EN ISO 20166-4:2021
(Main)Molecular in vitro diagnostic examinations - Specifications for preexamination processes for formalin-fixed and paraffin-embedded (FFPE) tissue - Part 4: In situ detection techniques (ISO 20166-4:2021)
Molecular in vitro diagnostic examinations - Specifications for preexamination processes for formalin-fixed and paraffin-embedded (FFPE) tissue - Part 4: In situ detection techniques (ISO 20166-4:2021)
This document specifies requirements and gives recommendations for the collection, handling, documentation, transport, storage and processing during the pre-examination phase of formalin-fixed and paraffin-embedded (FFPE) tissue specimens intended for qualitative and/or (semi-)quantitative in situ examination of the morphology and of biomolecules, such as metabolites, proteins, DNA and/or RNA, on FFPE tissue sections by using different in situ detection techniques.
This document is applicable to in vitro diagnostic examinations using in situ detection techniques. These include laboratory developed tests performed by pathology laboratories (histopathology laboratories) as well as by molecular pathology laboratories and other medical laboratories. It is also intended to be used by laboratory customers, in vitro diagnostics developers and manufacturers, biobanks, as well as institutions and commercial organizations performing biomedical research, and regulatory authorities.
This document is not applicable to the pre-examination phase of RNA, proteins and DNA isolated from FFPE tissue for examination. These are covered in ISO 20166-1, ISO 20166-2 and ISO 20166-3, Molecular in vitro diagnostic examinations — Specifications for pre-examination processes for isolated RNA, proteins and DNA, respectively.
Different dedicated measures are taken for pre-examination processes for fine needle aspirates (FNAs). These are covered in CEN WI 00140128, CEN WI 00140126, and CEN WI 00140129, Molecular in vitro diagnostic examinations — Specifications for pre-examination processes for Fine Needle Aspirates (FNAs) isolated cellular RNA, isolated proteins, and isolated genomic DNA, respectively.
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 formalinfixierte und paraffineingebettete (FFPE-) Gewebeproben - Teil 4: In-situ-Detektionstechniken (ISO 20166-4:2021)
Dieses Dokument legt Anforderungen an die Entnahme, Handhabung, Dokumentation, den Transport sowie die Lagerung und Verarbeitung von formalinfixierten und paraffineingebetteten (FFPE )Gewebeproben während der präanalytischen Phase fest, die für die qualitative und/oder (semi )quantitative In situ-Untersuchung der Morphologie und von Biomolekülen, beispielsweise Metaboliten, Proteinen, DNA und/oder RNA, an FFPE Gewebeschnitten mittels verschiedener In situ-Nachweisverfahren vorgesehen sind, und enthält entsprechende Empfehlungen.
Dieses Dokument ist anzuwenden für In vitro-diagnostische Untersuchungen, bei denen In situ-Nachweisverfahren angewendet werden. Dazu zählen auch im Labor entwickelte Prüfverfahren, die von pathologischen Laboratorien (histopathologischen Laboratorien) sowie von molekularpathologischen Laboratorien und anderen medizinischen Laboratorien durchgeführt werden. Es soll auch von Laborkunden, Entwicklern und Herstellern von In vitro-Diagnostika, Biobanken, Einrichtungen und kommerziellen Organisationen, die in der biomedizinischen Forschung tätig sind, sowie Aufsichtsbehörden eingesetzt werden.
Dieses Dokument ist nicht anzuwenden für die präanalytische Phase der Untersuchung von RNA, Proteinen und DNA, die zu Untersuchungszwecken aus FFPE Gewebeproben isoliert wurden. Diese werden in ISO 20166 1, ISO 20166 2 und ISO 20166 3, Molecular in vitro diagnostic examinations — Specifications for pre-examination processes for isolated RNA, proteins and DNA behandelt.
Für die präanalytischen Prozesse von Feinnadelaspiraten (FNAs) sind andere zweckbestimmte Maßnahmen zu treffen. Diese werden in CEN WI 00140128, CEN WI 00140126 und CEN WI 00140129, Molecular in vitro diagnostic examinations — Specifications for pre-examination processes for Fine Needle Aspirates (FNAs) isolated cellular RNA, isolated proteins, and isolated genomic DNA behandelt.
ANMERKUNG Für bestimmte Bereiche, die in diesem Dokument behandelt werden, können auch internationale, nationale oder regionale Bestimmungen oder Anforderungen gelten.
