
==== Front
J Genet Eng Biotechnol
J Genet Eng Biotechnol
Journal of Genetic Engineering & Biotechnology
1687-157X
2090-5920
Academy of Scientific Research and Technology, Egypt

S1687-157X(24)00112-4
10.1016/j.jgeb.2024.100409
100409
Review Article
Potentials of cytokinesis blocked micronucleus assay in radiation triage and biological dosimetry
Tamizh Selvan G. drtamizhselvang@nitte.edu.in
a⁎
Venkatachalam P. b
a Central Research Laboratory, K.S. Hegde Medical Academy, NITTE (Deemed to be University), Deralakatte, Mangalore, Karnataka, India
b Department of Human Genetics, Sri Ramachandra Institute of Higher Education and Research (Deemed to be University), Chennai, India
⁎ Corresponding author. drtamizhselvang@nitte.edu.in
16 8 2024
12 2024
16 8 2024
22 4 10040930 11 2023
4 2 2024
6 8 2024
© 2024 The Authors
2024
https://creativecommons.org/licenses/by-nc-nd/4.0/ This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
The measurement of micronucleus (MN) in the cytokinesis-block arrested binucleated cells has been extensively used as a biomarker in many radiation biology applications in specific biodosimetry. Following radiation casualties, medical management of exposed individuals begins with triage and biological dosimetry. The cytokinesis blocked micronucleus (CBMN) assay is the alternate for the gold standard dicentric chromosome assay in radiation dose assessment. In recent years, the CBMN assay has become well-validated and emerged as a method of choice for evaluating occupational and accidental exposures scenario. It is feasible due to its cost-effective, simple, and rapid dose assessment rather than a conventional chromosome aberration assay. PubMed search tool was used with keywords of MN, biodosimetry, radiotherapy and restricted to human samples. Since Fenech and Morely developed the assay, it has undergone many technical and technological reforms as a biomarker of various applications. In this review, we have abridged recent developments of the CBMN assay in radiation triage and biodosimetry, focusing on (a) the influence of variables on dose estimation, (b) the importance of baseline frequency and reported dose–response coefficient values among different laboratories, (c) inter-laboratory comparison and (d) its limitations and means to overcome them.

Keywords

Micronucleus
Biomarker
Biodosimetry
Triage
Radiation
Abbreviations

BN Binucleated

CBMN Cytokinesis Blocked Micronucleus

CT Computed Tomography

Cyto-B Cytochalasin-B

DC Dicentric

DSBs Double-Strand Breaks

FBS Fetal Bovine Serum

FISH Fluorescence in-situ Hybridization

Gy Gray

IAEA International Atomic Energy Agency

ILC Inter-Laboratory Comparison

ISO International Organization for Standardization

MN Micronucleus

mSv milli Sievert

NBUDs Nuclear Buds

NPBs Nucleoplasmic Bridges

PBLs Peripheral Blood Lymphocytes

PCC Premature Chromosome Condensation

PHA Phytohemagglutinin

RPMI Roswell Park Memorial Institute
==== Body
pmc1 Background

Upon cellular exposure to ionizing radiation, traversal of electron track followed by energy deposition induces transient or permanent changes in the molecules within the cell. The changes in the exposed test system (cells/ molecules) are being used to monitor the exposure-related consequences, including dose estimation and management of the exposed. The earliest and most straightforward dose determination method following radiation exposure involves registering daily counts of different cell types circulating in the peripheral blood; the extent and duration of the decline and subsequent recovery have been shown to correlate well with dose.1 Consequently, other indicators like changes in enzymes, chromosomes, proteins, metabolic intermediates and gene expression and their regulators such as microRNA (miRNA) have been developed. Amongst the spectrum of changes, cytogenetic indicators are widely used for estimating the dose in accidental and suspected overexposed individuals.2 The radiation absorbed by the exposed cells can induce strand breaks on the chromosomes. While repair, the mis-repaired breaks can result in abnormal chromosome structures. Various types of abnormal chromosomes can be identified and related to the dose. Methods that are used to estimate the chromosomal changes are dicentric chromosomes (DC) assay,3 premature chromosome condensation (PCC) assay4 and cytokinesis blocked micronucleus (CBMN) assay.5 Of those three assay methods, though PCC assay can be performed even in the un-cultured cells (time-saving) rather than the DC assay, both assays require technical expertise and analysis requires longer time. Thus, CBMN assay (Fig. 1) is a widespread option for measuring radiation absorbed dose during radiation triage.Fig. 1 Schematic representation of CBMN assay usage in the current scenario.

The micronucleus (MN) are extra-nuclear bodies that originate from acentric chromosomes or whole chromosomes; the fragments that lag at the anaphase of dividing cells and are not included in the nucleus appear as a small extra nucleus in cells that have completed (Table 1) one cell division.6 Though considerably smaller in size, they are enveloped by the nuclear membrane and resemble the structure of the daughter nuclei. Acentric fragments are arising from double-stranded DNA breaks that are unrepaired before entry into anaphase. During anaphase, the malsegregation of chromosomes is usually triggered by mitotic spindle failure, centromeric DNA hypomethylation, kinetochore damage and defects in the cell cycle control system. Aside from MN, the CBMN cytome assay permit the detection of other indicators such as nucleoplasmic bridges (NBBs), nuclear buds (NBUDs), the proportion of dividing cells (cytostasis parameter), and cells undergoing apoptosis and necrosis (parameters of cytotoxicity).7 The well-established and standardized CBMN assay in peripheral blood lymphocytes (PBLs) remains a valuable biodosimetry technique for ionizing radiation exposure and a viable alternative to the DC assay8 because it permits the analysis of larger samples in lesser time. Despite those merits, the MN assay falls short of the DC assay's sensitivity and specificity. Regardless, owing to the advantages like time, simplicity, and associated sensitivity due to more cell count, the MN is considered an attractive assay in many biodosimetry laboratories.Table 1 Base-line MN frequency observed in various populations.

S.No	Country	No. of Donors	Age range (yrs)	Cells scored	No. of
MN	MN Frequency (±SE)	Reference	
1.	Great Britain	14	20–45	9000	119	0.013 (±0.001)	37	
2.	Germany	12	22–37	17,141	339	0.020 (±0.001)	34	
3.	India	25	NA*	25,000	265	0.011 (±0.001)	138	
4.	Portugal	1	29	3792	24	0.006 (±0.001)	35	
5.	UK	8	23–57	28,161	332	0.012 (±0.001)	139	
6.	Turkey	3	24–28	11,964	10	0.001 (±0.0002)	11	
7.	France	47	25–30	42,033	470	0.011 (±0.001)	19	
8.	India	21	28–46	21,000	255	0.012 (±0.001)	25	
SE-Standard error.

*NA – Not Available.

2 Need for biodosimetry

Individual exposure to ionizing radiation is inevitable in day-to-day life due to its background everywhere.9 However, the probability of additional higher exposure levels is not absent among the radiation workers as part of their occupation. At workplaces, those workers wear personnel monitoring dosimeter, allowing qualified experts to assess each worker's dose and apply the envisaged measures, a mandate and regulatory requirement. In contrast, neither is it a compulsion nor in practice that the general public would monitor with a personal monitoring device for radiation exposures. Many unfortunate incidents like nuclear terrorism or natural calamities can result in large radiation exposure to the general public.10 Segregation of population based on the quantum of radiation exposure and or quantification of absorbed dose in exposed individuals is paramount to medical management. In the absence of physical dosimeters, biological indicators have been adopted to quantify the absorbed dose and confirm the suspected over-exposures. It has been extensively reported that MN frequency obtained from blood lymphocytes of exposed individuals can be adopted for those applications.2, 11, 12, 13, 14 Using the CBMN assay to provide information on genotoxic, cytotoxic, and cytostatic effects suggests that the assay's predictive value could be improved, which merits further exploration. This systematic review emphasizes recent developments, significant limits, and steps that must be considered to achieve universal adoption of CBMN assay for dose assessment during radiation emergencies.

3 Methodology

The “PubMed” database (National Library of Medicine, National Institutes of Health, Bethesda, MD, USA) was used to search for the literature for this systematic review. The search approach was based on terms such as “MN,” “applications of MN and Radiation,” “MN and radiotherapy,” “MN scoring software,” “baseline frequency of MN,” “MN dose–response,“ “MN inter-laboratory comparisons,” and restricted to studies using human peripheral blood samples. Based on the obtained results, the assay's triage and routine biodosimetry applications and associated recent advancements have been classified according to their use and relevance to different radiation exposure scenarios.

3.1 Factors that influence reliable dose estimation

Biological dosimetry is useful for triaging casualties in the immediate aftermath of radiation emergencies and suspected radiation overexposures. Its main goal is to give a rapid dose assessment to aid in medical management and treatment decisions to handle a larger exposed population. MN frequency has shown a reliable biomarker in human populations, exposed to ionizing radiation therapeutically, occupationally, unintentionally or naturally. The majority of the studies found that exposed populations had considerably greater MN rates than controls.15 MN frequency obtained from an exposed person is adopted to calculate the absorbed radiation dose, which is subsequently extrapolated from the lab's standard in vitro reference dose–response curve using available software.16 Although the methodology for the CBMN is well established8 time of sample collection,2 transport of the collected sample to the laboratory,17 reagents used to culture and process the samples,18 culture methodology,19 scoring method20 and scorers skill21, 22 had been shown to influence the yield of MN and then dose estimation in addition to the well-established factors like dose, dose-rate, types of radionuclides and radio-sensitivity. Due to economic, logistical, and infrastructure-related constraints, blood cultures are the reference method for dose estimation during unintentional ionizing radiation exposure. Thus achieving reliable and accurate dose estimation from blood cultures.

