Molecular in vitro diagnostic examinations - Specifications for pre-examination processes for circulating tumor cells (CTCs) in venous whole blood - Part 1: Isolated RNA

This document gives guidelines on the handling, storage, processing and documentation of venous whole blood specimens intended for the examination of human cellular RNA isolated from Circulating Tumor Cells (CTCs) during the pre-examination phase before a molecular examination is performed.
This document is applicable to molecular in vitro diagnostic examinations including laboratory developed tests performed by medical laboratories. 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.
This document does not cover the isolation of cellular RNA directly from venous whole blood containing CTCs. This is covered in EN ISO 20186-1.
This document does not cover the isolation of specific blood cells and subsequent isolation of cellular RNA therefrom.
RNA in pathogens present in blood is not covered by this document.
NOTE International, national or regional regulations or requirements can also apply to specific topics covered in this document.

Molekularanalytische in-vitro-diagnostische Verfahren - Spezifikationen für präanalytische Prozesse für zirkulierende Tumorzellen (CTC) in venösen Vollblutproben - Teil 1: Isolierte RNA

Dieses Dokument gibt Empfehlungen zur Handhabung, Dokumentation, Lagerung und Verarbeitung von Proben venösen Vollbluts, die für die Untersuchung der aus zirkulierenden Tumorzellen (CTCs) isolierten RNA vorgesehen sind, während der präanalytischen Phase vor der Durchführung einer molekularen Analyse.
Dieses Dokument ist anwendbar auf molekulare in-vitro-diagnostische Untersuchungen, die in medizinischen Laboratorien durchgeführt werden, einschließlich vom Laboratorium selbst entwickelter Verfahren. Es ist darüber hinaus für die Verwendung durch Kunden von Laboratorien, Entwickler und Hersteller von In-vitro-Diagnostika, durch Biobanken, Institutionen und kommerzielle Organisationen, die biomedizinische Forschungen durchführen, sowie durch Arzneimittelagenturen bestimmt.
Dieses Dokument behandelt nicht die direkte Isolierung von zellulärer RNA aus venösen Vollblutproben, die CTCs enthalten. Dies wird in EN ISO 20186-1, Molekularanalytische in-vitro-diagnostische Verfahren — Spezifikationen für präanalytische Prozesse für venöse Vollblutproben — Teil 1: Isolierte zelluläre RNA behandelt.
Dieses Dokument behandelt nicht die Isolierung von bestimmten weißen Blutzellen (Leukozyten) und die anschließende Isolierung von zellulärer RNA aus diesen Zellen.
Dieses Dokument behandelt auch nicht die Anforderungen an den präanalytischen Arbeitsablauf zur Kryokonservierung und Kultivierung lebensfähiger CTCs.
ANMERKUNG 1 Die in diesem Dokument dargelegten Anforderungen können auch auf andere zirkulierende Zellen (z. B. fetale Zellen) angewendet werden.
ANMERKUNG 2 Internationale, nationale oder regionale Regelungen bzw. Anforderungen können ebenfalls für bestimmte Themen in diesem Dokument gelten.

Analyses de diagnostic moléculaire in vitro - Spécifications relatives aux processus préanalytiques pour les cellules tumorales circulantes (CTCs) dans le sang total veineux - Partie 1 : ARN extrait

Molekularne diagnostične preiskave in vitro - Specifikacije za predpreiskovalne procese za cirkulirajoče tumorske celice (CTC) v venski polni krvi - 1. del: Izolirana RNK

Ta dokument podaja smernice glede obravnave, shranjevanja, obdelave in dokumentiranja vzorcev venske polne krvi, namenjenih za analizo človeške celične RNK, izolirane iz cirkulirajočih tumorskih celic (CTC) med predpreiskovalno fazo, preden se izvede molekularna preiskava. Ta dokument se uporablja za molekularne diagnostične preiskave in vitro, vključno z laboratorijsko razvitimi preskusi, ki jih izvajajo v medicinskih laboratorijih. Namenjen je tudi temu, da ga uporabljajo laboratorijske stranke, razvijalci in proizvajalci diagnostike in vitro, biobanke, institucije in komercialne organizacije, ki izvajajo biomedicinske raziskave, ter regulativni organi. Ta dokument ne zajema izolacije celične RNK neposredno iz venske polne krvi, ki vsebuje cirkulirajoče tumorske celice. To je zajeto v standardu EN ISO 20186-1. Ta dokument ne zajema izolacije določenih krvnih celic in nadaljnje izolacije celične RNK iz njih. RNK v patogenih v krvi ni zajeta v tem dokumentu.
OPOMBA: Za določene teme, ki so zajete v tem dokumentu, lahko veljajo tudi mednarodni, nacionalni ali regionalni predpisi ali zahteve.

