Radiation Environment and Medicine 2023 Vol.12, No.2 121–139

ReportCytogenetic Biodosimetry in Radiation Emergency Medicine:
5. The Dicentric Chromosome and its Role in Biodosimetry
Donovan Anderson1, Yu Abe2, Valerie Goh Swee Ting3, Ryo Nakayama1, 4, Kai Takebayashi1, 4,
Mai Tran Thanh1, 4, 5, Yohei Fujishima1, Akifumi Nakata6, Kentaro Ariyoshi7, Kosuke Kasai4,
Mitsuaki A. Yoshida1, 8 and Tomisato Miura1*
Department of Risk Analysis and Biodosimetr y, Institute of Radiation Emergency Medicine, Hirosaki University,
66-1 Hon-cho, Hirosaki, Aomori 036-8564, Japan2
Department of Radiation Biology and Protection, Atomic Bomb Disease Institute, Nagasaki University,
1-12-4 Sakamoto, Nagasaki, Nagasaki 852-8523, Japan3
Department of Radiobiology, Singapore Nuclear Research and Safety Initiative, National University of Singapore,
1 Create Way, Singapore 138602, Singapore
4
Department of Bioscience and Laborator y Medicine, Hirosaki University Graduate School of Health Sciences,66-1 Hon-cho, Hirosaki, Aomori 036-8564, Japan
5
Biodosimetr y 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 f or Integrated Science and Humanities, Fukushima Medical University, 1 Hikariga-oka, Fukushima City, Fukushima 960-1295, Japan
8
Institute of Chromosome Life Science, 11-5-409, Fukuokachuo 2-Chome, Fujimino-shi, Saitama 356-0031, Japan
1

Received 3 October, 2022; revised 8 February, 2023; accepted 2 March, 2023

In the case of nuclear or radiological emergencies, biodosimetry has been used to estimate
radiation dose to exposed persons and provide information to physicians for clinical treatment
and counselling of possible future stochastic consequences. There are currently several biological
endpoints and techniques available for assessing partial or whole-body radiological exposure in
peripheral blood lymphocytes. However, the use of dicentric chromosomes (Dic) in biodosimetryis still recognized as the main dose-assessment method for estimating exposure to ionizing
radiation and has become a routine component of radiation protection. Dics are specific to radiation
exposure and the background level is low in non-exposed individuals, making them advantageous
in biodosimetry. Here, we provide a review of Dics and its role in biodosimetry as research efforts
on assay optimization and high-throughput have been published since the mid-1960s. Additionally,
we provide recommended technical information (e.g., colcemid addition, scoring, generating doseresponse curves) needed to implement the dicentric chromosome assay (DCA) in laboratories
and to allow comparable dose assessment following exposure to acute ionizing radiation. While
DCA has been optimized for nuclear or radiological emergencies, increased uncertainty in dose
estimation can be caused by the scoring of Dic and variation of calibration curves. Total dose, doserate, radiation quality, and sampling time after exposure are some of the factors that influence theresults of DCA. Future consideration is also needed as no single assay is sufficient for all radiation
scenarios.
 icentric, radiation, biodosimetry, assay, DNA damage
Key words: d*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.12.2_121
Copyright © 2023 by Hirosaki University. All rights reserved.

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1. Introduction
Biological dosimetry (henceforth biodosimetry) andchromosome rearrangements in human peripheral
blood lymphocytes are widely used to quantify exposure
to a variety of mutagenic agents, including ionizing
radiation1–4). Cytogenetic methods, such as the dicentricchromosome assay (DCA), have seen great improvements
because of the development of an initiator of mitosis incultures of normal human leukocytes in the 1960s5), which
made metaphase chromosomes accessible for research.
While the current techniques of radiation biomarkers fordose assessment are constantly improving for practicaland high-throughput use in biodosimetry, the analysis
of dicentric chromosomes (Dic) has become a routine
procedure in radiation protection as it has the sensitivity
to detect previous exposures to ionizing radiation6). DCA
in biodosimetry has also been used in both accidental andoccupational overexposures where physical dosimetermeasurements were not available2, 3, 7–10). In this paper,
we exclusively discuss the DCA as a valuable dose
assessment method, especially in acute exposures, as it isparamount to understand the molecular mechanisms of
Dic and its role as a radiation biomarker in biodosimetry.
1.1. Radiation-induced chromosomal rearrangements
Ionizing radiation consists of both subatomic particles
and high-energy electromagnetic waves with sufficientenergy to detach electrons from atoms or molecules.
Examples of these subatomic particles include alphaparticles, beta particles, and neutrons. Examples ofhigh-energy electromagnetic waves include X-rays andgamma rays. Due to its high energy, ionizing radiation
exposure causes cellular damage. Tissue reactions, suchas radiation burns and radiation sickness, are caused byhigh acute doses. Lower doses over a protracted period
may cause stochastic effects such as cancer. Radiation
can also cause direct damage to DNA or indirect damage
by the production of hydroxyl radicals, superoxides orother chemical species from ionized water molecules. If
the DNA is damaged, the cell will attempt to correct thedamage through DNA repair. However, if the DNA ismisrepaired, chromosomal rearrangements could occur.
Particularly, in a dividing cell, the misrepair of radiationinduced DNA damage can cause stable chromosome
aberrations (e.g., translocations) that are inherited bydaughter cells or unstable chromosome aberrations (e.g.,Dic) that will eventually lead to apoptosis11). In this series,
we will discuss the unstable Dic aberration in detail. For
more information about other chromosome aberrations
or mechanisms of radiation-induced DNA strand breaks,
please refer to Series 3 and 4 in the special issue11, 12).
As ionizing radiation is not detectable by human senses,
instruments such as personal or pocket dosimeters (i.e.,thermoluminescent dosimeter) must be used to detectand measure radiation exposure. However, dosimeters
may not be readily available or exposed persons may nothave been wearing dosimeters at the time of radiation
exposure. In this case or in other unplanned radiation
exposures, chromosomal rearrangements can bequantified in different assays to estimate the absorbed
dose and provide information for medical treatment.
Radiation dose can be estimated because in both acuteand chronic exposures, chromosomal rearrangements
are formed when radiation-induced single or DNA double
strand breaks fail to repair (Fig. 1). These chromosomal
rearrangements, or stable and unstable chromosome
aberrations, accumulate at different frequencies percell depending on the radiation dose and quality. As
aforementioned, the most widely used unstable aberrationis the Dic and it is currently regarded as one of the mostvalidated radiation biomarker1).
2. Dic aberrations
As previously mentioned, radiation can cause single or
DNA double strand breaks. In response, the cell will
undergo DNA repair to identify and correct the damage.
However, mistakes can happen during DNA repair
where a misrepair of two broken chromosome ends
might form a Dic11, 13), which is an unstable chromosomal
rearrangement with two centromeres on the same
chromosome (Fig. 1 and 2). The type of Dic, or thepattern, will depend on the two chromosomes involvedin the chromosomal rearrangement (Fig. 2), i.e., thepattern will change if the chromosomes are metacentric,
submetacentric, or acrocentric. An acentric fragment, ora chromosome with no centromere, will also be among
the products produced by the misrepair. During mitosis,
the unstable Dic forms an anaphase bridge, causing
chromosome breakage and apoptosis. Fragments areunable to migrate to either cell pole and will disappear
during anaphase.
In general, background Dic frequency is about 1 in1000 metaphase cells14, 15). At least 80% of this backgroundDic level in the human population can be attributed toenvironmental and medical radiation6). Therefore, Dic is abiomarker which is highly specific for ionizing radiationand can be studied to assess exposed subjects. Both acuteand chronic radiation exposures increase the probabilityof Dic formation, and higher radiation doses produce
a higher Dic frequency. Absorbed dose to irradiatedsubjects can be estimated by the frequency of Dic inperipheral blood lymphocytes.
If chromosomal rearrangements, such as Dics, arepresent during cell division, cell-cycle arrest at various
cell-cycle checkpoints will occur until DNA damageis repaired. If damaged chromosomes bypass theseDonovan Anderson et al./ Radiation Environment and Medicine 2023 Vol.12, No.2 121–139

