SIST EN ISO 20046:2021
(Main)Radiological protection - Performance criteria for laboratories using Fluorescence In Situ Hybridization (FISH) translocation assay for assessment of exposure to ionizing radiation (ISO 20046:2019)
Radiological protection - Performance criteria for laboratories using Fluorescence In Situ Hybridization (FISH) translocation assay for assessment of exposure to ionizing radiation (ISO 20046:2019)
The purpose of this document is to provide criteria for quality assurance (QA), quality control (QC) and evaluation of the performance of biological dosimetry by cytogenetic service laboratories.
This document addresses:
a) the responsibilities of both the customer and the laboratory;
b) the confidentiality of personal information, for the customer and the laboratory;
c) the laboratory safety requirements;
d) sample processing; culturing, staining and scoring, including the criteria for scoring for translocation analysis by FISH;
e) the calibration sources and calibration dose ranges useful for establishing the reference dose‑response curves that contribute to the dose estimation from chromosome aberration frequency and the detection limit;
f) the scoring procedure for translocations stained by FISH used for evaluation of exposure;
g) the criteria for converting a measured aberration frequency into an estimate of absorbed dose (also appears as "dose");
h) the reporting of results;
i) the QA and QC;
j) Annexes A to F containing sample instructions for the customer, sample questionnaire, sample datasheet for recording aberrations, sample of report and fitting of the low dose-response curve by the method of maximum likelihood and calculating the uncertainty of dose estimate.
Strahlenschutz - Leistungskriterien für Laboratorien, die den Fluoreszenz-in-situ-Hybridisierungs-(FISH)-Translokationstest zur Bewertung der Exposition gegenüber ionisierender Strahlung verwenden (ISO 20046:2019)
Der Zweck dieses Dokuments ist es, Kriterien für die Qualitätssicherung (QA), für die Qualitätskontrolle (QC) sowie für die Leistungsbewertung der biologischen Dosimetrie durch zytogenetische Service-Laboratorien bereitzustellen.
Dieses Dokument behandelt Folgendes:
a) Verantwortung des Kunden und des Laboratoriums;
b) vertraulicher Umgang mit personenbezogenen Daten beim Kunden und im Laboratorium;
c) Sicherheitsanforderungen im Laboratorium;
d) Durchführung der Probenahme; Kultivierung, Anfärbung und Bewertung, einschließlich der Kriterien für die Bewertung bei Translokationsanalysen mit FISH;
e) die für die Bestimmung der Referenzdosis-Kennlinien verwendeten Kalibrierungsquellen und –dosis-bereiche, mit denen aus der chromosomalen Veränderungshäufigkeit und der Nachweisgrenze die Dosis eingeschätzt wird;
f) Bewertungsverfahren für die durch FISH angefärbten Translokationen zur Beurteilung der Strahlen-einwirkung;
g) Kriterien für die Umwandlung einer gemessenen Veränderungshäufigkeit in eine Energiedosis-einschätzung (auch als „Dosis“ bezeichnet);
h) Angabe der Ergebnisse;
i) Qualitätssicherung und Qualitätskontrolle;
j) Anhang A bis Anhang F mit Anweisungen zur Probeentnahme für den Kunden, Musterfragebogen, Musterdatenblatt zur Aufzeichnung der Veränderungen, Musterbericht und Anpassung der Niedrig-dosis-Wirkungs-Kurve anhand des Verfahrens der maximalen Wahrscheinlichkeit sowie Berechnung der Unsicherheit von Dosiseinschätzungen.
Radioprotection - Critères de performance pour les laboratoires utilisant l'analyse des translocations visualisées par hybridation in situ fluorescente (FISH) pour évaluer l'exposition aux rayonnements ionisants (ISO 20046:2019)
L'objectif du présent document est de fournir des critères pour l'assurance de la qualité (AQ), le contrôle de la qualité (CQ) et l'évaluation des performances des laboratoires de service pratiquant la dosimétrie biologique par cytogénétique.
