SIST EN ISO 17099:2017

SLOVENSKI STANDARD
01-december-2017
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Radiological protection - Performance criteria for laboratories using the cytokinesis block
micronucleus (CBMN) assay in peripheral blood lymphocytes for biological dosimetry
(ISO 17099:2014)
Radioprotection - Critères de performance pour les laboratoires pratiquant la dosimétrie
biologique par le test des micronoyaux avec blocage de la cytodiérèse (CBMN) dans les
lymphocytes du sang périphérique (ISO 17099:2014)
Ta slovenski standard je istoveten z: EN ISO 17099:2017
ICS:
13.280 Varstvo pred sevanjem Radiation protection
71.040.10 Kemijski laboratoriji. Chemical laboratories.
Laboratorijska oprema Laboratory equipment
2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.

EN ISO 17099
EUROPEAN STANDARD
NORME EUROPÉENNE
October 2017
EUROPÄISCHE NORM
ICS 13.280
English Version
Radiological protection - Performance criteria for
laboratories using the cytokinesis block micronucleus
(CBMN) assay in peripheral blood lymphocytes for
biological dosimetry (ISO 17099:2014)
Radioprotection - Critères de performance pour les
laboratoires pratiquant la dosimétrie biologique par le
test des micronoyaux avec blocage de la cytodiérèse
(CBMN) dans les lymphocytes du sang périphérique
(ISO 17099:2014)
This European Standard was approved by CEN on 13 September 2017.

CEN members are bound to comply with the CEN/CENELEC Internal Regulations which stipulate the conditions for giving this
European Standard the status of a national standard without any alteration. Up-to-date lists and bibliographical references
concerning such national standards may be obtained on application to the CEN-CENELEC Management Centre or to any CEN
member.
This European Standard exists in three official versions (English, French, German). A version in any other language made by
translation under the responsibility of a CEN member into its own language and notified to the CEN-CENELEC Management
Centre has the same status as the official versions.

CEN members are the national standards bodies of Austria, Belgium, Bulgaria, Croatia, Cyprus, Czech Republic, Denmark, Estonia,
Finland, Former Yugoslav Republic of Macedonia, France, Germany, Greece, Hungary, Iceland, Ireland, Italy, Latvia, Lithuania,
Luxembourg, Malta, Netherlands, Norway, Poland, Portugal, Romania, Serbia, Slovakia, Slovenia, Spain, Sweden, Switzerland,
Turkey and United Kingdom.
EUROPEAN COMMITTEE FOR STANDARDIZATION
COMITÉ EUROPÉEN DE NORMALISATION

EUROPÄISCHES KOMITEE FÜR NORMUNG

CEN-CENELEC Management Centre: Avenue Marnix 17, B-1000 Brussels
© 2017 CEN All rights of exploitation in any form and by any means reserved Ref. No. EN ISO 17099:2017 E
worldwide for CEN national Members.

Contents Page
European foreword . 3

European foreword
The text of ISO 17099:2014 has been prepared by Technical Committee ISO/TC 85 “Nuclear energy,
nuclear technologies, and radiological protection” of the International Organization for Standardization
(ISO) and has been taken over as EN ISO 17099:2017 by Technical Committee CEN/TC 430 “Nuclear
energy, nuclear technologies, and radiological protection” the secretariat of which is held by AFNOR.
This European Standard shall be given the status of a national standard, either by publication of an
identical text or by endorsement, at the latest by April 2018, and conflicting national standards shall be
withdrawn at the latest by April 2018.
Attention is drawn to the possibility that some of the elements of this document may be the subject of
patent rights. CEN shall not be held responsible for identifying any or all such patent rights.
According to the CEN-CENELEC Internal Regulations, the national standards organizations of the
following countries are bound to implement this European Standard: Austria, Belgium, Bulgaria,
Croatia, Cyprus, Czech Republic, Denmark, Estonia, Finland, Former Yugoslav Republic of Macedonia,
France, Germany, Greece, Hungary, Iceland, Ireland, Italy, Latvia, Lithuania, Luxembourg, Malta,
Netherlands, Norway, Poland, Portugal, Romania, Serbia, Slovakia, Slovenia, Spain, Sweden, Switzerland,
Turkey and the United Kingdom.
Endorsement notice
The text of ISO 17099:2014 has been approved by CEN as EN ISO 17099:2017 without any modification.

