EN ISO 20186-2:2019

SLOVENSKI STANDARD
01-julij-2019
Nadomešča:
SIST-TS CEN/TS 16835-2:2015
Molekularne diagnostične preiskave in vitro - Specifikacije za predpreiskovalne
procese za vensko polno kri - 2. del: Iz genoma izolirana DNK (ISO 20186-2:2019)
Molecular in vitro diagnostic examinations - Specifications for pre-examination processes
for venous whole blood - Part 2: Isolated genomic DNA (ISO 20186-2:2019)
Molekularanalytische in-vitro-diagnostische Verfahren - Spezifikationen für
präanalytische Prozesse für venöse Vollblutproben - Teil 2: Isolierte genomische DNA
(ISO 20186-2:2019)
Analyses de diagnostic moléculaire in vitro - Spécifications relatives aux processus
préanalytiques pour le sang total veineux - Partie 2: ARN cellulaire extrait (ISO 20186-
2:2019)
Ta slovenski standard je istoveten z: EN ISO 20186-2:2019
ICS:
11.100.10 Diagnostični preskusni In vitro diagnostic test
sistemi in vitro systems
11.100.30 Analiza krvi in urina Analysis of blood and urine
2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.

EN ISO 20186-2
EUROPEAN STANDARD
NORME EUROPÉENNE
March 2019
EUROPÄISCHE NORM
ICS 11.100.10 Supersedes CEN/TS 16835-2:2015
English Version
Molecular in vitro diagnostic examinations - Specifications
for pre-examination processes for venous whole blood -
Part 2: Isolated genomic DNA (ISO 20186-2:2019)
Analyses de diagnostic moléculaire in vitro - Molekularanalytische in-vitro-diagnostische Verfahren
Spécifications relatives aux processus préanalytiques - Spezifikationen für präanalytische Prozesse für
pour le sang total veineux - Partie 2: ADN génomique venöse Vollblutproben - Teil 2: Isolierte genomische
extrait (ISO 20186-2:2019) DNA (ISO 20186-2:2019)
This European Standard was approved by CEN on 2 February 2019.

CEN members are bound to comply with the CEN/CENELEC Internal Regulations which stipulate the conditions for giving this
European Standard the status of a national standard without any alteration. Up-to-date lists and bibliographical references
concerning such national standards may be obtained on application to the CEN-CENELEC Management Centre or to any CEN
member.
This European Standard exists in three official versions (English, French, German). A version in any other language made by
translation under the responsibility of a CEN member into its own language and notified to the CEN-CENELEC Management
Centre has the same status as the official versions.

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, Norway, Poland, Portugal, 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
© 2019 CEN All rights of exploitation in any form and by any means reserved Ref. No. EN ISO 20186-2:2019 E
worldwide for CEN national Members.

Contents Page
European foreword . 3

European foreword
This document (EN ISO 20186-2:2019) has been prepared by Technical Committee ISO/TC 212 "Clinical
laboratory testing and in vitro diagnostic test systems" in collaboration with Technical Committee
CEN/TC 140 “In vitro diagnostic medical devices” the secretariat of which is held by DIN.
This European Standard shall be given the status of a national standard, either by publication of an
identical text or by endorsement, at the latest by September 2019, and conflicting national standards
shall be withdrawn at the latest by March 2022.
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.
This document supersedes CEN/TS 16835-2:2015.
According to the CEN-CENELEC Internal Regulations, the national standards organizations of the
following countries are bound to implement this European Standard: Austria, Belgium, Bulgaria,
Croatia, Cyprus, Czech Republic, Denmark, Estonia, Finland, Former Yugoslav Republic of Macedonia,
France, Germany, Greece, Hungary, Iceland, Ireland, Italy, Latvia, Lithuania, Luxembourg, Malta,
Netherlands, Norway, Poland, Portugal, Romania, Serbia, Slovakia, Slovenia, Spain, Sweden, Switzerland,
Turkey and the United Kingdom.
Endorsement notice
The text of ISO 20186-2:2019 has been approved by CEN as EN ISO 20186-2:2019 without any
modification.
INTERNATIONAL ISO
STANDARD 20186-2
First edition
2019-02
Molecular in vitro diagnostic
examinations — Specifications for
pre-examination processes for venous
whole blood —
Part 2:
Isolated genomic DNA
Analyses de diagnostic moléculaire in vitro — Spécifications relatives
aux processus préanalytiques pour le sang total veineux —
Partie 2: ADN génomique extrait
Reference number
ISO 20186-2:2019(E)
©
ISO 2019
ISO 20186-2:2019(E)
© ISO 2019
All rights reserved. Unless otherwise specified, or required in the context of its implementation, no part of this publication may
be reproduced or utilized otherwise in any form or by any means, electronic or mechanical, including photocopying, or posting
on the internet or an intranet, without prior written permission. Permission can be requested from either ISO at the address
below or ISO’s member body in the country of the requester.
ISO copyright office
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Phone: +41 22 749 01 11
Fax: +41 22 749 09 47
Email: copyright@iso.org
Website: www.iso.org
Published in Switzerland
ii © ISO 2019 – All rights reserved