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Fig. 1. (A) Ionizing radiation can cause breaks in DNA which may (B) successfully repair or (C) fail to repair,
inducing stable and unstable chromosomal aberrations. Misrepair can cause stable chromosome aberrations (e.g.,translocations) that are inherited by daughter cells and unstable chromosome aberrations (e.g., dicentric) that result
in apoptosis.

Fig. 2. (A) Dic in human cell and (B) different Dic patterns in humans. M = metacentric chromosome; SM =submetacentric chromosome; A = acrocentric chromosome.

checkpoints, the genetic information can be transmitted
to daughter cells after cell division. However, Dics areunlikely to be inherited by daughter cells because cellswith Dics are highly likely to undergo apoptosis becauseof their inability to progress to anaphase and telophase.
Thus, Dic is also known as an unstable aberration and istypically only scored for subjects irradiated by an acuteand recent radiation exposure.
While spontaneous Dic can be induced at low
frequencies (about 1 in 1000 cells), acute radiation willcause a greater Dic frequency as doses increase. However,the number of Dic is expected to decline after the single
radiation event if no other radiation events occur as cellswith Dic and other unstable aberrations are not able toproliferate and die16). The ability to observe lymphocyteswith Dic is generally limited to the lifespan of circulating
G0 lymphocytes as human peripheral blood is used for
DCA in biodosimetry. Generally, the stimulated cells byphytohemagglutinin (PHA) that enter cell division are

typically T lymphocytes17) which have an average halflife of about three years in healthy humans18, 19). However,
the true lymphocyte lifetime is still only estimated, andits lifespan could also be influenced because of radiationinduced cell damage and death. Studies have shown thatthe mean lymphocyte lifetime in patients who received
radiotherapy for cervical cancer was about 1.5 years20),
while a mean of 4.3 years was observed in patients who
received radiotherapy for ankylosing spondylitis18, 21). In
either case, abnormalities in circulating lymphocytes
in peripheral blood with a lifespan of 3-4 days would be
difficult to detect if months or years have elapsed after
exposure.
The half-life of Dic (i.e., a reduction of examined Dic
frequency to half of the initial Dic frequency) can begenerally assumed to be three years based on a single
exponential decline with a time factor correction observedin Chernobyl subjects 22). When the study comparedbiological doses estimated from Dic frequency to physical124

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dosimetry, the time interval between irradiation and blood
sampling needs to be considered, or it could lead to dose
underestimation. A similar methodology which evaluatedoccupational exposure to ionizing radiation in nuclear
power plant workers suggested a longer half-life of sevenyears might be more realistic23, 24). Further debate arises
as some researchers suggest that the decline is delayed
about a year after exposure21, 25). Dics in lymphocytes
several years after the initial irradiation are likely derived
from long-life stem cells or progenitor cells with radiationinduced chromosomal instabilities or caused by protracted
radiogenic environmental stressors. Thus, it has been
well established that Dic is best suited to evaluate recent
acute exposures, while delayed sampling will result in an
increased uncertainty of dose estimation.
3. DCA in biodosimetry
Circulating lymphocytes in the G 0 stage of the cell
cycle can be stimulated with a mitogenic agent to start
the process of DNA replication and enter cell division.
The metaphase lymphocytes can then be observed forchromosome aberrations, such as Dic. Based on theprinciple that Dic is primarily attributed to the misrepairof radiation-induced DNA double strand breaks, anassay quantifying Dic frequency in lymphocytes can be
performed to assess prior radiation exposures, i.e., theDCA. Dic frequency in peripheral blood lymphocytes in
DCA can give a good approximation of radiation dose
for as low as 0.1 Gy26), especially if blood sampling has
not been delayed. Manual Dic scoring in DCA is a timeconsuming process that necessitates highly trained
staff. Additionally, many factors will influence theestimated dose of the DCA, including total dose, doserate, percentage of body irradiated, radiation quality,
and sampling time after exposure. Human error ordiscrepancies in Dic identification and mis-scoring arealso known to influence results. Thus, it is important tounderstand these contributing factors when interpreting
Dic frequency for precise radiation dose estimation (seesection 3.8 for more details).
3.1. Modifications of DCA
Dic frequency has been used as a dose estimation tool forbiological dosimetry since the 1960s. The information inthis section about DCA was adopted from IAEA 20111) andis currently the international guideline for DCA in routine
assessment. However, from the period prior to, and after,
the IAEA 2011 publication, there have been few changes
worth mentioning while some parts are still relatively
debated. In general, the measured Dic frequency isrobust against culture and reagent protocol variations27, 28).
However, modifications to the protocol could cause
uncertainties in dose estimation during laboratory inter-comparisons29). Nonetheless, DCA still remains the gold
standard for radiation assessment and further research
will continue to allow the progression or modification of
the assay to extend the assay’s utility to multiple radiation
scenarios.
One of the main modifications to DCA analysis is thetype of aberration used to estimate dose. Originally, theassessment required both Dic and ring chromosomes(rings) for dose estimation. The total yield of Dic andrings are referenced to an appropriate dose-response
curve to estimate dose (i.e., Dic + ring per cell curves).
However, Giemsa-stained rings are difficult to identify andrequire significantly higher scorer expertise. In the IAEA
2011 publication, and as recommended by the authors,
only Dic frequency in cells should be used to estimate
dose. In either case, cross-checking and validationof scoring results is required, and scoring should be
performed by experienced personnel.
The other variable discussed in the IAEA 2011
publication is the appropriate length of time to add
demecolcine (colcemid) to the lymphocyte culture.
Colcemid, a synthetic analogue of colchicine, consistsof alkaloid compounds that inhibit spindle formation
during mitosis 30). Once added to a culture, the cells
are arrested during metaphase and cannot progress toanaphase. As IAEA 2011 publication states, colcemid(0.05-0.10 μg/mL) is typically added 2 or 3 hours before
cultures are completed, i.e., at 45-46 hours. However,
the authors and other researchers recommend adding alower concentration of colcemid (0.05 μg/mL) either at
the start of culture31–33) or at 24 hours1, 34, 35). Even thougha longer colcemid treatment could affect mitotic indicesand the quality of metaphase chromosomes observed,
i.e., too condensed or too elongated, earlier colcemid
addition will prevent cells from progressing beyond thefirst metaphase and avoid any analysis of second-division
metaphase cells. Please refer to section 3.3 for moredetails on the importance of first-division metaphaseanalysis.
3.2. DCA protocol
DCA follows the general procedures given in the IAEA
2011 publication, which have been adopted by cytogenetic
laboratories to assess human peripheral blood samples(full methodology details used by authors are provided inAppendix A and B). Briefly, heparinized peripheral blood is
obtained from individuals potentially exposed to radiation
following the recommended blood sampling procedures
and management discussed in Series 1 and 2 36, 37).
Then, whole blood or isolated mononuclear cells arestimulated by PHA to proliferate in culture (incubatedat 37°C , 5% CO2 for 48 hours). Cells are then harvested
and fixed, and first-division metaphases are spread on
glass slides. Metaphase chromosomes are stained andDonovan Anderson et al./ Radiation Environment and Medicine 2023 Vol.12, No.2 121–139

