EN ISO 20166-2:2018

SLOVENSKI STANDARD
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
2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.

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.

Contents Page
European foreword . 3

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.
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
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
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Phone: +41 22 749 01 11
Fax: +41 22 749 09 47
Email: copyright@iso.org
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Published in Switzerland
ii © ISO 2018 – All rights reserved

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 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

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.
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.
ISO 20166-2:2018(E)
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
2 © ISO 2018 – All rights reserved

ISO 20166-2:2018(E)
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.]
ISO 20166-2:2018(E)
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

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 ambient conditions should
also be documented.
In addition, the formalin fixation itself, as well as the subsequent FFPE tissue storage duration and
storage temperature causes modifications of biomolecules and leads to suboptimal performance of
[12]
protein isolated from FFPE tissues . This should be considered in the quality control and application
of molecular analytical tests. Analytical test optimization for FFPE tissues or the use of non-crosslinking
alternatives to standard buffered formalin solution are options to minimize this issue for molecular
examinations.
Safety instructions on transport and handling shall be considered and followed in accordance with
ISO 15189:2012, 5.2.3 and 5.4.5, and ISO 15190.
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.
If a commercial product is not used in accordance with the manufacturers' instructions, responsibility
for its use and performance lies with the user.
5  Outside the laboratory
5.1 Specimen collection
5.1.1  General
For the collection of the specimen, the requirements (e.g. disease condition, specimen size) for the
intended molecular examination (see also Clause 6) should be considered.
See also ISO 15189:2012, 5.4.4.
ISO 20166-2:2018(E)
5.1.2  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/patient [e.g. healthy, disease type, concomitant
disease, demographics (e.g. age and gender)];
b) the information about routine medical treatment and special treatment prior to tissue collection
(e.g. anaesthetics, medications, surgical or diagnostic procedures);
c) the appropriate consent from the specimen donor/patient.
5.1.3  Information about the specimen
The documentation shall include, but is not limited to:
a) the start of ischemia within the body (warm ischemia) by documentation of the ischemia-relevant
vessel ligation/clamping time point (usually arterial clamping time);
b) the time and date when tissue is removed from the body and the method of removal (e.g. core-
needle biopsy, resection, biopsy device used for the collection);
c) the description of tissue type and origin, tissue condition (e.g. diseased, unaffected by the disease),
including references to any marking applied in or outside the operating theatre made by surgeon,
radiologist or pathologist;
d) the documentation steps described under 6.2, if the formalin fixation starts outside the laboratory,
and also the documentation steps described under 6.3, if the evaluation of the pathology of the
specimen and selection of the sample(s) is also done outside the laboratory.
The documentation should also include the ID of the responsible person for collecting the specimen.
5.1.4 Specimen processing
The following steps shall be performed:
a) the documentation of any additions or modifications to the specimen after removal from the body
[e.g. labelling for the orientation of the specimen (e.g. ink-marking, stitches), incision(s)];
b) the selection and use of containers and packages (e.g. cooling box, box for storing and transportation,
vacuum packaging) according to applicable transport regulations;
c) the selection and use of stabilization procedures (e.g. cooling methods) for transport;
NOTE 1 Accidentally freezing the tissue (e.g. by using cool packs in a wrong manner) can lead to protein
degradation when the tissue thaws thereafter. It can also impact the morphological characterization.
NOTE 2 This step can be omitted, if the specimen is transferred directly into standard buffered formalin
solution (see 6.2 and notice the importance of volume of fixative and tissue sectioning to allow adequate
penetration of fixative).
d) the labelling of the container (e.g. registration-number, barcode (1D or 2D), specimen type, quantity,
and organ tissue of origin) and additional documentation [information as specified in 5.1.2, 5.1.3,
and 5.1.4 a) to c)].
Several specimens from the same patient/donor sharing similar features (macroscopic appearance,
tissue type, disease status and anatomical location) may be put into a single container/container
compartment.
6 © ISO 2018 – All rights reserved

