ISO/DTS 7552-3
(Main)Molecular in vitro diagnostic examinations — Specifications for pre-examination processes for circulating tumour cells (CTCs) in venous whole blood — Part 3: Preparations for analytical CTC staining
Molecular in vitro diagnostic examinations — Specifications for pre-examination processes for circulating tumour cells (CTCs) in venous whole blood — Part 3: Preparations for analytical CTC staining
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 performed and/or developed by medical laboratories, in vitro diagnostics developers and manufacturers, institutions and commercial organizations performing biomedical research. It is also intended to be used by laboratory customers including health institutions requesting examinations for their patients as well as biobanks 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.
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FINAL DRAFT
Technical
Specification
ISO/TC 212
Molecular in vitro diagnostic
Secretariat: ANSI
examinations — Specifications
Voting begins on:
for pre-examination processes for
2024-07-17
circulating tumour cells (CTCs) in
Voting terminates on:
venous whole blood —
2024-10-09
Part 3:
Preparations for analytical CTC
staining
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 SUPPOR TING DOCUMENTATION.
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BEING ACCEPTABLE FOR INDUSTRIAL, TECHNO-
ISO/CEN PARALLEL PROCESSING LOGICAL, COMMERCIAL AND USER PURPOSES, DRAFT
INTERNATIONAL STANDARDS MAY ON OCCASION HAVE
TO BE CONSIDERED IN THE LIGHT OF THEIR POTENTIAL
TO BECOME STAN DARDS TO WHICH REFERENCE MAY BE
MADE IN NATIONAL REGULATIONS.
Reference number
FINAL DRAFT
Technical
Specification
ISO/TC 212
Molecular in vitro diagnostic
Secretariat: ANSI
examinations — Specifications
Voting begins on:
for pre-examination processes for
circulating tumour cells (CTCs) in
Voting terminates on:
venous whole blood —
Part 3:
Preparations for analytical CTC
staining
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 SUPPOR TING DOCUMENTATION.
© ISO 2024
IN ADDITION TO THEIR EVALUATION AS
All rights reserved. Unless otherwise specified, or required in the context of its implementation, no part of this publication may
BEING ACCEPTABLE FOR INDUSTRIAL, TECHNO-
LOGICAL, COMMERCIAL AND USER PURPOSES, DRAFT
be reproduced or utilized otherwise in any form or by any means, electronic or mechanical, including photocopying, or posting on
INTERNATIONAL STANDARDS MAY ON OCCASION HAVE
the internet or an intranet, without prior written permission. Permission can be requested from either ISO at the address below
TO BE CONSIDERED IN THE LIGHT OF THEIR POTENTIAL
or ISO’s member body in the country of the requester.
TO BECOME STAN DARDS TO WHICH REFERENCE MAY BE
MADE IN NATIONAL REGULATIONS.
ISO copyright office
CP 401 • Ch. de Blandonnet 8
CH-1214 Vernier, Geneva
Phone: +41 22 749 01 11
Email: copyright@iso.org
Website: www.iso.org
Published in Switzerland Reference number
ii
Contents Page
Foreword .iv
Introduction .v
1 Scope . 1
2 Normative references . 1
3 Terms and definitions . 1
4 General Considerations . 5
5 Activities outside the laboratory. 6
5.1 Specimen collection .6
5.1.1 General .6
5.1.2 Information about the specimen donor/patient.6
5.1.3 Selection of the venous whole blood collection tube by the laboratory .6
5.1.4 Venous whole blood specimen collection from the patient/donor .7
5.2 Specimen storage and transport .7
5.2.1 General .7
5.2.2 Storage and transport using blood collection tubes with stabilizers .8
5.2.3 Storage and transport using blood collection tubes without stabilizers .8
6 Activities inside the laboratory . 8
6.1 Specimen reception .8
6.2 Specimen storage after transport and reception .9
6.3 Enrichment of CTCs .9
6.3.1 General .9
6.3.2 Using a commercial CTC enrichment system intended for diagnostic use .9
6.3.3 Using the laboratory developed CTC enrichment procedure .10
6.4 Quality of enriched CTCs .10
6.5 Storage of enriched CTCs .10
6.6 Preparation for CTC staining .10
6.6.1 General .10
6.6.2 Pretreatment for different staining techniques (antibody, colour staining, in
situ techniques) .11
Annex A (informative) Decision guideline for critical steps of the CTC pre-analytical workflow .12
Bibliography . 14
iii
Foreword
ISO (the International Organization for Standardization) is a worldwide federation of national standards
bodies (ISO member bodies). The work of preparing International Standards is normally carried out through
ISO technical committees. Each member body interested in a subject for which a technical committee
has been established has the right to be represented on that committee. International organizations,
governmental and non-governmental, in liaison with ISO, also take part in the work. ISO collaborates closely
with the International Electrotechnical Commission (IEC) on all matters of electrotechnical standardization.