Analyses de diagnostic moléculaire in vitro - Spécifications relatives aux processus préanalytiques pour les tissus fixés au formol et inclus en paraffine (FFPE) - Partie 4: Techniques de détection in situ (ISO 20166-4:2021)
Le présent document spécifie des exigences et formule des recommandations concernant le prélèvement, la manipulation, la documentation, le transport, le stockage et le traitement durant la phase préanalytique de prélèvements de tissus fixés au formol et inclus en paraffine (FFPE) destinés à l’analyse in situ qualitative et/ou (semi‑)quantitative de la morphologie et de biomolécules, telles que des métabolites, des protéines, l’ADN et/ou l’ARN sur des coupes de tissu FFPE à l’aide de différentes techniques de détection in situ.
Le présent document s’applique aux analyses de diagnostic in vitro, utilisant des techniques de détection in situ. Celles‑ci englobent les analyses développées en laboratoire réalisées par des laboratoires de pathologie (laboratoires d’histopathologie) ainsi que par des laboratoires de pathologie moléculaire et autres laboratoires de biologie médicale. Il est également destiné à être utilisé par des clients de laboratoires, des développeurs et fabricants de l’industrie du diagnostic in vitro, des biobanques, ainsi que par des institutions et des organismes commerciaux spécialisés en recherche biomédicale, de même que des autorités de réglementation.
Le présent document ne s’applique pas à la phase préanalytique de l’analyse d’ARN, de protéines et d’ADN extraits de tissus FFPE, ces biomolécules étant respectivement couvertes par les normes ISO 20166‑1, ISO 20166‑2 et ISO 20166‑3, Analyses de diagnostic moléculaire in vitro — Spécifications relatives aux processus préanalytiques pour l’ARN extrait, les protéines extraites et l’ADN extrait.
Des mesures spéciales différentes sont prises pour les processus préanalytiques applicables aux prélèvements de biopsie à l’aiguille fine (BAF). Les mesures applicables aux différents prélèvements BAF (ARN cellulaire extrait, protéines extraites, et ADN génomique extrait) sont respectivement traitées par les CEN WI 00140128, CEN WI 00140126, et CEN WI 00140129, Molecular in vitro diagnostic examinations — Specifications for pre‑examination processes for Fine Needle Aspirates (FNAs) isolated cellular RNA, isolated proteins, and isolated genomic DNA (disponibles en anglais seulement).
NOTE Des réglementations ou exigences internationales, nationales ou régionales peuvent également s’appliquer à des sujets spécifiques traités dans le présent document.
Molekularne diagnostične preiskave in vitro - Specifikacije za predpreiskovalne procese za tkiva, ki so fiksirana v formalinu ter položena v parafin - 4. del: Tehnike detekcije in situ (ISO 20166-4:2021)
General Information
Standards Content (Sample)
SLOVENSKI STANDARD
01-oktober-2021
Molekularne diagnostične preiskave in vitro - Specifikacije za predpreiskovalne
procese za tkiva, ki so fiksirana v formalinu ter položena v parafin - 4. del: Tehnike
detekcije in situ (ISO 20166-4:2021)
Molecular in vitro diagnostic examinations - Specifications for preexamination processes
for formalin-fixed and paraffin-embedded (FFPE) tissue - Part 4: In situ detection
techniques (ISO 20166-4:2021)
Molekularanalytische in-vitro-diagnostische Verfahren - Spezifikationen für
präanalytische Prozesse für formalinfixierte und paraffineingebettete (FFPE-)
Gewebeproben - Teil 4: In-situ-Detektionstechniken (ISO 20166-4:2021)
Analyses de diagnostic moléculaire in vitro - Spécifications relatives aux processus
préanalytiques pour les tissus fixés au formol et inclus en paraffine (FFPE) - Partie 4:
Techniques de détection in situ (ISO 20166-4:2021)
Ta slovenski standard je istoveten z: EN ISO 20166-4:2021
ICS:
11.100.10 Diagnostični preskusni In vitro diagnostic test
sistemi in vitro systems
2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.
EN ISO 20166-4
EUROPEAN STANDARD
NORME EUROPÉENNE
July 2021
EUROPÄISCHE NORM
ICS 11.100.10
English Version
Molecular in vitro diagnostic examinations - Specifications
for preexamination processes for formalin-fixed and
paraffin-embedded (FFPE) tissue - Part 4: In situ detection
techniques (ISO 20166-4:2021)
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 4: Techniques de détection in situ (ISO Gewebeproben - Teil 4: In-situ-Detektionstechniken
20166-4:2021) (ISO 20166-4:2021)
This European Standard was approved by CEN on 20 May 2021.
