SIST-TS CEN/TS 16826-1:2015
(Main)Molecular in vitro diagnostic examinations - Specifications for pre-examination processes for snap frozen tissue - Part 1: Isolated RNA
Molecular in vitro diagnostic examinations - Specifications for pre-examination processes for snap frozen tissue - Part 1: Isolated RNA
This Technical Specification recommends the handling, documentation and processing of frozen tissue specimens intended for RNA analysis 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).
RNA profiles in tissues can change significantly before and after collection and can change differently in different donors’ / patients’ tissues.
Therefore, special measures have to be taken to minimize the described profile changes and modifications within the tissue for subsequent RNA analysis.
Tissues that have undergone chemical stabilisation pre-treatment before freezing are not covered in this document.
Molekularanalytische in-vitro-diagnostische Verfahren - Spezifikationen für präanalytische Prozesse für gefrorenes Gewebe - Teil 1: Isolierte RNS
Diese Technische Spezifikation gibt Empfehlungen zur Handhabung, Dokumentation und Verarbeitung von aus gefrorenem Gewebe bestehendem und für die RNS Analyse vorgesehenem Untersuchungsmaterial 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).
RNS Profile in Geweben können sich vor und nach der Probenahme erheblich verändern und dies je nach Spender/Patient in unterschiedlichem Maß.
Daher ist es wichtig, dass besondere Maßnahmen getroffen werden, um die beschriebenen Profilveränderungen und modifikationen im Gewebe im Hinblick auf die anschließende RNS Analyse möglichst gering zu halten.
Gewebe, die vor dem Gefriervorgang einer chemischen Vorbehandlung zur Stabilisierung unterzogen wurden, sind nicht durch dieses Dokument abgedeckt.
Tests de diagnostic moléculaire in vitro - Spécifications relatives aux processus préanalytiques pour les tissus à congélation rapide - Partie 1: ARN extrait
La présente Spécification technique fournit des recommandations pour la manipulation, la documentation et le traitement des échantillons de tissus primaires congelés destinés à l’analyse de l’ARN 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 d'ARN dans les tissus peuvent considérablement changer avant et après le prélèvement et peuvent varier selon les tissus de différents donneurs/patients.
Il est donc essentiel de prendre des mesures particulières afin de réduire le plus possible les changements de profil décrits et les modifications dans le tissu décrits lors de l'analyse de l’ARN.
Le présent document ne couvre pas les tissus ayant subi un prétraitement de stabilisation chimique avant la congélation.
Molekularne diagnostične preiskave in vitro - Specifikacije za predpreiskovalne procese za hitro zamrznjena tkiva - 1. del: Izolirani RNK
Ta tehnična specifikacija vsebuje priporočila za obravnavo, dokumentiranje in obdelavo vzorcev zamrznjenih tkiv, namenjenih za analizo RNK 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 RNK v tkivih se lahko močno spremenijo pred zbiranjem in po njem ter se različno spremenijo pri tkivih različnih darovalcev/bolnikov.
Zato je treba sprejeti posebne ukrepe, da se zmanjšajo opisane spremembe profila v tkivu za nadaljnje analize RNK.
Tkiva, ki so pred zamrzovanjem prestala predobdelavo za kemično stabilizacijo, niso zajeta v tem dokumentu.
General Information
Relations
Standards Content (Sample)
SLOVENSKI STANDARD
SIST-TS CEN/TS 16826-1:2015
01-oktober-2015
0ROHNXODUQHGLDJQRVWLþQHSUHLVNDYHLQYLWUR6SHFLILNDFLMH]DSUHGSUHLVNRYDOQH
SURFHVH]DKLWUR]DPU]QMHQDWNLYDGHO ,]ROLUDQL51.