INTERNATIONAL ISO
STANDARD 17099
First edition
2014-11-15
Radiological protection —
Performance criteria for laboratories
using the cytokinesis block
micronucleus (CBMN) assay in
peripheral blood lymphocytes for
biological dosimetry
Radioprotection — Critères de performance pour les laboratoires
pratiquant la dosimétrie biologique par analyse des micronoyaux
par blocage de la cytokinèse (CBMN) dans les lymphocytes du sang
périphérique
Reference number
ISO 17099:2014(E)
©
ISO 2014
ISO 17099:2014(E)
© ISO 2014
All rights reserved. Unless otherwise specified, 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
Case postale 56 • CH-1211 Geneva 20
Tel. + 41 22 749 01 11
Fax + 41 22 749 09 47
E-mail copyright@iso.org
Web www.iso.org
Published in Switzerland
ii © ISO 2014 – All rights reserved

ISO 17099:2014(E)
Contents Page
Foreword .v
Introduction .vi
1 Scope . 1
2 Terms and definitions . 1
3 Micronucleus assay methodology used in this standard . 4
3.1 General . 4
3.2 General requirement of the laboratory . 4
3.3 Requests for analysis and blood sampling . 4
3.4 Cell culturing . 5
3.5 Staining . 5
3.6 Microscopy . 6
3.7 Visual scoring of slides . 6
3.8 Automated analysis . 7
4 Confidentiality of personal information . 7
4.1 Overview . 7
4.2 Applications of the principle of confidentiality . 7
5 Laboratory safety requirements . 8
5.1 Overview . 8
5.2 Microbiological safety requirements . 8
5.3 Chemical safety requirements . 9
5.4 Optical safety requirements .10
5.5 Safety plan .10
6 Calibration source(s), calibration curve, and minimum resolvable dose .10
6.1 Calibration source(s) .10
6.2 Calibration curve .10
6.3 Background micronucleus frequency .11
6.4 Minimum resolvable dose measurement .12
7 Responsibility of the customer .12
8 Responsibility of the CBMN laboratory .12
8.1 Setup and sustainment of the QA program .12
8.2 Responsibility during service .13
9 Accidental overexposure involving few individuals .14
9.1 Procedure for scoring micronuclei in binucleated cells .14
9.2 Criteria for converting a micronucleus yield into an estimate of absorbed dose .14
9.3 Reporting of Results .15
10 Population triage .17
10.1 General .17
10.2 Use of a CBMN assay network for large scale exposures .17
10.3 Procedure for scoring micronuclei in binucleated cells .17
10.4 Criteria for converting a micronucleus yield into an estimate of absorbed dose .17
10.5 Reporting of results .17
11 Quality assurance and quality control .17
11.1 Overview .17
11.2 Quality Assurance .17
11.3 Quality control .18
Annex A (informative) Sample data sheet for recording micronuclei in binucleated cells .20
Annex B (informative) Automation of micronuclei scoring .21
Annex C (informative) Instructions for customer (sample) .23
ISO 17099:2014(E)
Annex D (informative) Sample questionnaire .24
Annex E (informative) Sample of report for single assessment .26
Annex F (informative) Example group sample report .27
Bibliography .29
iv © ISO 2014 – All rights reserved

