SIST EN ISO 20166-2:2019

SLOVENSKI STANDARD
SIST EN ISO 20166-2:2019
01-maj-2019
1DGRPHãþD
SIST-TS CEN/TS 16827-2:2015
0ROHNXODUQHGLDJQRVWLþQHSUHLVNDYHLQYLWUR6SHFLILNDFLMH]DSUHGSUHLVNRYDOQH
SURFHVH]DWNLYDNLVRILNVLUDQDYIRUPDOLQXWHUSRORåHQDYSDUDILQGHO,]ROLUDQL
SURWHLQL,62
Molecular in vitro diagnostic examinations - Specifications for pre-examinations
processes for formalin-fixed and paraffin-embedded (FFPE) tissue - Part 2: Isolated
proteins (ISO 20166-2:2018)
Molekularanalytische in-vitro-diagnostische Verfahren - Spezifikationen für
präanalytische Prozesse für formalinfixierte und paraffineingebettete (FFPE)-
Gewebeproben - Teil 2: Isolierte Proteine (ISO 20166-2:2018)
Analyses de diagnostic moléculaire in vitro - Spécifications relatives aux processus
préanalytiques pour les tissus fixés au formol et inclus en paraffine (FFPE) - Partie 2:
Protéines extraites (ISO 20166-2:2018)
Ta slovenski standard je istoveten z: EN ISO 20166-2:2018
ICS:
11.100.10 'LDJQRVWLþQLSUHVNXVQL In vitro diagnostic test
VLVWHPLLQYLWUR systems
SIST EN ISO 20166-2:2019 en
2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.

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SIST EN ISO 20166-2:2019

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SIST EN ISO 20166-2:2019


EN ISO 20166-2
EUROPEAN STANDARD

NORME EUROPÉENNE

December 2018
EUROPÄISCHE NORM
ICS 11.100.10 Supersedes CEN/TS 16827-2:2015
English Version

Molecular in vitro diagnostic examinations - Specifications
for pre-examinations processes for formalin-fixed and
paraffin-embedded (FFPE) tissue - Part 2: Isolated
proteins (ISO 20166-2:2018)
Analyses de diagnostic moléculaire in vitro - Molekularanalytische in-vitro-diagnostische Verfahren
Spécifications relatives aux processus préanalytiques - Spezifikationen für präanalytische Prozesse für
pour les tissus fixés au formol et inclus en paraffine formalinfixierte und paraffineingebettete (FFPE)-
(FFPE) - Partie 2: Protéines extraites (ISO 20166- Gewebeproben - Teil 2: Isolierte Proteine (ISO 20166-
2:2018) 2:2018)
This European Standard was approved by CEN on 22 November 2018.

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
© 2018 CEN All rights of exploitation in any form and by any means reserved Ref. No. EN ISO 20166-2:2018 E
worldwide for CEN national Members.

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SIST EN ISO 20166-2:2019
EN ISO 20166-2:2018 (E)
Contents Page
European foreword . 3

2

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SIST EN ISO 20166-2:2019
EN ISO 20166-2:2018 (E)
European foreword
This document (EN ISO 20166-2:2018) 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 June 2019, and conflicting national standards shall be
withdrawn at the latest by December 2021.
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 16827-2:2015.
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 20166-2:2018 has been approved by CEN as EN ISO 20166-2:2018 without any
modification.


3

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SIST EN ISO 20166-2:2019
INTERNATIONAL ISO
STANDARD 20166-2
First edition
2018-12
Molecular in vitro diagnostic
examinations — Specifications for pre-
examinations processes for formalin-
fixed and paraffin-embedded (FFPE)
tissue —
Part 2:
Isolated proteins
Analyses de diagnostic moléculaire in vitro — Spécifications relatives
aux processus préanalytiques pour les tissus fixés au formol et inclus
en paraffine (FFPE) —
Partie 2: Protéines extraites
Reference number
ISO 20166-2:2018(E)
©
ISO 2018

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SIST EN ISO 20166-2:2019
ISO 20166-2:2018(E)

