SIST EN ISO 19238:2018

SLOVENSKI STANDARD
01-februar-2018
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Radiological protection - Performance criteria for service laboratories performing
biological dosimetry by cytogenetics (ISO 19238:2014)
Radioprotection - Critères de performance pour les laboratoires de service pratiquant la
dosimétrie biologique par cytogénétique (ISO 19238:2014)
Ta slovenski standard je istoveten z: EN ISO 19238:2017
ICS:
13.280 Varstvo pred sevanjem Radiation protection
17.240 Merjenje sevanja Radiation measurements
2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.

EN ISO 19238
EUROPEAN STANDARD
NORME EUROPÉENNE
October 2017
EUROPÄISCHE NORM
ICS 13.280; 17.240
English Version
Radiological protection - Performance criteria for service
laboratories performing biological dosimetry by
cytogenetics (ISO 19238:2014)
Radioprotection - Critères de performance pour les
laboratoires de service pratiquant la dosimétrie
biologique par cytogénétique (ISO 19238:2014)
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 19238:2017 E
worldwide for CEN national Members.

Contents Page
European foreword . 3

European foreword
The text of ISO 19238:2014 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 19238:2017 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 19238:2014 has been approved by CEN as EN ISO 19238:2017 without any modification.

INTERNATIONAL ISO
STANDARD 19238
Second edition
2014-02-01
Radiological protection —
Performance criteria for service
laboratories performing biological
dosimetry by cytogenetics
Radioprotection — Critères de performance pour les laboratoires de
service pratiquant la dosimétrie biologique par cytogénétique
Reference number
ISO 19238:2014(E)
©
ISO 2014
ISO 19238:2014(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 19238:2014(E)
Contents Page
Foreword .v
Introduction .vi
1 Scope . 1
2 Terms and definitions . 1
3 Dicentric assay . 3
4 Responsibility of the customer . 3
5 Responsibility of the service laboratory. 4
5.1 Setup and sustainment of the QA program . 4
5.2 Responsibility during service . 4
6 Confidentiality of personal information . 5
6.1 Overview . 5
6.2 Applications of the principle of confidentiality . 5
7 Laboratory safety requirements . 6
7.1 Overview . 6
7.2 Microbiological safety requirements . 6
7.3 Chemical safety . 6
7.4 Optical safety requirements . 8
7.5 Safety plan . 8
8 Calibration curve(s) . 9
8.1 Culturing . 9
8.2 Calibration source(s) .10
8.3 Establishment of calibration curve(s) .10
8.4 Minimum resolvable dose measurement .11
9 Scoring unstable chromosome aberrations .11
9.1 Procedure for scoring first-division metaphases .11
9.2 Criteria for scoring .11
10 Criteria for converting a measured aberration frequency into an estimate of
absorbed dose .12
10.1 Overview .12
10.2 Comparison with controls .12
10.3 Testing the distribution of aberrations per cell .12
10.4 Determination of estimated whole-body dose and confidence limits .12
10.5 Acute and non-acute exposure cases .13
10.6 Partial-body and prior-exposure cases .13
11 Reporting of results .15
11.1 General .15
11.2 Content of the report (see Annex C for a standard form) .15
11.3 Interpretation of the results .16
12 Quality assurance and quality control .16
12.1 Overview .16
12.2 Specific requirements .17
Annex A (informative) Sample instructions for customer .19
Annex B (informative) Sample questionnaire .20
Annex C (informative) Sample of report .22
Annex D (informative) Fitting of the low-LET dose-response curve by the method of maximum
likelihood and calculating the error of dose estimate .23
Annex E (informative) Odds ratio method for cases of suspected exposure to a low dose .26
ISO 19238:2014(E)
Annex F (informative) Sample data sheet for recording aberrations .27
Bibliography .28
iv © ISO 2014 – All rights reserved