The procedures used to develop this document and those intended for its further maintenance are described
in the ISO/IEC Directives, Part 1. In particular, the different approval criteria needed for the different types
of ISO documents should be noted. This document was drafted in accordance with the editorial rules of the
ISO/IEC Directives, Part 2 (see www.iso.org/directives).
ISO draws attention to the possibility that the implementation of this document may involve the use of (a)
patent(s). ISO takes no position concerning the evidence, validity or applicability of any claimed patent
rights in respect thereof. As of the date of publication of this document, ISO had received notice of (a)
patent(s) which may be required to implement this document. However, implementers are cautioned that
this may not represent the latest information, which may be obtained from the patent database available at
www.iso.org/patents. ISO shall not be held responsible for identifying any or all such patent rights.
Any trade name used in this document is information given for the convenience of users and does not
constitute an endorsement.
For an explanation of the voluntary nature of standards, the meaning of ISO specific terms and expressions
related to conformity assessment, as well as information about ISO's adherence to the World Trade
Organization (WTO) principles in the Technical Barriers to Trade (TBT), see www.iso.org/iso/foreword.html.
This document was prepared by Technical Committee ISO/TC 212, Clinical laboratory testing and in vitro
diagnostic test systems, in collaboration with the European Committee for Standardization (CEN) Technical
Committee CEN/TC 140, In vitro diagnostic medical devices, in accordance with the Agreement on technical
cooperation between ISO and CEN (Vienna Agreement).
A list of all parts in the ISO 7552 series can be found on the ISO website.
Any feedback or questions on this document should be directed to the user’s national standards body. A
complete listing of these bodies can be found at www.iso.org/members.html.
iv
Introduction
Solid tumours release cells and bioanalytes into blood and other body fluids. This has opened the option
of utilizing such body fluids (liquid biopsies) for a minimally-invasive procedure for tumour detection,
diagnosis and characterization. Liquid biopsies can enable earlier detection and diagnosis of cancers and
[19,20]
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 can reflect the disease complexity that evolves during
[21]
tumour progression, with distinct genetic, epigenetic and expression features.
Besides the prognostic role of CTC identification and enumeration in cancer progression, CTC identification
and analysis can improve disease outcome prediction, therapeutic guidance and post-treatment monitoring
[19]
of the patient.
CTCs are now considered as a surrogate of tumour tissue in cancer early development, progression and
[22]
metastatic phase.
Molecular characterization of CTCs can provide a strategy for monitoring cancer during systemic
[23] [24]
therapies, identifying mechanisms of disease progression, identifying novel targets for treatment and
[19]
selecting targeted therapies .
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 approximately 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. This 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 of the CTCs, such
as expression of surface molecules, or physical properties, such as size and density, 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
[25]
cell loss.
CTC enrichment is usually followed by their identification by conventional cytochemical or protein-targeted
staining procedures that allow detection of the cell traits.
Standardization includes all steps of the pre-examination process, including blood collection and
stabilization, transport, storage, CTC enrichment, and CTC isolation (if included). This pre-examination
standardization is crucial to ensure reliable examination results in current clinical use and is also critical to
[26]
develop new CTC based diagnostic examinations and to establish these in clinical healthcare.
An illustration of 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.
v
FINAL DRAFT Technical Specification ISO/DTS 7552-3:2024(en)
Molecular in vitro diagnostic examinations — Specifications
for pre-examination
...
