ISO/FDIS 18704
(Main)Molecular in vitro diagnostic examinations — Requirements and recommendations for pre-examination processes for urine and other body fluids — Isolated cell-free DNA
Molecular in vitro diagnostic examinations — Requirements and recommendations for pre-examination processes for urine and other body fluids — Isolated cell-free DNA
This document specifies requirements and provides recommendations for the pre-examination phase of cell free DNA (cfDNA) from body fluid specimens other than blood, including but not limited to the collection, handling, storage, transport, processing and documentation of human body fluids, such as urine, cerebrospinal fluid (CSF), pleural effusions and saliva, intended for cfDNA examination. Processing includes multiple steps, such as centrifugation for specimen cleaning and isolation of cfDNA. This document is applicable to molecular in vitro diagnostic examinations performed by medical laboratories. It is also intended to be used by health institutions including facilities collecting and handling specimen, laboratory customers, in vitro diagnostics developers and manufacturers, biobanks, institutions and commercial organizations performing biomedical research, and regulatory authorities. Dedicated measures that need to be taken for cytohistological analysis of body fluid derived nucleated cells are not described in this document. Neither are measures for preserving and handling of pathogens, and other bacterial or whole microbiome DNA in body fluids described. Different dedicated measures need to be taken for preserving circulating cell free DNA (ccfDNA) from blood. These are not described in this document, but are covered in ISO 20186-3. NOTE International, national or regional regulations or requirements can also apply to specific topics covered in this document.
Analyses de diagnostic moléculaire in vitro — Exigences et recommandations relatives aux processus préanalytiques pour l’urine et d’autres liquides corporels — ADN libre extrait
General Information
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Standards Content (Sample)
FINAL DRAFT
International
Standard
ISO/TC 212
Molecular in vitro diagnostic
Secretariat: ANSI
examinations — Requirements
Voting begins on:
and recommendations for pre-
2025-11-17
examination processes for urine
Voting terminates on:
and other body fluids — Isolated
2026-01-12
cell-free DNA
RECIPIENTS OF THIS DRAFT ARE INVITED TO SUBMIT,
WITH THEIR COMMENTS, NOTIFICATION OF ANY
RELEVANT PATENT RIGHTS OF WHICH THEY ARE AWARE
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ISO/CEN PARALLEL PROCESSING LOGICAL, COMMERCIAL AND USER PURPOSES, DRAFT
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TO BECOME STAN DARDS TO WHICH REFERENCE MAY BE
MADE IN NATIONAL REGULATIONS.
Reference number
FINAL DRAFT
International
Standard
ISO/TC 212
Molecular in vitro diagnostic
Secretariat: ANSI
examinations — Requirements
Voting begins on:
and recommendations for pre-
examination processes for urine
Voting terminates on:
and other body fluids — Isolated
cell-free DNA
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 2025
IN ADDITION TO THEIR EVALUATION AS
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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 requirements . 6
5 Outside the laboratory . 7
5.1 Specimen collection .7
5.1.1 Information about the patient or specimen donor .7
5.1.2 Selection of the body fluid collection device by the laboratory .7
5.1.3 Urine and other body fluid specimen collection from the patient or donor and
stabilization procedures .8
5.1.4 Information about the specimen storage requirements at the body fluid
collection facility .9
5.2 Transport requirements .11
5.2.1 General .11
5.2.2 Transport using urine and other body fluid collection devices with cfDNA
stabilizers .11
5.2.3 Transport using urine and other body fluid collection devices without cfDNA
stabilizers . 12
6 Inside the laboratory .12
6.1 Specimen or sample reception . 12
6.2 Specimen or sample storage after transport and reception . 12
6.3 Urine and other body fluid specimen or sample processing prior to cfDNA isolation . 13
6.4 Storage requirements for urine and other body fluid samples after processing . 13
6.5 Isolation of urine and other body fluid cfDNA .14
6.5.1 General .14
6.5.2 Using a commercial cfDNA isolation kit approved for diagnostic use .14
6.5.3 Using a laboratory developed cfDNA isolation procedure .14
6.6 Quantity and quality assessment of isolated cfDNA . 15
6.6.1 General . 15
6.6.2 Quantity assessment of cfDNA . 15
6.6.3 Quality assessment of cfDNA . 15
6.7 Storage of isolated urine and other body fluid cfDNA.16
6.7.1 General .16
6.7.2 Storage of isolated urine and other body fluid cfDNA, isolated with a
commercially available kit . .16
6.7.3 Storage of isolated urine and other body fluid cfDNA, isolated with the
laboratory’s own procedure.17
Annex A (informative) Effects of pre-examination storage of unstabilized urine on cfDNA .18
Annex B (informative) Effects of pre-examination storage of unstabilized and stabilized urine
on the amount of a specific cfDNA target sequence.22
Bibliography .24
iii
Foreword
ISO (the International Organization for Standardization) is a worldwide federation of national standards
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CEN/TC 140, In vitro diagnostic medical devices, in accordance with the Agreement on technical cooperation
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iv
Introduction
Molecular in vitro diagnostics has enabled significant progress in medicine. Further progress has been
achieved and is still expected by new technologies used to examine profiles of nucleic acids, proteins, and
metabolites in human tissues and body fluids (e.g. genomic, epigenomic, transcriptomic, proteomic and
metabolomic profiling). However, the profiles of these molecules can change drastically during specimen
collection, transport, storage and processing. This can make the outcome from diagnostics or research
unreliable or even result in failure because the subsequent examination will not measure the genuine profile
of nucleic acids, proteins or metabolites as it was in the patient, but a profile altered by the pre-examination
process. Therefore, specifying, developing, verifying and validating preanalytical workflows has become an
[21]
essential part of examination development.
Most of the DNA in the body is located within cells, but small amounts of DNA originating from cells can
also be found outside of cells (extracellular DNA). In case of circulating body fluids such as blood, this DNA
is called circulating cell-free DNA (ccfDNA) and in case of non-circulating body fluids such as urine, saliva,
cerebrospinal fluid, pleural effusion, ascites, and synovial fluid, the DNA is called cell-free DNA (cfDNA).
cfDNA is of specific interest, as for example cfDNA in urine originates from cells from the genitourinary
[22]
tract or from ccfDNA passing through glomerular filtration. cfDNA from cancerous or malignant cells in
[23],[24]
urine have been associated with cancer specific sequences, epigenetic and structural changes. Urine
is currently the most frequently used non-circulating body fluid for cfDNA examination because it is easily
obtained from patients. Although urine is often described as the major specimen type, in this document the
term body fluid is used for urine and other body fluids as defined in Clause 3.
Standardization of the entire workflow from specimen collection to the cfDNA examination is needed to
minimize post-collection release of DNA from cells into the fluid and degradation of cfDNA in the specimen,
which can change the original native cfDNA profile in the body fluid. Post collection microbial growth in
the specimen can further enhance the degradation of the cfDNA, e.g. in urine and saliva. Furthermore, the
isolation of cfDNA can lead to a cfDNA profile bias. Different methods to determine cfDNA yield and quality
can lead to additional variations and impacts.
Studies have been undertaken to determine the pre-examination sources of these and other variables,
as they can impact the cfDNA examination. The variables can compromise the specified examination
performance characteristics, such as sensitivity, specificity, linearity and reproducibility. They can also
impact the examination reliability which could lead to an erroneous examination result and misdiagnosis.
This document draws upon such work to codify and standardize the steps prior to cfDNA examination from
body fluids in what is referred to as the pre-examination process.
