1、BSI Standards PublicationPD CEN/TS 16827-2:2015Molecular in vitro diagnosticexaminations Specificationsfor pre-examination processesfor FFPE tissuePart 2: Isolated proteinsPD CEN/TS 16827-2:2015Incorporating corrigendum September 2015PD CEN/TS 16827-2:2015 PUBLISHED DOCUMENTNational forewordThis Pub
2、lished Document is the UK implementation ofCEN/TS 16827-2:2015.The UK participation in its preparation was entrusted to TechnicalCommittee CH/212, IVDs.A list of organizations represented on this committee can be obtainedon request to its secretary.This publication does not purport to include all th
3、e necessary provisionsof a contract. Users are responsible for its correct application. The British Standards Institution 2015.Published by BSI Standards Limited 2015ISBN 978 0 580 91720 2ICS 11.100.10Compliance with a British Standard cannot confer immunity from legal obligations.This British Stand
4、ard was published under the authority of the StandardsPolicy and Strategy Committee on 31 August 2015.Amendments/corrigenda issued since publicationDate Text affected30 September 2015 Implementation of CEN Correction Notice 28 August 2015: Annex A updatedPD CEN/TS 16827-2:2015TECHNICAL SPECIFICATION
5、 SPCIFICATION TECHNIQUE TECHNISCHE SPEZIFIKATION CEN/TS 16827-2 August 2015 ICS 11.100.10 English Version Molecular in vitro diagnostic examinations - Specifications for pre-examination processes for FFPE tissue - Part 2: Isolated proteins Tests de diagnostic molculaire in vitro - Spcifications pour
6、les processus pranalytiques pour tissu FFPE - Partie 2: Protines extraites Molekularanalytische in-vitro-diagnostische Verfahren - Spezifikationen fr pranalytische Prozesse fr FFPE-Gewebeproben - Teil 2: Isolierte Proteine This Technical Specification (CEN/TS) was approved by CEN on 6 July 2015 for
7、provisional application. The period of validity of this CEN/TS is limited initially to three years. After two years 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 an
8、nounce the existence of this CEN/TS in the same way as for an EN and to make the CEN/TS available promptly at national 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
9、 the CEN/TS into an EN is reached. CEN members are the national standards bodies of 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,
10、 Malta, Netherlands, Norway, Poland, Portugal, Romania, Slovakia, Slovenia, Spain, Sweden, Switzerland, Turkey and United Kingdom. EUROPEAN COMMITTEE FOR STANDARDIZATION COMIT EUROPEN DE NORMALISATION EUROPISCHES KOMITEE FR NORMUNG CEN-CENELEC Management Centre: Avenue Marnix 17, B-1000 Brussels 201
11、5 CEN All rights of exploitation in any form and by any means reserved worldwide for CEN national Members. Ref. No. CEN/TS 16827-2:2015 EPD CEN/TS 16827-2:2015CEN/TS 16827-2:2015 (E) 2 Contents Page European foreword .3 Introduction .4 1 Scope 5 2 Normative references 5 3 Terms and definitions .5 4
12、General considerations .7 5 Outside the laboratory 8 5.1 Primary tissue collection manual.8 5.1.1 Information about 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 9 5.2 Transport requirements 9 6 Inside the labor
13、atory .9 6.1 Information on the primary tissue sample receipt .9 6.2 Formalin fixation of the specimen 10 6.3 Evaluation of the pathology of the specimen and selection of the sample 11 6.4 Post-fixation of frozen samples 11 6.5 Processing and paraffin embedding. 12 6.6 Storage requirements . 12 6.7
14、Isolation of the total protein 13 6.7.1 General . 13 6.7.2 General information for protein isolation procedures 13 6.7.3 Using commercial kits 13 6.7.4 Using the laboratories own protocols . 13 6.8 Quantity and quality assessment of isolated RNA 14 6.9 Storage of isolated RNA . 14 Annex A (informati
15、ve) Quality control of RNA extracted from formalin fixed and paraffin embedded tissue samples: implications for RT-qPCR based analyses . 15 A.1 Introduction . 15 A.2 Example . 15 A.2.1 General . 15 A.2.2 Experimental procedures . 15 A.2.2.1 General . 15 A.2.2.2 Tissues . 16 A.2.2.3 Protein analysis
16、16 A.2.3 Results . 17 A.2.4 Further reading . 18 Bibliography . 19 PD CEN/TS 16827-2:2015CEN/TS 16827-2:2015 (E) 3 European foreword This document (CEN/TS 16827-2:2015) has been prepared by Technical Committee CEN/TC 140 “In vitro diagnostic medical devices”, the secretariat of which is held by DIN.
