1、BSI Standards PublicationMolecular in vitro diagnosticexaminations Specifications for pre-examination processes for venous whole bloodPart 3: Isolated circulating cell free DNA from plasmaPD CEN/TS 16835-3:2015National forewordThis Published Document is the UK implementation of CEN/TS 16835-3:2015.T
2、he UK participation in its 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 i
3、ts correct application. The British Standards Institution 2015.Published by BSI Standards Limited 2015ISBN 978 0 580 85027 1ICS 11.100.30 Compliance with a British Standard cannot confer immunity fromlegal obligations.This Published Document was published under the authority of theStandards Policy a
4、nd Strategy Committee on 31 October 2015. Amendments/corrigenda issued since publicationDate Text affectedPUBLISHED DOCUMENTPD CEN/TS 16835-3:2015TECHNICAL SPECIFICATION SPCIFICATION TECHNIQUE TECHNISCHE SPEZIFIKATION CEN/TS 16835-3 October 2015 ICS 11.100.30 English Version Molecular in vitro diagn
5、ostic examinations - Specifications for pre-examination processes for venous whole blood - Part 3: Isolated circulating cell free DNA from plasma Tests de diagnostic molculaire in vitro - Spcifications relatives aux processus pr-analytiques pour le sang total veineux - Partie 3: ADN libre circulant
6、extrait du plasma Molekularanalytische in-vitro-diagnostische Verfahren - Spezifikationen fr pranalytische Prozesse fr vense Vollblutproben - Teil 3: Aus Plasma isolierte zirkulierende zellfreie DNS This Technical Specification (CEN/TS) was approved by CEN on 31 August 2015 for provisional applicati
7、on. 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 comments, particularly on the question whether the CEN/TS can be converted into a European Standard. CEN members are required to announce the existence
8、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 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 E
9、N is reached. CEN members are the national standards bodies of 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,
10、Norway, Poland, Portugal, Romania, Slovakia, Slovenia, Spain, Sweden, Switzerland, Turkey andUnited 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 ex
11、ploitation in any form and by any means reserved worldwide for CEN national Members. Ref. No. CEN/TS 16835-3:2015 EPD CEN/TS 16835-3:2015CEN/TS 16835-3:2015 (E) 2 Contents Page European foreword . 3 Introduction 4 1 Scope 5 2 Normative references 5 3 Terms and definitions . 5 4 General consideration
12、s . 7 5 Outside the laboratory 7 5.1 Primary venous whole blood 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 collection from the patient and stabilization proc
13、edures . 8 5.1.4 Information on the primary blood sample and storage requirements at the blood collection facility . 9 5.2 Transport requirements . 9 6 Inside the laboratory 10 6.1 Primary sample reception 10 6.2 Storage requirements for venous whole blood sample 10 6.3 Plasma preparation . 10 6.4 S
14、torage requirements for plasma sample . 10 6.5 Isolation of the ccfDNA . 11 6.6 Quality assessment and quantity measurement of isolated ccfDNA . 12 6.7 Storage of isolated ccfDNA 12 Annex A (informative) Influence of isolation procedures on ccfDNA fragments lengths distribution pattern in plasma sam
15、ples . 13 Bibliography . 14 PD CEN/TS 16835-3:2015CEN/TS 16835-3:2015 (E) 3 European foreword This document (CEN/TS 16835-3:2015) 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
16、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 following countries are bound
17、 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, Norway, Poland, Port
18、ugal, Romania, Slovakia, Slovenia, Spain, Sweden, Switzerland, Turkey and the United Kingdom. PD CEN/TS 16835-3:2015CEN/TS 16835-3:2015 (E) 4 Introduction Molecular in vitro diagnostics has enabled a significant progress in medicine. Further progress is expected by new technologies analysing signatu
19、res 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 diagnostics or research unreliable or even impossible bec
20、ause 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 circulating cell free DNA (ccfDNA) analysis is needed. Stu
21、dies have been undertaken to determine the important influencing factors. This Technical Specification draws upon such work to codify and standardize the steps for circulating cell free DNA analysis from plasma prepared from human venous whole blood in what is referred to as the preanalytical phase.
