SIST-TS CEN/TS 16826-2:2015
(Main)Molecular in vitro diagnostic examinations - Specifications for pre-examination processes for snap frozen tissue - Part 2: Isolated proteins
Molecular in vitro diagnostic examinations - Specifications for pre-examination processes for snap frozen tissue - Part 2: Isolated proteins
This Technical Specification recommends the handling, documentation and processing of frozen 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, in vitro diagnostics developers and manufacturers, institutions and commercial organisations 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 different donors’ / patients’ tissues. 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.
Therefore, it is essential to take special measures to minimize the described profile changes and modifications within the tissue for subsequent protein analysis.
Tissues that have undergone chemical stabilization pre-treatment before freezing are not covered in this document. In addition this document is not applicable for protein analysis by immunohistochemistry.
Molekularanalytische in-vitro-diagnostische Verfahren - Spezifikationen für präanalytische Prozesse für gefrorene Gewebeproben - Teil 2: Isolierte Proteine
Diese Technische Spezifikation gibt Empfehlungen zur Handhabung, Dokumentation und Verarbeitung von aus gefrorenem 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 Umgebungsbedingungen nach der Gewebeentnahme aus dem Körper beeinflusst sein.
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.
Gewebe, die vor dem Gefriervorgang einer chemischen Vorbehandlung zur Stabilisierung unterzogen wurden, sind nicht durch dieses Dokument abgedeckt. Des Weiteren gilt dieses Dokument nicht für eine immun-histo¬chemische Proteinanalyse.
Tests de diagnostic moléculaire in vitro - Spécifications relatives aux processus préanalytiques pour les tissus à congélation rapide - Partie 2: Protéines extraites
La présente Spécification technique fournit des recommandations pour la manipulation, la documentation et le traitement des échantillons primaires de tissus congelés destinés à l’analyse des protéines extraites durant la phase préanalytique précédant la réalisation d’un essai moléculaire. La présente Spécification technique s’applique aux tests de diagnostic moléculaire in vitro (par exemple, laboratoires de diagnostic in vitro, clients de laboratoires, concepteurs et fabricants de tests de diagnostic in vitro, institutions et organisations commerciales de recherche biomédicale, biobanques et autorités réglementaires).
Les profils de protéines et les interactions entre protéines dans les tissus peuvent considérablement changer avant et après le prélèvement (par exemple, en raison de l'induction de gènes, de la régulation négative de l'expression de gènes, de la dégradation des protéines). Les quantités d'espèces protéiques peuvent varier dans les tissus des différents donneurs/patients. L'expression des gènes peut être influencée par le traitement donné ou l'intervention réalisée (chirurgie, biopsie) ou par les médicaments administrés pour l'anesthésie, voire pour le traitement d'une maladie concomitante, ainsi que par les différentes conditions environnementales après le prélèvement des tissus sur l'organisme.
Il est donc essentiel de prendre des mesures spéciales afin de réduire le plus possible les changements de profil décrits et les modifications dans le tissu lors de l'analyse subséquente des protéines.
Le présent document ne couvre pas les tissus ayant subi un prétraitement de stabilisation chimique avant la congélation. De plus, ce document n'est pas applicable pour l'analyse des protéines par immunohistochimie.
Molekularne diagnostične preiskave in vitro - Specifikacije za predpreiskovalne procese za hitro zamrznjena tkiva - 2. del: Izolirani proteini
Ta tehnična specifikacija vsebuje priporočila za obravnavo, dokumentiranje in obdelavo vzorcev zamrznjenih tkiv, 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.
Zato je nujno treba sprejeti posebne ukrepe, da se zmanjšajo opisane spremembe profila v tkivu za nadaljnje analize proteinov.
Tkiva, ki so pred zamrzovanjem prestala predobdelavo za kemično stabilizacijo, niso zajeta v tem dokumentu. Ta dokument se ne uporablja za analize proteinov z imunohistokemijo.
General Information
Relations
Standards Content (Sample)
SLOVENSKI STANDARD
SIST-TS CEN/TS 16826-2:2015
01-oktober-2015
0ROHNXODUQHGLDJQRVWLþQHSUHLVNDYHLQYLWUR6SHFLILNDFLMH]DSUHGSUHLVNRYDOQH
SURFHVH]DKLWUR]DPU]QMHQDWNLYDGHO ,]ROLUDQLSURWHLQL
Molecular in vitro diagnostic examinations - Specifications for pre-examination processes
for snap frozen tissue - Part 2: Isolated proteins
Molekularanalytische in-vitro-diagnostische Verfahren - Spezifikationen für
präanalytische Prozesse für gefrorene Gewebeproben - Teil 2: Isolierte Proteine
Tests de diagnostic moléculaire in vitro - Spécifications relatives aux processus
préanalytiques pour les tissus à congélation rapide - Partie 2: Protéines extraites
Ta slovenski standard je istoveten z: CEN/TS 16826-2:2015
ICS:
11.100.10 'LDJQRVWLþQLSUHVNXVQL In vitro diagnostic test
VLVWHPLLQYLWUR systems
SIST-TS CEN/TS 16826-2:2015 en,fr,de
2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.
