Radiological protection -- Minimum criteria for electron paramagnetic resonance (EPR) spectroscopy for retrospective dosimetry of ionizing radiation

The purpose of this document is to provide minimum criteria required for quality assurance and quality control, evaluation of the performance and to facilitate the comparison of measurements related to absorbed dose estimation obtained in different laboratories applying ex vivo X-band EPR spectroscopy with human tooth enamel. This document covers the determination of absorbed dose in tooth enamel (hydroxyapatite). It does not cover the calculation of dose to organs or to the body. This document addresses: a) responsibilities of the customer and laboratory; b) confidentiality and ethical considerations; c) laboratory safety requirements; d) the measurement apparatus; e) preparation of samples; f) measurement of samples and EPR signal evaluation; g) calibration of EPR dose response; h) dose uncertainty and performance test; i) quality assurance and control.

Radioprotection -- Critères minimaux pour la spectroscopie par résonance paramagnétique électronique (RPE) pour la dosimétrie rétrospective des rayonnements ionisants

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Publication Date
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INTERNATIONAL ISO
STANDARD 13304-2
First edition
2020-07
Radiological protection — Minimum
criteria for electron paramagnetic
resonance (EPR) spectroscopy for
retrospective dosimetry of ionizing
radiation —
Part 2:
Ex vivo human tooth enamel
dosimetry
Radioprotection — Critères minimaux pour la spectroscopie par
résonance paramagnétique électronique (RPE) pour la dosimétrie
rétrospective des rayonnements ionisants —
Partie 2: Dosimétrie ex vivo à partir de l’émail dentaire humain
Reference number
ISO 13304-2:2020(E)
ISO 2020
---------------------- Page: 1 ----------------------
ISO 13304-2:2020(E)
COPYRIGHT PROTECTED DOCUMENT
© ISO 2020

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
Email: copyright@iso.org
Website: www.iso.org
Published in Switzerland
ii © ISO 2020 – All rights reserved
---------------------- Page: 2 ----------------------
ISO 13304-2:2020(E)
Contents Page

Foreword ..........................................................................................................................................................................................................................................v

Introduction ................................................................................................................................................................................................................................vi

1 Scope ................................................................................................................................................................................................................................. 1

2 Normative references ...................................................................................................................................................................................... 1

3 Terms and definitions ..................................................................................................................................................................................... 1

4 Apparatus ..................................................................................................................................................................................................................... 5

4.1 Specifications for EPR spectrometer.................................................................................................................................... 5

4.2 Spectrometer sensitivity ................................................................................................................................................................. 5

4.3 Microwave bridge ................................................................................................................................................................................. 5

4.4 Magnetic field ........................................................................................................................................................................................... 5

4.5 Microwave resonator ......................................................................................................................................................................... 6

5 Preparation of tooth enamel samples ............................................................................................................................................ 6

5.1 General ........................................................................................................................................................................................................... 6

5.2 Applicable grain size .......................................................................................................................................................................... 7

6 Measurement of the EPR spectrum ................................................................................................................................................... 7

6.1 Description of spectrum ................................................................................................................................................................. 7

6.2 Applicable measurement parameters and conditions .......................................................................................... 8

6.2.1 General...................................................................................................................................................................................... 8

6.2.2 Microwave power ............................................................................................................................................................ 8

6.2.3 Magnetic centre field .................................................................................................................................................... 8

6.2.4 Magnetic field sweep width .................................................................................................................................... 8

6.2.5 Magnetic field sweep time ....................................................................................................................................... 8

6.2.6 Time constant of signal channel receiver ................................................................................................... 9

6.2.7 EPR spectrum resolution .......................................................................................................................................... 9

6.2.8 Conversion time of spectrum acquisition .................................................................................................. 9

6.2.9 Magnetic field modulation amplitude ........................................................................................................... 9

6.2.10 Number of spectrum accumulations .............................................................................................................. 9

6.2.11 Sample positioning and loading ......................................................................................................................10

6.2.12 Dependence of EPR signal intensity on sample mass ...................................................................10

6.2.13 Use of standard samples ........................................................................................................................................10

6.2.14 Number of measurement repetitions .........................................................................................................11

7 Assessment of the RIS intensity .........................................................................................................................................................11

7.1 General ........................................................................................................................................................................................................11

7.2 Intrinsic EPR signals from microwave resonator and sample tube .......................................................12

8 Irradiation of tooth enamel calibration samples for low linear energy transfer

(LET) exposure ....................................................................................................................................................................................................12

9 Conversion of the RIS intensity into an estimate of absorbed dose .............................................................13

10 Calculation of uncertainty on dose estimate ........................................................................................................................14