General Information

Status
Published
Public Enquiry End Date
04-Sep-2019
Publication Date
09-Feb-2020
Technical Committee
Current Stage
6060 - National Implementation/Publication (Adopted Project)
Start Date
04-Feb-2020
Due Date
10-Apr-2020
Completion Date
10-Feb-2020

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SLOVENSKI STANDARD
SIST-TS CEN/TS 17390-1:2020
01-marec-2020
Molekularne diagnostične preiskave in vitro - Specifikacije za predpreiskovalne
procese za cirkulirajoče tumorske celice (CTC) v venski polni krvi - 1. del: Izolirana
RNK
Molecular in vitro diagnostic examinations - Specifications for pre-examination processes
for circulating tumor cells (CTCs) in venous whole blood - Part 1: Isolated RNA
Molekularanalytische in-vitro-diagnostische Verfahren - Spezifikationen für
präanalytische Prozesse für zirkulierende Tumorzellen (CTC) in venösen Vollblutproben
- Teil 1: Isolierte RNA
Analyses de diagnostic moléculaire in vitro - Spécifications relatives aux processus
préanalytiques pour les cellules tumorales circulantes (CTCs) dans le sang total veineux
- Partie 1 : ARN extrait
Ta slovenski standard je istoveten z: CEN/TS 17390-1:2020
ICS:
11.100.10 Diagnostični preskusni In vitro diagnostic test
sistemi in vitro systems
SIST-TS CEN/TS 17390-1:2020 en,fr,de
2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.

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CEN/TS 17390-1
TECHNICAL SPECIFICATION

SPÉCIFICATION TECHNIQUE

January 2020
TECHNISCHE SPEZIFIKATION
ICS 11.100.10
English Version

Molecular in vitro diagnostic examinations - Specifications
for pre-examination processes for circulating tumor cells
(CTCs) in venous whole blood - Part 1: Isolated RNA
Analyses de diagnostic moléculaire in vitro - Molekularanalytische in-vitro-diagnostische Verfahren
Spécifications relatives aux processus préanalytiques - Spezifikationen für präanalytische Prozesse für
pour les cellules tumorales circulantes (CTCs) dans le zirkulierende Tumorzellen (CTC) in venösen
sang total veineux - Partie 1 : ARN extrait Vollblutproben - Teil 1: Isolierte RNA
This Technical Specification (CEN/TS) was approved by CEN on 27 October 2019 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, France, Germany, Greece, Hungary, Iceland, Ireland, Italy, Latvia, Lithuania, Luxembourg, Malta, Netherlands, Norway,
Poland, Portugal, Republic of North Macedonia, 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
© 2020 CEN All rights of exploitation in any form and by any means reserved Ref. No. CEN/TS 17390-1:2020 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 . 10
5 Outside the laboratory . 11
5.1 Specimen collection . 11
5.2 Transport requirements. 13
6 Inside the laboratory . 14
6.1 Specimen reception . 14
6.2 Storage requirements for the venous whole blood specimen . 14
6.3 Enrichment of CTCs . 14
6.4 Quality of enriched CTCs. 15
6.5 Storage of enriched CTCs . 15
6.6 Isolation of CTCs . 15
6.7 Processing of isolated CTCs . 16
6.8 Isolation of RNA from an enriched CTC sample. 17
6.9 Quantity and quality assessment of isolated RNA from enriched or isolated CTCs . 18
6.10 Storage of isolated RNA from enriched CTCs . 18
Annex A (informative) Decision guideline for critical steps of the CTC pre-analytical
workflow for RNA isolation . 20
Bibliography . 23

