# Activities of ISO Working Group 18: Biological and Physical Retrospective Dosimetry

Ruth Wilkins, Elizabeth A Ainsbury, G.A. Alsbeih, Adyabalam Balajee, William Blakely, Mercedes Moreno Domene, David Endersfelder, Michael Fenech, Eric Gregoire, J.F. Herrold, et al.

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## To cite this version:

Ruth Wilkins, Elizabeth A Ainsbury, G.A. Alsbeih, Adyabalam Balajee, William Blakely, et al. Activities of ISO Working Group 18: Biological and Physical Retrospective Dosimetry. Disaster Medicine and Public Health Preparedness, 2025, 19, pp.e251. 10.1017/dmp.2025.10095. irsn-04920662v2

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Original Research

**Cite this article:** Wilkins R, Ainsbury EA, Alsbeih GA, Balajee A, Blakely WF, Deminge M, Endesfelder D, Fenech M, Gregoire E, Herrold J, Marrale M, Martinez JS, Miura T, Moreno Domene M, Oestreicher U, Pecoskie S, Prieto MJ, Regulla D, Romanyukha A, Sommer S, Suto Y, Swartz HM, Terzoudi G, Toyoda S and Trompier F (2025). Activities of ISO Working Group 18: Biological and Physical Retrospective Dosimetry. *Disaster Medicine and Public Health Preparedness*, **19**, e251, 1–11
https://doi.org/10.1017/dmp.2025.10095

Received: 11 December 2024

Revised: 11 December 2024

Accepted: 05 February 2025

**Keywords:**
biodosimetry; EPR; radiation; Standardization;
Cytogenetics

**Corresponding author:**
Ruth Wilkins;
Email: ruth.wilkins@hc-sc.gc.ca

Activities of ISO Working Group 18: Biological
and Physical Retrospective Dosimetry

Ruth Wilkins<fnref n="1" /> ID, Elizabeth A. Ainsbury², Ghazi A. Alsbeih³, Adayabalam Balajee⁴,
William F. Blakely⁵, Mayra Deminge⁶, David Endesfelder⁷, Michael Fenech⁸,
Eric Gregoire⁹, Jim Herrold¹⁰, Maurizio Marrale¹¹, Juan S. Martinez⁹,
Tomisato Miura¹², Mercedes Moreno Domene¹³, Ursula Oestreicher⁷,
Stephen Pecoskie¹⁴, Maria Jesus Prieto¹³, Deiter Regulla¹⁵,
Alexander Romanyukha⁵, Sylwester Sommer¹⁶, Yumiko Suto¹⁷, Harold M. Swartz¹⁸,
Georgia Terzoudi¹⁹, Shin Toyoda²⁰ and François Trompier⁹

Abstract

Biological and physical retrospective dosimetry for ionizing radiation exposure is a rapidly
growing field, and several methods for performing biological and physical retrospective dosim-
etry have been developed to provide absorbed dose estimates for individuals after occupational,
accidental, intentional, and incidental exposures to ionizing radiation. In large-scale radio-
logical/nuclear incidents, multiple retrospective dosimetry laboratories from several countries
may be involved in providing timely dose estimates for effective medical management of several
thousand exposed individuals. In such scenarios, the harmonization of methods among par-
ticipating laboratories is crucial for consistency in data analysis, dose estimation, and medical
decision-making. In this regard, ISO documents ensure that these practices are standardized
globally across the laboratories by providing quality assurance and quality control documenta-
tion that guide laboratories in maintaining high-quality performance for consistency. With the
intent of bringing standardization and harmonization of biological and physical retrospective
dosimetry methodologies across national and international laboratories, the ISO working group
18 (WG18) was established under ISO/TC85/SC2 (Technical Committee 85, Subcommittee
2-Radiation Protection) in 1999. This manuscript summarizes some of the past, current, and
future activities of WG18 on biological and physical retrospective dosimetry.

Introduction

Importance of Standards in Life Sciences and Industries

The International Organization for Standardization (ISO) facilitates the harmonization of operating procedures, such as laboratory protocols, to ensure that results are comparable and reproducible across different laboratories when the same methods are used. This harmonization of procedures is crucial for maintaining uniformity, reliability, consistency, and quality in industrial, medical, and educational operations. By providing guidelines and procedures, ISO standards not only help the quality control (QC) and quality assurance (QA) assessments but also improve the productivity of the organizations and industries by identifying and minimizing