COPYRIGHT PROTECTED DOCUMENT
© ISO 2018
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
CP 401 • Ch. de Blandonnet 8
CH-1214 Vernier, Geneva
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 2018 – All rights reserved

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SIST EN ISO 20166-2:2019
ISO 20166-2:2018(E)

Contents Page
Foreword .iv
Introduction .v
1 Scope . 1
2  Normative references . 1
3  Terms and definitions . 1
4  General considerations . 4
5  Outside the laboratory . 5
5.1 Specimen collection . 5
5.1.1 General. 5
5.1.2 Information about the specimen donor/patient . 6
5.1.3 Information about the specimen . 6
5.1.4 Specimen processing . 6
5.2 Transport requirements . 7
6  Inside the laboratory . 7
6.1 Information about the reception of the specimen . 7
6.2 Formalin fixation of the specimen or sample(s) . 7
6.3 Evaluation of the pathology of the specimen and selection of the sample(s) . 9
6.4 Post-fixation of frozen samples . 9
6.5 Processing and paraffin embedding .10
6.6 Storage requirements .10
6.7 Isolation of the total protein.11
6.7.1 General.11
6.7.2 General information for protein isolation procedures .11
6.7.3 Using commercial kits .11
6.7.4 Using the laboratories’ own protocols .11
6.8 Quality assessment of isolated proteins .12
6.9 Storage of isolated total protein .13
Annex A (informative) Examination of protein demonstrates changes of protein amounts
during cold ischemia .14
Bibliography .18
© ISO 2018 – All rights reserved iii

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SIST EN ISO 20166-2:2019
ISO 20166-2:2018(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 20166 series can be found on the ISO website.
iv © ISO 2018 – All rights reserved

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SIST EN ISO 20166-2:2019
ISO 20166-2:2018(E)