11 Minimum detectable dose .......................................................................................................................................................................15

12 Confidentiality and ethical considerations ............................................................................................................................16

13 Laboratory safety requirements .......................................................................................................................................................16

13.1 General ........................................................................................................................................................................................................16

13.2 Magnetic field safety requirements....................................................................................................................................16

13.3 Electromagnetic frequency requirements....................................................................................................................17

13.4 Chemical safety requirements ................................................................................................................................................17

13.5 Health risks from tooth samples ......... ..................................................................................................................................17

13.6 Optical safety requirements .....................................................................................................................................................17

14 Responsibility of the customer ...........................................................................................................................................................17

© ISO 2020 – All rights reserved iii
---------------------- Page: 3 ----------------------
ISO 13304-2:2020(E)

15 Responsibility of the service laboratory...................................................................................................................................17

16 Quality assurance and quality control (QA and QC) .....................................................................................................17

16.1 General ........................................................................................................................................................................................................17

16.2 Performance checks.........................................................................................................................................................................18

16.2.1 General...................................................................................................................................................................................18

16.2.2 Performance checks by inter-laboratory comparisons ...............................................................18

16.2.3 Performance checks of sample preparation ..........................................................................................18

16.2.4 Performance checks of general measurement laboratory conditions ............................19

16.2.5 Performance checks of EPR spectrometer .............................................................................................19

17 Collection/selection and identification of samples ......................................................................................................19

18 Transportation and storage of samples ....................................................................................................................................20

19 Minimum documentation requirements ..................................................................................................................................20

Bibliography .............................................................................................................................................................................................................................21

iv © ISO 2020 – All rights reserved
---------------------- Page: 4 ----------------------
ISO 13304-2:2020(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 of 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 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.
A list of all parts in the ISO 13304 series can be found on the ISO website.

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 2020 – All rights reserved v
---------------------- Page: 5 ----------------------
ISO 13304-2:2020(E)
Introduction

Electron paramagnetic resonance (EPR) or electron spin resonance (ESR) is an approach for

retrospective dosimetry of exposure to ionizing radiation in any situation where dosimetric

information is potentially incomplete or unknown for an individual. EPR is a tool for retrospective

evaluation of doses, pertinent as well for acute and protracted exposures in the past or recently. Doses

estimated with EPR were used to correlate the biological effect of ionizing radiation to received dose, to

validate other dosimetry techniques or methodologies, to manage casualties, or for forensic expertise

for judicial authorities.

EPR dosimetry is based on the fundamental properties of ionizing radiation: the generation of unpaired

electron species (e.g., radicals) proportional to absorbed dose. The technique of EPR specifically and

sensitively detects the unpaired electrons that have sufficient stability to be observed after their

generation. The amount of the detectable unpaired electrons is proportional to the total amount that

were generated, and hence to the absorbed dose. These species can interact with microwaves generating

the EPR signal, and therefore the relationship between the intensity of the EPR signal and the radiation

dose should be established.

The most extensive use of EPR in retrospective dosimetry has been with calcified tissue, especially

with enamel from teeth. EPR dosimetry is one of the methods of choice for retrospective evaluation

of doses to the involved populations from the atomic weapon exposures in Japan, after the Chernobyl

accident and radioactive releases of the Mayak facilities in the Southern Urals.

This document provides a guideline to perform the ex vivo measurements of human tooth enamel

samples by X-band EPR for dose assessment using documented and validated procedures. The

minimum requirements for reconstructing the absorbed dose in enamel, by defining precisely the

technical aspects of preparing enamel samples, recording EPR spectra, assessment of radiation induced

EPR signal, converting EPR yield to dose and performing proficiency tests, are described. Retrospective

dose assessment using EPR has relevance in radiation effect research, validating radio-epidemiological

dosimetry systems, medical management, 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 “Use of electron paramagnetic resonance dosimetry with tooth enamel for retrospective dose

[1]

assessment” . However, this document expands and standardizes the measurement and dose

reconstruction procedures and the evaluation of performance.
[2]

This document is compliant with ISO 13304-1 with particular consideration given to the specific

needs of X-band EPR dosimetry using human tooth enamel.
vi © ISO 2020 – All rights reserved
---------------------- Page: 6 ----------------------
INTERNATIONAL STANDARD ISO 13304-2:2020(E)
Radiological protection — Minimum criteria for electron
paramagnetic resonance (EPR) spectroscopy for
retrospective dosimetry of ionizing radiation —
Part 2:
Ex vivo human tooth enamel dosimetry
1 Scope

The purpose of this document is to provide minimum criteria required for quality assurance and quality

control, evaluation of the performance and to facilitate the comparison of measurements related to

absorbed dose estimation obtained in different laboratories applying ex vivo X-band EPR spectroscopy

with human tooth enamel.