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CAMBRIDGE
UNIVERSITY PRESS

<footnote n="1">¹Consumer and Clinical Radiation Protection Bureau, Health Canada, Ottawa, Canada; ²UK Health Security Agency, Radiation, Chemicals and Environmental Hazards Directorate, Chilton, Oxfordshire, United Kingdom; ³Biomedical Physics Department, Radiation Biology Section, King Faisal Specialist Hospital & Research Centre, Riyadh, Saudi Arabia; ⁴Cytogenetic Biodosimetry Laboratory, Radiation Emergency Assistance Center/Training Site, Oak Ridge Institute for Science and Education, Oak Ridge Associated Universities, Oak Ridge, TN, USA; ⁵Armed Forces Radiobiology Research Institute, Uniformed Services University of the Health Sciences, Bethesda, MD, USA; ⁶Biological Dosimetry Laboratory, Nuclear Regulatory Authority (ARN), Buenos Aires, Argentina; ⁷Federal Office for Radiation Protection, BfS, Oberschleissheim, Germany; ⁸Health and Biomedical Innovation, UniSA Clinical and Health Sciences, University of South Australia, Adelaide, Australia; ⁹Institut de Radioprotection et de Sûreté Nucléaire (IRSN), PSE-SANTE/SERAMED/LRAcc, F-92260, Fontenay-aux-Roses, France; ¹⁰Safety Office, University of Wyoming, Laramie, WY, USA; ¹¹Department of Physics and Chemistry “Emilio Segrè,” University of Palermo, Palermo, Italy; ¹²Department of Risk Analysis and Biodosimetry, Institute of Radiation Emergency Medicine, Hirosaki University, Hirosaki, Japan; ¹³Hospital General Universitario Gregorio Marañón; Servicio de Oncología Radioterápica; Laboratorio de dosimetria biológica, Madrid, Spain; ¹⁴Canadian Nuclear Laboratories, Chalk River, Canada; ¹⁵Institute for Clinical Radiology, University of Munich, Munich, Germany; ¹⁶Institute of Nuclear Chemistry and Technology, Warsaw, Poland; ¹⁷National Institutes for Quantum Science and Technology, Chiba, Japan; ¹⁸Department of Radiology, Geisel School of Medicine at Dartmouth, Hanover, NH, USA; ¹⁹National Centre for Scientific Research “Demokritos,” Health Physics, Radiobiology & Cytogenetics Laboratory, Agia Paraskevi, Greece and ²⁰Institute of Palaeontology and Geochronology, Okayama University of Science, 1-1 Ridai, Kita-ku, Okayama, Japan</footnote>
inefficiencies through implementation of corrective action plans. ISO documents are available for the management of industries, environment, health and safety, and food safety, among others, and these documents are developed and established by the scientific wisdom of subject matter experts working as a group. There are almost 300 technical committees that are currently involved in developing and establishing ISO standards for different disciplines.

The standards established by ISO have been in routine use in the industrial world for several decades and have found their way into the biological sciences, where standards are crucial for reliability of procedures, quality assurance, data comparison, and data validation. Radiation biological and physical retrospective dosimetry is an actively emerging field of science where several tools have been and continue to be developed for estimating the absorbed radiation dose in potentially exposed humans. ISO standards are needed for each of the biological and physical retrospective dosimetry assays to establish consensus methodologies for dose estimation either by individual or national/inter-national dosimetry network laboratories, specifically for use in the aftermath of mass-casualty radiological or nuclear incidents/accidents. In such a scenario, ISO standards for harmonizing the protocols among the laboratories are imperative to get consistent and reliable results for fair data comparison and validation of dose estimates.¹ Additionally, these ISO standards may be useful for accreditation of newly developed retrospective dosimetry laboratories. Realizing the importance of ISO standards for these laboratories, a technical committee (ISO/TC85/SC2) working group (WG18) was established in 1999 to develop standards for some of the established biological dosimetry tools. This was later expanded to include retrospective physical dosimetry through the establishment of a subworking group focused on electron paramagnetic resonance (EPR). This manuscript summarizes some of the past, current, and future activities of the WG18 on biological and physical retrospective dosimetry standards.

## Background on biological and physical retrospective dosimetry

Biological and physical retrospective dosimetry is the measurement of absorbed radiation dose based on the response of biological indicators such as DNA, RNA, proteins, lipids as well as a variety of metabolic byproducts or physical measurements in biological material such as EPR in teeth or bones. Retrospective dosimetry can be used to estimate the absorbed radiation dose when individuals without personal dosimeters are exposed to unknown doses of ionizing radiation. It can also be used to confirm the radiation dose on a personal dosimeter in the case of radiation/nuclear workers and to gain information, in some instances, about the nature and level of exposures. Biological dosimetry was developed in the mid-20th century as it became evident that chromosomal aberrations increased in a dose-responsive manner after exposure to ionizing radiation and could be used as an indicator of dose to the exposed individual. The use of biological dosimetry after the “Recuplex” criticality accident in Hanford, USA,² and after a radiation accident in 1969 involving industrial radiographers in the UK³ is an early example of its application. Throughout the second half of the 20th century, biological dosimetry was used for diverse nuclear accidents and incidents, which allowed for not only the dose assessment but also the improvements of the existing techniques.⁴⁻⁹

In the case of large-scale radiological/nuclear incidents/accidents, several hundreds and thousands of people could be exposed to substantial doses of ionizing radiation. The LD₅₀/₆₀ (the lethal dose that will kill 50% of the exposed individuals in 60 days) value

ranges from 3.5 Gy to 4 Gy without supportive care and more than 7 Gy with supportive care. Therefore, timely assessment of absorbed radiation dose will be useful for saving human lives.¹⁰ Figure 1 summarizes the methods that can be used, alone or in combination, to perform radiation dose estimates in these scenarios.

## Overview of ISO and WG18

### Life Cycle of Developing ISO Standards

The ISO standards in most cases contain tested and validated procedures or protocols for the best practices in any business, industrial, and educational/research organizations. There are 2 basic steps for the development of standards: (1) consensus among the subject matter experts about the added value an ISO standard would bring in a specific area and (2) acceptance of a standard at the national level.

Once there is consensus among experts in the field that a new standard would be of value, a proposal is then submitted to the relevant ISO technical committee composed of representatives from various countries and stakeholder groups. Bringing an ISO standard to life is a well-defined process, with each stage acting as a crucial checkpoint toward publication. While the timeframe might vary (18, 24, or 36 months), the core stages remain essential for every standard, ensuring thoroughness, consensus, and global relevance. The life cycle of an ISO standard is depicted in Figure 2.

