Molecular in vitro diagnostic examinations - Specifications for pre-examination processes for exosomes and other extracellular vesicles in venous whole blood - DNA, RNA and proteins

This document gives guidelines  on the handling, storage, processing and documentation of venous whole blood specimens intended for DNA, RNA and protein examination from exosomes and other extracellular vesiclesduring the pre-examination phase before a molecular examination is performed.  This document covers specimens collected in venous
whole blood collection tubes.
The pre-examination process described in this document results in isolated DNA, RNA and proteins from enriched exosomes and other extracellular vesicles.
This document is applicable to molecular in vitro diagnostic examinations performed by medical laboratories. It is also intended to be used by laboratory customers, in vitro diagnostics developers and manufacturers, biobanks, institutions and commercial organizations performing biomedical research, and regulatory authorities.
Different dedicated measures are taken during the pre-examination phase for venous whole blood circulating cell-free RNA (ccfRNA) examination and for venous whole blood circulating cell-free DNA (ccfDNA) examination, both without prior enrichment of exosomes and other extracellular vesicles. These are not described in this document but are covered in prEN ISO 20186-3, Molecular in-vitro diagnostic examinations — Specifications for pre-examination processes for venous whole blood — Part 3: Isolated circulating cell free DNA from plasma and CEN/PWI, Molecular in vitro diagnostic examinations — Specifications for pre-examination processes for venous whole blood — Isolated circulating cell free RNA from plasma.
NOTE   International, national or regional regulations or requirements can also apply to specific topics covered in this document.

Molekularanalytische in-vitro-diagnostische Verfahren - Spezifikationen für präanalytische Prozesse für Exosomen und andere extrazelluläre Vesikel im venösen Vollblut - DNA, RNA und Proteine

Dieses Dokument gibt Empfehlungen zur Handhabung, Lagerung, Verarbeitung und Dokumentation von Proben venösen Vollbluts, die für die Untersuchung von DNA, RNA und Proteinen aus Exosomen und anderen extrazellulären Vesikeln während der präanalytischen Phase vor der Durchführung einer molekularen Analyse vorgesehen sind. Dieses Dokument betrifft Untersuchungsmaterial, das mit Blutentnahmeröhrchen für venöses Vollblut entnommen wurde.
Mit dem in diesem Dokument beschriebenen präanalytischen Verfahren werden DNA, RNA und Proteine aus angereicherten Exosomen und anderen extrazellulären Vesikeln isoliert.
Dieses Dokument ist anwendbar auf molekulare in vitro-diagnostische Untersuchungen, die in medizinischen Laboratorien durchgeführt werden. Es ist darüber hinaus für die Verwendung durch Gesundheits¬einrichtungen, einschließlich Einrichtungen, die Untersuchungsmaterial entnehmen und handhaben, Laborkunden, Entwickler und Hersteller von In vitro-Diagnostika, Biobanken, in der biomedizinischen Forschung tätige Einrichtungen und kommerzielle Organisationen sowie Aufsichtsbehörden, vorgesehen.
Für die während der präanalytischen Phase durchgeführte Untersuchung von zirkulierender zellfreier RNA (ccfRNA, en: circulating cell-free RNA) und zirkulierender zellfreier DNA (ccfDNA, en: circulating cell-free DNA) aus venösem Vollblut, jeweils ohne vorherige Anreicherung von Exosomen und anderen extrazellulären Vesikeln, werden spezielle andere Maßnahmen angewendet. Diese werden nicht in diesem Dokument beschrieben, sind jedoch Gegenstand von EN ISO 20186-3, Molekularanalytische in-vitro-diagnostische Verfahren - Spezifikationen für präanalytische Prozesse für venöse Vollblutproben - Teil 3: Aus Plasma isolierte zirkulierende zellfreie DNA und CEN/TS (WI 00140134), Molecular in vitro diagnostic examinations - Specifications for pre-examination processes for venous whole blood - Isolated circulating cell free RNA from plasma.
ANMERKUNG Internationale, nationale oder regionale Regelungen bzw. Anforderungen können ebenfalls für bestimmte Themen in diesem Dokument gelten.

Analyses de diagnostic moléculaire in vitro - Spécifications relatives aux processuses préanalytiques pour exosomes et autres vésicules extracellulaires dans le sang total veineux - ADN, ARN et protéines

Molekularne diagnostične preiskave in vitro - Specifikacije za predpreiskovalne procese za eksosome in druge zunajcelične vezikle v vensko polni krvi - DNK, RNK in proteini

Ta dokument podaja smernice glede obravnave, shranjevanja, obdelave in dokumentiranja vzorcev venske polne krvi, namenjenih za analizo DNK, RNK in beljakovin iz eksosomov in drugih zunajceličnih veziklov v predpreiskovalnem procesu, preden se izvede molekularna preiskava.  Ta dokument zajema vzorce, zbrane v epruvete za odvzem polne venske krvi.
V predpreiskovalnem procesu, opisanem v tem dokumentu, se pridobijo izolirana DNK, RNK in beljakovine iz obogatenih eksosomov ter drugih zunajceličnih veziklov.
Ta dokument se uporablja za molekularne diagnostične preiskave in vitro, ki jih izvajajo v medicinskih laboratorijih. Namenjen je tudi temu, da ga uporabljajo laboratorijske stranke, razvijalci in proizvajalci diagnostike in vitro, biobanke, institucije in komercialne organizacije, ki izvajajo biomedicinske raziskave, ter regulativni organi.
Med predpreiskovalnim procesom se izvajajo različni namenski ukrepi za preiskavo cirkulirajoče brezcelične RNK (ccfRNA) polne krvi in za preiskavo cirkulirajoče brezcelične DNK (ccfDNA) venske krvi, pri obeh brez predhodne obogatitve eksosomov in drugih zunajceličnih veziklov. Ti niso opisani v tem dokumentu, ampak jih vsebuje standard prEN ISO 20186-3, Molekularne diagnostične preiskave in vitro – Specifikacije za predpreiskovalne procese za vensko polno kri – 3. del: Iz plazme izolirana cirkulirajoča brezcelična RNK in CEN/PWI, Molekularne diagnostične preiskave in vitro – Specifikacije za predpreiskovalne procese za vensko polno kri – Iz plazme izolirana cirkulirajoča brezcelična RNK.
OPOMBA:   Za določene teme, ki so zajete v tem dokumentu, lahko veljajo tudi mednarodni, nacionalni ali regionalni predpisi ali zahteve.