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.
v
FINAL DRAFT International Standard ISO/FDIS 18704:2025(en)
Molecular in vitro diagnostic examinations — Requirements
and recommendations for pre-examination processes for
urine and other body fluids — Isolated cell-free DNA
1 Scope
This document specifies requirements and provides recommendations for the pre-examination process
of cell-free DNA (cfDNA) from body fluid specimens other than blood, including but not limited to the
collection, handling, storage, transport, processing and documentation of human body fluids, such as urine,
pleural effusions, ascites, cerebrospinal fluid (CSF), and saliva, intended for cfDNA examination. Processing
includes multiple steps, such as centrifugation for specimen purification and isolation of cfDNA.
This document does not cover dedicated measures for cytohistological analysis of nucleated cells derived
from body fluid, nor measures for preserving and handling of pathogens, and other bacterial or whole
microbiome DNA in body fluids described.
Dedicated measures for preserving circulating cell-free DNA (ccfDNA) from blood are covered in ISO 20186-3.
This document is applicable to medical laboratories, health institutions including facilities collecting and
handling specimens, laboratory customers, in vitro diagnostic examination developers and manufacturers,
biobanks, institutions and organizations performing biomedical research, and regulatory authorities.
NOTE 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 13485, Medical devices — Quality management systems — Requirements for regulatory purposes
3 Terms and definitions
For the purposes of this document, the terms and definitions given in ISO 15189, ISO 13485 and the
following 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 that are uniform in structure and composition. Tissues are
heterogeneous and therefore cannot be aliquoted.
[SOURCE: ISO 20166-1:2018, 3.1, modified — “that are uniform in structure and composition ” was added in
Note 1 to entry.]
3.2
analyte
component represented in the name of a measurable quantity
[SOURCE: ISO 17511:2020, 3.1, modified — The example was removed.]
3.3
ascites
abnormal buildup of fluid in the abdomen that can cause swelling
Note 1 to entry: In late-stage cancer, tumour cells can be found in the fluid in the abdomen.
Note 2 to entry: Ascites also occurs in patients with liver disease.
Note 3 to entry: This definition was adapted from Reference [28].
3.4
body fluid
natural fluid or secretion that is produced by the body including, but not limited to, urine (3.26), saliva,
semen, mucus, vaginal secretions, breast milk, amniotic fluid, cerebrospinal fluid, synovial fluid, ascites
(3.3), pleural effusions and pericardial fluid
Note 1 to entry: For the purpose of this document blood and faeces are not included.
[SOURCE: ISO/TR 19591:2018, 3.23, modified — Blood and faeces were deleted from the definition, and
saliva, ascites and pleural effusion were added; Note 1 to entry was added.]
3.5
body fluid collection device
tube or other container in which the body fluid (3.4) (e.g. urine (3.26)) specimen (3.23) is collected
3.6
cfDNA
cell-free DNA
extracellular human DNA (3.12) present in body liquids such as urine (3.26)
[29]
Note 1 to entry: cfDNA can include DNA present in vesicles such as exosomes.
3.7
cfDNA profile
cell-free DNA profile
amount of different cell-free DNA (cfDNA) (3.6) molecules, present in a body fluid (3.4) that can be measured
in the absence of any losses, inhibition and interference
3.8
cfDNA stabilizer
cell-free DNA stabilizer
compound, solution or mixture that is designed to minimize degradation and fragmentation of cell-free DNA
(cfDNA) (3.6) as well as release of genomic DNA (3.16) from nucleated cells
3.9
closed system
non-modifiable system provided by the vendor including all necessary components for the analysis (i.e.,
hardware, software, procedures and reagents)
3.10
collection device manufacturer
entity that manufactures in vitro diagnostic or research devices, intended for the collection of specimens (3.23)
3.11
collection facility
area where a human specimen (3.23) is collected, such as physician’s office, patient’s home, hospital and clinic
3.12
DNA
deoxyribonucleic acid
polymer of deoxyribonucleotides occurring in a double-stranded (dsDNA) or single-stranded (ssDNA) form
[SOURCE: ISO 22174:2024, 3.1.6]
3.13
examination
set of operations having the objective of determining the numerical value, text value or characteristics of a
property
Note 1 to entry: An examination may be the total of a number of activities, observations or measurements required to
determine a value or characteristic.
Note 2 to entry: Laboratory examinations that determine a numerical value of a property are called “quantitative
examinations”; those that determine the characteristics of a property are called “qualitative examinations”.
Note 3 to entry: Laboratory examinations are also called “assays” or “tests”.
Note 4 to entry: For the purpose of this document, examination starts with the isolated cell-free DNA (cfDNA) (3.6).
[SOURCE: ISO 15189:2022, 3.8 — Note 4 to entry was added.]
3.14
examination manufacturer
entity that manufactures in vitro diagnostic or research examination devices, including measurement
systems, instruments, reagents, and instructions for use for a specific examination (3.13)
Note 1 to entry: Adapted from ISO 20166-4:2021, 3.16.
3.15
examination performance
analytical test performance
analytical performance
accuracy, precision, specificity, sensitivity and limit of detection of a test to examine the analyte (3.2) of
interest
Note 1 to entry: Other test performance characteristics such as robustness and repeatability may apply as well.
Note 2 to entry: Examination performance is determined from examination performance studies used to assess the
ability of an in vitro diagnostic examination (3.13) procedure to measure or detect a particular analyte.
Note 3 to entry: Examination performance includes such characteristics as analytical sensitivity, detection limit,
analytical specificity (interference and cross-reactivity), trueness, precision and linearity.
Note 4 to entry: Adapted from ISO 20184-1:2018, 3.4.
3.16
genomic DNA
gDNA
DNA (3.12) from the nuclear and mitochondrial genomes containing all coding (exon) and non-coding (intron
and other) sequences
[SOURCE: ISO 20186-2:2019, 3.12, modified — Note 1 to entry was removed.]
3.17
interfering substance
endogenous or exogenous substance that can be present in specimens (3.23) and that can alter an examination
(3.13) result
EXAMPLE Stabilization solution.
3.18
microorganism
entity of microscopic size, encompassing bacteria, fungi, protozoa and viruses
[SOURCE: ISO 11139:2018, 3.176]
3.19
pre-examination process
pre-analytical phase
pre-analytical workflow
pre-examination phase
process that starts, in chronological order, from the user’s request and includes the examination (3.13)
request, preparation and identification of the patient, collection of the specimen(s) (3.23), transportation to
and within the laboratory, ending when the examination begins
Note 1 to entry: For the purpose of this document, pre-examination process ends with the isolated cell-free DNA
(cfDNA) (3.6).
[SOURCE: ISO 15189:2022, 3.24, modified — The preferred terms “pre-analytical phase”, “pre-analytical
workflow” and “pre-examination phase” were added; Note 1 to entry was added.]
3.20
proficiency test
evaluation of participant performance against pre-established criteria by means of interlaboratory
comparisons
[SOURCE: ISO/IEC 17043:2023, 3.7, modified — The term was changed from “proficiency testing” to
“proficiency test”; Note 1 to entry was removed.]
3.21
room temperature
temperature in the range of 18 °C to 25 °C
Note 1 to entry: Local or national regulations can have different definitions.
[SOURCE: ISO 20166-1:2018, 3.22]
3.22
sample
one or more parts taken from a specimen (3.23)
[SOURCE: ISO 15189:2022, 3.28]
3.23
specimen
primary sample
discrete portion of a body fluid (3.4) or tissue or other sample (3.22) associated with the human body taken
for examination (3.13), 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 was removed.]