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European foreword
This document (CEN/TS 17390-1:2020) 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 17390 consists of the following parts, under the general title Molecular in vitro diagnostic
examinations — Specifications for pre-examination processes for Circulating Tumor Cells (CTCs) in venous
whole blood:
— Part 1: Isolated RNA
— Part 2: Isolated DNA
— Part 3: Preparations for analytical CTC staining
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, France, Germany, Greece, Hungary, Iceland,
Ireland, Italy, Latvia, Lithuania, Luxembourg, Malta, Netherlands, Norway, Poland, Portugal, Republic of
North Macedonia, Romania, Serbia, Slovakia, Slovenia, Spain, Sweden, Switzerland, Turkey and the
United Kingdom.
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Introduction
Solid tumours release cells and bioanalytes into blood and other body fluids. This has opened the option
of minimally-invasive tumour detection, diagnosis and characterization from venous whole blood
(liquid biopsies). Liquid biopsies are expected to enable earlier detection and diagnosis of cancers and
advance personalized patient treatment. These applications have become one of the fastest growing
segments of the entire diagnostic market.
Circulating tumour cells (CTCs) in venous whole blood reflect the disease complexity that evolves
during tumour progression, with distinct genetic, epigenetic and expression features. Besides the
prognostic role of CTC identification and/or enumeration in cancer progression, CTC molecular
characterization can improve e.g. disease outcome prediction, therapeutic guidance and post-treatment
monitoring of the patient.
CTCs are now considered as a surrogate of tumour tissue in cancer early development, progression and
metastatic phase.
Molecular characterization of CTCs can provide for example a strategy for monitoring cancer genotypes
during systemic therapies [1], identification of mechanisms of disease progression, identification of
novel targets for treatment [2] and to select targeted therapies. Moreover, CTC single-cell sequencing is
emerging as an important tool for tumour genomic heterogeneity analysis [3] [4] [5].
CTCs are fragile and tend to degrade within a few hours when collected in conventional blood collection
tubes, e.g. EDTA containing tubes, without dedicated CTC stabilizers. CTCs are extremely rare, especially
7
in early disease, e.g. less than 10 cells per 10 ml of blood, representing a ratio of approx. 1:10 CTCs to
white blood cells (WBCs). This low ratio represents a significant challenge to CTC enrichment required
for examination.
RNA profiles of CTCs resemble gene expression profiles of tumours. For RNA profile analysis, measures
need to be taken to get rid of the WBCs in order to obtain sufficiently enriched CTC-specific RNA.
RNA profiles can change significantly after blood collection, during CTC enrichment and isolation.
Therefore, special measures need to be taken to obtain good quality CTC samples and good quality
isolated RNA for gene expression analysis [4] [6].
Consequently, standardization of all steps of the pre-examination process is required. This includes
blood collection and stabilization, transport, storage, CTC enrichment, CTC isolation (if required), and
RNA isolation. A decision guideline for the critical steps of the CTC pre-analytical workflow for RNA
isolation is provided in Annex A.
This document describes special measures that need to be taken to obtain appropriate quality and
quantity of RNA from CTC containing blood specimens 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.
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1 Scope
This document gives guidelines on the handling, storage, processing and documentation of human
venous whole blood specimens intended for the examination of RNA isolated from circulating tumour
cells (CTCs) during the pre-examination phase before a molecular examination is performed.
This document is applicable to molecular in vitro diagnostic examinations including laboratory
developed tests performed by medical laboratories. 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.
This document does not cover the isolation of cellular RNA directly from venous whole blood containing
CTCs. This is covered in EN ISO 20186-1, Molecular in vitro diagnostic examinations - Specifications for
pre-examination processes for venous whole blood – Part 1: Isolated cellular RNA.
This document does not cover the isolation of specific white blood cells and subsequent isolation of
cellular RNA therefrom.
This document does not cover pre-analytical workflow requirements for viable CTC cryopreservation
and culturing.
NOTE 1 The requirements given in this document can also be applied to other circulating rare cells (e.g. fetal
cells).
NOTE 2 International, national or regional regulations or requirements can also apply to specific topics
covered in this document.
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 and the following
terms and definitions 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 https://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 References [7], [8] and [9].
[SOURCE: EN ISO 20166-3:2019, 3.1]
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3.2
ambient temperature
unregulated temperature of the surrounding air
[SOURCE: EN ISO 20166-3:2019, 3.2]
3.3
analyte
component represented in the name of a measurable quantity
[SOURCE: EN ISO 17511:2003, 3.2, modified — EXAMPLE has been removed.]
3.4
analytical test performance
accuracy, precision, specificity 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.
[SOURCE: EN ISO 20184-1:2018, 3.4]
3.5
blood collection set
intravenous device specialized for venipuncture consisting of a stainless steel bevelled needle and tube
(tubing) with attached plastic wings and fitting connector
Note 1 to entry: The connector attaches to an additional blood collection device, e.g. a blood collection tube.
3.6
blood collection tube
tube used for blood collection, usually in a vacuum which forces blood from the vein through the needle
and into the tube
3.7
backflow
flow of a liquid opposite to the usual or desired direction
3.8
circulating tumor cells
CTCs
cells present in blood, originating from a primary and/or metastatic site of a tumor
3.9
CTC enrichment
any method that is able to increase the ratio of CTCs to other cells
3.10
CTC isolation
any method resulting in a sample containing CTCs without any other cell types
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3.11
diagnosis
identification of a disease from its signs and 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
[SOURCE: EN ISO 20184-1:2018, 3.6]
3.12
DNase
deoxyribonuclease
enzyme that catalyzes the degradation of DNA into smaller components
[SOURCE: EN ISO 20186-1:2019, 3.11]
3.13
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 — Notes to entry 1 to 3 have been removed, Note 1 to
entry has been added and “analytical test” has been added as a preferred term.]
3.14
examination performance
analytical test performance
analytical performance
ability of an examination procedure to measure or detect a particular analyte
Note 1 to entry: Analytical performance is determined from analytical performance studies used to assess the
ability of an in vitro diagnostic examination procedure to measure or detect a particular analyte.
Note 2 to entry: Analytical performance includes such characteristics as analytical sensitivity, detection limit,
analytical specificity (interference and cross-reactivity), trueness, precision and linearity.
[SOURCE: ISO/TS 17822-1:2014, 3.2, modified — “analytical test performance” and “analytical
performance” have been added as preferred terms.]
3.15
examination manufacturer
analytical test manufacturer
group or company that provides the specific analytical test
3.16
needle holder
barrel used in routine venipuncture procedures to hold the blood collection tube in place and to protect
the phlebotomist from direct contact with blood
[SOURCE: EN ISO 20186-1:2019, 3.18]
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3.17
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),