ISO 19238:2014(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. 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. 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.
The committee responsible for this document is ISO/TC 85, Nuclear energy, nuclear technologies, and
radiological protection, Subcommittee SC 2, Radiological protection.
This second edition cancels and replaces the first edition (ISO 19238:2004), of which it constitutes a
minor revision.
ISO 19238:2014(E)
Introduction
The wide use of ionising radiations for medical, industrial, agricultural, research, and military purposes
increases the risk of overexposure of radiation workers and individuals of the general population.
Biological dosimetry, based on the study of chromosomal aberrations, mainly the dicentric assay, has
become a routine component of accidental dose assessment. Experience with its application in hundreds
of cases of suspected or verified overexposures has proved the value of this method and also defined its
limitations. It should be emphasized that cytogenetic analysis is used as a dosimeter and provides one
input into the compendium of information needed for assessment of a radiological accident.
Many studies in animals and man have shown that one can establish a good correlation between the
results obtained in vivo and in vitro, so that in vitro established dose-effect relationships from irradiated
blood samples can be used as calibration curves. The dicentric yield is dependent on radiation quality
and dose rate so that information about these variables needs to be established for each investigation.
If known, these exposure characteristics are important for refining the dose estimates. The specificity
of this technique is enhanced by the fact that generally 1 dicentric is observed per 1 000 metaphase
spreads in the normal population, and that this frequency is approximatively independent of age and
sex. The precision of the technique thus depends on the number of cells observed, the background level,
and the calibration curve used. Theoretically, it is possible to detect exposure as low as 0,01 Gy. However,
for these very low doses, it is necessary to analyse tens of thousands of metaphase spreads. In practice,
this level of detection is neither feasible nor necessary. The upper limits to dose detection extend well
into the range of doses that are lethal to humans.
The primary purpose of this International Standard is to provide a guideline to all laboratories in order
to perform the dicentric assay using documented and validated procedures. Secondly, it can facilitate
the comparison of results obtained in different laboratories, particularly for international collaborations
or intercomparisons. Finally, laboratories newly commissioned to carry out the dicentric assay should
conform to this International Standard in order to perform it reproducibly and accurately.
This International Standard is written in the form of procedures to be adopted for biological dosimetry
for overexposures involving, at most, a few casualties. The criteria required for such measurements
will usually depend upon the application of the results: radiation protection management, medical
management when appropriate, record keeping, and legal requirements. In the special situation of a mass
radiation casualty and limited resources, the technique can be applied for emergency triage analysis.
The standard recommended scoring criteria would then be relaxed as appropriate to the situation.
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, with particular consideration
given to the specific needs of biological dosimetry. The expression of uncertainties in dose estimations
given in this International Standard comply with the ISO guide to the expression of uncertainty
in measurement (ISO/IEC Guide 98-1) and the ISO 5725 on accuracy (trueness and precision) of
measurement methods and results.
vi © ISO 2014 – All rights reserved

INTERNATIONAL STANDARD ISO 19238:2014(E)
Radiological protection — Performance criteria for
service laboratories performing biological dosimetry by
cytogenetics
1 Scope
This International Standard provides criteria for quality assurance and quality control, evaluation of
the performance, and the accreditation of biological dosimetry by cytogenetic service laboratories.
This International Standard addresses
a) the confidentiality of personal information, for the customer and the service laboratory,
b) the laboratory safety requirements,
c) the calibration sources and calibration dose ranges useful for establishing the reference dose-effect
curves that contribute to the dose estimation from chromosome aberration frequency and the
minimum resolvable doses,
d) the scoring procedure for unstable chromosome aberrations used for biological dosimetry,
e) the criteria for converting a measured aberration frequency into an estimate of absorbed dose,
f) the reporting of results,
g) the quality assurance and quality control,
h) informative annexes containing sample instructions for customer, sample questionnaire, sample of
report, fitting of the low dose-response curve by the method of maximum likelihood and calculating
the error of dose estimate, odds ratio method for cases of suspected exposure to a low dose, and
sample data sheet for recording aberrations.
2 Terms and definitions
For the purposes of this document, the following terms and definitions apply.
2.1
acentric
terminal or interstitial chromosome fragment of varying size, referred to as an excess acentric fragment
when it is formed independently of a dicentric or centric ring chromosome aberration
2.2
background level
spontaneous frequency (or number) of chromosome aberrations recorded in control samples or
individuals
2.3
bias
statistical sampling or testing error caused by systematically favouring some outcomes over others
2.4
centric ring
aberrant circular chromosome resulting from the joining of two breaks on separate arms of the same
chromosome
Note 1 to entry: It is generally accompanied by an acentric fragment.
ISO 19238:2014(E)
2.5
centromere
specialized constricted region of a chromosome that appears during mitosis and joins together the
chromatid pair
2.6
confidence interval
statistical range about an estimated quantity within which the value of the quantity is expected to occur,
with a specified probability
2.7
chromosome
structure that comprises discrete packages of DNA and proteins that carries genetic information which
condense to form characteristically shaped bodies during nuclear division
2.8
chromatid
either of the two strands of a duplicated chromosome that are joined by a single centromere and separate
during cell division to become individual chromosomes
2.9
dicentric
aberrant chromosome bearing two centromeres derived from the joining of parts from two broken
chromosomes
Note 1 to entry: It is generally accompanied by an acentric fragment.
2.10
FISH
fluorescence in situ hybridization
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
2.11
interphase
period of a cell cycle between the mitotic divisions
2.12
LET
linear energy transfer
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
2.13
lower threshold of dose
smallest measurable amount (e.g. frequency or dose) that is detected with a probability β of non-
detection (Type II error) while accepting a probability α of erroneously deciding that a positive (non-
zero) quantity is present in an appropriate background sample (Type I error)
2.14
metaphase
stage of mitosis when the nuclear membrane is dissolved, the chromosomes condensed to their minimum
lengths and aligned for division
2.15
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 © ISO 2014 – All rights reserved

