ISO/DTS 7552-1

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

This document gives guidelines on the handling, storage, processing and documentation of human venous whole blood specimens intended for the examination of RNA isolated from circulating tumour cells (CTCs) during the pre-examination phase before a molecular examination is performed. This document is applicable to molecular in vitro diagnostic examinations 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 the isolation of cellular RNA directly from venous whole blood containing CTCs. This is covered in ISO 20186-1, Molecular in vitro diagnostic examinations - Specifications for pre-examination processes for venous whole blood – Part 1: Isolated cellular RNA. This document does not cover the isolation of specific white blood cells and subsequent isolation of cellular RNA therefrom. This document does not cover pre-analytical workflow requirements for viable CTC cryopreservation and culturing. NOTE 1 The requirements given in this document can also be applied to other circulating rare cells (e.g. fetal cells). NOTE 2 International, national or regional regulations or requirements can also apply to specific topics covered in this document.

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

General Information

• Status: Not Published
• ICS: 11.100.10 - In vitro diagnostic test systems
• Technical Committee: ISO/TC 212 - Clinical laboratory testing and in vitro diagnostic test systems
• Drafting Committee: ISO/TC 212 - Clinical laboratory testing and in vitro diagnostic test systems
• Current Stage: 5020 - FDIS ballot initiated: 2 months. Proof sent to secretariat
• Start Date: 15-Jul-2024
• Completion Date: 15-Jul-2024

Relations

• Consolidates: ISO/FDIS 23779 - Shot blasting machinery — Safety and environmental requirements
• Effective Date: 12-Nov-2022

Standards Content (Sample)

FINAL DRAFT
Technical
Specification
ISO/TC 212
Molecular in vitro diagnostic
Secretariat: ANSI
examinations — Specifications
Voting begins on:
for pre-examination processes for
2024-07-15
circulating tumour cells (CTCs) in
Voting terminates on:
venous whole blood —
2024-10-07
Part 1:
Isolated RNA
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.
IN ADDITION TO THEIR EVALUATION AS
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 1:
Isolated RNA
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-
ISO/CEN PARALLEL PROCESSING
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: [phone]
Email: [e-mail]
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 .7
5.1.4 Venous whole blood specimen collection from the patient/donor .7
5.2 Specimen storage and transport .8
5.2.1 General .8
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 .9
6 Activities inside the laboratory . 9
6.1 Specimen reception .9
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 .10
6.3.3 Using the laboratory-developed CTC enrichment procedure.10
6.4 Quality of enriched CTCs .11
6.5 Storage of enriched CTCs .11
6.6 Isolation of CTCs .11
6.6.1 General .11
6.6.2 Using a commercial CTC isolation system intended for diagnostic use . 12
6.6.3 Using the laboratory-developed CTC isolation procedure . 12
6.7 Isolation of RNA from an enriched CTC sample . 12
6.7.1 General . 12
6.7.2 Using a commercial RNA isolation kit intended for diagnostic use . 12
6.7.3 Using a laboratory-developed CTC RNA isolation procedure . 13
6.8 Quantity and quality assessment of isolated RNA from enriched or isolated CTCs . 13
6.8.1 General . 13
6.8.2 Quantity assessment of CTC RNA . 13
6.8.3 Quality assessment CTC RNA .14
6.9 Storage of isolated RNA from enriched CTCs .14
6.9.1 General .14
6.9.2 Storage of RNA isolated with a commercially available kit intended for
diagnostic use . 15
6.9.3 Storage of RNA isolated with the laboratory-developed procedure . 15
Annex A (informative) Decision guideline for critical steps of the CTC pre-examination
workflow . 16
Bibliography .18

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 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.

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
[20,21]
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
[41]
tumour progression, with distinct genetic, epigenetic and gene expression biomarkers.
Besides the prognostic role of CTC identification and enumeration in cancer progression, CTC molecular
characterization can improve disease outcome prediction, therapeutic guidance and post-treatment
[39]
monitoring of the patient.
CTCs are now considered as a surrogate of tumour tissue in cancer early development, progression and
[23]
metastatic phase .
Molecular characterization of CTCs can provide a strategy for monitoring cancer genotypes during systemic
[24]
therapies, identifying mechanisms of disease progression, identifying novel targets for biological
[25] [39]
treatment and selecting targeted therapies .
Moreover, CTC single-cell sequencing is emerging as an important tool for
...