transportation to and within the medical or pathology laboratory, isolation of analytes, and end 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 — “pre-analytical workflow” has been added as a
preferred term, Note 1 to entry has been added and the definition has been extended.]
3.18
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 — Notes to entry 1 to 3 have been removed.]
3.19
proficiency testing
evaluation of participant performance against pre-established criteria by means of inter-laboratory
comparisons
[SOURCE: EN ISO/IEC 17043:2010, 3.7, modified — Notes to entry 1 to 3 have been removed.]
3.20
RNA
ribonucleic acid
polymer of ribonucleotides occurring in a double-stranded or single-stranded form
[SOURCE: EN ISO 22174:2005, 3.1.3]
3.21
RNA proficiency testing program
proficiency testing for RNA based examinations
3.22
RNA profile
amounts of different RNA molecules present in a specimen or sample, that can be measured in the
absence of any losses, inhibition and interference
3.23
RNase
ribonuclease
enzyme that catalyzes the degradation of RNA into smaller components
[SOURCE: EN ISO 20186-1:2019, 3.24]
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3.24
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.
3.25
sample
one or more parts taken from a primary sample
[SOURCE: EN ISO 15189:2012, 3.24, modified — EXAMPLE has been removed.]
3.26
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 RNA.
[SOURCE: ISO Guide 30:2015, 2.1.15, modified — The words “reference material” were replaced by
“sample material”.]
3.27
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.
[SOURCE: EN ISO 20166-3:2019, 3.24]
3.28
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 — Notes to entry 1 to 3 have been removed.]
3.29
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.
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
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— reviewing documents prior to issue.
[SOURCE: EN ISO 9000:2015, 3.8.12, modified — Notes to entry 1 and 2 have been removed.]
3.30
workflow
series of activities necessary to complete a task
[SOURCE: EN ISO 20166-3:2019, 3.29]
4 General considerations
For general statements on medical laboratory quality management systems and in particular on
specimen collection and handling (including avoidance of cross contaminations), see
EN ISO 15189:2012, 4.2, 5.4.4, or EN ISO/IEC 17020:2012, 7.2 and 8. 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 specimen and/or sample storage conditions shall be verified and
validated. Workflow steps which cannot always be controlled 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.
NOTE CTCs RNA profiles can change significantly after blood collection, e.g. by gene induction, gene down
regulation, RNA degradation [10].
Before or during the design of an examination, it should be investigated and ensured that the specific
RNA profile/s intended to be analysed is/are not compromised in a manner impacting the examination
performance.
CTC analysis usually involves a CTC enrichment step (e.g. by size immunomagnetic-, or microfluidic-
based approaches) prior to RNA isolation. Depending on the requirements of the intended RNA
examination, enriched CTCs can undergo additional steps such as staining and counting for further
characterization. Additionally, the examination can require the isolation of CTCs from the enriched
sample containing an unknown number of CTCs.
Due to the complexity of the pre-analytical workflow, appropriate measures shall be taken to obtain
suitable quantity and quality of enriched CTC RNA.
Safety regulations 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 use and performance lies with the user.
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5 Outside the laboratory
5.1 Specimen collection
5.1.1 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 medical treatment and special treatment prior to blood collection;
c) the type and purpose of the proposed examination requested;
d) the appropriate consent from the specimen donor/patient.
See also EN ISO 15189:2012, 5.4.4.
5.1.2 Selection of the venous whole blood collection tube by the laboratory
Due to the low number of CTCs, a high recovery rate is required during enrichment. This can be
hampered by the potential instability of CTCs during transport and storage, leading to a reduction of the
CTC number in the specimen or reduced compatibility with the enrichment system [11]. Also gene
expression profile can change within minutes after blood collection.
Therefore, venous whole blood should be collected in appropriate collection tubes with stabilizers
maintaining the integrity of the CTCs and the CTCs RNA profile (CTC/RNA stabilizer).
Where the CTC examination manufacturer requires usage of a dedicated blood collection tube, this shall
be used. Where the CTC examination manufacturer does not specify a dedicated blood collection tube,
but the CTC isolation manufacturer does, this shall be used.
Where requirements on selecting a dedicated blood collection tube are neither available from the CTC
examination manufacturer nor from the CTC isolation manufacturer, the CTC enrichment
manufacturer’s requirements shall be followed.
Where requirements for a dedicated blood collection tube are not available, the laboratory shall specify,
verify and validate the usage of an appropriate blood collection tube. This shall include the verification
of the blood collection tube’s compatibility with the intended CTC enrichment, isolation and
examination method.
Blood collection tubes not containing any CTC/RNA stabilizer should only be used, if the specific RNA
molecule or the RNA profile to be examined is stable after blood draw during the entire pre-
examination process or if the requested examination allows the use of such tubes.
Where the laboratory decides to use a blood collection tube without a CTC/RNA stabilizer, the blood
specimen should be processed without delay.
NOTE There are also alternatives to conventional blood collection-based CTC enrichments. These systems
allow for in vivo and ex vivo CTC sampling from larger blood volumes [12] [13].
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5.1.3 Venous whole blood specimen collection from the donor/patient and stabilization
procedures
1. The identity of the person collecting the specimen and the date and time of blood collection
according to EN ISO 15189:2012, 5.4.4.3, f) shall be documented.
2. For the labelling/tagging (sample/specimen 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 techniques can be used. Steps for preventing possible backflow into the
donor's/patient’s body can be required. The manufacturer's instructions for using the blood
collection tubes shall be followed. A blood collection set and needle holder can be required where
using CTC/RNA stabilizer containing tubes. In this case, the instructions of the collection set and
needle holder manufacturer shall be followed.
NOTE 1 The integrity of CTCs can be influenced by inadequate venous whole blood collection procedures.
4. Blood collection tubes shall be filled in accordance to the manufacturer's instructions and attention
shall be drawn to the correct positioning of the collection tube during the blood draw as well as the
required blood volume.
5. The blood collection tube manufacturer's instructions for mixing or inverting the tube immediately
after blood collection shall be followed. Mixing or inverting the blood collection tube shall be done
gently.
NOTE 2 If the additives in the blood collection tubes are not homogenously mixed with the specimen, the
CTCs can be impacted, which can affect the validity and reliability of the examination results.
6. Any tampering with and/or additions to the specimen shall be documented.
5.1.4 Information on the specimen and storage requirements at the blood collection facility
5.1.4.1 General
As CTCs including their RNA profiles can change significantly after blood collection, thereby affecting
the validity and reliability of the examination result, the documentation regarding the specimen shall
include the date and time of blood collection.
The storage conditions (i.e. storage duration and temperature) shall be documented.
The temporary storage duration in the blood col
...