3.2 Cell culture reagents

To prepare the binucleated cells from the blood samples, widely used chemicals are culture medium, growth factors from serum and mitogens to stimulate the cells into cycling. It is well-known that composition/nutrient sources differ among culture media, particularly ascorbate, folic acid, and vitamin B12. This was because few researchers used modified McCoy's culture media,23 whereas most of the laboratories throughout the world used RPMI 1640 medium.24 The results demonstrated that the culture medium did not affect base-line micronucleated cell frequency when isolated lymphocytes were used for culture separately in the two types of media on the same day. It would be acceptable for the standard technique to be based on culture using RPMI 1640 medium to retain consistency with previous data. It would be essential to confirm if the results acquired are statistically identical.23, 24 Similar to that culture medium, the concentration of foetal bovine serum (FBS) used as growth factors in the culture varies among the studies, like 10 %, 20 % of total culture volume.19, 25 Though there has been no coordinated examination of the effects of different methodological features on the outcome of MN frequency, many inter-lab comparison studies have been published that recommend modifications to the standard techniques.26, 27, 28 The findings acquired using McCoy's medium (R=0.4, p < 0.01) were significantly correlated with the data obtained using RPMI 1640.23

3.3 Temperature

Blood samples occasionally need to be transported or stored and preserved, and it's important to know how this impacts the sensitivity of the lymphocytes used in biomonitoring. Although blood samples can be kept at a reduced temperature (4 °C), it results in a loss of cell viability.29 It has been reported that the yield of MN depends on temperature and storage time. Before, during, and after irradiations, the sample can be subjected/processed at a temperature ranging from 4 to 37 °C. It has been reported that the yield of DC depends on temperature, time30; and storage of exposed blood at refrigerated conditions or 20 °C (48 h) leads to the accumulation of damages.31 Irradiated blood kept at 22 °C or 5 °C, showed no changes in MN yield and also been reported that storage of blood at 4 °C and 20 °C for 48hr with phytohemaggultinin (PHA) did not alter cell viability and proliferation32 as well as reduction in viability.2 Cell viability is observed maximum of 100 % immediately upon sampling, then reduces upon storage at a low temperature with and without mitogenic stimulation.32 Except for the samples maintained at 4 °C and 20 °C in the presence of PHA (98.0 ± 0.70 % and 99.0 ± 1.15 %), showed a decline in cell viability of samples stored at 37 °C with and without PHA after 72 h shows a significant decrease compared to samples stored at 4 °C or 20 °C with and without PHA (82.0 ± 2.82 % and 75.0 ± 1.75 % compared to 90.0 ± 3.40 % to 96.5 ± 0.70 %, respectively). Cell viability values observed after 96 h of storage in samples kept at 4 °C with or without PHA (96.8 ± 1.15 % and 96.5 ± 0.71 %, respectively) demonstrated high percentages of cell viability.32 In comparison to other samples (58.0 ± 2.12 % to 69.0 ± 2.08 %), the optimal storage condition for sustaining cell viability was at 4 °C in the presence of PHA (86.0 ± 3.05 %). To prevent apoptosis and maintain cell proliferation, keeping at 4 °C for about 96 h in the presence of PHA is ideal. It was also recommended to stimulate lymphocytes with PHA immediately after venous blood collection and keep them below 20 °C to halt lymphocytes from transforming and progressing through the cell cycle until they are incubated at 37 °C.2

3.4 Culture time

Human peripheral lymphocytes, a representative cell population synchronized in G0 stage, desynchronize progressively on mitogenic stimulation, with varied dynamics for each individual. It is reported33 that a mean cycle time of roughly 12 h for peripheral lymphocytes in RPMI 1640 medium follows the first mitosis after stimulation. Evidence suggesting the medium and temperature impact the length of the cell cycle in culture.18 Cultures were carried out for various durations, followed by adding different concentrations of Cytochalasin-B (cyto-B) to determine the optimum concentration and harvest time at which cyto-B is most efficient in yielding binucleate cells.19

3.5 Chemicals/Reagents

The cyto-B is a mycotoxin used to arrest the cells at the cytokinesis stage had been shown as an essential factor in determining the percentage of binucleated cells. Both the concentration and cell harvest duration followed by the addition of cyto-B were reported to vary among the studies; concentration of cyto-B varied between 3 to 6 µg/ml and cultures were harvested 24 to 28hr after the cyto-B addition.5, 34, 35 Surrallés et al.,36 have reported that 6 µg/ml concentration of cyto-B is more in halting cytokinesis than 3 µg/ml; however, it resulted in a decreased frequency of MN. In contrast, found no differences in MN frequency between the two concentrations.37 When dealing with the cytokinesis block approach, the findings of monitoring proliferation in various individuals show that procedures with defined durations for applying cyto-B may be risky. Individual donor’s responses to PHA stimulation vary in promptness; therefore this is a factor to consider.34 The formation of MN is increased by prolonged culture of lymphocytes with PHA at varied time intervals.38, 39 An in vitro study reported delayed mitogenic stimulation decreases DNA damage or prolonged peripheral blood storage without mitogenic stimulation could lead to interphase cell death.17 Prolonged culture time with PHA, delayed mitogenic stimulation and late arising first division metaphase followed by exposure of human PBL in G0 influenced chromosomal aberrations and MN frequency.38, 39, 40

3.6 Scoring of aberrations

Guidelines to adopt the number of cells to analyze and interpret various aberrations are described in detail.2 However, identifying aberrations is an objective phenomenon that can be influenced by many parameters such as quality of slides, stain used, scorer ability, and experience to classify the aberration.21 Many inter-laboratory exercises have been conducted to maintain consistency and minimize such variations.21, 26, 27 To determine the extent of residual variation when laboratories scored cells from the same cultures using the same set of standard scoring criteria, an inter-laboratory slide-scoring exercise was performed among 34 laboratories from 21 countries with 51 slide scorers involved. This study shows that even under these optimized conditions, there is a variation in the MN frequency obtained by individual laboratories and scorers. It has been proved that there is an intra- and inter-lab and scorer variation in the scoring of MN.

3.7 Background frequency of MN and gender

The first step in biological dosimetry using any biomarker is constructing a reference dose–response curve. Generally, a linear-quadratic dose–response has been reported in the majority of the studies for CA and MN aberration frequency when it was scored from blood samples exposed to low LET radiations. It was emphasized that the number of cells and samples used to obtain background aberration frequency could influence the co-efficient of the reference dose–response curve. The existing literature consistently shows that the MN frequencies in a selected age group for healthy subjects vary between 1.4 fold and 2.3 fold depending on age and gender. Thus the minimum and maximum values for MN frequency in any particular age group differed, with the most significant differences occurring in the 40–59 year age range23 in a population (n = 62) consisting of healthy adults of both genders. Another study further supported the results in which a significant gender effect on baseline MN favors females. At the same time, yields of radiation-induced MN did not differ significantly between genders.41 Thus, age and gender are the most important demographic variables affecting the MN index; frequency is higher in females than males by 1.2 to 1.6, depending on the age group.23, 42 The baseline MN frequency observed in various populations around the globe is mentioned in Table 1. It's also critical to develop standardized protocols that allow more reliable data comparisons between laboratories.

3.8 Lifestyle factors

Unhealthy lifestyle factors contribute to cardiovascular disease, diabetes, obesity43 and cancer44; as MN biomarker has been prospectively connected with these lifestyle diseases, lifestyle choices that cause these diseases may be linked to MN frequency in PBL. The lifestyle practices include exercise, consumption of alcohol, smoking, working hours, sleeping hours, balanced nutrition and mental stress.45 Physical activity has been shown to influence MN in PBL in six trials.45, 46, 47, 48, 49, 50 In a study involving 208 healthy adult male Japanese hard-metal workers aged 19–59 years, Huang et al.,45 observed the impact of lifestyle practices. It generally ranges from a reduction in MN in fit participants following moderate activity or a triathlon event45, 50 to null effects,47, 49 and even a rise in MN in healthy volunteers following acute exhausting exercise.46, 48 Many demographic studies have looked into the effect of tobacco smoking on the frequency of MN in human lymphocytes. A total of 5710 participants were included in the database, including 3501 nonsmokers, 1409 current smokers, and 800 past smokers from occupational and environmental surveys. When compared to non-smokers, the overall outcome of the re-analysis indicated a slight non-significant decrease in MN frequencies in current and previous smokers.51 Although when the interaction with occupational exposure is considered, heavy smokers were the only group exhibiting a substantial increase in genotoxic damage as determined by the CBMN assay in lymphocytes. Alcohol use is associated with greater MN frequency in PBL, according to studies52, 53, 54 that have specifically and thoroughly explored the link between consumption of alcohol and MN formation. Using centromere probes, one study investigated the type of MN generated by alcoholism. It determined that the increase in MN frequency in alcoholics compared to controls is primarily due to MN originating from complete chromosomes.55 The micronutrient concentrations required as cofactors in DNA synthesis and repair are strongly influenced by the meals and supplements consumed and the amount consumed. The relationship between MN and nutrition was initially found in erythrocytes in studies of anemia induced by a folate and vitamin B12 deficiency.56 The MN frequency in PBL is strongly interconnected with either dietary consumption levels or plasma concentrations of vitamin B12, folate, riboflavin, pantothenate, biotin, vitamin E, beta-carotene, retinol and calcium, according to in vivo investigations in humans.57, 58, 59 As concluding remarks from these study groups, mental stress and drinking alcohol had no effect on MN frequency, whereas, smoking, nutritional deficiencies, a lack of regular exercise, poor sleep, and extra work all contributed in the increased MN frequency. These data suggests that bad lifestyle practices increase MN frequency in human lymphocytes considerably.

3.9 Suitability of MN as a biological indicator for accidental exposure

The CBMN approach has been proposed for use in a mass screening role for quick biological dosimetry to corroborate the initial triage (sorting) of vast numbers of people who may have been exposed following a severe radiation event. However, less evidence is available for its use for such a purpose has been found. The speed with which data can be acquired is thought to benefit. Individualized, early and definitive assessments of radiation doses are required to give medical aid in the days following a disaster in potential radiation exposure scenarios involving huge loss of life. Because of its simplicity and rapid quantification, MN has shown to be a promising perspective tool for triage in the medical management of a nuclear disaster. However, due to a spontaneous MN frequency of 0.002–0.036/cell, its sensitivity is just 0.25 Gy2. CBMN assay was employed in the Chernobyl nuclear disaster60 and the Istanbul bombing.61.

Nonetheless, during operation, a fault at the entrance into the depleted uranium-shielding device of a 192Ir source exposed an industrial radiography worker. MN frequency was not increased above the laboratory's control value of MN background frequency of unexposed individuals due to the absorbed dose falling below the lower detection limit of 0.3 Gy photon-equivalent whole-body exposure62. In triage mode, biological dosimetry must respond as rapidly as feasible. A preliminary dose estimate is sufficient since it divides victims into three groups for medical follow-up (less than 1 Gray (Gy), 1–2 Gy, and more than 2 Gy).63 To meet the triage requirements, other solutions are being developed that involve automation in scoring and sharing the workload with other laboratories approaches have been proposed. The available results confirm the efficacy of the automated CBMN assay for fast population triage in a multicentric setting, in the case of large radiation accidents64, 65, 66, 67, 68.