22、 PD CEN/TS 16835-3:2015CEN/TS 16835-3:2015 (E) 5 1 Scope This Technical Specification recommends the handling, documentation and processing of venous whole blood specimens intended for circulating cell free DNA (ccfDNA) analysis during the preanalytical phase before a molecular assay is performed. T
23、his 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 and manufacturers, ins
24、titutions and commercial organizations performing biomedical research, biobanks, and regulatory authorities). Blood ccfDNA profiles can change significantly after blood collection from the donor (e.g. release of genomic DNA from white blood cells, ccfDNA fragmentation and ccfDNA quantity change). Sp
25、ecial measures need to be taken to secure good quality blood samples for ccfDNA analysis and storage. Different dedicated measures need to be taken for preserving blood genomic DNA. These are not described in this Technical Specification. Blood genomic DNA is covered in CEN/TS 16835-2, Molecular in
26、vitro diagnostic examinations Specifications for pre-examination processes for venous whole blood Part 2: Isolated genomic DNA NOTE CcfDNA obtained from blood by the procedures suggested in this document can contain DNA present in exosomes 3 4. DNA from pathogens present in blood is not covered by t
27、his Technical Specification. 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
28、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 terms and
29、 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 kind of parameter testing or chemical manipulation for quantitative or qualitati
30、ve analysis 3.3 ccfDNA circulating cell free DNA extracellular human DNA present in blood, serum and plasma Note 1 to entry: ccfDNA can include DNA present in vesicles such as exosomes 3 4. PD CEN/TS 16835-3:2015CEN/TS 16835-3:2015 (E) 6 3.4 ccfDNA profile/s circulating cell free DNA profile/s amoun
31、ts of different ccfDNA molecules, that are present in blood and plasma, that can be measured in the absence of any losses, inhibition and interference 3.5 cryo-precipitates insoluble residue when frozen plasma is thawed 3.6 DNA deoxyribonucleic acid polymer of deoxyribonucleotides occurring in a dou
32、ble-stranded (dsDNA) or single-stranded (ssDNA) form SOURCE: EN ISO 22174:2005, 3.1.2 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
33、 of the patient, collection of the primary sample(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 mo
34、re details were included. Note 1 to entry: The 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 mor
35、e 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. 3.9 room temperature temperature which is defined as 18 C to 25 C for the purposes of this document 3.10 stability ability of a sample
36、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 the
37、 purpose of this document is ccfDNA. PD CEN/TS 16835-3:2015CEN/TS 16835-3:2015 (E) 7 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 verif
38、ied 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 workflow can influence the final analytical performance, the entire workflow comprising the preanalytical steps, including information on sample stabili
39、ty and storage conditions, and analytical steps should be verified and validated (see EN ISO 15189). Blood circulating cell free DNA profiles can change significantly after blood collection and plasma separation. The release of genomic DNA from white blood cells can change the ccfDNA profile signifi
40、cantly. This can impact the validity of the analytical test results. Additional post-collection effects can also occur e.g. ccfDNA fragmentation 5, 6, 7, 8. These changes can vary individually in different donors / patients blood depending on pathophysiological conditions 5 9, 10, 11. The stability
41、of the specific blood ccfDNA profile of interest should be investigated throughout the complete preanalytical workflow e.g. by applying the intended analytical test in time course studies reflecting the individual preanalytical workflow steps such as transport and storage. Before or during the desig
42、n of the analytical test system, it should be investigated and ensured that the blood ccfDNA profile/s intended to be analysed in the analytical test is/are not affected by the envisioned entire pre-analytical workflow. If a commercial product is not used in accordance with the manufacturers instruc
43、tions, responsibility for its validation, verification, use and performance lies with the user. Safety regulations on facilities, transport 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
44、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 and relevant lifestyle factors of the blood donor (e.g. healthy, gender, age, disease type, gestationa
45、l age); NOTE In particular e.g. cancer, inflammation, diabetes, hepatic disease, coronary disease, respiratory syndrome, trauma, after exhaustive exercise 5, in elderly patients suffering from acute or chronic disease, first trimester of pregnancy, placental disorders as pre-term labour, pre-eclamps
46、ia and malimplantation have been reported to affect both blood ccfDNA quantity and fragmentation 5, 9, 10, 11. c) the information about medical treatment and special treatment prior to blood collection (e.g. anaesthetics, medications, fasting status); d) the type and purpose of the proposed analytic
47、al test requested. See also EN ISO 15189:2012, 5.4.4. PD CEN/TS 16835-3:2015CEN/TS 16835-3:2015 (E) 8 5.1.2 Selection of the venous whole blood collection tube by the laboratory The blood ccfDNA profile can be influenced by inadequate blood collection procedures and inappropriate storage/shipping co
48、nditions, plasma separation as well as by ccfDNA isolation procedures. Specifically, the post-collection release of genomic DNA from white blood cells can change the ccfDNA profile significantly. This can impact the validity of the analytical test results. Venous whole blood should be collected in a
49、ppropriate collection devices. Blood Collection tubes containing ccfDNA profile stabilizers are recommended when post collection release of genomic DNA from blood cells or other ccfDNA profile changes can cause impacts on the intended analytical test. If blood collection tubes without ccfDNA profile stabilizers are used, EDTA blood collection tubes should be used in preference to other collection tubes 7, 12. Induced clotting process in serum tubes can lead to a leucocytes lysis and therefore the us
