SIST-TS CEN/TS 16827-3:2015
(Main)Molecular in vitro diagnostic examinations - Specifications for pre-examination processes for FFPE tissue - Part 3: Isolated DNA
Molecular in vitro diagnostic examinations - Specifications for pre-examination processes for FFPE tissue - Part 3: Isolated DNA
This Technical Specification recommends the handling, documentation and processing of FFPE tissue specimens intended for DNA 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 organizations performing biomedical research, biobanks, and regulatory authorities).
DNA integrity in tissues can change before and during formalin-fixation, processing and storage. Chemical modifications introduced into DNA during tissue fixation might lead to sequence alterations [1], changes in the methylation status or even structural changes which can lead to e.g., spurious copy number changes in array-CGH profiles [2]. These modifications of the DNA molecules can impact the validity and reliability of the analytical test results. Therefore, special measures have to be taken to minimize the described modifications for subsequent DNA analysis.
Molekularanalytische in-vitro-diagnostische Verfahren - Spezifikationen für präanalytische Prozesse für FFPE-Gewebe - Teil 3: Isolierte DNS
Diese Technische Spezifikation gibt Empfehlungen zur Handhabung, Dokumentation und Verarbeitung von aus FFPE Gewebe bestehendem und für die DNS 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).
Die DNS Integrität in Geweben kann sich vor und nach der Formalinfixierung, der Verarbeitung und der Lagerung verändern. Während der Gewebefixierung in die DNS eingebrachte chemische Modifikationen können zu Fragmentierungen und Sequenzänderungen [1], Veränderung des Methylierungszustands oder sogar zu Struktur¬veränderungen führen, die z. B. störende Kopienanzahlveränderungen in Array CGH Profilen bewirken können [2]. Diese Modifikationen der DNS Moleküle können die Gültigkeit und Zuverlässigkeit der analytischen Prüfergebnisse beeinträchtigen. Daher ist es wichtig, dass besondere Maßnahmen getroffen werden, um die beschriebenen Modifikationen im Hinblick auf die anschließende DNS Analyse möglichst gering zu halten.
Tests de diagnostic moléculaire in vitro - Spécifications relatives aux processus préanalytiques pour les tissus FFPE - Partie 3: ADN
La présente Spécification technique fournit des recommandations pour la manipulation, la documentation et le traitement des spécimens de tissus FFPE destinés à l’analyse de l’ADN durant la phase préanalytique précédant la réalisation d’un essai moléculaire. La présente Spécification technique s’applique aux examens de diagnostic moléculaire in vitro (par exemple, laboratoires de diagnostic in vitro, clients de laboratoires, concepteurs et fabricants de diagnostic in vitro, institutions et organisations commerciales de recherche biomédicale, biobanques et autorités réglementaires).
L’intégrité de l’ADN dans les tissus peut changer avant et pendant la fixation au formol, le traitement et le stockage. Les modifications chimiques apportées à l’ADN durant la fixation des tissus peuvent causer une fragmentation et une altération des séquences [1], des changements de l’état de méthylation ou même des changements structuraux susceptibles de conduire, par exemple, à des modifications erronées du nombre de copies dans les profils d’hybridation génomique comparative sur puce à ADN [2]. Ces modifications des molécules d’ADN peuvent avoir une incidence sur la validité et la fiabilité des résultats d’essais analytiques. Il est donc essentiel de prendre des mesures spéciales afin de réduire le plus possible les modifications décrites lors de l'analyse subséquente de l’ADN.
Molekularne diagnostične preiskave in vitro - Specifikacije za predpreiskovalne procese za FFPE tkiva - 3. del: Izolirani DNK
Ta tehnična specifikacija vsebuje priporočila za obravnavo, dokumentiranje in obdelavo vzorcev tkiv FFPE, namenjenih za analizo DNK 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).
Integriteta DNK v tkivih se lahko spremeni pred fiksacijo v formalinu, obdelavo in shranjevanjem ter med temi postopki. Kemijske spremembe DNK med fiksacijo tkiva lahko pripeljejo do sprememb zaporedja [1], sprememb v stanju metilacije ali celo strukturnih sprememb, kar lahko vodi do npr. sprememb števila napačnih kopij pri molekularni kariotipizaciji aCGH [2]. Te spremembe molekul DNK lahko vplivajo na veljavnost in zanesljivost rezultatov analitičnih preskusov. Zato je treba sprejeti posebne ukrepe za zmanjšanje opisanih sprememb pri nadaljnjih analizah DNK.
General Information
Relations
Standards Content (Sample)
SLOVENSKI STANDARD
SIST-TS CEN/TS 16827-3:2015
01-oktober-2015
0ROHNXODUQHGLDJQRVWLþQHSUHLVNDYHLQYLWUR6SHFLILNDFLMH]DSUHGSUHLVNRYDOQH
SURFHVH]D))3(WNLYDGHO ,]ROLUDQL'1.