Le présent document aborde:
a) les responsabilités du demandeur et du laboratoire;
b) la confidentialité des informations personnelles pour le demandeur et le laboratoire;
c) les exigences de sécurité du laboratoire;
d) le traitement des échantillons, la culture, la coloration et le dénombrement, y compris les critères de dénombrement pour l'analyse des translocations par FISH;
e) les sources de calibration et les gammes de doses de calibration utiles pour établir les courbes dose-effet de référence qui contribuent à l'estimation dosimétrique à partir de la fréquence des aberrations chromosomiques et la limite de détection;
f) la procédure de dénombrement des translocations colorées par FISH utilisée pour l'évaluation de l'exposition;
g) les critères pour convertir une fréquence mesurée d'aberrations en une estimation de dose absorbée (également dénommée «estimation dosimétrique»);
h) la présentation des résultats;
i) l'AQ et le CQ;
j) les Annexes A à F contenant des exemples d'instructions pour le traitement du prélèvement par le demandeur, de questionnaire, de tableau type pour le dénombrement des aberrations chromosomiques, de rapport et d'ajustement de la courbe dose-effet par la méthode du maximum de vraisemblance et de calcul de l'incertitude sur l'estimation de la dose absorbée.
Radiološka zaščita - Merila za delovanje laboratorijev, ki uporabljajo preskus translokacije fluorescenčne hibridizacije in-situ (FISH) za oceno izpostavljenosti ionizirnemu sevanju (ISO 20046:2019)
General Information
Standards Content (Sample)
SLOVENSKI STANDARD
SIST EN ISO 20046:2021
01-april-2021
Radiološka zaščita - Merila za delovanje laboratorijev, ki uporabljajo preskus
translokacije fluorescenčne hibridizacije in-situ (FISH) za oceno izpostavljenosti
ionizirnemu sevanju (ISO 20046:2019)
Radiological protection - Performance criteria for laboratories using Fluorescence In Situ
Hybridization (FISH) translocation assay for assessment of exposure to ionizing radiation
(ISO 20046:2019)
Strahlenschutz - Leistungskriterien für Laboratorien, die den Fluoreszenz-in-situ-
Hybridisierungs-(FISH)-Translokationstest zur Bewertung der Exposition gegenüber
ionisierender Strahlung verwenden (ISO 20046:2019)
Radioprotection - Critères de performance pour les laboratoires utilisant l'analyse des
translocations visualisées par hybridation in situ fluorescente (FISH) pour évaluer
l'exposition aux rayonnements ionisants (ISO 20046:2019)
Ta slovenski standard je istoveten z: EN ISO 20046:2021
ICS:
13.280 Varstvo pred sevanjem Radiation protection
SIST EN ISO 20046:2021 en,fr,de
2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.
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SIST EN ISO 20046:2021
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SIST EN ISO 20046:2021
EN ISO 20046
EUROPEAN STANDARD
NORME EUROPÉENNE
February 2021
EUROPÄISCHE NORM
ICS 13.280
English Version
Radiological protection - Performance criteria for
laboratories using Fluorescence In Situ Hybridization
(FISH) translocation assay for assessment of exposure to
ionizing radiation (ISO 20046:2019)
Radioprotection - Critères de performance pour les Strahlenschutz - Leistungskriterien für Laboratorien,
laboratoires utilisant l'analyse des translocations die den Fluoreszenz-in-situ-Hybridisierungs-(FISH)-
visualisées par hybridation in situ fluorescente (FISH) Translokationstest zur Bewertung der Exposition
pour évaluer l'exposition aux rayonnements ionisants gegenüber ionisierender Strahlung verwenden (ISO
(ISO 20046:2019) 20046:2019)
This European Standard was approved by CEN on 18 January 2021.
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, France, Germany, Greece, Hungary, Iceland, Ireland, Italy, Latvia, Lithuania, Luxembourg, Malta, Netherlands, Norway,
Poland, Portugal, Republic of North Macedonia, 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: Rue de la Science 23, B-1040 Brussels
© 2021 CEN All rights of exploitation in any form and by any means reserved Ref. No. EN ISO 20046:2021 E
worldwide for CEN national Members.
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SIST EN ISO 20046:2021
EN ISO 20046:2021 (E)
Contents Page
European foreword . 3
2
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SIST EN ISO 20046:2021
EN ISO 20046:2021 (E)
European foreword
The text of ISO 20046:2019 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 20046:2021 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 August 2021, and conflicting national standards shall
be withdrawn at the latest by August 2021.
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, France, Germany, Greece, Hungary, Iceland,
Ireland, Italy, Latvia, Lithuania, Luxembourg, Malta, Netherlands, Norway, Poland, Portugal, Republic of
North Macedonia, Romania, Serbia, Slovakia, Slovenia, Spain, Sweden, Switzerland, Turkey and the
United Kingdom.
