EN 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

• Status: Published
• Publication Date: 27-Jul-2021
• ICS: 11.100.10 - In vitro diagnostic test systems
• Technical Committee: CEN/TC 140 - In vitro diagnostic systems
• Drafting Committee: CEN/TC 140/WG 3 - Quality management in the medical laboratory
• Current Stage: 6060 - Definitive text made available (DAV) - Publishing
• Start Date: 28-Jul-2021
• Completion Date: 28-Jul-2021
• Ref Project: SIST EN 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)

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: [phone]
Email: [e-mail]
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
...