The rigorous review process is the foundation of ISO standards' credibility and trust. By incorporating diverse perspectives and expertise, the standards reflect the global needs of the field they serve. Subsequent sections of this manuscript will delve into the specific applications of ISO standards in retrospective dosimetry, which is a specialty under the working group ISO/TC 85/SC 2/WG18: biological and physical retrospective dosimetry.

### History of Identifying the Need for Standardized Biological and Physical Retrospective Dosimetry Techniques

In the 1990s, there were several accidental exposures to ionizing radiation that had no accompanying physical dosimetry and, at that time, only a few biological dosimetry laboratories were available for conducting the analysis. Although biological dosimetry was available, there were issues with the acceptance of the results, particularly if they were not as expected. One solution to address the lack of confidence in the results was to establish an international inter-comparison program. The first of these was conducted in 1995 with 2 laboratories, the Nuclear Safety and Protection Institute (IPSN, now Institut de Radioprotection et de Sûreté Nucléaire, IRSN, France) and what was then known as the National Radiological Protection Board (NRPB, now UK Health Security Agency, UKHSA). A second intercomparison was held in 2002 with 15 laboratories and included exposure to both neutrons and gamma rays.¹¹ Even though the estimations from 11 of the 15 participants fell within the ±30% of the physical reference dose, the results indicated some discrepancies in the rate of dicentrics enumerated among participants. As fixed cells were transported to each laboratory, 1 explanation was that the quality of metaphases was affected by the transportation of the samples. Additionally, slide-making procedures may have varied between laboratories. The differences in dicentric scoring among participants could be also due to the type of radiation used and inexperience at scoring highly damaged cells. With high-LET radiations, some metaphases may have contained numerous chromosomal aberrations such as multicentric
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<figure><img src="image_5.png" /><figcaption>**Figure 1.** Representation of different physical, biophysical, biological, and clinical dosimetry methods for radiation dose assessment.</figcaption></figure>

<figure><img src="image_6.png" /><figcaption>**Figure 2.** Depiction of the stages of an ISO document from proposal through to publication.</figcaption></figure>

chromosomes and centric rings which can make the scoring difficult. Another issue with the dose estimates could have been the dose-response curve used. Few of the participant laboratories had a neutron dose response curve, while others used their gamma or X-ray dose-response curves. However, smaller variations were observed in the dose estimations as compared to the frequency of aberrations, highlighting that experimental conditions such as slide making and scoring can be laboratory dependent.

A second solution to address the lack of confidence was to establish a process for laboratory accreditation by an independent international body.¹² At that time, there were only 2 applicable standards for laboratories:

* 9001:1987—Quality systems—model for quality assurance in design/development, production, installation and servicing (now 9001:2015 Quality management systems—requirements)¹³

* ISO/IEC Guide 25:1990 General requirements for the competence of calibration and testing laboratories (now 17025:2017)¹⁴

It was clear that there was a need to create standards specific to biological dosimetry techniques. In 1999 the ISO working group 18 “Biological Dosimetry” was formed under Technical Committee 85 (Nuclear energy, nuclear technologies, and radiation protection)/Subcommittee 2 (Radiological Protection). The WG consisted of 13 specialists from 11 countries who met for the first time at IRSN and started drafting the first standard on the dicentric assay.

Since its conception, the WG has expanded to include physical retrospective dosimetry and in 2021 changed its name to “Biological and physical retrospective dosimetry” reflecting the addition of standards on EPR. The committee has now developed a suite of standards including guidance on conducting the dicentric assay, the
<table><caption>**Figure 3.** Overview of activities of ISO WG18.</caption><caption>Overview of ISO WG18 Activities: 2002-2024</caption><thead><tr><th colspan="2">Cytogenetic Biodosimetry</th><th colspan="2">EPR Dosimetry</th></tr></thead><tbody><tr><td>Dicentric assay<br>(reference with<br>manual scoring)<br>ISO 19238</td><td>CBMN assay<br>ISO 17099</td><td colspan="2">Minimum criteria for electron<br>paramagnetic resonance (EPR)<br>spectroscopy for retrospective dosimetry<br>ISO 13304-1</td></tr><tr><td>Dicentric assay<br>(triage)<br>ISO 21243</td><td>FISH translocation<br>Assay<br>ISO 20046</td><td colspan="2">Ex vivo human<br>tooth enamel<br>dosimetry<br>ISO 13304-2</td></tr><tr><td colspan="2">PCC assay<br>ISO 22699 (pending)</td><td colspan="2">Ex vivo human<br>clipped nails<br>dosimetry<br>(pending)</td></tr></tbody></table>

cytokinesis-block micronucleus assay, translocation analysis using FISH and EPR (Figure 3). A list of these standards can be found in Table 1.

A typical standard for a specific assay will contain detailed technical information on various procedural steps, starting from sample collection, followed by assay procedure, data generation,

data analysis, dose estimation, and report generation. A detailed description of statistical analysis for dose estimation will also be included in the standard. It will also contain details on quality assurance and quality control procedures specific to the assay. Often examples of useful documents are included in the Annexes (e.g., instructions for requestors, questionnaires, reporting templates).