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, 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
© 2021 CEN All rights of exploitation in any form and by any means reserved Ref. No. EN ISO 20166-4:2021 E
worldwide for CEN national Members.
Contents Page
European foreword . 3
European foreword
This document (EN ISO 20166-4:2021) 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 January 2022, and conflicting national standards shall
be withdrawn at the latest by July 2024.
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/national committee. A complete listing of these bodies can be found on the CEN websites.
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, 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.
Endorsement notice
The text of ISO 20166-4:2021 has been approved by CEN as EN ISO 20166-4:2021 without any
modification.
INTERNATIONAL ISO
STANDARD 20166-4
First edition
2021-07
Molecular in vitro diagnostic
examinations — Specifications
for preexamination processes for
formalin-fixed and paraffin-embedded
(FFPE) tissue —
Part 4:
In situ detection techniques
Analyses de diagnostic moléculaire in vitro — Spécifications relatives
aux processus préanalytiques pour les tissus fixés au formol et inclus
en paraffine (FFPE) —
Partie 4: Techniques de détection in situ
Reference number
ISO 20166-4:2021(E)
©
ISO 2021
ISO 20166-4:2021(E)
© ISO 2021
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
Email: copyright@iso.org
Website: www.iso.org
Published in Switzerland
ii © ISO 2021 – All rights reserved
ISO 20166-4:2021(E)
Contents Page
Foreword .iv
Introduction .v
1 Scope . 1
2 Normative references . 1
3 Terms and definitions . 1
4 General considerations . 8
5 Activities outside the laboratory .10
5.1 Specimen collection .10
5.1.1 General.10
5.1.2 Information about the patient/specimen donor .10
5.1.3 Information about the specimen .10
5.1.4 Specimen processing, intermediate storage and preparation for transport .11
5.2 Transport .12
6 Activities inside the laboratory .12
6.1 Specimen reception .12
6.2 Formalin fixation of the specimen or sample(s) .12
6.3 Evaluation of the pathology of the specimen and selection of the sample(s) .13
6.3.1 General.13
6.3.2 Macroscopic evaluation .14
6.3.3 Selection of the sample(s).14
6.4 Post-fixation of frozen samples from intraoperative consultation .15
6.5 Sample decalcification/softening .15
6.6 Tissue processing and paraffin embedding .16
6.6.1 General.16
6.6.2 Dehydration and clearing .16
6.6.3 Impregnation with paraffin and paraffin embedding .17
6.7 Storage of FFPE tissue blocks .17
6.8 Sectioning of FFPE tissue blocks and storage of slide-mounted sections .18
6.9 Deparaffinization and rehydration of slide-mounted sections .19
6.10 Pretreatment of slide-mounted sections for in situ detection techniques .19
6.10.1 General.19
6.10.2 Pretreatment for antibody- or other affinity binder-based in situ detection
techniques .19
6.10.3 Pretreatment for hybridization-based in situ detection techniques.20
6.10.4 Pretreatment for other in situ detection techniques .20
6.11 Quality assessment of the pre-analytical part of in situ detection .20
Annex A (informative) Recommendations relating to verification and validation of
laboratory developed in situ detection tests . .21
Annex B (informative) Example protocol for tissue processing .22
Bibliography .23
ISO 20166-4:2021(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.
A list of all parts in the ISO 20166 series can be found on the ISO website.
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.
iv © ISO 2021 – All rights reserved
ISO 20166-4:2021(E)
Introduction
Molecular in vitro diagnostics, including molecular pathology, has enabled significant progress
in medicine. Further progress is expected by new technologies analyzing tissue morphology and
biomolecules, such as (e.g. proteins, DNA, RNA and/or metabolites (e.g. glucose) in human tissues and
body fluids.
In pathology, the majority of diagnoses are based on in situ staining of formalin-fixed and paraffin-
embedded (FFPE) tissue sections. In the context of personalized medicine, classical histological staining
(e.g. hematoxylin and eosin) for morphological evaluation is increasingly complemented by additional
in situ detection techniques, such as immunohistochemistry or in situ hybridization, as well as
molecular analysis of isolated biomolecules. For example, many regulatory bodies approved companion
diagnostics in oncology are based on in situ detection techniques applied on FFPE tissue sections.