SLOVENSKI STANDARD
kSIST-TS FprCEN/TS 17390-1:2019
01-september-2019
Molekularne diagnostične preiskave in vitro - Specifikacije za predpreiskovalne
procese za cirkulirajoče tumorske celice (CTC) v venski polni krvi - 1. del: Izolirani
RNK
Molecular in vitro diagnostic examinations - Specifications for pre-examination processes
for circulating tumor cells (CTCs) in venous whole blood - Part 1: Isolated RNA
Molekularanalytische in-vitro-diagnostische Verfahren - Spezifikationen für
präanalytische Prozesse für zirkulierende Tumorzellen (CTC) in venösen Vollblutproben
- Teil 1: Isolierte RNA
Ta slovenski standard je istoveten z: FprCEN/TS 17390-1
ICS:
11.100.10 Diagnostični preskusni In vitro diagnostic test
sistemi in vitro systems
kSIST-TS FprCEN/TS 17390-1:2019 en,fr,de
2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.

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kSIST-TS FprCEN/TS 17390-1:2019

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kSIST-TS FprCEN/TS 17390-1:2019


FINAL DRAFT
TECHNICAL SPECIFICATION
FprCEN/TS 17390-1
SPÉCIFICATION TECHNIQUE

TECHNISCHE SPEZIFIKATION

July 2019
ICS 11.100.10
English Version

Molecular in vitro diagnostic examinations - Specifications
for pre-examination processes for circulating tumor cells
(CTCs) in venous whole blood - Part 1: Isolated RNA
 Molekularanalytische in-vitro-diagnostische Verfahren
- Spezifikationen für präanalytische Prozesse für
zirkulierende Tumorzellen (CTC) in venösen
Vollblutproben - Teil 1: Isolierte RNA


This draft Technical Specification is submitted to CEN members for Vote. It has been drawn up by the Technical Committee
CEN/TC 140.

CEN members are the national standards bodies of Austria, Belgium, Bulgaria, Croatia, Cyprus, Czech Republic, Denmark, Estonia,
Finland, France, Germany, Greece, Hungary, Iceland, Ireland, Italy, Latvia, Lithuania, Luxembourg, Malta, Netherlands, Norway,
Poland, Portugal, Republic of North Macedonia, Romania, Serbia, Slovakia, Slovenia, Spain, Sweden, Switzerland, Turkey and
United Kingdom.

Recipients of this draft are invited to submit, with their comments, notification of any relevant patent rights of which they are
aware and to provide supporting documentation.