ISO 20186-2:2019(E)
Contents Page
Foreword .iv
Introduction .v
1 Scope . 1
2  Normative references . 1
3  Terms and definitions . 1
4 General considerations . 5
5 Outside the laboratory . 6
5.1 Specimen collection . 6
5.1.1 Information about the specimen donor/patient . 6
5.1.2 Selection of the venous whole blood collection tube by the laboratory . 6
5.1.3 Venous whole blood specimen collection from the donor/patient and
stabilization procedures . . 6
5.1.4 Information about the specimen and storage requirements at the blood
collection facility . 7
5.2 Transport requirements . 8
6 Inside the laboratory . 8
6.1 Specimen reception . 8
6.2 Storage requirements . 8
6.3 Isolation of the genomic DNA .10
6.3.1 General.10
6.3.2 Examination provider's instructions available .10
6.3.3 Examination provider's instructions not available .10
6.4 Quantity and quality assessment of isolated genomic DNA .11
6.5 Storage of isolated genomic DNA .11
6.5.1 General.11
6.5.2 Genomic DNA isolated with commercially available kits . .11
6.5.3 Genomic DNA isolated with the laboratory's own protocols .12
Annex A (informative) Impact of pre-examination process steps on venous whole blood
genomic DNA quality .13
Bibliography .19
ISO 20186-2:2019(E)
Foreword
ISO (the International Organization for Standardization) is a worldwide federation of national standards
bodies (ISO member bodies). The work of preparing International Standards is normally carried out
through ISO technical committees. Each member body interested in a subject for which a technical
committee has been established has the right to be represented on that committee. International
organizations, governmental and non-governmental, in liaison with ISO, also take part in the work.
ISO collaborates closely with the International Electrotechnical Commission (IEC) on all matters of
electrotechnical standardization.
The procedures used to develop this document and those intended for its further maintenance are
described in the ISO/IEC Directives, Part 1. In particular, the different approval criteria needed for the
different types of ISO documents should be noted. This document was drafted in accordance with the
editorial rules of the ISO/IEC Directives, Part 2 (see www .iso .org/directives).
Attention is drawn to the possibility that some of the elements of this document may be the subject of
patent rights. ISO shall not be held responsible for identifying any or all such patent rights. Details of
any patent rights identified during the development of the document will be in the Introduction and/or
on the ISO list of patent declarations received (see www .iso .org/patents).
Any trade name used in this document is information given for the convenience of users and does not
constitute an endorsement.
For an explanation of the voluntary nature of standards, the meaning of ISO specific terms and
expressions related to conformity assessment, as well as information about ISO's adherence to the
World Trade Organization (WTO) principles in the Technical Barriers to Trade (TBT) see www .iso
.org/iso/foreword .html.
This document was prepared by Technical Committee ISO/TC 212, Clinical laboratory testing and in
vitro diagnostic test systems.
Any feedback or questions on this document should be directed to the user’s national standards body. A
complete listing of these bodies can be found at www .iso .org/members .html.
A list of all parts in the ISO 20186 series can be found on the ISO website.
iv © ISO 2019 – All rights reserved