Endorsement notice
The text of ISO 20046:2019 has been approved by CEN as EN ISO 20046:2021 without any modification.
3
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SIST EN ISO 20046:2021
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SIST EN ISO 20046:2021
INTERNATIONAL ISO
STANDARD 20046
First edition
2019-03
Radiological protection —
Performance criteria for laboratories
using Fluorescence In Situ
Hybridization (FISH) translocation
assay for assessment of exposure to
ionizing radiation
Radioprotection — Critères de performance pour les laboratoires
utilisant l'analyse des translocations visualisées par hybridation in
situ fluorescente (FISH) pour évaluer l'exposition aux rayonnements
ionisants
Reference number
ISO 20046:2019(E)
©
ISO 2019
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SIST EN ISO 20046:2021
ISO 20046:2019(E)
COPYRIGHT PROTECTED DOCUMENT
© ISO 2019
All rights reserved. Unless otherwise specified, or required in the context of its implementation, no part of this publication may
be reproduced or utilized otherwise in any form or by any means, electronic or mechanical, including photocopying, or posting
on the internet or an intranet, without prior written permission. Permission can be requested from either ISO at the address
below or ISO’s member body in the country of the requester.
ISO copyright office
CP 401 • Ch. de Blandonnet 8
CH-1214 Vernier, Geneva
Phone: +41 22 749 01 11
Fax: +41 22 749 09 47
Email: copyright@iso.org
Website: www.iso.org
Published in Switzerland
ii © ISO 2019 – All rights reserved
---------------------- Page: 8 ----------------------
SIST EN ISO 20046:2021
ISO 20046:2019(E)
Contents Page
Foreword .v
Introduction .vi
1 Scope . 1
2 Normative references . 1
3 Terms and definitions . 1
4 Translocation assay by FISH . 5
4.1 General . 5
4.2 Culturing and fixation . 5
4.3 Types of staining. 5
4.4 Scoring . . 6
4.5 General requirement of the laboratory . 6
5 Responsibility of the customer . 6
6 Responsibility of the laboratory . 7
6.1 Setup and sustainment of the QA program . 7
6.2 Responsibility during service . 7
7 Confidentiality of personal information . 8
7.1 Overview . 8
7.2 Applications of the principle of confidentiality . 8
7.2.1 Delegation of responsibilities within the laboratory. 8
7.2.2 Requests for analysis . 9
7.2.3 Transmission of confidential information . 9
7.2.4 Anonymity of samples . 9
7.2.5 Reporting of results . 9
7.2.6 Storage of data and results . 9
8 Laboratory safety requirements . 9
8.1 Overview . 9
8.2 Microbiological safety requirements .10
8.3 Chemical safety requirements .10
8.4 Optical safety requirements .11
8.5 Safety plan .11
9 Sample processing .11
9.1 Culturing and staining .11
9.2 Scoring . .12
9.2.1 Criteria for scoring .12
9.2.2 Conversion of translocation frequencies to genome equivalence .12
10 Background levels of translocations .13
11 Calibration curves .14
11.1 Calibration source(s) .14
11.2 Establishment of calibration curve(s) .14
12 Criteria for converting a measured aberration frequency into an estimate of
absorbed dose .16
12.1 Determination of estimated whole-body absorbed dose and confidence limits .16
12.1.1 General.16
12.1.2 Comparison with the background level: Characterisation of the minimum
detectable dose.16
12.1.3 Confidence limits on the number of translocations .19
12.1.4 Adjustment for background yield .20
12.1.5 Calculation of absorbed dose .21
12.1.6 Calculation of uncertainty on absorbed dose .22
© ISO 2019 – All rights reserved iii
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SIST EN ISO 20046:2021
ISO 20046:2019(E)
12.1.7 Acute and non-acute exposure cases.22
12.1.8 Other exposure scenarios .23
13 Reporting of results .23
13.1 General .23
13.2 Content of the report (see Annex C for an example of a standard form) .23
13.3 Interpretation of the results .24
14 Quality assurance and quality control .24
14.1 Overview .24
14.2 Specific requirements .24
14.2.1 General.24
14.2.2 Performance checks by inter-laboratory comparisons .24
14.2.3 Performance check of scorer qualification .25
14.2.4 Performance checks of sample transport integrity .25
14.2.5 Performance checks of sample integrity by service laboratory .26
14.2.6 Performance checks of instrumentation .26
14.2.7 Performance checks of sample protocol .26
14.2.8 Performance checks of sample scoring .26
14.2.9 Performance checks of result report generation .26
Annex A (informative) Sample instructions for customer .27
Annex B (informative) Sample questionnaire .29
Annex C (informative) Sample of report .31
Annex D (informative) Sample data sheets for recording painted aberrations .32
Annex E (informative) Fitting of the dose response-curve by the method of maximum
likelihood and calculating the uncertainty of the absorbed dose estimate .34
Annex F (informative) Process for dose estimation.35
Bibliography .40
iv © ISO 2019 – All rights reserved
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SIST EN ISO 20046:2021
ISO 20046:2019(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 voluntary nature of standards, the meaning of ISO specific terms and
expressions related to conformity assessment, as well as information about ISO's adherence to the
World Trade Organization (WTO) principles in the Technical Barriers to Trade (TBT) see the following
URL: www .iso .org/iso/foreword .html.
