CEN/TS 16826-3:2018

SLOVENSKI STANDARD
01-september-2018
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Molecular in vitro diagnostic examinations - Specifications for pre-examination processes
for snap frozen tissue - Part 3: Isolated DNA
Molekularanalytische in-vitro-diagnostische Verfahren - Spezifikationen für
präanalytische Prozesse für schockgefrorene Gewebeproben - Teil 1: Isolierte DNA
Tests de diagnostic moléculaire in vitro - Spécifications relatives aux processus
préanalytiques pour les tissus à congélation rapide - Partie 3: ADN isolé
Ta slovenski standard je istoveten z: CEN/TS 16826-3:2018
ICS:
11.100.10 'LDJQRVWLþQLSUHVNXVQL In vitro diagnostic test
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2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.

CEN/TS 16826-3
TECHNICAL SPECIFICATION
SPÉCIFICATION TECHNIQUE
July 2018
TECHNISCHE SPEZIFIKATION
ICS 11.100.10
English Version
Molecular in vitro diagnostic examinations - Specifications
for pre-examination processes for snap frozen tissue - Part
3: Isolated DNA
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 3: ADN isolé schockgefrorene Gewebeproben - Teil 3: Isolierte DNA
This Technical Specification (CEN/TS) was approved by CEN on 16 April 2018 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, Serbia, 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: Rue de la Science 23, B-1040 Brussels
© 2018 CEN All rights of exploitation in any form and by any means reserved Ref. No. CEN/TS 16826-3:2018 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 . 9
5 Outside the laboratory . 9
5.1 Specimen collection . 9
5.1.1 General . 9
5.1.2 Information about the specimen donor/patient . 9
5.1.3 Information about the specimen . 10
5.1.4 Specimen processing . 10
5.2 Fresh tissue transport requirements. 11
5.2.1 General . 11
5.2.2 Preparations for the transport . 11
5.2.3 During transport . 11
6 Inside the laboratory . 11
6.1 Information about the reception of the specimen . 11
6.2 Evaluation of the pathology of the specimen and selection of the sample(s) . 12
6.3 Freezing of the specimen or sample(s) . 13
6.4 Storage requirements . 15
6.5 DNA isolation . 15
6.5.1 General . 15
6.5.2 Using commercial kits . 16
6.5.3 Using the laboratory's own protocols . 16
6.6 Quantity and quality assessment of isolated DNA . 16
6.7 Storage of isolated DNA . 17
Bibliography . 18

European foreword
This document (CEN/TS 16826-3:2018) 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 shall not be held responsible for identifying any or all such patent rights.
CEN/TS 16826 consists of the following parts:
— 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 2: Isolated proteins;
— Molecular in vitro diagnostic examinations — Specifications for pre-examination processes for snap
frozen tissue — Part 3: Isolated DNA.
According to the CEN/CENELEC Internal Regulations, the national standards organisations 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, Serbia, Slovakia, Slovenia, Spain, Sweden, Switzerland,
Turkey and the United Kingdom.
Introduction
Molecular in vitro diagnostics, including molecular pathology, has enabled a significant progress in
medicine. Further progress is expected with new technologies analysing nucleic acids, proteins, and
metabolites in human tissues and body fluids. However, integrity of these molecules can change during
specimen collection, transport, storage, and processing, thus making the outcome from diagnostics or
research unreliable or even impossible because the subsequent examination assay will not determine
the situation in the patient but an artificial pattern generated during the pre-examination process.
Therefore, a standardization of the entire process from specimen collection to the DNA examination is
needed. Studies have been undertaken to determine the important influencing factors. This document
draws upon such work to codify and standardize the steps for frozen tissue with regard to DNA
examination in what is referred to as the pre-examination phase.
DNA integrity in tissues can change during processing and storage. Modifications of the DNA molecules
can impact the validity and reliability of the examination test results. Therefore, it is essential to take
special measures to minimize the described DNA changes and modifications for subsequent
examination.
In this document, the following verbal forms are used:
— “shall” indicates a requirement;
— “should” indicates a recommendation;
— “may” indicates a permission;
— “can” indicates a possibility or a capability.
1 Scope
This document gives recommendations for the handling, storage, processing and documentation of
frozen tissue specimens intended for DNA examination during the pre-examination phase before a
molecular examination is performed.
This document is applicable to molecular in vitro diagnostic examination including laboratory
developed tests performed by medical laboratories and molecular pathology laboratories that evaluate
DNA isolated from frozen tissue. It is also intended to be used by laboratory customers, in vitro
diagnostics developers and manufacturers, biobanks, institutions and commercial organizations
performing biomedical research, and regulatory authorities.
Tissues that have undergone chemical stabilization pre-treatment before freezing are not covered in
this document.
NOTE International, national or regional regulations or requirements can also apply to specific topics
covered in this document.