ISO 17099:2014(E)
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).
Attention is drawn to the possibility that some of the elements of this document may be the subject of
patent rights. ISO shall not be held responsible for identifying any or all such patent rights. Details of any
patent rights identified during the development of the document will be in the Introduction and/or on
the ISO list of patent declarations received (see www.iso.org/patents).
Any trade name used in this document is information given for the convenience of users and does not
constitute an endorsement.
For an explanation on the meaning of ISO specific terms and expressions related to conformity
assessment, as well as information about ISO’s adherence to the WTO principles in the Technical Barriers
to Trade (TBT), see the following URL: Foreword — Supplementary information.
The committee responsible for this document is ISO/TC 85, Nuclear energy, nuclear technologies, and
radiological protection, Subcommittee SC 2, Radiological protection.
ISO 17099:2014(E)
Introduction
The purpose of this International Standard is to define the use of the cytokinesis block micronucleus
(CBMN) assay with human peripheral blood lymphocytes for biological dosimetry of exposure to
ionizing radiation. This assay is intended to be applied for accidental or malevolent exposures involving
a) up to a few casualties to provide individual full dose estimates or b) in a triage mode to populations
to provide interim dose estimates for individuals.
The CBMN assay is an alternative cytogenetic technique, which is possibly simpler and faster to perform
than the dicentric assay (ISO 19238:2014, ISO 21243:2008). It is also routinely used to demonstrate
exposure to genotoxic agents, other than ionizing radiation, which is not covered in this International
Standard. Although culture of the blood samples is slightly longer than for dicentrics, the scoring of
micronuclei in binucleated lymphocytes is easier.
As was done with the dicentric assay, the CBMN assay has been adapted for the emergency triage of
large-scale multi casualty radiation accidents. The blood volume required for sufficient number of
scorable binucleated cells is similar than required for the dicentric assay. Again, the faster counting
speed for micronuclei compensates for the extended culture time. In addition, the CBMN assay can be
performed in an automated mode.
This International Standard provides a guideline on how to perform the CBMN assay for dose assessment
using documented and validated procedures. Dose assessment using the CBMN assay has relevance
in medical management, radiation-protection management, record keeping, and medical/legal
requirements. This International Standard is divided into two parts, according to the use of CBMN
assay: radiation exposure of a few individuals or population triage in a large radiological event.
A part of the information in this International Standard is contained in other international guidelines and
scientific publications, primarily in the International Atomic Energy Agency’s (IAEA) technical reports
series on biological dosimetry. However, this International Standard expands and standardizes the
quality assurance and quality control, the criteria of accreditation and the evaluation of performance.
This International Standard is generally compliant with ISO/IEC 17025 “General requirements for the
competence of testing and calibration laboratories” with particular consideration given to the specific
needs of biological dosimetry. The expression of uncertainties in dose estimations given in this
International Standard complies with the “ISO-guide for the expression of uncertainty in measurement”
(former GUM) and the ISO 5725-all parts.
vi © ISO 2014 – All rights reserved