Warning : This document is not a Technical Specification. It is distributed for review and comments. It is subject to change
without notice and shall not be referred to as a Technical Specification.


EUROPEAN COMMITTEE FOR STANDARDIZATION
COMITÉ EUROPÉEN DE NORMALISATION

EUROPÄISCHES KOMITEE FÜR NORMUNG

CEN-CENELEC Management Centre: Rue de la Science 23, B-1040 Brussels
© 2019 CEN All rights of exploitation in any form and by any means reserved Ref. No. FprCEN/TS 17390-1:2019 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 . 10
5 Outside the laboratory . 11
5.1 Specimen collection . 11
5.1.1 Information about the specimen donor/patient . 11
5.1.2 Selection of the venous whole blood collection tube by the laboratory . 11
5.1.3 Venous whole blood specimen collection from the donor/patient and stabilization
procedures . 12
5.1.4 Information on the specimen and storage requirements at the blood collection
facility . 12
5.2 Transport requirements. 13
5.2.1 General . 13
5.2.2 Using blood collection tubes with stabilizers . 13
5.2.3 Using blood collection tubes without stabilizers. 13
6 Inside the laboratory . 14
6.1 Specimen reception . 14
6.2 Storage requirements for the venous whole blood specimen . 14

6.3 Enrichment of CTCs . 14
6.3.1 General . 14
6.3.2 Using a commercial CTC enrichment system . 14
6.3.3 Using laboratory’s own CTC enrichment protocols . 15
6.4 Quality of enriched CTCs. 15
6.5 Storage of enriched CTCs . 15
6.6 Isolation of CTCs . 15
6.6.1 General . 15
6.6.2 Using a commercial CTC isolation system . 16
6.6.3 Using the laboratory’s own CTC isolation procedure . 16
6.7 Processing of isolated CTCs . 16
6.8 Isolation of RNA from an enriched CTC sample. 17
6.8.1 General . 17
6.8.2 Using a commercial RNA isolation kit . 17
6.8.3 Using the laboratory’s own RNA isolation procedure . 17
6.9 Quantity and quality assessment of isolated RNA from enriched or isolated CTCs . 18
6.10 Storage of isolated RNA from enriched CTCs . 18
6.10.1 General . 18
6.10.2 RNA isolated with a commercially available kit . 19
6.10.3 RNA isolated with the laboratory's own protocols . 19
Annex A (informative) Decision guideline for critical steps of the CTC pre-analytical
workflow for RNA isolation . 20
Bibliography . 23
2

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European foreword
This document (FprCEN/TS 17390-1:2019) has been prepared by Technical Committee CEN/TC 140
“In vitro diagnostic medical devices”, the secretariat of which is held by DIN.
This document is currently submitted to the Vote on TS.
CEN/TS 17390 consists of the following parts, under the general title Molecular in vitro diagnostic
examinations — Specifications for pre-examination processes for Circulating Tumor Cells (CTCs) in whole
blood:
— Part 1: Isolated RNA
— Part 2: Isolated DNA
— Part 3: Preparations for analytical CTC staining
3

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Introduction
Solid tumours release cells and bioanalytes into blood and other body fluids. This has opened the option
of minimally-invasive tumour detection, diagnosis and characterization from venous whole blood
(liquid biopsies). Liquid biopsies are expected to enable earlier detection and diagnosis of cancers and
advance personalized patient treatment. These applications have become one of the fastest growing
segments of the entire diagnostic market.
Circulating tumour cells (CTCs) in venous whole blood reflect the disease complexity that evolves
during tumour progression, with distinct genetic, epigenetic and expression features. Besides the
prognostic role of CTC identification and/or enumeration in cancer progression, CTC molecular
characterization can improve e.g. disease outcome prediction, therapeutic guidance and post-treatment
monitoring of the patient.
CTCs are now considered as a surrogate of tumour tissue in cancer early development, progression and
metastatic phase.
Molecular characterization of CTCs can provide for example a strategy for monitoring cancer genotypes
during systemic therapies [1], identification of mechanisms of disease progression, identification of
novel targets for treatment [2] and to select targeted therapies. Moreover, CTC single-cell sequencing is
emerging as an important tool for tumour genomic heterogeneity analysis [3] [4] [5].
CTCs are fragile and tend to degrade within a few hours when collected in conventional blood collection
tubes, e.g. EDTA containing tubes, without dedicated CTC stabilizers. They are extremely rare,
7
especially in early disease, e.g. less than 10 cells per 10 ml of blood, representing a ratio of approx. 1:10
CTCs to white blood cells (WBCs). This low ratio represents a significant challenge to CTC enrichment
required for examination.
RNA profiles of CTCs resemble gene expression profiles of tumours. For RNA profile analysis, measures
need to be taken to get rid of the WBCs in order to obtain sufficiently enriched CTC specific RNA.
RNA profiles can change significantly after blood collection, during CTC enrichment and isolation.
Therefore, special measures need to be taken to obtain good quality CTC samples and good quality
isolated RNA for gene expression analysis [4] [6].
Consequently, standardization of all steps of the pre-examination process is required. This includes
blood collection and stabilization, transport, storage, CTC enrichment, CTC isolation (if required), and
RNA isolation. A decision guideline for the critical steps of the CTC pre-analytical workflow for RNA
isolation is provided in Annex A.
This document describes special measures that need to be taken to obtain appropriate quality and
quantity of RNA from CTC containing blood specimens 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.
4

