CEN/TS 16827-2:2015
(Main)Molecular in vitro diagnostic examinations - Specifications for pre-examination processes for FFPE tissue - Part 2: Isolated proteins
Molecular in vitro diagnostic examinations - Specifications for pre-examination processes for FFPE tissue - Part 2: Isolated proteins
This Technical Specification gives recommendations for the handling, documentation and processing of FFPE tissue specimens intended for the analysis of extracted proteins during the preanalytical phase before a molecular assay is performed. This Technical Specification is applicable to molecular in vitro diagnostic examinations (e.g., in vitro diagnostic laboratories, laboratory customers, developers and manufacturers of in vitro diagnostics, institutions and commercial organizations performing biomedical research, biobanks, and regulatory authorities).
Protein profiles and protein-protein interactions in tissues can change drastically before and after collection (due to e.g., gene induction, gene down regulation, protein degradation). Protein species amounts can change differently in tissues from different donors / patients. The expression of genes can be influenced by the given treatment or intervention (surgery, biopsy), or drugs administered for anaesthesia or even treatment of concomitant disease as well as by the different environment conditions after the tissue removal from the body.
Furthermore, the formalin fixation and paraffin embedding process leads to modifications of the protein molecules, which can impact the validity and reliability of the analytical test results.
Therefore, it is essential to take special measures to minimize the described profile changes and modifications within the tissue for subsequent protein analysis.
This document is not applicable for protein analysis by immunohistochemistry.
Molekularanalytische in-vitro-diagnostische Verfahren - Spezifikationen für präanalytische Prozesse für FFPE-Gewebeproben - Teil 2: Isolierte Proteine
Diese Technische Spezifikation gibt Empfehlungen zur Handhabung, Dokumentation und Verarbeitung von aus FFPE Gewebe bestehendem und für die Analyse isolierter Proteine vorgesehenem Untersuchungs-material während der präanalytischen Phase vor Beginn der molekularen Analyse. Diese Technische Spezifikation gilt für molekulare in vitro diagnostische Untersuchungen (z. B. In vitro Diagnostik Labore, Kunden dieser Labore, Entwickler und Hersteller von In vitro Diagnostika, Einrichtungen und kommerzielle Organisationen, die in der biomedizinischen Forschung tätig sind, Biobanken und Aufsichtsbehörden).
Proteinprofile und Protein Protein Interaktionen in Geweben können sich vor und nach der Probenahme drastisch verändern (z. B. aufgrund von Geninduktion, Herabregulation von Genen, Proteinabbau). Die Mengen der Proteinspezies können sich je nach Gewebespender/Patient unterschiedlich verändern. Die Genexpression kann durch die jeweilige Behandlung oder den Eingriff (Operation, Biopsie) oder die zur Anästhesie oder Behandlung von Begleiterkrankungen verabreichten Medikamente sowie abhängig von unterschiedlichen Umgebungs¬bedingungen nach der Gewebeentnahme aus dem Körper beeinflusst sein.
Die Fixierung mit Formalin und die Einbettung in Paraffin können außerdem zu Modifikationen der Proteinmoleküle führen, was sich auf die Gültigkeit und Zuverlässigkeit der Ergebnisse analytischer Prüfungen auswirken kann.
Daher ist es wichtig, dass besondere Maßnahmen getroffen werden, um die beschriebenen Profilveränderungen und modifikationen im Gewebe im Hinblick auf die anschließende Proteinanalyse möglichst gering zu halten.
Dieses Dokument gilt nicht für eine immunhistochemische Proteinanalyse.
