Radiat Environ Biophys

DOI 10.1007/s00411-017-0696-3

ORIGINAL ARTICLE

Cytogenetic biodosimetry and dose-rate effect after radioiodine

therapy for thyroid cancer
Igor K. Khvostunov1,2 • Vladimir A. Saenko2 • Valeri Krylov1 • Andrei Rodichev1 •

Shunichi Yamashita2

Received: 23 November 2016 / Accepted: 11 May 2017

Ó Springer-Verlag Berlin Heidelberg 2017

Abstract This study set out to investigate chromosomal the cumulative whole-body dose by the factor ranging from
damage in peripheral blood lymphocytes of thyroid cancer 2.6 to 6.8. Elevated frequency of chromosomal aberrations
patients receiving 131I for thyroid remnant ablation or observed in re-treated patients before radioiodine therapy
treatment of metastatic disease. The observed chromoso- allows estimation of a cumulative dose received from all
mal damage was further converted to the estimates of previous treatments.
whole-body dose to project the adverse side effects.
Chromosomal aberration analysis was performed in 24 Keywords Radioiodine therapy
Thyroid cancer

patients treated for the first time or after multiple courses. Chromosomal aberrations
Biological dosimetry
Dose-
Blood samples were collected before treatment and 3 or rate effect
4 days after administration of 2–4 GBq of 131I. Both con-
ventional cytogenetic and chromosome 2, 4 and 12 painting List of abbreviations
assays were used. To account for dose-rate effect, a dose- ace Acentric fragment
protraction factor was applied to calculate the whole-body BNC Binuclear cell
dose. The mean dose was 0.62 Gy (95% CI: 0.44–0.77 Gy) CI Confidence interval
in the subgroup of patients treated one time and 0.67 Gy DAPI 4,6-diamidino-2-phenylindole
(95% CI: 0.03–1.00 Gy) in re-treated patients. These dose dic Dicentric chromosome
estimates are about 1.7-fold higher than those disregarding DNA Deoxyribonucleic acid
the effect of exposure duration. In re-treated patients, the DTC Differentiated thyroid cancer
neglected dose-rate effect can result in underestimation of FISH Fluorescence in situ hybridization
FITC Fluorescein isothiocyanate
GE Genome equivalent
IAEA The International Atomic Energy Agency
ins Insertion
Electronic supplementary material The online version of this MIRD Medical Internal Radiation Dose
article (doi:10.1007/s00411-017-0696-3) contains supplementary MN Micronucleus (micronuclei)
material, which is available to authorized users. PHA Phytohemagglutinin
& Igor K. Khvostunov
rc Centric ring
igor.khvostunov@gmail.com RIT Radioiodine therapy
SEM Standard error of the mean
1
A.F. Tsyb Medical Radiological Research Center, Branch of tc Complete translocation
the National Medical Research Radiological Centre, Russian
ti Incomplete translocation
Ministry of Health Care, Koroliova str. 4, Obninsk,
Kaluga Region, Russia 249036 TNM Tumor node metastasis, tumor staging system
2 TSH Thyroid stimulating hormone
Department of Radiation Molecular Epidemiology, Atomic
Bomb Disease Institute, Nagasaki University, 1-12-4
Sakamoto, Nagasaki, Japan

