1、BSI Standards Publication PD CEN/TS 16827-3:2015 Molecular in vitro diagnostic examinations Specifications for pre-examination processes for FFPE tissue Part 3: Isolated DNAPD CEN/TS 16827-3:2015 PUBLISHED DOCUMENT National foreword This Published Document is the UK implementation of CEN/TS 16827-3:
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 85032 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 16827-3:2015TECHNICAL SPECIFICATION SPCIFICATION TECHNIQUE TECHNISCHE SPEZIFIKATION CEN/TS 16827-3 August 2015 ICS 11.100.10 English Version Molecular in vitro diagnostic examin
5、ations - Specifications for pre-examination processes for FFPE tissue - Part 3: Isolated DNA Tests de diagnostic molculaire in vitro - Spcifications relatives aux processus pranalytiques pour les tissus FFPE - Partie 3: ADN isol Molekularanalytische in-vitro-diagnostische Verfahren - Spezifikationen
6、 fr pranalytische Prozesse fr FFPE- Gewebeproben - Teil 3: Isolierte DNS 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 request
7、ed 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. I
8、t 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, Denma
9、rk, 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 COMMITTEE
10、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 16827-3:2015 EPD CEN/TS 168
11、27-3:2015 CEN/TS 16827-3:2015 (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 7 5.1 Primary tissue collection manual.7 5.1.1 Information about the primary sample donor .7 5.1.2
12、 Information on the primary tissue sample 8 5.1.3 Information on the primary tissue sample processing 8 5.2 Transport requirements 8 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 spec
13、imen 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 . 11 6.7 Isolation of DNA 12 6.7.1 General . 12 6.7.2 General information for DNA isolation procedures 12 6.7.3 Using commercial kits 12 6.7.4 Using the labor
14、atories own protocols . 13 6.8 Quantity and quality assessment of isolated RNA 13 6.9 Storage of isolated RNA . 14 Annex A (informative) Impact of the storage temperature on DNA Integrity in FFPE blocks of tissue 15 A.1 Introduction . 15 A.2 Results . 15 A.3 Conclusions 15 Bibliography . 16 PD CEN/T
15、S 16827-3:2015 CEN/TS 16827-3:2015 (E) 3 European foreword This document (CEN/TS 16827-3:2015) 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 some of the elements of this d
16、ocument 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 announce this Technical Specifi
17、cation: 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, Romania, Slovakia, Slovenia,
18、Spain, Sweden, Switzerland, Turkey and the United Kingdom. PD CEN/TS 16827-3:2015 CEN/TS 16827-3:2015 (E) 4 Introduction Molecular in vitro diagnostics has enabled a significant progress in medicine. Further progress is expected by new technologies analysing signatures of nucleic acids, proteins, an
19、d 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 impossible because the subseque
20、nt analytical assay will not determine the situation in the patient but an artificial molecular pattern generated during the pre-examination process. Studies have been undertaken to determine the influencing factors for DNA analysis from formalin fixed and paraffin embedded (FFPE) tissue. These stud
21、ies demonstrated that a standardization of the entire process from primary sample collection to DNA analysis is needed. This Technical Specification draws upon such work to codify and standardize the steps for FFPE tissue with regard to DNA analysis in what is referred to as the preanalytical phase.
