SIST EN ISO 17099:2017
(Main)Radiological protection - Performance criteria for laboratories using the cytokinesis block micronucleus (CBMN) assay in peripheral blood lymphocytes for biological dosimetry (ISO 17099:2014)
Radiological protection - Performance criteria for laboratories using the cytokinesis block micronucleus (CBMN) assay in peripheral blood lymphocytes for biological dosimetry (ISO 17099:2014)
ISO 17099:2014 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.
Strahlenschutz - Durchführungskriterien für Laboratorien für die Anwendung des zytokinese-geblockten Mikrokerntests (cytokinesis-blocked micronucleus, CBMN) in Lymphozyten des menschlichen peripheren Blutes im Rahmen der biologischen Dosimetrie (ISO 17099:2014)
Diese Internationale Norm behandelt Folgendes:
a) die Vertraulichkeit von persönlichen Angaben in Bezug auf den Kunden und das Laboratorium;
b) Labor-Sicherheitsanforderungen;
c) Strahlungsquellen, Dosisleistungen und -bereiche, die für die Erstellung der Referenz-Dosis-Wirkungs-Kurven für die Kalibrierung verwendet werden, die eine Schätzung der Dosis aus Ausbeuten des CBMN-Tests und der kleinsten auflösbaren Dosis ermöglichen;
d) Durchführung von Blutentnahme, Kultivierung, Ernte und Probenvorbereitung für die Bewertung mittels CBMN-Test;
e) Bewertungskriterien;
f) Umwandlung der Häufigkeit von Kleinkernen in zweikernigen Zellen in einen Schätzwert der absorbierten Dosis;
g) Berichterstattung der Ergebnisse;
h) Qualitätssicherung und Qualitätsteuerung;
i) informative Anhänge, die Beispiele für einen Fragebogen, Anweisungen für Kunden, ein Datenblatt für die mikroskopische Bewertung, einen Musterbericht und Hinweise zu Stärken und Grenzen von derzeitigen automatisierten Systemen für die automatisierte Bewertung von Kleinkernen enthalten.
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)
L'ISO 17099:2014 porte sur:
a) la confidentialité des informations personnelles pour le demandeur et le laboratoire;
b) les exigences de sécurité du laboratoire;
c) les sources de rayonnements, les débits de doses et les gammes utilisées pour établir les courbes dose-effet d'étalonnage de référence qui permettent d'estimer les doses à partir des résultats du test des CBMN, ainsi que la dose minimum détectable;
d) le prélèvement de sang, la mise en culture, le recueil des cellules après culture et la préparation des échantillons pour le test des CBMN;
e) les critères de dénombrement;
f) la conversion de la fréquence des micronoyaux dans les cellules binucléées en estimation de dose absorbée;
g) la présentation des résultats;
h) l'assurance et le contrôle de la qualité;
i) les annexes informatives contenant des exemples de questionnaire, d'instructions d'utilisation, de tableau de dénombrement au microscope et de rapport, ainsi qu'une synthèse des avantages et limites des systèmes automatisés actuellement disponibles pour le dénombrement des micronoyaux.
Radiološka zaščita - Merila za delovanje laboratorijev, ki za biološko dozimetrijo uporabljajo analizo tvorjenja mikrojeder s citokinetskim blokom v perifernih krvnih limfocitih (ISO 17099:2014)
Standard ISO 17099:2014 obravnava:
a) zaupnost osebnih podatkov stranke in laboratorija;
b) varnostne zahteve laboratorija;
c) vire sevanja, hitrosti odmerka in razpone, ki se uporabljajo za določanje referenčnih krivulj učinkov odmerka za umerjanje ter omogočajo ocenjevanje odmerka na podlagi rezultatov preizkusov CBMN in minimalnega razločljivega odmerka;
d) učinkovitost zbiranja krvi ter gojenja, zajemanja in pripravo vzorcev za ocenjevanje rezultatov preizkusov CBMN;
e) merila ocenjevanja;
f) pretvorbo pogostosti mikrojeder v dvojedrnih celicah v približek absorbiranega odmerka;
g) poročanje rezultatov;
h) zagotavljanje in nadzor kakovosti;
i) informativne dodatke, ki vsebujejo primere vprašalnika, navodil za stranke, tehničnega lista za ocenjevanje pri opazovanju z mikroskopom, vzorčnega poročila ter nasvet o prednostih in omejitvah trenutnih avtomatiziranih sistemov za ocenjevanje mikrojeder.
General Information
Standards Content (Sample)
SLOVENSKI STANDARD
SIST EN ISO 17099:2017
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
SIST EN ISO 17099:2017 en,fr,de
2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.
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SIST EN ISO 17099:2017
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SIST EN ISO 17099:2017
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.
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SIST EN ISO 17099:2017
EN ISO 17099:2017 (E)
Contents Page
European foreword . 3
2
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SIST EN ISO 17099:2017
EN ISO 17099:2017 (E)
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.
3
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SIST EN ISO 17099:2017
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SIST EN ISO 17099:2017
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
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SIST EN ISO 17099:2017
ISO 17099:2014(E)
COPYRIGHT PROTECTED DOCUMENT
© 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
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SIST EN ISO 17099:2017
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 2014 – All rights reserved iii
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SIST EN ISO 17099:2017
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
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SIST EN ISO 17099:2017
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 2014 – All rights reserved v
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SIST EN ISO 17099:2017
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
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SIST EN ISO 17099:2017
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 2014 – All rights reserved 1
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SIST EN ISO 17099:2017
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
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SIST EN ISO 17099:2017
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
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SIST EN ISO 17099:2017
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 inc
...
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