ISO 20166-2:2018(E)
Specimens should be transferred without delay into the container after the removal from the body. The
container then should be kept on wet-ice or at 2 °C to 8 °C in order to minimize protein profile changes.
The temperatures of the container’s surroundings during cold ischemia (e.g. temperatures in different
rooms; transport) should be documented. If the temperature cannot be measured, the temperature
range should be estimated by classification as ambient temperature, room temperature, or at 2 °C to 8 °C.
5.2  Transport requirements
The laboratory in collaboration with the clinical or surgery department shall establish a protocol for
the transport procedure of the specimen.
Temperature monitoring should be applied in a suitable manner.
If the specimen is not already placed into standard buffered formalin solution, it should be transported
on wet-ice or at 2 °C to 8 °C without delay in order to minimize changes to the protein profile.
NOTE There is evidence that proteins in tissues can be stabilized in plastic bags under vacuum when kept
[13]
at 0 °C to 4 °C during transport before the samples are archived for biobanks or used for histopathological
evaluation.
If the specimen is already placed into standard buffered formalin solution outside the laboratory, the
temperature during transport should not exceed room temperature.
The conformity with the protocol for the transport procedure shall be documented. Any deviations
from the protocol shall be described and documented.
6  Inside the laboratory
6.1  Information about the reception of the specimen
The ID or name of the person receiving the specimen shall be documented. The specimen arrival
date and time, and conditions (e.g. labelling, transport conditions including temperature, tissue type
and quantity of the specimen, leaking/breaking of the container) of the received specimens shall be
documented. Any deviations from the established protocol for the transport procedure (see 5.2) shall
be documented.
The correct identity of the specimen shall be checked. This should include the clinical information
(see 5.1.1 and 5.1.3) of the specimen, hospital admission number and/or donor/patient ID, name of the
patient, date of birth of the patient.
6.2  Formalin fixation of the specimen or sample(s)
This procedure is applicable to the specimen, and, in case that one or more parts are taken from a
specimen to the resulting sample(s).
The fixative used should be standard buffered formalin solution.
NOTE 1 In some countries, standard buffered formalin solution is referred to as neutral buffered formalin (NBF).
NOTE 2 There is evidence that the yield of certain proteins, as determined by Western blot examination, can
[14]
be increased by ultrasound mediated acceleration of tissue fixation .
The pH-value of the standard buffered formalin solution should be checked at least once per week
and before use or with every new batch as formalin is not stable (e.g. formaldehyde has a tendency to
[15]
oxidize to formic acid) .
ISO 20166-2:2018(E)
The following steps shall be performed:
a) the consultation of the manufacturer's Safety Data Sheet (SDS) before handling standard buffered
formalin solution;
NOTE Formaldehyde is a carcinogenic and hazardous compound that penetrates the tissue and
chemically modifies biomolecules. However, there are potential different local classifications.
b) the documentation of the time point of placing the tissue specimen or sample into standard buffered
formalin solution;
NOTE The total formalin fixation duration can have an impact on further examinations, e.g.
immunohistochemical techniques, nucleic acid based molecular examinations. The optimal formalin fixation
duration can vary depending on tissue type and size. For larger surgical specimens, e.g. a resected stomach,
inhomogeneous fixation can occur before the grossing process due to slow penetration of formaldehyde
from the surface of the tissue to the interior. Formalin fixation for more than 24 h can lead to a crosslinking
intensity that can impact the protein examination test. It has been shown that the protein yield decreased
[16][17]
with increasing fixation duration .
EXAMPLE For tissue pieces with a thickness of 5 mm, fixation durations between 12 h and 24 h are in
most cases reasonable for an appropriate penetration and fixation. See also 6.7.2.
c) the selection of container(s):
1) the capacity of the containers should be such that the specimen can be completely submerged
into the standard buffered formalin solution. The minimum standard buffered formalin
solution to tissue ratio depends on the tissue concerned, but should be at least 10:1 (volume to
[18]
volume) . To ensure complete formalin fixation of larger specimens, a special tissue handling
such as incision(s) of solid organs or opening of hollow organs should be performed.
Larger specimens may need to be bisected and appropriate portions selected to ensure
adequate fixative penetration. In this case, the standard buffered formalin solution shall be
changed periodically.
2) when using containers pre-filled with standard buffered formalin solution, provider’s product
instructions shall be followed;
3) the container shall be securely closable;
d) the labelling of the container [e.g. by using self-adhesive labels, handwriting, Radio Frequency
Identification Devices (RFID), pre-labelled containers, bar codes] shall ensure appropriate
traceability of specimens or samples. Therefore, the container labelling shall provide the minimum
information of:
1) the patient/donor ID, unique specimen/sample ID and date when the sample was collected,
which all can be in the form of a code (unique for every sample);
2) the basic specimen information, e.g. the tissue type, tissue condition, and related additional
information such as affected (e.g. tumour) or unaffected, unless a sample tracking system can
supply this information coupled to the identification of the specimen or sample used in 6.2 d) 1);
3) the unique numbering of each container, which can be included in 6.2 d) 1);
e) the documentation of types, quantity and description of specimen or samples.
It should be considered that under some disease conditions, such as tumours, molecular features
may not be present homogeneously in the specimen or tissue sample. Therefore, it is important that
the part of the actual specimen or tissue sample used for molecular examination is evaluated by a
medically qualified (e.g. board certified) pathologist (see 6.3). In this context it should be documented
which features of a disease are actually reflected in the tissue specimen or sample used for molecular
examination (e.g. different molecular mechanisms can be activated in the centre and at the invasion
front of the tumour, also tumours can be composed of areas showing different differentiation grades).
8  © ISO 2018 – All rights reserved