Introduction
Molecular in vitro diagnostics, including molecular pathology, has enabled a significant progress in
medicine. Further progress is expected with new technologies analyzing nucleic acids, proteins, and
metabolites in human tissues and body fluids. However, the profiles and/or integrity of these molecules
can change drastically during specimen collection, transport, storage, and processing, thus making
the outcome from diagnostics or research unreliable or even impossible because the subsequent
examination assay will not determine the situation in the patient but an artificial molecular pattern
generated during the pre-examination process.
Although originally thought as being impossible due to the crosslinking activities of formaldehyde,
protein isolation techniques from formalin-fixed and paraffin-embedded (FFPE) tissues have been
much improved in recent years. Heat-induced reversal of formaldehyde-induced crosslinks has been
[5][6]
demonstrated as an essential step in the protein isolation procedures . Currently, most investigators
[7]
accept that proteins isolated from FFPE tissue are suitable for downstream proteomic examination .
Protein profiles, protein integrities, and protein–protein interactions in tissues can change drastically
before, during and after collection (due to, e.g. gene induction, gene down regulation, protein
degradation). Protein species amounts can change differently in different donors’/patients’ tissues.
The expression of genes can be influenced by the given treatment or intervention (surgery, biopsy), or
drugs administered for anaesthesia or even treatment of concomitant disease as well as by the different
environmental conditions after the tissue removal from the body.
Furthermore, the formalin-fixation and paraffin-embedding processes lead to modifications of the
protein molecules, which can impact the validity and reliability of the examination test results.
Therefore, it is essential to take special measures to minimize the described protein profile changes
and modifications within tissues for subsequent examination.
A standardization of the entire process from specimen collection to the protein examination is needed.
Studies have been undertaken to determine the important influencing factors. This document draws
upon such work to codify and standardize the steps for FFPE tissue with regard to protein 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.
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SIST EN ISO 20166-2:2019
INTERNATIONAL STANDARD  ISO 20166-2:2018(E)
Molecular in vitro diagnostic examinations —
Specifications for pre-examinations processes for
formalin-fixed and paraffin-embedded (FFPE) tissue —
Part 2:
Isolated proteins
1 Scope
This document gives guidelines on the handling, documentation, storage and processing of formalin-
fixed and paraffin-embedded (FFPE) tissue specimens intended for the examination of isolated proteins
during the pre-examination phase before a molecular assay is performed.
This document is applicable to molecular in vitro diagnostic examinations including laboratory
developed tests performed by medical laboratories and molecular pathology 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.
This document is not applicable for protein examination by immunohistochemistry.
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.
ISO 15189:2012, Medical laboratories — Requirements for quality and competence
ISO 15190, Medical laboratories — Requirements for safety
ISO/IEC 17020:2012, Conformity assessment — Requirements for the operation of various types of bodies
performing inspection
3  Terms and definitions
For the purposes of this document, the terms and definitions given in 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 http: //www .electropedia .org/
3.1
aliquot
portion of a larger amount of homogeneous 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.
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Note 2 to entry: The definition is derived from References [28], [29], and [30].
3.2
ambient temperature
unregulated temperature of the surrounding air
3.3
analyte
component represented in the name of a measurable quantity
[SOURCE: ISO 17511:2003, 3.2 — EXAMPLE has been removed.]
3.4
analytical test performance
accuracy, precision, and sensitivity of a test to measure the analyte (3.3) of interest
Note 1 to entry: Other test performance characteristics such as robustness, repeatability can apply as well.
3.5
cold ischemia
condition after removal of the tissue from the body until stabilization or fixation
3.6
diagnosis
identification of a health or disease state from its signs and/or symptoms, where the diagnostic process
can involve examinations (3.7) and tests for classification of an individual's condition into separate and
distinct categories or subclasses that allow medical decisions about treatment and prognosis to be made
3.7
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 and include all kinds of parameter testing or
chemical manipulation for quantitative or qualitative examination.
[SOURCE: ISO 15189:2012, 3.7, modified — Notes to entry 1 to 3 have been removed, Note 1 to entry has
been added and “analytical test” has been added as a preferred term.]
3.8
formalin
saturated aqueous formaldehyde solution which at 100 % contains 37 % formaldehyde by mass
(corresponding to 40 % by volume)
3.9
formalin fixation
treatment of a sample with standard buffered formalin solution (3.21) for stabilization
3.10
grossing
gross examination
inspection of pathology specimens with the bare eye to obtain diagnostic information, while being
processed for further microscopic examination
3.11
paraffin embedding
process in which a tissue sample (3.19) is placed in paraffin to achieve a hard surrounding matrix so
that thin microscopic sections can be cut
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3.12
pre-examination process
pre-analytical phase
pre-analytical workflow
process that starts, in chronological order, from the clinician’s request and includes the examination
request, preparation and identification of the patient, collection of the primary sample(s), transportation
to and within the medical or pathology laboratory, isolation of analytes, and ends when the analytical
examination begins
Note 1 to entry: The pre-examination phase includes preparative processes, e.g. protein isolation procedures,
which influence the outcome of the intended examination.
[SOURCE: ISO 15189:2012, 3.15, modified — “pre-analytical workflow” has been added as a preferred
term, Note 1 to entry has been added and the definition has been extended.]
3.13
primary sample
specimen
discrete portion of a body fluid, breath, hair or tissue taken for examination (3.7), 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 1 to 3 have been removed.]
3.14
protein
type of biological macromolecule composed of one or more chains with a defined sequence of amino
acids connected through peptide bonds
3.15
protein profile
amounts of the individual protein (3.14) molecules that are present in a sample and that can be measured
in the absence of any losses, inhibition and interference
3.16
protein species
amounts of a chemically clearly-defined protein corresponding to one spot on a high-performance two-
dimensional gel electrophoresis pattern
Note 1 to entry: The definition is taken from Reference [7].
3.17
post-translational modification
chemical alterations to a primary protein structure, often crucial for conferring biological activity on
a protein
Note 1 to entry: The definition is taken from Reference [8].
3.18
room temperature
for the purposes of this document, temperature in the range of 18 °C to 25 °C
Note 1 to entry: Local or national regulations can have different definitions.
3.19
sample
one or more parts taken from a primary sample (3.13)
[SOURCE: ISO 15189:2012, 3.24, modified — EXAMPLE has been removed.]
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3.20
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
Note 1 to entry: The analyte for the purpose of this document is isolated protein.
[SOURCE: ISO Guide 30:2015, 2.1.15, modified — “reference material” has been replaced by “sample
material”, “characteristic” has been replaced by “ability” and Note 1 to entry has been changed.]
3.21
standard buffered formalin solution
neutral buffered formalin
NBF
10 % formalin (3.8) solution in water with a mass fraction of 3,7 % (corresponding to a volume fraction
of 4 %) formaldehyde, buffered to pH 6,8 to pH 7,2
Note 1 to entry: Standard buffered formalin solutions often contain small amounts of methanol to inhibit
oxidation and polymerization of formaldehyde.
3.22
storage
prolonged interruption of the pre-analytical workflow (3.12) of a sample or analyte respectively, or of
their derivatives, such as stained sections or tissue blocks, under appropriate conditions in order to
preserve their properties
Note 1 to entry: Long-term storage typically occurs in laboratory archives or in biobanks.
3.23
tissue processor
automated instrument where tissue fixation, dehydration, clearing and paraffin infiltration occurs
3.24
validation
confirmation, throughout the provision of objective evidence, that the requirements for a specific
intended use or application have been fulfilled
Note 1 to entry: “Validated” is used to designate the corresponding status.
[SOURCE: ISO 9000:2015, 3.8.13, modified — Notes to entry 1 and 3 have been removed.]
3.25
warm ischemia
condition before the tissue is removed from the body, but where it is deprived of its normal blood supply
3.26
workflow
series of activities necessary to complete a task
3.27
homogeneous
uniform in structure and composition
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, Clause 8 and 7.2. The requirements on
laboratory equipment, reagents, and consumables in accordance with 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.
4 © ISO 2018 – All rights reserved

