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 gives requirements 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 examinations of morphology and biomolecules, such as metabolites, proteins, DNA and/or RNA in situ on FFPE tissue sections by using different in situ detection techniques.
This document is applicable to routine and molecular diagnostic examinations using in situ detection techniques including laboratory developed tests performed by routine pathology laboratories (histology laboratories) as well as molecular pathology laboratories and other medical laboratories. It is also intended to be used by laboratory customers, in vitro diagnostics developers and manufacturers, as well as institutions and commercial organizations performing biomedical research, biobanks, and regulatory authorities.
This document is not applicable for the examination of isolated biomolecules such as proteins, DNA and RNA that cannot be mapped with a defined region of a FFPE section.
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)

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)

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Current Stage
6060 - Definitive text made available (DAV) - Publishing
Due Date
28-Jul-2021
Completion Date
28-Jul-2021

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SLOVENSKI STANDARD
SIST EN ISO 20166-4:2021
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
SIST EN ISO 20166-4:2021 en,fr,de

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

---------------------- Page: 1 ----------------------
SIST EN ISO 20166-4:2021
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SIST EN ISO 20166-4:2021
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.
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SIST EN ISO 20166-4:2021
EN ISO 20166-4:2021 (E)
Contents Page

European foreword ....................................................................................................................................................... 3

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SIST EN ISO 20166-4:2021
EN ISO 20166-4:2021 (E)
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.
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SIST EN ISO 20166-4:2021
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SIST EN ISO 20166-4:2021
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
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SIST EN ISO 20166-4:2021
ISO 20166-4:2021(E)
COPYRIGHT PROTECTED DOCUMENT
© 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
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SIST EN ISO 20166-4:2021
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 2021 – All rights reserved iii
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SIST EN ISO 20166-4:2021
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
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SIST EN ISO 20166-4:2021
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 2021 – All rights reserved v
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SIST EN ISO 20166-4:2021
ISO 20166-4:2021(E)
— "may" indicates a permission;
— "can" indicates a possibility or a capability.
vi © ISO 2021 – All rights reserved
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SIST EN ISO 20166-4:2021
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 2021 – All rights reserved 1
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SIST EN ISO 20166-4:2021
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
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SIST EN ISO 20166-4:2021
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 2021 – All rights reserved 3
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SIST EN ISO 20166-4:2021
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 paraffi
...

SLOVENSKI STANDARD
oSIST prEN ISO 20166-4:2020
01-september-2020
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/DIS 20166-4:2020)

Molecular in vitro diagnostic examinations - Specifications for preexamination processes

for formalin-fixed and paraffin-embedded (FFPE) tissue - Part 4: In situ detection

techniques (ISO/DIS 20166-4:2020)
Molekularanalytische in-vitro-diagnostische Verfahren - Spezifikationen für
präanalytische Prozesse für formalinfixierte und paraffineingebettete (FFPE-)
Gewebeproben - Teil 4: In-situ-Detektionstechniken (ISO/DIS 20166-4:2020)

Analyses de diagnostic moléculaire in vitro - Spécifications relatives aux processus

préanalytiques pour les tissus fixés au formol et inclus en paraffine (FFPE) - Partie 4:

Titre manque (ISO/DIS 20166-4:2020)
Ta slovenski standard je istoveten z: prEN ISO 20166-4
ICS:
11.100.10 Diagnostični preskusni In vitro diagnostic test
sistemi in vitro systems
oSIST prEN ISO 20166-4:2020 en,fr,de

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

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oSIST prEN ISO 20166-4:2020
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oSIST prEN ISO 20166-4:2020
DRAFT INTERNATIONAL STANDARD
ISO/DIS 20166-4
ISO/TC 212 Secretariat: ANSI
Voting begins on: Voting terminates on:
2020-07-13 2020-10-05
Molecular in vitro diagnostic examinations —
Specifications for preexamination processes for formalin-
fixed and paraffin-embedded (FFPE) tissue —
Part 4:
In situ detection techniques
ICS: 11.100.10
THIS DOCUMENT IS A DRAFT CIRCULATED
This document is circulated as received from the committee secretariat.
FOR COMMENT AND APPROVAL. IT IS
THEREFORE SUBJECT TO CHANGE AND MAY
NOT BE REFERRED TO AS AN INTERNATIONAL
STANDARD UNTIL PUBLISHED AS SUCH.
IN ADDITION TO THEIR EVALUATION AS
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ISO/DIS 20166-4:2020(E)
RECIPIENTS OF THIS DRAFT ARE INVITED
TO SUBMIT, WITH THEIR COMMENTS,
NOTIFICATION OF ANY RELEVANT PATENT
RIGHTS OF WHICH THEY ARE AWARE AND TO
PROVIDE SUPPORTING DOCUMENTATION. ISO 2020
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COPYRIGHT PROTECTED DOCUMENT
© ISO 2020

All rights reserved. Unless otherwise specified, or required in the context of its implementation, no part of this publication may

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oSIST prEN ISO 20166-4:2020
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Contents Page

Foreword ........................................................................................................................................................................................................................................iv

Introduction ..................................................................................................................................................................................................................................v

1 Scope ................................................................................................................................................................................................................................. 1

2 Normative references ...................................................................................................................................................................................... 1

3 Terms and definitions ..................................................................................................................................................................................... 1

4 General considerations .................................................................................................................................................................................. 8

5 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 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 Macroscopic evaluation ..........................................................................................................................................14

6.3.2 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

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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 2020 – All rights reserved
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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 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 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 standard 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;
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— "may" indicates a permission;
— "can" indicates a possibility or a capability.
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oSIST prEN ISO 20166-4:2020
DRAFT INTERNATIONAL STANDARD ISO/DIS 20166-4:2020(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 gives requirements and 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, -2 and -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

ISO/IEC 17020, 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.

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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
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 (3.37) (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 (e.g. 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

2 © ISO 2020 – All rights reserved
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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
3.11
deviation

departure from an approved instruction, procedure and/or method or established standard

[SOURCE: ISO 15378:2017(en), 3.7.5 modified — The words “approved (3.7.1) standard operating

procedure (SOP) (3.7.10)” were replaced by “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 (3.36)/samples (3.42) 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]
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3.19
formalin fixation

treatment of a sample (3.42) 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 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

provides 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 (3.37),

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

(3.37)) 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)
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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 (3.37) 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 its 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 (3.38) or DNA (3.13) in biological tissues or cells to visualize nucleic

acids (e.g. DNA (3.13) or RNA (3.38)) using brightfield or fluorescence microscope

3.31
nonconformity
nonfulfillment of a requirement
[SOURCE: ISO 9000:2005, 3.6.2]
3.32
paraffin
paraffin wax

product obtained from distillates, consisting essentially of a mixture of saturated hydrocarbons, solid

at room temperatures (3.41) used for paraffin embedding (3.33) of formalin-fixed tissue specimens

(3.36)/samples (3.42)
3.33
paraffin embedding
embedding in paraffin

process following tissue processing (3.47) in which a tissue sample (3.42), 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 (3.37), catalysed by enzymes, after its translation by ribosomes is complete,

that can be the addition of a functional group covalently to a protein (3.37) such as phosphorylation

and neddylation, the proteolytic processing and/or folding processes necessary for a protein (3.37) to

mature functionally
[SOURCE: Nature.com, modified – The two sentences were summarized in one [1]]
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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 primary sample(s) (3.36),

transportation to and within the medical laboratory, evaluation, tissue processing (3.47), embedding,

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
primary sample
specimen

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 — Th
...

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