Developments in personalized medicine and new technologies, such as multi-label immunostaining
and computer-based analysis of digital images (e.g. generated by using a slide scanner) pose new
requirements on standardization of pre-analytical procedures to obtain reproducible qualitative and
quantitative results.
Profiles and/or integrity of biomolecules and their in situ localization, amount and accessibility for
in situ detection in tissues can change drastically during the pre-examination process comprising
specimen collection, tissue processing, embedding, transport, storage, sectioning and pretreatment for
in situ detection. This makes the outcome from in situ detection in diagnostics or research unreliable or
even impossible because the subsequent examination will not represent the in vivo state of molecules,
but instead, an artificial profile or morphology generated during the pre-examination process.
Therefore, a standardization of the entire pre-examination process of FFPE tissue specimens intended
for in situ examinations of morphology and biomolecules on FFPE tissue sections by using different in
situ detection techniques, is needed.
There is multiple scientific evidence that several factors of the pre-examination phase influence the
outcome (e.g. quality or quantity in terms of specificity or sensitivity) of in situ detection and, thus, can
have major impact on the diagnostic results.
This document draws upon such work to organize and standardize the steps for formalin-fixed and
paraffin-embedded (FFPE) tissue with regard to various in situ detection techniques in what is referred
to as the pre-examination phase. This document is for the pre-examination phase of in situ detection
techniques and is applicable to the whole spectrum of in situ detection techniques.
These include but are not limited to:
— Classical histological staining, e.g. Hematoxylin & Eosin staining (H&E);
— Histochemical techniques, e.g. Lipid staining, Periodic Acid Schiff (PAS) reaction, Perls’ Prussian
Blue reaction, Feulgen’s reaction, enzyme histochemistry;
— Immunohistochemical staining (IHC) or immunofluorescence staining using antibodies (polyclonal,
monoclonal or recombinant antibodies) or other affinity binders;
— Hybridization-based techniques such as RNA or DNA in situ hybridization (ISH) techniques, e.g.
fluorescence in situ hybridization (FISH), chromogenic in situ hybridization (CISH), or silver
enhanced in situ hybridization (SISH);
— Molecular analysis of isolated biomolecules that can be mapped to a defined region of an FFPE
section (by e.g. in situ sequencing, imaging mass spectrometry).
In this document, the following verbal forms are used:
— "shall" indicates a requirement;
— "should" indicates a recommendation;
ISO 20166-4:2021(E)
— "may" indicates a permission;
— "can" indicates a possibility or a capability.
vi © ISO 2021 – All rights reserved
INTERNATIONAL STANDARD ISO 20166-4:2021(E)
Molecular in vitro diagnostic examinations —
Specifications for preexamination processes for formalin-
fixed and paraffin-embedded (FFPE) tissue —
Part 4:
In situ detection techniques
1 Scope
This document specifies requirements and gives recommendations for the collection, handling,
documentation, transport, storage and processing during the pre-examination phase of formalin-fixed
and paraffin-embedded (FFPE) tissue specimens intended for qualitative and/or (semi-)quantitative
in situ examination of the morphology and of biomolecules, such as metabolites, proteins, DNA and/or
RNA, on FFPE tissue sections by using different in situ detection techniques.
This document is applicable to in vitro diagnostic examinations using in situ detection techniques. These
include laboratory developed tests performed by pathology laboratories (histopathology laboratories)
as well as by molecular pathology laboratories and other medical laboratories. It is also intended to be
used by laboratory customers, in vitro diagnostics developers and manufacturers, biobanks, as well as
institutions and commercial organizations performing biomedical research, and regulatory authorities.
This document is not applicable to the pre-examination phase of RNA, proteins and DNA isolated from
FFPE tissue for examination. These are covered in ISO 20166-1, ISO 20166-2 and ISO 20166-3, Molecular
in vitro diagnostic examinations — Specifications for pre-examination processes for isolated RNA,
proteins and DNA, respectively.
Different dedicated measures are taken for pre-examination processes for fine needle aspirates (FNAs).
These are covered in CEN WI 00140128, CEN WI 00140126, and CEN WI 00140129, Molecular in vitro
diagnostic examinations — Specifications for pre-examination processes for Fine Needle Aspirates
(FNAs) isolated cellular RNA, isolated proteins, and isolated genomic DNA, respectively.