ISO 20186-2:2019(E)
Introduction
Molecular in vitro diagnostics has enabled 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 processes.
Genomic DNA can fragment or degrade after blood collection. Therefore, special measures need to be
taken to secure good quality specimens for genomic DNA examination. This is particularly relevant for
examination test procedures requiring high molecular weight DNA (HMW DNA).
Standardization of the entire workflow from specimen collection to the genomic DNA examination is
needed due to genomic DNA degradation and fragmentation after blood collection. Studies have been
undertaken to determine the important influencing factors. This document draws upon such work to
codify and standardize the steps for venous whole blood genomic DNA examination 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.
INTERNATIONAL STANDARD ISO 20186-2:2019(E)
Molecular in vitro diagnostic examinations —
Specifications for pre-examination processes for venous
whole blood —
Part 2:
Isolated genomic DNA
1 Scope
This document gives guidelines on the handling, storage, processing and documentation of venous
whole blood specimens intended for genomic DNA examination during the pre-examination phase
before a molecular examination is performed. This document covers specimens collected in venous
whole blood collection tubes.
This document is applicable to any molecular in vitro diagnostic examination 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 for stabilizing blood cell free circulating DNA, which are not
described in this document.
NOTE Circulating cell free DNA in blood is covered in ISO 20186-3.
Different dedicated measures are taken for collecting, stabilizing, transporting and storing capillary
blood as well as for collecting and storing blood by paper based technologies or other technologies
generating dried blood. These are not described in this document.
This document does not cover the isolation of specific blood cells and subsequent isolation of genomic
DNA therefrom.
DNA in pathogens present in blood is not covered by 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.
ISO 15189:2012, Medical laboratories — Requirements for quality and competence
3  Terms and definitions
For the purposes of this document, the following terms and definitions 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 http: //www .electropedia .org/
ISO 20186-2:2019(E)
3.1
analyte
component represented in the name of a measurable quantity
[SOURCE: ISO 17511:2003, 3.2]
3.2
backflow
flow of a liquid opposite to the usual or desired direction
3.3
blood collection set
intravenous device specialized for venepuncture consisting of a stainless steel bevelled 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).
3.4
blood collection tube
tube used for blood collection, usually in a vacuum which forces blood from the vein through the needle
into the tube
3.5
blood genomic DNA stabilizers
compounds, solutions or mixtures that are designed to minimize degradation and fragmentation of
genomic DNA (3.12) in blood
3.6
closed system
non-modifiable system provided by the vendor including all necessary components for the examination
(i.e. hardware, software, procedures and reagents)
3.7
deoxyribonucleic acid
DNA
polymer of deoxyribonucleotides occurring in a double-stranded (dsDNA) or single-stranded
(ssDNA) form
[SOURCE: ISO 22174:2005, 3.1.2]
3.8
DNase
deoxyribonuclease
enzyme that catalyses the degradation of DNA into smaller components
3.9
examination
analytical test
set of operations having the object of determining the value or characteristics of a property
Note 1 to entry: Processes that start with the isolated analyte (3.1) 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.]
2 © ISO 2019 – All rights reserved

ISO 20186-2:2019(E)
3.10
examination performance
analytical test performance
analytical performance
ability of an examination procedure to measure or detect a particular analyte (3.1)
Note 1 to entry: Analytical performance is determined from analytical performance studies used to assess the
ability of an in vitro diagnostic examination procedure to measure or detect a particular analyte.
Note 2 to entry: Analytical performance includes such characteristics as analytical sensitivity, detection limit,
analytical specificity (interference and cross-reactivity), trueness, precision and linearity.
[SOURCE: ISO/TS 17822-1:2014, 3.2, modified — Two terms have been added.]
3.11
examination provider
analytical test provider
entity that provides the specific analytical test
3.12
genomic DNA
DNA from the nuclear and mitochondrial genomes containing all coding (exon) and non-coding (intron
and other) sequences
Note 1 to entry: In this document, reference is only made to genomic DNA present in cells in blood, excluding
circulating cell free DNA.
3.13
high molecular weight DNA
HMW DNA
DNA with an average double strand size larger than 50 kb on a pulsed field electrophoresis gel for the
purpose of this document
3.14
interfering substances
endogenous or exogenous substances in clinical specimens (3.17)/samples (3.23) 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.
3.15
needle holder
barrel used in routine venepuncture procedures to hold the blood collection tube (3.4) in place and to
protect the phlebotomist from direct contact with blood
3.16
pre-examination processes
preanalytical phase
preanalytical workflow
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) (3.17),
transportation to and within the medical laboratory, isolation of analytes, and end when the analytical
examination begins
Note 1 to entry: The pre-examination phase includes preparative processes, e.g. DNA isolation procedures, which
influence the outcome of the intended examination.
[SOURCE: ISO 15189:2012, 3.15, modified — An additional term has been added and more details have
been included.]
ISO 20186-2:2019(E)
3.17
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 — Notes to entry have been omitted.]
3.18
primary sample collection device
apparatus specifically intended by an IVD manufacturer to obtain, contain and preserve a body fluid or
tissue for in vitro diagnostic examination
[SOURCE: ISO 18113-1:2009, 3.55]
Note 1 to entry: Includes devices intended to store a specimen prior to examination.
Note 2 to entry: Includes both vacuum and non-vacuum specimen collection devices.
3.19
proficiency testing
evaluation of participant performance against pre-established criteria by means of inter-laboratory
comparisons
[SOURCE: ISO 17043:2010, 3.7, modified — Term and definition are used here without the original notes.]
3.20
RNA
ribonucleic acid
polymer of ribonucleotides occurring in a double-stranded or single-stranded form
[SOURCE: ISO 22174:2005, 3.1.3]
3.21
RNase
ribonuclease
enzyme that catalyses the degradation of RNA into smaller components
3.22
room temperature
temperature in the range of 18 °C to 25 °C
Note 1 to entry: Local or national regulations can have different definitions.
3.23
sample
one or more parts taken from a primary sample (3.17)
[SOURCE: ISO 15189:2012, 3.24, modified — The example has been omitted.]
3.24
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”.]
4 © ISO 2019 – All rights reserved