This document covers the determination of absorbed dose in tooth enamel (hydroxyapatite). It does not

cover the calculation of dose to organs or to the body.
This document addresses:
a) responsibilities of the customer and laboratory;
b) confidentiality and ethical considerations;
c) laboratory safety requirements;
d) the measurement apparatus;
e) preparation of samples;
f) measurement of samples and EPR signal evaluation;
g) calibration of EPR dose response;
h) dose uncertainty and performance test;
i) quality assurance and control.
2 Normative references

The following documents are referred to in the text in such a way that some or all of their content

constitutes requirements of this document. For dated references, only the edition cited applies. For

undated references, the latest edition of the referenced document (including any amendments) applies.

ISO/IEC 17025, General requirements for the competence of testing and calibration laboratories

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 http:// www .electropedia .org/
© ISO 2020 – All rights reserved 1
---------------------- Page: 7 ----------------------
ISO 13304-2:2020(E)

NOTE Definitions of terms used in this document that pertain to radiation measurement and dosimetry are

[3]
compatible with ICRU 60 .
3.1
air kerma

sum of the initial kinetic energies of all the charged particles liberated by uncharged ionizing radiation

per unit mass of air

Note 1 to entry: This quantity is recommended for calibrating the reference photon radiation fields and reference

[4]
instruments .

Note 2 to entry: The unit of the air kerma is given in gray (Gy), which is equal to 1 J/kg.

3.2
absorbed dose

quantity of ionizing radiation energy imparted per unit mass of a specific material

Note 1 to entry: The unit of the absorbed dose is given in gray (Gy), which is equal to 1 J/kg.

3.3
background signal
BGS
signal in the EPR spectrum not generated by ionizing radiation

Note 1 to entry: The background signal (BGS) is not equivalent to the signal component of the radiation induced

signal (RIS) (3.25), which is generated by environmental background radiation.
3.4
bias
deviation of results or interferences from the true value and the estimator
3.5
calibration curve

mathematical description of the dose response relation derived by the in vitro irradiation (3.16) of

tooth enamel samples to known doses
3.6
confidence interval

range within which the true value of a statistical quantity lies, given a value of the probability

3.7
decision threshold

critical value of a measurand quantifying absorbed dose (3.2) in a sample above which exposure can be

identified
3.8
detection limit

smallest true value of a measurand quantifying absorbed dose (3.2) in a sample above which irradiation

can be identified with given probability
3.9
electron paramagnetic resonance
EPR
electron spin resonance
ESR

magnetic resonance technique detecting the net spin (magnetic moment) of unpaired electrons of

paramagnetic centres (3.22) in matter

Note 1 to entry: The terms EPR and ESR are equivalent and are widely used. The term electron magnetic

resonance (EMR) also sometimes is used because it is analogous to nuclear magnetic resonance (NMR).

2 © ISO 2020 – All rights reserved
---------------------- Page: 8 ----------------------
ISO 13304-2:2020(E)
3.10
EPR peak-to-peak line width

difference in the applied magnetic field values between the minimum and the maximum of the first

derivative of a single EPR signal
3.11
EPR signal

first derivative of the electron paramagnetic resonant microwave absorption of a specific paramagnetic

centre (3.22) measured as function of the applied magnetic field

Note 1 to entry: The area under the absorption curve is proportional to the amount of unpaired spins of the

paramagnetic centre. Hence, the amount of spins is proportional to the double integral of the EPR signal (EPR

signal intensity) or the product of EPR signal amplitude and the square of the EPR peak-to-peak line width.

3.12
EPR signal amplitude
peak-to-peak amplitude of the EPR signal (3.11)
3.13
EPR signal intensity

quantity proportional to the amount of paramagnetic centres that generated the EPR signal (3.11)

Note 1 to entry: The signal intensity can be evaluated by numerical double integration of the EPR signal by the

extension of the signal along the magnetic field. The signal intensity of a specific paramagnetic centre can also

be evaluated by comparing with a reference spectrum of the specific centre using least square method. The

reference spectrum may result from measurement of a sample including the specific paramagnetic centre or by

mathematical simulation of the spectrum.
3.14
EPR spectrometer

apparatus to measure the resonant absorption of electromagnetic energy (microwaves) resulting from

the transition of the spin of unpaired electrons between different energy levels, upon application of

microwave-frequencies to a paramagnetic substance in the presence of a magnetic field

3.15
EPR spectrum fitting

linear least squares curve fitting of an EPR spectrum using a set of reference EPR spectra of specific

paramagnetic centres
3.16
in vitro irradiation/measurement

irradiation/measurement carried out on tooth enamel samples outside the human body