2 Normative references
The following documents are referred to in the text in such a way that some or all of their content
constitutes requirements of this document. 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)
EN ISO/IEC 17020:2012, Conformity assessment — Requirements for the operation of various types of
bodies performing inspection (ISO/IEC 17020:2012)
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 and the following
apply.
ISO and IEC maintain terminological databases for use in standardization at the following addresses:
• IEC Electropedia: available at http://www.electropedia.org/
• ISO Online browsing platform: available at http://www.iso.org/obp
3.1
aliquot
portion of a larger amount of homogenous material, assumed to be taken with negligible sampling error
Note 1 to entry: The term is usually applied to fluids. Tissues are heterogeneous and therefore cannot be
aliquoted.
Note 2 to entry: The definition is derived from the Compendium of Chemical Terminology Gold Book.
International Union of Pure and Applied Chemistry. Version 2.3.3., 2014; the PAC, 1990,62,1193 (Nomenclature
for sampling in analytical chemistry (Recommendations 1990)) p. 1206; and the PAC 1990, 62, 2167 (Glossary of
atmospheric chemistry terms (Recommendations 1990)) p. 2173.
3.2
ambient temperature
unregulated temperature of the surrounding air
3.3
analyte
component represented in the name of a measurable quantity
[SOURCE: EN ISO 17511:2003, 3.2, modified — The example was not taken over.]
3.4
analytical test performance
accuracy, precision, and sensitivity of a test to measure the analyte of interest
Note 1 to entry: Other test performance characteristics such as robustness, repeatability can apply as well.
3.5
cold ischemia
condition after removal of the tissue from the body until its stabilization or fixation
3.6
diagnosis
identification of a health or disease state from its signs and/or symptoms, where the diagnostic process
can involve examinations and tests for classification of an individual's condition into separate and
distinct categories or subclasses that allow medical decisions about treatment and prognosis to be
made
3.7
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]
3.8
DNase
deoxyribonuclease
enzyme that catalyzes the degradation of DNA into smaller components
3.9
examination
analytical test
set of operations having the object of determining the value or characteristics of a property
Note 1 to entry: Processes that start with the isolated analyte and include all kinds of parameter testing or
chemical manipulation for quantitative or qualitative examination.
[SOURCE: EN ISO 15189:2012, 3.7, modified — The term and definition are used here without the
original notes.]
3.10
grossing
gross examination
inspection of pathology specimens with the bare eye to obtain diagnostic information, while being
processed for further microscopic examination
3.11
homogeneous
uniform in structure and composition
3.12
interfering substance
endogenous substance of a specimen/sample or exogenous substance (e.g. stabilization solution) that
can alter an examination result
3.13
pre-examination process
preanalytical phase
preanalytical workflow
process that start in chronological order, from the clinician’s request and include the examination
request, preparation and identification of the patient, collection of the primary sample(s),
transportation to and within the medical or pathology laboratory, isolation of analytes, and ends when
the analytical examination begins
Note 1 to entry: The pre-examination phase includes preparative processes that influence the outcome of the
intended examination.
[SOURCE: EN ISO 15189:2012, 3.15, modified — An additional term was added and details were
adjusted to the scope of this document.]
3.14
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 are used here without the
original notes.]
3.15
proficiency test
evaluation of participant performance against pre-established criteria by means of inter-laboratory
comparisons
[SOURCE: EN ISO/IEC 17043:2010, 3.7, modified — The term and definition are used here without the
original notes.]
3.16
room temperature
for the purposes of this document, temperature in the range of 18 °C to 25 °C
Note 1 to entry: Local or national regulations can have different definitions.
3.17
sample
one or more parts taken from a primary sample
[SOURCE: EN ISO 15189:2012, 3.24, modified — The example was not taken over.]
3.18
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
Note 1 to entry: The analyte for the purpose of this document is DNA.
[SOURCE: ISO Guide 30:2015, 2.1.15, modified — The words “reference material” were replaced by
“sample material”.]
3.19
storage
prolonged interruption of the preanalytical workflow of a sample or analyte respectively, or of their
derivatives e.g., stained sections or tissue blocks, under appropriate conditions in order to preserve
their properties
Note 1 to entry: Long-term storage typically occurs in laboratory archives or in biobanks.
3.20
validation
confirmation, throughout the provision of objective evidence, that the requirements for a specific
intended use or application have been fulfilled
Note 1 to entry: The term “validated” is used to designate the corresponding status.
[SOURCE: EN ISO 9000:2015, 3.8.13, modified — Note 1 and Note 3 were not taken over.]
3.21
verification
confirmation, through provision of objective evidence, that specified requirements have been fulfilled
Note 1 to entry: The term “verified” is used to designate the corresponding status.
[SOURCE: EN ISO 9000:2015, 3.8.12, modified — Note 1 and Note 2 were not taken over.]
Note 2 to entry: Confirmation can comprise activities such as:
— performing alternative calculations,
— comparing a new design specification with a similar proven design specification,
— undertaking tests and demonstrations, and
— reviewing documents prior to issue.