This document was prepared by Technical Committee ISO/TC 85, Nuclear energy, nuclear technologies
and radiological protection, Subcommittee SC 2, Radiological protection.
Any feedback or questions on this document should be directed to the user’s national standards body. A
complete listing of these bodies can be found at www .iso .org/members .html.
© ISO 2019 – All rights reserved v
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SIST EN ISO 20046:2021
ISO 20046:2019(E)
Introduction
The purpose of this document is to define the use of fluorescent in situ hybridization (FISH) for
chromosome translocation analysis on human peripheral blood lymphocytes for biological dosimetry
of exposure to ionizing radiation. Biological dosimetry, based on the study of chromosomal aberrations,
mainly the dicentric assay, has become a routine component of accidental dose assessment. Dicentric
aberrations, however, disappear with time after exposure, making this assay useful only in the short
term after exposure. Translocations, however, are more stable, allowing dose estimates to be made long
times after exposure or after protracted exposures.
This document provides a guideline for performing the translocation assay by FISH for dose assessment
using documented and validated procedures. The minimum requirements for testing translocation
yield in peripheral blood lymphocytes, by precisely defining the technical aspects of staining
chromosomes (number of chromosomes and types of painting), selecting types of aberrations and
cells, scoring aberrations, converting aberration yield to dose, statistical considerations, problems
related to heterogeneous, chronic or delayed exposures and extrapolation to full genome are described.
Dose assessment using the FISH assay has relevance in medical management, radiation-protection
management, record keeping, and medical/legal requirements.
A part of the information in this document 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 document expands and standardizes the quality assurance and
quality control and the evaluation of performance.
vi © ISO 2019 – All rights reserved
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SIST EN ISO 20046:2021
INTERNATIONAL STANDARD ISO 20046:2019(E)
Radiological protection — Performance criteria for
laboratories using Fluorescence In Situ Hybridization
(FISH) translocation assay for assessment of exposure to
ionizing radiation
1 Scope
The purpose of this document is to provide criteria for quality assurance (QA), quality control (QC) and
evaluation of the performance of biological dosimetry by cytogenetic service laboratories.
This document addresses:
a) the responsibilities of both the customer and the laboratory;
b) the confidentiality of personal information, for the customer and the laboratory;
c) the laboratory safety requirements;
d) sample processing; culturing, staining and scoring, including the criteria for scoring for
translocation analysis by FISH;
e) the calibration sources and calibration dose ranges useful for establishing the reference
dose-response curves that contribute to the dose estimation from chromosome aberration
frequency and the detection limit;
f) the scoring procedure for translocations stained by FISH used for evaluation of exposure;
g) the criteria for converting a measured aberration frequency into an estimate of absorbed dose
(also appears as “dose”);
h) the reporting of results;
i) the QA and QC;
j) Annexes A to F containing sample instructions for the customer, sample questionnaire, sample
datasheet for recording aberrations, sample of report and fitting of the low dose-response curve by
the method of maximum likelihood and calculating the uncertainty of dose estimate.
2 Normative references
There are no normative references in this document.
3 Terms and definitions
For the purposes of this document, the following terms and definitions apply.