125

Fig. 3. Hypotonic solution of 75 mM potassium chloride is added for cell swelling and to reduce metaphase
chromosome overlap. During slide preparation, fixative is slowly pipetted on the slide to ensure cells are dispersed
into a uniform suspension.

analyzed for chromosome aberrations. After Dic scoring,dose estimates are evaluated with Dic frequency from thesample with calibration curves of similar radiation quality.
3.3. Mitotic arrest and signi f icance of f irst-division
metaphases
As mentioned previously, DCA requires a culturingmethod to ensure that f irst-division metaphases
are scored. This is achieved by adding colcemid tosynchronize and arrest cell division to mid-M phase. In
other words, colcemid inhibits the anaphase movementof chromosomes and halts other stages of the mitotic
cycle. As recommended by the authors, colcemid is addedfrom the start of culture or 24 h after the start of culture
to ascertain first-division metaphase analysis. If colcemid
timing and concentration are not considered, the firstand second metaphases must be distinguished using
Fluorescence Plus Giemsa (FPG) staining38).
First-division metaphases (46-48 h after initiation of
the culture) must be analyzed because Dic is an unstable
chromosome aberration, and cells with these aberrant
chromosomes often fail to divide normally and die duringmitosis. If the cell-cycle progresses, cells with Dics may
die, decreasing the overall Dic frequency and ultimately
underestimating radiation dose. It was previously
shown that the Dic frequency of second-division cells
decreased by 46% as compared to first-division cells39). In
other words, about half of the cells containing unstable
aberrations will be eliminated after each mitosis.
Therefore, it is necessary to arrest dividing cells in the
beginning or the middle of the first cycle of mitosis after
PHA stimulation to ensure acute biodosimetry evaluation.

3.4. Cell spreading and cell suspension improvement
After blood culture, cells are first treated with hypotonic
solution (75 mM potassium chloride) for cell swelling andfixed in 3:1 methanol:acetic acid. After fixation, cells are
spread onto a pre-cleaned slide glass (Fig. 3). It should be
noted that the quality of metaphase chromosomes can beaffected by humidity, temperature and cell concentration(Fig. 4). Lack of consideration for optimized cell swelling
may make it difficult to distinguish chromosomes (seesection 3.8 for Dic identification). It is also recommended
to check the metaphase quality on a test slide before
preparing multiple slides for DCA evaluation. Laboratories
can also use automatic sample preparation systems, suchas the HANABI-PVI Metaphase Spreader (ADSTEC,Japan), to control environmental conditions for optimized
metaphase spreading on slides.
3.5. Slide staining
Generally, slides are stained with Giemsa in Gurr buffer(pH 6.8) in DCA because of its reduced cost and time
for slide preparation. However, depending on the type of
microscopes (e.g., bright-field, fluorescence microscopes)
available in the lab and other endpoints analyzed (e.g.,centromere identification), slides may be stained withother reagents such as 4 ,6-diamidino-2-phenylindole(DAPI). Other techniques can also be used to visualize
and score Dic, such as those that highlight centromeres(C-banding) or use of fluorescent probes to detect andlocalize specific DNA sequences on chromosomes(fluorescence in situ hybridization using peptide
nucleic acid probes; PNA-FISH). In this case, C-banding
techniques will typically be adapted from protocols

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Fig. 4. Humidity, temperature and cell concentration can affect the quality of metaphase spreads on slides. Before preparing
multiple slides for DCA analysis, it is a good habit to check how the metaphase chromosomes spread on a test slide. If the test
slide shows poor metaphase quality, adjust the above conditions again until optimal metaphases are obtained.

