to protein analysis is needed. Studies have been undertaken to determine the important influencing factors. This Technical Specification draws upon such work to codify and standardize the steps for frozen tissue with regard to protein analysis in what is referred to as the preanalytical phase. PD C
22、EN/TS 16826-2:2015CEN/TS 16826-2:2015 (E) 5 1 Scope This Technical Specification gives recommendations for the handling, documentation and processing of frozen tissue specimens intended for the analysis of extracted proteins during the preanalytical phase before a molecular assay is performed. This
23、Technical Specification is applicable to molecular in vitro diagnostic examinations (e.g., in vitro diagnostic laboratories, laboratory customers, developers and manufacturers of in vitro diagnostics, institutions and commercial organisations performing biomedical research, biobanks, and regulatory
24、authorities). Protein profiles and protein-protein interactions in tissues can change drastically before and after collection (due to e.g., gene induction, gene down regulation, protein degradation). Protein species amounts can change differently in tissues from different donors / patients. The expr
25、ession of genes can be influenced by the given treatment or medical intervention (surgery, biopsy), or drugs administered for anaesthesia or even treatment of concomitant disease as well as by the different environment conditions after the tissue removal from the body. Therefore, it is essential to
26、take special measures to minimize the described profile changes and modifications within the tissue for subsequent protein analysis. Tissues that have undergone chemical stabilization pre-treatment before freezing are not covered in this document. In addition this document is not applicable for prot
27、ein analysis by immunohistochemistry. 2 Normative references The following documents, in whole or in part, are normatively referenced in this document and are indispensable for its application. For dated references, only the edition cited applies. For undated references, the latest edition of the re
28、ferenced document (including any amendments) applies. EN ISO 15189:2012, Medical laboratories Requirements for quality and competence (ISO 15189:2012, Corrected version 2014-08-15) ISO 15190, Medical laboratories Requirements for safety 3 Terms and definitions For the purposes of this document, the
29、terms and definitions given in EN ISO 15189:2012 and the following apply. 3.1 ambient temperature unregulated temperature of the surrounding air 3.2 analytical phase processes that start with the isolated analyte and include all kinds of parameter testing or chemical manipulation for quantitative or
30、 qualitative analysis 3.3 cold ischemia condition after removal of the tissue from the body until its stabilization or fixation PD CEN/TS 16826-2:2015CEN/TS 16826-2:2015 (E) 6 3.4 pre-examination processes preanalytical phase preanalytical workflow processes that start, in chronological order, from
31、the clinicians request and include the examination request, preparation and identification of the patient, surgical procedure, collection of the primary sample(s), temporary storage, transportation to and within the analytical laboratory, aliquoting, retrieval, isolation of analytes, and end when th
32、e analytical examination begins SOURCE: EN ISO 15189:2012, definition 3.15, modified An additional term was added and more details were included. Note 1 to entry: The preanalytical phase may include preparative processes that may influence the outcome of the intended examination. 3.5 primary sample
33、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: EN ISO 15189:2012, 3.16, modified The term and definition is used here without the original notes. 3.6 protein typ
34、e of biological macromolecules composed of one or more chains with a defined sequence of amino acids connected through peptide bonds 3.7 protein profile amounts of the individual protein molecules that are present in a sample and that can be measured in the absence of any losses, inhibition and inte
35、rference 3.8 protein species amounts of a chemically clearly-defined protein corresponding to one spot on a high-performance 2-dimensional gel electrophoresis pattern SOURCE: Jungblut et. al.1996 3.9 PTM post translational modifications chemical alterations to a primary protein structure, often cruc
36、ial for conferring biological activity on a protein SOURCE: Encyclopedia of Psychopharmacology, 2010 3.10 room temperature temperature which is defined as 18 C to 25 C for the purposes of this document 3.11 sample one or more parts taken from a primary sample SOURCE: EN ISO 15189:2012, 3.24, modifie
37、d The example was not taken over. PD CEN/TS 16826-2:2015CEN/TS 16826-2:2015 (E) 7 3.12 stability ability of a sample material, when stored under specified conditions, to maintain a stated property value within specified limits for a specified period of time SOURCE: ISO Guide 30:1992, 2.7 Note 1 to e
38、ntry: The measured constituent for the purpose of this document is isolated protein. 3.13 warm ischemia warm Ischemia is the condition where the tissue is deprived of its normal blood supply containing oxygen and nutrients while the tissue is at body temperature 4 General considerations For general
39、statements on primary sample collection and handling (including avoidance of cross contaminations) see EN ISO 15189:2012, 5.4.4, 5.2.6. Consumables including kits shall be verified before use in examination (see EN ISO 15189:2012, 5.3.2.3); EN ISO 15189:2012, 5.5.1.2 and 5.5.1.3 can also apply. As a
40、ll steps of a diagnostic workflow can influence the final analytical performance, the entire workflow comprising the preanalytical steps, including information on biomolecule stability and storage conditions, and analytical steps should be verified and validated (see EN ISO 15189). The stability of
41、the specific protein(s) of interest and their posttranslational modifications (if important for the assay) should be investigated throughout the complete preanalytical workflow prior to the development and implementation of an analytical test. Before tissues are stabilized by freezing; protein amoun
42、ts, 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
43、 temperature before freezing. In addition, those effects can vary in tissues from different donors / patients. Generally, the longer the warm and cold ischemia times and the higher the ambient temperature before freezing the tissue specimen, the higher is the risk that changes in the protein profile
44、 can occur. NOTE Prolonged cold ischemia times result in changes of protein (e.g., cytokeratin 18) and phosphoprotein (e.g., phospho-p42/44) amounts 1, 2. Keeping the specimen on wet-ice diminishes this effect 3. Protein amounts as well as posttranslational modifications can also vary during the pre
45、analytical phase, depending on the origin and type of tissue, the underlying disease, the surgical procedure, the drug regime, 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 isch
46、emia cannot be easily standardized, its time and duration should be documented. When it is not possible to avoid cold ischemia, its time of onset and duration shall be documented and the temperatures of the specimen transport containers surroundings should be documented. Where the specimen is transp
47、orted to another facility for freezing, the transport duration shall be documented and the ambient conditions should also be documented. Safety regulations on transport and handling shall be considered (see EN ISO 15189:2012, 5.2.3 and 5.4.5 and ISO 15190). During the whole preanalytical workflow pr
48、ecautions shall be taken to avoid cross contamination between different samples. If a commercial product is not used in accordance with the manufacturers instructions, responsibility for its use and performance lies with the user. PD CEN/TS 16826-2:2015CEN/TS 16826-2:2015 (E) 8 5 Outside the laborat
49、ory 5.1 Primary tissue collection manual 5.1.1 Information about the primary sample donor The documentation should include, but is not limited to: a) the primary donor / patient ID, which can be in the form of a code; b) the health status of the primary sample donor (e.g., healthy, disease type, concomitant disease); c) the information about routine medical treatment and special treatment prior to tissue collection (e.g., anaesthetics, medications, surgical or diagnostic procedures (e.g., biopsy device used for the collection); d) the st