<table><caption>**Table 1.** List of standards published by WG18</caption><thead><tr><th>ISO #</th><th>Standard title</th><th>Date first published</th><th>Date of latest revision</th></tr></thead><tbody><tr><td>19238</td><td>Radiation protection - Performance criteria for service laboratories performing biological dosimetry by cytogenetics</td><td>2004</td><td>2023</td></tr><tr><td>21243</td><td>Radiation Protection- Performance criteria for laboratories performing initial cytogenetic dose assessment of mass casualties in radiological or nuclear emergencies — General principles and application to dicentric assay</td><td>2008</td><td>2022</td></tr><tr><td>17099</td><td>Radiological protection - Performance criteria for laboratories using the cytokinesis block micronucleus (CBMN) assay in peripheral blood lymphocytes for biological dosimetry</td><td>2014</td><td>2024</td></tr><tr><td>20046</td><td>Radiological protection — Performance criteria for laboratories using Fluorescence In Situ Hybridization (FISH) translocation assay for assessment of exposure to ionizing radiation</td><td>2019</td><td>2019</td></tr><tr><td>13304-1</td><td>Radiological protection — Minimum criteria for electron paramagnetic resonance (EPR) spectroscopy for retrospective dosimetry of ionizing radiation — Part 1: General principles</td><td>2013</td><td>2020</td></tr><tr><td>13304-2</td><td>Radiological protection — Minimum criteria for electron paramagnetic resonance (EPR) spectroscopy for retrospective dosimetry of ionizing radiation — Part 2: ex vivo human tooth enamel dosimetry</td><td>2020</td><td>2020</td></tr></tbody></table>

## ISO Standards for Biological and Physical Retrospective Dosimetry on Biological Samples

Ionizing radiation (IR) induces a wide spectrum of lesions on chromosomal DNA. DNA double-strand breaks are critical lesions which, when mis-rejoined, can result in microscopically detectable structural chromosome alterations. For this reason, various cytogenetic methods for detecting the structural chromosome aberrations have been developed and routinely used for radiation cytogenetic biological dosimetry. Methods for retrospective analysis using physical dosimetry have also been developed. Table 2 lists the biological and physical retrospective dosimetry assays that have ISO standards or are under consideration for standardization. A brief account of these assays is provided below, with the associated standards listed in Table 1:

### Dicentric chromosome assay (DCA)<sup>15,16</sup>

The DCA is a cytogenetic assay that is considered the gold standard of biological dosimetry and is the most used method.<sup>1,4</sup> This assay is performed either on whole peripheral blood cells or on isolated lymphocyte cultures and involves the scoring of dicentric chromosomes that are formed after the mis-rejoining of 2 broken chromosomes with intact centromeres and can be easily visualized in metaphase cells (Figure 4A). Identification of dicentric chromosomes can also be optimized by the addition of peptide nucleic acid (PNA) probe specific for centromeres of all the human chromosomes. Dicentric-bearing lymphocytes are unstable when passing through mitosis; therefore, they disappear with time.<sup>17,18</sup> Their persistence in the human body is estimated to be between 6 and 12 months after exposure<sup>19</sup> and therefore DCA is most suitable for acute recent exposures. Since *in vitro* culturing of lymphocytes for 48 h is required for chromosome preparation, the turnaround time for DCA-based dose estimation is usually 72-96 h. The DCA has been used in many instances of dose reconstruction for both small and large numbers of exposed individuals.<sup>4-9</sup>

In the case of a radiological emergency, triage methods shorten the analysis time and provide timely information to the medical
<table><caption>**Table 2.** Comparison of assays used for dose assessment (modified from the IAEA cytogenetic manual.†)</caption><thead><tr><th rowspan="2">Assays</th><th rowspan="2">Endpoints</th><th colspan="3">Radiation scenarios</th><th rowspan="2">Acute photon dose range, Gy</th><th colspan="2">Dose applications</th><th rowspan="2">ISO standard</th></tr><tr><th>Acute</th><th>Protracted</th><th>Prior†</th><th>Partial-body</th><th>Triage</th></tr></thead><tbody><tr><td>Dicentric chromosome assay (DCA)</td><td>Dicentrics (and rings)</td><td>Yes</td><td>Yes</td><td>Not ideal</td><td>0.1–6</td><td>Yes</td><td>Yes</td><td>Yes</td></tr><tr><td>Cytokinesis-blocked micronucleus (CBMN)</td><td>Micronuclei in binucleated cells, nucleoplasmic bridges</td><td>Yes</td><td>Yes</td><td>Not ideal</td><td>0.3–5</td><td>No</td><td>Yes</td><td>Yes</td></tr><tr><td>Fluorescence in situ hybridization (FISH)</td><td>Dicentrics (and rings), translocations</td><td>Yes</td><td>Yes</td><td>Yes</td><td>0.25–5</td><td>No</td><td>No</td><td>Yes</td></tr><tr><td>Premature chromosome condensation (PCC)‡</td><td>Excess fragments, rings, dicentrics, and length ratio</td><td>Yes</td><td>Yes</td><td>Not ideal</td><td>~0.2–30 (depending on endpoint)</td><td>Yes</td><td>Yes</td><td>Pending</td></tr><tr><td>Electron paramagnetic resonance (EPR)</td><td>EPR signal from nail clippings, intact nails (in vivo). Tooth (in vivo), or tooth enamel biopsies, tooth enamel extracted teeth</td><td>Yes</td><td>Yes</td><td>Yes</td><td>0.05 (tooth enamel, extracted teeth)<br/>>0.4 (enamel biopsies)<br/>>2 (nail clippings, nails in vivo, teeth in vivo)</td><td>Yes</td><td>Yes</td><td>Yes (for ex vivo tooth)</td></tr></tbody></table>