3.24
stability
ability of a sample (3.22) material, when stored under specified conditions, to maintain a stated property
value within specified limits for a specified period of time
Note 1 to entry: The measurand constituent for the purpose of this document is isolated DNA (3.12).
[SOURCE: ISO Guide 30:2015, 2.1.15, modified — In the definition, “characteristic” was changed to “ability”;
“specified property value” was changed to “stated property value”; Note 1 to entry was removed and a new
note was added.]
3.25
storage
prolonged interruption of the pre-examination process (3.19) of a sample (3.22) or analyte (3.2) respectively,
or of their derivatives, 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 20184-1:2018, 3.22, modified — The examples of derivatives “stained sections or tissue
blocks” were removed from the definition.]
3.26
urine
liquid product of the human excretory system produced by the kidneys and expelled through the urethra via
urination
[SOURCE: ISO 30500:2025, 3.1.2.3, modified — Note 1 to entry was removed.]
3.27
validation
confirmation of plausibility for a specific intended use or application through the provision of objective
evidence that specified requirements have been fulfilled
Note 1 to entry: Objective evidence can be obtained through observation, measurement, examination (3.23) or by
other means.
Note 2 to entry: The word “validated” is used to designate the corresponding status.
[SOURCE: ISO 15189:2022, 3.31, modified — Note 3 to entry was removed.]
3.28
verification
confirmation of truthfulness, through the provision of objective evidence, that specified requirements have
been fulfilled
EXAMPLE 1 Confirmation that design input specifications of a measuring system are achieved.
EXAMPLE 2 Confirmation that a target measurement uncertainty can be met.
Note 1 to entry: Verification is the process that intends to confirm that the established performance claims of a
measuring system, e.g. trueness, precision, reportable range, can be replicated in the laboratory before human sample
examination (3.23) is performed.
Note 2 to entry: The objective evidence needed for a verification can be the result of a regulatory authority assessment,
an inspection or other forms of determination, such as performing alternative calculations or reviewing documents.
Note 3 to entry: The word “verified” is used to designate the corresponding status.
[SOURCE: ISO 15189:2022, 3.32, modified — In Example 1 “performance” was changed to “design input”;
Notes 1 and 2 to entry have been revised; Note 3 to entry has been removed.]
3.29
workflow
series of activities necessary to complete a task
[SOURCE: ISO 20166-1:2018, 3.30]
4 General requirements
For general statements on medical laboratory quality management systems and in particular on
specimen collection, reception and handling (including avoidance of cross contaminations) see ISO 15189
and ISO/IEC 17020 or ISO/IEC 17025. ISO 20658 and ISO 20387 (for biobanking) may also apply. The
requirements on laboratory equipment, reagents, and consumables according to ISO 15189 shall be followed;
ISO/IEC 17020 and ISO/IEC 17025 may also apply. For IVD developers and manufacturers ISO 13485 may
apply instead.
All steps of the pre-examination, examination and post-examination processes (i.e. the entire workflow)
can influence the diagnosis or research study results, thus, this entire workflow shall be specified, verified
and validated during the development of the examination, including the development of in vitro diagnostic
(IVD) medical devices. This includes explicitly all pre-examination process steps such as the examination
request, preparation and identification of the patient, collection of the specimen(s), transport to and within
the laboratory, storage and isolation of analytes.
The stability of the cfDNA profile should be investigated throughout the complete pre-examination process.
The verification and validation shall account for the variability of the body fluid specimen’s quality. cfDNA
profiles can change significantly after body fluid collection. The post-collection release of genomic DNA from
cells in the body fluid can change the cfDNA profile (see Reference [31]). In some body fluids, such as urine,
post-collection growth of bacteria can cause additional changes to the cfDNA profile such as contaminating
the human cfDNA with bacterial DNA and causing degradation of target cfDNA. Post-collection conditions
such as large temperature variations and prolonged storage or transportation times, or both, can also result
in cfDNA degradation. Post-collection changes can vary individually in specimens from different donors
or patients, and they can also depend on pathophysiological conditions. This can impact the validity and
reliability of the examination results.
During the design and development of a cfDNA based examination, an appropriate risk assessment shall
be performed (see also ISO 14971, ISO 22367, ISO 35001). Mitigation measures for eliminating or reducing
identified risks shall be established where required for ensuring the performance of the examination. It shall
be investigated and ensured that any change to the cfDNA profile(s) introduced during the pre-examination
process does not lead to a change of the examination result. To ensure the cfDNA profile is not compromised,
it can be necessary to characterize whether or how, or both, the profile intended to be examined changes
during the pre-examination process steps (e.g. degradation of target cfDNA, and the post-collection release
of genomic DNA from present cells). This can be done, for example by applying the intended examination
to specimens or samples that have been subjected to pre-examination steps such as transport and storage
in time course studies (see Annex A). Measures can be implemented to prevent or reduce impacts by the
identified pre-examination variables, e.g. by using body fluid collection devices with cfDNA stabilizers (see
Annex B).
During the whole pre-examination process, precautions shall be taken to avoid cross contamination between
different specimens or samples (e.g. by using single-use material whenever feasible or appropriate cleaning
procedures between processing of different specimens or samples), and to avoid mixing up of specimens or
samples.
Safety instructions for the whole pre-examination process shall be in place and followed. Safety regulations
on specimen or sample transport and handling shall be considered (see ISO 15189, ISO 15190 and ISO 20658).
If transport is required over public areas, corresponding regulations or laws for packaging and transport
apply [e.g. International Air Transport Association (IATA) for air transport].
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 Outside the laboratory
5.1 Specimen collection
5.1.1 Information about the patient or specimen donor
The documentation shall include the identity of the patient or specimen donor, which may be in the form of
the name or a code.
The documentation should include, but is not limited to:
a) the relevant health status (e.g. healthy, disease type, concomitant disease) and demographics of the
patient or specimen donor (e.g. age and sex);
b) the information about medical treatment and special treatment prior to body fluid collection (e.g.
anaesthetics, medications, fasting status, surgical or diagnostic procedures);
c) the type and the purpose of the examination requested;
d) the appropriate consent from the patient or specimen donor.
NOTE See ISO 15189 for further information. ISO 20658 provides additional general guidance on the entire
collection process.
5.1.2 Selection of the body fluid collection device by the laboratory
The cfDNA profile of body fluids can be influenced by e.g. inadequate collection procedures, inappropriate
storage or transport conditions, separation of contaminating cells as well as by cfDNA isolation procedures.
Specifically, the post-collection degradation can significantly change the cfDNA profile in body fluids, e.g. in
[32],[33]
urine. This can impact the validity of the examination results.
In order to prevent cfDNA degradation, bacterial growth and, where required, release of genomic DNA from
cells and DNA from microorganisms, body fluid collection devices with cfDNA profile stabilizers, or devices
without stabilizers with immediate post-collection addition of stabilizers should be used. These stabilizers
should also allow the separation of nucleated cells from cfDNA in the body fluid, e.g. by centrifugation. Body
fluid collection devices without cfDNA profile stabilizers and workflows without immediate post-collection
addition of cfDNA stabilizers should only be used if the ordered examination specifications allow the non-
use of stabilizers.
The examination manufacturer’s instructions for use for the specimen collection shall be followed. Where
the cfDNA examination manufacturer requires usage of a dedicated body fluid collection device or body
fluid cfDNA stabilizer, these shall be used. This can include a transfer into a secondary container with a
stabilizer. The device’s and stabilizers’ catalogue and lot numbers shall be documented.