Tests de diagnostic moléculaire in vitro - Spécifications pour les processus préanalytiques pour tissu FFPE - Partie 2: Protéines extraites
La présente Spécification technique fournit des recommandations concernant la manipulation, la documentation et le traitement de spécimens de tissu FFPE destinés à l’analyse des protéines extraites lors de la phase préanalytique, avant la réalisation d’un essai moléculaire. La présente Spécification technique est applicable aux tests de diagnostic moléculaire in vitro (par exemple, par les laboratoires de diagnostic in vitro, les clients de laboratoires, les développeurs et fabricants de tests de diagnostic in vitro, les institutions et les organisations commerciales faisant de la recherche biomédicale, les biobanques et les autorités réglementaires).
Les profils protéiques et les interactions protéine-protéine dans les tissus peuvent varier considérablement avant et après le prélèvement (par exemple, en raison de l’induction d’un gène, de la régulation négative d’un gène, de la dégradation des protéines). Le nombre d’espèces de protéines peut évoluer différemment dans différents tissus de donneurs/patients. L’expression des gènes peut être influencée par le traitement administré, l’intervention (chirurgie, biopsie), les médicaments administrés pour l’anesthésie, ou même par le traitement d’une maladie concomitante, ainsi que par les différentes conditions environnementales une fois le tissu prélevé.
En outre, le processus de fixation au formol et d’inclusion en paraffine provoque des modifications des molécules de protéines, qui peuvent avoir une incidence sur la validité et la fiabilité des résultats de l’essai analytique.
C’est pourquoi il est essentiel de prendre des mesures particulières pour réduire au minimum les changements de profil et les modifications au sein du tissu décrits ci-dessus pour l’analyse consécutive des protéines.
Le présent document n’est pas applicable à l’analyse des protéines par immunohistochimie.
Molekularne diagnostične preiskave in vitro - Specifikacije za predpreiskovalne procese za FFPE tkiva - 2. del: Izolirani proteini
Ta tehnična specifikacija vsebuje priporočila za obravnavo, dokumentiranje in obdelavo vzorcev tkiv FFPE, namenjenih za analizo ekstrahiranih proteinov med predanalizno fazo, preden se izvede molekularni preskus. Ta tehnična specifikacija se uporablja za molekularne diagnostične preiskave in vitro (npr. diagnostični laboratoriji in vitro, laboratorijske stranke, razvijalci in proizvajalci diagnostike in vitro, institucije in komercialne organizacije, ki izvajajo biomedicinske raziskave, biobanke ter regulativni organi).
Profili proteinov in interakcije protein-protein v tkivih se lahko močno spremenijo pred zbiranjem in po njem (npr. zaradi genske indukcije, znižanja izražanja genov, degradacije proteinov). Količine vrst proteinov se lahko različno spreminjajo pri tkivih različnih darovalcev/bolnikov. Na izražanje genov je mogoče vplivati z zdravljenjem ali posegom (operacija, biopsija), anestetiki ali celo z zdravljenjem sočasne bolezni kot tudi z različnimi okoljskimi pogoji po odstranitvi tkiva iz telesa.
Fiksacija v formalinu in postopek vklopa v parafin vodita tudi do sprememb proteinskih molekul, kar lahko vpliva na veljavnost in zanesljivost rezultatov analitičnih preskusov.
Zato je nujno treba sprejeti posebne ukrepe, da se zmanjšajo opisane spremembe profila v tkivu za nadaljnje analize proteinov.
Ta dokument se ne uporablja za analize proteinov z imunohistokemijo.
General Information
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Standards Content (Sample)
SLOVENSKI STANDARD
01-oktober-2015
0ROHNXODUQHGLDJQRVWLþQHSUHLVNDYHLQYLWUR6SHFLILNDFLMH]DSUHGSUHLVNRYDOQH
SURFHVH]D))3(WNLYDGHO ,]ROLUDQLSURWHLQL
Molecular in vitro diagnostic examinations - Specifications for pre-examination processes
for FFPE tissue - Part 2: Isolated proteins
Molekularanalytische in-vitro-diagnostische Verfahren - Spezifikationen für
präanalytische Prozesse für FFPE-Gewebe - Teil 2: Isolierte Proteine
Tests de diagnostic moléculaire in vitro - Spécifications pour les processus
préanalytiques pour tissu FFPE - Partie 2: Protéines extraites
Ta slovenski standard je istoveten z: CEN/TS 16827-2:2015
ICS:
11.100.10 'LDJQRVWLþQLSUHVNXVQL In vitro diagnostic test
VLVWHPLLQYLWUR systems
2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.