ISO and IEC maintain terminological databases for use in standardization at the following addresses:
— ISO Online browsing platform: available at https: //www .iso .org/obp
— IEC Electropedia: available at https: //www .electropedia .org/
© ISO 2019 – All rights reserved 1
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SIST EN ISO 20046:2021
ISO 20046:2019(E)
3.1
absorbed dose
D
quantity of ionizing radiation energy imparted per unit mass of a specified material
3.2
acentric
terminal or interstitial chromosome fragment of varying size lacking a centromere, referred to as an
excess acentric fragment when it is formed independently of a dicentric or centric ring chromosome
aberration
3.3
anticoagulant
drug which prevents blood from clotting
3.4
background frequency/level
spontaneous frequency (or number) of chromosome aberrations recorded in a general population
3.5
buffy coat
layer of an anticoagulated blood sample after centrifugation that contains most of the white blood cells
3.6
calibration curve
graphical or mathematical description of the dose effect relation derived by the in vitro irradiation of
blood samples to known absorbed doses
Note 1 to entry: The curve is used to determine, by interpolation, the absorbed radiation dose to a potentially
exposed individual.
3.7
centromere
specialized constricted region of a chromosome that appears during mitosis and joins together the
chromatid pair
3.8
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
3.9
chromosome
structure comprised of discrete packages of DNA and proteins that carries genetic information, which
condense to form characteristically shaped bodies during nuclear division
3.10
chromosome aberration
change in the normal structure of a chromosome involving both chromatids of a single chromosome at
the same locus as observed in metaphase
3.11
colcemid
alkaloid compound that inhibits spindle formation during cell division
Note 1 to entry: It is used to collect a large number of metaphase cells by preventing them from progressing to
anaphase.
2 © ISO 2019 – All rights reserved
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SIST EN ISO 20046:2021
ISO 20046:2019(E)
3.12
complex aberration
aberration involving three or more breaks in two or more chromosomes and is characteristically
induced after exposure to densely ionizing radiation or high doses of sparsely ionizing radiation
3.13
confidence interval
range within which the true value of a statistical quantity lies with a specified probability
3.14
covariance
measure of the correlation of the variance between two (or more) dependent sets of data or parameters
3.15
decision threshold
value of the estimator of the measurand, which when exceeded by the result of the actual measurement
using a given measurement procedure of a measurand quantifying a physical effect, one decides that
the physical effect is present
Note 1 to entry: The decision threshold is defined such that in cases where the measurement result, y, exceeds
the decision threshold, y*, the probability that the true value of the measurand is zero is less or equal to a chosen
probability, α.
Note 2 to entry: If the result, y, is below the decision threshold, y*, the result cannot be attributed to the physical
effect; nevertheless it cannot be concluded that it is absent.
3.16
detection limit
smallest true value of the measurand which ensures a specified probabil
...
SLOVENSKI STANDARD
oSIST prEN ISO 20046:2020
01-november-2020
Radiološka zaščita - Merila za delovanje laboratorijev, ki uporabljajo preskus
translokacije fluorescenčne hibridizacije in-situ (FISH) za oceno izpostavljenosti
ionizirajočemu sevanju (ISO 20046:2019)
Radiological protection - Performance criteria for laboratories using Fluorescence In Situ
Hybridization (FISH) translocation assay for assessment of exposure to ionizing radiation
(ISO 20046:2019)
Strahlenschutz - Leistungskriterien für Laboratorien, die den Fluoreszenz-in-situ-
Hybridisierungs-(FISH)-Translokationstest zur Bewertung der Exposition gegenüber
ionisierender Strahlung verwenden (ISO 20046:2019)
Radioprotection - Critères de performance pour les laboratoires utilisant l'analyse des
translocations visualisées par hybridation in situ fluorescente (FISH) pour évaluer
l'exposition aux rayonnements ionisants (ISO 20046:2019)
Ta slovenski standard je istoveten z: prEN ISO 20046
ICS:
13.280 Varstvo pred sevanjem Radiation protection
oSIST prEN ISO 20046:2020 en,fr,de
2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.
---------------------- Page: 1 ----------------------
oSIST prEN ISO 20046:2020
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oSIST prEN ISO 20046:2020
INTERNATIONAL ISO
STANDARD 20046
First edition
2019-03
Radiological protection —
Performance criteria for laboratories
using Fluorescence In Situ
Hybridization (FISH) translocation
assay for assessment of exposure to
ionizing radiation
Radioprotection — Critères de performance pour les laboratoires
utilisant l'analyse des translocations visualisées par hybridation in
situ fluorescente (FISH) pour évaluer l'exposition aux rayonnements
ionisants
Reference number
ISO 20046:2019(E)
©
ISO 2019
---------------------- Page: 3 ----------------------
oSIST prEN ISO 20046:2020
ISO 20046:2019(E)
COPYRIGHT PROTECTED DOCUMENT
© ISO 2019
All rights reserved. Unless otherwise specified, or required in the context of its implementation, no part of this publication may
be reproduced or utilized otherwise in any form or by any means, electronic or mechanical, including photocopying, or posting
on the internet or an intranet, without prior written permission. Permission can be requested from either ISO at the address
below or ISO’s member body in the country of the requester.
