SIST EN ISO 20166-1:2019

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 International Standard recommends 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 International Standard 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, but also pertains institutions and commercial organisations performing biomedical research, biobanks, and regulatory authorities.
The formalin fixation and the paraffin embedding process 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 during collection and change differently in different tissue 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.
NOTE   International, national or regional regulations or requirements may also apply to specific topics covered in this International Standard.

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
• Public Enquiry End Date: 14-Sep-2016
• Publication Date: 08-Apr-2019
• ICS: 11.100.10 - In vitro diagnostic test systems
• Technical Committee: VAZ - Healthcare
• Current Stage: 6060 - National Implementation/Publication (Adopted Project)
• Start Date: 15-Jan-2019
• Due Date: 22-Mar-2019
• Completion Date: 09-Apr-2019
• Ref Project: EN ISO 20166-1:2018 - 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)

Relations

• Replaces: SIST-TS CEN/TS 16827-1:2015 - Molecular in vitro diagnostic examinations - Specifications for pre-examination processes for FFPE tissue - Part 1: Isolated RNA
• Effective Date: 01-May-2019
• Replaces: SIST-TS CEN/TS 16827-1:2015 - Molecular in vitro diagnostic examinations - Specifications for pre-examination processes for FFPE tissue - Part 1: Isolated RNA
• Effective Date: 08-Jun-2022

Standards Content (Sample)

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

2

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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.


3

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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: [phone]
Fax: [phone]
Email: [e-mail]
Website: www.iso.org
Published in Switzerland
ii © ISO 2018 – All rights reserved

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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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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
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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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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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]
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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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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
...