SIST-TS CEN/TS 17390-3:2020

SLOVENSKI STANDARD
SIST-TS CEN/TS 17390-3:2020
01-marec-2020
Molekularne diagnostične preiskave in vitro - Specifikacije za predpreiskovalne
procese za cirkulirajoče tumorske celice (CTC) v venski polni krvi - 3. del: Priprave
za analitično barvanje CTC
Molecular in vitro diagnostic examinations - Specifications for pre-examination processes
for circulating tumor cells (CTCs) in venous whole blood - Part 3: Preparations for
analytical CTC staining
Molekularanalytische in-vitro-diagnostische Verfahren - Spezifikationen für
präanalytische Prozesse für zirkulierende Tumorzellen (CTC) in venösen Vollblutproben
- Teil 3: Vorbereitungen für die analytische CTC-Färbung
Analyses de diagnostic moléculaire in vitro - Spécifications relatives aux processus
préanalytiques pour les cellules tumorales circulantes (CTC) du sang total veineux -
Partie 3 : Préparations pour l’analyse par coloration des CTC
Ta slovenski standard je istoveten z: CEN/TS 17390-3:2020
ICS:
11.100.10 Diagnostični preskusni In vitro diagnostic test
sistemi in vitro systems
SIST-TS CEN/TS 17390-3:2020 en,fr,de
2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.

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SIST-TS CEN/TS 17390-3:2020

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SIST-TS CEN/TS 17390-3:2020


CEN/TS 17390-3
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 3: Preparations for
analytical CTC staining
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 (CTC) du sang zirkulierende Tumorzellen (CTC) in venösen
total veineux - Partie 3 : Préparations pour l'analyse Vollblutproben - Teil 3: Vorbereitungen für die
par coloration des CTC analytische CTC-Färbung
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 17390-3:2020 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 . 9
5 Outside the laboratory . 9
5.1 Specimen collection . 9
5.2 Transport requirements. 11
6 Inside the laboratory . 12
6.1 Specimen reception . 12
6.2 Storage requirements for the venous whole blood specimen . 12
6.3 Enrichment of the CTCs . 13
6.4 Storage of enriched CTCs . 13
6.5 Preparation for CTC staining . 14
Annex A (informative) Decision guideline for critical steps of the CTC pre-analytical
workflow for staining . 16
Bibliography . 18