<figure><img src="image_7.png" /><figcaption>**Figure 4.** Representative images illustrating damage detection by some of the commonly used biological dosimetry assays. (A) Metaphase spread from the dicentric chromosome assay showing a dicentric (blue arrow) and 2 acentric fragments (red arrows). (B) A symmetrical translocation (white arrow) detected by FISH using a cocktail of fluorescently labeled chromosome-specific DNA probes. (C) A binucleated cell with 1 micronuclei detected by the cytokinesis-block micronucleus assay. (D) A G2-phase cell after chemically induced PCC assay showing a dicentric (blue arrow), 2 rings (red arrows), and 3 acentric fragments (yellow arrows). (E) PCC chromosome (red arrow) along with metaphase CHO chromosomes (blue arrow) from the PCC cell fusion assay and (F) 2 EPR spectra of dental enamel (irradiated and non-irradiated) and included g-values.</figcaption></figure>

<footnote>†Prior: An assessment of dose when blood sampling is performed greater than 3 months after radiation exposure.</footnote>

<footnote>‡With or without the use of centromeric and/or whole-chromosome specific hybridization probes.</footnote>
team to assist in the care of potentially exposed victims. In cytogenetics-based biological dosimetry, triage analysis includes manually scoring either 50 metaphases or 30 dicentrics (whichever is reached first), allowing dose estimates within ±1 Gy of the absorbed dose.⁴ This margin of uncertainty is sufficient to guide the medical teams in the initial treatment. Another triage option is to conduct semiautomatic analysis of 500 to 1000 metaphases, which could potentially shorten the analysis time.⁵

### Translocation analysis²⁰

Similar to the DCA, translocation analysis is performed on mitotic lymphocytes; however, the translocations, which involve the exchange of parts of the chromosomes between 2 or more chromosomes, are detected by fluorescence *in situ* hybridization (FISH) using probes for a specific set of chromosomes or the whole complement (Figure 4B). Symmetrical translocations, in which each resultant chromosome has 1 centromere, are considered stable aberrations that can pass through mitosis (unlike dicentric chromosomes) and are more easily transmitted to progeny cells. This allows them to persist in the body for many years (much longer than dicentric chromosomes) and hence are useful for retrospective biological dosimetry several years after radiation exposure. These translocations, however, increase with age and other lifestyle factors and should be corrected for when calculating dose estimates.²¹ Some notable examples of cases where FISH-based biological dosimetry was employed include the follow-up of victims from the Hiroshima-Nagasaki bombings²²; children living close to the Chernobyl nuclear accident site²³,²⁴; victims of the Goiânia accident²⁵,²⁶; victims of the Tallin incident²⁷,²⁸; and the workers from the Mayak nuclear weapon manufacturing site in the Techa river region (Russia), which was contaminated mainly by radioactive strontium in the 1950s.²⁹

### Cytokinesis-block micronucleus (CBMN) assay³⁰

In the lymphocyte cytokinesis-block micronucleus assay, micronuclei resulting from mal-segregation of whole chromosomes or chromosome fragments that are excluded from the main nuclei are scored *ex vivo* in cytokinesis-blocked binucleate cells (Figure 4C).⁴ Unlike dicentric chromosomes which are specific for radiation exposure, micronuclei can be induced by a variety of genotoxic agents including ionizing radiation. The CBMN assay has been applied to several accidental scenarios for retrospective dose reconstruction.⁷,³¹⁻³³

### Premature chromosome condensation (PCC) assay

This assay involves the use of protein kinase inhibitors (calyculin A and okadaic acid) to prematurely condense the chromosomes in the G2 phase of the cell cycle prior to reaching mitosis. This allows the visualization of chromosomes in heavily damaged cells that are permanently arrested in the G2-phase and are unable to progress to mitosis (Figure 4D).³⁴ Alternatively, G0-PCC can be induced where the irradiated cells (usually G0-human lymphocytes) are fused with mitotic cells of Chinese Hamster Ovary using polyethylene glycol (PEG) as fusogen (Figure 4E).³⁵ G0-PCC is advantageous because it can be performed directly after blood collection without the need for lymphocyte culturing as in the case of G2-PCC. One of the great advantages of the PCC assay is its capability to detect higher doses of exposure, thus being complementary to the DCA. In addition, its shorter culture time allows for a rapid dose estimation (few hours). It was notably used for victims of the Tokai-Mura criticality accident, where 2 individuals were exposed to doses higher than 6 Gy³⁶,³⁷ and in the Taiyuan radionuclide manufacturing accident

of 2008 where it was concluded that 5 employees exposed to gamma irradiation received doses ranging from 2 to 12 Gy.³⁸ This method is being proposed for the next standard to be drafted in WG18.

### Electron paramagnetic resonance (EPR) dosimetry³⁹,⁴⁰

Electron paramagnetic resonance (EPR) dosimetry can be defined as a method of radiation dose determination based on the measurements of concentration of radiation-induced radicals in matter with EPR spectroscopy. EPR dosimetry is used to measure radiation doses in solid tissues of humans or animals (*teeth* *in vitro* and *in vivo*, *bone* *in vitro*, *finger- and toenails* *in vitro* and *in vivo*) but also personal items such as cell phone screens, watch glasses, clothes, etc. (not addressed in the framework of WG18). This method has the advantage of being non-destructive to the material being measured, so that repeated measurements on the same sample can be made. The other attractive features of the EPR dosimetry include linear dose dependence for most materials and tissues, the possibility to do dose measurements immediately after irradiation, based on a physical process that is not affected by biological processes such as stress or simultaneous insults/damage that are likely to occur with irradiation such as wounds and burns. The timeframe for measurements can be made at any interval immediately after irradiation up to at least 2 weeks (fingernails), indefinitely (*teeth*),⁴¹ or a range of times for other EPR dosimetry materials. Furthermore, the EPR signal is unaffected by dose rate and can be used for dose measurements after partial body exposures.

