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)

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

Status
Published
Public Enquiry End Date
30-Nov-2020
Publication Date
21-Feb-2021
Technical Committee
Current Stage
6060 - National Implementation/Publication (Adopted Project)
Start Date
18-Feb-2021
Due Date
25-Apr-2021
Completion Date
22-Feb-2021

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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

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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.

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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
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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
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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

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
...

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