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European foreword
This document (CEN/TS 17390-3:2020) 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 identification and
analysis can improve e.g. disease outcome prediction, therapeutic guidance and post-treatment
monitoring of the patient.
CTCs are now considered as a surrogate sample of tumour tissue, both in cancer early development 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
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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 identification and examination as tumour-derived cells.
Furthermore, CTC morphology and biomolecules can change during the pre-examination process. These
can lead to changes in protein quantity, integrity, modification, conformation and localization within the
cell. This can impact the validity and reliability of the examination result.
CTC examination usually requires a CTC enrichment step (e.g. based on biological properties, such as
expression of surface molecules, or physical properties, such as size and density, of the CTCs or their
combination) prior to cytomorphological examination or immunofluorescent staining. CTC enrichment
technologies can provide CTCs attached on a solid surface, ready for cytological examination, or CTCs in
suspension requiring extra processing steps prior to the examination. This can lead to potential cell
loss. [6]
CTC enrichment is usually followed by their identification by conventional cytochemical or protein-
targeted staining procedures that allow detection of the cell traits.
Standardization of all steps of the pre-examination process is required. This includes blood collection
and stabilization, transport, storage, CTC enrichment, and CTC isolation (if required). A decision
guideline for the critical steps of the pre-analytical workflow for CTC staining is provided in Annex A.
This document describes measures to standardize the pre-examination process to obtain appropriate
CTC staining.
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 specifies guidelines on the handling, storage, processing and documentation of human
venous whole blood specimens intended for staining of 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 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 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)
ISO 15190, Medical laboratories — Requirements for safety
3 Terms and definitions
For the purposes of this document, 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 bibliographical references [7], [8] and [9].
[SOURCE: EN ISO 20166-3:2019, 3.1]
3.2
ambient temperature
unregulated temperature of the surrounding air
[SOURCE: EN ISO 20166-3:2019, 3.2]
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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, modified — “specificity” was added.]
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, which are derived from a primary and/or metastatic site of a tumor
3.9
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]
3.10
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 CTC staining 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.]
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3.11
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.]
3.12
examination manufacturer
analytical test manufacturer
group or company that provides the specific analytical test
3.13
immunocytochemistry
in situ detection technique that uses the principle of antibodies binding specifically to antigens in or on
cells to detect the antigens (e.g. proteins) using brightfield microscopy
3.14
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, 3.24]
3.15
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.]
3.16
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.]
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3.17
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.]
3.18
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.
3.19
sample
one or more parts taken from a primary sample
[SOURCE: EN ISO 15189:2012, 3.24, modified — EXAMPLE has been removed.]
3.20
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”.]
3.21
storage
prolonged interruption of the pre-analytical 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]
3.22
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.]
3.23
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,
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— comparing a new design specification with a similar proven design specification,
— undertaking tests and demonstrations, and
— reviewing documents prior to issue.
[SOURCE: EN ISO 9000:2015, 3.8.12, modified — Notes to entry 1 and 2 have been removed.]
3.24
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.
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.
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;
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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 [10].
Therefore, venous whole blood should be collected in appropriate collection tubes with stabilizers
maintaining the integrity of the CTCs.
Where the CTC examination manufacturer requires usage of a dedicated blood collection tube, this shall
be used.
Where there are no requirements available from the CTC examination 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 and examination method.
Where the laboratory decides to use a blood collection without a CTC stabilizer, the blood specimen
should be processed without delay.
NOTE 1 Studies have shown that CTC detection is possible in EDTA-collected venous whole blood within 4
hours after blood draw from patients with different tumour types [11–16].
NOTE 2 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. [17],[18].
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 (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 technique can be used. Steps for preventing possible backflow into the
donor's/patient’s body can 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 a CTC stabilizer. 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 manufacturers’ 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.
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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 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 collection facility contributes to the total duration for
storage.
5.1.4.2 Using blood collection tubes with stabilizers
As for storage, the specimens collected in commercial blood collection tubes with CTC stabilizers, the
blood collection tube manufacturer’s instructions on storage conditions shall be followed (e.g.
temperature and storage duration).
Where the CTC examination manufacturer’s instructions are available, these shall be followed. If these
are not available, but the CTC enrichment system manufacturer’s instructions are, these shall be
followed. The storage conditions (i.e. temperature and storage duration) shall be documented.
5.1.4.3 Using blood collection tubes without stabilizers
Where the CTC examination manufacturer's instructions on storage conditions are available, these shall
be followed.
Where the CTC examination manufacturer's instructions are not available, but the CTC enrichment
system manufacturer’s instructions are, these shall be followed.
The storage conditions (i.e. temperature and storage duration) shall be documented.
Where there are no requirements on the storage conditions available, the specimens should be
processed without delay to maximize the CTC recovery. The required storage condition specifications
(i.e. temperature and storage duration) shall be verified and documented.
Where the tube cannot be processed immediately, it should be stored at room temperature, but the
compatibility with the CTC enrichment procedure needs to be verified. The storage duration should be
as short as possible.
5.2 Transport requirements
5.2.1 General
The required transport conditions shall be followed and documented including any deviations
therefrom.
Temperature monitoring should be applied in a suitable manner (e.g. temperature tracking).
5.2.2 Using blood collection tubes with stabilizers
Where using blood collection tubes with CTC stabilizers, the dedicated tube manufacturer’s instructions
on transport conditions shall be followed (e.g. duration and temperature).
Where the CTC examination manufacturer’s instructions are available, these shall be followed.
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If these not are available, but the CTC enrichment system manufacturer’s instructions are, these shall be
followed.
5.2.3 Using blood collection tubes without stabilizers
Where CTC examination manufacturer's instructions on transport conditions are available, these shall
be followed.
Where CTC examination manufacturer's instructions are not available, but the CTC enrichment system
manufacturer’s instructions are available, these shall be followed.
Where there are no instructions on transport conditions available, t
...