Table 1. Typical DCA assessment of the reference, triage and QuickScan methods.
MethodNumber of metaphase spreads or Dic needed
Centromere count required
Reference1, 45)
1000 metaphases or 100 Dic
Yes
Triage46, 47)
50 metaphases or 30 Dic
Yes
QuickScan48)
50 metaphases
No*Found in ISO documents and consensus-based International Standards

referenced from Sumner et al.40) and Prosser et al.41), while
PNA-FISH can be adapted from Finnon et al.42) and Shi etal.43). For routine DCA with Giemsa, adequate distinctionof centromeres and chromosome arms are required toreduce misidentifications. Slide staining with C-banding or
PNA-FISH may assist in resolving the ambiguous images
and provide a better identification of suspected Dic.
3.6. Metaphase imaging
Stained slides are typically observed using an upright
optical microscope. High objective oil lens of 63× (60×)
or 100× are used to observe chromosome structures.
Metaphase spreads should first be located using a 10×
magnification objective lens, and then imaged with a 63×or 100× lens for manual chromosome aberration analysis.
A metaphase location software 44) can also be used
more efficient metaphase imaging as several thousand
metaphases can be captured automatically, which canthen be saved as digital images and accessed for future
scorer analysis.
3.7. Dic scoring and number of cells for analysis
It is recommended to score at least 500 to 1000
metaphases for accurate dose estimation as increasing
the number of scored cells will improve resolution,
support initial assessments, and provide evidence of

ISO*
Yes
Yes
No

inhomogeneous exposure45). It is not necessary to score
beyond 100 Dic if the aberration yield is high or if there
exists a high abundance of Dic in a few metaphasespreads. In the low dose range or when assessing radiation
doses years after exposure, thousands of metaphases mayneed to be scored to confirm an exposure. The numberof scored metaphases should be decided based on theradiation scenario and assay’s objective. In emergency
triage, the number of metaphases analyzed need to beaccurate enough to provide an estimated dose to victims
for physicians to provide treatment in a timely manner.
DCA analysis typically includes the reference method of
500 to 1000 complete metaphases45), a triage technique of
50 complete metaphases46, 47), or the “QuickScan method”48)
of 50 metaphases (Table 1). Reference and triage
methods also require centromeres to be counted toensure metaphases are complete. The QuickScan scoring
technique was devised by the Biodosimetry Laboratory
at Atomic Energy of Canada Ltd. (Chalk River, ON). It isan alternative rapid-scoring approach to the conventional
triage technique which rapidly examines metaphases for
obvious damage without centromere scoring48). However,this technique is only used by experienced scorers for
initial screening, rapid scoring for emergency exposures
or mass casualty radiation emergency and evaluating theability of scorers across laboratories. It is also importantDonovan Anderson et al./ Radiation Environment and Medicine 2023 Vol.12, No.2 121–139

127

Fig. 5. Metaphases that do not meet selection criteria should be excluded during Dic scoring; (A) Two or more
metaphases, (B) unclear or extended chromosomes, (C) too few chromosomes, (D) overlapped chromosomes, and(E) separated centromeres or sister chromatids.

to note that all three methods are found in the IAEA 2011
publication1), but only the reference and triage method arerecognized by the worldwide International Organizationfor Standardization (ISO)45).
3.8. Dic scoring criteria and chromosome staining features
Accurate Dic identification in selected metaphases isrequired to determine the true Dic frequency in exposed
subjects1). Prior to performing DCA, scoring personnel
need to be highly and frequently trained to control interlaboratory variability and eliminate possibility of scorer
error. If Dic frequency is not appropriately quantified,absorbed dose will either be over- or under-estimated.
Dic is only scored in metaphases selected based onrecommendations from the IAEA 2011 publication.
Metaphases must be complete, i.e., to contain 46 (or45) centromeres, not overlap, and chromosome arms
must be distinguishable. Examples of metaphases thatshould not be scored are provided in Figure 5. Somebiodosimetry laboratories will also accept metaphaseswith 45 centromeres in the metaphase selection criteria
because the missing centromere cannot form a Dic (i.e.,
not influence Dic frequency).
It is often remarked that DCA requires experienced
scorers and appropriate slide preparations. This isbecause incorrect Dic scoring is often the highest causeof uncertainty in dose estimations. Careful observationis needed because a Dic with two adjacent centromeres
may be difficult to distinguish from a monocentricchromosome. Understanding chromosome structure
and morphology will help in Dic identification. For
example, acrocentric chromosomes (i.e., #13, 14, 15, 21,
and 22 chromosomes) tend to be arranged with satellite
association (Fig. 6), which can help determine if a Dic has
been formed with group D or G chromosome (Fig. 7).
In addition to understanding chromosome structure,
staining patterns associated with Giemsa-stained
chromosomes can also help to identify Dic. Centromeresor satellite regions tend to be more lightly stained (i.e.,light gray or white). Both long and short chromosome
arms will appear dark gray or black. However, if thechromosome arms are twisted or if chromosomes overlap,
the color will appear darker than the rest of the body orother chromosomes (Fig. 8). As previously mentioned,
metaphases with overlap should not be counted. However,if the scorer can appropriately distinguish the arms andcentromeres using the respective staining features, thecell can be scored. In particular, misinterpretation ofstaining features should be avoided for Dic when scoring,such that a twisted chromatid arm is not identified as asecond centromere (Fig. 9). Scorers should reconfirm thatthe metaphase is complete and distinguish any fragments
before concluding if Dic is present. Images analyzed
should be stored, especially those containing suspected
Dic, to allow for future validation if needed and provide
reproducible results.

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Fig. 6. (A) Acrocentric chromosomes tend to be arranged withsatellite association. This helps determine if a Dic has been formed
with a group D or G chromosome. (B) An image of a pattern 2
Dic (metacentric chromosome or a submetacentric chromosome
mis-repaired with an acrocentric chromosome). (A’) and (B’) aremagnified images of (A) and (B), respectively.

Fig. 7. Example of a female donor’s metaphase that includes satellite
association with Dic and a D chromosome. Dic is characterized
as pattern 2. There are 5 group D chromosomes and 4 group G
chromosomes.

Fig. 8. Chromosome arms are dark gray or black while centromeres are a lighter shade in grayscale images. It
is important to clearly identify the staining features of a chromosome during scoring. Chromosomes may twist or
overlap causing darker shades and may appear as a Dic. If the staining feature can be identified, then the cell may
be scored. If not, we recommend the cell is not used in DCA analysis.

3.9. Dose-response curve and dose estimation
After analyzing an adequate number of lymphocyte
metaphases, Dic frequency (i.e., Dic per cell) can be used
to estimate a biological absorbed dose by referencing adose-response calibration curve. This in vitro curve isestablished by radiation exposure of peripheral blood
to doses of the appropriate radiation quality following

recommended guidelines1). More recently, separate curves
were constructed for low dose-rates (Table 2), as themolecular mechanisms and the kinetics of Dic induction
are influenced by radiation dose, period of exposure, andquality of radiation. Thus, it is recommended that curves
used for dose estimation are developed at the laboratory
performing the dose assessment for a range of radiation

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129

Fig. 9. Example of a Giemsa-stained metaphase spread with a twisted chromosome and overlapping chromosomes.
The twisted chromosome may be wrongly identified as a Dic.