3.22
warm ischemia
condition before the tissue is removed from the body, but where it is deprived of its normal blood
supply
3.23
workflow
series of activities necessary to complete a task
4 General considerations
For general statements on medical laboratory quality management systems and in particular on
specimen collection, reception and handling (including avoidance of cross contaminations)
see EN ISO 15189:2012, 4.2, 5.4.4, 5.4.6 or EN ISO/IEC 17020:2012, Clause 8 and 7.2. The requirements
on laboratory equipment, reagents, and consumables according to EN ISO 15189:2012, 5.3 shall be
followed; EN ISO 15189:2012, 5.5.1.2 and 5.5.1.3 and EN ISO/IEC 17020:2012, 6.2 can also apply.
All steps of a diagnostic workflow can influence the final analytical test result. Thus, the entire workflow
including biomolecule stability and sample storage conditions shall be verified and validated. Workflow
steps which cannot always be controlled (e.g. warm ischemia) shall be documented. A risk assessment
of non-controllable workflow steps including their potential impact on the analytical test performance
shall be performed and mitigation measures shall be established to enable the required analytical test
performance.
In contrast to RNA or proteins, DNA in tissue is relatively stable during warm and cold ischemia.
Changes of DNA sequence or copy numbers (e.g. comparative genomic hybridization (CGH) profiles)
due to longer warm and cold ischemia durations are unknown [6]. However, DNA methylation patterns
may change in response to ischemia. [7] The duration until the specimen is frozen should be kept as
short as possible in order to avoid enzymatic degradation of DNA. The duration before freezing shall be
documented and the temperature before freezing should be documented [6].
Safety requirements 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 pre-examination process, precautions shall be taken to avoid cross contamination
between different specimens/samples, e.g. by using single-use material whenever feasible or
appropriate cleaning procedures between processing of different specimens/samples.
If a commercial product is not used in accordance with the manufacturer's instructions, responsibility
for its validation, verification, use and performance lies with the user.
5 Outside the laboratory
5.1 Specimen collection
5.1.1 General
For the collection of the specimen, the requirements (e.g. disease condition, specimen size) for the
intended molecular examination (see also Clause 6) should be considered.
See also EN ISO 15189:2012, 5.4.4.
5.1.2 Information about the specimen donor/patient
The documentation shall include the ID of the specimen donor/patient, which can be in the form of a
code.
The documentation should include, but is not limited to:
a) the relevant health status of the specimen donor/patient (e.g. healthy, disease type, concomitant
disease, demographics (e.g. age and gender);
b) the information about routine medical treatment and special treatment prior to tissue collection
(e.g. anaesthetics, medications, surgical or diagnostic procedures);
c) the appropriate consent from the specimen donor/patient.
5.1.3 Information about the specimen
The documentation shall include, but is not limited to:
a) the start of ischemia within the body (warm ischemia) by documentation of the ischemia-relevant
vessel ligation/clamping time point (usually arterial clamping time);
NOTE Not needed where small tissue biopsy resection for freezing is performed.
b) the time and date when tissue is removed from the body and the method of removal (e.g. core-
needle biopsy, resection, biopsy device used for the collection);
c) the description of tissue type and origin, tissue condition (e.g. diseased, unaffected by the disease),
including references to any marking applied in or outside the operating theatre made by surgeon,
radiologist or pathologist;
If the freezing of the tissue is performed outside the laboratory, the documentation steps described
under 6.2, where pathology evaluation is required, and 6.3 shall be performed.
The documentation should also include the ID of the responsible person collecting the specimen.
5.1.4 Specimen processing
Tissue(s) that need to be frozen for diagnostic purposes can originate from a large specimen or can be
small specimen(s) e.g. biopsies taken by endoscopy or taken from patients during a surgical procedure
where fast frozen section diagnosis is required.
1) Any additions or modifications to the specimen after removal from the body (e.g. labelling for the
orientation of the specimen (e.g. ink-marking, stitches, incision(s)) shall be documented.
Where a pathology diagnosis is required on the specimen, sampling shall be performed by or under
supervision or guidance of a medically qualified (e.g. board certified) pathologist (see 6.2).
Where the specimen was removed without the requirement of pathology diagnosis, the evaluation,
selection and documentation of specimens may be done by other qualified persons than
pathologists.
2) The freezing of the specimen or samples taken from the specimen can be performed outside the
laboratory or inside the laboratory.
Cold ischemia can influence the DNA pattern (e.g. fragmentation); therefore immediate freezing
according to 6.3.2 should be preferred.
a) In case the specimen or sample is frozen outside the laboratory, proceed with 6.2 without
delay.
b) In case the specimen or sample is frozen inside the laboratory, fresh tissue specimens need to
be transported to the laboratory. The steps described under 5.2 for fresh tissue transport shall
be performed without delay.
5.2 Fresh tissue tra
...