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, Medical laboratories — Requirements for quality and competence
ISO 15190, Medical laboratories — Requirements for safety
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/
ISO 20166-4:2021(E)
3.1
affinity binder
molecules including affibodies, peptides, antibody (3.3) fragments or other small molecules that interact
with biomolecules (3.6) and structures in a cell, and can be used in in situ detection (3.27) techniques
3.2
ambient temperature
unregulated temperature of the surrounding air
[SOURCE: ISO 20166-1:2018, 3.2]
3.3
antibody
protein (3.36) (immunoglobulin) produced and secreted by B lymphocytes in response to a molecule
recognized as foreign (antigen (3.4)) and which is capable of binding to that specific antigen (3.4)
[SOURCE: ISO 16577:2016, 3.10, modified — “Note 1 to entry” has been deleted.]
3.4
antigen
substance that stimulates the production of antibodies (3.3) and reacts with them
[SOURCE: ISO 15089:2000, 3.5]
3.5
antigen retrieval
epitope retrieval
procedure(s) to unmask antigens (3.4)/epitopes (3.14) and restore their binding properties for antibodies
(3.3) used in immunohistochemistry (3.25) by neutralizing the modifications introduced by formalin
fixation (3.19), tissue processing (3.47) and paraffin embedding (3.33) of tissue
3.6
biomolecule
organic molecule produced in living organisms that is involved in the maintenance and metabolic
processes of organisms
Note 1 to entry: The examples of organic molecule are protein (3.37), carbohydrate, lipid, or nucleic acid.
3.7
clearing
process step in tissue processing (3.47) in which formalin-fixed tissue is transferred from dehydration
(3.10) reagent to clearing agent (for example, xylene) to prepare the tissue for impregnation (3.26)
3.8
cold ischemia
condition after removal of the tissue from the body until its stabilization or fixation
[SOURCE: ISO 20166-1:2018, 3.5]
3.9
decalcification
technique using chemical agents for removal of mineral (inorganic calcium) from bone or other calcified
tissue to adjust the hard tissue components to the softness of paraffin (3.32) for sectioning
3.10
dehydration
process step in tissue processing (3.47) for removal of water from formalin-fixed tissue by immersing
the tissue in a series of dehydrating reagent solutions of increasing concentration finishing with water
free (100 %) solution
2 © ISO 2021 – All rights reserved
ISO 20166-4:2021(E)
3.11
deviation
departure from an approved instruction, procedure and/or method
[SOURCE: ISO 15378:2017, 3.7.5 modified — “approved (3.7.1) standard operating procedure (SOP)
(3.7.10)” replaced by “approved instruction, procedure and/or method”.]
3.12
diagnosis
identification of a health or disease state from its signs and/or symptoms, where the diagnostic process
can involve examinations (3.15) 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
[SOURCE: ISO 20166-1:2018, 3.7]
3.13
DNA
deoxyribonucleic acid
polymer of deoxyribonucleotides occurring in a double-stranded (dsDNA) or single-stranded (ssDNA)
form
[SOURCE: ISO 22174:2005, 3.1.2]
3.14
epitope
antibody (3.3) binding site on a biomolecule (3.6) that is an antigen (3.4)
3.15
examination
analytical test
set of operations with the objective of determining the value or characteristics of a property
Note 1 to entry: Processes that start with the in situ detection (3.27) using antibodies (3.3), nucleic acid probes
or dyes 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.]
3.16
examination manufacturer
analytical test manufacturer
entity that manufactures and/or produces the specific analytical test (3.15)
3.17
FFPE tissue
formalin-fixed, paraffin-embedded tissue
tissue specimens (3.42)/samples (3.41) having undergone fixation in formalin (3.18, 3.19), tissue
processing (3.47), and paraffin embedding (3.33) in a tissue cassette
3.18
formalin
saturated aqueous formaldehyde solution which at 100 % contains 37 % formaldehyde by mass
(corresponding to 40 % by volume)
[SOURCE: ISO 20166-1:2018, 3.11]
ISO 20166-4:2021(E)
3.19
formalin fixation
treatment of a sample (3.41) with standard buffered formalin solution (3.45) for stabilization
[SOURCE: ISO 20166-1:2018, 3.12]
3.20
formalin pigment
acid formalin haematin pigment acid
hematin
black to brown amorphous to microcrystalline granules representing an artefact in histologic sections
prepared from tissues fixed in formalin (3.18, 3.19) having an increased formic acid concentration,
which is produced by acid acting upon haemoglobin
3.21
grossing
gross examination
inspection of pathology specimens (3.42) with the bare eye to obtain diagnostic information, while
being processed for further microscopic examination (3.15)
[SOURCE: ISO 20166-1:2018, 3.13]
3.22
histochemical technique(s)
in situ detection (3.27) technique(s) for the visualization and characterization of biomolecules (3.6) that
involves chemical reactions with specific groups, radicals, or chemical bonds in biomolecules (3.6) and
provide(s) information on the biomolecules’ (3.6) in situ localization in tissue sections (3.49)
Note 1 to entry: The examples of biomolecules (3.6) are carbohydrates, lipids, other metabolites, proteins (3.36),
amino acids, nucleic acids, pigments, or enzymes etc.