ISO 13304-1:2020 provides a general guidance on sample collection, their preparation for the dose measurements, EPR measurements in the samples under investigation, calibration of the EPR radiation response in the radiation dose units, and some other useful information related to the application of this technique. This standard covers *in vitro* and *in vivo* EPR dose measurements in biological tissues (mostly teeth and nails) of the person, personal items, and objects located in the immediate environment to the human. It usually employs X-band frequency (8-12 GHz), but higher and lower frequencies are also being considered. Specifically, this International Standard proposes a methodological frame and recommendations to set up, validate, and apply protocols from sample collection to dose reporting. However, this standard does not provide any specificity to the type of the sample (e.g., teeth, nails, and other applicable materials) used for the dose measurements. ICRU reports 68 and 94 give an analysis of the most commonly available methods of retrospective dosimetry, including EPR.⁴²,⁴³ These 2 reports provide extensive lists of the references on the methodology and applications of EPR dosimetry.

Standard ISO 13304-2:2020 provides specific guidelines to perform the *ex vivo* measurements of human tooth enamel samples by X-band EPR for dose assessment (Figure 4F). This technique is the most developed EPR dosimetry method, which has been applied to reconstruct radiation doses received by Hiroshima and Nagasaki A-bombardment survivors, population and emergency personnel affected by the Chernobyl accident, Southern Ural population in Russia residing in the areas contaminated by the Russian nuclear weapon plant Mayak, and in many other radio-epidemiological studies.⁴²,⁴⁴ ISO 13304-2:2020 expands and standardizes the measurement and dose reconstruction procedures and the evaluation of performance. This document is compliant with ISO 13304-1 "Radiological protection—minimum criteria for electron paramagnetic resonance (EPR) spectroscopy for retrospective dosimetry of ionizing radiation—Part 1: General principles" with particular
consideration given to the specific needs of X-band EPR dosimetry using human tooth enamel.

In the context of radiological accidents, this method has been mostly used on biopsies of bone tissues, providing very pertinent information when irradiation is localized to a small volume of the body and to a less extent to biopsies of tooth enamel.⁴³ *Ex vivo* tooth enamel dosimetry (on whole tooth) has been largely used in support of epidemiological studies of ancient and/or prolonged exposure.⁴²

## Factors involved in radiation dose assessment

There are many aspects of radiation dose assessment by biological or physical retrospective dosimetry that are addressed within the standards:

(1) Radiation exposure parameters can vary from one exposure scenario to the next: level of dose, radiation quality, dose rate including single or fractionated doses, and whether a person is entirely or partially exposed. Each of these factors will affect the dose estimates made and these are addressed in the standards. The amount of radiation one is exposed to can determine the number of cells required to be analyzed for the cytogenetic assays. Different qualities of radiation affect the shape of the dose response curves and advice is provided on the range and number of doses required when creating a dose response curve for high- and low-LET radiation. The dose rate also affects the shape of the dose response curve and advice is provided on how to apply an acute dose-response curve to non-acute exposures. Finally, it is of great importance to understand whether an individual has been exposed to a whole- or partial-body exposure, as the medical intervention will differ in these 2 cases. The standards describe methods to determine whether an exposure is partial body.

(2) There are continuous improvements to the assays that must reach a certain level of maturity and uptake before being incorporated into the standards. These are assessed each time a standard comes up for review. One example is the development of software tools for performing data and statistical analysis for cytogenetic assays. Although specific software is not mentioned, they are mentioned as an acceptable tool. Automation is another improvement that is becoming widely used but has not yet been described in detail in a standard.

(3) The type of dose assessment methodology to be employed may depend on the exposure scenario. For early response situations, the DCA, CBMN, or EPR standards might be followed, whereas for a retrospective analysis, at longer times postexposure, translocation analysis, or EPR would be applicable (Table 2). Depending on the number of casualties requiring analysis, one might consider employing triage scoring that has been described in ISO 21243,⁴⁵ which outlines scoring methodology for rapid throughput and activation of networks for sample sharing to establish surge capacity when a laboratory's capacity has been exceeded. This standard could be applied to any of the other standards.

(4) ISO 21243 also includes guidance on preparedness of a laboratory network prior to an event including organization, harmonization of protocols, quality assurance and control, training and intercomparison exercises to demonstrate capacities, and capabilities of network laboratories to maintain performance criteria and continuous exchange of scientific and technical information.

## Use and impact of standards/achievements

### Networks

In the case of large-scale accidents, the number of victims can easily exceed the capacity of single laboratories or organizations. Networking between experienced laboratories has been identified as a useful and important strategy in emergency management to overcome this bottleneck by sharing the workload and providing mutual assistance between laboratories. Besides increasing capacity, networking allows the broadening of the spectrum of methods and the choice of the most suitable approach for a particular scenario. In recent years, several laboratories around the world have joined forces and set up regional and/or global networks either on a formal or informal basis.⁴⁶ Global networks have been established by the World Health Organization (WHO) (BioDoseNet),⁴⁷,⁴⁸ the Radiation Emergency Medical Preparedness Assistance Network (REMPAN), and the International Atomic Energy Agency (IAEA) Response and Assistance Network (RANET).