INTERNATIONAL STANDARD ISO 17099:2014(E)
Radiological protection — Performance criteria for
laboratories using the cytokinesis block micronucleus
(CBMN) assay in peripheral blood lymphocytes for
biological dosimetry
1 Scope
This International Standard addresses the following:
a) confidentiality of personal information for the customer and the laboratory;
b) laboratory safety requirements;
c) radiation sources, dose rates, and ranges used for establishing the calibration reference dose-effect
curves allowing the dose estimation from CBMN assay yields and the minimum resolvable dose;
d) performance of blood collection, culturing, harvesting, and sample preparation for CBMN assay scoring;
e) scoring criteria;
f) conversion of micronucleus frequency in binucleated cells into an estimate of absorbed dose;
g) reporting of results;
h) quality assurance and quality control;
i) informative annexes containing examples of a questionnaire, instructions for customers, a
microscope scoring data sheet, a sample report and advice on strengths and limitations of current
automated systems for automated micronucleus scoring.
2 Terms and definitions
For the purposes of this document, the following terms and definitions apply.
2.1
acentric
chromosome fragment of varying size
Note 1 to entry: When it is formed independently of a dicentric or centric ring chromosome aberration, it is usually
referred to as an excess acentric.
2.2
background level
spontaneous yield (or number) of micronuclei recorded in control samples or individuals
2.3
bias
statistical sampling or testing error caused by systematically favouring some outcomes over others
2.4
binucleated cells
cells that have completed one nuclear division after mitogen stimulation and cell type in which
micronuclei are scored
Note 1 to entry: These cells are accumulated in culture using cytochalasin-B which is an inhibitor of cytokinesis.
ISO 17099:2014(E)
2.5
CBMN laboratory
laboratory performing biological dosimetry measurements using the CBMN assay
2.6
centric ring
aberrant circular chromosome resulting from the joining of two breaks on separate arms of the same
chromosome, generally accompanied by one acentric fragment
2.7
centromere
specialized constricted region of a chromosome that appears during mitosis joining together the two
sister chromatids
2.8
chromosome
structure that carries genetic information
Note 1 to entry: Normally, 46 such structures are contained in the human cell nucleus. During nuclear division,
they condense to form characteristically-shaped bodies.
2.9
chromatid
either of the two strands of a duplicated chromosome that are joined by a single centromere
Note 1 to entry: Chromatids separate during mitosis to become individual chromosomes.
2.10
confidence interval
statistical range about an estimated quantity within which the value of the quantity is expected to occur,
with a specified probability
2.11
cytochalasin-B
Cyto-B
reagent used to block cytokinesis in dividing cells allowing once-divided cells to be identified as
binucleated cells
Note 1 to entry: The binucleated cells are the cells in which micronuclei are specifically scored.
2.12
dicentric
aberrant chromosome bearing two centromeres derived from the joining of parts from two broken
chromosomes, generally accompanied by an acentric fragment
2.13
fluorescence in situ hybridization
FISH
technique that uses specific sequences of DNA as probes to particular parts of the genome, allowing
the chromosomal regions to be highlighted or “painted” in different colours by attachment of various
fluorochromes
Note 1 to entry: This technique permits the detection of damage involving exchanges between differently painted
pieces of DNA (usually whole chromosomes).
2.14
interphase
period of the cell cycle between the mitotic divisions
2 © ISO 2014 – All rights reserved