For specimens intended for extended storage in a biobank the individual human cfDNA examinations needed
are not always known in advance of extended storage. Therefore, body fluid collection devices with cfDNA
profile stabilizers or workflows with immediate post-collection addition of cfDNA stabilizers should be used
to enable the use of a wider range of examinations.
5.1.3 Urine and other body fluid specimen collection from the patient or donor and stabilization
procedures
5.1.3.1 General
The examination manufacturer shall specify, verify and validate the body fluid collection device for the
examination and shall provide instructions for use for the specimen collection procedure, either with or
where appropriate without stabilizers.
NOTE In some countries it is not a requirement of the examination manufacturer to provide such instructions for
use and to specify, verify and validate the body fluid collection device for the examination.
A cfDNA stabilizer should be used, to prevent any cfDNA profile changes during the pre-examination
process. This may be a collection device prefilled with a stabilizer or an external stabilizer may be added to
the collected urine or body fluid, e.g. from a bulk solution.
Where the specimen collection device is intended for self-collection or home collection, or both, the collection
device manufacturer shall validate this device for these purposes. This shall include mitigating any potential
risks for the patient or donor from the cfDNA stabilizer where appropriate (see Clause 4).
The laboratory shall have written or visual instructions, or both, in place for body fluid collection. These
shall follow the examination manufacturer’s instructions for use where provided.
Where the examination manufacturer’s instructions are not provided (e.g. due to less stringent legal
frameworks), or where, for justified reasons (e.g. unmet patient needs), they require modifications, the
laboratory shall specify, verify and validate the body fluid collection device and collection procedure for
the intended examination and document this according to its quality management system requirements.
This shall include specifications for specimen storage and transport as required for the examination (see
5.1.4 and 5.2). Where the selected body fluid collection device manufacturer provides specified and verified
instructions (see 5.1.3.2 and 5.1.3.3) for the body fluid collection, these can serve as a basis for the laboratory
to verify this device for the examination.
Where the specifications given in the instructions for use of the selected body fluid collection device do not
meet the examination requirements during verification, they shall be modified to be fit for purpose, verified
and documented by the laboratory.
Where specimen self-collection is possible (e.g. saliva, urine), written visual instructions, or both, either
from the body fluid collection device manufacturer or the laboratory shall be supplied to the patient or
donor. Laboratory instructions shall be based on the body fluid collection device manufacturer’s or the
examination manufacturer’s requirements, or both.
The instructions for body fluid specimen collection shall include:
a) Requirements and recommendations to follow before the collection, in particular related to e.g. drinking
or fasting, or both, before collection, or collection time (e.g. first midstream urine of the morning).
b) All requirements for patient identification, collection, storage and transport of the specimen to the
laboratory. For collection this shall include requirements:
1) to collect the specimen within the specified volume range;
2) to mix the specimen with the stabilizer(s) if required by the manufacturer, e.g. by immediate
shaking or inverting.
For self-collection, the patient or donor shall be provided with an appropriate body fluid collection device
(e.g. container, tube), identity tag(s) [(e.g. label, radio frequency identification (RFID)], and in general
anything needed for the specimen collection, preservation, storage and transport procedure for returning
the specimen to laboratory.
The patient or donor shall also be provided with an option to confirm compliance with the supplied
instructions for the body fluid specimen collection, e.g. electronic, paper based.
The patient or donor or person collecting the specimen from the patient or donor shall confirm compliance
with the supplied instructions for the body fluid specimen collection.
The identity of any person other than the patient or donor collecting the specimen shall be documented. The
date and time of body fluid collection shall be documented.
For the labelling (sample or specimen identification) of the body fluid collection device, a routine procedure
(for example ISO 15189 for medical laboratories or ISO 20387 for biobanks) or a procedure with additional
information (e.g. 2D-barcode) shall be used.
Any alterations or additions to the specimen shall be documented.
5.1.3.2 Urine and other body fluid collection with cfDNA stabilizers
The cfDNA stabilizer manufacturer and, where the stabilizer is incorporated into a collection device, the
collection device manufacturer, shall specify, verify and validate the instructions for use for body fluid
collection and stabilization.
The specimen collection device manufacturer shall also specify and verify the specimen collection device
quality parameters, e.g. interference of device materials, stability of stabilizer during product shelf-life and
tightness of closures or caps.
The examination manufacturer shall determine the cfDNA target stability (see Clause 4).
The examination manufacturer’s instructions and thereon built laboratory’s instructions for use may refer
to the instructions of the body fluid collection device or stabilizer, or both, provided by the manufacturer.
The person collecting the specimen from the patient or donor, or the patient or donor self-collecting the
specimen, shall follow the laboratory’s written instructions for use (see 5.1.3.1).
5.1.3.3 Urine and other body fluid collection without cfDNA stabilizers
The cfDNA profile can change rapidly post-collection and can thus impact the intended performance
characteristics of the examination. Therefore such collection devices shall only be used if they have been
specified, verified and validated for the specific examination.
The examination manufacturer’s instructions and thereon built laboratory’s instructions for use may refer
to the instructions of the body fluid collection device without stabilizer provided by the manufacturer.
Where the manufacturer of the body fluid collection device without stabilizer does not provide such
instructions, the laboratory shall specify, verify and validate the instructions for use of the device for body
fluid collection for cfDNA examination. The laboratory shall write instructions for use and follow them.
The instructions may include cooling of the specimen to 2 °C to 8 °C or placing on wet ice, and transportation
to the laboratory without delay within a verified period of time. Otherwise, microbial growth, cfDNA
degradation and contamination with genomic DNA released from cells present in the body fluid can happen
and impact examination test results.
The person collecting the specimen from the patient or donor, or the patient or donor self-collecting the
specimen, shall follow the laboratory’s written instructions for use (see 5.1.3.1).
5.1.4 Information about the specimen storage requirements at the body fluid collection facility
5.1.4.1 General
The examination manufacturer shall specify, verify and validate the storage requirements and shall provide
instructions for specimen storage, either with (see 5.1.4.2) or where appropriate without (see 5.1.4.3)
stabilizers.
NOTE In several countries it is not a requirement to provide such instructions for use.
This can be accomplished with time course studies, e.g. by storing the specimen for different storage
durations and temperatures. As an example, see Annex A.
The laboratory shall have written instructions in place for body fluid storage. These should follow the
examination manufacturer’s instructions for use where provided. Where these are not provided, the
laboratory shall specify, verify and validate the body fluid specimen storage conditions (e.g. temperature,
duration) for the intended examination and document this according to its quality management system
requirements. This can be accomplished by time course studies (see Annex A).
Where provided, the body fluid collection device manufacturer’s instructions may serve as a basis for the
laboratory’s own specification and verification for the intended examination.
The do
...
2025-08-31
ISO/TC 212/ WG 4
Secretariat: ANSI
Date: 2025-10-31
Molecular in vitro diagnostic examinations — Requirements and
recommendations for pre-examination processes for urine and other
body fluids — Isolated cell-free DNA
FDIS stage
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.org
Published in Switzerland
ii
Contents
Foreword . Error! Bookmark not defined.