ISO copyright office
CP 401 • Ch. de Blandonnet 8
CH-1214 Vernier, Geneva
Phone: +41 22 749 01 11
Fax: +41 22 749 09 47
Email: copyright@iso.org
Website: www.iso.org
Published in Switzerland
ii © ISO 2019 – All rights reserved
---------------------- Page: 4 ----------------------
oSIST prEN ISO 20046:2020
ISO 20046:2019(E)
Contents Page
Foreword .v
Introduction .vi
1 Scope . 1
2 Normative references . 1
3 Terms and definitions . 1
4 Translocation assay by FISH . 5
4.1 General . 5
4.2 Culturing and fixation . 5
4.3 Types of staining. 5
4.4 Scoring . . 6
4.5 General requirement of the laboratory . 6
5 Responsibility of the customer . 6
6 Responsibility of the laboratory . 7
6.1 Setup and sustainment of the QA program . 7
6.2 Responsibility during service . 7
7 Confidentiality of personal information . 8
7.1 Overview . 8
7.2 Applications of the principle of confidentiality . 8
7.2.1 Delegation of responsibilities within the laboratory. 8
7.2.2 Requests for analysis . 9
7.2.3 Transmission of confidential information . 9
7.2.4 Anonymity of samples . 9
7.2.5 Reporting of results . 9
7.2.6 Storage of data and results . 9
8 Laboratory safety requirements . 9
8.1 Overview . 9
8.2 Microbiological safety requirements .10
8.3 Chemical safety requirements .10
8.4 Optical safety requirements .11
8.5 Safety plan .11
9 Sample processing .11
9.1 Culturing and staining .11
9.2 Scoring . .12
9.2.1 Criteria for scoring .12
9.2.2 Conversion of translocation frequencies to genome equivalence .12
10 Background levels of translocations .13
11 Calibration curves .14
11.1 Calibration source(s) .14
11.2 Establishment of calibration curve(s) .14
12 Criteria for converting a measured aberration frequency into an estimate of
absorbed dose .16
12.1 Determination of estimated whole-body absorbed dose and confidence limits .16
12.1.1 General.16
12.1.2 Comparison with the background level: Characterisation of the minimum
detectable dose.16
12.1.3 Confidence limits on the number of translocations .19
12.1.4 Adjustment for background yield .20
12.1.5 Calculation of absorbed dose .21
12.1.6 Calculation of uncertainty on absorbed dose .22
© ISO 2019 – All rights reserved iii
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oSIST prEN ISO 20046:2020
ISO 20046:2019(E)
12.1.7 Acute and non-acute exposure cases.22
12.1.8 Other exposure scenarios .23
13 Reporting of results .23
13.1 General .23
13.2 Content of the report (see Annex C for an example of a standard form) .23
13.3 Interpretation of the results .24
14 Quality assurance and quality control .24
14.1 Overview .24
14.2 Specific requirements .24
14.2.1 General.24
14.2.2 Performance checks by inter-laboratory comparisons .24
14.2.3 Performance check of scorer qualification .25
14.2.4 Performance checks of sample transport integrity .25
14.2.5 Performance checks of sample integrity by service laboratory .26
14.2.6 Performance checks of instrumentation .26
14.2.7 Performance checks of sample protocol .26
14.2.8 Performance checks of sample scoring .26
14.2.9 Performance checks of result report generation .26
Annex A (informative) Sample instructions for customer .27
Annex B (informative) Sample questionnaire .29
Annex C (informative) Sample of report .31
Annex D (informative) Sample data sheets for recording painted aberrations .32
Annex E (informative) Fitting of the dose response-curve by the method of maximum
likelihood and calculating the uncertainty of the absorbed dose estimate .34
Annex F (informative) Process for dose estimation.35
Bibliography .40
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ISO 20046:2019(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 voluntary nature of standards, the meaning of ISO specific terms and
expressions related to conformity assessment, as well as information about ISO's adherence to the
World Trade Organization (WTO) principles in the Technical Barriers to Trade (TBT) see the following
URL: www .iso .org/iso/foreword .html.