ISO 17099:2014(E)
2.15
linear energy transfer
LET
quotient of dE/dl, as defined by the International Commission on Radiation Units and Measurements
(ICRU), where dE is the average energy locally imparted to the medium by a charged particle of specific
energy in traversing a distance of dl
2.16
metaphase
second stage of mitosis when the nuclear membrane is dissolved, the chromatids are condensed to their
minimum lengths and are aligned for division at the metaphase plate
2.17
micronucleus or micronuclei
MN
small nucleus that arises from lagging acentric chromosome fragments or whole chromosomes during
nuclear division and chromosome segregation at mitosis during anaphase/telophase
Note 1 to entry: More that 90 % of the micronuclei induced by ionizing radiation arise from lagging acentric
chromosome fragments.
2.18
minimum detection level
MDL
smallest measurable amount (e.g. yield or dose) that is detected with a probability β of non-detection
(Type II error) while accepting probability α of erroneously deciding that a positive (non-zero) quantity
is present in an appropriate background sample (Type I error)
2.19
minimum resolvable dose
lowest additional dose for which the lower 95 % poisson confidence limit is greater than 0, so that there
is a 97,5 % chance that the dose received in excess of normal background is greater than 0
2.20
nuclear division index
index in the CBMN assay that is calculated from the relative frequencies of mononucleated, binucleated,
and multinucleated cells
Note 1 to entry: This index provides a measure of inhibition of nuclear division.
2.21
precision
dispersion of measurements with respect to a measure of location or central tendency
2.22
quality assurance
planned and systematic actions necessary to provide adequate confidence that a process, measurement,
or service has satisfied given requirements for quality
EXAMPLE Dose specified in a licence.
2.23
quality control
part of quality assurance intended to verify that systems and components correspond to pre-
determined requirements
ISO 17099:2014(E)
3 Micronucleus assay methodology used in this standard
3.1 General
In this International Standard, the frequency of micronuclei in cytokinesis block binucleated lymphocytes
in cultured human peripheral blood lymphocytes scored by microscopy is used for dose estimation after
suspected exposure to ionizing radiation.
Lymphocytes are cultured by a method that permits once-divided cytokinesis block cells to be recognized
by their binucleated appearance for analysis. This requires whole blood or lymphocytes separated
from the other blood components to be incubated in culture medium with a mitogen that would enable
scoring of micronuclei in first-generation binucleated cells. A cytokinesis blocking agent, cytochalasin-B,
is added at least 6 h before the first mitosis commences to arrest dividing lymphocytes at the binucleated
cell stage after nuclear division is completed. The duration of the cell culture and the timing of addition
of the arresting agent are optimised to ensure an adequate frequency of binucleated cells.
Binucleated cells are recovered from the cultures by centrifugation, placing in a hypotonic salt solution
and fixing in a mixture of methanol and acetic acid. Fixed cells are placed on microscope slides and
stained. In the case of isolated lymphocytes, it is also acceptable to prepare slides by cytocentrifugation
of cells onto slides, followed by air-drying, fixation with methanol, and staining. The exact protocol
for cell culture, harvesting binucleated cells and staining employed by a CBMN laboratory should be
formally documented.
Microscope slides containing stained cells are methodically scanned to identify suitable binucleated
cells. The frequency of micronuclei observed in an appropriate number of scored binucleated cells is
converted to an estimate of radiation dose by reference to calibration data.
3.2 General requirement of the laboratory
The laboratory should be well-equipped with the required bio-hazard units, tissue culture, and standard
laboratory equipment for lymphocyte tissue culture, cell separation, slide preparation, and microscopy
scoring of cells and subcellular structures, such as micronuclei. The laboratory should maintain quality
assurance documents including those describing periodic calibration of the equipment used for cell
culture such as laminar flow hoods, pipettes, incubator, etc.
3.3 Requests for analysis and blood sampling
Depending on national regulations, the request for an analysis should normally be made by a doctor
representing the patient, by the patient him/herself, or could be requested due to legal claims. In all
cases where it is normally possible, the blood sampling for micronuclei analysis shall be made with the
patient’s informed consent. It is advisable that the laboratory head maintain the record of the patient’s
informed consent and the patient should also indicate who they will allow to receive the data. For minors,
the informed consent should be obtained from the parent/guardian.
It is the responsibility of the medical staff (e.g. doctor, nurse, etc.) to schedule blood draw and shipping
so as to ensure that the blood sample is received by the laboratory in the best possible conditions. The
purpose is to avoid having the blood sample sit for several hours from time of blood draw and before
sample pickup for transportation.
The blood sample is collected using lithium or sodium heparin anticoagulant, maintained at room
temperature (at approximately 20 °C) and cultured as soon as possible, but before 72 h. In some
unavoidable circumstances involving a delay beyond 72 h, good sample preparation is still possible if the
blood samples are stored with due precautions, such as using room temperature gel packs to maintain
a temperature of 20 °C.
4 © ISO 2014 – All rights reserved