Molecular in vitro diagnostic examinations - Specifications for pre-examination processes
for snap frozen tissue - Part 1: Isolated RNA
Molekularanalytische in-vitro-diagnostische Verfahren - Spezifikationen für
präanalytische Prozesse für gefrorenes Gewebe - Teil 1: Isolierte RNS
Tests de diagnostic moléculaire in vitro - Spécifications relatives aux processus
préanalytiques pour les tissus à congélation rapide - Partie 1: ARN extrait
Ta slovenski standard je istoveten z: CEN/TS 16826-1:2015
ICS:
11.100.10 'LDJQRVWLþQLSUHVNXVQL In vitro diagnostic test
VLVWHPLLQYLWUR systems
SIST-TS CEN/TS 16826-1:2015 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 16826-1:2015
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SIST-TS CEN/TS 16826-1:2015
TECHNICAL SPECIFICATION
CEN/TS 16826-1
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 1:
Isolated RNA
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 1: ARN extrait schockgefrorene Gewebeproben - Teil 1: Isolierte RNS
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 16826-1:2015 E
worldwide for CEN national Members.
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SIST-TS CEN/TS 16826-1:2015
CEN/TS 16826-1:2015 (E)
Contents Page
European foreword .3
Introduction .4
1 Scope .5
2 Normative references .5
3 Terms and definitions .5
4 General considerations .6
5 Outside the laboratory .7
5.1 Primary tissue collection manual.7
5.1.1 Information about the primary sample donor .7
5.1.2 Information on the primary tissue sample .7
5.1.3 Information on the primary tissue sample processing .8
5.2 Transport requirements .8
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 .9
6.4 Storage requirements . 10
6.5 Isolation of the total RNA . 11
6.5.1 General information for RNA isolation procedures . 11
6.5.2 Using commercial kits . 11
6.5.3 Using the laboratories' own protocols . 12
6.6 Quality assessment of isolated RNA . 12
6.7 Storage of isolated RNA . 12
Annex A (informative) Impact of preanalytical variables on RNA profiles obtained from frozen
liver tissue samples collected during and after routine surgery . 13
A.1 Comparison of stable and unstable genes identified under ischemic conditions . 13
A.2 Recommendations based on the results . 15
Bibliography . 16
2
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SIST-TS CEN/TS 16826-1:2015
CEN/TS 16826-1:2015 (E)
European foreword
This document (CEN/TS 16826-1:2015) 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.
3
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SIST-TS CEN/TS 16826-1:2015
CEN/TS 16826-1:2015 (E)
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 profile generated during the pre-examination process. Therefore, a standardization of
the entire process from primary sample collection to RNA 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 RNA analysis in what is referred to as the
preanalytical phase.
4
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CEN/TS 16826-1:2015 (E)
1 Scope
This Technical Specification gives recommendations for the handling, documentation and processing of frozen
tissue specimens intended for RNA analysis 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).
RNA profiles in tissues can change significantly before and after collection and can change differently in
tissues from different donors / patients.
Therefore, it is essential to take special measures to minimize the described profile changes and modifications
within the tissue for subsequent RNA analysis.
Tissues that have undergone chemical stabilisation pre-treatment before freezing are not covered in this
document.
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
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
5
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SIST-TS CEN/TS 16826-1:2015
CEN/TS 16826-1:2015 (E)
[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
quantitative RNA profile
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 and interference
3.7
RNA
ribonucleic acid
polymer of ribonucleotides occurring in a double-stranded or single-stranded form
[SOURCE: EN ISO 22174:2005, 3.1.3]
3.8
room temperature
temperature which is defined as 18 °C to 25 °C for the purposes of this document
3.9
sample
one or more parts taken from a primary sample
[SOURCE: EN ISO 15189:2012, 3.24, modified — The example was not taken over.]
3.10
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 RNA.
3.11
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.
6
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SIST-TS CEN/TS 16826-1:2015
CEN/TS 16826-1:2015 (E)
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 quantitative RNA profile(s) of interest should be investigated throughout the entire
preanalytical workflow prior to the development and implementation of an analytical test.
Before tissues stabilized by freezing, quantitative RNA profile can change e.g., by gene induction, gene down
regulation and RNA degradation. These effects depend on the duration of warm and cold ischemia and the
ambient temperature before freezing. In addition, the described 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 RNA profile can occur.