This document was prepared by Technical Committee ISO/TC 85, Nuclear energy, nuclear technologies
and radiological protection, Subcommittee SC 2, Radiological protection.
Any feedback or questions on this document should be directed to the user’s national standards body. A
complete listing of these bodies can be found at www .iso .org/members .html.
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ISO 20046:2019(E)
Introduction
The purpose of this document is to define the use of fluorescent in situ hybridization (FISH) for
chromosome translocation analysis on human peripheral blood lymphocytes for biological dosimetry
of exposure to ionizing radiation. Biological dosimetry, based on the study of chromosomal aberrations,
mainly the dicentric assay, has become a routine component of accidental dose assessment. Dicentric
aberrations, however, disappear with time after exposure, making this assay useful only in the short
term after exposure. Translocations, however, are more stable, allowing dose estimates to be made long
times after exposure or after protracted exposures.
This document provides a guideline for performing the translocation assay by FISH for dose assessment
using documented and validated procedures. The minimum requirements for testing translocation
yield in peripheral blood lymphocytes, by precisely defining the technical aspects of staining
chromosomes (number of chromosomes and types of painting), selecting types of aberrations and
cells, scoring aberrations, converting aberration yield to dose, statistical considerations, problems
related to heterogeneous, chronic or delayed exposures and extrapolation to full genome are described.
Dose assessment using the FISH assay has relevance in medical management, radiation-protection
management, record keeping, and medical/legal requirements.
A part of the information in this document 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 document expands and standardizes the quality assurance and
quality control and the evaluation of performance.
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oSIST prEN ISO 20046:2020
INTERNATIONAL STANDARD ISO 20046:2019(E)
Radiological protection — Performance criteria for
laboratories using Fluorescence In Situ Hybridization
(FISH) translocation assay for assessment of exposure to
ionizing radiation
1 Scope
The purpose of this document is to provide criteria for quality assurance (QA), quality control (QC) and
evaluation of the performance of biological dosimetry by cytogenetic service laboratories.
This document addresses:
a) the responsibilities of both the customer and the laboratory;
b) the confidentiality of personal information, for the customer and the laboratory;
c) the laboratory safety requirements;
d) sample processing; culturing, staining and scoring, including the criteria for scoring for
translocation analysis by FISH;
e) the calibration sources and calibration dose ranges useful for establishing the reference
dose-response curves that contribute to the dose estimation from chromosome aberration
frequency and the detection limit;
f) the scoring procedure for translocations stained by FISH used for evaluation of exposure;
g) the criteria for converting a measured aberration frequency into an estimate of absorbed dose
(also appears as “dose”);
h) the reporting of results;
i) the QA and QC;
j) Annexes A to F containing sample instructions for the customer, sample questionnaire, sample
datasheet for recording aberrations, sample of report and fitting of the low dose-response curve by
the method of maximum likelihood and calculating the uncertainty of dose estimate.
2 Normative references
There are no normative references in this document.
3 Terms and definitions
For the purposes of this document, the following terms and definitions apply.
ISO and IEC maintain terminological databases for use in standardization at the following addresses:
— ISO Online browsing platform: available at https: //www .iso .org/obp
— IEC Electropedia: available at https: //www .electropedia .org/
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3.1
absorbed dose
D
quantity of ionizing radiation energy imparted per unit mass of a specified material
3.2
acentric
terminal or interstitial chromosome fragment of varying size lacking a centromere, referred to as an
excess acentric fragment when it is formed independently of a dicentric or centric ring chromosome
aberration
3.3
anticoagulant
drug which prevents blood from clotting
3.4
background frequency/level
spontaneous frequency (or number) of chromosome aberrations recorded in a general population
3.5
buffy coat
layer of an anticoagulated blood sample after centrifugation that contains most of the white blood cells
3.6
calibration curve
graphical or mathematical description of the dose effect relation derived by the in vitro irradiation of
blood samples to known absorbed doses
Note 1 to entry: The curve is used to determine, by interpolation, the absorbed radiation dose to a potentially
exposed individual.
3.7
centromere
specialized constricted region of a chromosome that appears during mitosis and joins together the
chromatid pair
3.8
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
3.9
chromosome
structure comprised of discrete packages of DNA and proteins that carries genetic information, which
condense to form characteristically shaped bodies during nuclear division
3.10
chromosome aberration
change in the normal structure of a chromosome involving both chromatids of a single chromosome at
the same locus as observed in metaphase
3.11
colcemid
alkaloid compound that inhibits spindle formation during cell division
Note 1 to entry: It is used to collect a large number of metaphase cells by preventing them from progressing to
anaphase.
