Molecular in vitro diagnostic examinations - Specifications for pre-examination processes for formalin-fixed and paraffin-embedded (FFPE) tissue - Part 1: Isolated RNA (ISO 20166-1:2018)

This document gives guidelines on the handling, documentation, storage and processing of formalin-fixed and paraffin-embedded (FFPE) tissue specimens intended for RNA examination during the pre-examination phase before a molecular assay is performed.
This document is applicable to molecular in vitro diagnostic examinations including laboratory developed tests performed by medical laboratories and molecular pathology laboratories. It is also intended to be used by laboratory customers, in vitro diagnostics developers and manufacturers, biobanks, institutions and commercial organizations performing biomedical research, and regulatory authorities.
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 1: Isolierte RNS (ISO 20166-1:2018)

Analyses de diagnostic moléculaire in vitro - Spécifications relatives aux processus préanalytiques pour les tissus fixés au formol et inclus en paraffine (FFPE) - Partie 1: ARN extrait (ISO 20166-1:2018)

Le présent document fournit des lignes directrices concernant la manipulation, la documentation, le stockage et le traitement de prélèvements tissus fixés au formol et inclus en paraffine (FFPE) destinés à l'analyse de l'ARN durant la phase préanalytique précédant la réalisation d'une analyse moléculaire.
Le présent document s'applique aux analyses de diagnostic moléculaire in vitro, y compris les analyses développées en laboratoire réalisées par des laboratoires de biologie médicale et des laboratoires de pathologie moléculaire. Il est également destiné à être utilisé par des clients de laboratoires, des développeurs et fabricants de l'industrie du diagnostic in vitro, ainsi que par des biobanques, des institutions et des organismes commerciaux spécialisés en recherche biomédicale, de même que des autorités de réglementation.
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 - 1. del: Izolirani RNK (ISO 20166-1:2018)

Ta mednarodni standard vsebuje priporočila za obravnavo, dokumentiranje, shranjevanje in obdelavo vzorcev tkiv, ki so fiksirana v formalinu ter položena v parafin (FFPE), namenjenih za analizo RNK med predpreiskovalno fazo, preden se izvede molekularni preskus. Ta mednarodni standard se uporablja za molekularne diagnostične preiskave in vitro, vključno z laboratorijsko razvitimi preskusi, ki jih izvajajo v medicinskih laboratorijih in laboratorijih za molekularno patologijo. Uporabljali naj bi ga tudi uporabniki laboratorijev, razvijalci in proizvajalci diagnostike in vitro, nanaša pa se tudi na institucije in komercialne organizacije, ki izvajajo biomedicinske raziskave, biobanke ter regulativne organe. Fiksacija v formalinu in postopek vstavitve v parafin vodita do sprememb molekul RNK, kar lahko vpliva na veljavnost in zanesljivost rezultatov preskusov preiskav. Profili RNK v tkivih se lahko močno spremenijo med zbiranjem in se različno spremenijo pri tkivih različnih darovalcev/bolnikov. Zato je nujno treba sprejeti posebne ukrepe, da se zmanjšajo opisane spremembe profila RNK v tkivu za nadaljnje preiskave.  OPOMBA:   Za določene teme, ki so zajete v tem mednarodnem standardu, lahko veljajo tudi mednarodni, nacionalni ali regionalni predpisi ali zahteve.

General Information

Status
Published
Publication Date
18-Dec-2018
Current Stage
6060 - Definitive text made available (DAV) - Publishing
Due Date
19-Dec-2018
Completion Date
19-Dec-2018

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SLOVENSKI STANDARD
SIST EN ISO 20166-1:2019
01-maj-2019
1DGRPHãþD
SIST-TS CEN/TS 16827-1:2015
0ROHNXODUQHGLDJQRVWLþQHSUHLVNDYHLQYLWUR6SHFLILNDFLMH]DSUHGSUHLVNRYDOQH

SURFHVH]DWNLYDNLVRILNVLUDQDYIRUPDOLQXWHUSRORåHQDYSDUDILQGHO,]ROLUDQL

51. ,62

Molecular in vitro diagnostic examinations - Specifications for pre-examination processes

for formalin-fixed and paraffin-embedded (FFPE) tissue - Part 1: Isolated RNA (ISO

20166-1:2018)
Molekularanalytische in-vitro-diagnostische Verfahren - Spezifikationen für
präanalytische Prozesse für formalinfixierte und paraffineingebettete (FFPE)-
Gewebeproben - Teil 1: Isolierte RNS (ISO 20166-1:2018)