Introduction . v
1 Scope . 1
2 Normative references . 1
3 Terms and definitions . 1
4 General requirements . 6
5 Outside the laboratory . 7
5.1 Specimen collection . 7
5.2 Transport requirements . 12
6 Inside the laboratory . 13
6.1 Specimen or sample reception . 13
6.2 Specimen or sample storage after transport and reception . 13
6.3 Urine and other body fluid specimen or sample processing prior to cfDNA isolation . 14
6.4 Storage requirements for urine and other body fluid samples after processing . 14
6.5 Isolation of urine and other body fluid cfDNA . 15
6.6 Quantity and quality assessment of isolated cfDNA . 16
6.7 Storage of isolated urine and other body fluid cfDNA . 17
Annex A (informative) Effects of pre-examination storage of unstabilized urine on cfDNA . 19
Annex B (informative) Effects of pre-examination storage of unstabilized and stabilized urine
on the amount of a specific cfDNA target sequence . 24
Bibliography . 27
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'sISO’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).
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
Molecular in vitro diagnostics has enabled significant progress in medicine. Further progress has been
achieved and is still expected by new technologies used to examine profiles of nucleic acids, proteins, and
metabolites in human tissues and body fluids (e.g. genomic, epigenomic, transcriptomic, proteomic and
metabolomic profiling). However, the profiles of these molecules can change drastically during specimen
collection, transport, storage and processing. This can make the outcome from diagnostics or research
unreliable or even result in failure because the subsequent examination will not measure the genuine profile
of nucleic acids, proteins or metabolites as it was in the patient, but a profile altered by the pre-examination
process. Therefore, specifying, developing, verifying and validating preanalytical workflows has become an
[21[1] ]
essential part of examination development. .
Most of the DNA in the body is located within cells, but small amounts of DNA originating from cells can also
be found outside of cells (extracellular DNA). In case of circulating body fluids such as blood, this DNA is called
circulating cell-free DNA (ccfDNA) and in case of non-circulating body fluids such as urine, saliva,
cerebrospinal fluid, pleural effusion, ascites, and synovial fluid, the DNA is called cell-free DNA (cfDNA). cfDNA
is of specific interest, as for example cfDNA in urine originates from cells from the genitourinary tract or from
[22[2] ]
ccfDNA passing through glomerular filtration. . cfDNA from cancerous or malignant cells in urine have
[23],[24[3],[4] ]
been associated with cancer specific sequences, epigenetic and structural changes. . Urine is
currently the most frequently used non-circulating body fluid for cfDNA examination because it is easily
obtained from patients. Although urine is often described as the major specimen type, in this document the
term body fluid is used for urine and other body fluids as defined in Clause 3Clause 3.
Standardization of the entire workflow from specimen collection to the cfDNA examination is needed to
minimize post-collection release of DNA from cells into the fluid and degradation of cfDNA in the specimen,
which can change the original native cfDNA profile in the body fluid. Post collection microbial growth in the
specimen can further enhance the degradation of the cfDNA, e.g. in urine and saliva. Furthermore, the isolation
of cfDNA can lead to a cfDNA profile bias. Different methods to determine cfDNA yield and quality can lead to
additional variations and impacts.
Studies have been undertaken to determine the pre-examination sources of these and other variables, as they
can impact the cfDNA examination. The variables can compromise the specified examination performance
characteristics, such as sensitivity, specificity, linearity and reproducibility. ItThey can also impact the
examination reliability which could lead to an erroneous examination result and misdiagnosis.
This document draws upon such work to codify and standardize the steps prior to cfDNA examination from
body fluids in what is referred to as the pre-examination phaseprocess.
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.
v
Molecular in vitro diagnostic examinations — Requirements and
recommendations for pre-examination processes for urine and other
body fluids — Isolated cell-free DNA
1 Scope
This document specifies requirements and provides recommendations for the pre-examination phaseprocess
of cell-free DNA (cfDNA) from body fluid specimens other than blood, including but not limited to the
collection, handling, storage, transport, processing and documentation of human body fluids, such as urine,
pleural effusions, ascites, cerebrospinal fluid (CSF), and saliva, intended for cfDNA examination. Processing
includes multiple steps, such as centrifugation for specimen purification and isolation of cfDNA.
This document does not cover dedicated measures for cytohistological analysis of nucleated cells derived from
body fluid, nor measures for preserving and handling of pathogens, and other bacterial or whole microbiome
DNA in body fluids described.
Dedicated measures for preserving circulating cell-free DNA (ccfDNA) from blood are covered in ISO 20186-
3.
This document is applicable to medical laboratories, health institutions including facilities collecting and
handling specimens, laboratory customers, in vitro diagnostic examination developers and manufacturers,
biobanks, institutions and organizations performing biomedical research, and regulatory authorities.
NOTE International, national or regional regulations or requirements maycan 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 13485, Medical devices — Quality management systems — Requirements for regulatory purposes
3 Terms and definitions
For the purposes of this document, the terms and definitions given in ISO 15189, ISO 13485 and the following
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 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 that are uniform in structure and composition. Tissues are
heterogeneous and therefore cannot be aliquoted.
[SOURCE: ISO 20166-1:2018, 3.1 —, modified — “that are uniform in structure and composition-” was added
in Note 1 to entry was modified].]
3.2 3.2
analyte
component represented in the name of a measurable quantity
[SOURCE: ISO 17511:2020, 3.1, modified — The example was removed.]
3.3 3.3
ascites
abnormal buildup of fluid in the abdomen that can cause swelling
Note 1 to entry: In late-stage cancer, tumour cells can be found in the fluid in the abdomen.
Note 2 to entry: Ascites also occurs in patients with liver disease.
[8]
Note 3 to entry: This definition was derivedadapted from Reference [28 .].
3.4 3.4
body fluid
natural fluid or secretion that is produced by the body including, but not limited to, urine (3.26,), saliva, semen,
mucus, vaginal secretions, breast milk, amniotic fluid, cerebrospinal fluid, synovial fluid, ascites (3.3,), pleural
effusions and pericardial fluid
Note 1 to entry: For the purpose of this document blood and faeces are not included.
[SOURCE: ISO/TR 19591:2018, 3.23, modified — Blood and faeces were deleted from the definition, and
saliva, ascites and pleural effusion were added.; Note 1 to entry was added.]
3.5 3.5
body fluid collection device
tube or other container in which the body fluid (3.4) (e.g. urine (3.26))) specimen (3.23) is collected
3.6 3.6
cfDNA
cell-free DNA
extracellular human DNA (3.12) present in body liquids such as urine (3.26)
[ [ ].]
Note 1 to entry: cfDNA can include DNA present in vesicles such as exosomes. 29 9
3.7 3.7
cfDNA profile
cell-free DNA profile
amount of different cell-free DNA (cfDNA) (3.6 (3.6)) molecules, present in a body fluid (3.4) that can be
measured in the absence of any losses, inhibition and interference
3.8 3.8
cfDNA stabilizer
cell-free DNA stabilizer
compound, solution or mixture that is designed to minimize degradation and fragmentation of cell-free DNA
(cfDNA) (3.6 (3.6)) as well as release of genomic DNA (3.16(3.16)) from nucleated cells
3.9 3.9
closed system
non-modifiable system provided by the vendor including all necessary components for the analysis (i.e.,
hardware, software, procedures and reagents)
3.10 3.10
collection device manufacturer
entity that manufactures in vitro diagnostic or research devices, intended for the collection of specimens (3.23)
3.11 3.11
collection facility
area where a human specimen (3.23) is collected, such as physician’s office, patient’s home, hospital and clinic
3.12 3.12
DNA
deoxyribonucleic acid
polymer of deoxyribonucleotides occurring in a double-stranded (dsDNA) or single-stranded (ssDNA) form
[SOURCE: ISO 22174:2024, 3.1.6]
3.13 3.13
examination
set of operations having the objective of determining the numerical value, text value or characteristics of a
property
Note 1 to entry: An examination may be the total of a number of activities, observations or measurements required to
determine a value or characteristic.
