Radiation Environment and Medicine 2022 Vol.11, No.2 91–103

ReportCytogenetic Biodosimetry in Radiation Emergency Medicine:
4. Overview of Cytogenetic Biodosimetry
Ryo Nakayama1, 2, Yu Abe3, Valerie Goh Swee Ting4, Kai Takebayashi1, 2, Mai Tran Thanh1, 2, 5,
Yohei Fujishima1, Akifumi Nakata6, Kentaro Ariyoshi7, Kosuke Kasai2, Donovan Anderson1,
Hiroyuki Hanada8, Mitsuaki A. Yoshida1, 9, Katsuhiro Ito8 and Tomisato Miura1*
Department of Risk Analysis and Biodosimetry, Institute of Radiation Emergency Medicine, Hirosaki University,
66-1 Hon-cho, Hirosaki, Aomori 036-8564, Japan2
Department of Bioscience and Laboratory Medicine, Hirosaki University Graduate School of Health Sciences,66-1 Hon-cho, Hirosaki, Aomori 036-8564, Japan3
Department of Radiation Biology and Protection, Atomic Bomb Disease Institute, Nagasaki University,
1-12-4 Sakamoto, Nagasaki, Nagasaki 852-8523, Japan
4
Department of Radiobiology, Singapore Nuclear Research and Safety Initiative, National University of Singapore,
1 Create Way, Singapore 138602, Singapore
5Biodosimetry Group, Centre of Radiation Technology and Biotechnology, Dalat Nuclear Research Institute,
1 Nguyen Tu Luc, Ward 8, Dalat City, Lamdong Province, Vietnam
6
Faculty of Pharmaceutical Sciences, Hokkaido University of Science, 15-4-1, Maeda 7-jo, Teine-ku, Sapporo, Hokkaido 006-8585, Japan
7
Center for Integrated Science and Humanities, Fukushima Medical University,
1 Hikariga-oka, Fukushima City, Fukushima 960-1295, Japan
8
Advanced Emergency and Critical Care Center, Hirosaki University Hospital, Hirosaki University,
53 Hon-cho, Hirosaki, Aomori 036-8563, Japan
9
Institute of Chromosome Life Science, 11-5-409, Fukuokachuo 2-Chome, Fujimino-shi, Saitama 356-0031, Japan
1

Received 21 April 2022; revised 18 May 2022; accepted 20 May 2022

Dose estimation is performed to provide physicians individual doses of patients exposed toradiation for medical treatment in radiation emergency medicine. Cytogenetic dose assessment
plays an important role in radiation medicine because it directly analyzes the in vivo responseof exposed patients and accurately estimates acute whole-body exposure. The endpoint used asa biological dosimeter in cytogenetic dose assessment fulfils three requirements: (1) specificity,(2) stability, and (3) dose-dependency. Dicentric chromosome (Dic) assay (DCA) is recognized
as the gold standard in biodosimetry because Dic is an excellent endpoint that meets all threerequirements of a biological dosimeter. In addition to DCA, premature chromosome condensation
assay, cytokinesis block micronucleus assay, and translocation assay are used in cytogenetic dose
assessment. As the endpoints of each assay are different, the most suitable method is selectedaccording to the exposure scenario in terms of partial/whole-body exposure and applicable dose
range. This article outlines the characteristics of cytogenetic dose assessment methods, reagentsused for blood culture, and precautions for harvesting and spreading in chromosome preparation.
Key words: cytogenetic biodosimetry, application, selection, blood culture, harvesting, chromosome
spreading*Tomisato Miura: Department of Risk Analysis and Biodosimetry, Institute of Radiation Emergency Medicine, Hirosaki University, 66-1 Hon-cho, Hirosaki, Aomori 036-8564,
JapanE-mail: tomisato@hirosaki-u.ac.jp
https://doi.org/10.51083/radiatenvironmed.11.2_91
Copyright © 2022 by Hirosaki University. All rights reserved.