Note 1 to entry: The term ex vivo dosimetry refers to samples measured in vitro but were irradiated within the

human body.
3.17
linear energy transfer
LET
dE/dl

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
© ISO 2020 – All rights reserved 3
---------------------- Page: 9 ----------------------
ISO 13304-2:2020(E)
3.18
magnetic field
magnetic flux density (induction)
Note 1 to entry: SI unit Tesla (T) replaced the Gauss (G). 1 T = 10 000 G.
3.19
microwave bridge

apparatus to generate microwaves that are provided to the microwave resonator and to detect

microwaves that were reflected at the resonator
3.20
microwave resonator

resonator for electromagnetic waves consisting of a metal box with appropriate dimensions that

confines the electromagnetic fields in the microwave range and allows formation of standing waves

Note 1 to entry: For EPR measurement the sample is located inside of the microwave resonator. The term

microwave cavity is equivalent to microwave resonator.
3.21
microwave resonator working volume

volume inside the resonator extending along the vertical resonator axis around the centre, within which

the local sensitivity does not decrease more than 25 % relative to the maximal sensitivity at the centre

3.22
paramagnetic centre
species with unpaired electron(s)

Note 1 to entry: Paired electrons have the same quantum state but opposite spin orientation; unpaired electrons

do not have a “partner” with the opposite spin. When the unpaired spin is on a molecule, it is termed a radical;

when the unpaired electron is in a solid, it is termed electron or electron defect (hole) centre.

3.23
quality assurance

planned and systematic actions necessary to provide adequate confidence that a process, measurement,

or service satisfies given requirements for quality
3.24
quality control

planned and systematic actions intended to verify that systems and components conform with

predetermined requirements
3.25
radiation induced signal
RIS

EPR signal (3.11) resulting from paramagnetic centres (3.22) generated by ionizing radiation

3.26
reference spectrum

unit EPR spectrum of a specific paramagnetic centre (3.22) used to evaluate the intensity of the EPR

spectrum of this centre in a sample under investigation

Note 1 to entry: The unit spectrum is reconstructed from EPR measurement of a sample containing the specific

paramagnetic centre or by mathematical simulation.
3.27
retrospective dosimetry
dosimetry to assess dose coming from past exposures
4 © ISO 2020 – All rights reserved
---------------------- Page: 10 ----------------------
ISO 13304-2:2020(E)
3.28
standard sample
sample used to verify the performance stability of the EPR spectrometer

Note 1 to entry: The EPR signal of the standard sample shall be stable to allow reproducible measurements over

extended periods.
3.29
tooth enamel calibration samples

tooth enamel powder samples prepared from whole teeth exposed in vitro to defined absorbed doses

(3.2) or from unexposed teeth with in vitro exposure of the powder to calibrate the RIS dose response

4 Apparatus
4.1 Specifications for EPR spectrometer
The specifications of the apparatus provided by the manufacturer include
a) sensitivity,
b) range of frequency and power of the applicable microwaves,

c) range and stability, scan range and spatial homogeneity of the applicable magnetic field,

d) magnetic field modulation amplitude and frequency, and
e) unloaded quality factor (Q value) of the microwave resonator.
4.2 Spectrometer sensitivity

Commercial X-band EPR spectrometers have typically the sensitivity (indicated by the minimum

14 [5]

detectable spin number/signal half-width) of less than 1 × 10 spins/T . This corresponds to the

amount of CO -radicals generated in 100 mg of tooth enamel by absorbed radiation dose of less than

[6]
1 mGy .
4.3 Microwave bridge

The frequencies of microwaves provided by X-band microwave bridges from different suppliers are in

the range of 9 GHz to10 GHz depending on the types of attached microwave resonators. A microwave

bridge equipped with an auto frequency control (AFC) is recommended. The maximal power provided

by microwave bridges lies typically in the range of 100 mW to 200 mW. For EPR measurement of tooth

[7]

enamel, the microwave bridge should be able to provide microwave power from 0,5 mW to 25 mW .

4.4 Magnetic field

For measurement of tooth enamel, the static magnetic field (centre field) should be set to a value that is

equivalent to a Landé factor of g = 2,00 (350 mT at microwave frequency of 9,8 GHz). Typical values for

[1][7]
the magnetic field scan range from 5 mT to 10 mT .

The resolution of applied magnetic field, its stability over time and homogeneity over sample volume,

determine the maximal degree of the EPR signal distortion (variation of signal line width). With up-to-

date EPR spectrometers, values for field resolution, stability per hour and homogeneity over sample

volume are all better than 5 µT. Hence, an EPR line with width of 0,5 mT, as e.g., the g EPR signal

component of the CO -radical in tooth enamel can be recorded with distortion of less than 1 % for

several measurements within one hour.