General Information

Status
Published
Public Enquiry End Date
23-Jan-2022
Publication Date
12-May-2022
Technical Committee
Current Stage
6060 - National Implementation/Publication (Adopted Project)
Start Date
04-May-2022
Due Date
09-Jul-2022
Completion Date
13-May-2022

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Standards Content (Sample)

SLOVENSKI STANDARD
SIST-TS CEN/TS 17747:2022
01-junij-2022
Molekularne diagnostične preiskave in vitro - Specifikacije za predpreiskovalne
procese za eksosome in druge zunajcelične vezikle v vensko polni krvi - DNK, RNK
in proteini
Molecular in vitro diagnostic examinations - Specifications for pre-examination processes
for exosomes and other extracellular vesicles in venous whole blood - DNA, RNA and
proteins
Molekularanalytische in-vitro-diagnostische Verfahren - Spezifikationen für
präanalytische Prozesse für Exosomen und andere extrazelluläre Vesikel im venösen
Vollblut - DNA, RNA und Proteine
Analyses de diagnostic moléculaire in vitro - Spécifications relatives aux processuses
préanalytiques pour exosomes et autres vésicules extracellulaires dans le sang total
veineux - ADN, ARN et protéines
Ta slovenski standard je istoveten z: CEN/TS 17747:2022
ICS:
11.100.10 Diagnostični preskusni In vitro diagnostic test
sistemi in vitro systems
SIST-TS CEN/TS 17747:2022 en,fr,de
2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.

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SIST-TS CEN/TS 17747:2022


CEN/TS 17747
TECHNICAL SPECIFICATION

SPÉCIFICATION TECHNIQUE

April 2022
TECHNISCHE SPEZIFIKATION
ICS 11.100.10
English Version

Molecular in vitro diagnostic examinations - Specifications
for pre-examination processes for exosomes and other
extracellular vesicles in venous whole blood - DNA, RNA
and proteins
Analyses de diagnostic moléculaire in vitro - Molekularanalytische in-vitro-diagnostische Verfahren
Spécifications relatives aux processuses préanalytiques - Spezifikationen für präanalytische Prozesse für
pour exosomes et autres vésicules extracellulaires Exosomen und andere extrazelluläre Vesikel im
dans le sang total veineux - ADN, ARN et protéines venösen Vollblut - DNA, RNA und Proteine
This Technical Specification (CEN/TS) was approved by CEN on 13 March 2022 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 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. 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, Croatia, Cyprus, Czech Republic, Denmark, Estonia,
Finland, France, Germany, Greece, Hungary, Iceland, Ireland, Italy, Latvia, Lithuania, Luxembourg, Malta, Netherlands, Norway,
Poland, Portugal, Republic of North Macedonia, Romania, Serbia, Slovakia, Slovenia, Spain, Sweden, Switzerland, Turkey and
United Kingdom.





EUROPEAN COMMITTEE FOR STANDARDIZATION
COMITÉ EUROPÉEN DE NORMALISATION

EUROPÄISCHES KOMITEE FÜR NORMUNG

CEN-CENELEC Management Centre: Rue de la Science 23, B-1040 Brussels
© 2022 CEN All rights of exploitation in any form and by any means reserved Ref. No. CEN/TS 17747:2022 E
worldwide for CEN national Members.