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Introduction
Radioiodine therapy (RIT) has been used for treatment of
differentiated thyroid cancer (DTC) for more than 70 years. In
this setting, RIT is highly efficient for postsurgical ablation of
thyroid remnants, eradication of nodal disease and of iodine-
avid metastases that are not surgically accessible, such as
miliary pulmonary metastases in young patients (Robbins
et al. 2005). The specificity of 131I metabolism, its biophysical
properties and risk-to-benefit ratio of RIT are the subject of
on-going investigations (Van Nostrand et al. 2009).
Despite obvious advantages, possible drawbacks of RIT
should be taken into account. The main long-term life-
threatening side effects are bone marrow suppression,
aplastic anemia, pulmonary fibrosis and increased inci-
dence of second primary malignancies (Van Nostrand et al.
2009). A meta-analysis of 1607 studies found that the risk
of second malignancies in DTC patients treated with
radioiodine was just slightly higher than in those not
receiving RIT (Sawka et al. 2009). However, a significantly
increased risk of leukemia was reported for administered
cumulative activities exceeding 18.5 GBq, especially in
patients treated previously with external-beam radiation
therapy (Robbins et al. 2005).
To evaluate the risk of side effects, an estimate of the
individual whole-body dose (or a dose to the blood) is
required. Because no serious hematological side effects have
been observed in patients who had received 2 Gy or less to
the blood, which is the proxy for the bone marrow, this
empirical cutoff has been used as a guideline for nuclear
medicine physicians (Benua and Leeper 1986). According to
the radioiodine therapy manual (Luster et al. 2008), the local
131
I doses to the thyroid and other internal organs should be
determined using 131I activity measured in patients and
taking into account biokinetic parameters that describe iso-
tope intake, uptake and excretion (Brill et al. 2006). Unfor-
tunately, the inter-individual variation of these parameters is
substantial. In fact, the actual whole-body dose received by
DTC patients administered a prescribed activity of 11.1, 7.4
and even 3.7 GBq may exceed 2 Gy (Kulkarni et al. 2006).
Optimum administration of 131I has to ensure the elim-
ination of tumor cells while avoiding excessive whole-
body exposure. Achieving treatment goals requires patient-
specific planning strategy and personalization of therapy
(Buckley et al. 2009; Stabin and Brill 2008). Therefore, in
addition to the estimates using radioactivity measurements,
biological dosimetry based on chromosomal aberrations in
blood lymphocytes can be effectively used (IAEA 2011).
In contrast to most physical models (Stabin and Brill 2008),
biological dosimetry assesses individual whole-body dose
without relying on the averaged parameters of a ‘‘refer-
ence’’ man. Besides, biological dose to the blood may be a
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Radiat Environ Biophys
better indicator of treatment success, as opposed to
administered 131I activity (Verburg et al. 2011).
Numerous studies in DTC patients sought to examine
whether the induced frequency of micronuclei (MN) and/or
chromosomal aberrations in blood lymphocytes provides a
reliable estimate of the absorbed dose (Lloyd et al. 1976;
M’Kacher et al. 1996, 1997, 1998; Watanabe et al.
1998, 2004; Monteiro Gil et al. 2000; Nascimento et al. 2010;
Catena et al. 2000; Monsieurs et al. 1999, 2001; Serna et al.
2008). Conditions potentially affecting chromosome damage,
such as an adaptive response (Monsieurs et al. 2000; Monteiro
Gil et al. 2002; Monfared et al. 2004), recombinant human
TSH administration (Frigo et al. 2009), and time of follow-up
(Hernández-Jardines et al. 2010; Puerto et al. 2000; Gutierez
et al. 1995; Federico et al. 2008; Gutiérrez et al. 1999) have
been investigated. Also, Gundy et al. (1996) explored chro-
mosomal aberrations in blood lymphocytes of DTC patients
induced by an external and radionuclide radiation therapy.
Biological dosimetry links the observed biological
response to the calibration dose–response curve established
in vitro (IAEA 2011). The most frequently used for dose
estimation linear-quadratic model describes dose–response
and accounts for dose-rate effect (Brenner et al. 1998; Dale
1985). In case of radionuclide therapy, the importance of
dose rate has been extensively discussed (Lassman et al.
2005; Watanabe et al. 2004; Serna et al. 2008). Recently, a
pronounced dose-rate effect was demonstrated using fluo-
rescent in situ hybridization (FISH) in murine lymphocytes
after low dose-rate exposure to 137Cs delivered in vitro either
by irradiation or by contamination of the medium (Roch-
Lefevre et al. 2016). In addition, Bertucci et al. (2016)
obtained chronic and acute exposure curves for the
micronuclei induced in human lymphocytes and reported a
clear dose-rate effect for the doses above 2 Gy. Therefore,
the significance of dose rate for in vivo dose formation in
DTC patients during RIT needs further careful investigation.
The aim of this study was to investigate the frequency of
chromosomal aberrations in DTC patients after one-time or
multiple therapeutic administrations of 131I. The correla-
tion between the cumulative administered activity of 131I
and induced cytogenetic damage was evaluated. Taking
into account a dose-rate effect, the absorbed dose was
calculated using unstable and stable chromosomal aberra-
tions in blood lymphocytes.
Methods
Patients
Characteristics of the participants of the study are sum-
marized in Table 1. A total of 2 males and 22 females aged
Radiat Environ Biophys

