SIST-TS 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 recommends 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, in vitro diagnostics developers and manufacturers, 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 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.
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-Gewebe - 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
Relations
Standards Content (Sample)
SLOVENSKI STANDARD
SIST-TS CEN/TS 16827-2:2015
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
SIST-TS CEN/TS 16827-2:2015 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 16827-2:2015
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SIST-TS CEN/TS 16827-2:2015
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.
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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 .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
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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.
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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.
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.
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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
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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, 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.
3.9
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.]
3.10
protein
type of biological macromolecules composed of one or more chains with a defined sequence of amino acids
connected through peptide bonds
3.11
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
3.12
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]
3.13
PTM
post translational modifications
chemical alterations to a primary protein structure, often crucial for conferring biological activity on a protein
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[SOURCE: Encyclopedia of Psychopharmacology, 2010]
3.14
room temperature
temperature which is defined as 18 °C to 25 °C for the purposes of this document
3.15
sample
one or more parts taken from a primary sample
[SOURCE: EN ISO 15189:2012, 3.24, modified — The example was not taken over.]
3.16
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.
3.17
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.
3.18
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 fixed in standard buffered formalin solution, 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 formalin fixation. 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
fixation the tissue specimen, the higher is the risk that changes in the protein profile can occur.
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NOTE Prolonged cold ischemia times result in changes of protein (e.g., cytokeratin 18) and phosphoprotein (e.g.,
phospho-p42/44) amounts [4], [5]. Keeping the specimen on wet-ice diminishes this effect [6]. Proteins amounts as well as
the protein modifications can 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 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 formalin fixation, the transport duration shall be documented and the ambient conditions
should also be documented.
In addition, formalin fixation causes modifications of biomolecules and leads to suboptimal performance of
protein extracted from FFPE tissues [7] that should be considered in quality control and application of
molecular assays. Assay optimization for FFPE tissues or the use of non-crosslinking alternatives to standard
buffered formalin solution is an option to minimize this issue for molecular analyses.
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
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.
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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
protein 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 protein 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 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 protein profile.
NOTE There is evidence that proteins in tissues can be stabilised in plastic bags under vacuum when kept at 0 °C to
4 °C during transport [8] before the samples are archived for biobanks or used for histopathological evaluation.
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., labelling, transport conditions including temperature, tissue type and quantity of the
primary sample, leaking/breaking of the container) of the received samples shall be documented. Any
deviations from the established protocol for the transport procedure (see 5.2) shall be documented.
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6.2 Formalin fixation of the specimen
The fixative used should be standard buffered formalin solution.
The pH-value and concentration of the standard buffered formalin solution should be checked regularly as
formalin is not stable (e.g., it has a tendency to be oxidized to formic acid [6]).
The following steps shall be performed:
1. the consultation with the manufacturer's material safety data sheet (MSDS) before handling standard
buffered formalin solution;
NOTE Formaldehyde is a hazardous compound that chemically modifies biomolecules.
2. the documentation of the time point of placing the tissue sample into standard buffered formalin solution;
NOTE The total formalin fixation duration can impact further analyses, e.g., immunohistochemical techniques, nucleic
acid based molecular analyses. The optimal formalin fixation duration can vary depending on tissue type and size.
For larger surgical specimen inhomogeneous fixation can occur before the grossing process due to the slow
penetration rate of formaldehyde from the surface of the tissue to the interior. Formalin fixation for more than 24 h
can lead to a crosslinking intensity that can impact the protein analytical test. It has been shown that the protein yield
decreased with increasing fixation times [10], [11].
EXAMPLE For tissue pieces with a maximum thickness of 5 mm fixation times between 12 h and 24 h are
reasonable in most cases. See also 6.7.2.
3. the selection of collection container/s:
a) the capacity of the collection containers should be such that the primary sample can be completely
submerged into the standard buffered formalin solution. The minimum standard buffered formalin
solution to tissue ratio depends on the tissue concerned, but should be at least 4:1, (volume to
volume). To ensure complete formalin fixation of larger specimens a special tissue handling such as
incision(s) of solid organs or opening of hollow organs can be necessary.
NOTE The fixative-to-tissue-volume-ratio influences the progression of the fixation. There is evidence that a ratio of
10:1 can be necessary [9]. If the container has a small volume, standard buffered formalin solution can be changed
periodically, because formalin reacts with tissue components in a time-dependent way.
b) when using containers pre-filled with standard buffered formalin solution, provider’s product
instructions shall be followed;
c) the container shall be securely closable;
4. the labelling of the container (e.g., by using self-adhesive labels, handwriting, radio frequency
identification devices (RFID), pre-labelled containers, bar codes) shall give the minimum information of:
a) the patient ID, which can be in the form of a code;
b) the basic information on e.g., the tissue type, tissue condition, and related additional information such
as affected (e.g., tumour) or unaffected, unless a sample tracking system can supply this information
coupled to the identification of the sample used in 6.2, 4. a);
c) the unique numbering of each container;
5. the documentation of types, quantity and description of samples.
It should be considered that under some disease conditions, such as tumours, molecular features may not be
present homogeneously in the tissue sample. Therefore, it is important that the part of the actual tissue
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SIST-TS CEN/TS 16827-2:2015
CEN/TS 16827-2:2015 (E)
sample used for molecular analysis is evaluated by a medically qualified (e.g., board certified) pathologist. In
this context it should be documented which features of a disease are actually reflected in the tissue sample
used for molecular analysis (e.g., different molecular mechanisms can be activated at the centre and at the
invasion front of the tumour; also tumours can be composed of areas showing different differentia
...
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