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1. Introduction
In radiation medicine, it is important to estimate theradiation dose of an exposed patient to predict possible
radiation-induced diseases and prepare a medical
treatment plan. External radiation dose can be easily
estimated with personal dosimeters. In the case ofradiation workers, they are obliged to carry personal
dosimeters to monitor and record occupational exposure
doses. However, personal dosimeters alone are unable
to evaluate doses from internal exposure. If radioactive
substances are ingested or inhaled into the body,whole body counters, thyroid monitors, lung monitors,
radiochemical analysis, and bioassays are performed
instead1).
On the other hand, if ordinary residents who do
not usually equip personal dosimeters are exposed
to radiation, different methods are used to assess theexternal exposure dose due to ionizing radiation. Physicaldose assessment is first performed by evaluating the riskof exposure based on radiation sources and/or radioactive
substances and calculating external dose from actual
measurements of the ambient dose-rate at the accident
site. However, as physical dose assessment is unable todirectly measure the exposure dose of an exposed patient,biodosimetry is then performed to accurately estimate anindividualʼs exposure as requested by a physician.
Biodosimetry directly measures in vivo response
induced by radiation exposure. There are two main
methods in biodosimetry. The first method is peripheral
blood cell count analysis detecting a decrease in thenumber of peripheral blood leukocytes. In exposed
patients, leukocytes, especially radiation-sensitive
lymphocytes, decrease in a dose-dependent manner afterradiation exposure2). Peripheral blood leukocyte countmeasurement of exposed patients can estimate a roughexposure dose range. However, fluctuations in blood cellcount vary greatly among individuals and is therefore
unreliable as an accurate dosimeter. The second method
is cytogenetic biodosimetry, where the frequency ofchromosome aberrations shows a positive correlation
with the exposure dose and is effective for precise
individual exposure dose assessment. In cytogenetic
dose assessment, chromosome aberrations in peripheral
blood lymphocytes collected from exposed patients areanalyzed, and a pre-constructed dose-response curve iscommonly used to calculate an individualʼs exposed dose3).
There are various assays analyzing different aberration
endpoints in cytogenetic biodosimetry and each assay
is selected according to type of radiation exposure
scenario. Dicentric chromosome assay (DCA)3-5) uses
dicentric chromosomes (Dic), premature chromosome
condensation (PCC) assay6-10) uses ring chromosomes(rings) in G1- or G 2/M-PCC cells, cytokinesis block

micronucleus (CBMN) assay 11-13) uses micronuclei
in binucleated cells, and translocation assay uses
chromosome translocations as endpoints 14, 15). This
paper outlines the preparation of blood cultures for
cytogenetic biodosimetry, the selection of cytogenetic
dose assessment method and the characteristics of eachmethod.
2. Role of biodosimetry in radiation medicine
It is generally accepted that tissues with actively dividing
cells are highly sensitive to radiation exposure16). The
bone marrow, which is a hematopoietic tissue, is very
susceptible to radiation because hematopoietic stem cells
are constantly undergoing cell division. In addition, thegonads that produce gametes and the intestine, whichhave a rapid turnover, are also highly radiation-sensitive
tissues/organs17). On the other hand, as the cell turnoverrate and cell division frequency are low in muscles andthe nervous system, the radiation sensitivity is lower.
Thus, deterministic effects or delayed health outcomesmay vary as particular tissue/organ dose thresholds are
exceeded.
Dose assessments are performed with the aim ofproviding physicians individual exposed doses to develop
treatment plans for exposed patients. Cytogenetic dose
assessment plays an important role in radiation medicine
because it directly analyzes the in vivo response of
exposed patients and accurately estimates the whole-body
exposure dose. However, it is difficult to estimate theabsorbed dose immediately after the patient is admitted
into a medical facility and to estimate an extreme local
exposure dose in the event of a partial body exposure.
Through dose assessment, exposed patients and theirfamilies can better understand their condition andmedical treatment. In addition, it should be noted thatdose assessment can also reduce unnecessary anxiety
when patients and their families are informed that they
were not exposed to any health hazards.
3. Peripheral blood culture
Lymphocyte chromosomes needed for cytogenetic dose
assessment in radiation emergency medicine can beobtained from peripheral blood, which can be collectedfrom exposed patients using optimal blood collection
and management procedures18). However, chromosomescannot be directly observed as peripheral blood
lymphocytes are in G0 phase and are not undergoingcell division. Therefore, in order to analyze chromosome
aberrations in peripheral blood lymphocytes, it isnecessary to stimulate the lymphocytes with a mitogensuch as phytohemagglutinin (PHA) to induce cell
division. Whole blood or freshly isolated peripheral blood

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Fig. 2. Reference ranges for leukocyte fraction in human peripheral
blood. The reference ranges stated for leukocyte fraction are citedfrom the literature21, 22).

