ISO/TS 7552-3:2024

Technical
Specification
ISO/TS 7552-3
First edition
Molecular in vitro diagnostic
2024-11
examinations — Specifications
for pre-examination processes for
circulating tumour cells (CTCs) in
venous whole blood —
Part 3:
Preparations for analytical CTC
staining
Analyses de diagnostic moléculaire in vitro — Spécifications
relatives aux processus préanalytiques pour les cellules tumorales
circulantes (CTC) dans le sang total veineux —
Partie 3: Préparations pour l’analyse par coloration des CTC
Reference number
© ISO 2024
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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 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
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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, Medical laboratories and in vitro diagnostic
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
Technical Specification ISO/TS 7552-3:2024(en)
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
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 pre-examination phase before an 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.
Different dedicated measures are taken for stabilizing CTCs genomic DNA and RNA that are not described in
this document; they are covered in ISO 7552-1 and ISO 7552-2.
NOTE 1 The requirements given in this document can also be applied to other circulating rare cells (e.g. foetal 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.
ISO 15189, Medical laboratories — Requirements for quality and competence
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 terminology databases for use in standardization at the following addresses:
— ISO Online browsing platform: available at https:// www .iso .org/ obp
— IEC Electropedia: available at https:// www .electropedia .org/

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.
[SOURCE: ISO 20166-3:2018, 3.1]
3.2
analyte
component represented in the name of a measurable quantity
[SOURCE: ISO 17511:2020, 3.1, modified — The example has been removed.]
3.3
backflow
flow of a liquid opposite to the usual or desired direction
3.4
blood collection set
intravenous device specialized for venipuncture consisting of a stainless steel beveled 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 (3.5).
3.5
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
3.6
circulating tumour cells
CTCs
cells present in blood, originating from a primary or metastatic site(s) of a tumour
3.7
closed system
non-modifiable system provided by the vendor including all necessary components for the analysis (i.e.
hardware, software, procedures and reagents)
[SOURCE: ISO 20186-2:2019, 3.6]
3.8
CTC enrichment
method that is able to increase the ratio of CTCs (3.6) to other cells including white blood cells in a sample (3.21)
3.9
CTC isolation
method resulting in a sample (3.21) containing CTCs (3.6) without any other cell types
3.10
diagnosis
identification of a health or disease state from its signs and symptoms, where the diagnostic process can
involve examinations (3.11) 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: ISO 20184-1:2018, 3.6]

3.11
examination
analytical test
set of operations having the objective of determining the numerical value, text value or characteristics of a
property
Note 1 to entry: An examination includes the processes that start with CTC staining and include all kinds of parameter
testing or chemical manipulation for quantitative or qualitative examination.
[SOURCE: ISO 15189:2022, 3.8, modified — The original Notes to entry have been removed, and a new Note 1
to entry has been added; “analytical test” has been added as a preferred term.]
3.12
examination performance
analytical test performance
analytical performance
ability of an examination (3.11) procedure to measure or detect a particular analyte (3.2)
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 20186-3:2019, 3.11]
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
manufacturer
entity that is legally responsible for manufacturing a specific workflow (3.26) component
Note 1 to entry: For the purpose of this document, manufacturers can be examination (3.11) manufacturers, collection
device manufacturers, CTC enrichment (3.8) and isolation manufacturers, nucleic acid isolation manufacturers.
3.15
needle holder
barrel used in routine venipuncture procedures to hold the blood collection tube (3.5) in place and to protect
the phlebotomist from direct contact with blood
[SOURCE: ISO 20186-1:2019, 3.16]
3.17
pre-examination process
pre-analytical phase
pre-analytical workflow
process that starts, in chronological order, from the clinician’s request and includes the examination
(3.11) request, preparation and identification of the patient, collection of the primary sample(s) (3.18),
transportation to and within the laboratory, cell enrichment, and isolation of analytes (3.2), ending 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: ISO 15189:2022, 3.24, modified — “pre-analytical phase” and “pre-analytical workflow” have
been added as preferred terms; in the definition, “user’s request ” has been changed to “clinician’s request ”;
“cell enrichment, isolation of analytes” has been added to the definition; Note 1 to entry has been added.]