3.23
histological staining
in situ detection (3.27) technique undertaken to prepare tissue sections (3.49) by using histological
stains (e.g. Haematoxylin-Eosin (HE), Chromotrop-Anilinblue (CAB)) to highlight features of the tissue
section (3.49) and enhance tissue section (3.49) contrast
3.24
homogeneous
uniform in structure and composition
[SOURCE: ISO 20166-1:2018, 3.31]
3.25
immunohistochemistry
IHC
in situ detection (3.27) technique that uses the principle of antibodies (3.3) binding specifically to
antigens (3.4) / epitopes (3.14) in biological tissues or cells to visualize antigens (3.4) (e.g. proteins
(3.36)) using brightfield microscopy
3.26
impregnation
impregnation with paraffin
process step in tissue processing (3.47) for replacement of clearing agent and infiltration of tissue with
molten paraffin (3.32)
4 © ISO 2021 – All rights reserved
ISO 20166-4:2021(E)
3.27
in situ detection
technique that allows for precise localization of a specific biomolecule (3.6) within a slide-mounted
section
EXAMPLE Immunohistochemistry (3.25) for protein (3.36) detection, in situ hybridization (3.30) for nucleic
acid detection, histological staining (3.23), histochemical techniques (3.22), MALDI imaging mass spectrometry,
imaging mass cytometry, in situ Raman spectroscopy.
3.28
in situ detection system
detection system
set of reagents used to visualize the binding of molecules to their target in situ within a tissue section
(3.49)
Note 1 to entry: Examples of molecules are antibody (3.3), affinity binder (3.1), and nucleic acid.
Note 2 to entry: Most common for in situ detection (3.27) are enzyme- and fluorophore-based detection systems.
3.29
in situ examination
analytical in situ test
examination (3.15) based on in situ detection (3.27)
3.30
in situ hybridization
ISH
in situ detection (3.27) technique that uses the principle of complementary nucleic acid probes binding
specifically to segments of RNA (3.37) or DNA (3.13) in biological tissues or cells to visualize nucleic
acids (e.g. DNA (3.13) or RNA (3.37)) using brightfield or fluorescence microscope
3.31
nonconformity
nonfulfillment of a requirement
[SOURCE: ISO 9000:2015, 3.6.9 modified — Note 1 removed.]
3.32
paraffin
paraffin wax
product obtained from distillates, consisting essentially of a mixture of saturated hydrocarbons, solid
at room temperatures (3.40) used for paraffin embedding (3.33) of formalin-fixed tissue specimens
(3.42)/samples (3.41)
3.33
paraffin embedding
embedding in paraffin
process following tissue processing (3.47) in which a tissue sample (3.41), is placed in melted paraffin
(3.32) to achieve a hard surrounding matrix so that thin microscopic sections can be cut
3.34
post-translational modification
modifications on a protein (3.36), catalysed by enzymes, after its translation by ribosomes is complete
that can be the addition of a functional group covalently to a protein (3.36) such as phosphorylation
and neddylation, the proteolytic processing and/or folding processes necessary for a protein (3.36) to
mature functionally
ISO 20166-4:2021(E)
3.35
pre-examination process
pre-analytical workflow
pre-analytical phase
pre-examination phase
process that starts, in chronological order, from the clinician’s request and includes the examination
(3.15) request, preparation and identification of the patient, collection of the speciment(s) (3.42),
transportation to and within the medical laboratory, evaluation, tissue processing (3.47), paraffin
embedding (3.33), FFPE block storage (3.46) and retrieval, sectioning (including mounting onto glass
slides and drying), storage (3.46) of unstained slide-mounted tissue sections (3.49) (if applicable), and
pre-treatment steps for in situ detection (3.27) techniques, and ends when the analytical examination
(3.15) begins
Note 1 to entry: The pre-examination phase for in situ detection (3.27) includes preparative processes, e.g.
antigen/epitope retrieval (3.5) and pre-hybridization procedures, which influence the outcome of the intended
examination (3.15).