Fig. 1. Cell-cycle progression of peripheral blood lymphocytes
during blood culture in DCA.

mononuclear cells (PBMCs) stimulated by PHA aretransformed to blast cells and enter the S phase throughthe G1 phase. It takes more than 24 hours after PHA
stimulation to enter the S phase19, 20). Subsequently, thecell-cycle of lymphocytes progresses to the S phase (6to 8 hours), the G2 phase (4 to 6 hours), and then to themetaphase3). Therefore, cytogenetic dose assessment
requires a blood culture for 34 hours or longer and isgenerally performed for 48 hours. In DCA, colcemid orcolchicine is added to the culture medium to arrest thecell-cycle in metaphase, and metaphases from the firstcell-cycle after PHA stimulation are analyzed (Fig. 1).
In blood culture, serum that supports cell growth
and antibiotics that suppress bacterial growth are also
added to the basal medium suitable for blood culture. An
appropriate volume of peripheral blood is added to the
supplemented culture medium with a suitable mitogen.
Cells can be cultured in two methods with peripheral
blood collected using heparin as an anticoagulant. The
first is to directly culture whole blood to a concentrationof about 10% into culture medium, and the second is toisolate PBMCs from whole blood and culture.
3.1. Isolation of PBMCs from whole blood
Whole blood cultures contain nucleated cells such as
neutrophils, lymphocytes and monocytes, and a large
number of red blood cells. Lymphocytes make up 20 –40% of white blood cells (Fig. 2)21). In addition, T cells
make up 56.5 – 84.7% of peripheral blood lymphocytes,
and the proportion of T cells in all nucleated cells is 11.3 –
33.9%22). Therefore, if whole blood is cultured, a relatively
low frequency of metaphases is observed after DCA. To

increase metaphase frequency, isolated PBMCs withdensity gradient centrifugation with an isotonic density
solution with a density of 1.077 g/mL can be cultured
under PHA stimulation as reported by Hayata et al.23).
Although the PBMC culture method involves additional
separation steps before blood culture, an increase in MI
contributes to an improved efficiency of chromosome
observation and metaphase image capturing.
3.2. Preparation of culture medium
In cell culture, serum and antibiotics are added to an
aseptically prepared basal medium. In general, theprepared culture medium should be stored in 4 °Crefrigerator and used within 1 month.
3.2.1. Culture medium
For chromosome analysis in peripheral blood lymphocyte
culture, the cell culture medium developed by Roswell
Park Memorial Institute (RPMI) modified from McCoyʼs
5A medium is recommended (RPMI 1640). Sodium
bicarbonate in RPMI 1640 helps to maintain osmoticpressure and neutralize culture medium pH during the
incubation period (i.e. the bicarbonate salt equilibrates CO2
gas from CO2 incubator and carbonate ions in the culture
medium). 4-(2-hydroxyethyl)-1-piperazineethanesulfonicacid (C8H18N2O4S; HEPES), one of the twenty widelyused Goodʼs buffering agents, may also be added toincrease the buffer capacity of culture media between
the pH range of 6.8 to 8.2. The addition of HEPES isuseful because the pH of the medium changes due to cell
metabolism and aerobic respiration altering CO2 levels
in PBMC culture. It is thus highly recommended to use
RPMI 1640 with HEPES for long culture periods (e.g., 72hour culture) as it is commercially available.
3.2.2. Serum
Animal-derived sera rich in growth factors and nutrients

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Fig. 3. Comparison of non-inactivated (both leftmost conical tubes)
and heat-inactivated (both rightmost conical tubes) sera in whole
blood cultures. Displayed is human peripheral blood cultured in using
media supplemented with PHA, kanamycin sulfate, and 20% noninactivated fetal bovine serum (FBS) or heat-inactivated FBS for 48 h.

are used to support lymphocyte growth in eukaryotic
cell culture (e.g., fetal bovine serum). Animal-derived
sera are provided unique lot numbers from a single
manufacturer, and it is highly recommended each lot
number is preliminary tested for optimal cell growth prior
to purchase. In peripheral blood cultures, serum is added
at a final concentration of 15 – 20%3). As the commerciallyavailable serum used for cell culture has been sterilized,
sterilization of the serum is not necessary. If non-sterile
serum is used, the serum should be sterilized with a
0.45 or 0.22 µm membrane filter. In addition, as thephysiological substances in the serum are inactivated athigh temperatures, serum should not be treated at hightemperatures with an autoclave.
Furthermore, serum contains complement proteins. As
complement proteins have opsonin and cell-lytic activities,
it is necessary to inactivate them by heating (56 °C for
30 minutes) before using it for cell culture. Temperatureand treatment time must be appropriately controlled
because the physiological substances in the serum could
be inactivated due to prolonged treatment. In addition,complement proteins are involved in blood coagulation24).
If whole blood culture is used for chromosome aberration
analysis, blood coagulation may occur if serum isuntreated. To prevent this from occurring, heatinactivated serum should be used (Fig. 3).
3.2.3. Antibiotics
Blood is basically sterile in vivo. However, when bloodis collected from a septic patient or when blood iscontaminated at the time of collection, the bacteria will
proliferate during the blood culture and hinder cell
division, greatly reducing metaphases for chromosome
aberration analysis. T herefore, in blood culture,