EPR spectrometers exist with maximal values of the field modulation frequency of 50 kHz or 100 kHz.

For measurement of tooth enamel, maximal available modulation frequency should be used with typical

[1][7]
values of the field modulation amplitudes in the range of 0,15 mT to 0,5 mT .
© ISO 2020 – All rights reserved 5
---------------------- Page: 11 ----------------------
ISO 13304-2:2020(E)
4.5 Microwave resonator
A microwave resonator is charact
...

FINAL
INTERNATIONAL ISO/FDIS
DRAFT
STANDARD 13304-2
ISO/TC 85/SC 2
Radiological protection — Minimum
Secretariat: AFNOR
criteria for electron paramagnetic
Voting begins on:
2020-04-17 resonance (EPR) spectroscopy for
retrospective dosimetry of ionizing
Voting terminates on:
2020-06-12
radiation —
Part 2:
Ex vivo human tooth enamel
dosimetry
Radioprotection — Critères minimaux pour la spectroscopie par
résonance paramagnétique électronique (RPE) pour la dosimétrie
rétrospective des rayonnements ionisants —
Partie 2: Dosimétrie ex vivo à partir de l?émail dentaire humain
RECIPIENTS OF THIS DRAFT ARE INVITED TO
SUBMIT, WITH THEIR COMMENTS, NOTIFICATION
OF ANY RELEVANT PATENT RIGHTS OF WHICH
THEY ARE AWARE AND TO PROVIDE SUPPOR TING
DOCUMENTATION.
IN ADDITION TO THEIR EVALUATION AS
Reference number
BEING ACCEPTABLE FOR INDUSTRIAL, TECHNO-
ISO/FDIS 13304-2:2020(E)
LOGICAL, COMMERCIAL AND USER PURPOSES,
DRAFT INTERNATIONAL STANDARDS MAY ON
OCCASION HAVE TO BE CONSIDERED IN THE
LIGHT OF THEIR POTENTIAL TO BECOME STAN-
DARDS TO WHICH REFERENCE MAY BE MADE IN
NATIONAL REGULATIONS. ISO 2020
---------------------- Page: 1 ----------------------
ISO/FDIS 13304-2:2020(E)
COPYRIGHT PROTECTED DOCUMENT
© ISO 2020

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 2020 – All rights reserved
---------------------- Page: 2 ----------------------
ISO/FDIS 13304-2:2020(E)
Contents Page

Foreword ..........................................................................................................................................................................................................................................v

Introduction ................................................................................................................................................................................................................................vi

1 Scope ................................................................................................................................................................................................................................. 1

2 Normative references ...................................................................................................................................................................................... 1

3 Terms and definitions ..................................................................................................................................................................................... 1

4 Apparatus ..................................................................................................................................................................................................................... 5

4.1 Specifications for EPR spectrometer.................................................................................................................................... 5

4.2 Spectrometer sensitivity ................................................................................................................................................................. 5

4.3 Microwave bridge ................................................................................................................................................................................. 5

4.4 Magnetic field ........................................................................................................................................................................................... 5

4.5 Microwave resonator ......................................................................................................................................................................... 6

5 Preparation of tooth enamel samples ............................................................................................................................................ 6

5.1 General ........................................................................................................................................................................................................... 6

5.2 Applicable grain size .......................................................................................................................................................................... 7

6 Measurement of the EPR spectrum ................................................................................................................................................... 7

6.1 Description of spectrum ................................................................................................................................................................. 7

6.2 Applicable measurement parameters and conditions .......................................................................................... 8

6.2.1 General...................................................................................................................................................................................... 8

6.2.2 Microwave power ............................................................................................................................................................ 8

6.2.3 Magnetic center field .................................................................................................................................................... 8

6.2.4 Magnetic field sweep width .................................................................................................................................... 8

6.2.5 Magnetic field sweep time ....................................................................................................................................... 8

6.2.6 Time constant of signal channel receiver ................................................................................................... 9

6.2.7 EPR spectrum resolution .......................................................................................................................................... 9

6.2.8 Conversion time of spectrum acquisition .................................................................................................. 9

6.2.9 Magnetic field modulation amplitude ........................................................................................................... 9

6.2.10 Number of spectrum accumulations .............................................................................................................. 9

6.2.11 Sample positioning and loading ......................................................................................................................10

6.2.12 Dependence of EPR signal intensity on sample mass ...................................................................10

6.2.13 Use of standard samples ........................................................................................................................................10

6.2.14 Number of measurement repetitions .........................................................................................................11

7 Assessment of the RIS intensity .........................................................................................................................................................11