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Contents Page
European foreword . 4
Introduction . 4
1 Scope . 6
2 Normative references . 6
3 Terms and definitions . 7
4 General Requirements . 11
5 Outside the laboratory . 13
5.1 Specimen collection . 13
5.1.1 General . 13
5.1.2 Information about the patient/specimen donor . 13
5.1.3 Selection of the venous whole blood collection tube by the laboratory . 13
5.1.4 Venous whole blood specimen collection from the patient/donor . 14
5.2 Specimen storage and transport . 15
5.2.1 General . 15
5.2.2 Storage and transport using blood collection tubes with stabilizers . 15
5.2.3 Storage and transport using blood collection tubes without stabilizers . 15
6 Inside the laboratory . 16
6.1 Specimen reception . 16
6.2 Specimen storage after transport and reception . 16
6.3 Plasma preparation . 16
6.4 Storage requirements for plasma samples . 17
6.5 Enrichment of EVs from specimen . 17
6.5.1 General . 17
6.5.2 Using a commercial EV enrichment system . 18
6.5.3 Using the laboratory’s own EV enrichment procedure . 18
6.5.4 Quality of enriched EVs . 18
6.5.5 Storage of enriched EVs . 19
6.6 Isolation of analyte of interest from EVs . 19
6.6.1 General . 19
6.6.2 Using a commercial kit for EV DNA, RNA, protein isolation intended for diagnostic
use . 19
6.6.3 Using a laboratory developed isolation procedure . 20
6.7 Quantity and quality assessment of isolated analyte of interest . 21
6.7.1 General . 21
6.7.2 Quantity assessment of EV DNA and RNA . 21
6.7.3 Quality assessment of DNA and RNA . 22
6.7.4 Quantity and quality assessment of EV proteins . 22
6.8 Storage of isolated analyte of interest . 23
6.8.1 General . 23
6.8.2 Storage of DNA . 23
6.8.3 Storage of RNA . 24
6.8.4 Storage of EV protein . 25
Annex A (informative) Example of a typical protocol for plasma preparation for EV
enrichment from unstabilized blood . 26
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Annex B (informative) Overview of different enrichment procedures . 27
B.1 General . 27
B.2 EV enrichment by centrifugation . 27
B.3 EV enrichment by filtration . 27
B.4 EV enrichment by chromatography . 28
B.5 EV enrichment by precipitation . 28
B.6 EV enrichment by immunoaffinity . 28
B.7 EV enrichment by microfluidics . 28
Bibliography . 29
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European foreword
This document (CEN/TS 17747:2022) 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 document may be the subject of
patent rights. CEN shall not be held responsible for identifying any or all such patent rights.
Any feedback and questions on this document should be directed to the users’ national standards body.
A complete listing of these bodies can be found on the CEN website.
According to the CEN/CENELEC Internal Regulations, the national standards organisations of the
following countries are bound to announce this Technical Specification: Austria, Belgium, Bulgaria,
Croatia, Cyprus, Czech Republic, Denmark, Estonia, Finland, France, Germany, Greece, Hungary, Iceland,
Ireland, Italy, Latvia, Lithuania, Luxembourg, Malta, Netherlands, Norway, Poland, Portugal, Republic of
North Macedonia, Romania, Serbia, Slovakia, Slovenia, Spain, Sweden, Switzerland, Turkey and the United
Kingdom.
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Introduction
Molecular in vitro diagnostics has enabled a significant progress in medicine. Further progress is expected
by new technologies analysing profiles of nucleic acids, proteins, and metabolites in human tissues and
body fluids. However, the profiles of these molecules can change drastically during the pre-examination
process, including the specimen collection, transport, storage and processing.
Consequently, this makes the outcome from diagnostics or research unreliable or even impossible
because the subsequent examination might not determine the real situation in the patient but an artificial
profile generated during the pre-examination process.
Besides cell free circulating nucleic acids, circulating tumour cells (CTCs) and other rare cells, exosomes
and other extracellular vesicles represent another key component of liquid biopsies. Therefore, there is
a strongly increasing interest in research and emerging diagnostics in exosomes and other extracellular
vesicles.
The pre-examination process described in this document results in enriched extracellular vesicles (EV)
(e.g. exosomes) or DNA, RNA and proteins isolated therefrom.
New additional extracellular vesicles can be released and existing extracellular vesicles can be lost after
blood collection, thus changing the overall EV DNA/RNA/protein profiles. Also, different anticoagulants
in different types of blood collection tubes can influence the release of EVs from different cells present in
blood, including those from platelets. Further factors can influence the post collection changes of the
entire blood EV composition, such as storage and transport temperature and duration, centrifugation
parameters, etc.
Standardization of the entire workflow from the specimen collection to the EV surface protein and
isolated DNA, RNA and protein examination from EVs is therefore needed. Studies have been undertaken
to determine the important influencing factors. This document draws upon such work to codify and
standardize the steps of EV surface protein examination and of DNA, RNA and protein examination from
EVs in what is referred to as the pre-examination phase.
In this document, the following verbal forms are used:
— “shall” indicates a requirement;
— “should” indicates a recommendation;
— “may” indicates a permission;
— “can” indicates a possibility or a capability.
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1 Scope
This document gives guidelines on the handling, storage, processing and documentation of venous whole
blood specimens intended for DNA, RNA and protein examination from exosomes and other extracellular
vesicles during the pre-examination phase before a molecular examination is performed. This document
covers specimens collected in venous whole blood collection tubes.
The pre-examination process described in this document results in isolated DNA, RNA and proteins from
enriched exosomes and other extracellular vesicles.
This document is applicable to molecular in vitro diagnostic examinations performed by medical
laboratories. It is also intended to be used by health care institutions including facilities collecting and
handling specimen, laboratory customers, in vitro diagnostics developers and manufacturers, biobanks,
institutions and commercial organizations performing biomedical research, and regulatory authorities.
Different dedicated measures are taken during the pre-examination phase for venous whole blood
circulating cell-free RNA (ccfRNA) examination and for venous whole blood circulating cell-free DNA
(ccfDNA) examination, both without prior enrichment of exosomes and other extracellular vesicles.
These are not described in this document but are covered in EN ISO 20186-3, Molecular in vitro diagnostic
examinations - Specifications for pre-examination processes for venous whole blood - Part 3: Isolated
circulating cell free DNA from plasma and CEN/TS 17742, Molecular in vitro diagnostic examinations -
Specifications for pre-examination processes for venous whole blood - Isolated circulating cell free RNA from
plasma.
NOTE International, national or regional regulations or requirements can also apply to specific topics covered
in this document.
2 Normative references
The following documents are referred to in the text in such a way that some or all of their content
constitutes requirements of this document. 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, Medical laboratories - Requirements for quality and competence (ISO 15189)
ISO 15190, Medical laboratories — Requirements for safety
ISO/TS 20658, Medical laboratories — Requirements for collection, transport, receipt, and handling of
samples
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3 Terms and definitions
For the purposes of this document, the terms and definitions given in EN ISO 15189 and the following
apply.
ISO and IEC maintain terminological databases for use in standardization at the following addresses:
— ISO Online browsing platform: available at https://www.iso.org/obp
— IEC Electropedia: available at https://www.electropedia.org/
3.1
aliquot
portion of a larger amount of homogenous material, assumed to be taken with negligible sampling error
Note 1 to entry: The term is usually applied to fluids. Tissues are heterogeneous and therefore cannot be
aliquoted.
Note 2 to entry: The definition is derived from the Compendium of Chemical Terminology Gold Book.
International Union of Pure and Applied Chemistry. Version 2.3.3., 2014; the PAC, 1990,62,1193 (Nomenclature for
sampling in analytical chemistry (Recommendations 1990)) p. 1206; and the PAC 1990, 62, 2167 (Glossary of
atmospheric chemistry terms (Recommendations 1990)) p. 2173.
3.2
analyte
component represented in the name of a measurable quantity
[SOURCE: ISO 17511:2020, 3.2 — Deleted Example.]
3.3
blood collection set
intravenous device specialized for venipuncture consisting of a stainless steel beveled needle and tube
(tubing) with attached plastic wings and fitting connector
Note 1 to entry: The connector attaches to an additional blood collection device, e.g. a blood collection tube.
3.4
blood collection tube
tube used for blood collection, usually with a vacuum which forces blood from the vein through the needle
into the tube
3.5
closed system