ISO 20166-2:2018(E)
6.3  Evaluation of the pathology of the specimen and selection of the sample(s)
The evaluation and documentation of the pathology of the specimen and the selection of the sample(s)
from the specimen for further processing shall be done by or under supervision or responsibility of a
medically qualified (e.g. board certified) pathologist.
Local, national or regional regulations can apply.
Options to select the sample(s) for protein examination:
a) The selection of appropriate parts of the specimen for molecular and histopathological
examinations as well as for further research purposes shall be done by or under supervision of a
medically qualified (e.g. board certified) pathologist to ensure that the collection of the sample(s)
for protein examination does not compromise the histopathological examination. For molecular
examination, suitable tissue parts should be selected, whereas parts potentially compromising the
molecular examination, such as bleeding and necrotic parts, should be avoided where appropriate.
Microdissection of tissue should be considered to select or enrich for certain cellular features of a
disease.
NOTE 1 Depending on local procedures, the selection of appropriate parts of the specimen can also be
done outside of the laboratory, e.g. in the operating theatre (see 5.1.3).
In the context of the macroscopic evaluation of the surgical specimen before and/or after formalin
fixation, the clinical information (see 5.1.2 and 5.1.3), of the specimen (e.g. type, size, number),
hospital admission number and/or pathology case number and/or donor/patient ID, name of the
patient, date of birth of the patient and type of tissue should be checked. The surgical specimen and
all findings shall be described appropriately according to the guidelines of the respective medical
societies, e.g. societies of pathology, and in correlation with the clinical information and questions,
e.g. patient record or clinician’s request. The anatomic localization represented in the specimen
shall be described, resection margins and other important areas may be marked if necessary
and helpful for later microscopic evaluation; photographs may be taken. Representative samples
for microscopic evaluation shall be taken (i.e. grossing) according to the organ/disease specific
guidelines from the respective medical societies.
NOTE 2 The above described evaluation or documentation can also be done outside of the laboratory, e.g.
in the operating theatre.
b) Where the tissue specimen was removed from the body without the requirement of a
histopathological diagnosis, the documentation of this specimen, the evaluation, selection and
documentation of the samples may be done by other qualified persons than pathologists.
The documentation can include photographs. The size of the samples shall be appropriate for the
tissue cassette (maximum of approximately 3 cm × 2 cm × 0,5 cm). If the specimen is not yet fixed
appropriately, post-fixation can be performed within the tissue cassette. Each tissue cassette shall be
labelled with a unique identifi
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