7.1 General ........................................................................................................................................................................................................11

7.2 Intrinsic EPR signals from microwave resonator and sample tube .......................................................12

8 Irridiation of tooth enamel calibration samples for low linear energy transfer (LET)

exposure .....................................................................................................................................................................................................................12

9 Conversion of the RIS intensity into an estimate of absorbed dose .............................................................13

10 Calculation of uncertainty on dose estimate ........................................................................................................................14

11 Minimum detectable dose .......................................................................................................................................................................15

12 Confidentiality and ethical considerations ............................................................................................................................16

13 Laboratory safety requirements .......................................................................................................................................................16

13.1 General ........................................................................................................................................................................................................16

13.2 Magnetic field safety requirements....................................................................................................................................16

13.3 Electromagnetic frequency requirements....................................................................................................................17

13.4 Chemical safety requirements ................................................................................................................................................17

13.5 Health risks from tooth samples ......... ..................................................................................................................................17

13.6 Optical safety requirements .....................................................................................................................................................17

14 Responsibility of the customer ...........................................................................................................................................................17

© ISO 2020 – All rights reserved iii
---------------------- Page: 3 ----------------------
ISO/FDIS 13304-2:2020(E)

15 Responsibility of the service laboratory...................................................................................................................................17

16 Quality assurance and quality control (QA and QC) .....................................................................................................17

16.1 General ........................................................................................................................................................................................................17

16.2 Performance checks.........................................................................................................................................................................18

16.2.1 General...................................................................................................................................................................................18

16.2.2 Performance checks by inter-laboratory comparisons ...............................................................18

16.2.3 Performance checks of sample preparation ..........................................................................................18

16.2.4 Performance checks of general measurement laboratory conditions ............................19

16.2.5 Performance checks of EPR spectrometer .............................................................................................19

17 Collection/selection and identification of samples ......................................................................................................19

18 Transportation and storage of samples ....................................................................................................................................20

19 Minimum documentation requirements ..................................................................................................................................20

Bibliography .............................................................................................................................................................................................................................21

iv © ISO 2020 – All rights reserved
---------------------- Page: 4 ----------------------
ISO/FDIS 13304-2:2020(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 of 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 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.
A list of all parts in the ISO 13304 series can be found on the ISO website.

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/FDIS 13304-2:2020(E)
Introduction

Electron paramagnetic resonance (EPR) or electron spin resonance (ESR) is an approach for

retrospective dosimetry of exposure to ionizing radiation in any situation where dosimetric

information is potentially incomplete or unknown for an individual. EPR is a tool for retrospective

evaluation of doses, pertinent as well for acute and protracted exposures in the past or recently. Doses

estimated with EPR were used to correlate the biological effect of ionizing radiation to received dose, to

validate other dosimetry techniques or methodologies, to manage casualties, or for forensic expertise

for judicial authorities.

EPR dosimetry is based on the fundamental properties of ionizing radiation: the generation of unpaired

electron species (e.g., radicals) proportional to absorbed dose. The technique of EPR specifically and

sensitively detects the unpaired electrons that have sufficient stability to be observed after their

generation. The amount of the detectable unpaired electrons is proportional to the total amount that

were generated, and hence to the absorbed dose. These species can interact with microwaves generating

the EPR signal, and therefore the relationship between the intensity of the EPR signal and the radiation

dose should be established.

The most extensive use of EPR in retrospective dosimetry has been with calcified tissue, especially

with enamel from teeth. EPR dosimetry is one of the methods of choice for retrospective evaluation

of doses to the involved populations from the atomic weapon exposures in Japan, after the Chernobyl

accident and radioactive releases of the Mayak facilities in the Southern Urals.

This document provides a guideline to perform the ex vivo measurements of human tooth enamel

samples by X-band EPR for dose assessment using documented and validated procedures. The

minimum requirements for reconstructing the absorbed dose in enamel, by defining precisely the

technical aspects of preparing enamel samples, recording EPR spectra, assessment of radiation induced

EPR signal, converting EPR yield to dose and performing proficiency tests are described. Retrospective

dose assessment using EPR has relevance in radiation effect research, validating radio-epidemiological

dosimetry systems, medical management, 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 “Use of electron paramagnetic resonance dosimetry with tooth enamel for retrospective dose

[1]

assessment” . However, this document expands and standardizes the measurement and dose

reconstruction procedures and the evaluation of performance.
[2]

This document is compliant with ISO 13304-1 with particular consideration given to the specific

needs of X-band EPR dosimetry using human tooth enamel.
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FINAL DRAFT INTERNATIONAL STANDARD ISO/FDIS 13304-2:2020(E)
Radiological protection — Minimum criteria for electron
paramagnetic resonance (EPR) spectroscopy for
retrospective dosimetry of ionizing radiation —
Part 2:
Ex vivo human tooth enamel dosimetry
1 Scope

The purpose of this document is to provide minimum criteria required for quality assurance and quality

control, evaluation of the performance and to facilitate the comparison of measurements related to

absorbed dose estimation obtained in different laboratories applying ex vivo X-band EPR spectroscopy

with human tooth enamel.