Fig. 4. Differences in PHA responsiveness between lymphocyte
subsets during cell culture. PBMCs were cultured in the presenceof PHA for indicated periods. After cultivation, cells were stainedwith propidium iodide and cell-cycle status was analyzed using a flow
cytometer.

antibiotics are added to the culture medium in orderto suppress bacterial growth. Even though antibiotics
have their own antibacterial spectrum, antibiotics whichshow antibacterial activity against a wide range ofbacterial species are used in blood cultures. In general,a mixed solution of penicillin G potassium salt, whichis effective against gram-positive bacteria due to its
cell wall synthesis inhibitory action, and streptomycin
sulfate, which is effective against gram-positive and gramnegative bacteria due to its protein synthesis inhibitory
action, is used for mammalian cell culture. However,as penicillin and streptomycin are ineffective against
mycoplasma, the authors recommend kanamycin sulfate,
which has antibacterial activity against gram-positive andgram-negative bacteria and mycoplasma25). When using
these antibiotics, they should be used at appropriate
concentrations which are not cytotoxic.
3.3. MitogenA lectin is a protein or glycoprotein that exists in plants,
animals, and microorganisms, which has specific binding
activity to sugar chains. Some plant-derived lectinsbind to sugar chains on the surface of lymphocytes andhave lymphocyte stimulating activity. The discoveryof phytohemagglutinin (PHA), a type of lectin, greatly
facilitated human chromosome analysis 26-28). PHA
has both hemagglutination activity and lymphocyte
stimulating activity due to its protein structure29-31).
PHA is a tetrameric glycoprotein made up of different
combinations of E-subunits with hemagglutination activity
and L subunits with lymphocyte-stimulating activity.
Although the details of the lymphocyte stimulation
mechanism by PHA remain unclear, PHA preferably
stimulates T cell division in peripheral blood cultures(Fig. 4). Therefore, in cytogenetic biodosimetry inwhich peripheral blood is cultured in the presence of
PHA, chromosome aberrations in T cells assumed to beRyo Nakayama et al./ Radiation Environment and Medicine 2022 Vol.11, No.2 91–103

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Table 1. Comparison of mitogens with lymphocyte stimulating activity
Mitogens

Stimulated lymphocytes

SourcesClassification

Phytohemagglutinin (PHA)

T cellBean of Phaseolus vulgaris

Lectin (glycoprotein)

Concanavalin A (ConA)

T cellBean of Canavalia ensiformi

Lectin (glycoprotein)

Pokeweed mitogen (PWM)

T and B cells

Roots of Phytolacca americana

Lectin (glycoprotein)

Lipopolysaccharide (LPS)

T and B cells

Cell wall of gram-negative bacteria, such as Escherichia
coli, Salmonella etc.

Endotoxin

Fig. 5. Ventilation and test tube angle during blood culture.

analyzed. In addition to PHA, other mitogens may be used
depending on the experiment, such as other lectins ofconcanavalin A and Pokeweed mitogen, and the cell wallcomponent of gram-positive bacteria lipopolysaccharide(Table 1).
3.4. Culture condition
When performing blood culture for cytogenetic
biodosimetry, it is convenient to culture cells in acentrifuge tube (e.g., 15 ml conical tubes) that can becentrifuged immediately after the culture is completed.
Blood should also be cultured in a vessel with gas
exchange. However, as 15 ml conical tubes withventilation caps are not commercially available, cells
should be cultured in a semi-open state without closing
the cap completely. In addition, conical tubes during blood
culture can be tilted diagonally to increase the surface
area of the culture solution in contact with CO2 gas tosustain a neutral pH environment suitable for cell culture(Fig. 5).
4. Methods in cytogenetic biodosimetry
The endpoint used as a biological dosimeter in cytogenetic
biodosimetry has the following three requirements: (1)specificity: a reaction that occurs specifically in radiation
exposure, (2) stability: stable detection is possible for acertain period after accident and (3) dose-dependency:
the exposure dose and the reaction amount (frequency)
show a correlation (Fig. 6). DCA is recognized as the gold
standard in biodosimetry of acute exposures because Dic

Fig. 6. Three requirements as a biological dosimeter in biodosimetry.

