CEN/TS 16835-3:2015

SLOVENSKI STANDARD
01-december-2015
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Molecular in vitro diagnostic examinations - Specifications for pre-examination processes
for venous whole blood - Part 3: Isolated circulating cell free DNA from plasma
Molekularanalytische in-vitro-diagnostische Verfahren - Spezifikationen für
präanalytische Prozesse für venöses Vollblutproben - Teil 3: Aus Plasma isolierte
zirkulierende zellfreie DNS
Tests de diagnostic moléculaire in vitro - Spécifications relatives aux processus pré-
analytiques pour le sang total veineux - Partie 3: ADN libre circulant extrait du plasma
Ta slovenski standard je istoveten z: CEN/TS 16835-3:2015
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 16835-3
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 3: Isolated circulating cell free DNA from plasma
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 3: ADN libre circulant extrait du venöse Vollblutproben - Teil 3: Aus Plasma isolierte
plasma zirkulierende zellfreie 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 16835-3:2015 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 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 . 9
5.2 Transport requirements . 9
6 Inside the laboratory . 10
6.1 Primary sample reception . 10
6.2 Storage requirements for venous whole blood sample. 10
6.3 Plasma preparation . 10
6.4 Storage requirements for plasma sample . 10
6.5 Isolation of the ccfDNA . 11
6.6 Quality assessment and quantity measurement of isolated ccfDNA . 12
6.7 Storage of isolated ccfDNA . 12
Annex A (informative) Influence of isolation procedures on ccfDNA fragments' lengths
distribution pattern in plasma samples . 13
Bibliography . 14

European foreword
This document (CEN/TS 16835-3:2015) 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.
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. Therefore, a standardization of the entire process from primary sample collection to
circulating cell free DNA (ccfDNA) 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 circulating cell free DNA analysis from plasma prepared from human venous
whole blood 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 circulating cell free DNA (ccfDNA) 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 ccfDNA profiles can change significantly after blood collection from the donor (e.g. release of
genomic DNA from white blood cells, ccfDNA fragmentation and ccfDNA quantity change). Special
measures need to be taken to secure good quality blood samples for ccfDNA analysis and storage.
Different dedicated measures need to be taken for preserving blood genomic DNA. These are not
described in this Technical Specification. Blood genomic DNA is covered in CEN/TS 16835-2, Molecular
in vitro diagnostic examinations — Specifications for pre-examination processes for venous whole
blood — Part 2: Isolated genomic DNA
NOTE CcfDNA obtained from blood by the procedures suggested in this document can contain DNA present
in exosomes [3] [4].
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
ccfDNA
circulating cell free DNA
extracellular human DNA present in blood, serum and plasma
Note 1 to entry: ccfDNA can include DNA present in vesicles such as exosomes [3] [4].
3.4
ccfDNA profile/s
circulating cell free DNA profile/s
amounts of different ccfDNA molecules, that are present in blood and plasma, that can be measured in
the absence of any losses, inhibition and interference
3.5
cryo-precipitates
insoluble residue when frozen plasma is thawed
3.6
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]
3.7
pre-examination processes
preanalytical phase
preanalytical workflow
processes that start, in chronological order, from the clinicians' 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 purposes 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 ccfDNA.
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 analytical steps should be verified and validated (see EN ISO 15189).
Blood circulating cell free DNA profiles can change significantly after blood collection and plasma
separation. The release of genomic DNA from white blood cells can change the ccfDNA profile
significantly. This can impact the validity of the analytical test results. Additional post-collection
effects can also occur e.g. ccfDNA fragmentation [5], [6], [7], [8]. These changes can vary individually in
different donors’ / patients’ blood depending on pathophysiological conditions [5] [9], [10], [11].
The stability of the specific blood ccfDNA profile of interest should be investigated throughout the
complete preanalytical workflow e.g. by applying the intended analytical test in time course studies
reflecting the individual preanalytical workflow steps such as transport and storage.
Before or during the design of the analytical test system, it should be investigated and ensured that the
blood ccfDNA profile/s intended to be analysed in the analytical test is/are not affected by the
envisioned entire pre-analytical workflow.
If a commercial product is not used in accordance with the manufacturers' instructions, responsibility
for its validation, verification, 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, gender, age, disease
type, gestational age);
NOTE In particular e.g. cancer, inflammation, diabetes, hepatic disease, coronary disease, respiratory
syndrome, trauma, after exhaustive exercise [5], in elderly patients suffering from acute or chronic disease,
first trimester of pregnancy, placental disorders as pre-term labour, pre-eclampsia and malimplantation
have been reported to affect both blood ccfDNA quantity and fragmentation [5], [9], [10], [11].
c) the information about medical treatment and special treatment prior to blood collection (e.g.
anaesthetics, medications, fasting status);
d) the type and purpose of the proposed 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 blood ccfDNA profile can be influenced by inadequate blood collection procedures and
inappropriate storage/shipping conditions, plasma separation as well as by ccfDNA isolation
procedures. Specifically, the post-collection release of genomic DNA from white blood cells can change
the ccfDNA profile significantly. This can impact the validity of the analytical test results.
Venous whole blood should be collected in appropriate collection devices.
Blood Collection tubes containing ccfDNA profile stabilizers are recommended when post collection
release of genomic DNA from blood cells or other ccfDNA profile changes can cause impacts on the
intended analytical test.
If blood collection tubes without ccfDNA profile stabilizers are used, EDTA blood collection tubes
should be used in preference to other collection tubes [7], [12].
Induced clotting process in serum tubes can lead to a leucocytes lysis and therefore the use of serum
tubes should be avoided [13].
— EDTA prevents clotting and allows for extended storage compared to other anticoagulants.
Consult the specifications by the analytical test provider for details.
— Venous whole blood collection tubes containing specific blood ccfDNA profile stabilizers can be
able to stabilize the ccfDNA profile for up to several days at room temperature or 2 °C to 8 °C [6],
[7], [12]. Consult also the specifications by the analytical test provider for details.
5.1.3 Primary venous whole blood 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 venipuncture 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 ccfDNA profile 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 blood ccfDNA
profile. 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 t
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