non-modifiable system provided by the vendor including all necessary components for the examination
(i.e. hardware, software, procedures and reagents)
3.6
deoxyribonucleic acid
DNA
polymer of deoxyribonucleotides occurring in a double-stranded (dsDNA) or single-stranded (ssDNA)
form
[SOURCE: EN ISO 22174:2005, 3.1.2]
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3.7
deoxyribonuclease
DNase
enzyme that catalyzes the degradation of DNA into smaller components
3.8
examination
analytical test
set of operations having the object of determining the value or characteristics of a property
Note 1 to entry: Processes (i.e. set of operations) that start with the isolated analyte and include all kinds of
parameter testing or chemical manipulation for quantitative or qualitative examination.
[SOURCE: EN ISO 15189:2012, 3.7, modified — Term and definition are used here without the original
notes; an additional term was added.]
3.9
examination manufacturer
analytical test manufacturer
entity that manufactures and/or produces a specific analytical test
Note 1 to entry: For the purpose of this document, an EV DNA, RNA and protein examination manufacturer is meant.
3.10
examination performance
analytical test performance
analytical performance
accuracy, precision, and sensitivity of a test to measure the analyte of interest
Note 1 to entry: Other test performance characteristics such as robustness, repeatability can apply as well.
[SOURCE: EN ISO 20184-1:2018, 3.4]
3.11
extracellular vesicle
EV
particle naturally released from the cell that are delimited by a lipid bilayer and cannot replicate, i.e. does
not contain a functional nucleus
EXAMPLE Exosomes, endosomes, oncosomes, apoptotic bodies
[SOURCE: [1]]
3.12
EV stabilizer
chemical formulation that increases the stability of EVs, including the overall EV populations, their cargo
(e.g. DNA, RNA and proteins) and EV surface proteins in a specimen or sample
3.13
interfering substance
endogenous or exogenous substances in clinical specimens/samples that can alter an examination result
Note 1 to entry: Examples of endogenous substances are blood components and acidic polysaccharides.
Note 2 to entry: Examples of exogenous substances are talc and anticoagulant.
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3.14
needle holder
barrel used in routine venepuncture procedures to hold the blood collection tube in place and to protect
the phlebotomist from direct contact with blood
Note 1 to entry: Examples of endogenous substances are blood components and acidic polysaccharides.
Note 2 to entry: Examples of exogenous substances are talc and anticoagulant.
3.15
pre-examination processes
preanalytical phase
preanalytical workflow
pre-examination phase
processes that start, in chronological order, from the clinician’s request and include the examination
request, preparation and identification of the patient, collection of the primary sample(s), transportation
to and within the medical laboratory, storage, isolation of analytes, and end when the analytical
examination begins
Note 1 to entry: The pre-examination phase includes preparative processes, e.g. RNA isolation procedures, which
influence the outcome of the intended examination.
[SOURCE: EN ISO 15189:2012, 3.15, modified — An additional term was added and more detail was
included.]
3.16
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 15189:2012, 3.16, modified — The term and definition are used here without the
original notes.]
3.17
proficiency testing
evaluation of participant performance against pre-established criteria by means of interlaboratory
comparisons
[SOURCE: EN ISO/IEC 17043:2010, 3.7, modified — Term and definition are used here without the
original notes.]
3.18
protein
type of biological macromolecules composed of one or more chains with a defined sequence of amino
acids connected through peptide bonds
3.19
ribonucleic acid
RNA
polymer of ribonucleotides occurring in a double-stranded or single-stranded form
[SOURCE: EN ISO 22174:2005, 3.1.3]
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3.20
ribonuclease
RNase
enzyme that catalyses the degradation of RNA into smaller components
3.21
room temperature
temperature in the range of 18 °C to 25 °C
Note 1 to entry: Local or national regulations can have different definitions.
[SOURCE: EN ISO 20166-1:2018, 3.22]
3.22
sample
one or more parts taken from a primary sample
[SOURCE: EN ISO 15189:2012, 3.24, modified — Example has been removed.]
3.23
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 phrase “reference material” has been replaced by
“sample material”.]
3.24
storage
prolonged interruption of the pre-analytical workflow of a sample or analyte respectively, or of their
derivatives, under appropriate conditions in order to preserve their properties
Note 1 to entry: Long-term storage typically occurs in laboratory archives or in biobanks.
[SOURCE: EN ISO 20184-1:2018, 3.21, modified — Example has been removed.]
3.25
validation
confirmation, through the provision of objective evidence, that the requirements for a specific intended
use or application have been fulfilled
Note 1 to entry: The term “validated” is used to designate the corresponding status.
[SOURCE: ISO 9000:2015, 3.8.13, modified — Note 1 and Note 3 have been omitted.]
3.26
venous whole blood
blood collected after directly puncturing a vein, usually with a needle and syringe, or other collection
device
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3.27
verification
confirmation, through the provision of objective evidence, that specified requirements have been fulfilled
Note 1 to entry: The term “verified” is used to designate the corresponding status.
Note 2 to entry: Confirmation can comprise activities such as:
— performing alternative calculations;
— comparing a new design specification with a similar proven design specification;
— undertaking tests and demonstrations;
— reviewing documents prior to issue.
[SOURCE: ISO 9000:2015, 3.8.12, modified — Note 1 and Note 2 have been omitted.]
3.28
workflow
series of activities necessary to complete a task
[SOURCE: EN ISO 20166-1:2018, 3.30]
4 General Requirements
For general statements on medical laboratory quality management systems and in particular on primary
sample collection, reception and handling (including avoidance of cross contaminations) see
EN ISO 15189 and EN ISO/IEC 17020 or EN ISO/IEC 17025. ISO/TS 20658 and EN ISO 20387 (for
biobanking) can also apply. The requirements on laboratory equipment, reagents, and consumables
according to EN ISO 15189 shall be followed; EN ISO/IEC 17020 and EN ISO/IEC 17025 can also apply.
All steps of the pre-examination, examination and post-examination processes (i.e. the entire workflow)
can influence the diagnosis or research study results.
Thus, this entire workflow shall be specified, verified and validated during the development of the
examination, including the development of in vitro diagnostic (IVD) medical devices. This includes
specifically all pre-examination process steps such as the examination request, preparation and
identification of the patient, collection of the primary sample(s), transport to and within the medical
laboratory, storage and isolation of analytes.
The EV profile composition can change significantly during or after blood collection, for example by
releasing additional EVs from cells and by aggregation of EVs with cells or amongst each other [1]. In
addition, enriched or isolated EVs can be lost upon storage by adhering to the surface of the storage
vessel. Thus, also the total EV DNA, RNA and protein profiles in blood and enriched fraction of EVs or
isolated EVs can change significantly after blood collection and during storage of blood and enriched or
isolated EVs. These changes can vary individually in blood from different donors or patients, and can
impact the validity and reliability of examination results.
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During the design and development of an EV DNA, RNA and protein based examination, a risk assessment
shall be performed (see also EN ISO 14971). Mitigation measures for eliminating or reducing identified
risks shall be established where required for ensuring the performance of the examination. It shall
especially be investigated and ensured that the EV DNA, RNA and protein profile(s) intended to be
analysed is/are not compromised in a manner impacting the examination performance. This includes
investigations on whether and/or how the profile intended to be examined changes during all pre-
analytical process steps. This can be done, e.g. by applying the intended examination to
specimens/samples which underwent time course studies reflecting the individual pre-examination
process steps such as transport and storage and by implementing measures to prevent or reduce impacts
by the identified pre-analytical variables, e.g. by using blood collection tubes with stabilizers.
NOTE A time course study involves repeated observations of the same variables at specific intervals over a
relevant time-period (e.g. time 0, 12 h, 24 h, 36 h, 48 h). This reflects any knowledge on the stability of the analyte(s)
of interest. Typically, this involves multiple aliquots from the same donor taken from the same blood draw repeated
for several donors.
The EV enrichment and/or isolation procedure can impact the type and purity of the EVs obtained. Also,
EV surface proteins can undergo changes during the EV enrichment procedure. The methods used for
DNA, RNA and protein isolation from EVs can also lead to profile changes of the analytes (e.g.
fragmentation of DNA, degradation of RNA, modification
...