Analyses de diagnostic moléculaire in vitro - Spécifications relatives aux processus

préanalytiques pour les tissus fixés au formol et inclus en paraffine (FFPE) - Partie 1:

ARN extrait (ISO 20166-1:2018)
Ta slovenski standard je istoveten z: EN ISO 20166-1:2018
ICS:
11.100.10 'LDJQRVWLþQLSUHVNXVQL In vitro diagnostic test
VLVWHPLLQYLWUR systems
SIST EN ISO 20166-1:2019 en

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

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SIST EN ISO 20166-1:2019
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SIST EN ISO 20166-1:2019
EN ISO 20166-1
EUROPEAN STANDARD
NORME EUROPÉENNE
December 2018
EUROPÄISCHE NORM
ICS 11.100.10 Supersedes CEN/TS 16827-1:2015
English Version
Molecular in vitro diagnostic examinations - Specifications
for pre-examination processes for formalin-fixed and
paraffin-embedded (FFPE) tissue - Part 1: Isolated RNA
(ISO 20166-1:2018)

Analyses de diagnostic moléculaire in vitro - Molekularanalytische in-vitro-diagnostische Verfahren

Spécifications relatives aux processus préanalytiques - Spezifikationen für präanalytische Prozesse für

pour les tissus fixés au formol et inclus en paraffine formalinfixierte und paraffineingebettete (FFPE)-

(FFPE) - Partie 1: ARN extrait (ISO 20166-1:2018) Gewebeproben - Teil 1: Isolierte RNS (ISO 20166-

1:2018)
This European Standard was approved by CEN on 22 November 2018.

This European Standard was corrected and reissued by the CEN-CENELEC Management Centre on 30 January 2019.

CEN members are bound to comply with the CEN/CENELEC Internal Regulations which stipulate the conditions for giving this

European Standard the status of a national standard without any alteration. Up-to-date lists and bibliographical references

concerning such national standards may be obtained on application to the CEN-CENELEC Management Centre or to any CEN

member.

This European Standard exists in three official versions (English, French, German). A version in any other language made by

translation under the responsibility of a CEN member into its own language and notified to the CEN-CENELEC Management

Centre has the same status as the official versions.

CEN members are the national standards bodies of Austria, Belgium, Bulgaria, Croatia, Cyprus, Czech Republic, Denmark, Estonia,

Finland, Former Yugoslav Republic of Macedonia, France, Germany, Greece, Hungary, Iceland, Ireland, Italy, Latvia, Lithuania,

Luxembourg, Malta, Netherlands, Norway, Poland, Portugal, Romania, Serbia, Slovakia, Slovenia, Spain, Sweden, Switzerland,

Turkey and United Kingdom.
EUROPEAN COMMITTEE FOR STANDARDIZATION
COMITÉ EUROPÉEN DE NORMALISATIO N
EUROPÄISCHES KOMITEE FÜR NORMUN G
CEN-CENELEC Management Centre: Rue de la Science 23, B-1040 Brussels

© 2018 CEN All rights of exploitation in any form and by any means reserved Ref. No. EN ISO 20166-1:2018 E

worldwide for CEN national Members.
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SIST EN ISO 20166-1:2019
EN ISO 20166-1:2018 (E)
Contents Page

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

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SIST EN ISO 20166-1:2019
EN ISO 20166-1:2018 (E)
European foreword

This document (EN ISO 20166-1:2018) has been prepared by Technical Committee ISO/TC 212 "Clinical

laboratory testing and in vitro diagnostic test systems" in collaboration with Technical Committee

CEN/TC 140 “In vitro diagnostic medical devices” the secretariat of which is held by DIN.

This European Standard shall be given the status of a national standard, either by publication of an

identical text or by endorsement, at the latest by June 2019, and conflicting national standards shall be

withdrawn at the latest by December 2021.

Attention is drawn to the possibility that some of the elements of this document may be the subject of

patent rights. CEN shall not be held responsible for identifying any or all such patent rights.

This document supersedes CEN/TS 16827-1:2015.