131
Table 1 Characteristics of DTC patients treated with I
131 131
Patient Age, Gender Stage (TNM) Type of One-time activity of I, Cumulative activity I, Number of previous
no. (y) (F/M) therapy GBq (mCi) GBq (mCi) administrations

P1 15 M pT3mN1aM0 One time 2.0 (54) 0 0

P2 8 F pT3mN1bM1 One time 2.0 (54) 0 0
P3 9 F pT4aN1bM1 One time 2.0 (54) 0 0
P4 10 F hN1bN1bM1 One time 2.0 (54) 0 0
P5 11 F pT4N1M0 One time 2.22 (60) 0 0
P6 14 F hN2N1bM0 One time 2.22 (60) 0 0
P7 13 F pT3(m)N0M0 One time 2.22 (60) 0 0
P8 11 F pT1bN1aM0 One time 2.22 (60) 0 0
P9 15 F pT4aN1M0 One time 2.22 (60) 0 0
P10 17 F hN2N1fM0 One time 2.6 (70) 0 0
P11 62 F pT2N0M1 One time 3.0 (81) 0 0
P12 64 F pT2N0M0 One time 3.0 (81) 0 0
P13 19 F hN3N1M0 One time 3.0 (81) 0 0
P14 67 F pT1N0M0 One time 3.0 (81) 0 0
P15 7 M pT3mN1bM1 Multiple 2.0 (54) 1.48 (40) 1 for 1 year
P16 15 F pT3mN1bM1 Multiple 2.6 (70) 25.9 (699) 13 for 7 year
P17 26 F pT2mN1bM1 Multiple 3.0 (81) 42.8 (1156) 17 for 14 year
P18 13 F pT3N1bM0 Multiple 3.0 (81) 11.1 (300) 5 for 3 year
P19 15 F pT4N1bM1 Multiple 3.0 (81) 26.9 (725) 13 for 7 year
P20 18 F pT3N1bM1 Multiple 3.33 (90) 21.7 (585) 7 for 4 year
P21 69 F pT2N0M1 Multiple 4.0 (108) 34.6 (936) 9 for 5 year
P22 68 F pT3N0M1 Multiple 4.0 (108) 23.0 (621) 6 for 3 year
P23 48 F pT3N1bM1 Multiple 4.0 (108) 39.0 (1053) 10 for 5 year
P24 41 F pT4N0M0 Multiple 5.0 (135) 23.0 (621) 6 for 4 year

from 7 to 69 years (mean 27 y.o.) treated for DTC in
Medical Radiological Research Center (Obninsk) were
enrolled. All patients underwent total thyroidectomy; the
diagnosis was confirmed by postsurgical histopathology.
None of the patients had been previously treated with
external-beam radiotherapy. Thyroid hormone withdrawal
was performed 4 weeks before RIT. Peripheral blood
samples collected from the median cubital vein of each
patient before (day 0) and on day 3 or 4 after RIT were
used for cytogenetic analysis. The study was performed in
accordance with national ethical regulations; all study
participants provided informed consent.
In vitro irradiation
To establish in vitro dose response curves, blood samples
(heparinized) obtained from 3 healthy male and 2 female
donors aged 30–45 years were irradiated with the doses
ranging 0.035–4.27 Gy for unstable and 0.25–3.0 Gy for
stable aberration analysis at a dose rate of 0.5 Gy/min
(60Co c-rays, Luch-1, VNIITFA, RosAtom, Russia).
Physical dosimetry was performed with LiF TLD-100
chips (Harshaw Chemical Company, USA) and an ioniza-
tion chamber equipped with c-detector. Blood samples
were irradiated at room temperature and then incubated at
37 °C for 1 h to allow DNA repair before cell culturing.
The number of analyzed cells per dose point ranged from
7799 to 300 for conventional assay and from 3346 to 700
for FISH analysis (Khvostunov et al. 2011, 2015a).
Lymphocyte culture and chromosome preparation
Cell culturing and chromosome preparation were per-
formed using the standard methods routinely used in our
laboratory (IAEA 2011; Sevan’kaev et al. 2005; Khvos-
tunov et al. 2015a). In brief, heparinized peripheral blood
was added to a 10-ml Carrel flask, and lymphocyte cultures
were set up in Minimal Essential Medium (Gibco) sup-
plemented with 20% heat inactivated fetal calf serum
(Gibco), phytohemagglutinin (PHA M-form, Gibco), L-
glutamine (Sigma) and penicillin/streptomycin (Gibco).
BrdU was not added to the medium since this culture
protocol usually results in \5% of second division meta-
phases (Sevan’kaev et al. 1995, 2005; Khvostunov et al.