3.10 MN as a biological indicator for occupational radiation exposure

Technicians and physicians who operate with X-ray equipment, industries, and mines personnel are regularly exposed to low levels of radiation. MN is not radiation specific because various clastogenic agents can cause it in the form of acentric chromosome fragments and aneugenic agents in entire chromosomes. As a result, the CBMN assay is frequently employed in general toxicological testing6. The CBMN assay has proven to be a reliable, wholly established, and standardized technique in radiation biology since ionizing radiation is a robust clastogenic agent and hence a significant inducer of MN. The number of radiation-induced MN is closely connected with radiation dose and is dependent on the radiation quality.69, 70, 71, 72 MN yields in PBLs of diverse groups of patients treated with fractionated partial body radiotherapy, such as cervical cancer, prostate cancer, or Hodgkin's disease, were examined to determine the applicability of the CBMN assay for biological dosimetry. The doses obtained using MN analysis are similar to the averaged whole-body doses calculated using radiation treatment plans.73, 74, 75, 76, 77 Studies performed in thyroid cancer patients undergoing radioiodine treatment further demonstrated that the CBMN assay is sensitive enough to detect low average whole-body doses from internal exposure scenarios.78, 79, 80 The CBMN assay was used in specific radiation disaster investigations to determine protracted exposure owing to populations in the area of the Chernobyl nuclear power station81 and the Semipalatinsk nuclear test site ingesting long-lived radionuclides.82 The estimated internal absorbed dosage was significantly linked with MN frequencies measured in many residents.

Radiation employees, such as nuclear power plant personnel and hospital staff, are the subjects of large-scale biomonitoring studies, have shown that the CBMN assay, particularly when combined with fluorescence in-situ hybridization (FISH) staining for centromeres, can detect radiation-induced chromosomal damage to the population level for accumulated doses received during their occupation exceeding 50 milliSivert (mSv). These biomonitoring investigations, which looked at a broad group of radiation workers (between 70 and 270 participants) who were exposed to accumulated doses ranging from 10 to 248 mSv, revealed a clear relationship between MN development and the accumulated dose83, 84, 85, 86. In nuclear power plant workers, the biomonitoring investigation revealed 0.03 MN/1000 BN cells/mSv87 and 0.025 MN/ 1000 BN cells/mSv84 of exposure. On the other hand, medical radiation has become a significant source of radiation exposure for the general public. It has also been shown that dosages received during radiology imaging and interventional procedures induce considerable DNA damage in patients' cells88 and healthcare personnel89. The usage of MN not only ends with biomonitoring investigations in industries and cancer patients; it also extends its application to estimating absorbed dose during diagnostic radiological procedures90, 91. The biological effects of modest dosages received during diagnostic imaging are under investigation; immediate and long-term effects are unknown. The CBMN assay has found a substantial increase in MN in PBLs from patients with plain or contrast CT scans90. Infants who got a second computed tomography (CT) scan after 48 h had a higher MN, implying that earlier CT imaging improved cellular responses to successive CT scans92. Medical radiation professionals exposed to low-dose ionizing radiation PBLs were monitored for cytogenetic changes and revealed a significant increase in MN.93

3.11 Recent advances and developments in biodosimetry using CBMN assay

Potential scenarios of radiation exposure resulting in mass casualties require individual, early, and definitive radiation dose assessment to provide medical aid within days of a disaster. The essential steps in triage medical management are preliminary dose assessment and segregation of exposed and non-exposed people. Furthermore, first responders must be checked on a regular basis to verify that the dose levels they are exposed to during evacuation are safe. Alternative ways are being developed to satisfy the need, as traditional cytogenetic procedures by manual scoring are time-consuming; current improvements in biodosimetry include task sharing among expert groups, automation of analytical processes, and early indicators to ionizing radiation. Table 2 shows α and β coefficients obtained with MN dose–response curve for different types of radiations in various laboratories irrespective of scoring methods.Table 2 α and β coefficients obtained with MN dose–response curve for different types of radiations in various laboratories.

S.No	Type of Radiation	Dose range (Gy) and Dose rate (Gy/min)	Scoring method	α ± SE x 10-2 Gy- 1	β ± SE x 10-2 Gy- 1	Reference	
1.	60 Co-gamma	0.6	Manual	9.5 ± 1.7	4.8 ± 0.6	140	
2.	137Cs	0–2 & 0.9	Flow cytometry	1.84	0.91	108	
3.	60 Co	0–4 & 0.5	Manual	1.9 ± 1.1	5.7 ± 0.45	138	
4.	60Co	0–5 & 0.5	Manual	9.3 ± 0.9	13.3 ± 1.4	35	
5.	60Co	0–4 & 0.5	Manual	5.24 ± 0.98	3.15 ± 0.35	101	
6.	60Co	0–4 & 0.43	Manual	4.29 ± 0.74	1.87 ± 0.23	
7.	60Co	0–6 & 0.09	Manual	7.5 ± 2.23	1.76 ± 0.55	
8.	60Co	0–4 & 0.7	Manual	1.48 ± 0.55	3.33 ± 0.22	
9.	60Co	0–5 & 0.45	Manual	0.44 ± 0.2	1.16 ± 0.1	11	
10.	60Co (conventional method)	0–4 & 0.75	Manual	6.39 ± 1.2	3.22 ± 0.41	141
	
11.	60Co (synchronized method)	0–4 & 0.75	Manual	14.70 ± 0.96	2.48 ± 0.28	141	
12.	60Co	0–4 & 0.5	Manual	7.06 ± 1.30	2.79 ± 0.43	19	
13.	60Co	0–4 & 0.75	Manual	6.39 ± 1.29	3.23 ± 0.42	25	
14.	60Co	0.5	Manual	7.1	2.8	142	
15.	60Co	0–5 & 2.05	Manual	7.5 ± 0.2	2.8 ± 0.06	143	
16.	60Co		Manual	7.5 ± 1.1	1.6 ± 0.3	144	
17.	60Co		Automated	6.14 ± 1.11	1.6 ± 0.42	26	
18.	60Co		Semi automated	4.56 ± 0.90	3.22 ± 0.37	
19.	60Co		Manual	7.67 ± 1.8	4.18 ± 0.73	
20.	60Co		Automated	1.84 ± 0.13	2.67 ± 0.07	
21.	137Cs	0–8 & 6.0	Automated	92.28	−0.22	145	
22.	137Cs		Manual	7.18 ± 4.07	4.73 ± 9.63	26	
23.	X rays (250kVp)	0–5& 1.0	Manual	11.7 ± 0.6	0.8 ± 1.6	37	
24.	X rays	0–4	Manual	13.9 ± 4.4	3.68 ± 1.8	146	
25.	X rays (220kVp)	0–4 & 0.5	Manual	9.77 ± 0.95	4.78 ± 0.33	147	
26.	X rays	0–4	Manual	4.09 ± 2.23	4.33 ± 0.6	148	
27.	X rays (240kVp)	1.0	Manual	0.120	0.023	34	
28.	X rays (250kVp)	0–4 & 0.5	Manual	6.3 ± 1.8	4.3 ± 0.7	138	
29.	X rays (250kVp)	0–5& 2.0	Manual	4.95 ± 1.6	4.61 ± 0.6	139	
30.	X rays	0–4 & 1.0	Manual	5.96 ± 1.10	3.92 ± 0.36	149	
31.	X-rays		Automated	4.94	4.1	150	
32.	X ray (240kVp)	1.0	Automated	3.7 ± 1.14	3.6 ± 0.44	26	
33.	X ray (240kVp)	1.0	Semi automated	5.47 ± 0.96	1.66 ± 0.02	
34.	X ray (240kVp)	1.0	Automated	5.35 ± 0.66	1.54 ± 0.12	
35.	X ray (240kVp)	1.0	Automated	5.89 ± 1.16	−--	
36.	X ray (240kVp)		Manual	9.23 ± 1.59	6.01 ± 0.58	
37.	X rays (250kVp)	0–4 & 1.3	Imaging flow cytometry	0.06	0.98	151	
38.	2.27 MeV β particles	0–2	Manual	2.48 ± 1.3	3.8 ± 1.0	139	
39.	Electrons		Manual	8.4 ± 1.6	2.1 ± 0.3	144	
40.	X-rays
(225kVp)		Manual	8.2 ± 0.9	2.3 ± 0.6	152	
X-rays
(3 MV)		Manual	7.4 ± 3.9	1.8 ± 1.3	
X-rays
(6 MV)		Manual	6.0 ± 1.4	1.8 ± 0.5	
α − Linear coefficient; β − Quadratic coefficient; SE − Standard error.

