ISO/DTS 7552-1

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
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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

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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
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The procedures used to develop this document and those intended for its further maintenance are described
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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.
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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:
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— “shall” indicates a requirement;
— “should” indicates a recommendation;
— “may” indicates a permission;
— “can
...