[SOURCE: ISO 15189:2012, 3.15, modified — An additional term was added, and more details were
included.]
3.36
protein
type of biological biomolecules (3.6) composed of one or more chains with a defined sequence of amino
acids connected through peptide bonds
[SOURCE: ISO 20166-2:2018, 3.14, modified – The terms "biological macromolecules" were replaced
with "biological biomolecules"]
3.37
RNA
ribonucleic acid
polymer of ribonucleotides occurring in a double-stranded or single-stranded form
[SOURCE: ISO 20166-1:2018, 3.20]
3.38
RNA profile
amounts of the individual RNA (3.37) molecules that are present in a sample (3.41) and that can be
measured in the absence of any losses, inhibition and interference
[SOURCE: ISO 20166-1:2018, 3.19]
3.39
RNase
ribonuclease
enzyme that catalyzes the degradation of RNA (3.37) into smaller components
[SOURCE: ISO 20166-1:2018, 3.21]
3.40
room temperature
for the purpose 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.
[SOURCE: ISO 20166-1:2018, 3.22]
3.41
sample
one or more parts taken from a specimen (3.42)
[SOURCE: ISO 15189:2012, 3.24, modified — EXAMPLE has been removed.]
6 © ISO 2021 – All rights reserved
ISO 20166-4:2021(E)
3.42
specimen
primary sample
discrete portion of a body fluid, breath, hair or tissue taken for examination (3.15), 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 is used here without the original
notes.]
3.43
specimen container
container into which the tissue specimen (3.42) is transferred after its collection for further pre-
analytical workflow (3.35) steps (e.g. transport and/or fixation)
3.44
stability
ability of a sample (3.41) 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 analytes for the purpose of this document are biomolecules (3.6) located in situ/in the tissue.
[SOURCE: ISO 20166-1:2018, 3.24, modified — Note 1 to entry has been deleted and new Note 1 to entry
has been added.]
3.45
standard buffered formalin solution
neutral buffered formalin
NBF
10 % formalin (3.18) 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.
[SOURCE: ISO 20166-1:2018, 3.25]
3.46
storage
maintenance of biological material under appropriate conditions for intended use
3.47
tissue processing
process during the generation of FFPE tissue (3.17) consisting of the four consecutive steps tissue
fixation, dehydration (3.10), clearing (3.7), and impregnation with paraffin (3.26)
3.48
tissue processor
automated instrument where tissue fixation, dehydration (3.10), clearing (3.7) and impregnation (with
paraffin) (3.26) occur
[SOURCE: ISO 20166-1:2018, 3.27]
3.49
tissue section
very thin slice of tissue generated by cutting tissue with a dedicated section cutting device (i.e.
microtome)
Note 1 to entry: FFPE tissue (3.17) is cut using a microtome to generate (µm-) thin sections that are mounted onto
glass slides for performing in situ detection (3.27) techniques.
ISO 20166-4:2021(E)
3.50
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: The word "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.]
3.51
verification
confirmation, through 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, and
— reviewing documents prior to issue.
[SOURCE: ISO 9000:2015, 3.8.4]
3.52
warm ischemia
condition before the tissue is removed from the body, but deprived of its normal blood supply
Note 1 to entry: Disruption of normal blood supply varies significantly case by case. Therefore, warm ischemia
duration and its effects depend on individual cases and it also can depend on the anatomy of the arterial blood
supply.
[SOURCE: ISO 20166-1:2018, 3.29]
3.53
workflow
structured series of activities necessary to complete a task
[SOURCE: ISO 20166-1:2018, 3.30, modified - The word “structured” was added]
4 General considerations
For general statements on medical laboratory quality management systems and in particular on
specimen collection, receipt, and handling (including avoidance of cross contaminations) see ISO 15189,
ISO/IEC 17025 or ISO/IEC 17020. ISO/TS 20658 and ISO 20387 (for biobanking) can also apply. The
requirements on laboratory equipment, reagents, and consumables according to ISO 15189 shall be
followed; ISO/IEC 17025 and ISO/IEC 17020 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 verified and
validated. 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), transportation to and
within the medical laboratory, fixation, processing, embedding, sectioning and storage.