On the regional level, the key networks are the European Network for Biological and Retrospective Physical Dosimetry (RENEB), the European Radiation Dosimetry Group (EURADOS), the Latin American Biological Dosimetry Network (LBDNet), the North American Network, and the Asian Radiation Dosimetry Group (ARADOS). The RENEB network of biological and physical retrospective dosimetry laboratories has been operating in Europe since 2017. The network currently includes 16 voting members and 43 associate members from Europe and Asia.⁴⁹ The North American network is an informal network established first in Canada in 2002 and has now expanded to include several US laboratories.⁵⁰,⁵¹ LBDNet was founded in 2007 and originally comprised 7 countries.⁵² ARADOS⁵³ was established in October 2015 by researchers from China, South Korea, and Japan. It consists of a host institute (China, Korea, or Japan, alternately) and 4 working groups (WGs) on Internal Dosimetry, External Dosimetry, Biological Dosimetry, and Computational Dosimetry. In addition, India has also established a national biological dosimetry network involving 7 laboratories.⁵⁴

An essential requirement to achieve comparable results between the laboratory in a network is the harmonization of protocols and standardization of procedures. ISO 21243⁴⁵ provides excellent guidance on the aspects of establishing a network, while the ISO standards on individual techniques provide invaluable guidance for harmonizing procedures across laboratories.⁵⁵

### Inter-laboratory comparisons

The organization of exercises and interlaboratory comparisons (ILC) are important tools to guarantee high-quality results and to improve and optimize a network's performance. Coordination of infrastructure, logistical aspects, data management, and communication are essential for managing large-scale accidents in biological and physical retrospective dosimetry to increase the throughput and to validate the workflow in a sustainable and operational network. Training and education programs and activities help to improve the ability of network members to provide reliable dose estimates. Furthermore, differences in the experimental setup or the radiation source used by the organizing institutions can help to identify potential issues and to improve and optimize the performance of the network.⁴⁹ International ILCs have been mainly run through RENEB,⁵⁶⁻⁵⁹ EURADOS,⁵⁵,⁶⁰ LBDNet,⁵² ARADOS,⁵³ or Canada.⁶¹ Typically, dose reconstruction is performed using several methods: manual and automatic dicentric chromosome analysis,
manual and automatic micronucleus assay, translocation analysis, histone γH2AX foci assay, PCC fusion, gene expression analysis, and EPR. These ILCs are highly dependent on the use of protocols based on ISO standards developed in the frame of WG18, IAEA technical publications,⁴ and scientific publications. The focus of the RENEB exercises was variable and included tele-scoring exercises, triage, and full mode scoring for biological dosimetry methods, irradiation with different sources, homogeneous and heterogeneous irradiations, a field exercise, and comparisons between different assays.⁵⁵ There have also been several ILCs where performance of the EPR dosimetry in tooth enamel at different laboratories was compared. The most comprehensive one was carried out in 2010.⁶² Based on the results obtained by each participant, critical dose and detection limits were calculated by the organizers. The most recent comprehensive RENEB ILC was conducted in 2022⁶³ in which the performance quality was compared between the established cytogenetic assays, molecular biological assays (γH2AX foci, gene expression), and physical dosimetry-based assays (EPR, optically or thermally stimulated luminescence). The results of all ILCs have contributed to harmonization of SOPs and statistical methods for uncertainty assessment within the RENEB network and EURADOS that have also fed back into revision of ISO standards for biological dosimetry.

## Future Perspectives

### New Standards Under Consideration

Premature chromosome condensation (PCC) is under consideration for the next biological dosimetry standard. It has been adopted widely by the biological dosimetry community and has advantages over other cytogenetic techniques as described above. A standard dedicated to automation of different aspects of the biological dosimetry methods is also under consideration. This could potentially include both sample processing and scoring of aberrations.

### Emerging Assays for Future Consideration

There are several assays that are emerging as biological dosimeters but have not yet reached the maturity for standardization, for example, the γH2AX foci assay. The histone H2AX, a variant of histone H2A, which is a part of the histone octamer on which DNA is wound and is one of the first proteins that responds to DNA damage after radiation exposure through phosphorylation at serine 139 to the γH2AX form.⁶⁴ Fluorescent conjugated antibodies are commercially available for easy visualization of the γH2AX foci that form at the sites of DNA double-strand breaks in a radiation dose-dependent manner. Although the histone γH2AX assay is considered a novel biological dosimetric method, with sensitivity in the low dose range (in the order of tens of mSv) and applicability up to at least 10 Gy,⁶⁵ many technical and methodological problems must still be solved, most notably the fast appearance (optimal peak time is 30 minutes to an hour after exposure) and disappearance of histone foci over time with only about 20% of the initial number of foci remain after 24 h.⁶⁶ This means that dose estimation is limited to a short time after the event necessitating the immediate shipment of samples, preferably on ice, to the biological dosimetry laboratories for analysis. Nevertheless, γH2AX assay can still be effectively used as a biological dosimeter if appropriate *in vitro* calibration curves are constructed for different post-exposure times to account for the disappearance of γH2AX foci.