ISO 19238:2014(E)
2.16
precision
concept employed to describe dispersion of measurements with respect to a measure of location or
central tendency
2.17
quality assurance
planned and systematic actions necessary to provide adequate confidence that a process, measurement,
or service satisfies given requirements for quality in, for example, those specified in a licence
2.18
quality control
part of quality assurance intended to verify that systems and components conform to predetermined
requirements
2.19
service laboratory
laboratory performing biological dosimetry measurements
3 Dicentric assay
The frequency of unstable chromosomal aberrations seen at metaphase in cultured human peripheral
blood lymphocytes is the recommended method for biological dosimetry. The chromosome aberrations
to be used are dicentrics or dicentrics and centric rings. For the application of this International Standard,
the service laboratory shall choose which type of aberrations to score for the purpose of assessing dose
estimates and shall be consistent throughout. Hereafter, chromosome aberrations are referred to as
dicentrics but may include centric rings if determined by the service laboratory.
Lymphocytes are cultured by a method that permits first-division metaphases to be recognized for
analysis (see 9.1). This requires whole blood, or lymphocytes separated from the other blood components,
to be incubated in a culture medium that would enable scoring of first-generation metaphase cells. A
mitotic blocking agent, colcemid or colchicine, is added to arrest dividing lymphocytes in metaphase.
The duration of the cell culture and the timing of addition of the arresting agent are optimised to ensure
an adequate mitotic index and predominance of first-division metaphases.
Metaphases are recovered from the cultures by centrifugation, placing in a hypotonic salt solution and
fixing in a mixture of alcohol and acetic acid. Fixed cells are placed on microscope slides and stained. The
exact protocol for cell culture, harvesting metaphases, and staining employed by a service laboratory
shall be formally documented (see Clause 12).
Microscope slides containing stained cells are methodically scanned to identify suitable first-division
metaphases to score dicentric aberrations (see 9.2). The frequency of dicentrics observed in an
appropriate number of scored metaphases is converted to an estimate of radiation dose by reference to
calibration data (see Clause 10).
4 Responsibility of the customer
This clause includes items that are not controlled by the service laboratory. Prior to blood sampling,
coordination between the customer and the service laboratory should occur. Essential requirements
should be explained to the customer and this may be by a standardised instruction sheet as illustrated
in Annex A. The essential features are:
a) Blood sampling should use the collection system containing lithium heparin as anticoagulant which
has been sent or specified by the service laboratory.
b) Blood should be collected (ideally about 10 ml), labelled accurately and unambiguously, maintained
at room temperature (around 20 °C), and sent to the service laboratory as soon as possible.
ISO 19238:2014(E)
c) Precautions to ensure the integrity of the container and prevent leakage during shipment shall be
observed. Blood samples should be kept cool during shipping (i.e. 6° C to 30 °C). A temperature
recording could be included to document that the temperature during shipment is controlled.
Packaging and labelling shall conform to national and international regulations. If air transportation
is involved, a physical dosimeter could be included to monitor whether the sample was irradiated in
transit.
d) A questionnaire provided by the service laboratory should be completed and returned promptly.
e) The service laboratory should be alerted of biologically contaminated samples.
5 Responsibility of the service laboratory
5.1 Setup and sustainment of the QA program
The service laboratory shall establish and maintain a QA program (see Clause 12), which covers all
aspects of the service. The QA program should address the following issues:
a) The laboratory’s QA program shall include periodic internal checks of equipment operations, reagent
suitability, and various performance checks (i.e. intracomparison exercises, operator qualifications,
sample protocol, scoring, dose estimations, report generation, etc.).
b) The laboratory’s QA program shall include periodic external checks of the laboratory’s operations.
The external audits shall include a review of the service laboratory’s documentation of equipment
operations, reagent suitability, and various performance checks (i.e. intercomparison exercises,
operator qualifications, sample transport integrity, etc.).
5.2 Responsibility during service
The service laboratory shall provide necessary guidance, procedures, and reporting to provide dose
assessment by cytogenetics in response to a request for service. The service activities shall address the
following issues:
a) The service laboratory shall have documentation, reviewed and endorsed by a qualified expert (i.e.
service laboratory radiobiologist or equivalent), which includes the following:
1) an instruction sheet to be sent to the customer describing shipping procedures (Annex A);
2) a questionnaire that shall elicit patient consent and information on whole or partial body
exposure, source and quality of the radiation, circumstances of the exposure, exposure location
(country, city, company, etc.), date and time of exposure, previous occupational or medical
exposures to radiation, intake of pharmaceuticals, infection, smoking habit, and significant
exposures to any other DNA damaging agents (such as organic solvents or heavy metals)
(Annex B);
3) step-by-step procedures for processing the blood sample from receipt of the sample to reporting
of the dose.
b) If required, a blood collection system (10 ml) containing lithium heparin as the anticoagulant
shall be sent to the customer with the appropriately labelled and addressed packaging material
for the return of the sample to the service laboratory. The packaging shall conform to national
and/or international regulations for the transit of potentially infectious pathological specimens
(see 12.2.4).
c) After receipt of the blood sample, the following steps shall be performed:
1) Document the receipt of the blood sample (date, time, consignee).
2) Code the blood sample.
4 © ISO 2014 – All rights reserved