3.12 Need for the network for biodosimetry

In recent years, the arrangements to undertake triage in radiation mass casualty scenarios have been a key new development. A biological dosimetry laboratory's response to a rapid rise in cases is discussed, including using assays in a triage mode, speeding up analysis with computer-assisted microscopy, and networking with other labs. International recommendations on quality assurance, quality control, and participation in inter-laboratory comparison (ILC) exercises have coincided with this enhanced provision for collaborative emergency response work.2, 94 Biodosimetry laboratories can only handle a certain number of victims at a time. Sharing the workload among the experts is an attractive option for handling a considerable sample size. Thus, in the event of a mass-casualty radiation disaster, when the management of hundreds of victims is required, national and/or international networking is required. On the other hand, networking must be founded on the ability to produce consistent outcomes.95, 96 This means that to get consistent dose estimate/results from the participating laboratories, all the labs should adopt a similar culture methodology, reagents used to culture cells, sample processing and scoring methods.97 International accreditation bodies like the International Organization for Standardization (ISO) have contributed immensely to this goal by providing standard performance criteria (pertaining to quality assurance and quality control) for cytogenetic service laboratories undertaking biodosimetry98. It is now widely agreed that networking should include regular international ILC exercises exhibiting various scenarios, ensuring a faster reaction and higher dose estimate reliability.99

ILC also aids in the harmonisation of culture conditions, scoring standards, and statistical analysis techniques. If laboratories build up networks to respond to a mass casualty event, this harmonisation is required. The number of potentially exposed individuals to be analyzed the response capabilities of the local responders100. The mutual assistance of several laboratories is required to increase the number of samples to be processed and achieve faster results availability. In 2007, the WHO initiated “BioDoseNet,” a network of more than 30 laboratories worldwide and implemented revised regulations pertaining to human health, including the field of radio-nuclear incidents.97 The approach for increasing throughput is the development of networks among laboratories available on a global scale. Several networks have been known to improve the rate of dose estimation, such as the Latin American Biological Dosimetry Network101, the National Biological Dosimetry Response Plan (NBDRP) in Canada102, the Chromosome Network in Japan103, the European Network, Realizing the European Network of Biodosimetry (RENEB)104, NATO biodosimetry group26, India105 and in China106 for the DCA; as it requires more time and remains the sensitive assay. However, compared to DCA, ILC using CBMN assay is limited21, 26, 28. The goal of ILCs is to determine problems that participants have and develop solutions, such as harmonization, training, and dose estimation activities. Garcia et al.,101 reported that 11 of the 15 estimates of dose based on DC and 9 of the 12 based on MN fell within ± 30 % of the correct dose. The outcome of the ILCs showed that MN is suitable for dose estimation during radiation triage. However, it has been reported that differences in scoring criteria, analysis of frequencies, dose rate and the radiation source for dose estimation and lack of homogeneity in calculating the uncertainties were observed as limits and pitfalls of ILCs.107

3.13 Automation

In addition to the ILC exercise among the participant laboratories to handle larger samples, rapid technological advancement has been made to score the MN with increased efficiency and faster automation. Several automated platforms, Flow cytometry,108 automated microscope,109 imaging flow cytometry,110 IMSTAR PathfinderTM,111 iCyte® automated imaging cytometer112 and Rapid Automated Biodosimetry Tool (RABiT)113 have been established to execute the CBMN assay and analysis. These platforms have advantages and limitations in robust, high-throughput, sensitivity, specificity, and visual confirmation of MN (Table 3).Table 3 Comparison of different automated platforms available for robustness in MN analysis during triage.

Platform	Advantages	Disadvantages	
Microscopy analysis of CBMN cells stained with Giemsa	• Slides can be stored for longer time and can be reanalysed for verification

	• At times artefacts due to non-specific staining can be counted as MN

	
Fluorescence microscopy analysis of CBMN cells stained with DAPI/PI	• Better sensitivity than giemsa stained preparations

	• Fluorescence microscope is expensive

• Slides can’t be used for reanalysis or verification

	
Flow cytometry	• Cells measured in fluid content

• Rapid analysis

	• Not suitable for biodosimetry

	
Image flow cytometry	• Cells measured in fluid content

• Robust analysis

• High-resolution image

	• Necessitates expert knowledge

• Rigorous optimization is required

• Cannot re-locatable

• Sensitivity > 1 Gy

	
MetaSystems	• Slide based analysis

• Manual correction is possible

• Easily re-locatable

• Suitable for Giemsa and fluorescent dyes

• More than 100 slides per day

• No expertise required

	• Sensitivity > 0.25 Gy

	
IMSTAR Pathfinder	• Based on unique algorithms

• It enables the MN scoring in mono- bi- or poly nucleated-cells

	• Necessitates expert knowledge

• Not yet used for biodosimetry purpose

	
iCyte® automated imaging cytometer, CompuCyte	• It works by combining of flow and image cytometry

• 10–20 slides per day

	• Not yet commercialized

	
Rapid Automated Biodosimetry Tool (RABiT)	• Completely automated (right from sampling to analysis)

• High-throughput

• Increases scoring efficiency and handling capacity

	• Not yet commercialized

• Cost factor

	

3.14 Flow cytometry-based micronucleus (FCMN) assay

Developing an FCMN assay to measure the MN frequency can improve reproducibility and reduce turn-around time compared with the traditional microscopy-based analysis. Flow cytometry is a technology that simultaneously measures and analyses multiple physical characteristics of single particles, usually cells, as they flow in a fluid stream through a beam of light; properties measured include a particle’s relative size, granularity, or internal complexity and fluorescence intensity. These characteristics are determined using an optical-to-electronic coupling system that records how the cell or particle scatters incident laser light and emits fluorescence. Any suspended particle or cell from 0.2–150 μm in size is suitable for analysis. Therefore, FCMN was considered as a high throughput and potential for triage. Speed is an inherent advantage of flow cytometry; however each particle present in the suspension produces a signal and is registered separately, making it difficult or sometimes impossible to discriminate between MN from the nonspecific background.108

Moreover, DNA content in MN lower than 2 % of the G1-phase cells could not be discriminated from debris. An improvement has been made to separate debris from the MN according to their different physical properties to increase sensitivity and reproducibility and to measure the number and size distribution of MN.108 Even though this technique agreed well with microscopy, difficulties were observed in differentiating true MN from other DNA positive events.114, 115 The existing modern flow cytometry approach retains the capability to acquire data from thousands of cells in a limited time and permits automatic analysis of all data. The rapidity of this technique and the requirement for nominal operator intervention are attractive features for toxicology testing. Avlasevich et al.,116 and Byrce et al.,117 had made substantial developments to the procedure by combining fluorescent dyes to overcome the problems encountered when distinguishing cellular debris, MN and free chromosomes from mitotic cells. Research engaging various well-known clastogens and aneugens have confirmed that statistically significant increases in MN frequency can be detected compared with controls using In Vitro MicroFlow Kit (Litron Laboratories, Rochester, NY).116, 117 Inter- and intra-individual variations of spontaneous and radiation-induced MN frequencies, the minimum detection limit has been reported as above the dose of 1 Gy.117, 118, 119 However, the lack of cellular visualization (Mono, bi and poly nucleated cells) is the major drawback of conventional flow cytometry methods. It is difficult to confirm that MN identified are genuine. An additional disadvantage is that cell membranes need to be lysed to release both the main nuclei and MN from a cell, leading to debris such as individual apoptotic bodies. Regardless of the contemporary developments in the approach, it is difficult to make a decisive difference between MN and DNA positive debris.112 To overcome this, Rodrigues120 developed a microscopic-based method called an imaging flow cytometer, which can combine the high-throughput nature of traditional flow cytometry with microscopy's high-resolution imaging. The IDEAS® software was used to make a rapid and automated MN assay based on high throughput picture capture and feature-based image analysis using imaging flow cytometry with the ImageStream®. The Amnis® AI software was used to construct a deep-learning algorithm based on convolutional neural networks to score imaging flow cytometry data in both the cytokinesis-blocked and unblocked versions of the MN test to overcome the complexity and rigidity of feature-based image analysis.110 However, optimization and validation are required for the next step in the development of automated MN assay.

3.15 Automated microscope for MN scoring

Despite the potential for rapid scoring of MN with flow cytometry, difficulty in discriminating MN from artifacts arising from either cell processing or as a consequence of apoptosis and necrosis leads to a false-positive interpretation119 and compromise in the sensitivity; furthermore, difficulty in analyzing nucleoplasmic bridges (NPBs) and nuclear buds (NUBDs), sample preservation and re-analysis are added limitations.22 Automated image cytometry is preferred because improved computer algorithms allow more advanced image analysis on a cell-by-cell basis with higher sensitivity. Tates et al.,121 reported that 63 % of the BN cells and 57 % of the MN within these BN cells and exposure could be detected automatically. Castelain et al.,122 were able to detect 67 % of the BN cells and 33 % of the MN in these BN cells automatically with the help of the Magiscan Image Analysis System; thus though the speed was increased using automated scoring, it is capable of detecting 1/2th of the BN cells and 3/4th of the MN in these cells. It was attributed to that relatively high inaccuracy in the classification of the BN cells. Of late, systems like MetaSystems Metafer MNScore,109 IMSTAR PathfinderTM Screentox Auto-MN111 and Compucyte iCyte® Laser Scanning cytometer,112 which are commercially available to increase the scoring speed of MN with better accuracy in identifying the MN and BN cells.

3.16 MetaSystems

MetaSystems developed and commercialized a scoring system with a software module (Metaphase finder platform Metafer4), which allows automatic scoring of MN in BN cells.109 This image analysis software detects the BN cells based on the similarity of the daughter nuclei [Fig. 2]. However, one should keep it in mind that the MetaSystems software employed to analyze the MN does not use the cytoplasmic boundary to identify BN cells, critical criteria adopted in manual scoring; further, if required, the cytoplasmic boundary should be visualized using phase-contrast microscopy123 to verify the accuracy of BN cell detection. The first commercially available and widely used system is the Metafer MNScore (MetaSystems) platform in cancer research,124 biomonitoring studies in the field of air pollution,125 cell lines in in vitro studies126 and radiation biodosimetry.14 The comparison of manual and automated scoring was 23.6 % different, with an efficiency of 24.9 % in the obtained MN frequencies. Slides stained with fluorescent dyes are better for automated scoring than Giemsa-stain.14 This MN technique allows reliable dose reconstruction after high radiation doses with a method adapted for automated high-speed sample processing systems.127 The manually scored slides can eliminate false positives, quantify cytotoxicity, and conduct mechanistic research. The classifier setting should be used when performing MN scoring on the semi-automated Metafer system to account for chemical or cell line-specific morphological alterations and avoid false findings (positive and negative). These semi-automated and completely automated platforms can thus be employed for dose–response analysis since they can score a much larger number of cells, allowing for more statistical power. Future studies should focus on addressing these problems and assessing inter-laboratory repeatability for these methodologies to be more extensively employed for MN scoring and dose–response analyses.Fig. 2 Images of cytochalasin-B blocked binucleated cells from human peripheral blood lymphocytes obtained using Metafer microscope. (A) Giemsa-stained slides −binucleated cells with cytoplasm (B) propidium iodide stained slides − binucleated cells.