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2015b, 2015c). Lymphocytes were harvested 48 h later
after the final 0.2 lg/ml Colcemid block (Sigma) for 3 h
according to the standard procedure (IAEA 2011). All
slides were coded to avoid scorer bias.
Conventional cytogenetics
Giemsa-stained slides were scored for unstable chromoso-
mal aberrations, namely dicentrics (dic), centric rings (rc)
and excess acentric fragments (ace). Scoring and recording
were performed according to the IAEA manual (IAEA
2011) using an Eclipse E200F light microscope (Nikon,
Japan). Depending on the slide quality, from 300 to 1186
(mean 526) cells per person were analyzed. Only complete
metaphases (46 centromeres) were considered resulting in
a total of 25,241 cells in this work.
Fluorescence in situ hybridization
To establish the calibration curve or analyze the samples,
the slides were hybridized with either Texas Red-labeled
DNA probes (Metasystems, Germany) to chromosomes 2,
4 and 12 according to the manufacturer’s protocol or
biotinylated probes to chromosomes 2, 3 and 8 followed by
incubation with fluorescein isothiocyanate (FITC)-conju-
gated avidin D and biotinylated goat anti-avidin D anti-
bodies (all reagents from Vector Laboratories) (Table 2).
Counterstaining was performed with 4,6-diamidino-2-
phenylindole (DAPI).
The slides were visualized under a Leica DM5000 B TL
fluorescent microscope (Leica Mikrosysteme Vertrieb
GmbH, Germany), and translocations and insertions were
scored. The chromosomes were analyzed by the direct
microscopy without discrimination between stable and
unstable cells. Only well-spread metaphases with all
painted chromosomes present, and with fluorochrome
labeling sufficiently bright to detect exchange events were
considered. Bicolored chromosomes were classified either
as complete or incomplete symmetrical translocations or
insertions. A complete translocation (tc) was scored when
two bicolored monocentric chromosomes (i.e., with
exchanged counterparts) were present. An incomplete
translocation (ti) was scored when only one bicolored
monocentric chromosome was present. An insertion (ins)
was scored when a chromosome had exactly two color
junctions on the same side of the centromere with the
flanking parts of the same color.
From 312 to 1200 (mean 950), cells per person were
analyzed by FISH technique in a total of 11,397 metaphases.
Dose assessment
The original biodosimetry approach using linear-quadratic
dose–response of chromosomal aberration frequency has
been applied (IAEA 2011). This was further refined by
considering a dose-rate effect. The incomplete-repair
model was used to account for dose-rate effect as described
in Supplementary materials. The results were compared
with the predictions of mono-exponential and instanta-
neous models of irradiation recommended by IAEA (IAEA
2011). Considering that biokinetic parameters of DTC
patients drastically differ from those of a ‘‘reference’’ man
because of thyroidectomy (Stabin and Brill 2008), the
effective decay coefficient, k, has to be estimated using
individual 131I retention in DTC patients measured by a
whole-body scan. In the present work, k = 0.061 h-1 was
used based on the published data (Willegaignon et al.
2006). Assuming the repair time constant for lesions in
human lymphocytes as l = 0.438 h-1 (Bhat and Rao
2003), the probability u(t) and the dose-protraction factor
G(T) were calculated using Eqs. (2), (3) and (4) as shown
in Supplementary materials. The probability u(t) was

