1、BSI Standards PublicationPD CEN/TS 16827-1:2015Molecular in vitro diagnosticexaminations Specificationsfor pre-examination processesfor FFPE tissuePart 1: Isolated RNAPD CEN/TS 16827-1:2015 PUBLISHED DOCUMENTNational forewordThis Published Document is the UK implementation of CEN/TS16827-1:2015.The
2、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 responsible for its
3、correctapplication. The British Standards Institution 2015. Published by BSI StandardsLimited 2015ISBN 978 0 580 85029 5ICS 11.100.10Compliance with a British Standard cannot confer immunity fromlegal obligations.This Published Document was published under the authority of theStandards Policy and St
4、rategy Committee on 31 August 2015.Amendments issued since publicationDate Text affectedPD CEN/TS 16827-1:2015TECHNICAL SPECIFICATION SPCIFICATION TECHNIQUE TECHNISCHE SPEZIFIKATION CEN/TS 16827-1 August 2015 ICS 11.100.10 English Version Molecular in vitro diagnostic examinations - Specifications f
5、or pre-examination processes for FFPE tissue - Part 1: Isolated RNA Tests de diagnostic molculaire in vitro - Spcifications relatives aux processus pranalytiques pour les tissus FFPE - Partie 1: ARN extrait Molekularanalytische in-vitro-diagnostische Verfahren - Spezifikationen fr pranalytische Proz
6、esse fr FFPE-Gewebeproben - Teil 1: Isolierte RNS This 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 years the members of CEN will be requested to submit their comm
7、ents, 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 national level in an appropriate form. It is permissible to kee
8、p 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, Croatia, Cyprus, Czech Republic, Denmark, Estonia, Finland, F
9、ormer 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 United Kingdom. EUROPEAN COMMITTEE FOR STANDARDIZATION COM
10、IT 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. CEN/TS 16827-1:2015 EPD CEN/TS 16827-1:2015CEN/TS 16827-1
11、: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 about the primary sample donor .8 5.1.2 Information on the prim
12、ary 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 Formalin fixation of the specimen .9 6.3 Evaluation of the pathology of the specimen and selection of th
13、e sample 10 6.4 Post-fixation of frozen samples 11 6.5 Processing and paraffin embedding. 11 6.6 Storage requirements . 12 6.7 Isolation of the total RNA . 12 6.7.1 General . 12 6.7.2 General information for RNA isolation procedures 12 6.7.3 Using commercial kits 13 6.7.4 Using the laboratories own
14、protocols . 13 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 Summary 15 A.2 Results . 15 A.2.1 T
15、ime dependency of RNA integrity . 15 A.2.2 Impact of formalin-fixation on cDNA synthesis efficiency . 16 A.2.3 Fixation and storage introduces major gene-to-gene variations in RT-qPCR . 17 A.2.4 Impact of storage conditions of FFPE blocks on RNA Integrity 18 A.3 Conclusions 18 A.4 Further reading .
16、19 Bibliography . 20 PD CEN/TS 16827-1:2015CEN/TS 16827-1:2015 (E) 3 European foreword This document (CEN/TS 16827-1:2015) has been prepared by Technical Committee CEN/TC 140 “In vitro diagnostic medical devices”, the secretariat of which is held by DIN. Attention is drawn to the possibility that so
17、me 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 the following countries are bound to ann
18、ounce 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, Netherlands, Norway, Poland, Portugal, R
19、omania, Slovakia, Slovenia, Spain, Sweden, Switzerland, Turkey and the United Kingdom. PD CEN/TS 16827-1:2015CEN/TS 16827-1:2015 (E) 4 Introduction Molecular in vitro diagnostics has enabled a significant progress in medicine. Further progress is expected by new technologies analysing signatures of
20、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 diagnostics or research unreliable or even imp
21、ossible because the subsequent analytical assay will not determine the situation in the patient but an artificial profile generated during the pre-examination process. Therefore, a standardization of the entire process from primary sample collection to RNA analysis is needed. Studies have been under
22、taken to determine the important influencing factors. This Technical Specification draws upon such work to codify and standardize the steps for formalin fixed and paraffin embedded (FFPE) tissue with regard to RNA analysis in what is referred to as the preanalytical phase. PD CEN/TS 16827-1:2015CEN/
23、TS 16827-1:2015 (E) 5 1 Scope This Technical Specification gives recommendations for the handling, documentation and processing of FFPE tissue specimens intended for RNA analysis during the preanalytical phase before a molecular assay is performed. This Technical Specification is applicable to molec
24、ular 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, and regulatory authorities). The formalin fixation and the pa
25、raffin embedding process lead to modifications of the RNA 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 for subsequent RNA anal