ISO 17099:2014(E)
3.4 Cell culturing
The protocol for the CBMN assay shall be established and documented by each CBMN laboratory. The
protocol used for the calibration curve and for dose estimates of patient samples shall be identical. There
are several critical aspects that shall be adhered to.
a) Blood used to establish the calibration curves shall be incubated for 2 h at 37 °C immediately
following irradiation and prior to culture of samples.
b) Cultures should be set up in duplicate to allow the determination of the intra-experimental
coefficient of variation.
c) Cells shall be cultured at 37 °C ± 0,5 °C either as whole blood, enriched lymphocyte suspension
(buffy coat), or isolated lymphocytes.
d) Culture vessel shall be sterile and handled in a way to avoid microbial contamination.
e) Specific culture media that allow peripheral blood lymphocytes to proliferate shall be used.
EXAMPLE RPMI-1640, Ham’s F10, MEM, or McCoy supplemented with Foetal Bovine Serum (FBS)
−1 −1
between 10 % and 20 %, 200 mM L-glutamine, and penicillin/Streptomycin (100 IU ml /100 μg·ml ) is
commonly used.
f) Mitogen [e.g. phytohaemagglutinin (PHA)] shall be added to the media to stimulate lymphocytes
into mitosis.
g) Cytochalasin-B (Cyto-B) shall be added, 24 h to 44 h after mitogen stimulation at a concentration of
at least 3,0 μg/ml and no more than 6,0 μg/ml to the cell culture to block cytokinesis in cells during
their first nuclear division after mitogen stimulation.
h) The timing of harvest is crucial to maximize the number of binucleated cells and minimize the number
of mononucleated and multinucleated cells. It shall be adapted according to the standard culture
conditions for each CBMN laboratory. The recommended culture time after mitogen stimulation
for cell harvest is 72 h but under certain conditions (e.g. where mitotic delay is anticipated), longer
time might be required. Typically, binucleated cells are harvested 24 h to 48 h after addition of
cytochalasin-B.
i) Cells may be treated with a hypotonic solution such as 0,075 M KCl for 10 min to 15 min to swell the
cells prior to fixation.
j) Cells may be fixed in suspension and then transferred to slides or alternatively, they may be
transferred to slides by cyto-centrifugation and then fixed on the slide after air drying. In the former
case, cells shall be fixed in freshly prepared fixative solution (i.e. 5:1 methanol:acetic acid) while
agitating the cells to prevent clump formation and washed three times or four times with the same
fixative until the cell suspension is clear. In the latter case, cells shall be fixed in absolute methanol.
k) If storage of fixed cells is required, then cell suspensions shall be kept in a −20 °C freezer.
l) Slides shall be prepared to ensure integrity of the cell membrane and allow an unambiguous
identification of micronuclei in binucleated cells. Humidity and temperature conditions can be
adjusted to increase the quality of the spreading.
m) Duplicate cultures should be performed from each blood sample per individual.
3.5 Staining
Cells shall be stained appropriately so that nuclei and micronuclei can be clearly visualized. Commonly
used stains include, but are not limited to, Giemsa (for brightfield microscopy), DAPI, and acridine
orange (for fluorescence microscopy). The stain used shall be specific for nuclei and micronuclei to avoid
artefactual staining of other cellular structures that might resemble micronuclei (e.g. centrioles).
ISO 17099:2014(E)
3.6 Microscopy
Use a fluorescence or brightfield microscope depending on the stain used. Observation of cells at a
magnification of at least 400 × is required for scoring of cells and micronuclei. For optimal scoring,
however, a higher magnification (e.g. 1 000 ×) is recommended.
3.7 Visual scoring of slides
3.7.1 General
Each sample shall be scored by two individuals, each scoring at least 500 binucleated cells (for a total of
at least 1 000 binucleated cells) from different slides for the presence of micronuclei. Fewer binucleated
cells can be scored for high dose samples or in triage mode (see Clause 10). The distribution of micronuclei
amongst the binucleated cells should also be recorded. The slide scorers should be experienced in the
scoring of micronuclei in lymphocytes (see Clause 9).
3.7.2 Criteria for scoring
3.7.2.1 Criteria for selecting binucleated cells which can be scored for micronucleus frequency
The cytokinesis block cells that can be scored for MN frequency should have the following characteristics:
a) the cells shall be binucleated (BN);
b) the two nuclei in a BN cell shall have intact nuclear membranes and be situated within the same
cytoplasmic boundary;
c) the two nuclei in a BN cell shall be approximately equal in size, staining pattern, and staining intensity;
d) the two nuclei within a BN cell may be unconnected or may be attached by one or more fine
nucleoplasmic bridges, which are no wider than 1/4 of the nuclear diameter;
e) the two main nuclei in a BN cell may touch but ideally should not overlap each other. A cell with two
overlapping nuclei can be scored only if the nuclear boundaries of either nucleus are distinguishable;
f) the cytoplasmic boundary or membrane of a BN cell shall be intact and clearly distinguishable from
the cytoplasmic boundaries of adjacent cells.
3.7.2.2 Criteria for scoring micronuclei
MN are morphologically identical to, but smaller than, the main nuclei. They also shall have the following
characteristics:
a) the diameter of MN in human lymphocytes shall be between 1/16 and 1/3 of the mean diameter of
the main nuclei, which corresponds to 1/256 and 1/9 of the area of one of the main nuclei in a BN
cell, respectively;
b) MN shall not be linked or connected to the main nuclei;
c) MN may touch but shall not overlap the main nuclei and the micronuclear boundary shall be
distinguishable from the nuclear boundary;
d) MN usually have the same staining intensity as the main nuclei but occasionally, staining may be
less intense;
e) MN are non-refractile and can therefore be readily distinguished from artefacts such as staining
particles;
f) micronuclei lying above or below the daughter nuclei shall not be scored.
6 © ISO 2014 – All rights reserved