According to the CEN-CENELEC Internal Regulations, the national standards organizations of the

following countries are bound to implement this European Standard: Austria, Belgium, Bulgaria,

Croatia, Cyprus, Czech Republic, Denmark, Estonia, Finland, Former Yugoslav Republic of Macedonia,

France, Germany, Greece, Hungary, Iceland, Ireland, Italy, Latvia, Lithuania, Luxembourg, Malta,

Netherlands, Norway, Poland, Portugal, Romania, Serbia, Slovakia, Slovenia, Spain, Sweden, Switzerland,

Turkey and the United Kingdom.
Endorsement notice

The text of ISO 20166-1:2018 has been approved by CEN as EN ISO 20166-1:2018 without any

modification.
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SIST EN ISO 20166-1:2019
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SIST EN ISO 20166-1:2019
INTERNATIONAL ISO
STANDARD 20166-1
First edition
2018-12
Molecular in vitro diagnostic
examinations — Specifications for pre-
examination processes for formalin-
fixed and paraffin-embedded (FFPE)
tissue —
Part 1:
Isolated RNA
Analyses de diagnostic moléculaire in vitro — Spécifications relatives
aux processus préanalytiques pour les tissus fixés au formol et inclus
en paraffine (FFPE) —
Partie 1: ARN extrait
Reference number
ISO 20166-1:2018(E)
ISO 2018
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SIST EN ISO 20166-1:2019
ISO 20166-1:2018(E)
COPYRIGHT PROTECTED DOCUMENT
© ISO 2018

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

be reproduced or utilized otherwise in any form or by any means, electronic or mechanical, including photocopying, or posting

on the internet or an intranet, without prior written permission. Permission can be requested from either ISO at the address

below or ISO’s member body in the country of the requester.
ISO copyright office
CP 401 • Ch. de Blandonnet 8
CH-1214 Vernier, Geneva
Phone: +41 22 749 01 11
Fax: +41 22 749 09 47
Email: copyright@iso.org
Website: www.iso.org
Published in Switzerland
ii © ISO 2018 – All rights reserved
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SIST EN ISO 20166-1:2019
ISO 20166-1:2018(E)
Contents Page

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

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

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

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

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

4  General considerations .................................................................................................................................................................................. 5

5  Outside the laboratory ................................................................................................................................................................................... 6

5.1 Specimen collection ............................................................................................................................................................................ 6

5.1.1 General...................................................................................................................................................................................... 6

5.1.2 Information about the specimen donor/patient .................................................................................. 6

5.1.3 Information about the specimen ....................................................................................................................... 6

5.1.4 Specimen processing .................................................................................................................................................... 6

5.2 Transport requirements ................................................................................................................................................................. 7

6  Inside the laboratory ....................................................................................................................................................................................... 7

6.1 Information about the reception of the specimen .................................................................................................... 7

6.2 Formalin fixation of the specimen or sample(s) ........................................................................................................ 8

6.3 Evaluation of the pathology of the specimen and selection of the sample(s) .................................. 9

6.4 Post-fixation of frozen samples .............................................................................................................................................10

6.5 Decalcification ......................................................................................................................................................................................10

6.6 Processing and paraffin embedding ..................................................................................................................................10

6.7 Storage requirements .....................................................................................................................................................................10

6.8 Isolation of RNA ..................................................................................................................................................................................11

6.8.1 General...................................................................................................................................................................................11

6.8.2 General information for RNA isolation procedures ........................................................................11

6.8.3 Using commercial kits ..............................................................................................................................................12

6.8.4 Using the laboratories’ own protocols .......................................................................................................12

6.9 Quantity and quality assessment of isolated RNA .................................................................................................13

6.10 Storage of isolated RNA ................................................................................................................................................................13

6.10.1 General...................................................................................................................................................................................13

6.10.2 Using commercially available kits for RNA isolation .....................................................................14

6.10.3 Using the laboratory's own protocols for RNA isolation ............................................................14

Annex A (informative) Quality control of RNA extracted from formalin-fixed and paraffin-

embedded tissue samples: implications for RT-qPCR based examinations .........................................15

Bibliography .............................................................................................................................................................................................................................21

© ISO 2018 – All rights reserved iii
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SIST EN ISO 20166-1:2019
ISO 20166-1:2018(E)
Foreword

ISO (the International Organization for Standardization) is a worldwide federation of national standards

bodies (ISO member bodies). The work of preparing International Standards is normally carried out

through ISO technical committees. Each member body interested in a subject for which a technical

committee has been established has the right to be represented on that committee. International

organizations, governmental and non-governmental, in liaison with ISO, also take part in the work.

ISO collaborates closely with the International Electrotechnical Commission (IEC) on all matters of

electrotechnical standardization.