is an excellent endpoint that meets all three requirementsof a biological dosimeter 32).
4.1. Comparison and selection of cytogenetic dose assessment
methods
In addition to DCA, CBMN, PCC and chromosometranslocation assays are also used as cytogenetic dose
assessment methods (Table 2, Fig. 7). As each assay
differs in factors such as endpoints analyzed, applicable
dose range, and if the assay is suitable for partial bodydose estimation, it must be carefully selected according to
the exposure scenario. Furthermore, in DCA and CBMN
assays, international guidelines recommend scoringa reduced number of cells to report preliminary dose
estimates for triage4, 5, 33). The authors use the following
criteria when selecting a suitable cytogenetic dose
assessment method: (1) period from radiation exposure
to blood sampling, (2) exposure dose expected fromprodromal symptoms and (3) number of exposed persons
requiring cytogenetic biodosimetry (Fig. 8).
When performing DCA, blood needs to be collected
within 28 days after the accident because Dic is an
unstable chromosome aberration. As time passes after
the accident, the number of Dic-positive cells decreases
and the exposure dose is likely underestimated3, 34-36).
Therefore, if the blood is collected more than one monthafter the accident, the chromosome translocation assay
is the only applicable method for dose estimation withretrospective dosimetry. On the other hand, in high doseexposed patients, blood should be collected promptly as
the number of peripheral blood lymphocytes decreases

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Table 2. Comparison of cytogenetic biodosimetry assays
Cytogenetic biodosimetry assays
Dicentric chromosome
assay

PCC-ring assay

CBMN assay

FISH translocation
assay

Aberration endpoint

Dicentrics

Rings

Micronuclei

Secondary index (Cell-cycle
progression)

Mitotic index (MI)PCC index
Cell-cycle progression
index (CPI)

Nuclear division index(NDI)

Translocations

Radiation scenario
application

Acute
Protracted
Recent exposure

Acute
Recent exposure

AcuteProtracted
Recent exposure

AcuteProtracted
Old exposure

Dose range for whole-body
exposure application

0.1 – 5 Gy

5 – 20 Gy

0.3 – 4 Gy

0.25 – 4 Gy

Staining and labeling

・Giemsa staining
・Modified C-banding
・P N A - F I S H u s i n g
pa n -cent romere a nd
telomere probes

・Giemsa staining

・Giemsa staining
・FISH using whole・Diff-Quik staining
chromosome painting
・DAPI (DAPI/PI) staining
probes・Acridine Orange staining

Blood collection afterradiation exposure

24 h – 28 days

Asap

24 h – 28 days

24 h – (few years)

Blood storable period< 72 hAsap< 72 h< 72 hBlood culture time

48 h

4-6 h (fusion PCC)
48 h (chemical PCC)

48 or 72 h

48 h

Useful for partial-body
exposure application?

Yes

Yes

NA*

Yes

Useful for triage dose
assessment?

Yes

Yes

Yes

NA

Status of assay
standardization

ISO 19238: 20144)
ISO 21243: 2008 (triage)5)NA

ISO 17099: 201433)

ISO 20046: 201975)

Table was modified from IAEA EPR-Biodosimetry3)*NA: not applicable/not available.

Fig. 7. Representative images of chromosome aberrations observed in the irradiated human peripheral lymphocytes. A:
metaphase with dicentric chromosome (Dic) and acentric fragment (Frag), B: G2/M-PCC cell with PCC-rings, C: binucleatedcells with 2 micronuclei (MNs), and D: metaphase with reciprocal chromosome translocation between chromosomes #1 and #2.

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Fig. 8. Flowchart used when selecting a suitable assay for cytogenetic biodosimetry.

Fig. 9. Estimated time required for DCA if a small number of samples is processed after a radiological accident. The numbers indicate the time(h) required for each process.

immediately2). Moreover, cell division tends to arrest at
the G2 phase during the blood culture period, making
it difficult to obtain metaphase cells37). Therefore, inpatients exposed to high dose radiation, the PCC assay
is used where chromatin in interphase cells is artificiallycondensed6-10).
Unfortunately, the disadvantage in DCA is the longer
length of time required for chromosome analysis inindividual cells5). When a large-scale radiation exposure
occurs, the CBMN assay could be more effective as
micronuclei are easier to analyze and automated scoring
is available38, 39). However, the applicable lower limit
exposure dose differs depending on the spontaneous
frequency of each endpoint and the inf luence offactors other than radiation exposure. The frequencyof spontaneous occurrence of Dic is extremely low
and is not affected by age, gender and smoking40). The
applicable lower limit exposure dose for CBMN assay andchromosome translocation assay is higher than that for
DCA because micronuclei and chromosome translocations
are affected by age, gender and smoking41, 42).