26、ysis. 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 referenced document (including any
27、 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 terms and definitions given in E
28、N 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 qualitative analysis 3.3 cold i
29、schemia condition after removal of the tissue from the body until its stabilization or fixation 3.4 FFPE formalin fixation and paraffin embedding 3.5 FFPE tissues formalin fixed and paraffin embedded tissues 3.6 formalin saturated formaldehyde solution containing a mas fraction of 37 % (correspondin
30、g to a volume fraction of 40 %) formaldehyde, termed 100 % formalin PD CEN/TS 16827-1:2015CEN/TS 16827-1:2015 (E) 6 3.7 formalin fixation treatment of a sample with standard buffered formalin solution for stabilization 3.8 pre-examination processes preanalytical phase preanalytical workflow processe
31、s that start, in chronological order, from 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, retrie
32、val, isolation of analytes, and end when the 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 th
33、e intended examination. 3.9 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: EN ISO 15189:2012, 3.16, modified The term and definition is used here
34、without the original notes. 3.10 quantitative RNA profile RNA profile amounts of the individual RNA molecules that are present in a sample and that can be measured in the absence of any losses, inhibition and interference 3.11 RNA ribonucleic acid polymer of ribonucleotides occurring in a double-str
35、anded or single-stranded form SOURCE: EN ISO 22174:2005, 3.1.3 3.12 room temperature temperature which is defined as 18 C to 25 C for the purposes of this document 3.13 sample one or more parts taken from a primary sample SOURCE: EN ISO 15189:2012, 3.24, modified The example was not taken over. 3.14
36、 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 PD CEN/TS 16827-1:2015CEN/TS 16827-1:2015 (E) 7 Note 1 to entry: The measured constituent for the
37、 purpose of this document is RNA. 3.15 standard buffered formalin solution 10 % formalin solution containing a mass fraction of 3,7 % (corresponding to a volume fraction of 4 %) formaldehyde buffered to pH 6,8 to pH 7,2 Note 1 to entry: Standard buffered formalin solutions often contain methanol to
38、inhibit oxidation and polymerization of formaldehyde. 3.16 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 statements on primary sample co
39、llection 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 all steps of a diagnostic workfl
40、ow 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 the specific quantitative RNA p
41、rofile(s) of interest should be investigated throughout the entire preanalytical workflow prior to the development and implementation of an analytical test. Before tissues are fixed in standard buffered formalin solution, RNA profiles can change significantly depending on the duration of warm and co
42、ld ischemia and the temperature before formalin fixation (e.g., gene induction, gene down regulation, RNA degradation). In addition, those changes can vary in tissues from different donors / patients. Generally, the longer the warm and cold ischemia times and the higher the ambient temperature befor
43、e fixation of the tissue specimen, the higher is the risk that changes in the RNA profile can occur. NOTE Intraoperative warm ischemia can result in more pronounced changes of RNA profiles than in postoperative cold ischemia. RNA profiles can also vary, depending on the origin and type of tissue, th
44、e underlying disease, the surgical procedure, drugs administered for anaesthesia or treatment of concomitant disease, and on the different environmental conditions after the tissue removal from the body. As warm ischemia cannot be easily standardized, its time and duration should be documented. When
45、 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 transported to another facility for formalin fixation, the transport duration shall be doc
46、umented and the ambient conditions should also be documented. In addition, formalin fixation causes modifications of biomolecules and leads to suboptimal performance of RNA extracted from FFPE tissues that should be considered in quality control and application of molecular assays, especially in the
47、 context of gene expression studies (see 1, 2, 3). Assay optimization for FFPE tissues or the use of non-crosslinking alternatives to standard buffered formalin solution are options to minimize this issue for molecular analyses (see 4). Safety regulations on transport and handling shall be considere
48、d (see EN ISO 15189:2012, 5.2.3 and 5.4.5 and ISO 15190). PD CEN/TS 16827-1:2015CEN/TS 16827-1:2015 (E) 8 During the whole preanalytical workflow precautions shall be taken to avoid cross contamination between different samples. If a commercial product is not used in accordance with the manufacturer
49、s instructions, responsibility for its use and performance lies with the user. 5 Outside the laboratory 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, medicati