Pre-analytical workflow steps, which cannot always be controlled (e.g. duration and condition of warm
ischemia depending on the surgical procedure), shall be documented. A risk assessment of pre-analytical
8 © ISO 2021 – All rights reserved
ISO 20166-4:2021(E)
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.
NOTE 1 Disruption of normal blood supply varies significantly case by case. Therefore, warm ischemia
duration and its effects depend on individual cases and it can also depend on the anatomy of the arterial blood
supply.
The preservation of tissue morphology and the stability of the specific biomolecules of interest and
their post-translational modifications (if important for the analytical test) should be investigated
throughout the complete pre-examination process prior to the development and implementation of an
[2-4]
analytical test (e.g. by performing a time course experiment or study; see references ).
Before tissues are fixed in standard buffered formalin solution, biomolecule in situ/subcellular
localization, amounts, conformations and binding status can change, e.g. by biomolecule degradation,
washing out, crosslinking and altered synthesis following e.g. gene induction, gene down regulation,
RNA degradation, changes in post translational modification of proteins and changes of the biochemical
pathway and energy status. These effects depend on the duration of warm and cold ischemia, and the
temperature before formalin fixation. In addition, the described effects can vary in different patients’/
donors’ tissues.
Generally, the longer the durations of warm and cold ischemia and the higher the temperature before
fixation of the tissue specimen, the higher is the risk that changes in biomolecule profile and in situ/
subcellular localization can occur.
NOTE 2 Biomolecule amounts, modifications, and in situ/subcellular location 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. Prolonged cold ischemia results in changes of protein and
[5,6,48] [7]
phosphoprotein amounts . Keeping the specimen at low temperature (e.g. 4° C diminishes this effect. RNA
profiles can change significantly depending on the warm and cold ischemia duration and the temperature before
[8,9]
formalin fixation (e.g. gene induction, gene down regulation, RNA degradation) . Changes of DNA, sequence
or copy numbers (e.g. comparative genomic hybridization (CGH) profiles) due to longer warm and cold ischemia
[10] [11]
durations are unknown . However, DNA methylation patterns can change in response to ischemia .
Where relevant, warm ischemia duration shall be documented (e.g. for large tumor resections). Where
it is not possible to avoid cold ischemia (e.g. due to transport to the laboratory before formalin fixation),
its duration shall be documented, and the temperatures of the specimen container’s surroundings shall
be documented.
Formalin fixation (including quality of formalin solution, ratio of sample volume to formalin
solution volume, duration and temperature of fixation) and other pre-examination processes can
[12]
impact morphology, cause modifications of biomolecules, and lead to suboptimal analytical test
performance affecting results obtained by in situ detection. These pre-examination processes include
[13] [13] [13]
tissue processing, embedding, storage of FFPE tissue blocks, sectioning (including selection of
types of slides, section thickness, drying and storage of unstained slide mounted tissue sections). This
should be considered in the quality control and application of in situ detection tests. Optimization of
the analytical test for FFPE tissues and/or of the pretreatment of slide mounted FFPE tissue sections
(e.g. antigen retrieval, pre-hybridization; see 6.10.2 and 6.10.3) are options to minimize this issue for in
situ detection examinations.
Immunohistochemistry can be affected by modification or loss of immunoreactivity in FFPE tissue
[14]
due to denaturation of molecules, crosslinking of proteins, and thus loss or “masking” of epitopes or
[3,15]
steric interference of antibody binding to epitopes . Evidence shows that many antigens (epitopes)
[16,17]
can substantially, if not completely, be restored by antigen retrieval . Therefore, antigen retrieval
can be performed in order to restore immunoreactivity in FFPE tissue and/or to “retrieve” epitopes
“masked” or modified by formalin fixation, and tissue processing and embedding. Since optimal antigen
retrieval procedures depend on the pre-analytical conditions applied, antigen retrieval requirements
shall be verified in the context of the whole pre-analytical and analytical workflow.
NOTE 3 Extensive antigen retrieval can cause morphological artefacts.
ISO 20166-4:2021(E)
In situ hybridization can be affected by biomolecule properties such as nucleic acid integrity,
nucleic acid–nucleic acid interaction or nucleic acid–protein interaction, which can be reversed by
pre-hybridization procedures
...
Questions, Comments and Discussion
Ask us and Technical Secretary will try to provide an answer. You can facilitate discussion about the standard in here.
Loading comments...