CEN/TS 16835-2:2015

SLOVENSKI STANDARD
01-december-2015
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SURFHVH]DYHQVNRSROQRNULGHO'1$L]ROLUDQL]JHQRPD
Molecular in vitro diagnostic examinations - Specifications for pre-examination processes
for venous whole blood - Part 2: Isolated genomic DNA
Molekularanalytische in-vitro-diagnostische Verfahren - Spezifikationen für
präanalytische Prozesse für venöse Vollblutproben - Teil 2: Isolierte genomische DNA
Ta slovenski standard je istoveten z: CEN/TS 16835-2:2015
ICS:
11.100.10 'LDJQRVWLþQLSUHVNXVQL In vitro diagnostic test
VLVWHPLLQYLWUR systems
11.100.30 Analiza krvi in urina Analysis of blood and urine
2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.

CEN/TS 16835-2
TECHNICAL SPECIFICATION
SPÉCIFICATION TECHNIQUE
October 2015
TECHNISCHE SPEZIFIKATION
ICS 11.100.30
English Version
Molecular in vitro diagnostic examinations - Specifications
for pre-examination processes for venous whole blood -
Part 2: Isolated genomic DNA
Tests de diagnostic moléculaire in vitro - Spécifications Molekularanalytische in-vitro-diagnostische Verfahren
relatives aux processus pré-analytiques pour le sang - Spezifikationen für präanalytische Prozesse für
total veineux - Partie 2: ADN génomique extrait venöse Vollblutproben - Teil 2: Isolierte genomische
DNS
This Technical Specification (CEN/TS) was approved by CEN on 31 August 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 16835-2:2015 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 . 7
5.1 Primary venous whole blood collection manual . 7
5.1.1 Information about the primary sample donor . 7
5.1.2 Selection of the venous whole blood collection tube by the laboratory . 8
5.1.3 Primary venous whole blood sample collection from the patient and stabilization
procedures . 8
5.1.4 Information on the primary blood sample and storage requirements at the blood
collection facility . 8
5.2 Transport requirements. 9
6 Inside the laboratory . 10
6.1 Primary sample reception . 10
6.2 Storage requirements . 10
6.3 Isolation of the genomic DNA . 11
6.3.1 General . 11
6.3.2 Using commercial kits . 12
6.3.3 Using the laboratories own protocols . 12
6.4 Quantity and quality assessment of isolated genomic DNA . 12
6.5 Storage of isolated genomic DNA . 13
Annex A (informative) Impact of preanalytical workflow steps on venous whole blood
genomic DNA quality . 14
A.1 General information on operated experiments in Annex A . 14
A.2 Influence of preanalytical variables (blood storage duration and temperature, and
DNA isolation methods) on genomic DNA integrity . 14
A.3 Influence of blood storage time on the genomic DNA integrity. 15
A.4 Influence of genomic DNA integrity on an analytical test based on long PCR
amplicons . 17
A.5 Influence of blood storage conditions on the performance of PCR tests based on
short amplicons . 18
Bibliography . 20