Fig. 10. Dic in non-human biota; (A) mouse and (B) wild boar. 2n, number of chromosomes in somatic cells.

qualities and dose-rates. The estimated dose obtained
from interpolation of Dic frequency to a dose-response
curve represents an average absorbed dose to thelymphocytes. This dose is also assumed to approximate
a whole-body dose as peripheral blood lymphocytes aremobile and distributed around the body.
The type of radiation contributes to Dic frequency inlymphocytes. For example, high LET radiation, such as
fission neutrons and alpha particles, cause significantly
higher and complex damage to cells at acute doses
and this damage tends to have a linear effect as dose
increases. Thus, the dose-response relationship fits alinear regression model Y = α D, where Y is the Dic
frequency and D is dose. On the other hand, low LET
radiation, including X-rays and gamma rays, tends to havea linear-quadratic dose response and there is a good doseeffect relationship in the range of 0.1 to 5.0 Gy for acuteexposures. Low LET radiation is best fitted by a linear-quadratic model Y = C + α D +βD2.
In addition, Dic frequency follow Poisson statistics
for acute whole-body low LET radiation. Statisticalverification with dispersion index (i.e., ratio of varianceto mean) and suitable Poisson tests (e.g., u-test) will need
to be calculated before dose estimation. The objective of
curve fitting in a dose-response curve is to determine thevalues of coefficients C, α and β which best fit the data
points. Overall, multiple laboratories have developed doseresponse curves showing these tendencies with high andlow LET radiation (Table 2).
4. Current issues and applications in biodosimetry
While Dic is preferably used to investigate effects ofionizing radiation exposure and dose estimation, it isimportant to consider factors that could affect valid andreproducible results in different experimental designs.

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Table 2. Previously published constructed in vitro dose-response curves for DCA and dose estimation. The curves were based Dic frequency inhuman lymphocytes exposed to various ionizing radiation sources.
Reference
Bauchinger et al.49)
Martins et al.50)
Schmid et al.51)
Abe et al.52)
Mendes et al.53)
Lindholm et al.54)
Wong et al.55)
Top et al.56)
Koksal et al.57)
Bauchinger et al.49)
Beinke et al.58)
Haeri et al.59)
Voisin et al.60)
Prasanna et al.61)
Stricklin et al.62)
Bauchinger et al.49)
Prasanna et al.61)
Beinke et al.58)
Prasanna et al.61)
Lloyd et al.63)

High or
Low LET
Low
Low
Low
Low
Low
Low
Low
Low
Low
Low
Low
Low
Low
Low
Low
Low
Low
Low
High
High

Irradiation source

Dose rate(Gy min-1)

Co
Co
60
Co
60
Co
60
Co
60
Co
60
Co
60
Co
60
Co
60
Co
60
Co
60
Co
60
Co
60
Co
137
Cs
220 keV X-rays
220-250 keV X-rays
240 kV X-rays
Fission neutron (0.71 MeV)
Fission neutron (0.7 MeV)

0.017
0.018
0.033
0.026
0.05
0.24
0.41
0.425
0.457
0.5
0.64
0.8
1
1
0.4
0.5
1
1
0.25
0.5

60
60

DCA works well if an exposure is acute and uniform over
the whole body with no delay in blood sampling from theinitial exposure (i.e., blood sample is typically collected
after 24 hours after irradiation). However, if these factors
are altered, one should expect an increase of uncertainties
for dose estimations.
4.1. Issues
One of the limitations of DCA in nuclear or radiologicalemergencies is the time and expertise required to performthe scoring of 500 to 1,000 metaphases per sample (i.e.,
Reference method, Table 1) with an effective dose range
between 0.1-5 Gy. This range covers the dose range lower
than 0.5 Gy, where peripheral blood lymphocyte depletion
is observed and has sufficient sensitivity to evaluate
deterministic effects64). If the number of metaphases
scored is reduced, the range between the upper and lower
dose limit will be increased. More than 1,000 metaphases
should be scored if assessing exposures below 0.1 Gy or if
the exposure was received over a protracted period.
Even though high-throughput automated metaphase
image capture and Dic scoring have been developed
to quicken DCA analysis 44, 65, 66), the technology canbe quite expensive and might require trained scorers
to re-evaluate automated Dic frequency. Open-source
automated Dic scoring software or alternative staining
methods for easier Dic identification must be developed
for laboratories who are unable to afford such equipment

Dose-response curve equation(High LET, Y = c + α D; Low LET, Y = c + α D + βD2)c (±SE)α (±SE)β (±SE)
0.00038 (±0.00013)
0.0090 (±0.004)
0.0417 (±0.0028)
0.0011 (±0.0006)
0.0098 (±0.0036)
0.0489 (±0.002)
0.0003 (±0.0002)
0.0139 (±0.0052)
0.0304 (±0.0030)
0.0013 (±0.0005)
0.0067 (±0.0071)
0.0313 (±0.0091)
0.0014 (±0.0008)
0.0074 (±0.0069)
0.0449 (±0.0044)
0.00055 (±0.0024)
0.0135 (±0.0043)
0.0544 (±0.0034)
0.000 (±0.000)
0.026
0.03860.0006 (±0.0008)
0.034 (±0.00729)
0.0605 (±0.00282)
0.005 (±0.003)
0.0216 (±0.006)
0.0706 (±0.0025)
0.00039 (±0.00013)
0.0107 (±0.0041)
0.0555 (±0.0028)
0.000 (±0.000)
0.0306 (±0.0068)
0.0480 (±0.0036)
0.000 (±0.000)
0.012
0.04610.0004 (±0.0023)
0.0374 (±0.0127)
0.0549 (±0.0034)
0.000 (±0.000)
0.098 (±0.021)
0.044 (±0.009)
0.0025 (±0.0016)
0.013 (±0.007)
0.065 (±0.003)
0.000 (±0.000)
0.0404 (±0.03)
0.0598 (±0.017)
0.000 (±0.000)
0.059 (±0.0.0136)
0.029 (±0.0046)
0.0007 (±0.0002)
0.0432 (±0.00459)
0.0630 (±0.0039)
0.000 (±0.000)
0.677 (±0.003)
0.000 (±0.000)
0.8349 (±0.003)-

and software.
The other limitation is the presumably short halflife of Dic and its reduced sensitivity to low dose or
low dose-rate ionizing radiation. In general, unstable
chromosome aberrations like Dic have a shorter halflife than stable chromosome aberrations, as cells withunstable chromosome aberrations cannot proceed withcell division and are halted during cell-cycle checkpoints
or cause cell apoptosis. However, as mentioned in section
2, the estimated Dic half-life ranges from 1.5-3 years insome studies to 4-7 years in others. The authors suspect
the discrepancy may be due to the radiation type andquality that was investigated as a longer Dic halflife was estimated in Hiroshima and Nagasaki atomic
bomb survivors, while other acute exposure scenarios
showed a quicker decline of Dic67). Regardless, the halflife has raised concerns on whether Dic can be used
to interpret low dose and low dose-rate radiation. DCA
may not be feasible if more than ten years have passed
since the radiation event. In such cases and in low dosestudies, the analysis of stable chromosome aberrations
such as translocations is preferred as the endpoint has
the sensitivity and longevity to evaluate past irradiatedsubjects.
4.2. Environmental dosimetry for terrestrial animals
Other than humans, the scientific community has also
aimed to protect the environment and wildlife from