Another emerging technique is measuring changes in gene expression that can be measured in blood and lymphocytes post-exposure to ionizing radiation, showing promise for biological dosimetry and prediction of clinical outcomes.⁶⁷⁻⁶⁹ Typically, a panel of genes encoding proteins related to the DNA damage response (e.g., DDB2, CDKN1A, GADD45A), apoptosis (e.g., FDXR, BAX, BBC3), and the development of the inflammatory reaction (e.g., GDF15, TNFSF4) is examined, showing significant and repeatable expression changes in peripheral blood cells.⁷⁰⁻⁷² The research and literature data confirm that the level of transcripts of various genes in the blood shows a clear dependence on the absorbed radiation dose at specified time points after exposure in the range of at least 0.2 Gy to 4 Gy. The method of analyzing gene expression changes is a fast and low-cost method that can be used in the case of a large-scale radiation exposure scenario.

### Automation of the DCA and CBMN Assay

One of the main disadvantages of DCA is that manual scoring of dicentric chromosomes is laborious and time-consuming, limiting the sample throughput of a single laboratory that would quickly become overwhelmed in the event of a mass casualty incident. Automation of DCA has been developed over the last 20 years⁵,⁷³,⁷⁴ and has been successfully tested in the framework of the MULTI-BIODOSE EU FP7 project.⁷⁵,⁷⁶ To date, several automatic DCA analysis systems have been developed and are in development.⁷⁷⁻⁸¹ The use of artificial intelligence (AI) also offers new opportunities to increase capacity in biological dosimetry and to support current and future research in biological dosimetry.⁷³,⁸² Automation and semiautomation of the DCA and the CBMN assay have been briefly mentioned in both ISO 19238 and ISO 17099¹⁵,³⁰; however, due to the rapid development of automation algorithms and use of AI-based tools for automation, these aspects need to be carefully considered by ISO WG18 before incorporating them in the future standards.

Analysis of MN in CBMN is not as technically demanding as the DCA which makes it more amenable to automation, drastically reducing the scoring time. Automatic and semiautomatic MN analysis has been used in many laboratories around the world in recent years.⁸³⁻⁸⁶ Several of these automated CBMN methods are becoming mature enough to be considered for ISO standardization. As this method is also widely used outside of the biological dosimetry community, a standard on automation would be of use in the analysis of cytotoxic and genotoxic effects of environmental mutagens, carcinogens, and clastogens.

Regardless of the automation tool or application, these systems must be validated against the standardized techniques prior to the incorporation into an ISO standard.

### Electron Paramagnetic Resonance (EPR) Dosimetry
Standardization for Large-Scale Event

*In vivo* EPR of teeth and finger/toenails offers the advantage of performing onsite measurements of individuals after a small or large-scale radiation event. While its feasibility for triage for such events has been demonstrated, more development/refinement is needed for its acceptance as a standardized technique.⁸⁷⁻⁸⁹ In addition, *ex vivo* Q-band EPR on tooth enamel mini-biopsies has already been used for an actual radiological accident.⁹⁰ As the measurement takes less than 5 minutes after the biopsy is obtained and there are minimal requirements for sample preparation, this method has the potential to be implemented on a large scale, though
no organized efforts are in place at this time.⁹¹ Furthermore, AI
could help these retrospective EPR dosimetric techniques by auto-
mating data analysis, improving calibration accuracy, and facilitat-
ing standardization. Indeed, AI algorithms can be trained to
automatically identify and quantify EPR signals as well as AI-driven
calibration models can improve the accuracy and precision of EPR
measurements by accounting for various factors such as sample
variability, significantly reducing analysis time.⁹² Coordinated
efforts could allow for more rapid standardization of technique
also in case of adoption of AI tools.

Limitations of Standards

Although standardization of biological and physical dosimetry
methods is an essential step in harmonizing laboratories and ensur-
ing robust dose estimates, standards can also have some limitations
and downfalls. As the working group is comprised of international
experts from different laboratories, there can be difficulties in
reaching international consensus on the best procedures when
drafting the standards. These differences are usually resolved
through thoughtful discussions and compromise, often keeping
detailed instructions for crucial points and allowing flexibility
where possible so as not to be excessively restrictive.

The ISO standards should be the set of best practices, the foun-
dation for technical and organizational usage of the described
method but should also be easily applied. While retrospective dos-
imetry ISO standards for biological material are often used as a
reference because of their scientific credibility, only a few laboratories
around the world are accredited against these standards. Therefore, it
is also important to ensure that the requirements of the procedures
are not so prescriptive that they become too difficult to meet, making
accreditation against the standard a challenge. Furthermore, main-
tenance of experienced personnel and a well-equipped laboratory, as
required to comply with the standard, can be resource-intensive.

Conclusions

The ISO WG18: “Biological and Physical Retrospective Dosimetry”
has been an active working group since 1999. This WG, comprises
members from many countries, has developed and revised 6 stand-
ards related to biological and physical retrospective dosimetry,
5 focused on methodology, and 1 on operational processes for
large-scale events. These standards have been of great value to the
establishment of networks for harmonizing methods across the
network laboratories. Future developments in this area will focus
on the revision of current standards and on the development of new
and emerging techniques as they become sufficiently developed and
validated for standardization.

**Acknowledgments/Disclaimer.** Mention of any brand name products does not imply endorsement. The opinions, conclusions, and recommendations herein expressed are those of the authors and do not necessarily reflect the official policy or position of the Uniformed Services University or the United States Department of Defense. This work was prepared by a civilian employee of the US Government as part of the individual's official duties and therefore is in the public domain and does not possess copyright protection.

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