SLOVENSKI STANDARD
kSIST-TS FprCEN/TS 17747:2022
01-januar-2022
Molekularne diagnostične preiskave in vitro - Specifikacije za predpreiskovalne
procese za eksosome in druge zunajcelične vezikle v vensko polni krvi - DNK, RNK
in proteini
Molecular in vitro diagnostic examinations - Specifications for pre-examination processes
for exosomes and other extracellular vesicles in venous whole blood - DNA, RNA and
proteins
Molekularanalytische in-vitro-diagnostische Verfahren - Spezifikationen für
präanalytische Prozesse für Exosomen und andere extrazelluläre Vesikel im venösen
Vollblut - DNA, RNA und Proteine
Analyses de diagnostic moléculaire in vitro - Spécifications relatives aux processuses
préanalytiques pour exosomes et autres vésicules extracellulaires dans le sang total
veineux - ADN, ARN et protéines
Ta slovenski standard je istoveten z: FprCEN/TS 17747
ICS:
11.100.10 Diagnostični preskusni In vitro diagnostic test
sistemi in vitro systems
kSIST-TS FprCEN/TS 17747:2022 en,fr,de
2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.

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kSIST-TS FprCEN/TS 17747:2022


FINAL DRAFT
TECHNICAL SPECIFICATION
FprCEN/TS 17747
SPÉCIFICATION TECHNIQUE

TECHNISCHE SPEZIFIKATION

November 2021
ICS 11.100.10
English Version

Molecular in vitro diagnostic examinations - Specifications
for pre-examination processes for exosomes and other
extracellular vesicles in venous whole blood - DNA, RNA
and proteins
Analyses de diagnostic moléculaire in vitro - Molekularanalytische in-vitro-diagnostische Verfahren
Spécifications relatives aux processuses préanalytiques - Spezifikationen für präanalytische Prozesse für
pour exosomes et autres vésicules extracellulaires Exosomen und andere extrazelluläre Vesikel im
dans le sang total veineux - ADN, ARN et protéines venösen Vollblut - DNA, RNA und Proteine


This draft Technical Specification is submitted to CEN members for Vote. It has been drawn up by the Technical Committee
CEN/TC 140.

CEN members are the national standards bodies of Austria, Belgium, Bulgaria, Croatia, Cyprus, Czech Republic, Denmark, Estonia,
Finland, France, Germany, Greece, Hungary, Iceland, Ireland, Italy, Latvia, Lithuania, Luxembourg, Malta, Netherlands, Norway,
Poland, Portugal, Republic of North Macedonia, Romania, Serbia, Slovakia, Slovenia, Spain, Sweden, Switzerland, Turkey and
United Kingdom.