Donovan Anderson et al./ Radiation Environment and Medicine 2023 Vol.12, No.2 121–139

harmful effects of ionizing radiation 68). To advanceenvironmental protection and dosimetry for terrestrial
biota, it is necessary to have accurate probabilistic
framework for dose assessments of external (andinternal) doses for terrestrial animals. Similar to humans,
there is extreme diversity of non-human biota, and thiscreates a major challenge for such assessments. Likewise,
Dic can accumulate in animals following ionizing
radiation exposure (Fig. 10), and DCA has also beenused to estimate the extent of radiation damage afterradioactive contamination in areas near the Chernobyl
Nuclear Power Plant69) and Fukushima Dai-ichi Nuclear
Power Plant 70, 71). As mentioned earlier, DCA is moresuited for acute radiation exposures from 0.1 to 5 Gy. It
is still unclear if the assay is applicable to such cases ofchronic radiation exposure to accurately improve wildlife
prediction models.
5. Conclusion
In cytogenetic biodosimetry, DCA is the preferred
method for evaluating the effects of ionizing radiationand is a valuable dose assessment method. As DCA ishighly radiation specific and has an effective dose range
between 0.1-5 Gy, it is considered the gold standard in
biodosimetry. However, DCA analysis can be laborintensive, requires expert scorers and takes up a lot oftime. Over the years, improvements have been made to
reduce the time and expertise needed for Dic scoring.
Furthermore, published reports of dose-response curves
have shown that the differences exist in measured
Dic frequency among laboratories for the same dose.
It is currently recommended that each biodosimetrylaboratory establish its own Dic dose-response curveusing different radiation types, dose-rates, and doses.
There is still room for improvement to make DCA
more beginner-friendly and applicable for mass-casualty
accidents. Such could be addressed by developing
open-source AI scoring systems or optimizing staining
methodology to reduce scoring time and further improveautomated Dic identification.
Point 1. DCA is based on the principle thatradiation exposure causes DNA strand breaks.
• Dic can be formed due to a misrepair of DNA double
strand breaks, resulting in one chromosome withtwo centromeres.
• Dic is highly specific and sensitive to ionizing
radiation.
Point 2. Man y f act ors in f luence t he Dic
frequency.
• Total dose, dose-rate, percentage of body irradiated,131

and radiation quality will influence Dic frequency.
Sampling time of heparinized peripheral blood after
initial exposure will also influence DCA.
• Scorer’s ability and metaphase selection criteria
may affect Dic frequency and cause dose under- or
overestimation.
Point 3. Absorbed dose to exposed subjects is
estimated from Dic frequency and dose-response
calibration curves.
• Curves need to be established prior to the analyses
and have similar radiation quality to the exposure
scenario.
• S c o r i n g 5 0 0 t o 10 0 0 c e l l s ( o r 10 0 Di c ) i s
recommended for the reference method for accurate
dose estimation. 50 metaphases (or 30 Dic) can bealternatively scored for triage assessment.
Point 4. DCA issues and limitations• DC A is t he gold s t a nda rd f or c y t ogenet ic
biodosimetry. However, limitations such as theshorter half-life compared to stable aberrations, long
analysis time and high scorer expertise need to be
overcome.
• High-throughput automation including metaphaseimaging and Dic scoring do quicken DCA analysis.
The technology is expensive and still requires scorer
expertise for potential re-evaluation.
•

Conflict of Interest
The authors declare that they have no conflict ofinterests.
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Appendix
The dicentric chromosome assay (DCA) follows the general procedures provided by the IAEA manual
published in 20111) and ISO2, 3). The DCA procedures have been adopted by cytogenetic biodosimetrylaboratories to assess human blood samples for radiation exposures. Here are detailed protocols of DCA
used by the authors’ laboratory, which have been adopted and modified from the international protocols.
Please note that once the blood arrives at the laboratory, the researchers should decide if DCA will
be performed with isolated peripheral blood mononuclear cells (PBMC) or whole blood. The authors
recommend PBMC when possible. Please refer to (Appendix A) for PBMC DCA protocol and(Appendix B) for whole blood DCA protocol.
Appendix A: DCA protocol for PBMC culture
A.1 Materials and Reagent setup
A. Equipment
ID
Biological safety cabinet
Centrifuge
Aspiration system
CO2 incubator
Water bath
Vortex mixer
Hanabi harvester (optional)
Hanabi spreader (manual model)
15 mL polypropylene, sterile conical tubes
50 mL polypropylene, sterile conical tubes
Portable pipette gun
Serological pipettes, sterile, 10 mL and 5 mL
0- 20 µL micropipette + sterile 20 µL tips
0- 200 µL micropipette + sterile 200 µL tips
Sterile slugged Pasteur pipette 9”
Phase contrast microscope pre-cleaned slides, frosted
end, 76 x 26 mm
Premium cover glasses, Size: 50 x 24 mm
Coplin jar
Waste container
Latex gloves

Supplier

Cat. #ADSTECADSTEC

B. ReagentsID
Histopaque 1077
RPMI 1640 medium with L-glutamine, phenol red and 25mM HEPES
Fetal Bovine Serum, Prime, Heat-inactivatedKanamycin sulfate (100X), liquid
KaryoMAX Colcemid Solution in HBSSPHA Reagent Grade (HA15)
Potassium chloride (KCl)
MethanolAcetic acid, glacial
Giemsa’s azur eosin methylene blue solution
Gurr buffer tablets
Malinol 750cps

Supplier
Sigma-Aldrich
Gibco
Biosource
Gibco
Gibco
Remel
Sigma-AldrichCat. #
H8889-100ML
22400089
200P-500HI
15160054
15210040
R30852701
P5405-250G

Merck
Gibco
Muto Pure Chemicals

1.09204.0103
10582013
20093

Donovan Anderson et al./ Radiation Environment and Medicine 2023 Vol.12, No.2 121–139

C. Reagent setup
ID· Complete medium without PHA (CM)*RPMI 1640 medium supplemented with 20% FBS and Kanamycin sulfate.
· Washing solution (Wash buffer)*
RPMI 1640 medium supplemented with 2% FBS and Kanamycin sulfate.
· Hypotonic solution (75 mM KCl)
2.796 g KCl dissolved in 500 mL of distilled water
Sterilize using autoclave (121 ℃ for 20 min), store at room temperature. Warm hypotonic solution at 37 °C before cell
fixation.
· Fixative solution
Methanol: acetic acid = 3:1 (prepared on ice for PBMC)
Note: To freshly prepare fixative prepare 30 min to 2 h before harvest· Gurr Buffer
Dissolve 1 Gurr buffer tablet in 100 mL of deionized water, sterilize using autoclave (121 ℃ for 20 min), and store atroom temperature **· Giemsa staining solution
4% Giemsa solution in Gurr buffer (pH 6.8)
*FBS will need to be inactivated by heating at 56 ℃ for 30 min prior to making medium and buffer**The instructions should be modified depending on the type of Gurr tablets purchased.