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ISO 20166-2:2018(E)

All steps of a diagnostic workflow can influence the final analytical test result. Thus, the entire
workflow including biomolecule stability and sample storage conditions shall be verified and validated.
Workflow steps which cannot always be controlled (e.g. warm ischemia) shall be documented. A risk
assessment of non-controllable workflow steps including their potential impact on the analytical test
performance shall be performed and mitigation measures shall be established to enable the required
analytical test performance.
The stability of the specific protein(s) of interest and their post-translational modifications (if important
for the assay) should be investigated throughout the complete pre-examination process prior to the
development and implementation of an examination test (e.g. by performing a time course experiment
or study; see also Annex A and Reference [9]).
Before tissues are fixed in standard buffered formalin solution, protein amounts, conformations and
binding status can change, e.g. by protein degradation and altered synthesis following gene induction,
gene down regulation, RNA degradation, and changes of the biochemical pathway and energy status.
These effects depend on the duration of warm and cold ischemia and the ambient temperature before
formalin fixation. In addition, the described effects can vary in different donors’/patients’ tissues.
Generally, the longer the durations of warm and cold ischemia and the higher the ambient temperature
before fixation of the tissue specimen, the higher is the risk that changes in the protein profile can occur.
NOTE Prolonged cold ischemia results in changes of protein (e.g. cytokeratin 18) and phosphoprotein (e.g.
[9][10] [11]
phospho-p42/44) amounts . Keeping the specimen on wet-ice diminishes this effect . Protein amounts as
well as the protein modifications can also vary, depending on the origin and type of tissue, the underlying disease,
the surgical procedure, the drug regime, and drugs administered for anaesthesia or treatment of concomitant
disease, and on the different environmental conditions after the tissue removal from the body.
As warm ischemia cannot be easily standardized, its duration shall be documented. When it is not
possible to avoid cold ischemia, its duration shall be documented and the temperatures of the specimen
container's surroundings shall be documented. Where the specimen is transported to another facility
for formalin fixation, the transport duration shall be documented and the
...