Recipients of this draft are invited to submit, with their comments, notification of any relevant patent rights of which they are
aware and to provide supporting documentation.

Warning : This document is not a Technical Specification. It is distributed for review and comments. It is subject to change
without notice and shall not be referred to as a Technical Specification.


EUROPEAN COMMITTEE FOR STANDARDIZATION
COMITÉ EUROPÉEN DE NORMALISATION

EUROPÄISCHES KOMITEE FÜR NORMUNG

CEN-CENELEC Management Centre: Rue de la Science 23, B-1040 Brussels
© 2021 CEN All rights of exploitation in any form and by any means reserved Ref. No. FprCEN/TS 17747:2021 E
worldwide for CEN national Members.

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Contents Page

European foreword . 4
Introduction . 5
1 Scope . 6
2 Normative references . 6
3 Terms and definitions . 7
4 General Requirements . 11
5 Outside the laboratory . 13
5.1 Specimen collection . 13
5.1.1 General . 13
5.1.2 Information about the patient/specimen donor . 13
5.1.3 Selection of the venous whole blood collection tube by the laboratory . 13
5.1.4 Venous whole blood specimen collection from the patient/donor . 14
5.2 Specimen storage and transport . 15
5.2.1 General . 15
5.2.2 Storage and transport using blood collection tubes with stabilizers . 15
5.2.3 Storage and transport using blood collection tubes without stabilizers . 15
6 Inside the laboratory . 16
6.1 Specimen reception . 16
6.2 Specimen storage after transport and reception . 16
6.3 Plasma preparation . 16
6.4 Storage requirements for plasma samples . 17
6.5 Enrichment of EVs from specimen . 17
6.5.1 General . 17
6.5.2 Using a commercial EV enrichment system . 18
6.5.3 Using the laboratory’s own EV enrichment procedure . 18
6.5.4 Quality of enriched EVs . 18
6.5.5 Storage of enriched EVs . 19
6.6 Isolation of analyte of interest from EVs . 19
6.6.1 General . 19
6.6.2 Using a commercial kit for EV DNA, RNA, protein isolation intended for diagnostic
use . 19
6.6.3 Using a laboratory developed isolation procedure . 19
6.7 Quantity and quality assessment of isolated analyte of interest . 21
6.7.1 General . 21
6.7.2 Quantity assessment of EV DNA and RNA . 21
6.7.3 Quality assessment of DNA and RNA . 21
6.7.4 Quantity and quality assessment of EV proteins . 22
6.8 Storage of isolated analyte of interest . 22
6.8.1 General . 22
6.8.2 Storage of DNA . 23
6.8.3 Storage of RNA . 23
6.8.4 Storage of EV protein . 24
Annex A (informative) Example of a typical protocol for plasma preparation for EV
enrichment from unstabilized blood . 26
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Annex B (informative) Overview of different enrichment procedures . 27
B.1 General . 27
B.2 EV enrichment by centrifugation . 27
B.3 EV enrichment by filtration . 27
B.4 EV enrichment by chromatography . 28
B.5 EV enrichment by precipitation . 28
B.6 EV enrichment by immunoaffinity . 28
B.7 EV enrichment by microfluidics . 28
Bibliography . 29