3.17 IMSTAR PathfinderTM

The IMSTAR PathfinderTM Screentox Auto-MN, an automated imaging system for the CBMN assay, is based on advanced unique algorithms in successive steps; first, the cytoplasm of each cell within the whole sample, accurate recognition of the number and morphology characteristics of nuclei and MN within every cell; thus it enable the MN scoring in mono- bi- or poly nucleated-cells, the measurement of the cytokinesis block proliferation index (CBPI), as well as the validation by an expert/technician.111

3.18 iCyte® automated imaging cytometer, CompuCyte

Laser scanning cytometry (LSC) using iCyte® (CompuCyte Corporation, Westwood, MA, USA) is an alternative method suitable for automated MN scoring, which offers unique analytical capabilities that combine those of flow and image cytometry.128 This system has been validated to score propidium iodide (PI)-stained MN in BN cells in cell lines,129 PBL or bone marrow erythrocytes and buccal cells. The number of slides that can be analyzed per day is relatively low in the range of 10–20 per day and for Giemsa stain, this automated method is not validated. Of late, many types of equipment with automation platforms to score MN are developed and used by individual laboratories. They are not commercialized; for example, Cellomics,130 GE Healthcare,131 ROBIAS (Robotic Image Analysis System),132 RABiT (Rapid Automated Biodosimetry Tool).133

3.19 Rapid automated biodosimetry Tool (RABiT)

The RABiT system is designed as entirely automated, from the input of the capillary blood sample into the machine to the output of a dose estimate. Using RABiT, the blood pricked from the finger is sufficient to get all the data based on the complete automation of two well-characterized biodosimetric assays like the CBMN assay. The CBMN assay can be performed and are highly radiation-specific; the estimated blind doses were within 20 % of the actual dose in 97 % of the samples113. The obtained results using the high-throughput RABiT-IFC CBMN assay maintain the potential to increase scoring and handling capacity for triage biodosimetry during a large-scale radiological/nuclear event.134 Several studies have confirmed the reliability of the automated CBMN assay for high throughput population triage.113, 135, 136 Recently, a quick CBMN harvest protocol has been proposed for isolated peripheral blood mononuclear cells. It is a viable alternative to cytocentrifugation, as many scorable binucleated cells were obtained with routine biodosimetry.137 In summary, however, the advancements mentioned earlier might be helpful after the validation and standardization of the proposed technique.

4 Conclusion

The methodology described here can improve triage radiation biodosimetry response capacity using CBMN assay. The traditional CBMN assay (microscopic analysis of CBMN cells) has been thoroughly validated and standardized. The alternative approaches, such as centromere staining, NPB scoring, and MN scoring automation, have recently been optimized and are still being developed, making the CBMN assay more sensitive and specific for radiation dose estimations and a particular interest in large-scale screening applications. Comparable results can be obtained and the high throughput of automated MN scoring can be increased to allow a rapid response to large-scale radiation accidents by establishing and creating an international network of trained laboratories using similar equipment for MN automation and the same classifiers standardized fixation protocols, and so on.

To optimize the usage in retrospective biodosimetry and investigate cases of extended and partial-body exposure, the CBMN assay has to be refined further. Only a limited amount of data is available on MN's disappearance, and more research and validation are required. For more complex exposure scenarios, appropriate calibration curves must also be constructed. At last, a test system that combines the high-throughput, high-content, and multiplexing capabilities of flow cytometry with the image analysis benefits of re-validation and data storage would be a significant step toward creating a genuinely twenty-first-century method.

5 Consent for publication

Not Applicable.

6 Ethics approval and consent to participate

Not Applicable.

7 Author’s contributions

TG wrote the first draft of the manuscript and edited by PV. All authors discussed, edited and approved the final version of the manuscript.

Funding

This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.

Declaration of Competing Interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Acknowledgement

The authors are thankful to Dr. N K Chaudhury, Scientist G (Retd), INMAS, DRDO for his support and constructive input.
==== Refs
References