The estimated exposure dose assumes that the wholebody is homogeneously exposed to radiation exposure
although most exposure cases show non-uniform dose
distribution. In extremely biased exposure (partial body
exposure), dose calculation suitable for partial exposure
can be performed with DCA 43-45) and PCC assay 46).
However, CBMN assay is unable to accurately estimate
partial body exposure.
In cytogenetic dose assessment, G0 phase lymphocytes
are stimulated with PHA and cultured for chromosome
analysis. Apart from cell fusion-induced PCC (fusionPCC)47-49) assay, 48 to 72 hours of blood culture is requiredfor cytogenetic biodosimetry. As a result, 3 to 5 days after
blood collection is usually required to report estimateddoses.
4.2. Characteristics of DCA
A notable point of DCA is the low f requency of
spontaneous Dic occurrence. The number of spontaneous
Dic per 1000 metaphases is less than one and the radiation
exposure specificity is extremely high. In addition,

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consideration of factors such as age, gender, and smoking
history, are not necessary because Dic is a consequence
typically only induced by radiation exposure unlike
CBMN and chromosome translocation assays40-42).
Understanding the morphological characteristics ofhuman chromosomes is essential for Dic analysis. The
number of chromosomes in the chromosome group(s)of chromosomes involved in Dic formation need to beverified to confirm Dic scoring (e.g., if Dic is formed
with a D/G group chromosome, the number of D and G
group chromosomes need to be verified)15, 50). In recent
years, centromeres can be labelled with fluorescence insitu hybridization (FISH) using a centromere-specificfluorescence-labeled DNA painting probe, and it has
become possible to improve the detection accuracy of
Dic51, 52). However, in order to quickly score Dic by triage
and provide physicians with a preliminary estimateddose5), scoring on Giemsa-stained specimens is preferred
and training is essential to ensure accurate scoring.
Figure 9 shows the time required for a typical DCA
performed after radiation exposure. Blood is collected
24 hours after exposure18). After culturing the blood for
48 hours, the estimated dose is reported to the physician
through a process of cell harvesting (hypotonic treatment
and fixation), image capturing, chromosome aberration
analysis and dose calculation. The preliminary dose
report takes at least 78 hours from the occurrence of theradiation exposure accident (54 hours from blood receipt),and the final estimated dose report takes 129 to 153 hours(105 to 129 hours after receipt of blood).
4.3. Characteristics of PCC assay
The DCA is the preferred cytogenetic dose assessment
method in most exposure cases, but it is not recommended
at high dose exposures above the applicable dose limitof 5 Gy because severe DNA damage causes cell-cycle
arrest and/or delay. This causes a marked shortage of
metaphase cell numbers, making Dic analysis difficult53-55).
The PCC assay is thus preferred in high dose exposure
situations as chromatin in G1 and G2 cells is artificiallycondensed and chromosome aberrations, such as ring
chromosomes (PCC-ring, Fig. 7B), induced by radiation
exposure can be observed10). While this assay is capableof covering a wide dose range (0.2-20 Gy)3, 56-58), theapplicable dose range depends on the endpoint analyzed.
When PCC-ring is used as the endpoint, the applicable
dose range is 5 to 20 Gy57) (Table 2).
There are two types of PCC assays: the fusion-PCC
method and the chemically induced PCC (chemical-PCC)
method. In the former, a cell fusion agent (polyethylene
glycol) is used to fuse peripheral blood lymphocytes with
M-phase cells of mammalian cells (i.e., Chinese hamster
ovary cells (CHO)) to induce G1-PCC cells47-49). In thelatter, phosphatase inhibitors such as calyculin A induce

chromosome condensation in blast cells in each cell-cycle.
When high-dose exposure is suspected due to the accident
description or the patientʼs prodromal symptoms, both
DCA and PCC assay can be performed together for dose
assessment of a wide range of exposed doses6, 7, 10), suchas in the Tokai-mura JCO criticality nuclear accident59).
4.4. Characteristics of CBMN assay
In anaphase of cell division, kinetochore (centromere)
microtubules are attached to the centromere sequencespecific protein complex (kinetochore protein) of
chromosomes convened on the metaphase plate. The
chromatid is pulled to the polar side of the spindle
with the help of motor proteins. Chromatids separated
into both poles decondense and become the main
component of nuclei in daughter cells. However, aberrant
chromosomes such as acentric fragments and Dic are notnormally distributed and appear as micronuclei (MN) andnucleoplasmic bridges (NPB) in telophase, respectively60).
MN and NPB are difficult to detect because they areobserved in telophase and disappear with multiple cell
divisions61). By using cytochalasin B to inhibit cytokinesis
in CBMN assay, these chromosome abnormalities areeasily detected in binucleated cells (Fig. 7C).
As the CBMN assay targets cells after the telophasein which the nucleus is condensed, it is easier to analyze
than Dic and the development of automated technology
aids in fast analysis for cytogenetic dose assessment62).
MN also appears with genotoxicity and chromosome misseparation, and is used as a useful marker in the field ofcytotoxicity assessment and oncology63). Furthermore,
it was discovered that it reflects cell radiosensitivity
and is involved in the prognosis of tumor treatment64, 65).
The radiation exposure specificity in MN is lower thanthat of Dic, and it is necessary to correct the frequency
of MN occurrence due to factors other than radiation
exposure66, 67). Its applicable dose range is 0.3-4 Gy 3)(Table 2).
4.5. Characteristics of translocation assay
The mis-rejoining of ionizing radiation induced DNA
double-strand breaks results in (a) unstable chromosome
aberrations (e.g., Dic and ring chromosomes) and (b)stable chromosome aberrations (e.g., translocations).
Similar to Dic formations, translocations are proportional
to radiation dose, and theoretically, both aberrations
should have similar frequencies per cell. However, ahigher frequency of translocations occurs in humanperipheral blood lymphocytes68) because of the longer
half-life of translocations than unstable aberrations69-71).
Thus, translocations have been used as an endpoint for
retrospective dosimetry and cytogenetic dosimetry forchronic radiation exposure71-73).
Clinical laboratories routinely use banding methods

