1、BSI Standards PublicationMolecular in vitro diagnosticexaminations Specifications for pre-examination processes for venous whole bloodPart 2: Isolated genomic DNAPD CEN/TS 16835-2:2015National forewordThis Published Document is the UK implementation of CEN/TS 16835-2:2015.The UK participation in its
2、 preparation was entrusted to TechnicalCommittee CH/212, IVDs.A list of organizations represented on this committee can be obtained onrequest to its secretary.This publication does not purport to include all the necessary provisions ofa contract. Users are responsible for its correct application. Th
3、e British Standards Institution 2015.Published by BSI Standards Limited 2015ISBN 978 0 580 85033 2ICS 11.100.30Compliance with a British Standard cannot confer immunity fromlegal obligations.This Published Document was published under the authority of theStandards Policy and Strategy Committee on 31
4、 October 2015. Amendments/corrigenda issued since publicationDate Text affectedPUBLISHED DOCUMENTPD CEN/TS 16835-2:2015TECHNICAL SPECIFICATION SPCIFICATION TECHNIQUE TECHNISCHE SPEZIFIKATION CEN/TS 16835-2 October 2015 ICS 11.100.30 English Version Molecular in vitro diagnostic examinations - Specif
5、ications 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 in-vitro-diagnostische Verfahre
6、n - 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 to three years. After two years
7、 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 CEN/TS available promptly at natio
8、nal 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 Austria, Belgium, Bulgaria, Cro
9、atia, 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, Sweden, Switzerland, Turkey andU
10、nited 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 for CEN national Members. Ref. No.
11、CEN/TS 16835-2:2015 EPD 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 blood collection manual . 7 5.1.1 In
12、formation 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 sample and storage requirements at
13、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 laboratories own protocols 12 6.4 Quantity
14、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 Influence of preanalytical variable
15、s (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 Influence of blood storage conditio
16、ns 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 devices”, the secretariat of which i
17、s 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 Regulations, the national standar
18、ds 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, Ireland, Italy, Latvia, Lithuania,
19、 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 progress in medicine. Further progr
20、ess 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 thus making the outcome from diag
21、nostics 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 sample collection to genomic DNA anal
22、ysis 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 whole blood genomic DNA analysis in wh
23、at 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 preanalytical phase before a molecular
24、 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, in vitro diagnostics developers
25、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 blood samples for genomic DNA anal
26、ysis. 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 cell free DNA in blood is covered
27、 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, transporting and storing capillary blo
28、od 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 whole or in part, are normatively
29、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 laboratories Requirements for quality and
30、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 temperature unregulated temperature o
31、f 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 that are made to minimize degradati
32、on 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, 3.1.2 3.5 genomic DNA DNA from the gen
33、ome 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 than 50 kb for the purpose of this do
34、cument 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 sample(s), temporary storage, transp
35、ortation 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: The preanalytical phase may include
36、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 for the whole SOURCE: EN ISO 15189:
37、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 sample material, when stored under spec
38、ified 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 the purpose of this document is gen
39、omic 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 15189:2012, 5.3.2.3); EN ISO 1518
40、9:2012, 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 analytical steps should be verified and
41、 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 amount and size required for the analyt
42、ical 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, transport and handling shall be consider
43、ed (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 ID, which can be in the form of a
44、 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 purpose of the analytical test req
45、uested. 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 collection procedures, inappropriate stor
46、age/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 containing EDTA as an anticoagulant are
47、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 DNA eluate can cause inhibitions
48、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 whole blood sample collection from the
49、 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 required. The manufacturers inst