Note 2 to entry: Laboratory examinations that determine a numerical value of a property are called "“quantitative
examinations";”; those that determine the characteristics of a property are called "“qualitative examinations".”.
Note 3 to entry: Laboratory examinations are also called "“assays"” or "“tests".”.
Note 4 to entry: For the purpose of this document, examination starts with the isolated cell-free DNA (cfDNA) (3.6 (3.6).).
[SOURCE: ISO 15189:2022, 3.8 — Note 4 to entry was added.]
3.14 3.14
examination manufacturer
entity that manufactures in vitro diagnostic or research examination devices, including measurement systems,
instruments, reagents, and instructions for use for a specific examination (3.13(3.13))
Note 1 to entry: Adapted from ISO 20166-4:2021, 3.16.
3.15 3.15
examination performance
analytical test performance
analytical performance
accuracy, precision, specificity, sensitivity and limit of detection of a test to examine the analyte (3.2(3.2)) of
interest
Note 1 to entry: Other test performance characteristics such as robustness and repeatability may apply as well.
Note 2 to entry: Analytical Examination performance is determined from analyticalexamination performance studies
used to assess the ability of an in vitro diagnostic examination (3.13(3.13)) procedure to measure or detect a particular
analyte (3.2).
Note 3 to entry: Analytical Examination 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], ISO 7552-1:2024]
[SOURCE: ISO 20184-1:2018, 3.4, modified — The preferred terms “examination performance” and “analytical
performance” were added; “specificity” and “limit of detection” were added to the definition, and “measure”
was changed to “examine”; Note 2 and 3 added.]
3.16
Note 4 to entry: Adapted from ISO 20184-1:2018, 3.4.
3.16
genomic DNA
gDNA
DNA (3.12) from the nuclear and mitochondrial genomes containing all coding (exon) and non-coding (intron
and other) sequences[SOURCE: ISO 20186-2:2019, 3.12 — Note 1 to entry was removed.]
3.17
[SOURCE: ISO 20186-2:2019, 3.12, modified — Note 1 to entry was removed.]
3.17
interfering substance
endogenous or exogenous substance that can be present in specimens (3.23) and that can alter an examination
(3.13(3.13)) result
EXAMPLE Stabilization solution.
3.163.18 3.18
microorganism
entity of microscopic size, encompassing bacteria, fungi, protozoa and viruses
[SOURCE: ISO 11139:2018, 3.176]
3.173.19 3.19
pre-examination process
pre-analytical phase
pre-analytical workflow
pre-examination phase
process that starts, in chronological order, from the user’s request and includes the examination (3.13(3.13))
request, preparation and identification of the patient, collection of the primary samplespecimen(s)
(3.23(3.23),), transportation to and within the laboratory, ending when the examination (3.13) begins
Note 1 to entry: For the purpose of this document, pre-examination process ends with the isolated cell-free DNA (cfDNA)
(3.6 (3.6).).
[SOURCE: ISO 15189:2022, 3.24 —, modified — The preferred terms “pre-analytical phase”, “pre-analytical
workflow” and “pre-examination phase” were added; Note 1 to entry was added.]
3.183.20 3.20
proficiency test
evaluation of participant performance against pre-established criteria by means of interlaboratory
comparisons
[SOURCE: ISO/IEC 17043:2023, 3.7, modified — The term was changed from “proficiency testing” to
“proficiency test”; Note 1 to entry was removed.]
3.193.21 3.21
room temperature
temperature in the range of 18 °C to 25 °C
Note 1 to entry: Local or national regulations can have different definitions.
[SOURCE: ISO 20166--1:2018, 3.22]
3.203.22 3.22
sample
one or more parts taken from a specimen (3.23primary sample (3.23))
[SOURCE: ISO 15189:2022, 3.28]
3.213.23 3.23
specimen
primary sample
discrete portion of a body fluid (3.4) or tissue or other sample (3.22) associated with the human body taken
for examination (3.13(3.13),), 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 was removed.]
3.223.24 3.24
stability
ability of a sample (3.22) material, when stored under specified conditions, to maintain a stated property value
within specified limits for a specified period of time
Note 1 to entry: The measurand constituent for the purpose of this document is isolated DNA (3.12.).
[SOURCE: ISO Guide 30:2015, 2.1.15, modified — In the definition, “characteristic” was changed to “ability”;
“specified property value” was changed to “stated property value”; Note 1 to entry was removed and a new
note was added.]
3.233.25 3.25
storage
prolonged interruption of the pre-examination process (3.19analytical workflow) of a sample (3.22) or analyte
(3.2) respectively, or of their derivatives, 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 20184--1:2018, 3.22], modified — The examples of derivatives “stained sections or tissue
blocks” were removed from the definition.]
3.243.26 3.26
urine
liquid product of the human excretory system produced by the kidneys and expelled through the urethra via
urination
[SOURCE: ISO 30500:2025, 3.1.2.3], modified — Note 1 to entry was removed.]
3.253.27 3.27
validation
confirmation of plausibility for a specific intended use or application through the provision of objective
evidence that specified requirements have been fulfilled
Note 1 to entry: Objective evidence can be obtained through observation, measurement, examination (3.23) or by other
means.
Note 2 to entry: The word “validated” is used to designate the corresponding status.
[SOURCE: ISO 15189:2022, 3.31, modified — Note 3 to entry has been deletedwas removed.]
3.263.28 3.28
verification
confirmation of truthfulness, through the provision of objective evidence, that specified requirements have
been fulfilled
EXAMPLE 1 Confirmation that design input specifications of a measuring system are achieved.
EXAMPLE 2 Confirmation that a target measurement uncertainty can be met.
Note 1 to entry: Verification is the process that intends to confirm that the established performance claims of a measuring
system, e.g. trueness, precision, reportable range, can be replicated in the laboratory before human sample examination
(3.23) is performed.
Note 2 to entry: The objective evidence needed for a verification can be the result of a regulatory authority assessment,
an inspection or other forms of determination, such as performing alternative calculations or reviewing documents.
Note 3 to entry: The word “verified” is used to designate the corresponding status.
[SOURCE: ISO 15189:2022, 3.32, modified — EXAMPLE — In Example 1 has been revised.“performance” was
changed to “design input”; Notes 1 and 2 to entry have been revised.; Note 3 to entry has been removed.]
3.273.29 3.29
workflow
series of activities necessary to complete a task
[SOURCE: ISO 20166--1:2018, 3.30]
4 General requirements
For general statements on medical laboratory quality management systems and in particular on specimen
collection, reception and handling (including avoidance of cross contaminations) see ISO 15189 and
ISO/IEC 17020 or ISO/IEC 17025. ISO 20658 and ISO 20387 (for biobanking) may also apply. The
requirements on laboratory equipment, reagents, and consumables according to ISO 15189 shall be followed;
ISO/IEC 17020 and ISO/IEC 17025 may also apply. For IVD developers and manufacturers ISO 13485 may
apply instead.
All steps of the pre-examination, examination and post-examination processes (i.e. the entire workflow) can
influence the diagnosis or research study results, thus, this entire workflow shall be specified, verified and
validated during the development of the examination, including the development of in vitro diagnostic (IVD)
medical devices. This includes explicitly all pre-examination process steps such as the examination request,
preparation and identification of the patient, collection of the specimen(s), transport to and within the
laboratory, storage and isolation of analytes.