1 Vorobiev A.I. Acute radiation disease and biological dosimetry in 1993 Stem Cells 15 2009 269 274 10.1002/stem.5530150736
2 IAEA 2011 2011. https://www.iaea.org/publications/8735/cytogenetic-dosimetry-applications-in-preparedness-for-and-response-to-radiation-emergencies (accessed April 29, 2022).
3 Ohtaki K. Shimba H. Awa A.A. Sofuni T. Comparison of type and frequency of chromosome aberrations by conventional and G-staining methods in Hiroshima atomic bomb survivors J Radiat Res 23 1982 441 449 10.1269/jrr.23.441 7182494
4 Hittelman W.N. Rao P.N. Premature chromosome condensation. I. Visualization of x-ray-induced chromosome damage in interphase cells Mutat Res 23 1974 251 258 10.1016/0027-5107(74)90145-6 4836309
5 Fenech M. Morley A. Solutions to the kinetic problem in the micronucleus assay Cytobios 43 1985 233 246 4075848
6 Luzhna L. Kathiria P. Kovalchuk O. Micronuclei in genotoxicity assessment: from genetics to epigenetics and beyond Front Genet 4 2013 10.3389/fgene.2013.00131
7 Fenech M. Cytokinesis-block micronucleus assay evolves into a “cytome” assay of chromosomal instability, mitotic dysfunction and cell death Mutat Res 600 2006 58 66 10.1016/j.mrfmmm.2006.05.028 16822529
8 ISO 2014. ISO 17099:2014. ISO n.d. https://www.iso.org/cms/render/live/en/sites/isoorg/contents/data/standard/05/91/59141.html (accessed April 29, 2022).
9 UNSCEAR 1982 report n.d. https://www.unscear.org/unscear/en/publications/1982.html (accessed April 29, 2022).
10 Williams M. Armstrong L. Sizemore D.C. Biologic, Chemical, and Radiation Terrorism Review 2022 StatPearls Publishing StatPearls, Treasure Island (FL)
11 Bender M.A. Gooch P.C. Persistent chromosome aberrations in irradiated human subjects Radiat Res 16 1962 44 53 13867088
12 Koksal G. Pala F.S. Dalci D.O. In vitro dose-response curve for chromosome aberrations induced in human lymphocytes by 60Co gamma-radiation Mutat Res 329 1995 57 61 10.1016/0027-5107(95)00019-f 7770076
13 Bertho J.M. Roy L. A rapid multiparametric method for victim triage in cases of accidental protracted irradiation or delayed analysis Br J Radiol 82 2009 764 770 10.1259/bjr/49063618 19433485
14 Tamizh Selvan G. Chaudhury N.K. Venkatachalam P. Comparison of results of the manual and automated scoring of micronucleus frequencies in (60)Co-irradiated peripheral blood lymphocytes for triage dosimetry Appl Radiat Isot 97 2015 70 77 10.1016/j.apradiso.2014.12.018 25544665
15 Sari-Minodier I. Orsière T. Bellon L. Pompili J. Sapin C. Botta A. Cytogenetic monitoring of industrial radiographers using the micronucleus assay Mutat Res 521 2002 37 46 10.1016/s1383-5718(02)00213-9 12438002
16 Ainsbury E.A. Lloyd D.C. Dose estimation software for radiation biodosimetry Health Phys 98 2010 290 295 10.1097/01.HP.0000346305.84577.b4 20065696
17 Selvan GT, Bhavani M, Vijayalakshmi J, Solomon FDP, Chaudhury NK, Venkatachalam P. Delayed Mitogenic Stimulation Decreases DNA Damage Assessed by Micronucleus Assay in Human Peripheral Blood Lymphocytes after 60CO Irradiation. Dose-Response 2014;12:dose-response.1. doi: 10.2203/dose-response.13-060.Selvan.
18 Purrott R.J. Lloyd D.C. Vulpis N. Chromosome dosimetry: the influence of culture media on the proliferation of irradiated and unirradiated human lymphocytes Radiat Prot Dosim 1 1981 203 208
19 Paillole N. Voisin P. Is micronuclei yield variability a problem for overexposure dose assessment to ionizing radiation? Mutat Res 413 1998 47 56 10.1016/s1383-5718(98)00015-1 9602858
20 Brown J.K. Williams A. Withers H.R. Sources of variability in the determination of micronuclei in irradiated peripheral blood lymphocytes Mutat Res 389 1997 123 128 10.1016/s1383-5718(96)00127-1
21 Fenech M. Bonassi S. Turner J. Intra- and inter-laboratory variation in the scoring of micronuclei and nucleoplasmic bridges in binucleated human lymphocytes Mutation Research/genetic Toxicol Environ Mutagen 534 2003 45 64 10.1016/S1383-5718(02)00248-6
22 Fenech M. Kirsch-Volders M. Rossnerova A. HUMN project initiative and review of validation, quality control and prospects for further development of automated micronucleus assays using image cytometry systems Int J Hyg Environ Health 216 2013 541 552 10.1016/j.ijheh.2013.01.008 23507632
23 Fenech M. Important variables that influence base-line micronucleus frequency in cytokinesis-blocked lymphocytes-a biomarker for DNA damage in human populations Mutat Res 404 1998 155 165 10.1016/s0027-5107(98)00109-2 9729354
24 Surrallés J. Natarajan A.T. Human lymphocytes micronucleus assay in Europe. An International Survey Mutat Res 392 1997 165 174 10.1016/s0165-1218(97)00054-2 9269340
25 Venkatachalam P. Solomon F.D. Prabhu B.K. Mohankumar M.N. Gajendiran N. Jeevanram R.K. Estimation of dose in cancer patients treated with fractionated radiotherapy using translocation, dicentrics and micronuclei frequency in peripheral blood lymphocytes Mutat Res 429 1999 1 12 10.1016/s0027-5107(99)00096-2 10434020
26 Romm H. Barnard S. Boulay-Greene H. Laboratory Intercomparison of the Cytokinesis-Block Micronucleus Assay Radiat Res 180 2013 120 128 10.1667/RR3234.1 23862731
27 Wilkins R.C. Beaton-Green L.A. Lachapelle S. Evaluation of the annual Canadian biodosimetry network intercomparisons Int J Radiat Biol 91 2015 443 451 10.3109/09553002.2015.1012305 25670072
28 Depuydt J. Baeyens A. Barnard S. RENEB intercomparison exercises analyzing micronuclei (Cytokinesis-block Micronucleus Assay) Int J Radiat Biol 93 2017 36 47 10.1080/09553002.2016.1206231 27673504
29 Granath F. Darroudi F. Auvinen A. Retrospective dose estimates in Estonian Chernobyl clean-up workers by means of FISH Mutat Res 369 1996 7 12 10.1016/s0165-1218(96)90042-7 8700185
30 Virsik-Peuckert R.P. Harder D. Temperature and the formation of radiation-induced chromosome aberrations. II. The temperature dependence of lesion repair and lesion interaction Int J Radiat Biol Relat Stud Phys Chem Med 49 1986 673 681 10.1080/09553008514552921 3485609
31 Moroni MM, Krasnopolsky K, Subramanian U, Martin PR, Doherty KM, S PG, et al. *Corresponding Author: 2008.
32 Belloni P. Meschini R. Palitti F. Effects of storage conditions of human whole blood on the viability of lymphocytes Int J Radiat Biol 84 2008 613 619 10.1080/09553000802203630 18661377
33 Gonsebatt M.E. Mutchinick O. Human lymphocyte proliferation kinetics in Hanks’ BSS supplemented with autologous plasma and in synthetic medium Mutat Res 243 1990 255 258 10.1016/0165-7992(90)90140-f 2325691
34 Gantenberg H.W. Wuttke K. Streffer C. Müller W.U. Micronuclei in human lymphocytes irradiated in vitro or in vivo Radiat Res 128 1991 276 281 1961924
35 Silva M.J. Carothers A. Dias A. Luis J.H. Piper J. Boavida M.G. Dose dependence of radiation-induced micronuclei in cytokinesis-blocked human lymphocytes Mutat Res 322 1994 117 128 10.1016/0165-1218(94)00019-0 7519318
36 Surrallés J. Carbonell E. Marcos R. Degrassi F. Antoccia A. Tanzarella C. A collaborative study on the improvement of the micronucleus test in cultured human lymphocytes Mutagenesis 7 1992 407 410 10.1093/mutage/7.6.407 1474915
37 Prosser J.S. Moquet J.E. Lloyd D.C. Edwards A.A. Radiation induction of micronuclei in human lymphocytes Mutat Res 199 1988 37 45 10.1016/0027-5107(88)90228-x 3362163
38 Hoffmann G.R. Sayer A.M. Littlefield L.G. Higher frequency of chromosome aberrations in late-arising first-division metaphases than in early-arising metaphases after exposure of human lymphocytes to X-rays in G0 Int J Radiat Biol 78 2002 765 772 10.1080/09553000210152962 12428917
39 Krishnaja A.P. Sharma N.K. Differential radiation effects in smokers–culture time dependence of the yield of gamma ray-induced chromosome damage in first division metaphases Int J Radiat Biol 82 2006 363 377 10.1080/09553000600774097 16782654
40 Holmberg K. Meijer A.E. Harms-Ringdahl M. Lambert B. Chromosomal instability in human lymphocytes after low dose rate gamma-irradiation and delayed mitogen stimulation Int J Radiat Biol 73 1998 21 34 10.1080/095530098142671 9464474
41 Joksic G. Petrovic S. Ilic Z. Age-related changes in radiation-induced micronuclei among healthy adults Braz J Med Biol Res 37 2004 1111 1117 10.1590/S0100-879X2004000800002 15273813
42 Bolognesi C. Abbondandolo A. Barale R. Age-related increase of baseline frequencies of sister chromatid exchanges, chromosome aberrations, and micronuclei in human lymphocytes Cancer Epidemiol Biomarkers Prev 6 1997 249 256 9107430
43 Andreassi M.G. Barale R. Iozzo P. Picano E. The association of micronucleus frequency with obesity, diabetes and cardiovascular disease Mutagenesis 26 2011 77 83 10.1093/mutage/geq077 21164186
44 Metcalfe C. Davey Smith G. Macleod J. Hart C. The role of self-reported stress in the development of breast cancer and prostate cancer: a prospective cohort study of employed males and females with 30 years of follow-up Eur J Cancer 43 2007 1060 1065 10.1016/j.ejca.2007.01.027 17336053
45 Huang P. Huang B. Weng H. Nakayama K. Morimoto K. Effects of lifestyle on micronuclei frequency in human lymphocytes in Japanese hard-metal workers Prev Med 48 2009 383 388 10.1016/j.ypmed.2008.12.023 19463494
46 Schiffl C. Zieres C. Zankl H. Exhaustive physical exercise increases frequency of micronuclei Mutat Res 389 1997 243 246 10.1016/s1383-5718(96)00154-4 9093390
47 Hartmann A. Pfuhler S. Dennog C. Germadnik D. Pilger A. Speit G. Exercise-Induced DNA Effects in Human Leukocytes Are Not Accompanied by Increased Formation of 8-Hydroxy-2′-Deoxyguanosine or Induction of Micronuclei Free Radic Biol Med 24 1998 245 251 10.1016/S0891-5849(97)00249-9 9433899
48 Umegaki K. Higuchi M. Inoue K. Esashi T. Influence of one bout of intensive running on lymphocyte micronucleus frequencies in endurance-trained and untrained men Int J Sports Med 19 1998 581 585 10.1055/s-2007-971963 9877151
49 Pittaluga M. Parisi P. Sabatini S. Cellular and biochemical parameters of exercise-induced oxidative stress: relationship with training levels Free Radic Res 40 2006 607 614 10.1080/10715760600623015 16753838
50 Reichhold S. Neubauer O. Ehrlich V. Knasmüller S. Wagner K.-H. No acute and persistent DNA damage after an Ironman triathlon Cancer Epidemiol Biomarkers Prev 17 2008 1913 1919 10.1158/1055-9965.EPI-08-0293 18708380
51 Bonassi S. Neri M. Lando C. Effect of smoking habit on the frequency of micronuclei in human lymphocytes: results from the Human MicroNucleus project Mutat Res 543 2003 155 166 10.1016/s1383-5742(03)00013-9 12644185
52 Ishikawa H. Yamamoto H. Tian Y. Kawano M. Yamauchi T. Yokoyama K. Effects of ALDH2 gene polymorphisms and alcohol-drinking behavior on micronuclei frequency in non-smokers Mutat Res 541 2003 71 80 10.1016/s1383-5718(03)00179-7 14568296