TECHNICAL SPECIFICATION
CEN/TS 16827-2
SPÉCIFICATION TECHNIQUE
TECHNISCHE SPEZIFIKATION
August 2015
ICS 11.100.10
English Version
Molecular in vitro diagnostic examinations - Specifications for
pre-examination processes for FFPE tissue - Part 2: Isolated
proteins
Tests de diagnostic moléculaire in vitro - Spécifications pour Molekularanalytische in-vitro-diagnostische Verfahren -
les processus préanalytiques pour tissu FFPE - Partie 2: Spezifikationen für präanalytische Prozesse für FFPE-
Protéines extraites Gewebeproben - Teil 2: Isolierte Proteine
This Technical Specification (CEN/TS) was approved by CEN on 6 July 2015 for provisional application.
The period of validity of this CEN/TS is limited initially to three years. After two years the members of CEN will be requested to submit their
comments, particularly on the question whether the CEN/TS can be converted into a European Standard.
CEN members are required to announce the existence of this CEN/TS in the same way as for an EN and to make the CEN/TS available
promptly at national level in an appropriate form. It is permissible to keep conflicting national standards in force (in parallel to the CEN/TS)
until the final decision about the possible conversion of the CEN/TS into an EN is reached.
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, 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: Avenue Marnix 17, B-1000 Brussels
© 2015 CEN All rights of exploitation in any form and by any means reserved Ref. No. CEN/TS 16827-2:2015 E
worldwide for CEN national Members.
Contents Page
European foreword .3
Introduction .4
1 Scope .5
2 Normative references .5
3 Terms and definitions .5
4 General considerations .7
5 Outside the laboratory .8
5.1 Primary tissue collection manual.8
5.1.1 Information about the primary sample donor .8
5.1.2 Information on the primary tissue sample .8
5.1.3 Information on the primary tissue sample processing .9
5.2 Transport requirements .9
6 Inside the laboratory .9
6.1 Information on the primary tissue sample receipt .9
6.2 Formalin fixation of the specimen . 10
6.3 Evaluation of the pathology of the specimen and selection of the sample. 11
6.4 Post-fixation of frozen samples . 11
6.5 Processing and paraffin embedding. 12
6.6 Storage requirements . 12
6.7 Isolation of the total protein . 13
6.7.1 General . 13
6.7.2 General information for protein isolation procedures . 13
6.7.3 Using commercial kits . 13
6.7.4 Using the laboratories’ own protocols . 13
6.8 Quantity and quality assessment of isolated RNA . 14
6.9 Storage of isolated RNA . 14
Annex A (informative) Quality control of RNA extracted from formalin fixed and paraffin
embedded tissue samples: implications for RT-qPCR based analyses . 15
A.1 Introduction . 15
A.2 Example . 15
A.2.1 General . 15
A.2.2 Experimental procedures . 15
A.2.2.1 General . 15
A.2.2.2 Tissues . 16
A.2.2.3 Protein analysis . 16
A.2.3 Results . 17
A.2.4 Further reading . 18
Bibliography . 19
European foreword
This document (CEN/TS 16827-2:2015) has been prepared by Technical Committee CEN/TC 140 “In vitro
diagnostic medical devices”, the secretariat of which is held by DIN.
Attention is drawn to the possibility that some of the elements of this document may be the subject of patent
rights. CEN [and/or CENELEC] shall not be held responsible for identifying any or all such patent rights.
According to the CEN-CENELEC Internal Regulations, the national standards organizations of the following
countries are bound to announce this Technical Specification: 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, Slovakia, Slovenia, Spain, Sweden, Switzerland, Turkey and the United Kingdom.