NOTE Intraoperative warm ischemia can result in more pronounced changes of RNA profiles than during
postoperative cold ischemia. RNA profiles can also vary, 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, duration shall be documented and 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.
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:
7
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CEN/TS 16826-1:2015 (E)
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) will lead to
RNA 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 RNA profile changes.
The temperatures of the transport container's surroundings during cold ischemia time (e.g., temperatures in
different rooms, transport) should be documented. If the temperature cannot be measured, the temperature
range should be estimated by classification as ambient temperature, room temperature, or at 2 °C to 8 °C.
5.2 Transport requirements
The laboratory in partnership with the clinical or surgery department shall establish a protocol for the transport
procedure of the specimen.
If the primary tissue sample is not already f
...
SLOVENSKI STANDARD
kSIST-TS FprCEN/TS 16826-1:2015
01-maj-2015
0ROHNXODUQHGLDJQRVWLþQHSUHLVNDYHLQYLWUR6SHFLILNDFLMH]DSUHGSUHLVNRYDOQH
SURFHVH]DKLWUR]DPU]QMHQDWNLYDGHO 3RVDPLþQL51$
Molecular in vitro diagnostic examinations - Specifications for pre-examination processes
for snap frozen tissue - Part 1: Isolated RNA
Molekularanalytische in-vitro-diagnostische Verfahren - Spezifikationen für
präanalytische Prozesse für gefrorenes Gewebe - Teil 1: Isolierte RNS
Tests de diagnostic moléculaire in vitro - Spécifications relatives aux processus
préanalytiques pour les tissus à congélation rapide - Partie 1: ARN extrait
Ta slovenski standard je istoveten z: FprCEN/TS 16826-1
ICS:
11.100.10 'LDJQRVWLþQLSUHVNXVQL In vitro diagnostic test
VLVWHPLLQYLWUR systems
kSIST-TS FprCEN/TS 16826-1:2015 en,fr,de
2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.
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kSIST-TS FprCEN/TS 16826-1:2015
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kSIST-TS FprCEN/TS 16826-1:2015
TECHNICAL SPECIFICATION
FINAL DRAFT
FprCEN/TS 16826-1
SPÉCIFICATION TECHNIQUE
TECHNISCHE SPEZIFIKATION
March 2015
ICS 11.100.10
English Version
Molecular in vitro diagnostic examinations - Specifications for
pre-examination processes for snap frozen tissue - Part 1:
Isolated RNA
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 gefrorenes
congélation rapide - Partie 1: ARN extrait Gewebe - Teil 1: Isolierte RNS
This draft Technical Specification is submitted to CEN members for formal vote. It has been drawn up by the Technical Committee CEN/TC
140.
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.
Recipients of this draft are invited to submit, with their comments, notification of any relevant patent rights of which they are aware and to
provide supporting documentation.
Warning : This document is not a Technical Specification. It is distributed for review and comments. It is subject to change without notice
and shall not be referred to as a Technical Specification.
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. FprCEN/TS 16826-1:2015 E
worldwide for CEN national Members.
---------------------- Page: 3 ----------------------
kSIST-TS FprCEN/TS 16826-1:2015
FprCEN/TS 16826-1:2015 (E)
Contents Page
Foreword .3
Introduction .4
1 Scope .5
2 Normative references .5
3 Terms and definitions .5
4 General considerations .6
5 Outside the laboratory .7
5.1 Primary tissue collection manual.7
5.1.1 Information about the primary sample donor .7
5.1.2 Information on the primary tissue sample .7
5.1.3 Information on the primary tissue sample processing .8
5.2 Transport requirements .8
6 Inside the laboratory .8
6.1 Information on the primary tissue sample receipt .8
6.2 Evaluation of the pathology of the specimen and selection of the sample.9
6.3 Cryo-storage of the specimen .9
6.4 Storage requirements . 10
6.5 Isolation of the total RNA . 10
6.5.1 General information for RNA isolation procedures . 10
6.5.2 Using commercial kits . 11
6.5.3 Using the laboratories own protocols . 11
6.6 Quality assessment of isolated RNA . 12
6.7 Storage of isolated RNA . 12
Annex A (informative) Impact of preanalytical variables on RNA profiles obtained from frozen
liver tissue samples collected during and after routine surgery . 13
A.1 Comparison of stable and unstable genes identified under ischemic conditions . 13
A.2 Recommendations based on the results . 15
Bibliography . 16
2
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kSIST-TS FprCEN/TS 16826-1:2015
FprCEN/TS 16826-1:2015 (E)
Foreword
This document (FprCEN/TS 16826-1:2015) has been prepared by Technical Committee CEN/TC 140 “In vitro
diagnostic medical devices”, the secretariat of which is held by DIN.