ISO 19238:2014(E)
3) Document the place of storage until the setting up of cultures.
4) Set up cultures in parallel as soon as possible and document date, time, and operator.
5) Document all the reagents used for culturing with appropriate lot numbers.
6) Document the addition of reagents and the end of the culture (date, time, operator).
7) Document the short- and long-term storage of the sample until slide making.
8) Document the slide codes, number of slides, and location of storage.
9) Document the results from scoring.
10) Store the slides and case documents in an appropriate place for at least 30 years for possible
medico-legal re-evaluation of the case.
d) The service laboratory shall interpret the results and prepare reports (Annex C).
e) The service laboratory shall sustain a dialogue with the requestor, reprioritizing cases as required,
and providing results to the requestor.
6 Confidentiality of personal information
6.1 Overview
Biological dosimetry investigations made by a service laboratory shall be undertaken in accordance
with national regulations regarding confidentiality. This 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 1) written, electronic, or verbal communications between the laboratory and
the person/organization requesting the analysis and receiving the report, and 2) the secure protection
of confidential information held within the organization where the service laboratory is located.
6.2 Applications of the principle of confidentiality
6.2.1 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.
6.2.2 Requests for analysis
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, the blood sampling for chromosome analysis shall be made with the patient’s informed consent.
The laboratory head, depending on the national regulations, may be required to maintain the record of
the patient’s informed consent.
6.2.3 Transmission of confidential information
Whatever the chosen means of communication, confidentiality shall be ensured during the exchange of
information and reports between the service laboratory and the requestor of the analysis.
ISO 19238:2014(E)
The laboratory head needs to define all processes for information transmission and assurance of
confidentiality.
6.2.4 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 service 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 6.2.1. Decoding, interpretation
of results, and compiling the report are also to be performed by an authorized person.
6.2.5 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 copies may, with appropriate
approvals, be passed to other responsible persons.
6.2.6 Storage
The laboratory head shall define how 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 medico-legal re-evaluation of the case. Final disposal of documents shall be
by secure means such as shredding.
7 Laboratory safety requirements
7.1 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.
7.2 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 minimising culture failure due to microbial contamination. Use of sharps, e.g. 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 plasticware shall be sterilised, for example by
autoclaving or incineration, before final disposal.
Staff should be offered available vaccinations 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.
7.3 Chemical safety
Certain chemicals and pharmaceuticals are used routinely in the procedures covered in this International
Standard. When present in cultures or used in staining procedures, they are mostly used in small
volumes and in dilutions that generally present no health hazard. They are, however, prepared and
6 © ISO 2014 – All rights reserved