17、 Attention 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 organizatio
18、ns of 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, M
19、alta, Netherlands, Norway, Poland, Portugal, Romania, Slovakia, Slovenia, Spain, Sweden, Switzerland, Turkey and the United Kingdom. Annex (informative) Quantitative protein analysis demonstrates changes of protein amounts during cold ischemiaPD CEN/TS 16827-2:2015CEN/TS 16827-2:2015 (E) 2 Contents
20、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 about the primary sample donor .8 5.1.2 Information on the primary tissue sample 8 5
21、.1.3 Information on the primary tissue sample processing 9 5.2 Transport requirements 9 6 Inside the laboratory .9 6.1 Information on the primary tissue sample receipt .9 6.2 Formalin fixation of the specimen 10 6.3 Evaluation of the pathology of the specimen and selection of the sample 11 6.4 Post-
22、fixation of frozen samples 11 6.5 Processing and paraffin embedding. 12 6.6 Storage requirements . 12 6.7 Isolation of the total protein 13 6.7.1 General . 13 6.7.2 General information for protein isolation procedures 13 6.7.3 Using commercial kits 13 6.7.4 Using the laboratories own protocols . 13
23、6.8 Quantity and quality assessment of isolated RNA 14 6.9 Storage of isolated RNA . 14 Annex A (informative) Quality control of RNA extracted from formalin fixed and paraffin embedded tissue samples: implications for RT-qPCR based analyses . 15 A.1 Introduction . 15 A.2 Example . 15 A.2.1 General .
24、 15 A.2.2 Experimental procedures . 15 A.2.2.1 General . 15 A.2.2.2 Tissues . 16 A.2.2.3 Protein analysis 16 A.2.3 Results . 17 A.2.4 Further reading . 18 Bibliography . 19 PD CEN/TS 16827-2:2015CEN/TS 16827-2:2015 (E) 3 European foreword This document (CEN/TS 16827-2:2015) has been prepared by Tech
25、nical Committee CEN/TC 140 “In vitro diagnostic medical devices”, the secretariat of which is held by DIN. Attention 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
26、 such patent rights. According to the CEN-CENELEC Internal Regulations, the national standards organizations of the following countries are bound to announce this Technical Specification: Austria, Belgium, Bulgaria, Croatia, Cyprus, Czech Republic, Denmark, Estonia, Finland, Former Yugoslav Republic
27、 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 the United Kingdom. PD CEN/TS 16827-2:2015CEN/TS 16827-2:2015 (E) 4 Introduction
28、Molecular in vitro diagnostics has enabled a significant progress in medicine. Further progress is expected by 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 dras
29、tically during primary sample collection, transport, storage, and processing thus making the outcome from diagnostics 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
30、 the pre-examination process. Although originally thought as being impossible due to the crosslinking activities of formaldehyde, protein extraction techniques from formalin formalin fixed and paraffin embedded (FFPE) tissues have been much improved in recent years. Heat-induced reversal of formalde
31、hyde-induced crosslinks has been demonstrated as an essential step in the protein extraction procedures 1, 2. Currently, most investigators accept that proteins extracted from FFPE tissue are suitable for downstream proteomic analysis 3. However, a standardization of the entire process from primary
32、sample collection 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 standardise the steps for FFPE tissue with regard to protein analysis in what is referred to as the preanalyti
33、cal phase. PD CEN/TS 16827-2:2015CEN/TS 16827-2:2015 (E) 5 1 Scope This Technical Specification gives recommendations for the handling, documentation and processing of FFPE tissue specimens intended for the analysis of extracted proteins during the preanalytical phase before a molecular assay is per
34、formed. This 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 organizations performing biomedical research, biobanks, an
35、d regulatory 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 / patie
36、nts. 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 environment conditions after the tissue removal from the body. Furthermore, the formalin
37、 fixation and paraffin embedding process leads to modifications of the protein molecules, which can impact the validity and reliability of the analytical test results. Therefore, it is essential to take special measures to minimize the described profile changes and modifications within the tissue fo
38、r subsequent protein analysis. This document is not applicable for protein 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 edit
39、ion cited applies. For undated references, the latest edition of the referenced 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
40、 safety 3 Terms and definitions For the purposes of this document, the 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
41、kinds of parameter testing or chemical manipulation for quantitative or qualitative analysis 3.3 cold ischemia condition after removal of the tissue from the body until its stabilization or fixation 3.4 FFPE formalin fixation and paraffin embedding PD CEN/TS 16827-2:2015CEN/TS 16827-2:2015 (E) 4 Int
42、roduction Molecular in vitro diagnostics has enabled a significant progress in medicine. Further progress is expected by 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 c
43、hange drastically during primary sample collection, transport, storage, and processing thus making the outcome from diagnostics or research unreliable or even impossible because the subsequent analytical assay will not determine the situation in the patient but an artificial molecular pattern genera
44、ted during the pre-examination process. Although originally thought as being impossible due to the crosslinking activities of formaldehyde, protein extraction techniques from formalin formalin fixed and paraffin embedded (FFPE) tissues have been much improved in recent years. Heat-induced reversal o
45、f formaldehyde-induced crosslinks has been demonstrated as an essential step in the protein extraction procedures 1, 2. Currently, most investigators accept that proteins extracted from FFPE tissue are suitable for downstream proteomic analysis 3. However, a standardization of the entire process fro
46、m primary sample collection 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 standardise the steps for FFPE tissue with regard to protein analysis in what is referred to as the
47、preanalytical phase. PD CEN/TS 16827-2:2015CEN/TS 16827-2:2015 (E) 5 1 Scope This Technical Specification gives recommendations for the handling, documentation and processing of FFPE tissue specimens intended for the analysis of extracted proteins during the preanalytical phase before a molecular as
48、say is performed. This 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 organizations performing biomedical research, bi
49、obanks, and regulatory 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 expression of genes can be influenced by the given treatment or intervention (surgery, biopsy), or drugs administered fo