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SIST-TS CEN/TS 16826-2:2015
TECHNICAL SPECIFICATION
CEN/TS 16826-2
SPÉCIFICATION TECHNIQUE
TECHNISCHE SPEZIFIKATION
August 2015
ICS 11.100.10
English Version
Molecular in vitro diagnostic examinations - Specifications for
pre-examination processes for snap frozen tissue - Part 2:
Isolated proteins
Tests de diagnostic moléculaire in vitro - Spécifications Molekularanalytische in-vitro-diagnostische Verfahren -
relatives aux processus préanalytiques pour les tissus à Spezifikationen für präanalytische Prozesse für
congélation rapide - Partie 2: Protéines extraites schockgefrorene 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 16826-2:2015 E
worldwide for CEN national Members.
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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 .8
5.2 Transport requirements .9
6 Inside the laboratory .9
6.1 Information on the primary tissue sample receipt .9
6.2 Evaluation of the pathology of the specimen and selection of the sample.9
6.3 Cryo-storage of the specimen . 10
6.4 Storage requirements . 11
6.5 Isolation of total protein . 11
6.5.1 General . 11
6.5.2 Using commercial kits . 11
6.5.3 Using the laboratories' own protocols . 11
6.6 Quality assessment of isolated proteins . 12
6.7 Storage of isolated total protein . 12
Annex A (informative) Quantitative protein analysis demonstrates changes of protein amounts
during cold ischemia . 13
A.1 Introduction . 13
A.2 Example . 13
A.2.1 General . 13
A.2.2 Experimental procedures . 13
A.2.2.1 General . 13
A.2.2.2 Tissues . 14
A.2.2.3 Protein analysis . 14
A.2.3 Results . 15
A.2.4 Further reading . 16
Bibliography . 17
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European foreword
This document (CEN/TS 16826-2:2015) 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.
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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. Therefore, 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 standardize the steps for frozen tissue with regard to protein analysis in what is
referred to as the preanalytical phase.
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1 Scope
This Technical Specification gives recommendations for the handling, documentation and processing of frozen
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 organisations 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 medical 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.
Therefore, it is essential to take special measures to minimize the described profile changes and modifications
within the tissue for subsequent protein analysis.
Tissues that have undergone chemical stabilization pre-treatment before freezing are not covered in this
document. In addition 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
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3.4
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, surgical procedure, collection of the primary sample(s), temporary
storage, transportation to and within the analytical laboratory, aliquoting, retrieval, isolation of analytes, and
end when the analytical examination begins
[SOURCE: EN ISO 15189:2012, definition 3.15, modified — An additional term was added and more details
were included.]
Note 1 to entry: The preanalytical phase may include preparative processes that may influence the outcome of the
intended examination.
3.5
primary sample
specimen
discrete portion of a body fluid, breath, hair or tissue taken for examination, study or analysis of one or more
quantities or properties assumed to apply for the whole
[SOURCE: EN ISO 15189:2012, 3.16, modified — The term and definition is used here without the original
notes.]
3.6
protein
type of biological macromolecules composed of one or more chains with a defined sequence of amino acids
connected through peptide bonds
3.7
protein profile
amounts of the individual protein molecules that are present in a sample and that can be measured in the
absence of any losses, inhibition and interference
3.8
protein species
amounts of a chemically clearly-defined protein corresponding to one spot on a high-performance 2-
dimensional gel electrophoresis pattern
[SOURCE: Jungblut et. al.1996]
3.9
PTM
post translational modifications
chemical alterations to a primary protein structure, often crucial for conferring biological activity on a protein
[SOURCE: Encyclopedia of Psychopharmacology, 2010]
3.10
room temperature
temperature which is defined as 18 °C to 25 °C for the purposes of this document
3.11
sample
one or more parts taken from a primary sample
[SOURCE: EN ISO 15189:2012, 3.24, modified — The example was not taken over.]
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3.12
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
[SOURCE: ISO Guide 30:1992, 2.7]
Note 1 to entry: The measured constituent for the purpose of this document is isolated protein.