ISO 20186-2:2019(E)
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 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
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.
[SOURCE: ISO 9000: 2015, 3.8.12, modified — Note 1 and Note 2 have been omitted.]
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; and
— reviewing documents prior to issue.
3.28
workflow
series of activities necessary to complete a task
4 General considerations
For general statements on medical laboratory quality management systems and in particular on
specimen collection, reception and handling (including avoidance of cross contaminations) see
ISO 15189:2012, 4.2, 5.4.4, 5.4.6 or ISO/IEC 17020:2012, 7.2 and Clause 8. The requirements on
laboratory equipment, reagents, and consumables according to ISO 15189:2012, 5.3 shall be followed;
ISO 15189:2012, 5.5.1.2 and 5.5.1.3 and ISO/IEC 17020:2012, 6.2 can also apply.
All steps of a diagnostic workflow can influence the final examination result. Thus, the entire workflow,
including specimen/sample storage and transport conditions, and its impact on the stability of
biomolecules intended to be examined shall be verified and validated. Workflow steps which cannot
always be controlled shall be documented and their impact on the examination performance shall
be investigated and mitigation measures shall be established to enable the required examination
performance. In these cases, risk assessment is recommended.
The stability of the genomic DNA should be investigated throughout the complete pre-examination
[8]
workflow. Additional post-collection effects can also occur, e.g. genomic DNA fragmentation .
Before or during the design of an examination, it should be investigated and ensured that the genomic
DNA minimum amount and size required for the examination are not affected by the envisioned entire
pre-examination workflow.
Safety procedures for handling and transport shall be in place. Safety regulations on transport and
handling shall be considered (see ISO 15189:2012, 5.2.3 and 5.4.5, and ISO 15190).
ISO 20186-2:2019(E)
During the whole pre-examination process, precautions shall be taken to avoid cross contamination
between different samples/specimens, e.g. by using single-use material whenever feasible or
appropriate cleaning procedures between processing of different specimens/samples.
If a commercial product is not used in accordance with the manufacturer's instructions, responsibility
for its validation, verification, use and performance lies with the user.
NOTE International, national or regional regulations or requirements can also apply to specific topics
covered in this document.
5 Outside the laboratory
5.1 Specimen collection
5.1.1  Information about the specimen donor/patient
The documentation shall include the ID of the specimen donor/patient, which can be in the form of a code.
The documentation should include, but is not limited to:
a) the relevant health status of the specimen donor or patient [e.g. healthy, disease type, concomitant
disease, demographics (e.g. age and gender)];
b) the information about medical treatment and special treatment prior to blood collection (e.g.
anaesthetics, medications);
c) the type and the purpose of the proposed examination requested;
d) the appropriate consent from the specimen donor/patient.
See also ISO 15189:2012, 5.4.4.
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 venous whole
blood collection procedures, inappropriate storage/shipping conditions as well as DNA isolation
[9][10][11][12][13][14][15][16]
procedures .
Blood should be collected in appropriate venous whole blood collection tubes containing an
[17]
anticoagulant such as EDTA or acid citrate dextrose (ACD) , and their catalogue and lot number
should be documented.
NOTE Blood collection tubes containing EDTA as an anticoagulant are preferable for most genomic DNA
examination. 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 in examination technologies, such as PCR.
Specifically developed whole blood collection tubes, containing genomic DNA stabilizing reagents, are
also available, claiming to standardize blood collection, transport and storage and intended for DNA
examinations from whole blood.
5.1.3  Venous whole blood specimen collection from the donor/patient and stabilization
procedures
a) The identity of the person collecting the specimen and the time and date of blood collection
according to ISO 15189:2012, 5.4.4.3, f) shall be documented.
b) For the labelling (sample/specimen identification) of the blood collection tube a routine procedure
[ISO 15189:2012, 5.4.4.3, e)] or a procedure with additional information (e.g. 2D-barcode) shall
be used.
6 © ISO 2019 – All rights reserved

ISO 20186-2:2019(E)
c) Standard venepuncture technique can be used. Steps for preventing possible backflow into the
donor's/patient’s body can be required. The manufacturer's instructions for using the blood
collection tubes shall be followed. A blood collection set and needle holder can be required when
using blood genomic DNA stabilizer containing tubes. In this case, the instructions of the collection
set and needle holder manufacturer shall be followed.
NOTE 1 There is no known specific
...