ISO 17099:2014(E)
3.7.2.3 Criteria for accepting scores
a) Inter-scorer variability is one of the key sources of variation in the micronucleus assay. It is therefore
essential that the same scorers are maintained throughout a single assessment and ideally, two
scorers are used, each providing a count from each of the duplicate cultures and their mean values
calculated to take into account both experimental and scorer variation. However, it is also acceptable
to use a single scorer if two experienced scorers are not available.
b) For duplicate samples in which more than 100 MN per 1 000 BN cells are induced, CVs should be
less than 20 %.
3.7.3 Scoring data sheets
An example of a scoring data sheet is provided in Annex A.
3.8 Automated analysis
Several systems for automated image analysis for the CBMN assay have been developed. Automation at
present is beyond the scope of this International Standard. Efforts in this area are described in Annex B.
4 Confidentiality of personal information
4.1 Overview
Biological dosimetry investigations made by a CBMN laboratory shall be undertaken in accordance
with national regulations regarding confidentiality. It would normally include the maintenance of
confidentiality of the patient’s identity, medical data, and social status. In addition, the commercial
confidentiality of the patient’s employer and any other organizations involved in a radiological
accident/incident should be observed. This requirement extends to the following:
a) written, electronic, or verbal communications between the laboratory and the person/organization
requesting the analysis and receiving the report;
b) the secure protection of confidential information held within the organization where the CBMN
laboratory is located.
4.2 Applications of the principle of confidentiality
4.2.1 Delegation of responsibilities within the laboratory
The head of the laboratory may authorize a limited number of laboratory staff to deal with documents
related to the analysis. Persons with this authority shall have signed a commitment to confidentiality
regarding their duties within the laboratory.
The laboratory head shall maintain the signed confidentiality agreements and ensure the security and
safety of all confidential documents.
4.2.2 Requests for analysis
Depending on national regulations, the request for an analysis should normally be made by a doctor
representing the patient or by the patient him/herself (other requestors may be added here, especially
for networking). In all cases where it is normally possible, the blood sampling for micronucleus analysis
shall be made with the patient’s informed consent and the patient should also indicate who they permit
to receive the data. For minors, the informed consent shall be obtained from the parent/guardian. The
laboratory head, depending on the national regulations, might be required to maintain a record of the
patient’s informed consent.
ISO 17099:2014(E)
4.2.3 Transmission of confidential information
Whatever the chosen means of communication, confidentiality shall be ensured during the exchange of
information and reports between the CBMN laboratory and the requestor of the analysis.
The laboratory head needs to define all processes for information transmission and assurance of
confidentiality.
4.2.4 Anonymity of samples
The laboratory head needs to have established protocols for maintaining the anonymity of samples.
To avoid the identification of the patient while guaranteeing the traceability of the analysis, the
blood samples should be coded upon arrival in the CBMN laboratory. The coding is performed in an
unambiguous way according to a standard procedure. The same code is to be used for all the stages of
the analysis. The code is assigned by an authorized person, as defined in 8.2. Decoding, interpretation of
results, and compiling the report are also to be performed by an authorized person.
4.2.5 Reporting of results
The final report containing the results and their interpretation (when needed) is communicated to
the requestor of the analysis. Depending on national regulations, further copy may, with appropriate
approval, be passed to another responsible person.
4.2.6 Storage
The laboratory shall store the cultured cell pellet and slides to facilitate review/analysis by an external
expert or another laboratory in the event of any dispute regarding the analysis.
The laboratory head shall define how the data and results are stored. All laboratory documents relating
to a case and which could permit the patient and/or employer to be identified shall be stored in a place
only accessible to the authorized persons. Documents shall be retained in an appropriate place for at
least 30 years for possible medical/legal re-evaluation of the case. Final disposal of any records shall be
by secure means, such as shredding of paper records and complete deletion of electronic records.
5 Laboratory safety requirements
5.1 Overview
Staff shall conform to their national legislation and institutional regulations regarding safety in the
laboratories. There are some particular features concerning safety in service laboratories that are
worth highlighting. These include microbiological, chemical, and optical considerations.
5.2 Microbiological safety requirements
Handling human blood poses some risk of blood borne parasites and infections being transmitted
to laboratory staff. All specimens should be regarded as being potentially infectious even if they are
known to be derived from apparently healthy persons. Specimens shall be unpacked and manipulated
in a class 2 microbiological safety cabinet. Setting up cultures in such a cabinet has the added benefit of
minimizing culture failure due to microbial contamination. Use of hypodermic needles should be kept to
a minimum to reduce the risk of injuries. Suitable disinfectants shall be available to deal with spills. All
biological waste and used disposable plastic ware shall be sterilised (e.g. by autoclaving or incineration,
before final disposal).
Staff should be offered available vaccination against blood borne diseases. The legal and ethical position
regarding HIV testing of blood samples upon receipt differs between countries and researchers should
follow their national requirements. It should be noted that when blood samples are accepted from
abroad, depending on the country of origin, airlines might require the sender to provide a certificate
confirming that the samples have been tested and are HIV negative.
8 © ISO 2014 – All rights reserved

ISO 17099:2014(E)
5.3 Chemical safety
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