Molecular in vitro diagnostic examinations - Specifications for pre-examination processes
for FFPE tissue - Part 3: Isolated DNA
Molekularanalytische in-vitro-diagnostische Verfahren - Spezifikationen für
präanalytische Prozesse für FFPE-Gewebe - Teil 3: Isolierte DNS
Tests de diagnostic moléculaire in vitro - Spécifications relatives aux processus
préanalytiques pour les tissus FFPE - Partie 3: ADN
Ta slovenski standard je istoveten z: CEN/TS 16827-3:2015
ICS:
11.100.10 'LDJQRVWLþQLSUHVNXVQL In vitro diagnostic test
VLVWHPLLQYLWUR systems
SIST-TS CEN/TS 16827-3:2015 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 16827-3:2015
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SIST-TS CEN/TS 16827-3:2015
TECHNICAL SPECIFICATION
CEN/TS 16827-3
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 3: Isolated
DNA
Tests de diagnostic moléculaire in vitro - Spécifications Molekularanalytische in-vitro-diagnostische Verfahren -
relatives aux processus préanalytiques pour les tissus Spezifikationen für präanalytische Prozesse für FFPE-
FFPE - Partie 3: ADN isolé Gewebeproben - Teil 3: Isolierte DNS
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 16827-3:2015 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 .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 .8
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 Formalin fixation of the specimen .9
6.3 Evaluation of the pathology of the specimen and selection of the sample. 10
6.4 Post-fixation of frozen samples . 11
6.5 Processing and paraffin embedding. 11
6.6 Storage requirements . 11
6.7 Isolation of DNA . 12
6.7.1 General . 12
6.7.2 General information for DNA isolation procedures . 12
6.7.3 Using commercial kits . 12
6.7.4 Using the laboratories’ own protocols . 13
6.8 Quantity and quality assessment of isolated RNA . 13
6.9 Storage of isolated RNA . 14
Annex A (informative) Impact of the storage temperature on DNA Integrity in FFPE blocks of
tissue . 15
A.1 Introduction . 15
A.2 Results . 15
A.3 Conclusions . 15
Bibliography . 16
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European foreword
This document (CEN/TS 16827-3:2015) 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. Studies have been undertaken to
determine the influencing factors for DNA analysis from formalin fixed and paraffin embedded (FFPE) tissue.
These studies demonstrated that a standardization of the entire process from primary sample collection to
DNA analysis is needed. This Technical Specification draws upon such work to codify and standardize the
steps for FFPE tissue with regard to DNA 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 FFPE
tissue specimens intended for DNA 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 organizations performing biomedical research, biobanks, and regulatory
authorities).
DNA integrity in tissues can change before and during formalin fixation, processing and storage. Chemical
modifications introduced into DNA during tissue fixation might lead to fragmentation and sequence alterations
[1], changes in the methylation status or even structural changes which can lead to e.g., spurious copy
number changes in array-CGH profiles [2]. These modifications of the DNA molecules can impact the validity
and reliability of the analytical test results. Therefore, it is essential to take special measures to minimize the
described modifications for subsequent DNA analysis.
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
DNA
deoxyribonucleic acid
polymer of deoxyribonucleotides occurring in a double-stranded (dsDNA) or single-stranded (ssDNA) form
[SOURCE: EN ISO 22174:2005, 3.1.2]
3.5
FFPE
formalin fixation and paraffin embedding
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3.6
FFPE tissues
formalin fixed and paraffin embedded tissues
3.7
formalin
saturated formaldehyde solution containing a mas fraction of 37 % (corresponding to a volume fraction of 40
%) formaldehyde, termed 100 % formalin
3.8
formalin fixation
treatment of a sample with standard buffered formalin solution for stabilization
3.9
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.
3.10
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.]
3.11
room temperature
temperature which is defined as 18 °C to 25 °C for the purposes of this document
3.12
sample
one or more parts taken from a primary sample
[SOURCE: EN ISO 15189:2012, 3.24, modified — The example was not taken over.]
3.13
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 DNA.
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3.14
standard buffered formalin solution
10 % formalin solution containing 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 methanol to inhibit oxidation and polymerization of
formaldehyde.
3.15
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).
For samples intended to be analysed for DNA, the following specific aspects shall be considered.
In contrast to RNA or proteins, DNA in tissue is relatively stable during warm and cold ischemia times.
Changes of DNA, sequence or copy numbers (e.g., CGH profiles,) due to an extended duration of warm and
cold ischemia are unknown. The duration until the specimen is placed into standard buffered formalin solution
should be kept as short as possible in order to avoid enzymatic degradation of DNA. The duration before
fixation shall be documented and the temperature before fixation should be documented [3].
During the fixation, processing and storage, the DNA integrity can change depending on the kind of fixative,
fixation time and temperature, storage or archiving of the fixed paraffin embedded sample as well as the
method used for DNA isolation and purification. When using a fixative based on formaldehyde, temperature,
and fixation duration have a significant impact on DNA integrity. The longer the fixation duration and the
higher the temperature, the more chemical modifications and crosslinks are introduced, which can lead to
degradation or sequence alterations [1], [2], [4], [5].
Safety regulations on specimen 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;
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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 6.2, if the formalin fixation starts 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) fit for transport of formalin fixed tissue samples, if relevant;
3. the selection and use of stabilization 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
DNA degradation when the tissue thaws thereafter. It can also impact the morphological characterization.
NOTE 2 This step can be omitted, if the specimen is transferred directly into standard buffered formalin
solution (see 6.2).
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 DNA degradation.
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.
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If the primary tissue sample is not already placed into standard buffered formalin solution, it should be
transported on wet-ice or at 2 °C to 8 °C without delay in order to minimize changes to the DNA.
If the primary tissue sample is already placed into standard buffered formalin solution outside the laboratory,
the temperature during transport should not exceed room temperature.
The compliance with the protocol for the transport procedure shall be documented. Any deviations from the
protocol shall be described and documented.
6 Inside the laboratory
6.1 Information on the primary tissue sample receipt
The name of the person receiving the primary tissue sample shall be documented. The tissue sample arrival
time and conditions (e.g., labellin
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