ISO 19238:2014(E)
stored in concentrated stock solutions. The main reagents of concern and their internationally agreed
hazard statements (H-Statements) according to the GHS classification system are listed below:
Acetic acid H226, H290, H314
Benzylpenicillin H317, H334
Bromodeoxyuridine (BrdU) H351
Colcemid H300, H361
Cytochalasin B H300, H310, H330, H361
Giemsa stain H225, H301, H311, H331, H370
Heparin H315, H319, H334
Hoechst stain (Bisbenzimide) H302, H315, H319
Methanol H225, H301, H311, H331, H370
Phytohaemagglutinin H302, H317, H332
Streptomycin sulphate H302, H332, H317, H334, H361
ISO 19238:2014(E)
Keys
H225: Highly flammable liquid and vapour
H226 Flammable liquid and vapour
H290: May be corrosive to metals
H300: Fatal if swallowed
H301: Toxic if swallowed
H302: Harmful if swallowed
H310: Fatal in contact with skin
H311: Toxic in contact with skin
H314: Causes severe skin burns and eye damage
H315: Causes skin irritation
H317: May cause an allergic skin reaction
H319: Causes serious eye irritation
H330: Fatal if inhaled
H331: Toxic if inhaled
H332: Harmful if inhaled
H334: May cause allergy or asthma symptoms or breathing difficulties
if inhaled
H351: Suspected of causing cancer
H361: Suspected of damaging fertility or the unborn child
H370: Causes damage to organs
7.4 Optical safety requirements
When ultraviolet lamps are used in sterilising the interior of microbiological safety cabinets or exposing
slides during the fluorescence plus Giemsa (FPG) staining procedure, shielding and working procedures
shall be in place to avoid direct irradiation of the skin or eyes of laboratory staff.
7.5 Safety plan
The laboratory head shall define written safety procedures for protection against microbiological,
chemical, and optical hazards.
The laboratory head shall maintain a record of accidents and protocols or procedures to avoid repeating
similar accidents.
8 © ISO 2014 – All rights reserved

ISO 19238:2014(E)
8 Calibration curve(s)
8.1 Culturing
The same culturing conditions shall be used for establishing the calibration curve as for analysing
aberrations in a case of suspected overexposure.
The exact protocol for the dicentric assay shall be established by each service laboratory, and there are
several critical aspects that shall be adhered to as listed below:
a) Blood shall be incubated for a minimum of 2 h at 37 °C immediately following irradiation and prior
to culture.
b) Cells shall be cultured at 37 °C ± 0,5 °C either as whole blood, as an enriched lymphocyte suspension
(buffy coat), or as isolated lymphocytes.
c) The culture vessel shall be sterile and used in a way to avoid microbial contamination.
d) Specific culture media that allow peripheral blood lymphocytes to proliferate shall be used. These
are commonly supplemented with Foetal Bovine Serum (FBS) (between 10 % and 20 %), 200 mM
[8]
L-glutamine, and Penicillin/Streptomycin (100 IU·ml−1/100 μg·ml−1)(see Reference ).
e) The mitogen (e.g. phytohaemagglutinin or PHA) shall be added to the media to stimulate lymphocytes
into mitosis.
f) A method to ensure the scoring of first-division metaphases shall be used (see 9.1).
g) Colcemid or colchicine shall be added, at a time and concentration determined by the laboratory, to
the cell culture to block cells in mitosis.
h) The timing of harvest is crucial to maximize the number of cells in first-division metaphase and
shall be adapted according to the standard culture conditions for that service laboratory. The
recommended culture time is 48 h, but under certain conditions where mitotic delay is anticipated,
longer time might be required.
i) Cells are centrifuged in order to separate the cells from the medium. Thereafter, cells shall 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) After centrifugation, the supernatant shall be removed and cells shall be fixed in freshly prepared
fixative solution (i.e. 1:3 acetic acid:methanol) and washed three or four times with the same fixative
until the cell suspension is clear.
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 allow an unambiguous identification of chromosomal aberrations.
Humidity and temperature conditions can be adjusted to increase the quality of the spreading.
m) Slides should be allowed to dry for at least 1 h prior to staining. Slides shall be stained with Giemsa.
If the FPG method is being used for the identification of first-division metaphases, FPG staining has
to be performed with Hoechst 33258, followed by UV exposure.
n) To avoid fading, stained slides shall be stored in the dark at room temperature. For better
conservation, slides should be mounted with an appropriate mounting medium.
ISO 19238:2014(E)
8.2 Calibration source(s)
The service laboratory shall provide a report, reviewed and endorsed by a qualified expert (i.e. radiation
physicist or the service laboratory head), that addresses the following issues:
a) description [e.g. Philips X-ray machine with a 2,1 mmCu half value layer (HVL), 250 kVp, filament
current 12,5 mA, and a source-to-surface distance (SSD) of 50 cm] for all radiation calibration
source(s) used to generate in vitro calibration
...