1、BSI Standards Publication PD CEN/TS 16827-1:2015 Molecular in vitro diagnostic examinations Specifications for pre-examination processes for FFPE tissue Part 1: Isolated RNAPD CEN/TS 16827-1:2015 PUBLISHED DOCUMENT National foreword This Published Document is the UK implementation of CEN/TS 16827-1:
2、2015. The UK participation in its preparation was entrusted to Technical Committee CH/212, IVDs. A list of organizations represented on this committee can be obtained on request to its secretary. This publication does not purport to include all the necessary provisions of a contract. Users are respo
3、nsible for its correct application. The British Standards Institution 2015. Published by BSI Standards Limited 2015 ISBN 978 0 580 85029 5 ICS 11.100.10 Compliance with a British Standard cannot confer immunity from legal obligations. This Published Document was published under the authority of the
4、Standards Policy and Strategy Committee on 31 August 2015. Amendments issued since publication Date 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 examin
5、ations - Specifications for 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 - Spezifikatio
6、nen fr pranalytische Prozesse 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 requ
7、ested 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 national level in an appropriate form
8、. 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, Croatia, Cyprus, Czech Republic, De
9、nmark, 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 United Kingdom. EUROPEAN COMMITT
10、EE 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. CEN/TS 16827-1:2015 EPD CEN/TS
11、16827-1:2015 CEN/TS 16827-1:2015 (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.
12、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 Formalin fixation of the specimen .9 6.3 Evaluation of the pathology of the s
13、pecimen and selection of the 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
14、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 (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、15 A.2 Results . 15 A.2.1 Time 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 Conclusion
16、s 18 A.4 Further reading . 19 Bibliography . 20 PD CEN/TS 16827-1:2015 CEN/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 draw
17、n 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 the followin
18、g 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, Netherlands,
19、 Norway, Poland, Portugal, Romania, Slovakia, Slovenia, Spain, Sweden, Switzerland, Turkey and the United Kingdom. PD CEN/TS 16827-1:2015 CEN/TS 16827-1:2015 (E) 4 Introduction Molecular in vitro diagnostics has enabled a significant progress in medicine. Further progress is expected by new technolo
20、gies 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 diagnostics or re
21、search unreliable or even impossible 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 ne
22、eded. Studies have been undertaken 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 phas
23、e. PD CEN/TS 16827-1:2015 CEN/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 Specif
24、ication 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, and regulatory authorities). Th
25、e formalin fixation and the paraffin 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
26、tissue for subsequent RNA analysis. 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 refe
27、renced 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 te
28、rms 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 q
29、ualitative 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 3.5 FFPE tissues formalin fixed and paraffin embedded tissues 3.6 formalin saturated formaldehyde solution containing a mas
30、fraction of 37 % (corresponding to a volume fraction of 40 %) formaldehyde, termed 100 % formalin PD CEN/TS 16827-1:2015 CEN/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
31、preanalytical workflow processes 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
32、 laboratory, aliquoting, retrieval, 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
33、may influence the outcome of the 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 te
34、rm and definition is used here 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 ribonucleo
35、tides occurring in a double-stranded 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 e
36、xample was not taken over. 3.14 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 16827-1:2015 CEN/TS 16827-1:2015 (E) 7 Note 1 to entry:
37、The measured constituent for the 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 solu
38、tions often contain methanol to 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
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
40、all 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 quantitative RNA profile(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 dependin
42、g on the duration of warm and cold 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 high
43、er the ambient temperature before 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 t
44、he origin and type of tissue, the 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 dur
45、ation 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 transported to another facility for formalin fixation, the
46、 transport duration shall be documented 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 mol
47、ecular assays, especially in the 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
48、 and handling shall be considered (see EN ISO 15189:2012, 5.2.3 and 5.4.5 and ISO 15190). PD CEN/TS 16827-1:2015 CEN/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
49、 accordance with the manufacturers 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 collec
