1、BSI Standards Publication Molecular in vitro diagnostic examinations Specifications for pre-examination processes for venous whole blood Part 2: Isolated genomic DNA PD CEN/TS 16835-2:2015National foreword This Published Document is the UK implementation of CEN/TS 16835- 2:2015. The UK participation
2、 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 responsible for its correct app
3、lication. The British Standards Institution 2015. Published by BSI Standards Limited 2015 ISBN 978 0 580 85033 2 ICS 11.100.30 Compliance with a British Standard cannot confer immunity from legal obligations. This Published Document was published under the authority of the Standards Policy and Strat
4、egy Committee on 31 October 2015. Amendments/corrigenda issued since publication Date Text affected PUBLISHED DOCUMENT PD CEN/TS 16835-2:2015 TECHNICAL SPECIFICATION SPCIFICATION TECHNIQUE TECHNISCHE SPEZIFIKATION CEN/TS 16835-2 O c t o b e r 2 0 1 5 ICS 11.100.30 English Version Molecular in vitro
5、diagnostic examinations - Specifications for pre-examination processes for venous whole blood - Part 2: Isolated genomic DNA Tests de diagnostic molculaire in vitro - Spcifications relatives aux processus pr-analytiques pour le sang total veineux - Partie 2: ADN gnomique extrait Molekularanalytische
6、 in-vitro-diagnostische Verfahren - Spezifikationen fr pranalytische Prozesse fr vense Vollblutproben - Teil 2: Isolierte genomische DNS This Technical Specification (CEN/TS) was approved by CEN on 31 August 2015 for provisional application. The period of validity of this CEN/TS is limited initially
7、 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 announce the existence of this CEN/TS in the same way as for an EN and to make the CE
8、N/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 the CEN/TS into an EN is reached. CEN members are the national standards bodies of
9、 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, Spain,
10、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 2015 CEN All rights of exploitation in any form and by any means reserved worldwide fo
11、r CEN national Members. Ref. No. CEN/TS 16835-2:2015 E PD CEN/TS 16835-2:2015CEN/TS 16835-2:2015 (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 7 5.1 Primary venous whole bl
12、ood collection manual . 7 5.1.1 Information about the primary sample donor . 7 5.1.2 Selection of the venous whole blood collection tube by the laboratory . 8 5.1.3 Primary venous whole blood sample collection from the patient and stabilization procedures . 8 5.1.4 Information on the primary blood s
13、ample and storage requirements at the blood collection facility . 8 5.2 Transport requirements. 9 6 Inside the laboratory 10 6.1 Primary sample reception 10 6.2 Storage requirements . 10 6.3 Isolation of the genomic DNA . 11 6.3.1 General . 11 6.3.2 Using commercial kits 12 6.3.3 Using the laborator
14、ies own protocols 12 6.4 Quantity and quality assessment of isolated genomic DNA 12 6.5 Storage of isolated genomic DNA 13 Annex A (informative) Impact of preanalytical workflow steps on venous whole blood genomic DNA quality . 14 A.1 General information on operated experiments in Annex A . 14 A.2 I
15、nfluence of preanalytical variables (blood storage duration and temperature, and DNA isolation methods) on genomic DNA integrity . 14 A.3 Influence of blood storage time on the genomic DNA integrity. 15 A.4 Influence of genomic DNA integrity on an analytical test based on long PCR amplicons 17 A.5 I
16、nfluence of blood storage conditions on the performance of PCR tests based on short amplicons 18 Bibliography . 20 PD CEN/TS 16835-2:2015CEN/TS 16835-2:2015 (E) 3 European foreword This document (CEN/TS 16835-2:2015) has been prepared by Technical Committee CEN/TC 140 “In vitro diagnostic medical de
17、vices”, 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 such patent rights. According to the CEN-CENELEC Internal
18、 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 of Macedonia, France, Germany, Greece, Hungary, Iceland,
19、Ireland, Italy, Latvia, Lithuania, Luxembourg, Malta, Netherlands, Norway, Poland, Portugal, Romania, Slovakia, Slovenia, Spain, Sweden, Switzerland, Turkey and the United Kingdom. PD CEN/TS 16835-2:2015CEN/TS 16835-2:2015 (E) 4 Introduction Molecular in vitro diagnostics has enabled a significant p
20、rogress 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 of these molecules can change drastically during primary sample collection, transport, storage and processing
21、 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 profile generated during the pre-examination process. A standardization of the entire process from primary sam
22、ple collection to genomic DNA analysis is needed due to genomic DNA degradation and fragmentation after blood collection. Studies have been undertaken to determine the important influencing factors. This Technical Specification draws upon such work to codify and standardize the steps for venous whol
23、e blood genomic DNA analysis in what is referred to as the preanalytical phase. PD CEN/TS 16835-2:2015CEN/TS 16835-2:2015 (E) 5 1 Scope This Technical Specification recommends the handling, documentation and processing of venous whole blood specimens intended for genomic DNA analysis during the prea
24、nalytical phase before a molecular assay is performed. This Technical Specification covers specimens collected by venous whole blood collection tubes. This Technical Specification is applicable to molecular in vitro diagnostic examinations (e.g. in vitro diagnostic laboratories, laboratory customers
25、, in vitro diagnostics developers and manufacturers, institutions and commercial organizations performing biomedical research, biobanks, and regulatory authorities). Blood genomic DNA can fragment or degrade after blood collection. Therefore, special measures need to be taken to secure good quality
26、blood samples for genomic DNA analysis. This is particularly relevant for analytical test procedures requiring high molecular weight DNA. Different dedicated measures need to be taken for preserving blood circulating cell free DNA, which are not described in this Technical Specification. Circulating
27、 cell free DNA in blood is covered in CEN/TS 16835-3, Molecular in vitro diagnostic examinations Specifications for pre-examination processes for venous whole blood Part 3: Isolated circulating cell free DNA from plasma. Different dedicated measures need to be taken for collecting, stabilizing, tran
28、sporting and storing capillary blood as well as for blood collected and stored by paper based technologies. These are not described in this Technical Specification. DNA from pathogens present in blood is not covered by this Technical Specification. 2 Normative references The following documents, in
29、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) applies. EN ISO 15189:2012, Medical laborato
30、ries 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 and the following apply. 3.1 ambient tem
31、perature unregulated temperature of the surrounding air 3.2 analytical phase processes that start with the isolated analyte and include all kind of parameter testing or chemical manipulation for quantitative or qualitative analysis 3.3 blood genomic DNA stabilizers compounds, solutions or mixtures t
32、hat are made to minimize degradation and fragmentation of genomic DNA in a blood sample PD CEN/TS 16835-2:2015CEN/TS 16835-2:2015 (E) 6 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.