Table 2 Stable chromosomal aberrations detected by selective chromosome painting (FISH method) after in vitro irradiation of donor blood
samples with 60Co source
Dose, Gy No cells Tc Tc ? ti ins Chromosomes No GE cells F*G(tc ? ti)/100 GE cells±SEM
Observed Induced

0 3346 7 8 0 (2, 3, 8) 1077 0.74 ± 0.26 0.0 ± 0.53

0.25 2000 3 12 0 (2, 3, 8) 644 1.86 ± 0.54 1.12 ± 0.8
0.5 1625 10 18 0 (2, 4, 12) 509 3.54 ± 0.83 2.80 ± 1.10
0.5 1338 9 16 0 (2, 3, 8) 431 3.71 ± 0.93 2.97 ± 1.19
1.0 1000 16 23 0 (2, 3, 8) 322 7.14 ± 1.49 6.40 ± 1.75
1.0 1859 43 61 1 (2, 4, 12) 582 10.5 ± 1.3 9.74 ± 1.60
2.0 1709 112 141 2 (2, 4, 12) 535 26.4 ± 2.2 25.6 ± 2.5
2.0 712 40 65 0 (2, 3, 8) 229 28.4 ± 3.5 27.6 ± 3.8
3.0 700 111 163 1 (2, 4, 12) 219 74.4 ± 5.8 73.7 ± 6.1
* FG frequency of (tc ? ti) per 100 genome equivalent cells

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Radiat Environ Biophys
calculated using the incomplete-repair model for which the
model parameter was set to s = l-1 = 2.28 h (Bhat and
Rao 2003).
Statistical analysis
Data were summarized as a mean ± standard error of the
mean (SEM). SEM was calculated assuming Poisson dis-
tribution of aberrations in the cells using the U test proposed
by Papworth (Edwards et al. 1979). When the statistic
|U| [ 1.96, the distribution is non-Poisson. Student’s paired
t test was used to compare means with a threshold for sta-
tistical significance p = 0.05. The 95% confidence intervals
(CI) of absorbed dose were calculated using mathematic
algorithm recommended by the IAEA in which two com-
ponents contribute to the uncertainty in the dose: one from
the Poisson nature of the yield of aberrations and another
from uncertainties associated with the calibration curve
(IAEA 2011). The least squares method taking into account
SEM was used to construct the dose–response frequency of
translocations after in vitro exposure to 60Co c-rays.
To calculate genomic equivalence (GE), the Lucas’ for-
mula was used (Lucas et al. 1992). DNA content of chro-
mosomes 2, 3, 4, 8 and 12 was obtained from the earlier work
(Morton 1991). The GE frequency of translocations, FG, was
calculated as FG = FP/NGE, where FP is the frequency of
translocations measured by FISH, NGE is the number of GE
cells determined as NGE = fg N; fg = 2.05 fp (1-fp), where
fp is the fraction of genome covered by the three chromo-
somes, N is the number of cells actually analyzed. In the
current study fg = 0.313 for chromosomes 2, 4 and 12 and
0.322 for chromosomes 2, 3 and 8.
Results
Dose–response curves
The dose–response curve based on unstable aberration
analysis has been established earlier (Khvostunov et al.
Table 3 Calibration curve fitting using a linear-quadratic formula (Y = c ? a D ? b D2) for the frequency of unstable radiation markers
detected by conventional method and stable radiation markers detected by FISH method
Aberration type (c ± SEM), (a ± SEM),
aberrations/100 cells aberrations/100 cells, Gy-1
Dic observed 0.011 ± 0.012 1.40 ± 0.38
(dic ? rc) observed 0.012 ± 0.012 1.42 ± 0.42
(dic ? rc) induced 0 1.54 ± 0.44
(tc ? ti) observed 0.81 ± 0.25* 1.81 ± 1.25*
(tc ? ti) induced 0 1.58 ± 1.38*
* Scaled to genome according to (Lucas et al. 1992)
2015a, b). The result of stable aberration analysis using
selective chromosome (2, 4, 12) or (2, 3, 8) painting is
shown in Table 2. Adjusted parameters of linear-quadratic
dose curves that fit the frequency of all aberrations (ob-
served) or excess aberrations due to exposure (induced) are
presented in Table 3.
Patients
Twenty-four DTC patients were treated with 2–5 Gbq of
131
I, Table 1. Patients were classified into two subgroups,
one-time RIT or re-treatment. The first subgroup included
14 patients; the second—10 patients with 1–17 prior
cumulative administrations of 1.48–42.8 Gbq of 131I during
1–14 years. According to IAEA manual (IAEA 2011), the
cytogenetic examination of each DTC patient was per-
formed before and just after RIT.
Unstable aberrations detected by conventional
method
Table 4 demonstrates the numbers and frequencies of
dicentrics and centric rings, which are radiation markers.
DTC patients treated one time did not display excess of
aberrations as compared to spontaneous level before
treatment. In contrast, re-treated patients displayed a clear
evidence of previous exposure(s) before the last treatment.
For this reason, the dose received by patients treated one
time was calculated using the post-treatment frequency and
the dose curve for observed aberrations (Table 5). For re-
treated patients, we used an increment in frequency (sub-
traction of pre-value from post-value) and the dose curve
for radiation-induced aberrations (Table 6).
The dose-protraction factor, G(T), was calculated for
each patient using incomplete-repair model and the period
of isolated hospital stay (Tables 5 and 6). The mean whole-
body dose of patients treated one time ranged 0.4–0.96 Gy
with the average of 0.62 Gy. The mean whole-body dose of
re-treated patients ranged 0.11–1.22 Gy with the average
0.67 Gy. The dose calculated assuming instantaneous
(b ± SEM), References
aberrations/100 cells, Gy-2
7.28 ± 0.24 (Khvostunov et al. 2015b)
9.59 ± 0.27 (Khvostunov et al. 2015a)
9.55 ± 0.28 (Khvostunov et al. 2015a)
6.34 ± 0.73* This study
6.51 ± 0.82* This study
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 Radiat Environ Biophys