The stability of the cfDNA profile should be investigated throughout the complete pre-examination process.
The verification and validation shall account for the variability of the body fluid specimen'sspecimen’s quality.
cfDNA profiles can change significantly after body fluid collection. The post-collection release of genomic DNA
[11]
from cells in the body fluid can change the cfDNA profile (see Reference [31 ).]). In some body fluids, such
as urine, post-collection growth of bacteria can cause additional changes to the cfDNA profile such as
contaminating the human cfDNA with bacterial DNA and causing degradation of target cfDNA. Post-collection
conditions such as large temperature variations and prolonged storage and/or transportation times, or both,
can also result in cfDNA degradation. Post-collection changes can vary individually in specimens from different
donors or patients, and they can also depend on pathophysiological conditions. This can impact the validity
and reliability of the examination results.
During the design and development of a cfDNA based examination, an appropriate risk assessment shall be
performed (see also ISO 14971, ISO 22367, ISO 35001). Mitigation measures for eliminating or reducing
identified risks shall be established where required for ensuring the performance of the examination. It shall
be investigated and ensured that any change to the cfDNA profile(s) introduced during the pre-examination
process does not lead to a change of the examination result. To ensure the cfDNA profile is not compromised,
it can be necessary to characterize whether and/or how, or both, the profile intended to be examined changes
during the pre-examination process steps (e.g. degradation of target cfDNA, and the post-collection release of
genomic DNA from present cells). This can be done, for example by applying the intended examination to
specimens/ or samples that have been subjected to pre-examination steps such as transport and storage in
time course studies (see Annex AAnnex A).). Measures can be implemented to prevent or reduce impacts by
the identified pre-examination variables, e.g. by using body fluid collection devices with cfDNA stabilizers (see
Annex BAnnex B).).
During the whole pre-examination process, precautions shall be taken to avoid cross contamination between
different specimens/ or samples (e.g. by using single-use material whenever feasible or appropriate cleaning
procedures between processing of different specimens/ or samples), and to avoid mixing up of specimens/ or
samples.
Safety instructions for the whole pre-examination process shall be in place and followed. Safety regulations
on specimen/ or sample transport and handling shall be considered (see ISO 15189, ISO 15190 and
ISO 20658). If transport is required over public areas, corresponding regulations or laws for packaging and
transport apply ([e.g. International Air Transport Association (IATA) for air transport).].
The manufacturer'smanufacturer’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'smanufacturer’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'smanufacturer’s instructions, responsibility for its verification, validation, use and
performance lies with the laboratory.
5 Outside the laboratory
5.1 Specimen collection
5.1.1 Information about the patient or specimen donor
The documentation shall include the identity of the patient or specimen donor, which may be in the form of
the name or a code.
The documentation should include, but is not limited to:
a) a) the relevant health status (e.g. healthy, disease type, concomitant disease) and demographics
of the patient or specimen donor (e.g. age and sex);
b) b) the information about medical treatment and special treatment prior to body fluid collection
(e.g. anaesthetics, medications, fasting status, surgical or diagnostic procedures);
c) c) the type and the purpose of the examination requested;
d) d) the appropriate consent from the patient or specimen donor.
NOTE See ISO 15189 for further information. ISO 20658 provides additional general guidance on the entire
collection process.
5.1.2 Selection of the body fluid collection device by the laboratory
The cfDNA profile of body fluids can be influenced by e.g. inadequate collection procedures, inappropriate
storage/ or transport conditions, separation of contaminating cells as well as by cfDNA isolation procedures.
Specifically, the post-collection degradation can significantly change the cfDNA profile in body fluids, e.g. in
[ ],[ [12],[13] ]
urine. 32 33 . This can impact the validity of the examination results.
In order to prevent cfDNA degradation, bacterial growth and, where required, release of genomic DNA from
cells and DNA from microorganisms, body fluid collection devices with cfDNA profile stabilizers, or devices
without stabilizers with immediate post-collection addition of stabilizers should be used. These stabilizers
should also allow the separation of nucleated cells from cfDNA in the body fluid, e.g. by centrifugation. Body
fluid collection devices without cfDNA profile stabilizers and workflows without immediate post-collection
addition of cfDNA stabilizers should only be used if the ordered examination specifications allow the non-use
of stabilizers.
The examination manufacturer'smanufacturer’s instructions for use for the specimen collection shall be
followed. Where the cfDNA examination manufacturer requires usage of a dedicated body fluid collection
device or body fluid cfDNA stabilizer, these shall be used. This can include a transfer into a secondary container
with a stabilizer. The device'sdevice’s and stabilizers'stabilizers’ catalogue and lot numbers shall be
documented.
For specimens intended for extended storage in a biobank the individual human cfDNA examinations needed
are not always known in advance of extended storage. Therefore, body fluid collection devices with cfDNA
profile stabilizers or workflows with immediate post-collection addition of cfDNA stabilizers should be used
to enable the use of a wider range of examinations.
5.1.3 Urine and other body fluid specimen collection from the patient or donor and stabilization
procedures
5.1.3.1 General
The examination manufacturer shall specify, verify and validate the body fluid collection device for the
examination and shall provide instructions for use for the specimen collection procedure, either with or where
appropriate without stabilizers.
NOTE In some countries it is not a requirement of the examination manufacturer to provide such instructions for
use and to specify, verify and validate the body fluid collection device for the examination.
A cfDNA stabilizer should be used, to prevent any cfDNA profile changes during the pre-examination process.
This may be a collection device prefilled with a stabilizer or an external stabilizer may be added to the collected
urine or body fluid, e.g. from a bulk solution.
Where the specimen collection device is intended for self-collection or home collection, or both, the collection
device manufacturer shall validate this device for these purposes. This shall include mitigating any potential
risks for the patient or donor from the cfDNA stabilizer where appropriate (see Clause 4Clause 4).).
The laboratory shall have written and/or visual instructions, or both, in place for body fluid collection. These
shall follow the examination manufacturer'smanufacturer’s instructions for use where provided.
Where the examination manufacturer'smanufacturer’s instructions are not provided (e.g. due to less stringent
legal frameworks), or where, for justified reasons (e.g. unmet patient needs), they require modifications, the
laboratory shall specify, verify and validate the body fluid collection device and collection procedure for the
intended examination and document this according to its quality management system requirements. This shall
include specifications for specimen storage and transport as required for the examination (see 5.1.45.1.4 and
5.25.2).). Where the selected body fluid collection device manufacturer provides specified and verified
instructions (see 5.1.3.25.1.3.2 and 5.1.3.35.1.3.3)) for the body fluid collection, these can serve as a basis for
the laboratory to verify this device for the examination.
Where the specifications given in the instructions for use of the selected body fluid collection device do not
meet the examination requirements during verification, they shall be modified to be fit for purpose, verified
and documented by the laboratory.
Where specimen self-collection is possible (e.g. saliva, urine), written visual instructions, or both, either from
the body fluid collection device manufacturer or the laboratory shall be supplied to the patient or donor.
Laboratory instructions shall be based on the body fluid collection device manufacturer'smanufacturer’s or
the examination manufacturer'smanufacturer’s requirements, or both.
