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

This document gives guidelines on the handling, storage, processing and documentation of venous whole blood and the CTC (Circulating Tumor Cell) enrichment, CTC isolation and other preparations for analytical staining (i.e., conventional cytochemical and immunocytochemical staining) of CTCs during the pre-examination Phase before the cytopathological evaluation 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 specific staining procedures.
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 3: Vorbereitungen für die analytische CTC Färbung

Dieses Dokument spezifiziert Empfehlungen zur Handhabung, Lagerung, Verarbeitung und Dokumentation von Proben venösen Vollbluts, die für die Färbung der zirkulierenden Tumorzellen (CTCs) 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 Anforderungen an den präanalytischen Arbeitsablauf zur Kryokonser¬vierung 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 (CTC) du sang total veineux - Partie 3 : Préparations pour l’analyse par coloration des CTC

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

General Information

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Published
Publication Date
21-Jan-2020
Current Stage
6060 - Definitive text made available (DAV) - Publishing
Due Date
22-Jan-2020
Completion Date
22-Jan-2020

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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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CEN/TS 17390-3:2020 (E)
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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CEN/TS 17390-3:2020 (E)
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

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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CEN/TS 17390-3:2020 (E)
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
...

SLOVENSKI STANDARD
kSIST-TS FprCEN/TS 17390-3:2019
01-september-2019
Molekularne diagnostične preiskave in vitro - Specifikacije za predpreiskovalne

procese za cirkulirajoče tumorske celice (CTC) v venski polni krvi - 3. del: Priprave

za analitično CTC barvanje

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
Ta slovenski standard je istoveten z: FprCEN/TS 17390-3
ICS:
11.100.10 Diagnostični preskusni In vitro diagnostic test
sistemi in vitro systems
kSIST-TS FprCEN/TS 17390-3:2019 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 17390-3:2019
FINAL DRAFT
TECHNICAL SPECIFICATION
FprCEN/TS 17390-3
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 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

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 17390-3:2019 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 ................................................................................................................................. 9

5 Outside the laboratory .................................................................................................................................. 9

5.1 Specimen collection ........................................................................................................................................ 9

5.1.1 Information about the specimen donor/patient ................................................................................. 9

5.1.2 Selection of the venous whole blood collection tube by the laboratory .................................. 10

5.1.3 Venous whole blood specimen collection from the donor/patient and stabilization

procedures ...................................................................................................................................................... 10

5.1.4 Information on the specimen and storage requirements at the blood collection

facility ............................................................................................................................................................... 11

5.2 Transport requirements............................................................................................................................ 11

5.2.1 General ............................................................................................................................................................. 11

5.2.2 Using blood collection tubes with stabilizers .................................................................................... 11

5.2.3 Using blood collection tubes without stabilizers.............................................................................. 12

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

6.3.1 General ............................................................................................................................................................. 13

6.3.2 Using a commercial CTC enrichment system ..................................................................................... 13

6.3.3 Using laboratory’s own CTC enrichment protocols ......................................................................... 13

6.4 Storage of enriched CTCs ........................................................................................................................... 13

6.5 Preparation for CTC staining ................................................................................................................... 14

6.5.1 General ............................................................................................................................................................. 14

6.5.2 Pre-treatment for different staining techniques (antibody, colour staining, in situ

techniques) ..................................................................................................................................................... 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 (FprCEN/TS 17390-3:2019) 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
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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. They are extremely rare, 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 and identification 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 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]
3.2
ambient temperature
unregulated temperature of the surrounding air
[SOURCE: EN ISO 20166-3:2018, 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

the 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 20186-1:2018, 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: ISO 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 20166-3:2018, 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 20166-3:2018, 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.

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 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 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

(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

For storing, 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 dedicate
...

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