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1 Scope
This document gives guidelines on the handling, storage, processing and documentation of human
venous whole blood specimens intended for the examination of RNA isolated from circulating tumour
cells (CTCs) during the pre-examination phase before a molecular examination is performed.
This document is applicable to molecular in vitro diagnostic examinations including laboratory
developed tests performed by medical laboratories. 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.
This document does not cover the isolation of cellular RNA directly from venous whole blood containing
CTCs. This is covered in EN ISO 20186-1, Molecular in vitro diagnostic examinations - Specifications for
pre-examination processes for venous whole blood - Part 1: Isolated cellular RNA.
This document does not cover the isolation of specific white blood cells and subsequent isolation of
cellular RNA therefrom.
This document does not cover pre-analytical workflow requirements for viable CTC cryopreservation
and culturing.
NOTE 1 The requirements given in this document can also be applied to other circulating rare cells (e.g. fetal
cells).
NOTE 2 International, national or regional regulations or requirements can also apply to specific topics
covered in this document.
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 and the following
terms and definitions 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 References [7], [8] and [9].
[SOURCE: EN ISO 20166-3:2018, 3.1]
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3.2
ambient temperature
unregulated temperature of the surrounding air
[SOURCE: EN ISO 20166-3:2018, 3.2]
3.3
analyte
component represented in the name of a measurable quantity
[SOURCE: EN ISO 17511:2003, 3.2, modified — EXAMPLE has been removed.]
3.4
analytical test performance
accuracy, precision, specificity 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.
[SOURCE: EN ISO 20184-1:2018, 3.4]
3.5
blood collection set
intravenous device specialized for venipuncture consisting of a stainless steel bevelled needle and tube
(tubing) with attached plastic wings and fitting connector
Note 1 to entry: The connector attaches to an additional blood collection device, e.g. a blood collection tube.
3.6
blood collection tube
tube used for blood collection, usually in a vacuum which forces blood from the vein through the needle
and into the tube
3.7
backflow
flow of a liquid opposite to the usual or desired direction
3.8
circulating tumor cells
CTCs
cells present in blood, originating from a primary and/or metastatic site of a tumor
3.9
CTC enrichment
any method that is able to increase the ratio of CTCs to other cells
3.10
CTC isolation
any method resulting in a sample containing CTCs without any other cell types
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3.11
diagnosis
identification of a disease from its signs and 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
[SOURCE: EN ISO 20184-1:2018, 3.6]
3.12
DNase
deoxyribonuclease
enzyme that catalyzes the degradation of DNA into smaller components
[SOURCE: EN ISO 20186-1:2018, 3.11]
3.13
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 — Notes to entry 1 to 3 have been removed, Note 1 to
entry has been added and “analytical test” has been added as a preferred term.]
3.14
examination performance
analytical test performance
analytical performance
ability of an examination procedure to measure or detect a particular analyte
Note 1 to entry: Analytical performance is determined from analytical performance studies used to assess the
ability of an in vitro diagnostic examination procedure to measure or detect a particular analyte.
Note 2 to entry: Analytical performance includes such characteristics as analytical sensitivity, detection limit,
analytical specificity (interference and cross-reactivity), trueness, precision and linearity.
[SOURCE: ISO/TS 17822-1:2014, 3.2, modified — “analytical test performance” and “analytical
performance” have been added as preferred terms.]
3.15
examination manufacturer
analytical test manufacturer
group or company that provides the specific analytical test
3.16
needle holder
barrel used in routine venipuncture procedures to hold the blood collection tube in place and to protect
the phlebotomist from direct contact with blood
[SOURCE: EN ISO 20186-1:2018, 3.18]
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3.17
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),
transportation to and within the medical or pathology laboratory, isolation of analytes, and end 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 — “pre-analytical workflow” has been added as a
preferred term, Note 1 to entry has been added and the definition has been extended.]
3.18
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 — Notes to entry 1 to 3 have been removed.]
3.19
proficiency testing
evaluation of participant performance against pre-established criteria by means of inter-laboratory
comparisons
[SOURCE: ISO 17043:2010, 3.7, modified — Notes to entry 1 to 3 have been removed.]
3.20
RNA
ribonucleic acid
polymer of ribonucleotides occurring in a double-stranded or single-stranded form
[SOURCE: EN ISO 22174:2005, 3.1.3]
3.21
RNA proficiency testing program
proficiency testing for RNA based examinations
3.22
RNA profile
amounts of different RNA molecules present in a specimen or sample, that can be measured in the
absence of any losses, inhibition and interference
3.23
RNase
ribonuclease
enzyme that catalyzes the degradation of RNA into smaller components