European foreword
This document (CEN/TS 16835-2:2015) 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.
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 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.
A standardization of the entire process from primary sample collection to genomic DNA analysis is
needed due to genomic DNA degradation and fragmentation after blood collection. Studies have been
undertaken to determine the important influencing factors. This Technical Specification draws upon
such work to codify and standardize the steps for venous whole blood genomic DNA analysis in what is
referred to as the preanalytical phase.
1 Scope
This Technical Specification recommends the handling, documentation and processing of venous whole
blood specimens intended for genomic DNA analysis during the preanalytical phase before a molecular
assay is performed. This Technical Specification covers specimens collected by venous whole blood
collection tubes. 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).
Blood genomic DNA can fragment or degrade after blood collection. Therefore, special measures need to
be taken to secure good quality blood samples for genomic DNA analysis. This is particularly relevant
for analytical test procedures requiring high molecular weight DNA.
Different dedicated measures need to be taken for preserving blood circulating cell free DNA, which are
not described in this Technical Specification. Circulating cell free DNA in blood is covered in
CEN/TS 16835-3, Molecular in vitro diagnostic examinations — Specifications for pre-examination
processes for venous whole blood — Part 3: Isolated circulating cell free DNA from plasma.
Different dedicated measures need to be taken for collecting, stabilizing, transporting and storing
capillary blood as well as for blood collected and stored by paper based technologies. These are not
described in this Technical Specification.
DNA from pathogens present in blood is not covered by this Technical Specification.
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 kind of parameter testing or chemical
manipulation for quantitative or qualitative analysis
3.3
blood genomic DNA stabilizers
compounds, solutions or mixtures that are made to minimize degradation and fragmentation of
genomic DNA in a blood sample
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
genomic DNA
DNA from the genome containing all coding (exon) and non-coding (intron and other) sequences
Note 1 to entry: In this document it is always only referred to genomic DNA present in blood cells, excluding
circulating cell free DNA.
3.6
high molecular weight DNA
HMW DNA
DNA larger than 50 kb for the purpose of this document
3.7
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, 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, 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.
3.8
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.]
3.9
room temperature
temperature which is defined as 18 °C to 25 °C for the purpose of this document
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 30:2015, 2.1.15, modified — The words “reference material” were replaced by
“sample material".]
Note 1 to entry: The measured constituent for the purpose of this document is genomic DNA.
4 General considerations
For general statements on primary sample collection and handling (including avoidance of cross
contaminations), see EN ISO 15189:2012, 5.2.6, 5.4.4. 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 sample stability and storage conditions,
and the analytical steps should be verified and validated (see EN ISO 15189).
The stability of the genomic DNA should be investigated throughout the complete pre-analytical
workflow.
Before or during the design of the analytical test system it should be investigated and ensured that the
genomic DNA minimum amount and size required for the analytical test are not affected by the
envisioned entire preanalytical workflow.
If a commercial product is not used in accordance with the manufacturers' instructions, responsibility
for its use and performance lies with the user.
Safety regulations on facilities, transport and handling shall be considered (EN ISO 15189:2012, 5.2.3
and 5.4.5, and ISO 15190).
5 Outside the laboratory
5.1 Primary venous whole blood 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 and relevant lifestyle factors of the blood donor (e.g. healthy, disease type, gender,
age);
c) the information about medical treatment and special treatment prior to blood collection (e.g.
anaesthetics, medications);
d) the type and the purpose of the analytical test requested.
See also EN ISO 15189:2012, 5.4.4.
5.1.2 Selection of the venous whole blood collection tube by the laboratory
The quality of genomic DNA can be influenced (e.g., DNA fragmentation), by inadequate blood collection
procedures, inappropriate storage/shipping conditions and DNA isolation procedures, [3], [4], [5], [6],
[7], [8], [9], [10].
Blood should be collected in appropriate venous whole blood collection tubes containing an
anticoagulant such as EDTA or Acid Citrate Dextrose (ACD) [11].
NOTE Blood collection tubes containing EDTA as an anticoagulant are preferable for most genomic DNA
analysis. Blood collection tubes containing heparin as an anticoagulant can impact the purity of the isolated
genomic DNA, when using genomic DNA isolation methods not eliminating the heparin. Carrying over of heparin
into the genomic DNA eluate can cause inhibitions in analytical test technologies, such as PCR.
Specifically developed blood collection tubes, containing genomic DNA stabilizing reagents, are also
available aimed to standardize blood collection, transport and storage of venous whole blood.
5.1.3 Primary venous whole blood sample collection from the patient and stabilization
procedures
1. The identity of the person collecting the primary sample and the time of blood collection according
to EN ISO 15189:2012, 5.4.4.3, f) shall be documented.
2. For the labelling (sample identification) of the blood collection tube a routine procedure
(EN ISO 15189:2012, 5.4.4.3, e)) or a procedure with additional information (e.g. 2D-barcode) shall
be used.
3. Standard venepuncture technique can be used. Steps for preventing possible backflow may be
required. The manufacturers’ instructions for using the blood collection tubes shall be followed. A
blood collection set and needle holder can be required when using blood genomic DNA stabilizer
containing tubes. In this case, the instructions of the collection set and needle holder manufacturer
shall be followed.
NOTE There is no known specific effect of venous whole blood draw procedure on the genomic DNA.
Routine procedures can therefore be used.
4. Blood collection tubes shall be filled in accordance to the manufacturers’ instructions and attention
should be drawn to the correct positioning of the collection tube during the blood draw as well as
the required volume.
5. Blood collection tube manufacturers' instructions for mixing or inverting the tube immediately
after blood collection shall be followed.
NOTE Unless additives are homogenously mixed with the blood sample, the genomic DNA quality can be
compromised, which can impact the validity and reliability of the analytical test results.
6. Any tampering with and/or additions to the primary sample shall be documented.
5.1.4 Information on the primary blood sample and storage requirements at the blood
collection facility
5.1.4.1 General
As blood genomic DNA can fragment or degrade after blood collection (Figure A.1) and can thereby
affect the validity and reliability of the analytical test result (Figure A.3), the documentation on the
primary blood sample shall include the date and should also include the time of blood collection [11].
For samples dedicated to be archived in a biobank it is usually not known which individual genomic
DNA tests will be performed after archiving, therefore either tubes with genomic DNA stabilizers should
be used or, if using tubes without genomic DNA stabilizers, the recommendations for HMW DNA should
be followed (see Table 1 and 5.1.4.3.2).
The temporary storage duration in the blood collection facility contributes to the total duration for
storage.
5.1.4.2 Using blood collection tubes with stabilizers
For storing the primary blood samples collected in blood collection tubes with blood genomic DNA
stabilizers, the blood collection tube manufacturers' instructions on storage conditions shall be
followed (including the storage duration and temperature). Where the analytical test providers'
instructions are more stringent, these shall be followed. The storage conditions (including the storage
duration and temperature) shall be documented.
5.1.4.3 Using blood collection tubes without stabilizers
5.1.4.3.1 When using blood collection tubes without blood genomic DNA stabilizers, the analytical
test providers' instructions on storage conditions shall be followed. This can require documentation of
storage conditions.
5.1.4.3.2 When using blood collection tubes without blood genomic DNA stabilizers and no
requirements on the storage conditions are available from the analytical test provider, the primary
venous whole blood samples should be processed as soon as possible.
As blood collection tubes containing EDTA as an anticoagulant are most wide spread for genomic DNA
analysis the following recommendations (see also Table 1) refer to this blood collection tube type. For
analytical tests requiring High Molecular Weight DNA, the blood sample should be stored at room
temperature for not longer than one day or at 2 °C to 8 °C for not longer than three days. For a longer
storage the sample should be kept at −20 °C for not longer than 1 month, or at −70 °C or below for
longer storage. For the analysis of DNA variants not requiring HMW DNA analyses, the blood sample
should be stored at room temperature for up to 3 days or at 2 °C to 8 °C for up to 7 days (Figure A.4).
For a longer storage the sample should be kept at −20 °C for up to 3 months or at −70 °C or below for
longer st
...