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Fig. 10. Main processes from imaging to dose estimation in DCA.

(e.g., G-band or Q-band) to detect translocations inradiation exposed patients. However, as hundreds tothousands of cells need to be analyzed for cytogenetic
dosimetry, these banding methods are not recommended.
Instead, the FISH method using whole-chromosome
painting probes (WCP-FISH) is preferred. WCP for
autosomal chromosomes 1-22 and sex chromosomes X
and Y have been developed to help detect and distinguishchromosome exchanges74). The detectable dose range is
0.25 to 4 Gy3) and was standardized by the International
Organization for Standardization (ISO) in 201975) (Table 2).
It should be noted that the WCP-FISH method requiresa correction for the number of cells analyzed based on
the amount of stained chromosomal DNA as a percentage
of total genomic DNA. Furthermore, the genomic DNA
per chromosome differs slightly between males andfemales76). This is especially important for inter-laboratorycomparisons as the color and number of chromosomes
stained may vary. For example, if three-color FISH
probes specific for chromosomes 1, 2, and 4 are used77),
the number of cells analyzed would be equivalent to197-198 cells when 500 cells are available for analysis78).
Corrections for age and lifestyle habits (e.g., smoking) arenecessary because such factors influenced chromosometranslocation frequencies42, 79).
5. Cell harvest and chromosome spreading
Producing high quality chromosome specimens isparamount in cytogenetic dose assessment. Harvesting
and chromosome spreading are key processes inchromosome analysis where chromosomes packed
in lymphocytes with a diameter of about 8-10 µm areexpanded on a slide glass without overlapping.
5.1. Cell harvest: hypotonic treatment and fixation
DCA, PCC and chromosome translocation assays require
metaphases of moderately expanded chromosomes.
After blood culture, cells are first treated with hypotonic
solution for cell swelling and removal of cell membrane
and cytoplasm. They are then fixed to stop hypotonic

shock and autolysis. It should be noted that the hypotonic
treatment conditions are different between whole bloodand isolated PBMC culture. In addition, as CBMN assay
analyzes binucleated cells, it is necessary to optimize
harvest such that the cell membrane and cytoplasm are
maintained while still expanding the cell area13). Cells withruptured cell membrane and cytoplasm are excluded fromthe analysis because the origin of micronuclei induced byradiation exposure cannot be confirmed.
5.2. Chromosome spreading
After fixation, the cell suspension in the fixative isdropped onto a pre-cleaned slide glass. Chromosome
expansion is affected by temperature and humidity
in the laboratory or humidity chamber, as well as cellconcentration and volume of the cell suspension dropped
on the slide. As the temperature and humidity vary fromregion to region and even seasonally, it is recommended
to check the optimum condition for chromosome
spreading on a regular basis.
In order to adjust the cell concentration in the cellsuspension, cell concentration can be measured usinga hemocytometer or any other cell counting device.
This step can also be substituted by referencing to a
standardized suspension. The authors use a standardized
suspension by adjusting the McFarland standard solution
used in standardize microbial testing to an appropriate
concentration. Chromosome spreading should be
optimized by confirming the cell concentration and the
expanded state of the chromosome with a phase-contrast
microscope before air-drying and staining.
6. Chromosome observation and image capture
An upright optical microscope is typically used to observe
chromosome specimens stained with Giemsa for DCA.
A 60×(63×) or 100×objective lens is used to accurately
determine subtle structural changes. Metaphase cells
are first searched at a low magnification (usually 10×lens). The chosen metaphase is then viewed at a higher
magnification where the number of chromosomes and100