3.18
primary sample
specimen
discrete portion of a body fluid or tissue or other sample (3.21) associated with the human body taken for
examination (3.11), study or analysis of one or more quantities or characteristics to determine the character
of the whole
[SOURCE: ISO 15189:2022, 3.25, modified — Note 1 to entry has been removed.]
3.19
proficiency testing
PT
evaluation of participant performance against pre-established criteria by means of interlaboratory
comparisons
[SOURCE: ISO/IEC 17043:2023, 3.7, modified — Note 1 to entry has been removed.]
3.20
room temperature
temperature in the range of 18 °C to 25 °C
Note 1 to entry: Local or national regulations can have different definitions.
3.21
sample
one or more parts taken from a primary sample (3.18)
[SOURCE: ISO 15189:2022, 3.28.]
3.22
stability
ability of a sample (3.21) 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”; “specified” replaced by “stated” before “property value”. Note 1 to entry has been removed.]
3.23
storage
prolonged interruption of the pre-examination workflow (3.26) of a sample (3.21) or analyte (3.2)
respectively, or of their derivatives, such as 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: ISO 20166-3:2018, 3.21]
3.24
validation
confirmation, through 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: ISO 9000:2015, 3.8.13, modified — The original Notes 1 to 3 to entry have been removed.]
3.25
verification
confirmation, through the 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;
— comparing a new design specification with a similar proven design specification;
— undertaking tests and demonstrations;
— reviewing documents prior to issue.
[SOURCE: ISO 9000:2015, 3.8.12, modified — The original Notes 1 and 2 to entry have been removed and
Note 2 to entry has been added.]
3.26
workflow
series of activities necessary to complete a task
[SOURCE: ISO 20166-3:2018, 3.25]
4 General considerations
Refer to ISO 15189, ISO/IEC 17020 or ISO/IEC 17025 for general statements on medical laboratory quality
management systems. In vitro diagnostic (IVD) manufacturers should follow ISO 13485. General quality
management system requirements can be found in ISO 9001. For other general requirements on pre-
examination processes, including pre-collection activities, collection, transport, receipt, and handling of
specimen, see ISO 20658 and ISO 15189:2022, 7.2.
All steps of a diagnostic workflow can influence the final analytical test result. Thus, the entire workflow
including biomolecule stability and both specimen and sample storage conditions shall be specified, verified,
and validated during the development of the examination including the development of in vitro diagnostic
medical devices. A risk assessment of relevant 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. Guidance is provided in ISO 14971 and ISO 35001.
CTC analysis usually involves a CTC enrichment step (e.g. by size, immunomagnetic-, or microfluidic-based
approaches) prior to CTC staining. Due to the nature of the specimen/sample and the complexity of the
procedure potentially affecting the morphology and integrity of CTCs, appropriate measures shall be taken
during the pre-examination workflow to maintain the CTCs features required for the examination.
The degree of contamination of CTCs with WBCs or other cells is critical. The presence of WBCs in a CTC
enriched sample is unavoidable and can affect the performance of the examination e.g. the CTC staining due
to nonspecific binding of detection components to WBC count. To overcome this problem, an isolation step
can be necessary to obtain a pure CTC sample.
Safety instructions for the whole pre-examination process shall be in place and followed. They shall be in
accordance with requirements specified in ISO 15189 and ISO 15190.
During the whole pre-examination process, precautions shall be taken to avoid cross contamination
between either different specimens or samples, e.g. by using single-use material whenever feasible or by
using appropriate cleaning procedures between processing of either different specimens or samples.
The manufacturer's material safety data sheet shall be considered before first use of any potentially
hazardous material (e.g. chemicals in stabilizers).
For all pre-examination steps, the examination manufacturer's instructions shall be followed, if provided.
Where, for justified reasons (e.g. unmet patient needs), a commercial product is not used in accordance with
the manufacturer's instructions, responsibility for its verification, validation, use and performance lies with
the laboratory.
5 Activities outside the laboratory
5.1 Specimen collection
5.1.1 General
For the collection of the blood specimen, the requirements for the intended molecular examination (e.g. type
of blood collection tube, collection procedure) laid out in Clause 6 shall be followed.
5.1.2 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 such as age, sex, and gender);
b) the information about medical treatment and special treatment prior to blood collection;
c) the type and purpose of the examination requested;
d) the appropriate consent from the specimen donor/patient (see also ISO 15189);
e) time point of the blood draw where relevant (e.g. patients rest or active times).
NOTE A recent study demonstrated a higher CTC concentration in blood during the rest phase of breast cancer
[30]
patients.
5.1.3 Selection of the venous whole blood collection tube by the laboratory
The CTC staining can be influenced by inadequate venous whole blood collection procedures and
inappropriate storage/transport conditions, as well as by enrichment and isolation procedures.
Due to the low number of CTCs, a high recovery efficiency is required during enrichment. This can be
hampered by the potential instability of CTCs during transport and storage, leading to a reduction of the
[13]
CTC number in the specimen or reduced compatibility with the enrichment system.
Therefore, venous whole blood should be collected in appropriate collection tubes with stabilizers
maintaining the integrity of the CTCs (CTC stabilizer).
The examination manufacturer instructions should contain specifications on the blood collection tube(s) to
be used. Where the examination manufacturer specifies usage of dedicated blood collection tube(s), these
shall be used.
Where the examination manufacturer does not provide such specifications, but either the CTC enrichment
or the isolation manufacturer specifies a dedicated blood collection tube, this can serve as a basis for the
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