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European foreword
This document (FprCEN/TS 17747:2021) has been prepared by Technical Committee CEN/TC 140 “In
vitro diagnostic medical devices”, the secretariat of which is held by DIN.
This document is currently submitted to the Vote on TS.
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Introduction
Molecular in vitro diagnostics has enabled a significant progress in medicine. Further progress is expected
by new technologies analysing profiles of nucleic acids, proteins, and metabolites in human tissues and
body fluids. However, the profiles of these molecules can change drastically during the pre-examination
process, including the specimen collection, transport, storage and processing.
Consequently, this makes the outcome from diagnostics or research unreliable or even impossible
because the subsequent examination might not determine the real situation in the patient but an artificial
profile generated during the pre-examination process.
Besides cell free circulating nucleic acids, circulating tumour cells (CTCs) and other rare cells, exosomes
and other extracellular vesicles represent another key component of liquid biopsies. Therefore, there is
a strongly increasing interest in research and emerging diagnostics in exosomes and other extracellular
vesicles.
The pre-examination process described in this document results in enriched extracellular vesicles (EV)
(e.g. exosomes) or DNA, RNA and proteins isolated therefrom.
New additional extracellular vesicles can be released and existing extracellular vesicles can be lost after
blood collection, thus changing the overall EV DNA/RNA/protein profiles. Also, different anticoagulants
in different types of blood collection tubes can influence the release of EVs from different cells present in
blood, including those from platelets. Further factors can influence the post collection changes of the
entire blood EV composition, such as storage and transport temperature and duration, centrifugation
parameters, etc.
Standardization of the entire workflow from the specimen collection to the EV surface protein and
isolated DNA, RNA and protein examination from EVs is therefore needed. Studies have been undertaken
to determine the important influencing factors. This document draws upon such work to codify and
standardize the steps of EV surface protein examination and of DNA, RNA and protein examination from
EVs in what is referred to as the pre-examination phase.
In this document, the following verbal forms are used:
— “shall” indicates a requirement;
— “should” indicates a recommendation;
— “may” indicates a permission;
— “can” indicates a possibility or a capability.
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1 Scope
This document gives guidelines on the handling, storage, processing and documentation of venous whole
blood specimens intended for DNA, RNA and protein examination from exosomes and other extracellular
vesicles during the pre-examination phase before a molecular examination is performed. This document
covers specimens collected in venous whole blood collection tubes.
The pre-examination process described in this document results in isolated DNA, RNA and proteins from
enriched exosomes and other extracellular vesicles.
This document is applicable to molecular in vitro diagnostic examinations performed by medical
laboratories. It is also intended to be used by health care institutions including facilities collecting and
handling specimen, laboratory customers, in vitro diagnostics developers and manufacturers, biobanks,
institutions and commercial organizations performing biomedical research, and regulatory authorities.
Different dedicated measures are taken during the pre-examination phase for venous whole blood
circulating cell-free RNA (ccfRNA) examination and for venous whole blood circulating cell-free DNA
(ccfDNA) examination, both without prior enrichment of exosomes and other extracellular vesicles.
These are not described in this document but are covered in EN ISO 20186-3, Molecular in vitro diagnostic
examinations — Specifications for pre-examination processes for venous whole blood — Part 3: Isolated
circulating cell free DNA from plasma and CEN/TS (WI 00140134), Molecular in vitro diagnostic
examinations — Specifications for pre-examination processes for venous whole blood — Isolated circulating
cell free RNA from plasma.
NOTE International, national or regional regulations or requirements can also apply to specific topics covered
in this document.
2 Normative references
The following documents are referred to in the text in such a way that some or all of their content
constitutes requirements of this document. 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, Medical laboratories - Requirements for quality and competence (ISO 15189:2012, Corrected
version 2014-08-15)
ISO 15190, Medical laboratories — Requirements for safety
ISO/TS 20658, Medical laboratories — Requirements for collection, transport, receipt, and handling of
samples
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3 Terms and definitions
For the purposes of this document, the terms and definitions given in EN ISO 15189 and the following
apply.
ISO and IEC maintain terminological databases for use in standardization at the following addresses:
— ISO Online browsing platform: available at http://www.iso.org/obp
— IEC Electropedia: available at https://www.electropedia.org/
3.1
aliquot
portion of a larger amount of homogenous material, assumed to be taken with negligible sampling error
Note 1 to entry: The term is usually applied to fluids. Tissues are heterogeneous and therefore cannot be
aliquoted.
Note 2 to entry: The definition is derived from the Compendium of Chemical Terminology Gold Book.
International Union of Pure and Applied Chemistry. Version 2.3.3., 2014; the PAC, 1990,62,1193 (Nomenclature for
sampling in analytical chemistry (Recommendations 1990)) p. 1206; and the PAC 1990, 62, 2167 (Glossary of
atmospheric chemistry terms (Recommendations 1990)) p. 2173.
3.2
analyte
component represented in the name of a measurable quantity
[SOURCE: ISO 17511:2020, 3.2 — Deleted Example.]
3.3
blood collection set
intravenous device specialized for venipuncture consisting of a stainless steel beveled needle and tube
(tubing) with attached plastic wings and fitting connector
Note 1 to entry: The connector attaches to an additional blood collection device, e.g. a blood collection tube.
3.4
blood collection tube
tube used for blood collection, usually with a vacuum which forces blood from the vein through the needle
into the tube
3.5
closed system
non-modifiable system provided by the vendor including all necessary components for the examination
(i.e. hardware, software, procedures and reagents)
3.6
deoxyribonucleic acid
DNA
polymer of deoxyribonucleotides occurring in a double-stranded (dsDNA) or single-stranded (ssDNA)
form
[SOURCE: EN ISO 22174:2005, 3.1.2]
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3.7
deoxyribonuclease
DNase
enzyme that catalyzes the degradation of DNA into smaller components
3.8
examination
analytical test
set of operations having the object of determining the value or characteristics of a property
Note 1 to entry: Processes (i.e. set of operations) that start with the isolated analyte and include all kinds of
parameter testing or chemical manipulation for quantitative or qualitative examination.
[SOURCE: ISO 15189:2012, 3.7, modified — Term and definition are used here without the original notes;
an additional term was added.]
3.9
examination manufacturer
analytical test manufacturer
entity that manufactures and/or produces a specific analytical test
Note 1 to entry: For the purpose of this document, an EV DNA, RNA and protein examination manufacturer is meant.
3.10
examination performance
analytical test performance
analytical performance
accuracy, precision, and sensitivity of a test to measure the analyte of interest
Note 1 to entry: Other test performance characteristics such as robustness, repeatability can apply as well.
[SOURCE: EN ISO 20184-1:2018, 3.4]
3.11
extracellular vesicle
EV
particle naturally released from the cell that are delimited by a lipid bilayer and cannot replicate, i.e. does
not contain a functional nucleus
EXAMPLE Exosomes, endosomes, oncosomes, apoptotic bodies
[SOURCE: [1]]
3.12
EV stabilizer
chemical formulation that increases the stability of EVs, including the overall EV populations, their cargo
(e.g. DNA, RNA and proteins) and EV surface proteins in a specimen or sample
3.13
interfering substance