[SOURCE: EN ISO 20186-1:2018, 3.24]
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3.24
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.
3.25
sample
one or more parts taken from a primary sample
[SOURCE: EN ISO 15189:2012, 3.24, modified — EXAMPLE has been removed.]
3.26
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 RNA.
[SOURCE: ISO Guide 30:2015, 2.1.15, modified — The words “reference material” were replaced by
“sample material”.]
3.27
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.
[SOURCE: EN ISO 20166-3:2018, 3.24]
3.28
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 — Notes to entry 1 to 3 have been removed.]
3.29
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.
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
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— reviewing documents prior to issue.
[SOURCE: EN ISO 9000:2015, 3.8.12, modified — Notes to entry 1 and 2 have been removed.]
3.30
workflow
series of activities necessary to complete a task
[SOURCE: EN ISO 20166-3:2018, 3.29]
4 General considerations
For general statements on medical laboratory quality management systems and in particular on
specimen collection and handling (including avoidance of cross contaminations), see
EN ISO 15189:2012, 4.2, 5.4.4, or EN ISO/IEC 17020:2012, 7.2 and 8. 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 specimen and/or sample storage conditions shall be verified and
validated. Workflow steps which cannot always be controlled 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.
NOTE CTCs RNA profiles can change significantly after blood collection, e.g. by gene induction, gene down
regulation, RNA degradation [10].
Before or during the design of an examination, it should be investigated and ensured that the specific
RNA profile/s intended to be analysed is/are not compromized in a manner impacting the examination
performance.
CTC analysis usually involves a CTC enrichment step (e.g. by size immunomagnetic-, or microfluidic-
based approaches) prior to RNA isolation. Depending on the requirements of the intended RNA
examination, enriched CTCs can undergo additional steps such as staining and counting for further
characterization. Additionally, the examination can require the isolation of CTCs from the enriched
sample containing an unknown number of CTCs.
Due to the complexity of the pre-analytical workflow, appropriate measures shall be taken to obtain
suitable quantity and quality of enriched CTC RNA.
Safety regulations 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 use and performance lies with the user.
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5 Outside the laboratory
5.1 Specimen collection
5.1.1 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 medical treatment and special treatment prior to blood collection;
c) the type and purpose of the proposed examination requested;
d) the appropriate consent from the specimen donor/patient.
See also ISO 15189:2012, 5.4.4.
5.1.2 Selection of the venous whole blood collection tube by the laboratory
Due to the low number of CTCs, a high recovery rate is required during enrichment. This can be
hampered by the potential instability of CTCs during transport and storage, leading to a reduction of the
CTC number in the specimen or reduced compatibility with the enrichment system [11]. Also gene
expression can change within minutes after blood collection.
Therefore, venous whole blood should be collected in appropriate collection tubes with stabilizers
maintaining the integrity of the CTCs and the CTCs RNA profile (CTC/RNA stabilizer).
Where the CTC examination manufacturer requires usage of a dedicated blood collection tube, this shall
be used. Where the CTC examination manufacturer does not specify a dedicated blood collection tube,
but the CTC isolation manufacturer does, this shall be used.
Where requirements on selecting a dedicated blood collection tube are neither available from the CTC
examination manufacturer nor from the CTC isolation manufacturer, the CTC enrichment
manufacturer’s requirements shall be followed.
Where requirements for a dedicated blood collection tube are not available, the laboratory shall specify,
verify and validate the usage of an appropriate blood collection tube. This shall include the verification
of the blood collection tube’s compatibility with the intended CTC enrichment, isolation and
examination method.
Blood collection tubes not containing any CTC/RNA stabilizer should only be used, if the specific RNA
molecule or the RNA profile to be examined is stable after blood draw during the entire pre-
examination process or if the requested examination allows the use of such tubes.
Where the laboratory decides to use a blood collection tube without a CTC/RNA stabilizer, the blood
specimen should be processed without delay.
NOTE There are also alternatives to conventional blood collection-based CTC enrichments. These systems
allow for in vivo and ex vivo CTC sampling from larger blood volumes [12] [13].
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5.1.3 Venous whole blood specimen collection from the donor/patient and stabilization
procedures
1. The identity of the person collecting the specimen and the date and time of blood collection
according to ISO 15189:2012, 5.4.4.3, f) shall be documented.
2. For the labelling/tagging (sample/specimen identification) of the blood collection tube, a routine
procedure (ISO 15189:2012, 5.4.4.3, e) o
...

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