The procedures used to develop this document and those intended for its further maintenance are

described in the ISO/IEC Directives, Part 1. In particular, the different approval criteria needed for the

different types of ISO documents should be noted. This document was drafted in accordance with the

editorial rules of the ISO/IEC Directives, Part 2 (see www .iso .org/directives).

Attention is drawn to the possibility that some of the elements of this document may be the subject of

patent rights. ISO shall not be held responsible for identifying any or all such patent rights. Details of

any patent rights identified during the development of the document will be in the Introduction and/or

on the ISO list of patent declarations received (see www .iso .org/patents).

Any trade name used in this document is information given for the convenience of users and does not

constitute an endorsement.

For an explanation of the voluntary nature of standards, the meaning of ISO specific terms and

expressions related to conformity assessment, as well as information about ISO's adherence to the

World Trade Organization (WTO) principles in the Technical Barriers to Trade (TBT) see www .iso

.org/iso/foreword .html.

The committee responsible for this document is 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 2018 – All rights reserved
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SIST EN ISO 20166-1:2019
ISO 20166-1:2018(E)
Introduction

Molecular in vitro diagnostics, including molecular pathology, has enabled significant progress in

medicine. Further progress is expected with new technologies analysing nucleic acids, proteins, and

metabolites in human tissues and body fluids. However, the profiles and/or integrity of these molecules

can change drastically during specimen collection, transport, storage and processing, thus making

the outcome from diagnostics or research unreliable or even impossible because the subsequent

examination assay will not determine the situation in the patient but an artificial profile generated

during the pre-examination process.

Therefore, a standardization of the entire process from specimen collection to the RNA examination is

needed. Studies have been undertaken to determine the important influencing factors. This document

draws upon such work to codify and standardize the steps for formalin-fixed and paraffin-embedded

(FFPE) tissue with regard to RNA examination in what is referred to as the pre-examination phase.

The formalin-fixation and the paraffin-embedding processes lead to modifications of the RNA

molecules, which can impact the validity and reliability of the examination test results.

RNA profiles in tissues can change drastically before, during and after collection and change differently

in different donors'/patients' tissues. Therefore, it is essential to take special measures to minimize the

described RNA profile changes and modifications within the tissue for subsequent examination.

In this document, the following verbal forms are used:
— "shall" indicates a requirement;
— "should" indicates a recommendation;
— "may" indicates a permission;
— "can" indicates a possibility or a capability.
© ISO 2018 – All rights reserved v
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SIST EN ISO 20166-1:2019
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SIST EN ISO 20166-1:2019
INTERNATIONAL STANDARD  ISO 20166-1:2018(E)
Molecular in vitro diagnostic examinations —
Specifications for pre-examination processes for formalin-
fixed and paraffin-embedded (FFPE) tissue —
Part 1:
Isolated RNA
1 Scope

This document gives guidelines on the handling, documentation, storage and processing of formalin-

fixed and paraffin-embedded (FFPE) tissue specimens intended for RNA examination during the pre-

examination phase before a molecular assay is performed.

This document is applicable to molecular in vitro diagnostic examinations including laboratory

developed tests performed by medical laboratories and molecular pathology laboratories. It is also

intended to be used by laboratory customers, in vitro diagnostics developers and manufacturers,

biobanks, institutions and commercial organizations performing biomedical research, and regulatory

authorities.

NOTE International, national or regional regulations or requirements can also apply to specific topics

covered in this document.
2  Normative references

The following documents are referred to in the text in such a way that some or all of their content

constitutes requirements of this document. For dated references, only the edition cited applies. For

undated references, the latest edition of the referenced document (including any amendments) applies.

ISO 15189:2012, Medical laboratories — Requirements for quality and competence
ISO 15190, Medical laboratories — Requirements for safety

ISO/IEC 17020:2012, Conformity assessment — Requirements for the operation of various types of bodies

performing inspection
3  Terms and definitions

For the purposes of this document, the terms and definitions given in ISO 15189 and the following apply.

ISO and IEC maintain terminological databases for use in standardization at the following addresses:

— ISO Online browsing platform: available at https: //www .iso .org/obp
— IEC Electropedia: available at http: //www .electropedia .org/
3.1
aliquot

portion of a larger amount of homogeneous material, assumed to be taken with negligible sampling error

Note 1 to entry: The term is usually applied to fluids. Tissues are heterogeneous and therefore cannot be

aliquoted.
Note 2 to entry: The definition is derived from References [28], [29] and [30].
© ISO 2018 – All rights reserved 1
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SIST EN ISO 20166-1:2019
ISO 20166-1:2018(E)
3.2
ambient temperature
unregulated temperature of the surrounding air
3.3
analyte
component represented in the name of a measurable quantity
[SOURCE: ISO 17511:2003, 3.2, modified — EXAMPLE has been removed.]
3.4
analytical test performance

accuracy, precision, and sensitivity of a test to measure the analyte (3.3) of interest

Note 1 to entry: Other test performance characteristics such as robustness, repeatability can apply as well.