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3.12
complex aberration
aberration involving three or more breaks in two or more chromosomes and is characteristically
induced after exposure to densely ionizing radiation or high doses of sparsely ionizing radiation
3.13
confidence interval
range within which the true value of a statistical quantity lies with a specified probability
3.14
covariance
measure of the correlation of the variance between two (or more) dependent sets of data or parameters
3.15
decision threshold
value of the estimator of the measurand, which when exceeded by the result of the actual measurement
using a given measurement procedure of a measurand quantifying a physical effect, one decides that
the physical effect is present
Note 1 to entry: The decision threshold is defined such that in cases where the measurement result, y, exceeds
the decision threshold, y*, the probability that the true value of the measurand is zero is less or equal to a chosen
probability, α.
Note 2 to entry: If the result, y, is below the decision threshold, y*, the result cannot be attributed to the physical
effect; nevertheless it cannot be concluded that it is absent.
3.16
detection limit
smallest true value of the measurand which ensures a specified probability of being detectable by the
measurement procedure
Note 1 to entry: With the decision threshold (3.15), the detection limit is the smallest true value of the measurand
for which the probability of wrongly deciding that the true value of the measurand is zero is equal to a specified
value, β, when, in fact, the true value of the measurand is not zero
3.17
dicentric
aberrant chromosome bearing two centromeres derived from the joining of parts from two broken
chromosomes, generally accompanied by an acentric fragment
3.18
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
3.19
fluorochrome
molecules that are fluorescent when appropriately excited
Note 1 to entry: They are used for FISH cytogenetics to highlight specific chromosomal regions.
3.20
genome equivalent
number of translocations that would be observed with all chromosomes painted, calculated from the
number of translocations detected with a limited number of painted chromosomes
3.21
insertion
chromosome rearrangement in which a piece of one chromosome has been inserted within another
chromosome
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3.22
interphase
period of a cell cycle between the mitotic divisions
3.23
linear energy transfer
LET
radiation energy lost per unit length of path through a biological material
3.24
metaphase
stage of mitosis when the nuclear membrane is dissolved, the chromosomes condensed to their minimum
lengths and aligned for division
3.25
protocol for aberration identification and nomenclature terminology
PAINT
terminology used in FISH analysis for describing chromosomal aberrations
3.26
precision
concept employed to describe dispersion of measurements with respect to a measure of location or
central tendency
3.27
proficiency test
evaluation of participant performance against pre-established criteria by means of inter-laboratory
comparisons
3.28
quality assurance
QA
planned and systematic actions necessary to provide adequate confidence that a process, measurement
or service satisfies given requirements for quality
3.29
quality control
QC
part of quality assurance intended to verify that systems and components conform with predetermined
requirements
3.30
radiation-induced translocation
among the observed translocations, the ones that can be attributed to a radiation exposure i.e. not
translocations induced by other sources (e.g. age, lifestyle factors)
3.31
ring
aberrant circular chromosome resulting from the joining of two breaks within one chromosome
Note 1 to entry: Rings can be centric or acentric.
3.32
service laboratory
laboratory performing biological dosimetry measurements
3.33
stable aberration
aberration which is not lethal to the cell and can be passed on to daughter cells (e.g. simple translocation)
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3.34
stable cell
cell without unstable aberrations, that may be entirely undamaged or contain stable type aberrations
only, and are likely to survive division
3.35
translocation
stable chromosome aberration in which parts of two or more chromosomes are exchanged
3.36
unstable aberration
aberration which is lethal to the cell (e.g. dicentrics/centric rings/acentric fragments)
4 Translocation assay by FISH
4.1 General
The frequency of chromosomal aberrations seen at metaphase in cultured human peripheral blood
lymphocytes is used for absorbed dose estimation after suspected exposure to ionizing radiation. This
document focuses on retrospective evaluation of exposures which occurred in the past or protracted
exposures where the dicentric assay (see ISO 19238) and the cytokinesis block micronucleus assay
(see ISO 17099) are not applicable due to the decrease in this type of damage over time. The aberrations
to be used are translocations and insertions in stable cells. For the application of this document, the
service laboratory shall choose which type of aberrations to score for
...
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