ISO/TC 212/WG 4
Secretariat: ANSI
Date: 2024-04-3007-03
Molecular in vitro diagnostic examinations — Specifications for
pre-examination processes for circulating tumortumour cells
(CTCs) in venous whole blood — —
Part 3:
Preparations for analytical CTC staining
DTS stage
Warning for WDs and CDs
This document is not an ISO International Standard. It is distributed for review and comment. It is subject to
change without notice and may not be referred to as an International Standard.
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.
© ISO #### – All rights reserved
ISO #####-#:####(X)
2 © ISO #### – All rights reserved
ISO #####-#:####(X/DTS 7552-3:(en)
All rights reserved. Unless otherwise specified, or required in the context of its implementation, no part of this publication
may be reproduced or utilized otherwise in any form or by any means, electronic or mechanical, including photocopying,
or posting on the internet or an intranet, without prior written permission. Permission can be requested from either ISO
at the address below or ISO’s member body in the country of the requester.
ISO copyright office
CP 401 • Ch. de Blandonnet 8
CH-1214 Vernier, Geneva
Phone: + 41 22 749 01 11
EmailE-mail: copyright@iso.org
Website: www.iso.orgwww.iso.org
Published in Switzerland
© ISO #### 2024 – All rights reserved
iii
ISO #####-#:####(X/DTS 7552-3:(en)
Contents
Foreword . v
Introduction . vi
Part 3: Preparations for analytical CTC staining . 1
1 Scope . 1
2 Normative references . 1
3 Terms and definitions . 1
4 General Considerations . 5
5 Activities outside the laboratory . 6
5.1 Specimen collection . 6
5.1.1 General . 6
5.1.2 Information about the specimen donor/patient . 6
5.1.3 Selection of the venous whole blood collection tube by the laboratory . 7
5.1.4 Venous whole blood specimen collection from the patient/donor . 8
5.2 Specimen storage and transport . 8
5.2.1 General . 8
5.2.2 Storage and transport using blood collection tubes with stabilizers . 9
5.2.3 Storage and transport using blood collection tubes without stabilizers . 9
6 Activities inside the laboratory . 9
6.1 Specimen reception . 9
6.2 Specimen storage after transport and reception . 10
6.3 Enrichment of CTCs . 10
6.3.1 General . 10
6.3.2 Using a commercial CTC enrichment system intended for diagnostic use . 10
6.3.3 Using the laboratory developed CTC enrichment procedure . 11
6.4 Quality of enriched CTCs . 11
6.5 Storage of enriched CTCs . 11
6.6 Preparation for CTC staining . 11
6.6.1 General . 11
6.6.2 Pretreatment for different staining techniques (antibody, colour staining, in situ
techniques) . 12
Annex A (informative) Decision guideline for critical steps of the CTC pre-analytical workflow 14
Bibliography . 16
© ISO #### 2024 – All rights reserved
iv
ISO #####-#:####(X/DTS 7552-3:(en)
Foreword
ISO (the International Organization for Standardization) is a worldwide federation of national standards
bodies (ISO member bodies). The work of preparing International Standards is normally carried out through
ISO technical committees. Each member body interested in a subject for which a technical committee has been
established has the right to be represented on that committee. International organizations, governmental and
non-governmental, in liaison with ISO, also take part in the work. ISO collaborates closely with the
International Electrotechnical Commission (IEC) on all matters of electrotechnical standardization.
The procedures used to develop this document and those intended for its further maintenance are described
in the ISO/IEC Directives, Part 1. In particular, the different approval criteria needed for the different types of
ISO documents should be noted. This document was drafted in accordance with the editorial rules of the
ISO/IEC Directives, Part 2 (see www.iso.org/directives).
ISO draws attention to the possibility that the implementation of this document may involve the use of (a)
patent(s). ISO takes no position concerning the evidence, validity or applicability of any claimed patent rights
in respect thereof. As of the date of publication of this document, ISO had not received notice of (a) patent(s)
which may be required to implement this document. However, implementers are cautioned that this may not
represent the latest information, which may be obtained from the patent database available at
www.iso.org/patents. ISO shall not be held responsible for identifying any or all such patent rights.
Any trade name used in this document is information given for the convenience of users and does not
constitute an endorsement.