3.5
cold ischemia

condition after removal of the tissue from the body until stabilization or fixation

3.6
cDNA
complementary DNA

single-stranded DNA that is complementary to a given RNA synthesized in the presence of reverse

transcriptase to serve as a template for synthesis of DNA copies
[SOURCE: ISO 17822-1:2014, 3.12]
3.7
diagnosis

identification of a health or disease state from its signs and/or symptoms, where the diagnostic process

can involve examinations (3.10) and tests for classification of an individual's condition into separate and

distinct categories or subclasses that allow medical decisions about treatment and prognosis to be made

3.8
DNA
deoxyribonucleic acid

polymer of deoxyribonucleotides occurring in a double-stranded (dsDNA) or single-stranded

(ssDNA) form
[SOURCE: ISO 22174:2005, 3.1.2]
3.9
DNase
deoxyribonuclease
enzyme that catalyzes the degradation of DNA (3.8) into smaller components
3.10
examination
analytical test

set of operations having the object of determining the value or characteristics of a property

Note 1 to entry: Processes that start with the isolated analyte and include all kinds of parameter testing or

chemical manipulation for quantitative or qualitative examination.

[SOURCE: ISO 15189:2012, 3.7, modified — Notes to entry 1 to 3 have been removed, Note 1 to entry has

been added and “analytical test” has been added as a preferred term.]
3.11
formalin

saturated aqueous formaldehyde solution which at 100 % contains 37 % formaldehyde by mass

(corresponding to 40 % by volume)
2 © ISO 2018 – All rights reserved
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SIST EN ISO 20166-1:2019
ISO 20166-1:2018(E)
3.12
formalin fixation

treatment of a sample with standard buffered formalin solution (3.25) for stabilization

3.13
grossing
gross examination

inspection of pathology specimens with the bare eye to obtain diagnostic information, while being

processed for further microscopic examination
3.14
interfering substances

endogenous substances of a specimen (3.17)/sample (3.23) or exogenous substances (e.g. stabilization

solution) that can alter an examination result
3.15
paraffin embedding

process in which a tissue sample is placed in paraffin to generate a hard surrounding matrix so that

thin microscopic sections can be cut
3.16
pre-examination process
pre-analytical phase
pre-analytical workflow

process that starts, in chronological order, from the clinician’s request and includes the examination

request, preparation and identification of the patient, collection of the primary sample(s), transportation

to and within the medical or pathology laboratory, isolation of analytes, and ends when the analytical

examination begins

Note 1 to entry: The pre-examination phase includes preparative processes that influence the outcome of the

intended examination.

[SOURCE: ISO 15189:2012, 3.15, modified — “pre-analytical workflow” has been added as a preferred

term, Note 1 to entry has been added and the definition has been extended.]
3.17
primary sample
specimen

discrete portion of a body fluid, breath, hair or tissue taken for examination (3.10), study or analysis of

one or more quantities or properties assumed to apply for the whole

[SOURCE: ISO 15189:2012, 3.16, modified — Notes to entry 1 to 3 have been removed.]

3.18
proficiency test

evaluation of participant performance against pre-established criteria by means of inter-laboratory

comparisons

[SOURCE: ISO 17043:2010, 3.7, modified — Notes to entry 1 and 2 have been removed.]

3.19
RNA profile

amounts of the individual RNA molecules that are present in a sample and that can be measured in the

absence of any losses, inhibition or interference
3.20
RNA
ribonucleic acid

polymer of ribonucleotides occurring in a double-stranded or single-stranded form

[SOURCE: ISO 22174:2005, 3.1.3]
© ISO 2018 – All rights reserved 3
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SIST EN ISO 20166-1:2019
ISO 20166-1:2018(E)
3.21
RNase
ribonuclease
enzyme that catalyzes the degradation of RNA into smaller components
3.22
room temperature
for the purposes of this document, temperature in the range of 18 °C to 25 °C
Note 1 to entry: Local or national regulations can have different definitions.
3.23
sample
one or more parts taken from a primary sample (3.17)
[SOURCE: ISO 15189:2012, 3.24, modified — EXAMPLE has been removed.]
3.24
stability

ability of a sample material, when stored under specified conditions, to maintain a stated property

value within specified limits for a specified period of time
Note 1 to entry: The analyte for the purpose of this document is isolated RNA.

