1、BSI Standards Publication Molecular in vitro diagnostic examinations Specifications for pre-examination processes for venous whole blood Part 3: Isolated circulating cell free DNA from plasma PD CEN/TS 16835-3:2015National foreword This Published Document is the UK implementation of CEN/TS 16835- 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 85027 1 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
4、Standards Policy and Strategy Committee on 31 October 2015. Amendments/corrigenda issued since publication Date Text affected PUBLISHED DOCUMENT PD CEN/TS 16835-3:2015 TECHNICAL SPECIFICATION SPCIFICATION TECHNIQUE TECHNISCHE SPEZIFIKATION CEN/TS 16835-3 O c t o b e r 2 0 1 5 ICS 11.100.30 English V
5、ersion Molecular in vitro diagnostic 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 -
6、 Partie 3: ADN libre circulant 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
7、 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 requested to submit their comments, particularly on the question whether the CEN/TS can be converted into a European Standard. CEN members are requ
8、ired 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. It is permissible to keep conflicting national standards in force (in parallel to the CEN/TS) until the final decision about the possible con
9、version of the CEN/TS into an EN 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, L
10、uxembourg, Malta, Netherlands, Norway, Poland, Portugal, Romania, Slovakia, Slovenia, Spain, 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 Br
11、ussels 2015 CEN All rights of exploitation in any form and by any means reserved worldwide for CEN national Members. Ref. No. CEN/TS 16835-3:2015 E PD 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 defini
12、tions . 5 4 General considerations . 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 t
13、he patient and stabilization procedures . 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
14、6.3 Plasma preparation . 10 6.4 Storage 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 d
15、istribution pattern in plasma samples . 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. Atten
16、tion 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 standards organizations of
17、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, Luxembourg, Malta,
18、Netherlands, Norway, Poland, Portugal, 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 n
19、ew 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 diagnostics or research
20、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 circulating cell free DNA
21、 (ccfDNA) analysis is needed. Studies 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 referr
22、ed to as the preanalytical phase. 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
23、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, in vitro diagnostics
24、developers and manufacturers, institutions 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 fragmentatio
25、n and ccfDNA quantity change). Special 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
26、 in CEN/TS 16835-2, Molecular in 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 pre
27、sent in blood is not covered by this 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
28、latest edition of the referenced 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 purpose
29、s of this document, the terms 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 kind of parameter testing or chemical manipulat
30、ion for quantitative or qualitative 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 circulat
31、ing cell free DNA profile/s amounts 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 deoxyr
32、ibonucleotides occurring in a double-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 reques
33、t, preparation and identification 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
34、 additional term was 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. 3.8 primary sample specimen discrete portion of a body fluid, breath, hair or tissue taken for examination
35、, 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 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.
36、10 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 30:2015, 2.1.15, modified The words “reference material” were replaced by “sample material“. Note 1 to entry:
37、 The measured constituent for the 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. Consumab
38、les 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 all steps of a diagnostic workflow can influence the final analytical performance, the entire workflow comprising the preanalytical steps, includ
39、ing information on sample stability 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
40、change the ccfDNA profile significantly. 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 condi
41、tions 5 9, 10, 11. The stability 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 st
42、orage. Before or during the design 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 accordan
43、ce with the manufacturers instructions, 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 veno
44、us 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 code; b) the health status and relevant lifestyle factors of the blood donor (e.g. healthy, gend
45、er, age, disease type, gestational 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 disorder
46、s as pre-term labour, pre-eclampsia 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
47、 purpose of the proposed analytical 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
48、inappropriate storage/shipping conditions, 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 who
49、le blood should be collected in appropriate 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 leucocyt