ISO/TC 212/ WG 4
Secretariat: ANSI
Date: 2024-04-3007-01
Molecular in vitro diagnostic examinations — Specifications for
pre-examination processes for circulating tumortumour cells
(CTCs) in venous whole blood — —
Part 1:
Isolated RNA
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 #### – All rights reserved

ISO #####-#:####(X/DTS 7552-1:(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: [e-mail]
Website: www.iso.orgwww.iso.org
Published in Switzerland
© ISO #### 2024 – All rights reserved
iv
ISO #####-#:####(X/DTS 7552-1:(en)
Contents
Foreword . vi
Introduction . vii
1 Scope . 1
2 Normative references . 1
3 Terms and definitions . 1
4 General considerations . 6
5 Activities outside the laboratory . 7
5.1 Specimen collection . 7
5.1.1 General . 7
5.1.2 Information about the specimen donor/patient . 7
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 . 9
5.2.1 General . 9
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 . 10
6.1 Specimen reception . 10
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 . 11
6.3.3 Using the laboratory-developed CTC enrichment procedure . 11
6.4 Quality of enriched CTCs . 12
6.5 Storage of enriched CTCs . 12
6.6 Isolation of CTCs . 12
6.6.1 General . 12
6.6.2 Using a commercial CTC isolation system intended for diagnostic use . 13
6.6.3 Using the laboratory-developed CTC isolation procedure . 13
6.7 Isolation of RNA from an enriched CTC sample . 13
6.7.1 General . 13
6.7.2 Using a commercial RNA isolation kit intended for diagnostic use . 14
6.7.3 Using a laboratory-developed CTC RNA isolation procedure . 14
6.8 Quantity and quality assessment of isolated RNA from enriched or isolated CTCs . 15
6.8.1 General . 15
6.8.2 Quantity assessment of CTC RNA . 15
6.8.3 Quality assessment CTC RNA . 15
6.9 Storage of isolated RNA from enriched CTCs . 16
6.9.1 General . 16
6.9.2 Storage of RNA isolated with a commercially available kit intended for diagnostic use . 16
6.9.3 Storage of RNA isolated with the laboratory-developed procedure . 17
Annex A (informative) Decision guideline for critical steps of the CTC pre-examination
workflow . 18
Bibliography . 20

© ISO #### 2024 – All rights reserved
v
ISO #####-#:####(X/DTS 7552-1:(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
vi
ISO #####-#:####(X/DTS 7552-1:(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
[20,21 ]
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
[41 ]
during tumortumour progression, with distinct genetic, epigenetic and gene expression biomarkers. [3].
Besides the prognostic role of CTC identification and/or enumeration in cancer progression, CTC molecular
characterization can improve e.g. disease outcome prediction, therapeutic guidance and post-treatment
[39 ]
monitoring of the patient. [1].
CTCs are now considered as a surrogate of tumortumour tissue in cancer early development, progression and
[23 ]
metastatic phase [4]. .
Molecular characterization of CTCs can provide for example a strategy for monitoring cancer genotypes during
[24 ]
systemic therapies, [5], identification of identifying mechanisms of disease progression, identification
[25 ] [39 ]
ofidentifying novel targets for biological treatment [6] and to selectselecting targeted therapies [1]. .
Moreover, CTC single-cell sequencing is emerging as an important tool for tumortumour genomic
[26-28 ]
heterogeneity analysis. [7-9]. 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 examination.
RNA profiles of CTCs resemble gene expression profiles of tumorstumours. For RNA profile analysis, measures
need to be taken to get rid ofremove the WBCs are necessary in order to obtain sufficiently enriched CTC-
specific RNA.
RNA profiles can change significantly after blood collection, during CTC enrichment and isolation. Therefore,
special measures need to be takenare necessary to obtain CTC samples of adequate quality CTC samples and
[29
isolated RNA of appropriate quality isolated RNA for ensuring the specified RNA examination performance.
]
[10].
Standardization ofincludes all steps of the pre-examination process is required. This includes, including blood
collection and stabilization, transport, storage, CTC enrichment, CTC isolation (if requiredincluded), and RNA
isolation. This pre-examination standardization is crucial to ensure reliable examination results in current
clinical use and is also critical to develop new CTC based diagnostic examinations and to establish these in
[30 ]
clinical healthcare. . (11)
An illustration of critical steps of the CTC pre-analytical workflow is provided in Annex AAnnex A. .
This document describes special measures that need to be taken to obtain appropriate quality and quantity of
RNA from CTC-containing blood specimens for subsequent examination.
In this document, the following verbal forms are used:
© ISO #### 2024 – All rights reserved
vii
ISO #####-#:####(X/DTS 7552-1:(en)
— “shall” indicates a requirement;
— “should” indicates a recommendation;
— “may” indicates a permission;
— “can
...