[SOURCE: ISO Guide 30:2015, 2.1.15, modified — “reference material” has been replaced by “sample

material”, “characteristic” has been replaced by “ability” and Note 1 to entry has been changed.]

3.25
standard buffered formalin solution
neutral buffered formalin
NBF

10 % formalin (3.11) solution in water with a mass fraction of 3,7 % (corresponding to a volume fraction

of 4 %) formaldehyde buffered to pH 6,8 to pH 7,2

Note 1 to entry: Standard buffered formalin solutions often contain small amounts of methanol to inhibit

oxidation and polymerization of formaldehyde.
3.26
storage

prolonged interruption of the pre-analytical workflow of a sample or analyte, or of their derivatives,

such as stained sections or tissue blocks, under appropriate conditions in order to preserve their

properties

Note 1 to entry: Long-term storage typically occurs in laboratory archives or in biobanks.

3.27
tissue processor

automated instrument where tissue fixation, dehydration, clearing and paraffin infiltration occurs

3.28
validation

confirmation, throughout the provision of objective evidence, that the requirements for a specific

intended use or application have been fulfilled
Note 1 to entry: “Validated” is used to designate the corresponding status.

[SOURCE: ISO 9000:2015, 3.8.13, modified — Notes to entry 1 and 3 have been removed.]

3.29
warm ischemia

condition before the tissue is removed from the body, but deprived of its normal blood supply

4 © ISO 2018 – All rights reserved
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SIST EN ISO 20166-1:2019
ISO 20166-1:2018(E)
3.30
workflow
series of activities necessary to complete a task
3.31
homogeneous
uniform in structure and composition
4  General considerations

For general statements on medical laboratory quality management systems and in particular on

specimen collection, reception, and handling (including avoidance of cross contaminations) see

ISO 15189:2012, 4.2, 5.4.4, 5.4.6 or ISO/IEC 17020:2012, Clause 8, and 7.2. The requirements on

laboratory equipment, reagents, and consumables in accordance with ISO 15189:2012, 5.3 shall be

followed; ISO 15189:2012, 5.5.1.2 and 5.5.1.3, and ISO/IEC 17020:2012, 6.2 can also apply.

All steps of a diagnostic workflow can influence the final analytical test result. Thus, the entire

workflow including biomolecule stability and sample storage conditions shall be verified and validated.

Workflow steps which cannot always be controlled (e.g. warm ischemia) shall be documented. A risk

assessment of non-controllable workflow steps including their potential impact on the analytical test

performance shall be performed and mitigation measures shall be established to enable the required

analytical test performance.

Before or during the design of an analytical test, it should therefore be investigated and assured that the

RNA profile(s) intended to be analyzed is/are not compromized in a manner impacting the analytical

test performance (e.g. by performing a time course experiment or study; see also Annex A).

Before tissues are fixed in standard buffered formalin solution, the RNA profile(s) can change

significantly depending on the warm and cold ischemia duration and the temperature before formalin

fixation (e.g. gene induction, gene down regulation, RNA degradation). In addition, those changes can

vary in different donors’/patients’ tissues.

Generally, the longer the durations of warm and cold ischemia and the higher the ambient temperature

before fixation of the tissue specimen, the higher is the risk that changes in the RNA profile can occur.

NOTE Intraoperative warm ischemia can result in more pronounced changes of RNA profiles than in

[7][8]

postoperative cold ischemia . RNA profiles can also vary, depending on the origin and type of tissue, the

underlying disease, the surgical procedure, drugs administered for anaesthesia or treatment of concomitant

disease, and on the different environmental conditions after the tissue removal from the body.

As warm ischemia cannot be easily standardized, its duration shall be documented. When it is not

possible to avoid cold ischemia (e.g. transport to the laboratory before formalin fixation), its duration

shall be documented and the temperatures of the specimen container’s surroundings shall be

documented. Where the specimen is transported to another facility for formalin fixation, the transport

duration shall be documented and the ambient conditions should also be documented.

In addition, the formalin fixation itself as well as the subsequent FFPE tissue storage duration and

storage temperature cause modifications of biomolecules and leads to suboptimal a

...

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