33、2 3.5 genomic DNA DNA from the genome containing all coding (exon) and non-coding (intron and other) sequences Note 1 to entry: In this document it is always only referred to genomic DNA present in blood cells, excluding circulating cell free DNA. 3.6 high molecular weight DNA HMW DNA DNA larger tha
34、n 50 kb for the purpose of this document 3.7 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, collection of the primary sa
35、mple(s), temporary storage, transportation to and within the analytical laboratory, aliquotting, retrieval, isolation of analytes, and end when the analytical examination begins SOURCE: EN ISO 15189:2012, 3.15, modified An additional term was added and more details were included. Note 1 to entry: Th
36、e preanalytical phase may include preparative processes that may influence the outcome of the intended examination. 3.8 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 f
37、or the whole SOURCE: EN ISO 15189:2012, 3.16, modified The term and definition is used here without the original notes. 3.9 room temperature temperature which is defined as 18 C to 25 C for the purpose of this document PD CEN/TS 16835-2:2015CEN/TS 16835-2:2015 (E) 7 3.10 stability ability of a sampl
38、e material, when stored under specified conditions, to maintain a stated property value within specified limits for a specified period of time SOURCE ISO Guide 30:2015, 2.1.15, modified The words “reference material” were replaced by “sample material“. Note 1 to entry: The measured constituent for t
39、he purpose of this document is genomic DNA. 4 General considerations For general statements on primary sample collection and handling (including avoidance of cross contaminations), see EN ISO 15189:2012, 5.2.6, 5.4.4. Consumables including kits shall be verified before use in examination (see EN ISO
40、 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 sample stability and storage conditions, and the analy
41、tical steps should be verified and validated (see EN ISO 15189). The stability of the genomic DNA should be investigated throughout the complete pre-analytical workflow. Before or during the design of the analytical test system it should be investigated and ensured that the genomic DNA minimum amoun
42、t and size required for the analytical test are not affected by the envisioned entire preanalytical workflow. If a commercial product is not used in accordance with the manufacturers instructions, responsibility for its use and performance lies with the user. Safety regulations on facilities, transp
43、ort and handling shall be considered (EN ISO 15189:2012, 5.2.3 and 5.4.5, and ISO 15190). 5 Outside the laboratory 5.1 Primary venous whole blood 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
44、 ID, which can be in the form of a code; b) the health status and relevant lifestyle factors of the blood donor (e.g. healthy, disease type, gender, age); c) the information about medical treatment and special treatment prior to blood collection (e.g. anaesthetics, medications); d) the type and the
45、purpose of the analytical test requested. See also EN ISO 15189:2012, 5.4.4. PD CEN/TS 16835-2:2015CEN/TS 16835-2:2015 (E) 8 5.1.2 Selection of the venous whole blood collection tube by the laboratory The quality of genomic DNA can be influenced (e.g., DNA fragmentation), by inadequate blood collect
46、ion procedures, inappropriate storage/shipping conditions and DNA isolation procedures, 3, 4, 5, 6, 7, 8, 9, 10. Blood should be collected in appropriate venous whole blood collection tubes containing an anticoagulant such as EDTA or Acid Citrate Dextrose (ACD) 11. NOTE Blood collection tubes contai
47、ning EDTA as an anticoagulant are preferable for most genomic DNA analysis. Blood collection tubes containing heparin as an anticoagulant can impact the purity of the isolated genomic DNA, when using genomic DNA isolation methods not eliminating the heparin. Carrying over of heparin into the genomic
48、 DNA eluate can cause inhibitions in analytical test technologies, such as PCR. Specifically developed blood collection tubes, containing genomic DNA stabilizing reagents, are also available aimed to standardize blood collection, transport and storage of venous whole blood. 5.1.3 Primary venous whol
49、e blood sample collection from the patient and stabilization procedures 1. The identity of the person collecting the primary sample and the time of blood collection according to EN ISO 15189:2012, 5.4.4.3, f) shall be documented. 2. For the labelling (sample identification) of the blood collection tube a routine procedure (EN ISO 15189:2012, 5.4.4.3, e) or a procedure with additional information (e.g. 2D-barcode) shall be used. 3. Standard venepuncture technique can be used. Steps for preventing possible backflow may be req