22、 PD CEN/TS 16827-3:2015 CEN/TS 16827-3:2015 (E) 5 1 Scope This Technical Specification gives recommendations for the handling, documentation and processing of FFPE tissue specimens intended for DNA analysis during the preanalytical phase before a molecular assay is performed. This Technical Specific
23、ation 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). DNA
24、integrity in tissues can change before and during formalin fixation, processing and storage. Chemical modifications introduced into DNA during tissue fixation might lead to fragmentation and sequence alterations 1, changes in the methylation status or even structural changes which can lead to e.g.,
25、spurious copy number changes in array-CGH profiles 2. These modifications of the DNA molecules can impact the validity and reliability of the analytical test results. Therefore, it is essential to take special measures to minimize the described modifications for subsequent DNA analysis. 2 Normative
26、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 amendments) appli
27、es. 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 EN ISO 15189:2012 a
28、nd 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 ischemia condition
29、after removal of the tissue from the body until its stabilization or fixation 3.4 DNA deoxyribonucleic acid polymer of deoxyribonucleotides occurring in a double-stranded (dsDNA) or single-stranded (ssDNA) form SOURCE: EN ISO 22174:2005, 3.1.2 3.5 FFPE formalin fixation and paraffin embedding PD CEN
30、/TS 16827-3:2015 CEN/TS 16827-3:2015 (E) 6 3.6 FFPE tissues formalin fixed and paraffin embedded tissues 3.7 formalin saturated formaldehyde solution containing a mas fraction of 37 % (corresponding to a volume fraction of 40 %) formaldehyde, termed 100 % formalin 3.8 formalin fixation treatment of
31、a sample with standard buffered formalin solution for stabilization 3.9 pre-examination processes preanalytical phase preanalytical workflow processes that start, in chronological order, from the clinicians request and include the examination request, preparation and identification of the patient, s
32、urgical procedure, collection of the primary sample(s), temporary storage, transportation to and within the analytical 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
33、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. 3.10 primary sample specimen discrete portion of a body fluid, breath, hair or tissue taken for examination, study or analysis
34、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. 3.11 room temperature temperature which is defined as 18 C to 25 C for the purposes of this document 3.12 sample one or mo
35、re parts taken from a primary sample SOURCE: EN ISO 15189:2012, 3.24, modified The example was not taken over. 3.13 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 G
36、uide 30:1992, 2.7 Note 1 to entry: The measured constituent for the purpose of this document is DNA. PD CEN/TS 16827-3:2015 CEN/TS 16827-3:2015 (E) 7 3.14 standard buffered formalin solution 10 % formalin solution containing a mass fraction of 3,7 % (corresponding to a volume fraction of 4 %) formal
37、dehyde buffered to pH 6,8 to pH 7,2 Note 1 to entry: Standard buffered formalin solutions often contain methanol to inhibit oxidation and polymerization of formaldehyde. 3.15 warm ischemia warm Ischemia is the condition where the tissue is deprived of its normal blood supply containing oxygen and nu
38、trients while the tissue is at body temperature 4 General considerations For general 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
39、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 workflow can influence the final analytical performance, the entire workflow comprising the preanalytical steps, including information on biomolecule stability and storage conditions, and an
40、alytical steps should be verified and validated (see EN ISO 15189). For samples intended to be analysed for DNA, the following specific aspects shall be considered. In contrast to RNA or proteins, DNA in tissue is relatively stable during warm and cold ischemia times. Changes of DNA, sequence or cop
41、y numbers (e.g., CGH profiles,) due to an extended duration of warm and cold ischemia are unknown. The duration until the specimen is placed into standard buffered formalin solution should be kept as short as possible in order to avoid enzymatic degradation of DNA. The duration before fixation shall
42、 be documented and the temperature before fixation should be documented 3. During the fixation, processing and storage, the DNA integrity can change depending on the kind of fixative, fixation time and temperature, storage or archiving of the fixed paraffin embedded sample as well as the method used
43、 for DNA isolation and purification. When using a fixative based on formaldehyde, temperature, and fixation duration have a significant impact on DNA integrity. The longer the fixation duration and the higher the temperature, the more chemical modifications and crosslinks are introduced, which can l
44、ead to degradation or sequence alterations 1, 2, 4, 5. Safety regulations on specimen 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 precautions shall be taken to avoid cross contamination between different s
45、amples. If a commercial product is not used in 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 includ
46、e, but is not limited to: a) the primary donor / patient ID, which can be in the form of a code; PD CEN/TS 16827-3:2015 CEN/TS 16827-3:2015 (E) 8 b) the health status of the primary sample donor (e.g., healthy, disease type, concomitant disease); c) the information about routine medical treatment an
47、d special treatment prior to tissue collection (e.g., anaesthetics, medications, surgical or diagnostic procedures (e.g., biopsy device used for the collection); d) the start of ischemia within the body (warm ischemia) by documenting the ischemia-relevant vessel ligation/clamping time point (usually
48、 arterial clamping time). 5.1.2 Information on the primary tissue sample The documentation shall include, but is not limited to: a) the time point when tissue is removed from the body; b) the description of tissue type, tissue condition (e.g., diseased, unaffected by the disease) and organ tissue of
49、 origin, including references to any marking applied in the operating theatre made by surgeon, radiologist or pathologist; c) the documentation steps described under 6.2, if the formalin fixation starts outside the laboratory. 5.1.3 Information on the primary tissue sample processing The following steps shall be performed: 1. the documentation of any additions or modifications to the primary sample after removal from the body (e.g., labelling for the orientation of the specimen (e.g., ink-marking, stitches), incision(s); 2. the selec