For an explanation of the voluntary nature of standards, the meaning of ISO specific terms and expressions
related to conformity assessment, as well as information about ISO's adherence to the World Trade
Organization (WTO) principles in the Technical Barriers to Trade (TBT), see www.iso.org/iso/foreword.html.
This document was prepared by Technical Committee ISO/TC 212, Clinical laboratory testing and in vitro
diagnostic test systems., in collaboration with the European Committee for Standardization (CEN) Technical
Committee CEN/TC 140, In vitro diagnostic medical devices, in accordance with the Agreement on technical
cooperation between ISO and CEN (Vienna Agreement).
A list of all parts in the ISO 7552 series can be found on the ISO website.
Any feedback or questions on this document should be directed to the user’s national standards body. A
complete listing of these bodies can be found at www.iso.org/members.html.
© ISO #### 2024 – All rights reserved
v
ISO #####-#:####(X/DTS 7552-3:(en)
Introduction
Solid tumorstumours release cells and bioanalytes into blood and other body fluids. This has opened the
option of utilizing such body fluids (liquid biopsies) for a minimally-invasive procedure for tumortumour
detection, diagnosis and characterization. Liquid biopsies can enable earlier detection and diagnosis of
[19,20 ]
cancers and advance personalized patient treatment. [1,2].
These applications have become one of the fastest growing segments of the entire diagnostic market.
Circulating tumortumour cells (CTCs) in venous whole blood can reflect the disease complexity that evolves
[21 ]
during tumortumour progression, with distinct genetic, epigenetic and expression features. 3.
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
[19 ]
monitoring of the patient. [1].
CTCs are now considered as a surrogate sample of tumortumour tissue, both in cancer early development,
[22 ]
progression and metastatic phase. [4].
Molecular characterization of CTCs can provide for example a strategy for monitoring cancer during systemic
[23 ]
therapies, [5], identification of identifying mechanisms of disease progression, identification ofidentifying
[24 ] [19 ]
novel targets for treatment [6] and to selectselecting targeted therapies [1]. .
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.approximately 1:10 CTCs to
white blood cells (WBCs). This low ratio represents a significant challenge to CTC enrichment required for
identification and examination as tumortumour-derived cells.
Furthermore, CTC morphology and biomolecules can change during the pre-examination process. TheseThis
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 of the CTCs, 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
[25 ]
cell loss. [7].
CTC enrichment is usually followed by their identification by conventional cytochemical or protein-targeted
staining procedures that allow detection of the cell traits.
Standardization ofincludes all steps of the pre-examination process is required. This includes, including blood
collection and stabilization, transport, storage, CTC enrichment, and CTC isolation (if requiredincluded). This
pre-examination standardization is crucial to ensure reliable examination results in current clinical use and is
[26
also critical to develop new CTC based diagnostic examinations and to establish these in clinical healthcare.
]
[8].
An illustration of critical steps of the pre-analytical workflow for CTC staining is provided in Annex AAnnex
A.
This document describes measures to standardize the pre-examination process to obtain appropriate CTC
staining.
© ISO #### 2024 – All rights reserved
vi
ISO #####-#:####(X/DTS 7552-3:(en)
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.
The International Organization for Standardization (ISO) draws attention to the fact that it is claimed that
compliance with this document may involve the use of a patent.
ISO takes no position concerning the evidence, validity and scope of this patent right.
The holder of this patent right has assured ISO that he/she is willing to negotiate licences under reasonable
and non-discriminatory terms and conditions with applicants throughout the world. In this respect, the
statement of the holder of this patent right is registered with ISO. Information may be obtained from the patent
database available at .
Attention is drawn to the possibility that some of the elements of this document may be the subject of patent
rights other than those in the patent database. ISO shall not be held responsible for identifying any or all such
patent rights.
© ISO #### 2024 – All rights reserved
vii
Molecular in vitro diagnostic examinations — Specifications for pre-
examination processes for circulating tumortumour cells (CTCs) in
venous whole blood — —
Part 3:
Preparations for analytical CTC staining
1 Scope
This document specifies requirements and gives recommendations on the handling, storage, CTC enrichment,
preparation for CTC staining, and documentation of venous whole blood specimens intended for staining of
CTCs during the
...
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