53 Ishikawa H. Miyatsu Y. Kurihara K. Yokoyama K. Gene-environmental interactions between alcohol-drinking behavior and ALDH2 and CYP2E1 polymorphisms and their impact on micronuclei frequency in human lymphocytes Mutat Res 594 2006 1 9 10.1016/j.mrfmmm.2005.07.005 16126235
54 Ishikawa H. Ishikawa T. Yamamoto H. Fukao A. Yokoyama K. Genotoxic effects of alcohol in human peripheral lymphocytes modulated by ADH1B and ALDH2 gene polymorphisms Mutat Res 615 2007 134 142 10.1016/j.mrfmmm.2006.11.026 17207821
55 Maffei F. Forti G.C. Castelli E. Stefanini G.F. Mattioli S. Hrelia P. Biomarkers to assess the genetic damage induced by alcohol abuse in human lymphocytes Mutat Res 514 2002 49 58 10.1016/s1383-5718(01)00318-7 11815244
56 Dawson D.W. Bury H.P. The significance of Howell-Jolly bodies and giant metamyelocytes in marrow smears J Clin Pathol 14 1961 374 380 10.1136/jcp.14.4.374 13720318
57 Fenech M. Rinaldi J. The relationship between micronuclei in human lymphocytes and plasma levels of vitamin C, vitamin E, vitamin B12 and folic acid Carcinogenesis 15 1994 1405 1411 10.1093/carcin/15.7.1405 8033318
58 Titenko-Holland N. Jacob R.A. Shang N. Balaraman A. Smith M.T. Micronuclei in lymphocytes and exfoliated buccal cells of postmenopausal women with dietary changes in folate Mutation Res./Genet. Toxicol. Environ. Mutagen. 417 1998 101 114 10.1016/S1383-5718(98)00104-1
59 Schneider M. Diemer K. Engelhart K. Zankl H. Trommer W.E. Biesalski H.K. Protective effects of vitamins C and E on the number of micronuclei in lymphocytes in smokers and their role in ascorbate free radical formation in plasma Free Radic Res 34 2001 209 219 10.1080/10715760100300201 11264897
60 Mikhalevich L.S. De Zwart F.A. Perepetskaya G.A. Chebotareva N.V. Mikhalevich E.A. Tates A.D. Radiation effects in lymphocytes of children living in a Chernobyl contaminated region of Belarus Int J Radiat Biol 76 2000 1377 1385 10.1080/09553000050151655 11057746
61 Internationale Atomenergie-Organisation, Internationale Atomenergie-Organisation, editors. Cytogenetic analysis for radiation dose assessment: a manual. Vienna: IAEA; 2001.
62 Beinke C. Ben-Shlomo A. Abend M. Port M. A case report: cytogenetic dosimetry after accidental radiation exposure during (192)Ir industrial radiography testing Radiat Res 184 2015 66 72 10.1667/RR14013.1 26151173
63 Herate C. Sabatier L. Retrospective biodosimetry techniques: Focus on cytogenetics assays for individuals exposed to ionizing radiation Mutation Res/Rev Mutat Res 783 2020 108287 10.1016/j.mrrev.2019.108287
64 Willems P. August L. Slabbert J. Automated micronucleus (MN) scoring for population triage in case of large scale radiation events Int J Radiat Biol 86 2010 2 11 10.3109/09553000903264481 20070210
65 Ainsbury E.A. Al-Hafidh J. Bajinskis A. Inter- and intra-laboratory comparison of a multibiodosimetric approach to triage in a simulated, large scale radiation emergency Int J Radiat Biol 90 2014 193 202 10.3109/09553002.2014.868616 24289146
66 Lee Y. Jin Y.W. Seong K.M. Wilkins R.C. Jang S. Improving radiation dosimetry with an automated micronucleus scoring system: correction of automated scoring errors Radiat Environ Biophys 62 2023 349 356 10.1007/s00411-023-01030-7 37195317
67 Bertucci A. Wilkins R.C. Lachapelle S. Turner H.C. Brenner D.J. Garty G. Comparison of isolated lymphocyte and whole blood-Based CBMN assays for radiation triage Cytogenet Genome Res 2023 1 11 10.1159/000533488 37517400
68 Ting Goh V.S. Fujishima Y. Nakayama R. Manual scoring with shortened 48 h cytokinesis-block micronucleus assay feasible for triage in the event of a mass-casualty radiation accident Radiat Res 2023 199 10.1667/RADE-22-00191.1
69 Verhaegen F. Vral A. Sensitivity of micronucleus induction in human lymphocytes to low-LET radiation qualities: RBE and correlation of RBE and LET Radiat Res 139 1994 208 213 8052697
70 Vral A. Verhaegen F. Thierens H. De Ridder L. Micronuclei induced by fast neutrons versus 60Co gamma-rays in human peripheral blood lymphocytes Int J Radiat Biol 65 1994 321 328 10.1080/09553009414550381 7908311
71 Wuttke K. Müller W.U. Streffer C. The sensitivity of the in vitro cytokinesis-blocked micronucleus assay in lymphocytes for different and combined radiation qualities Strahlenther Onkol 174 1998 262 268 10.1007/BF03038719 9614955
72 Gersey B. Sodolak J. Hada M. Micronuclei induction in human fibroblasts exposed in vitro to Los Alamos high-energy neutrons Adv Space Res 40 2007 1754 1757 10.1016/j.asr.2007.03.018
73 Fenech M. Denham J. Francis W. Morley A. Micronuclei in cytokinesis-blocked lymphocytes of cancer patients following fractionated partial-body radiotherapy Int J Radiat Biol 57 1990 373 383 10.1080/09553009014552471 1968501
74 Thierens H. Vral A. Van Eijkeren M. Speleman F. De Ridder L. Micronucleus induction in peripheral blood lymphocytes of patients under radiotherapy treatment for cervical cancer or Hodgkin’s disease Int J Radiat Biol 67 1995 529 539 10.1080/09553009514550641 7775828
75 le Roux J. Slabbert J. Smit B. Blekkenhorst G. Assessment of the micronucleus assay as a biological dosimeter using cytokinesis-blocked lymphocytes from cancer patients receiving fractionated partial body-radiotherapy Strahlenther Onkol 174 1998 75 81 10.1007/BF03038479 9487369
76 Lee T.K. O’Brien K.F. Naves J.L. Micronuclei in lymphocytes of prostate cancer patients undergoing radiation therapy Mutat Res 469 2000 63 70 10.1016/s1383-5718(00)00072-3 10946243
77 Silva-Barbosa I. Pereira-Magnata S. Amaral A. Sotero G. Melo H.C. Dose assessment by quantification of chromosome aberrations and micronuclei in peripheral blood lymphocytes from patients exposed to gamma radiation Genet Mol Biol 28 2005 452 457 10.1590/S1415-47572005000300021
78 Livingston G.K. Foster A.E. Elson H.R. Effect of in vivo exposure to iodine-131 on the frequency and persistence of micronuclei in human lymphocytes J Toxicol Environ Health 40 1993 367 375 10.1080/15287399309531802 8230307
79 Monsieurs M.A. Bacher K. Brans B. Patient dosimetry for 131I-lipiodol therapy Eur J Nucl Med Mol Imaging 30 2003 554 561 10.1007/s00259-002-1108-7 12690415
80 Iarmarcovai G. Ceppi M. Botta A. Orsière T. Bonassi S. Micronuclei frequency in peripheral blood lymphocytes of cancer patients: a meta-analysis Mutat Res 659 2008 274 283 10.1016/j.mrrev.2008.05.006 18582599
81 Livingston G.K. Jensen R.H. Silberstein E.B. Radiobiological evaluation of immigrants from the vicinity of Chernobyl Int J Radiat Biol 72 1997 703 713 10.1080/095530097142861 9416793
82 Tanaka K. Iida S. Takeichi N. Unstable-type chromosome aberrations in lymphocytes from individuals living near semipalatinsk nuclear test site JRR 47 2006 A159 A164 10.1269/jrr.47.A159
83 Thierens H. Vral A. De Ridder L. A cytogenetic study of radiological workers: effect of age, smoking and radiation burden on the micronucleus frequency Mutat Res/Environ Mutagen Related Subject 360 1996 75 82 10.1016/0165-1161(95)00058-5
84 Thierens H. Vral A. Barbé M. Aousalah B. De Ridder L. A cytogenetic study of nuclear power plant workers using the micronucleus-centromere assay Mutat Res 445 1999 105 111 10.1016/s1383-5718(99)00134-5 10521696
85 Thierens H. Vral A. Morthier R. Aousalah B. De Ridder L. Cytogenetic monitoring of hospital workers occupationally exposed to ionizing radiation using the micronucleus centromere assay Mutagenesis 15 2000 245 249 10.1093/mutage/15.3.245 10792018
86 Thierens H. Inter-laboratory comparison of cytogenetic endpoints for the biomonitoring of radiological workers Int J Radiat Biol 75 1999 23 34 10.1080/095530099140771 9972788
87 Cytogenetic monitoring of Workers from a Nuclear Power Plant n.d. https://www.nnk.gov.hu/cejoem/Volume3/Vol3No1/ce971-4.html (accessed April 29, 2022).
88 Reddy A. Rebekha A. Modi P. Radiation-induced DNA damage in patients exposed to X-rays during cerebral angiography Int J Low Radiat 7 2010 10 19 10.1504/IJLR.2010.032767
89 Basheerudeen S.A.S. Murtaza S. Raavi V. Assessment of early and late DNA damages in interventional radiologists exposed to protracted low dose and dose rate of X-radiation Int J Low Radiat 10 2016 198 209 10.1504/IJLR.2016.081452
90 Kanagaraj K, Abdul Syed Basheerudeen S, Tamizh Selvan G, Jose MT, Ozhimuthu A, Panneer Selvam S, et al. Assessment of dose and DNA damages in individuals exposed to low dose and low dose rate ionizing radiations during computed tomography imaging. Mutat Res Genet Toxicol Environ Mutagen 2015;789–790:1–6. doi: 10.1016/j.mrgentox.2015.05.008.
91 Basheerudeen S.A.S. Kanagaraj K. Jose M.T. Entrance surface dose and induced DNA damage in blood lymphocytes of patients exposed to low-dose and low-dose-rate X-irradiation during diagnostic and therapeutic interventional radiology procedures Mutat Res/genetic Toxicol Environ Mutagenes 818 2017 1 6 10.1016/j.mrgentox.2017.04.001
92 Khattab M. Walker D.M. Albertini R.J. Frequencies of micronucleated reticulocytes, a dosimeter of DNA double-strand breaks, in infants receiving computed tomography or cardiac catheterization Mutat Res/genetic Toxicol Environ Mutagenes 820 2017 8 18 10.1016/j.mrgentox.2017.05.006
93 Tian X.-L. Lu X. Cai T.-J. Lyu Y.-M. Tian M. Liu Q.-J. Cytogenetic monitoring of peripheral blood lymphocytes from medical radiation professionals occupationally exposed to low-dose ionizing radiation Mutat Res/genet Toxicol Environ Mutagenes 867 2021 503370 10.1016/j.mrgentox.2021.503370
94 14:00-17:00. ISO 13528:2015. ISO n.d. https://www.iso.org/cms/render/live/en/sites/isoorg/contents/data/standard/05/61/56125.html (accessed April 29, 2022).
95 Kulka U. Ainsbury L. Atkinson M. Realising the European network of biodosimetry: RENEB-status quo Radiat Prot Dosimetry 164 2015 42 45 10.1093/rpd/ncu266 25205835
96 Voisin P. Standards in biological dosimetry: A requirement to perform an appropriate dose assessment Mutat Res Genet Toxicol Environ Mutagen 793 2015 115 122 10.1016/j.mrgentox.2015.06.012 26520381
97 Christie D.H. Chu M.C. Carr Z. Global networking for biodosimetry laboratory capacity surge in radiation emergencies Health Phys 98 2010 168 171 10.1097/HP.0b013e3181abaad4 20065679
98 Roy L. Buard V. Delbos M. International intercomparison for criticality dosimetry: the case of biological dosimetry Radiat Prot Dosimetry 110 2004 471 476 10.1093/rpd/nch349 15353693
99 Wojcik A. Lloyd D. Romm H. Roy L. Biological dosimetry for triage of casualties in a large-scale radiological emergency:capacity of the EU member states Radiat Prot Dosimetry 138 2010 397 401 10.1093/rpd/ncp279 19951985
100 Di Giorgio M. Barquinero J.F. Vallerga M.B. Biological dosimetry intercomparison exercise: an evaluation of triage and routine mode results by robust methods Radiat Res 175 2011 638 649 10.1667/RR2425.1 21306200
101 Garcia O.F. Ramalho A.T. Di Giorgio M. Intercomparison in cytogenetic dosimetry among five laboratories from Latin America Mutat Res 327 1995 33 39 10.1016/0027-5107(94)00066-e 7870095