This document covers the determination of absorbed dose in tooth enamel (hydroxyapatite). It does not

cover the calculation of dose to organs or to the body.
This document addresses:
a) responsibilities of the customer and laboratory;
b) confidentiality and ethical considerations;
c) laboratory safety requirements;
d) the measurement apparatus;
e) preparation of samples;
f) measurement of samples and EPR signal evaluation;
g) calibration of EPR dose response;
h) dose uncertainty and performance test;
i) quality assurance and control.
2 Normative references

The following documents are referred to in the text in such a way that some or all of their content

constitutes requirements of this document. For dated references, only the edition cited applies. For

undated references, the latest edition of the referenced document (including any amendments) applies.

ISO/IEC 17025, General requirements for the competence of testing and calibration laboratories

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 http:// www .electropedia .org/
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ISO/FDIS 13304-2:2020(E)

NOTE Definitions of terms used in this document that pertain to radiation measurement and dosimetry are

[3]
compatible with ICRU 60 .
3.1
air kerma

sum of the initial kinetic energies of all the charged particles liberated by uncharged ionizing radiation

per unit mass of air

Note 1 to entry: This quantity is recommended for calibrating the reference photon radiation fields and reference

[4]
instruments .

Note 2 to entry: The unit of the air kerma is given in gray (Gy), which is equal to 1 J/kg.

3.2
absorbed dose

quantity of ionizing radiation energy imparted per unit mass of a specific material

Note 1 to entry: The unit of the absorbed dose is given in gray (Gy), which is equal to 1 J/kg.

3.3
background signal
BGS
signal in the EPR spectrum not generated by ionizing radiation

Note 1 to entry: The background signal (BGS) is not equivalent to the signal component of the radiation induced

signal (RIS) (3.25) which is generated by environmental background radiation.
3.4
bias
deviation of results or interferences from the true value and the estimator
3.5
calibration curve

mathematical description of the dose response relation derived by the in vitro irradiation (3.16) of

tooth enamel samples to known doses
3.6
confidence interval

range within which the true value of a statistical quantity lies, given a value of the probability

3.7
decision threshold

critical value of a measurand quantifying absorbed dose (3.2) in a sample above which exposure can be

identified
3.8
detection limit

smallest true value of a measurand quantifying absorbed dose (3.2) in a sample above which irradiation

can be identified with given probability
3.9
electron paramagnetic resonance
EPR
electron spin resonance
ESR

magnetic resonance technique detecting the net spin (magnetic moment) of unpaired electrons of

paramagnetic centres (3.22) in matter

Note 1 to entry: The terms EPR and ESR are equivalent and are widely used. The term electron magnetic

resonance (EMR) also sometimes is used because it is analogous to nuclear magnetic resonance (NMR).

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ISO/FDIS 13304-2:2020(E)
3.10
EPR peak-to-peak line width

difference in the applied magnetic field values between the minimum and the maximum of the first

derivative of a single EPR signal
3.11
EPR signal

first derivative of the electron paramagnetic resonant microwave absorption of a specific paramagnetic

centre (3.22) measured as function of the applied magnetic field

Note 1 to entry: The area under the absorption curve is proportional to the amount of unpaired spins of the

paramagnetic centre. Hence, the amount of spins is proportional to the double integral of the EPR signal (EPR

signal intensity) or the product of EPR signal amplitude and the square of the EPR peak-to-peak line width.

3.12
EPR signal amplitude
peak-to-peak amplitude of the EPR signal (3.11)
3.13
EPR signal intensity

quantity proportional to the amount of paramagnetic centres which generated the EPR signal (3.11)

Note 1 to entry: The signal intensity can be evaluated by numerical double integration of the EPR signal by the

extension of the signal along the magnetic field. The signal intensity of a specific paramagnetic centre can also

be evaluated by comparing with a reference spectrum of the specific centre using least square method. The

reference spectrum may result from measurement of a sample including the specific paramagnetic centre or by

mathematical simulation of the spectrum.
3.14
EPR spectrometer

apparatus to measure the resonant absorption of electromagnetic energy (microwaves) resulting from

the transition of the spin of unpaired electrons between different energy levels, upon application of

microwave-frequencies to a paramagnetic substance in the presence of a magnetic field

