CEN/TS 16827-1:2015

SLOVENSKI STANDARD
01-oktober-2015
0ROHNXODUQHGLDJQRVWLþQHSUHLVNDYHLQYLWUR6SHFLILNDFLMH]DSUHGSUHLVNRYDOQH
SURFHVH]D))3(WNLYDGHO,]ROLUDQL51.
Molecular in vitro diagnostic examinations - Specifications for pre-examination processes
for FFPE tissue - Part 1: Isolated RNA
Molekularanalytische in-vitro-diagnostische Verfahren - Spezifikationen für
präanalytische Prozesse für FFPE-Gewebe - Teil 1: Isolierte RNS
Tests de diagnostic moléculaire in vitro - Spécifications relatives aux processus
préanalytiques pour les tissus FFPE - Partie 1: ARN extrait
Ta slovenski standard je istoveten z: CEN/TS 16827-1:2015
ICS:
11.100.10 'LDJQRVWLþQLSUHVNXVQL In vitro diagnostic test
VLVWHPLLQYLWUR systems
2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.

TECHNICAL SPECIFICATION
CEN/TS 16827-1
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 1: Isolated
RNA
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 1: ARN extrait 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 16827-1:2015 E
worldwide for CEN national Members.

Contents Page
European foreword .3
Introduction .4
1 Scope .5
2 Normative references .5
3 Terms and definitions .5
4 General considerations .7
5 Outside the laboratory .8
5.1 Primary tissue collection manual.8
5.1.1 Information about the primary sample donor .8
5.1.2 Information on the primary tissue sample .8
5.1.3 Information on the primary tissue sample processing .8
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 .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 . 12
6.7 Isolation of the total RNA . 12
6.7.1 General . 12
6.7.2 General information for RNA isolation procedures . 12
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 Summary . 15
A.2 Results . 15
A.2.1 Time dependency of RNA integrity . 15
A.2.2 Impact of formalin-fixation on cDNA synthesis efficiency . 16
A.2.3 Fixation and storage introduces major gene-to-gene variations in RT-qPCR . 17
A.2.4 Impact of storage conditions of FFPE blocks on RNA Integrity . 18
A.3 Conclusions . 18
A.4 Further reading . 19
Bibliography . 20

European foreword
This document (CEN/TS 16827-1:2015) 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.
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 formalin fixed and paraffin embedded (FFPE) tissue with regard to RNA analysis
in what is referred to as the preanalytical phase.
1 Scope
This Technical Specification gives recommendations for the handling, documentation and processing of FFPE
tissue specimens intended for 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 organizations performing biomedical research, biobanks, and regulatory
authorities).
The formalin fixation and the paraffin embedding process lead to modifications of the RNA 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 RNA 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
FFPE
formalin fixation and paraffin embedding
3.5
FFPE tissues
formalin fixed and paraffin embedded tissues
3.6
formalin
saturated formaldehyde solution containing a mas fraction of 37 % (corresponding to a volume fraction of 40
%) formaldehyde, termed 100 % formalin
3.7
formalin fixation
treatment of a sample with standard buffered formalin solution for stabilization
3.8
pre-examination processes
preanalytical phase
preanalytical workflow
processes that start, in chronological order, from the clinician’s request and include the examination request,
preparation and identification of the patient, 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
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
3.11
RNA
ribonucleic acid
polymer of ribonucleotides occurring in a double-stranded or single-stranded form
[SOURCE: EN ISO 22174:2005, 3.1.3]
3.12
room temperature
temperature which is defined as 18 °C to 25 °C for the purposes of this document
3.13
sample
one or more parts taken from a primary sample
[SOURCE: EN ISO 15189:2012, 3.24, modified — The example was not taken over.]
3.14
stability
ability of a sample material, when stored under specified conditions, to maint
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