102 Miller S.M. Ferrarotto C.L. Vlahovich S. Wilkins R.C. Boreham D.R. Dolling J.-A. Canadian cytogenetic emergency network (CEN) for biological dosimetry following radiological/nuclear accidents Int J Radiat Biol 83 2007 471 477 10.1080/09553000701370860 17538797
103 Yoshida M.A. Hayata I. Tateno H. The Chromosome Network for biodosimetry in Japan Radiat Meas 6–7 2007 1125 1127 10.1016/j.radmeas.2007.05.047
104 Kulka U. Ainsbury L. Atkinson M. Realising the European network of biodosimetry (RENEB) Radiat Prot Dosim 151 2012 621 625 10.1093/rpd/ncs157
105 Bhavani M. Tamizh Selvan G. Kaur H. Dicentric chromosome aberration analysis using giemsa and centromere specific fluorescence in-situ hybridization for biological dosimetry: an inter- and intra-laboratory comparison in Indian laboratories Appl Radiat Isot 92 2014 85 90 10.1016/j.apradiso.2014.06.004 25014548
106 Pan Y, Ruan J, Gao G, Wu L, Piao C, Liu J. Laboratory Intercomparison of Cytogenetic Dosimetry Among 38 Laboratories in China. Dose-Response 2019;17:155932581983347. doi: 10.1177/1559325819833473.
107 Gregoire E. Barquinero J.F. Gruel G. RENEB Inter-Laboratory comparison 2017: limits and pitfalls of ILCs Int J Radiat Biol 97 2021 888 905 10.1080/09553002.2021.1928782 33970757
108 Schreiber G.A. Beisker W. Braselmann H. Bauchinger M. Bögl K.W. Nüsse M. An automated flow cytometric micronucleus assay for human lymphocytes Int J Radiat Biol 62 1992 695 709 10.1080/09553009214552651 1362763
109 Schunck C. Johannes T. Varga D. Lörch T. Plesch A. New developments in automated cytogenetic imaging: unattended scoring of dicentric chromosomes, micronuclei, single cell gel electrophoresis, and fluorescence signals Cytogenet Genome Res 104 2004 383 389 10.1159/000077520 15162069
110 Rodrigues M.A. Probst C.E. Zayats A. The in vitro micronucleus assay using imaging flow cytometry and deep learning Npj Syst Biol Appl 7 2021 20 10.1038/s41540-021-00179-5 34006858
111 Decordier I. Papine A. Plas G. Automated image analysis of cytokinesis-blocked micronuclei: an adapted protocol and a validated scoring procedure for biomonitoring Mutagenesis 24 2008 85 93 10.1093/mutage/gen057 18854579
112 Darzynkiewicz Z. Smolewski P. Holden E. Laser scanning cytometry for automation of the micronucleus assay Mutagenesis 26 2011 153 161 10.1093/mutage/geq069 21164197
113 Repin M. Pampou S. Garty G. Brenner D.J. RABiT-II: A fully-automated micronucleus assay system with shortened time to result Radiat Res 191 2019 232 10.1667/RR15215.1 30657421
114 Wessels J.M. Nüsse M. Flow cytometric detection of micronuclei by combined staining of DNA and membranes Cytometry 19 1995 201 208 10.1002/cyto.990190303 7537648
115 Roman D. Locher F. Suter W. Cordier A. Bobadilla M. Evaluation of a new procedure for the flow cytometric analysis of in vitro, chemically induced micronuclei in V79 cells Environ Mol Mutagen 32 1998 387 396 9882014
116 Avlasevich S. Bryce S. De Boeck M. Flow cytometric analysis of micronuclei in mammalian cell cultures: past, present and future Mutagenesis 26 2011 147 152 10.1093/mutage/geq058 21164196
117 Bryce S.M. Avlasevich S.L. Bemis J.C. Interlaboratory evaluation of a flow cytometric, high content in vitro micronucleus assay Mutat Res/genet Toxicol Environ Mutagenes 650 2008 181 195 10.1016/j.mrgentox.2007.11.006
118 Nusse M. Kramer J. Flow cytometric analysis of micronuclei found in cells after irradiation Cytometry 5 1984 20 25 10.1002/cyto.990050105 6697820
119 Laingam S. Froscio S.M. Humpage A.R. Flow-cytometric analysis of in vitro micronucleus formation: comparative studies with WIL2-NS human lymphoblastoid and L5178Y mouse lymphoma cell lines Mutat Res/genetic Toxicol Environ Mutagen 656 2008 19 26 10.1016/j.mrgentox.2008.06.015
120 Rodrigues M.A. Automation of the in vitro micronucleus assay using the Imagestream ® imaging flow cytometer. In Vitro MN Assay by Imaging Flow Cytometry Cytometry 93 2018 706 726 10.1002/cyto.a.23493 30118149
121 Tates A.D. van Welie M.T. Ploem J.S. The present state of the automated micronucleus test for lymphocytes Int J Radiat Biol 58 1990 813 825 10.1080/09553009014552191 1977825
122 Castelain Ph. Van Hummelen P. Deleener A. Kirsch-Volders M. Automated detection of cytochalasin-B blocked binucleated lymphocytes for scoring micronuclei Mutagenesis 8 1993 285 293 10.1093/mutage/8.4.285 7690875
123 Eastmond D.A. Tucker J.D. Identification of aneuploidy-inducing agents using cytokinesis-blocked human lymphocytes and an antikinetochore antibody Environ Mol Mutagen 13 1989 34 43 10.1002/em.2850130104 2783409
124 Varga D. Michel I. Patino-Garcia B. Paiss T. Vogel W. Maier C. Radiosensitivity detected by the micronucleus test is not generally increased in sporadic prostate cancer patients Cytogenet Genome Res 111 2005 41 45 10.1159/000085668 16093719
125 Rossnerova A. Spatova M. Rossner P. Solansky I. Sram R.J. The impact of air pollution on the levels of micronuclei measured by automated image analysis Mutat Res/fundament Molecul Mechan Mutagenes 669 2009 42 47 10.1016/j.mrfmmm.2009.04.008
126 Doherty A.T. Hayes J. Fellows M. Kirk S. O’Donovan M. A rapid, semi-automated method for scoring micronuclei in mononucleated mouse lymphoma cells Mutat Res/genetic Toxicol Environ Mutagenes 726 2011 36 41 10.1016/j.mrgentox.2011.08.002
127 Pujol-Canadell M. Perrier J.R. Cunha L. Cytogenetically-based biodosimetry after high doses of radiation PLoS One 15 2020 e0228350 32320391
128 Henriksen M. Quantitative imaging cytometry: instrumentation of choice for automated cellular and tissue analysis Nat Methods 7 2010 i ii 10.1038/nmeth.f.302
129 Smolewski P. Ruan Q. Vellon L. Darzynkiewicz Z. Micronuclei assay by laser scanning cytometry Cytometry 45 2001 19 26 10.1002/1097-0320(20010901)45:1<19::aid-cyto1140>3.0.co;2-g 11598943
130 Diaz D. Scott A. Carmichael P. Shi W. Costales C. Evaluation of an automated in vitro micronucleus assay in CHO-K1 cells Mutat Res 630 2007 1 13 10.1016/j.mrgentox.2007.02.006 17446119
131 Shibai-Ogata A. Kakinuma C. Hioki T. Kasahara T. Evaluation of high-throughput screening for in vitro micronucleus test using fluorescence-based cell imaging Mutagenesis 26 2011 709 719 10.1093/mutage/ger037 21745803
132 Frieauff W. Martus H.J. Suter W. Elhajouji A. Automatic analysis of the micronucleus test in primary human lymphocytes using image analysis Mutagenesis 28 2013 15 23 10.1093/mutage/ges047 23042049
133 Garty G. Chen Y. Salerno A. The rabit: a rapid automated biodosimetry tool for radiological triage Health Phys 98 2010 209 217 10.1097/HP.0b013e3181ab3cb6 20065685
134 Wang Q. Rodrigues M.A. Repin M. Automated triage radiation biodosimetry: integrating imaging flow cytometry with high-throughput robotics to perform the cytokinesis-block micronucleus assay Radiat Res 191 2019 342 10.1667/RR15243.1 30779694
135 Rodrigues M.A. Beaton-Green L.A. Wilkins R.C. Validation of the cytokinesis-block micronucleus assay using imaging flow cytometry for high throughput radiation biodosimetry Health Phys 110 2016 29 36 10.1097/HP.0000000000000371 26606062
136 Rodrigues M.A. Beaton-Green L.A. Wilkins R.C. Fenech M.F. The potential for complete automated scoring of the cytokinesis block micronucleus cytome assay using imaging flow cytometry Mutat Res Genet Toxicol Environ Mutagen 836 2018 53 64 10.1016/j.mrgentox.2018.05.003 30389163
137 Goh V.S.T. Nakayama R. Blakely W.F. Improved harvest and fixation methodology for isolated human peripheral blood mononuclear cells in cytokinesis-block micronucleus assay Int J Radiat Biol 97 2021 194 207 10.1080/09553002.2021.1844338 33135957
138 Sreedevi and Rao 1994. Assay of Micronuclei in Peripheral Blood Lymphocytes as a Biological Indicator of Radiation Dose | Radiation Protection Dosimetry | Oxford Academic n.d. https://academic.oup.com/rpd/article-abstract/51/1/41/1602795?redirectedFrom=fulltext (accessed May 2, 2022).
139 Mill A.J. Wells J. Hall S.C. Butler A. Micronucleus induction in human lymphocytes: comparative effects of X rays, alpha particles, beta particles and neutrons and implications for biological dosimetry Radiat Res 145 1996 575 585 8619023
140 Mitchell J.C. Norman A. The induction of micronuclei in human lymphocytes by low doses of radiation Int J Radiat Biol Relat Stud Phys Chem Med 52 1987 527 535 10.1080/09553008714552031 3499407
141 Paul S.F.D. Venkatachalam P. Jeevanram R.K. A comparative study of synchronised and conventional culture methods on the micronucleus dose–response curve Mutat Res/Genet Toxicol Environ Mutagenes 391 1997 91 98 10.1016/S0165-1218(97)00038-4
142 Voisin P. Benderitter M. Claraz M. The cytogenetic dosimetry of recent accidental overexposure Cell Mol Biol (noisy-Le-Grand) 47 2001 557 564 11441964
143 Pala F.S. Alkaya F. Tabakçioğlu K. The Effects of micronuclei with whole chromosome on biological dose estimation Turk J Biol 32 2008 283 290
144 Acharya S. Sanjeev G. Bhat N.N. Siddappa K. Narayana Y. The effect of electron and gamma irradiation on the induction of micronuclei in cytokinesis-blocked human blood lymphocytes Radiat Environ Biophys 48 2009 197 203 10.1007/s00411-008-0209-5 19142652
145 Bolognesi 2011 MH& S. Micronucleus test for radiation biodosimetry in mass casualty events: Evaluation of visual and automated scoring accessed May 2 MetaSystems N.d. 2022 https://metasystems-international.com/en/publications/bolognesi11/
146 Kormos C. Köteles G.J. Micronuclei in X-irradiated human lymphocytes Mutat Res 199 1988 31 35 10.1016/0027-5107(88)90227-8 3362162
147 Littlefield L.G. Sayer A.M. Frome E.L. Comparisons of dose-response parameters for radiation-induced acentric fragments and micronuclei observed in cytokinesis-arrested lymphocytes Mutagenesis 4 1989 265 270 10.1093/mutage/4.4.265 2674605
148 Thierens H. Vral A. de Ridder L. Biological dosimetry using the micronucleus assay for lymphocytes: interindividual differences in dose response Health Phys 61 1991 623 630 10.1097/00004032-199111000-00005 1752745
149 Venkatachalam.P. Estimation of long term radiation exposure using translocation frequency in chromosoms. University; 1993.
150 Thierens H. Vral A. The micronucleus assay in radiation accidents Ann Ist Super Sanita 45 2009 260 264 19861730
151 Rodrigues M.A. Beaton-Green L.A. Kutzner B.C. Wilkins R.C. Automated analysis of the cytokinesis-block micronucleus assay for radiation biodosimetry using imaging flow cytometry Radiat Environ Biophys 53 2014 273 282 10.1007/s00411-014-0525-x 24604721
152 Tamizh Selvan G. Kanagaraj K. Venkatachalam P. The relative biological effectiveness of high-energy clinical 3 and 6 MV X-rays for micronucleus induction in human lymphocytes Int J Radiat Biol 97 2021 687 694 10.1080/09553002.2021.1906972 33798020