This document is currently submitted to the Formal Vote.
3
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kSIST-TS FprCEN/TS 16826-1:2015
FprCEN/TS 16826-1:2015 (E)
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 profile generated during the pre-examination process. Therefore, a standardisation of
the entire process from primary sample collection to RNA 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 RNA analysis in what is referred to as the
preanalytical phase.
4
---------------------- Page: 6 ----------------------
kSIST-TS FprCEN/TS 16826-1:2015
FprCEN/TS 16826-1:2015 (E)
1 Scope
This Technical Specification recommends the handling, documentation and processing of frozen tissue
specimens intended for RNA analysis 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).
RNA profiles in tissues can change significantly before and after collection and can change differently in
different donors’ / patients’ tissues.
Therefore, it is essential to take special measures to minimize the described profile changes and modifications
within the tissue for subsequent RNA analysis.
Tissues that have undergone chemical stabilisation pre-treatment before freezing are not covered in this
document.
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
terms and definitions 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 kind 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
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, aliquotting, 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.]
5
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kSIST-TS FprCEN/TS 16826-1:2015
FprCEN/TS 16826-1:2015 (E)
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
quantitative RNA profile
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 and interference
3.7
RNA
ribonucleic acid
polymer of ribonucleotides occurring in a double-stranded or single-stranded form
[SOURCE: EN ISO 22174:2005, 3.1.3]
3.8
room temperature
temperature which is defined as 18 °C to 25 °C for the purposes of this document
3.9
sample
one or more parts taken from a primary sample
[SOURCE: EN ISO 15189:2012, 3.24, modified — The example was not taken over.]
3.10
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 RNA.
3.11
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).
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The stability of the specific quantitative RNA profile(s) of interest should be investigated throughout the entire
preanalytical workflow prior to the development and implementation of an analytical test.
Before tissues stabilized by freezing, quantitative RNA profile can change e.g., by gene induction, gene down
regulation and RNA degradation. These effects depend on the warm and cold ischemia times and the ambient
temperature before freezing. In addition, the described effects can vary in different donors’ / patients’ tissues.
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 RNA profile can occur.
NOTE Intraoperative warm ischemia can result in more pronounced changes of RNA profiles than during
postoperative cold ischemia. RNA profiles can also vary, 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 environment 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, duration shall be documented and 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.
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 of
origin, including references to any marking applied in the operating theatre made by surgeon, radiologist
or pathologist;
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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) will lead to RNA
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 RNA profile changes.
The temperatures of the transport container's surroundings during cold ischemia time (e.g., temperatures in
different rooms, transport) should be documented. If the temperature cannot be measured, the temperature
range should be estimated by classification as ambient temperature, room temperature, or at 2 °C to 8 °C.
5.2 Transport requirements
The laboratory in partnership with the clinical or surgery department shall establish a protocol for the transport
procedure of the specimen.
If the primary tissue sample is not already frozen, it should be transported on wet-ice or at 2 °C to 8 °C without
delay in order minimize changes to the RNA profile.
NOTE There is evidence that RNA in tissues can be stabilised in plastic bags under vacuum when kept at 0 °C to 4
C° during transport [1] before the samples are archived for biobanks or used for histopathological evaluatio
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