3.15
EPR spectrum fitting

linear least squares curve fitting of an EPR spectrum using a set of reference EPR spectra of specific

paramagnetic centres
3.16
in vitro irradiation/measurement

irradiation/measurement carried out on tooth enamel samples outside the human body

Note 1 to entry: The term ex vivo dosimetry refers to samples measured in vitro but were irradiated within the

human body.
3.17
linear energy transfer
LET
dE/dl

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
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ISO/FDIS 13304-2:2020(E)
3.18
magnetic field
magnetic flux density (induction)
Note 1 to entry: SI unit Tesla (T) replaced the Gauss (G). 1 T = 10 000 G.
3.19
microwave bridge

apparatus to generate microwaves that are provided to the microwave resonator and to detect

microwaves that were reflected at the resonator
3.20
microwave resonator

resonator for electromagnetic waves consisting of a metal box with appropriate dimensions that

confines the electromagnetic fields in the microwave range and allow formation of standing waves

Note 1 to entry: At EPR measurement the sample is located inside of the microwave resonator. The term

microwave cavity is equivalent to microwave resonator.
3.21
microwave resonator working volume

volume inside the resonator extending along the vertical resonator axis around the center, within which

the local sensitivity does not decrease more than 25 % relative to the maximal sensitivity at the center

3.22
paramagnetic centre
species with unpaired electron(s)

Note 1 to entry: Paired electrons have the same quantum state except for opposite spin orientation; unpaired

electrons do not have a “partner” with the opposite spin. When the unpaired spin is on a molecule, it is termed a

radical; when the unpaired electron is in a solid, it is termed electron or electron defect (hole) centre.

3.23
quality assurance

planned and systematic actions necessary to provide adequate confidence that a process, measurement,

or service satisfies given requirements for quality
3.24
quality control

planned and systematic actions intended to verify that systems and components conform with

predetermined requirements
3.25
radiation induced signal
RIS

EPR signal (3.11) resulting from paramagnetic centres (3.22) generated by ionizing radiation

3.26
reference spectrum

unit EPR spectrum of a specific paramagnetic centre (3.22) used to evaluate the intensity of the EPR

spectrum of this centre in a sample under investigation

Note 1 to entry: The unit spectrum is reconstructed from EPR measurement of a sample containing the specific

paramagnetic centre or by mathematical simulation.
3.27
retrospective dosimetry
dosimetry to assess dose coming from past exposures
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ISO/FDIS 13304-2:2020(E)
3.28
standard sample
sample used to verify the performance stability of the EPR spectrometer

Note 1 to entry: The EPR signal of the standard sample shall be stable to allow reproducible measurements over

extended periods.
3.29
tooth enamel calibration samples

tooth enamel powder samples prepared from whole teeth exposed in vitro to defined absorbed doses

(3.2) or from unexposed teeth with in vitro exposure of the powder to calibrate the RIS dose response

4 Apparatus
4.1 Specifications for EPR spectrometer
The specifications of the apparatus provided by the manufacturer include
a) sensitivity,
b) range of frequency and power of the applicable microwaves,

c) range and stability, scan range and spatial homogeneity of the applicable magnetic field,

d) magnetic field modulation amplitude and frequency, and
e) unloaded quality factor (Q value) of the microwave resonator.
4.2 Spectrometer sensitivity

Commercial X-band EPR spectrometers have typically the sensitivity (indicated by the minimum

14 [5]

detectable spin number/signal half-width) of less than 1 × 10 spins/T . This corresponds to the

amount of CO -radicals generated in 100 mg of tooth enamel by absorbed radiation dose of less than

[6]
1 mGy .
4.3 Microwave bridge

The frequencies of microwaves provided by X-band microwave bridges from different suppliers are in

the range of 9 GHz to10 GHz depending on the types of attached microwave resonators. A microwave

bridge equipped with an auto frequency control (AFC) is recommended. The maximal power provided

by microwave bridges lies typically in the range of 100 mW to 200 mW. For EPR measurement of tooth

[7]

enamel, the microwave bridge should be able of providing microwave power from 0,5 mW to 25 mW .

4.4 Magnetic field

At measurement of tooth enamel, the static magnetic field (center field) should be set to a value that is

equivalent to a Landé factor of g = 2,00 (350 mT at microwave frequency of 9,8 GHz). Typical values for

[1][7]
the magnetic field scan range from 5 mT to 10 mT .

The resolution of applied magnetic field, its stability over time and homogeneity over sample volume

determine the maximal degree of the EPR signal distortion (variation of signal line width). With up-

to-date EPR spectrometers, values for field resolution, stability per hour and homogeneity over sample

volume are all better
...

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