endogenous or exogenous substances in clinical specimens/samples that can alter an examination result
Note 1 to entry: Examples of endogenous substances are blood components and acidic polysaccharides.
Note 2 to entry: Examples of exogenous substances are talc and anticoagulant.
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3.14
needle holder
barrel used in routine venepuncture procedures to hold the blood collection tube in place and to protect
the phlebotomist from direct contact with blood
Note 1 to entry: Examples of endogenous substances are blood components and acidic polysaccharides.
Note 2 to entry: Examples of exogenous substances are talc and anticoagulant.
3.15
pre-examination processes
preanalytical phase
preanalytical workflow
pre-examination phase
processes that start, in chronological order, from the clinician’s request and include the examination
request, preparation and identification of the patient, collection of the primary sample(s), transportation
to and within the medical laboratory, storage, isolation of analytes, and end when the analytical
examination begins
Note 1 to entry: The pre-examination phase includes preparative processes, e.g. RNA isolation procedures, which
influence the outcome of the intended examination.
[SOURCE: ISO 15189:2012, 3.15, modified — An additional term was added and more detail was
included.]
3.16
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: ISO 15189:2012, 3.16, modified — The term and definition are used here without the original
notes.]
3.17
proficiency testing
evaluation of participant performance against pre-established criteria by means of interlaboratory
comparisons
[SOURCE: EN ISO/IEC 17043:2010, 3.7, modified — Term and definition are used here without the
original notes.]
3.18
protein
type of biological macromolecules composed of one or more chains with a defined sequence of amino
acids connected through peptide bonds
3.19
ribonucleic acid
RNA
polymer of ribonucleotides occurring in a double-stranded or single-stranded form
[SOURCE: EN ISO 22174:2005, 3.1.3]
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3.20
ribonuclease
RNase
enzyme that catalyses the degradation of RNA into smaller components
3.21
room temperature
temperature in the range of 18 °C to 25 °C
Note 1 to entry: Local or national regulations can have different definitions.
[SOURCE: EN ISO 20166-1:2018, 3.22]
3.22
sample
one or more parts taken from a primary sample
[SOURCE: ISO 15189:2012, 3.24, modified — Example has been removed.]
3.23
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 phrase “reference material” has been replaced by
“sample material”.]
3.24
storage
prolonged interruption of the pre-analytical workflow of a sample or analyte respectively, or of their
derivatives, under appropriate conditions in order to preserve their properties
Note 1 to entry: Long-term storage typically occurs in laboratory archives or in biobanks.
[SOURCE: EN ISO 20184-1:2018, 3.21, modified — Example has been removed.]
3.25
validation
confirmation, through the provision of objective evidence, that the requirements for a specific intended
use or application have been fulfilled
Note 1 to entry: The term “validated” is used to designate the corresponding status.
[SOURCE: ISO 9000:2015, 3.8.13, modified — Note 1 and Note 3 have been omitted.]
3.26
venous whole blood
blood collected after directly puncturing a vein, usually with a needle and syringe, or other collection
device
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3.27
verification
confirmation, through the provision of objective evidence, that specified requirements have been fulfilled
Note 1 to entry: The term “verified” is used to designate the corresponding status.
Note 2 to entry: Confirmation can comprise activities such as:
— performing alternative calculations;
— comparing a new design specification with a similar proven design specification;
— undertaking tests and demonstrations;
— reviewing documents prior to issue.
[SOURCE: ISO 9000:2015, 3.8.12, modified — Note 1 and Note 2 have been omitted.]
3.28
workflow
series of activities necessary to complete a task
[SOURCE: EN ISO 20166-1:2018, 3.30]
4 General Requirements
For general statements on medical laboratory quality management systems and in particular on primary
sample collection, reception and handling (including avoidance of cross contaminations) see
EN ISO 15189 and EN ISO/IEC 17020 or EN ISO/IEC 17025. ISO/TS 20658 and EN ISO 20387 (for
biobanking) can also apply. The requirements on laboratory equipment, reagents, and consumables
according to EN ISO 15189 shall be followed; EN ISO/IEC 17020 and EN ISO/IEC 17025 can also apply.
All steps of the pre-examination, examination and post-examination processes (i.e. the entire workflow)
can influence the diagnosis or research study results.
Thus, this entire workflow shall be specified, verified and validated during the development of the
examination, including the development of in vitro diagnostic (IVD) medical devices. This includes
specifically all pre-examination process steps such as the examination request, preparation and
identification of the patient, collection of the primary sample(s), transport to and within the medical
laboratory, storage and isolation of analytes.
The EV profile composition can change significantly during or after blood collection, for example by
releasing additional EVs from cells and by aggregation of EVs with cells or amongst each other [1]. In
addition, enriched or isolated EVs can be lost upon storage by adhering to the surface of the storage
vessel. Thus, also the total EV DNA, RNA and protein profiles in blood and enriched fraction of EVs or
isolated EVs can change significantly after blood collection and during storage of blood and enriched or
isolated EVs. These changes can vary individually in blood from different donors or patients, and can
impact the validity and reliability of examination results.
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During the design and development of a EV DNA, RNA and protein based examination, a risk assessment
shall be performed (see also EN ISO 14971). Mitigation measures for eliminating or reducing identified
risks shall be established where required for ensuring the performance of the examination. It shall
especially be investigated and ensured that the EV DNA, RNA and protein profile(s) intended to be
analysed is/are not compromised in a manner impacting the examination performance. This includes
investigations on whether and/or how the profile intended to be examined changes during all pre-
analytical process steps. This can be done, e.g. by applying the intended examination to
specimens/samples which underwent time course studies reflecting the individual pre-examination
process steps such as transport and storage and by implementing measures to prevent or reduce impacts
by the identified pre-analytical variables, e.g. by using blood collection tubes with stabilizers.
NOTE A time course study involves repeated observations of the same variables at specific intervals over a
relevant time-period (e.g. time 0, 12 h, 24 h, 36 h, 48 h). This reflects any knowledge on the stability of the analyte(s)
of interest. Typically, this involves multiple aliquots from the same donor taken from the same blood draw repeated
for several donors.
The EV enrichment and/or isolation procedure can impact the type and purity of the EVs obtained. Also,
EV surface proteins can undergo changes during the EV enrichment procedure. The methods used for
DNA, RNA and protein isolation from EVs can also lead to profile changes of the analytes (e.g.
fragmentation of DNA, degradation of RNA, modification of proteins).
In addition, analyte profiles can change during long-term storage or archiving.
During the whole pre-examination process, precautions shall be taken to avoid cross contamination
between different specimens/samples (e.g. by using single-use material whenever feasible or
appropriate cleaning procedures between processing of different specimens/samples) and to avoid
mixing up of specimens/samples.
Safety instructions for the whole pre-examination process shall be in place and followed. Safety
regulations on specimen/sample transport and handling shall be considered (see EN ISO 15189,
ISO 15190 and ISO/TS 20658). If transport is required over public areas, corresponding regulations or
laws for packaging and transport apply (e.g. International Air Transport Association (IATA) for air
transport).
The manufacturer's material safety data sheet should be considered be
...

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