The instructions for body fluid specimen collection shall include:
a) a) Requirements and recommendations to follow before the collection, in particular related to e.g.
drinking or fasting, or both, before collection, or collection time, (e.g. first midstream urine of the
morning.).
b) b) All requirements for patient identification, collection, storage and transport of the specimen to
the laboratory. For collection this shall include requirements:
1) 1) to collect the specimen within the specified volume range;
2) 2) to mix the specimen with the stabilizer(s) if required by the manufacturer, e.g. by immediate
shaking or inverting.
For self-collection, the patient or donor shall be provided with an appropriate body fluid collection device (e.g.
container, tube), identity tag(s) ([(e.g. label, radio frequency identification (RFID),)], and in general anything
needed for the specimen collection, preservation, storage and transport procedure for returning the specimen
to laboratory.
The patient or donor shall also be provided with an option to confirm compliance with the supplied
instructions for the body fluid specimen collection, e.g. electronic, paper based.
The patient or donor or person collecting the specimen from the patient or donor shall confirm compliance
with the supplied instructions for the body fluid specimen collection.
The identity of any person other than the patient or donor collecting the specimen shall be documented. The
date and time of body fluid collection shall be documented.
For the labelling (sample or specimen identification) of the body fluid collection device, a routine procedure
(for example ISO 15189 for medical laboratories or ISO 20387 for biobanks) or a procedure with additional
information (e.g. 2D-barcode) shall be used.
Any alterations or additions to the specimen shall be documented.
5.1.3.2 Urine and other body fluid collection with cfDNA stabilizers
The cfDNA stabilizer manufacturer and, where the stabilizer is incorporated into a collection device, the
collection device manufacturer, shall specify, verify and validate the instructions for use for body fluid
collection and stabilization.
The specimen collection device manufacturer shall also specify and verify the specimen collection device
quality parameters, e.g. interference of device materials, stability of stabilizer during product shelf-life and
tightness of closures or caps.
The examination manufacturer shall determine the cfDNA target stability (see Clause 4Clause 4).).
The examination manufacturer'smanufacturer’s instructions and thereon built laboratory’s instructions for
use may refer to the instructions of the body fluid collection device or stabilizer, or both, provided by the
manufacturer.
The person collecting the specimen from the patient or donor, or the patient or donor self-collecting the
specimen, shall follow the laboratory’s written instructions for use (see 5.1.3.15.1.3.1).).
5.1.3.3 Urine and other body fluid collection without cfDNA stabilizers
The cfDNA profile can change rapidly post-collection and can thus impact the intended performance
characteristics of the examination. Therefore such collection devices shall only be used if they have been
specified, verified and validated for the specific examination.
The examination manufacturer'smanufacturer’s instructions and thereon built laboratory’s instructions for
use may refer to the instructions of the body fluid collection device without stabilizer provided by the
manufacturer.
Where the manufacturer of the body fluid collection device without stabilizer does not provide such
instructions, the laboratory shall specify, verify and validate the instructions for use of the device for body
fluid collection for cfDNA examination. The laboratory shall write instructions for use and follow them.
The instructions may include cooling of the specimen to 2 °C to 8 °C or placing on wet ice, and transportation
to the laboratory without delay within a verified period of time. Otherwise, microbial growth, cfDNA
degradation and contamination with genomic DNA released from cells present in the body fluid can happen
and impact examination test results.
The person collecting the specimen from the patient or donor, or the patient or donor self-collecting the
specimen, shall follow the laboratory’s written instructions for use (see 5.1.3.15.1.3.1).).
5.1.4 Information about the specimen storage requirements at the body fluid collection facility
5.1.4.1 General
The examination manufacturer shall specify, verify and validate the storage requirements and shall provide
instructions for specimen storage, either with (see 5.1.4.25.1.4.2)) or where appropriate without (see
5.1.4.35.1.4.3)) stabilizers.
NOTE In several countries it is not a requirement to provide such instructions for use.
This can be accomplished with time course studies, e.g. by storing the specimen for different storage durations
and temperatures. As an example, see Annex AAnnex A.
The laboratory shall have written instructions in place for body fluid storage. These should follow the
examination manufacturer'smanufacturer’s instructions for use where provided. Where these are not
provided, the laboratory shall specify, verify and validate the body fluid specimen storage conditions (e.g.
temperature, duration) for the intended examination and document this according to its quality management
system requirements. This can be accomplished by time course studies (see Annex AAnnex A).).
Where provided, the body fluid collection device manufacturer’s instructions may serve as a basis for the
laboratory'slaboratory’s own specification and verification for the intended examination.
The documentation regarding the specimen shall include the date and time of specimen collection and the
storage conditions (i.e. storage duration and temperature).
Temperature monitoring should be applied in a suitable manner in case the specified storage conditions
cannot be ensured.
It shall be documented that the required storage conditions have been followed. The temporary storage
duration in the body fluid collection facility contributes to the total duration for storage.
The patient or donor, or any person other than the patient or donor storing the specimen, shall confirm
compliance with the supplied storage instructions.
5.1.4.2 Storage of urine and other body fluid collected with cfDNA stabilizers
The examination manufacturer'smanufacturer’s instructions for use of the body fluid collection device or
cfDNA stabilizers, or both, shall be followed for specimen storage.
The examination manufacturer'smanufacturer’s instructions for use of the body fluid collection device or
cfDNA stabilizers, or both, may refer to the instructions of the body fluid collection device manufacturer or
stabilizer manufacturer. If a laboratory self-prepares a collection device with stabilizer, the laboratory should
specify, verify and validate the ability of the device to meet storage requirements.
Where the examination manufacturer'smanufacturer’s instructions are not provided (e.g. due to less stringent
legal frameworks), but the body fluid collection device manufacturer provides specified and verified
instructions, these can serve as a basis for the laboratory'slaboratory’s examination specific verification.
Where these instructions cannot be successfully verified with the examination or where no such instructions
are provided, the storage conditions shall be specified, verified and documented by the laboratory.
Instructions for use shall be written accordingly and followed.
Where the examination is not yet known (e.g. in biobanks), the specified storage instructions given by the
body fluid collection device or stabilizer manufacturer, or both, should be followed.
The storage conditions (temperature, duration, etc.) shall be documented.
5.1.4.3 Storage of urine and other body fluid collected without cfDNA stabilizers
The examination manufacturer'smanufacturer’s instructions for use of the body fluid collection device shall
be followed for specimen storage.
Where storing body fluids collected without cfDNA stabilizers, the examination manufacturer'smanufacturer’s
instructions on storage conditions shall be followed. The required storage conditions shall be documented
including any deviations therefrom.
Where the examination manufacturer does not provide such instructions (e.g. due to less stringent legal
frameworks), the storage procedure shall be specified, verified and documented by the laboratory.
Instructions shall be written accordingly for the user and followed. This may include immediate specimen
cooling to 2 °C to 8 °C or placing on wet ice in order to minimize cfDNA profile changes.
NOTE Some studies determined a maximum storage duration at 2 °C to 8 °C of 2 h to minimize chemical reactions,
[23],[34[3],[14] ]
enzymatic activities and bacterial growth in urine specimens to ensure cfDNA integrity. .
Body fluid specimen should not be frozen to avoid cell disruption upon ice crystal formation as this can lead
to the release of genomic DNA from cells, thus contaminating the native cfDNA profile. Other verified methods
for enabling freezing without cell disruption may also be used.
5.2 Transport requirements
5.2.1 General
The examination manufacturer shall specify, verify and validate the transport requirements and should
provide instructions for specimen transport, either with (see 5.2.25.2.2)) or where appropriate without (see
5.2.3) stabilizers (see 5.2.3).
NOTE 1: In several countries it is not a requirement to provide such i
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