3.13
warm ischemia
warm Ischemia is the condition where the tissue is deprived of its normal blood supply containing oxygen and
nutrients while the tissue is at body temperature
4 General considerations
For general statements on primary sample collection and handling (including avoidance of cross
contaminations) see EN ISO 15189:2012, 5.4.4, 5.2.6. Consumables including kits shall be verified before use
in examination (see EN ISO 15189:2012, 5.3.2.3); EN ISO 15189:2012, 5.5.1.2 and 5.5.1.3 can also apply.
As all steps of a diagnostic workflow can influence the final analytical performance, the entire workflow
comprising the preanalytical steps, including information on biomolecule stability and storage conditions, and
analytical steps should be verified and validated (see EN ISO 15189).
The stability of the specific protein(s) of interest and their posttranslational modifications (if important for the
assay) should be investigated throughout the complete preanalytical workflow prior to the development and
implementation of an analytical test.
Before tissues are stabilized by freezing; protein amounts, conformations and binding status can change e.g.,
by protein degradation and altered synthesis following gene induction, gene down regulation, RNA
degradation, and changes of the biochemical pathway and energy status. These effects depend on the
duration of warm and cold ischemia and the ambient temperature before freezing. In addition, those effects
can vary in tissues from different donors / patients.
Generally, the longer the warm and cold ischemia times and the higher the ambient temperature before
freezing the tissue specimen, the higher is the risk that changes in the protein profile can occur.
NOTE Prolonged cold ischemia times result in changes of protein (e.g., cytokeratin 18) and phosphoprotein (e.g.,
phospho-p42/44) amounts [1], [2]. Keeping the specimen on wet-ice diminishes this effect [3]. Protein amounts as well as
posttranslational modifications can also vary during the preanalytical phase, depending on the origin and type of tissue,
the underlying disease, the surgical procedure, the drug regime, and 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 time and duration should be documented. When it is not
possible to avoid cold ischemia, its time of onset and duration shall be documented and the temperatures of
the specimen transport container's surroundings should be documented. Where the specimen is transported
to another facility for freezing, the transport duration shall be documented and the ambient conditions should
also be documented.
Safety regulations on transport and handling shall be considered (see EN ISO 15189:2012, 5.2.3 and 5.4.5
and ISO 15190).
During the whole preanalytical workflow precautions shall be taken to avoid cross contamination between
different samples.
If a commercial product is not used in accordance with the manufacturers' instructions, responsibility for its
use and performance lies with the user.
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5 Outside the laboratory
5.1 Primary tissue collection manual
5.1.1 Information about the primary sample donor
The documentation should include, but is not limited to:
a) the primary donor / patient ID, which can be in the form of a code;
b) the health status of the primary sample donor (e.g., healthy, disease type, concomitant disease);
c) the information about routine medical treatment and special treatment prior to tissue collection (e.g.,
anaesthetics, medications, surgical or diagnostic procedures (e.g., biopsy device used for the collection));
d) the start of ischemia within the body (warm ischemia) by documenting the ischemia-relevant vessel
ligation/clamping time point (usually arterial clamping time).
5.1.2 Information on the primary tissue sample
The documentation shall include, but is not limited to:
a) the time point when tissue is removed from the body;
b) the description of tissue type, tissue condition (e.g., diseased, unaffected by the disease) and organ
tissue of origin, including references to any marking applied in the operating theatre made by surgeon,
radiologist or pathologist;
c) the documentation steps described under 6.3, if freezing is performed outside the laboratory.
5.1.3 Information on the primary tissue sample processing
The following steps shall be performed:
1. the documentation of any additions or modifications to the primary sample after removal from the body
(e.g., labelling for the orientation of the specimen (e.g., ink-marking, stitches), incision(s));
2. the selection and use of transport containers and packages (e.g., cooling box, box for storing and
transportation, vacuum packaging);
3. the selection and use of stabilisation procedures (e.g., cooling methods) for transport;
NOTE 1 Accidentally freezing and thawing the tissue (e.g., by using cool packs in a wrong manner) can lead to
protein degradation when the tissue thaws thereafter. It can also impact the morphological characterisation.
NOTE 2 This step can be omitted, if the specimen is directly frozen (see 6.3.)
4. the labelling of the transport container (e.g., registration-number, barcode (1D or 2D), primary sample
type, quantity, and organ tissue of origin) and additional documentation (information as specified in 5.1.1,
5.1.2, and 5.1.3, 1. to 3.). If a single sample container contains several aliquots of the same specimen,
and the aliquots represent different features (e.g., tissue type, disease status, location) this shall be
documented.
Specimens should be transferred without delay into the transport container after the removal from the body.
The container should then be kept on wet-ice or at 2 °C to 8 °C in order to minimize protein profile changes.
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The temperatures of the transport
...
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