Ryo Nakayama et al./ Radiation Environment and Medicine 2022 Vol.11, No.2 91–103

centromeres are counted and various chromosome
abnormalities are recorded.
To speed up the process of Dic scoring, a device
has been developed which scans metaphase cells witha low-magnification objective lens and automaticallyphotographs selected metaphase cells with a highmagnification objective lens. The widespread usageof this device in biodosimetry laboratories globallyhas significantly reduced the time required to capture
metaphase images. In addition, as the image files havebeen digitized, image files can be easily shared between
biodosimetry laboratories around the world.
T he main steps f rom image capturing to dose
estimation in DCA are provided in Figure 10. The image
files taken with the high-magnification objective lens are
expanded on a PC monitor and metaphases that meet theconditions to be analyzed are selected by first checking
the thumbnail images. Then, on selected metaphases,
centromeres are scored and various chromosome
aberrations are recorded. Finally, the estimated dose iscalculated from the dose-response curve based on the Dic
frequency.
The preparation of good quality chromosome spreads
is thus of utmost importance in the accurate analysis ofchromosome aberrations. The condition of chromosome
spreads should be observed on the glass slide with aphase contrast microscope frequently as the environmentof each biodosimetry laboratory could change and affectmetaphase quality.

7. Conclusion
In cytogenetic biodosimetry where endpoints analyzed
are chromosome aberrations caused by radiation
exposure, peripheral blood lymphocytes are stimulatedwith PHA in blood culture for cell-cycle entry. Therefore,a culture solution suitable for lymphocyte proliferation
and reagents for supporting lymphocyte proliferation
should be appropriately prepared. Cytogenetic dose
assessment can be performed in DCA, PCC, CBMN andchromosome translocation assays. An assay suitablefor dose assessment must be selected depending on
the radiation exposure scenario, such as the time from
accident to blood sampling, exposure dose estimatedfrom prodrome, and number of patients requiring dose
assessment.
In cytogenetic dose assessment, it is essential toprepare high quality chromosome spreads for accurate
dose estimation. Optimal conditions for preparing
chromosome spreads va r y w it h en v ironment a lconditions. At the time of specimen preparation, a phase
contrast microscope is highly recommended to confirm
chromosome spreads, cell concentration and the presenceor absence of cytoplasm.
Dose assessment in radiation medicine aims to provide
physicians with dose information to develop treatment
plans for exposed patients. Exposed patients and theirfamilies can understand their condition and treatmentusing dose assessment. In addition, dose assessment canalso reduce anxiety when patients and their families are
informed their exposure will not cause deterministic
health effects.

Point 1. Role of cytogenetic biodosimetry• Cytogenetic biodosimetry provides physicians withdose information to develop medical treatment plansfor exposed patients.
• Exposed patients and their families can understandtheir condition and medical treatment through dose
assessment.
• Dose assessment can also reduce unnecessary anxiety
if patient is assessed to have no/low exposure.
Point 2. Requirements for biological dosimeter• Specificity: a reaction that occurs specifically inradiation exposure.
• Stability: stable detection is possible for a certain
period after accident.
• Dose-dependency: the exposure dose and theendpoint frequency show a correlation.
Point 3. Selection of cytogenetic dose assessment
methods• DCA is the gold standard for biodosimetry.
• Applicable assay(s) is selected from DCA, PCC,
CBMN and chromosome translocation assays
depending on the type of exposure scenarios (1) to(3).
(1)Period from accident to blood sampling(2)Exposure dose estimated f rom prodromal
symptoms(3)Number of patients requiring dose assessment
Point 4. Blood culture• Blood culture medium is supplemented with serum
and antibiotics for blood culture.
• Serum should be inactivated by heating at 56 ℃ for
30 minutes.
• Antibiotics with a broad antibacterial spectrum is
recommended.
• PHA is added to stimulate the proliferation of
peripheral blood lymphocytes.
Point 5. Harvesting, spreading and chromosome
observation• Hypotonic treatment conditions differ between whole
blood culture and isolated PBMC culture.

Ryo Nakayama et al./ Radiation Environment and Medicine 2022 Vol.11, No.2 91–103

CBMN assay requires the preservation of cell
membranes and cytoplasm.
• Cell expansion conditions vary depending on thetemperature and humidity in the laboratory.
• It is important to adjust the final cell concentration of
the fixed cell suspension.
• Good quality specimen preparation is essential for
accurate cytogenetic biodosimetry.
• Status of chromosome spreading should befrequently observed under the microscope to ensure
consistently good metaphase spreads.
•

Conflict of Interest
The authors declare that they have no conflict ofinterests.
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