Introduction
Molecular in vitro diagnostics has enabled a significant progress in medicine. Further progress is expected by
new technologies analysing signatures of nucleic acids, proteins, and metabolites in human tissues and body
fluids. However, the profiles and/or integrity of these molecules can change drastically during primary sample
collection, transport, storage, and processing thus making the outcome from diagnostics or research
unreliable or even impossible because the subsequent analytical assay will not determine the situation in the
patient but an artificial molecular pattern generated during the pre-examination process.
Although originally thought as being impossible due to the crosslinking activities of formaldehyde, protein
extraction techniques from formalin formalin fixed and paraffin embedded (FFPE) tissues have been much
improved in recent years. Heat-induced reversal of formaldehyde-induced crosslinks has been demonstrated
as an essential step in the protein extraction procedures [1], [2]. Currently, most investigators accept that
proteins extracted from FFPE tissue are suitable for downstream proteomic analysis [3].
However, a standardization of the entire process from primary sample collection to protein analysis is needed.
Studies have been undertaken to determine the important influencing factors. This Technical Specification
draws upon such work to codify and standardise the steps for FFPE tissue with regard to protein analysis in
what is referred to as the preanalytical phase.
1 Scope
This Technical Specification gives recommendations for the handling, documentation and processing of
FFPE tissue specimens intended for the analysis of extracted proteins during the preanalytical phase before a
molecular assay is performed. This Technical Specification is applicable to molecular in vitro diagnostic
examinations (e.g., in vitro diagnostic laboratories, laboratory customers, developers and manufacturers of in
vitro diagnostics, institutions and commercial organizations performing biomedical research, biobanks, and
regulatory authorities).
Protein profiles and protein-protein interactions in tissues can change drastically before and after collection
(due to e.g., gene induction, gene down regulation, protein degradation). Protein species amounts can change
differently in tissues from different donors / patients. The expression of genes can be influenced by the given
treatment or intervention (surgery, biopsy), or drugs administered for anaesthesia or even treatment of
concomitant disease as well as by the different environment conditions after the tissue removal from the body.
Furthermore, the formalin fixation and paraffin embedding process leads to modifications of the protein
molecules, which can impact the validity and reliability of the analytical test results.
Therefore, it is essential to take special measures to minimize the described profile changes and modifications
within the tissue for subsequent protein analysis.
This document is not applicable for protein analysis by immunohistochemistry.
2 Normative references
The following documents, in whole or in part, are normatively referenced in this document and are
indispensable for its application. For dated references, only the edition cited applies. For undated references,
the latest edition of the referenced document (including any amendments) applies.
EN ISO 15189:2012, Medical laboratories — Requirements for quality and competence (ISO 15189:2012,
Corrected version 2014-08-15)
ISO 15190, Medical laboratories — Requirements for safety
3 Terms and definitions
For the purposes of this document, the terms and definitions given in EN ISO 15189:2012 and the following
apply.
3.1
ambient temperature
unregulated temperature of the surrounding air
3.2
analytical phase
processes that start with the isolated analyte and include all kinds of parameter testing or chemical
manipulation for quantitative or qualitative analysis
3.3
cold ischemia
condition after removal of the tissue from the body until its stabilization or fixation
3.4
FFPE
formalin fixation and paraffin embedding
3.5
FFPE tissues
formalin fixed and paraffin embedded tissues
3.6
formalin
saturated formaldehyde solution containing a mas fraction of 37 % (corresponding to a volume fraction of 40
%) formaldehyde, termed 100 % formalin
3.7
formalin fixation
treatment of a sample with standard buffered formalin solution for stabilization
3.8
pre-examination processes
preanalytical phase
preanalytical workflow
processes that start, in chronological order, from the clinician’s request and include the examination request,
preparation and identification of the patient
...
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