A.2 Protocol
A. Blood culture and PHA stimulation
Please refer to Series 44) for more information regarding culture procedures including the choice of
culture medium, serum, and mitogen.
1. M ononuclear cells can be isolated from whole blood by directly layering onto Histopaque 1077
or other ready-to-use tubes for such lymphocyte separation (e.g., BD Vacutainer® CPT™ Cell
Preparation tubes, Stemcell Technologies Sepmate™ PBMC isolation tubes). If ready-to-use
tubes are used, manufacturer’s instructions should be followed.
The authors recommend the following steps for PBMC isolation if blood is directly layered on
Histopaque 1077 in a 15 mL centrifuge tube:a.	2.5 mL of Histopaque 1077 into 15 mL centrifuge tube and bring to room temperature.
b.	Mix 1.8-2.4 mL whole blood with 2 mL washing solution (i.e., 1:1 whole blood to washingsolution)5).
c.	Carefully layer the mixed sample (4 mL) onto Histopaque 1077.
d.	Centrifuge at 600 x g for 30 min at 20 °C in a horizontal rotor according to the
manufacturer’s instruction.
e.	After centrifuge, the PBMC layer is then carefully transferred to a sterile conical tube
containing 6.5 mL of washing solution.
f.	
Centrifuge at 400 x g for 5 min and aspirate the supernatant.
g. Then, proceed to step (2).
2. R
 esuspend isolated PBMC cell pellet with 1 mL of CM (warmed), pipette well to mix.
3. T ransfer 1 mL of PBMC cell suspension into new 15 mL conical tube with 5 mL of CM(warmed).
4. F
 or 6 mL culture, add 30 µl colcemid (final colcemid concentration of 0.03 µg/mL) and 120 µl
PHA (final concentration of 180 µg/ml for PHA).
5. Mix gently by vortex.
6. Loosely close the tube cap and start the culture in 5% CO2 incubator for 48 h.

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B. Hypotonic treatment and fixation
1. After blood culture, centrifuge at 400 x g for 5 min at 20 °C.
2. Carefully aspirate the supernatant.
3. Resuspend cell pellet with 2 mL of hypotonic solution (37 °C) while vortexing.
4. Incubate at 37 °C for 20 min.
5. Add 200 µL of cold fixative solution, invert the tube 5 to 6 times, and keep on ice for 5 min.
6. Centrifuge at 400 x g for 5 min at 4 °C.
7. After centrifugation, carefully aspirate the supernatant.
8. G ently tap the bottom of the conical tube to break up the cell pellet.
9. R
 esuspend cell pellet with 5 mL of cold fixative solution while vortexing in the same conical
tube.
10. C
 entrifuge at 400 x g for 5 min at 4 °C.
11. After centrifugation, carefully aspirate the supernatant.
12. R esuspend in 5 mL of cold fixative solution while vortexing.
13. C entrifuge at 400 x g for 5 min at 4 °C.
14. R epeat steps 11-13 twice, discard supernatant and resuspend cell pellet in an optimum volume
of cold fixative solution. (Store at -30 °C)
C. Slide preparation
1. Adjust the cell concentration with freshly prepared cold fixative.
2. D rop the 13-17 µl cell suspension onto a clean slide (or use Hanabi spreader to spread cells in acontrolled environment).
3. D ry the slide for at least 30 min prior to staining. Slides can be dried quickly in a 50-60 °C ovenor dried overnight at room temperature.
Note: Preparation of clean slide
Pre-cleaned slides were kept in 100% Et-OH over 1 week (if necessary).
D. Giemsa staining
1. P repare the staining solution (4% Giemsa in Gurr buffer, pH 6.8). Note: The concentration of
Giemsa may be modified depending on the reagents purchased.
2. S
 et the slide into an appropriate staining jar.
3. S tain the slide for 10 min at room temperature.
4. After 10 min, gently remove the oxidized Giemsa film formed on the surface of the staining
solution.
5. R inse slides with water.
6. A uthors recommend that the processed slides should fully dry before mounting. If dose
estimate needs to be reported in a short period (i.e., triage), slides can be dried in 60 °C for 1 h
before mounting or immersion oil can be dropped directly on fully dried slides for microscopeviewing. In all other cases, slides should be dried at 37 °C for 24 h.
E. Mounting
Mounting media, such as Malinol, are needed for making permanent slides. This ensures that thecells are protected after Giemsa staining and allows for long-term slide storage.
1. D
 rop the mounting medium (Malinol) on the cover slip.
2. M ount the cover slip gently. Do not make any bubbles.
3. K eep the slide on a flat surface and place it in a clean environment for 2 h to allow the coverslip to firmly adhere to slide.

Donovan Anderson et al./ Radiation Environment and Medicine 2023 Vol.12, No.2 121–139

Appendix B: DCA protocol for whole blood culture
B.1 Materials and Reagent setup
A. Equipment
ID
Biological safety cabinet
Centrifuge
Aspiration system
CO2 incubator
Water bath
Vortex mixer
Hanabi harvester (optional)
Hanabi spreader (manual model)
15 mL polypropylene, sterile conical tubes
50 mL polypropylene, sterile conical tubes
Portable pipette gun
Serological pipettes, sterile, 10 mL and 5 mL
0- 20 µL micropipette + sterile 20 µL tips
0- 200 µL micropipette + sterile 200 µL tips
Sterile slugged Pasteur pipette 9”
Phase contrast microscope pre-cleaned slides, frosted end, 76 x 26 mm
Premium cover glasses, Size: 50 x 24 mm
Coplin jar
Waste container
Latex gloves

Supplier

Cat. #ADSTECADSTEC

B. ReagentsID
RPMI 1640 medium with L-glutamine, phenol red and 25mM HEPES
Fetal Bovine Serum, Prime, Heat-inactivatedKanamycin sulfate (100X), liquid
KaryoMAX Colcemid Solution in HBSSPHA Reagent Grade (HA15)
Potassium chloride (KCl)
MethanolAcetic acid, glacial
Giemsa’s azur eosin methylene blue solution
Gurr buffer tablets
Malinol 750cps

Supplier
Gibco
Biosource
Gibco
Gibco
Remel
Sigma-AldrichCat. #
22400089
200P-500HI
15160054
15210040
R30852701
P5405-250G

Merck
Gibco
Muto Pure Chemicals

1.09204.0103
10582013
20093

C. Reagent setup
ID· Complete medium without PHA (CM)*RPMI 1640 medium supplemented with 20% FBS and Kanamycin sulfate.
· Washing solution (Wash buffer)*
RPMI 1640 medium supplemented with 2% FBS and Kanamycin sulfate.
· Hypotonic solution (75 mM KCl)
2.796 g KCl dissolved in 500 mL of distilled water
Sterilize using autoclave (121 ℃ for 20 min), store at room temperature. Warm hypotonic solution at 37 °C before cell
fixation.
· Fixative solution
Methanol: acetic acid = 3:1 (kept at room temperature for whole blood)
Note: To freshly prepare fixative prepare 30 min to 2 h before harvest· Gurr Buffer
Dissolve 1 Gurr buffer tablet in 100 ml of deionized water, sterilize using autoclave (121 ℃ for 20 min), and store atroom temperature**· Giemsa staining solution
4% Giemsa solution in Gurr buffer (pH 6.8)
*FBS will need to be inactivated by heating at 56 ℃ for 30 min prior to making medium and buffer**The instructions should be modified depending on the type of Gurr tablets purchased.

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B.2 Protocol
A. Blood culture and PHA stimulation
Please refer to Series 44) for more information regarding culture procedures including the choiceof culture medium, serum, and mitogen.
1. 0 .5 mL of whole blood can be directly transferred into a 15 mL sterile conical tube with 5.5 mLof warmed Complete Medium (CM), then proceed to step (1).
2. F
 or 6 mL culture, add 30 µl colcemid (final colcemid concentration of 0.03 µg/mL) and 120 µl
PHA (final concentration of 180 µg/ml for PHA).
3. M
 ix gently by vortex.
4. L
 oosely close the tube cap and start the culture in 5% CO2 incubator for 48 h.
B. Hypotonic treatment and fixation
1. After blood culture, centrifuge at 400 x g for 5 min at 20 °C.
2. C
 arefully aspirate the supernatant.
3. R esuspend cell pellet with 5 mL of hypotonic solution (37 °C) and gently invert tube 5-6 times
to mix.
4. I ncubate at 37 °C for 20 min.
5. C entrifuge at 400 x g for 5 min at 20 °C.
6. Carefullyaspirate the supernatant.
7. Resuspendcell pellet again with 5 mL of hypotonic solution (37 °C) while vortexing.
8. Add 200 µL of room temperature fixative solution and invert the tube 5 to 6 times.
9. C entrifuge at 400 x g for 5 min at 20 °C.
10. After centrifugation, carefully aspirate the supernatant.
11. G ently tap the bottom of the conical tube to break up the cell pellet.
12. R
 esuspend cell pellet with 5 mL of fixative solution while vortexing in the same conical tube.
13. C entrifuge at 400 x g for 5 min at 4 °C.
14. After centrifugation, carefully aspirate the supernatant.
15. C entrifuge at 400 x g for 5 min at 4 °C.
16. R epeat Steps 11-13 twice, discard supernatant and resuspend cell pellet in an optimum volumeof room temperature fixative solution. (Store at -30 °C)
C. Slide preparation
1. Adjust the cell concentration with freshly prepared cold fixative.
2. D rop the 13-17 µL cell suspension onto a clean slide (or use Hanabi spreader to spread cells ina controlled environment).
3. D ry the slide for at least 30 min prior to staining. Slides can be dried quickly in a 50-60 °C ovenor dried overnight at room temperature.
 ote: Preparation of clean slideNPre-cleaned slides were kept in 100% Et-OH over 1 week (if necessary).
D. Giemsa staining
1. P repare the staining solution (4% Giemsa in Gurr buffer, pH 6.8).
2. S
 et the slide into an appropriate staining jar.
3. S tain the slide for 10 min at room temperature.
4. After 10 min, gently remove the oxidized Giemsa film formed on the surface of the staining
solution.
5. R inse slides with water.
6. A uthors recommend that the processed slides should fully dry before mounting. If dose
estimate needs to be reported in a short period (i.e., triage), slides can be dried in 60 °C for 1 h
before mounting or immersion oil can be dropped directly on fully dried slides for microscopeviewing. In all other cases, slides should be dried at 37 °C for 24 h.
E. Mounting
Mounting media, such as Malinol, are needed for making permanent slides. This ensures that thecells are protected after Giemsa staining and allows for long-term slide storage.

Donovan Anderson et al./ Radiation Environment and Medicine 2023 Vol.12, No.2 121–139

1. D
 rop the mounting medium (Malinol) on the cover slip.
2. M ount the cover slip gently. Do not make any bubbles.
3. K eep the slide on a flat surface and place it in a clean environment for 2 h to allow the coverslip to firmly adhere to slide.
References
1.

IAEA. Cytogenetic Dosimetry: Applications in Preparedness for and Response to Radiation Emergencies. Vienna: International
Atomic Energy Agency; 2011.

2.

International Organization for Standardization. [Internet] ISO 19238 - Radiological Protection— Performance Criteria for
Service Laboratories Performing Biological Dosimetry by Cytogenetics [updated 2014; cited 2022 Aug]. Available from;https://www.iso.org/standard/52157.html.

3.

International Organization for Standardization. [Internet] ISO 21243. - Radiation Protection— Performance Criteria for
Laboratories Performing Cytogenetic Triage for Assessment of Mass Casualties in Radiological or Nuclear Emergencies —General Principles and Application to Dicentric Assay [updated 2008; cited 2022 Aug]. Available from; https://www.iso.org/
standard/40088.html.

4.

Nakayama R, Abe Y, Goh V, Takebayashi K, Thanh MT, Fujishima Y, et al. Cytogenetic Biodosimetry in Radiation Emergency
Medicine: 4. Overview of cytogenetic biodosimetry. Radiat Environ Med. 2022;11:91–103.

5.

Takebayashi K, Echizenya K, Kameya Y, Nakajima D, Nakayama R, Fujishima Y, et al. Mitotic index maximization with noeffect on radiation-induced dicentric chromosome frequency. Int J Radiat Biol. 2023;99(5):750–9.

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