Table 4 Unstable chromosomal aberrations in blood lymphocytes of DTC patients detected by the conventional method before and after RIT
Patient no. Date, days No of cells ace rc dic ? rc (dic ? rc)/100 Distribution (dic ? rc) r2/Y* U**
cells ± SEM in the cells

P1 0 500 1 0 0 0.00 ± 0.36 500/0 0.00 0.00

P1 3 1131 8 1 11 0.97 ± 0.29 1120/11/0 0.99 -0.22
P2 0 500 3 0 1 0.20 ± 0.20 499/1/0 1.00 0.00
P2 3 610 14 0 9 1.48 ± 0.49 601/9/0 0.99 -0.24
P3 0 500 2 1 1 0.20 ± 0.20 499/1/0 1.00 0.00
P3 4 500 32 2 11 2.20 ± 0.66 489/11/0 0.98 -0.33
P4 0 500 0 0 0 0.00 ± 0.36 500/0 0.00 0.00
P4 3 500 7 1 18 3.60 ± 0.85 484/14/2/0 1.19 3.07
P5 0 500 0 0 0 0.00 ± 0.36 500/0 0.00 0.00
P5 3 300 4 1 4 1.33 ± 0.67 296/4/0 0.99 -0.14
P6 0 500 3 0 0 0.00 ± 0.36 500/0 0.00 0.00
P6 3 500 13 2 7 1.40 ± 0.53 493/7/0 0.99 -0.21
P7 0 500 0 0 0 0.00 ± 0.36 500/0 0.00 0.00
P7 3 500 6 1 7 1.40 ± 0.53 493/7/0 0.99 -0.21
P8 0 500 1 0 0 0.00 ± 0.36 500/0 0.00 0.00
P8 3 500 11 0 8 1.60 ± 0.57 492/8/0 0.99 -0.24
P9 0 500 0 0 1 0.20 ± 0.20 499/1/0 1.00 0.00
P9 3 500 6 2 10 2.00 ± 0.63 490/10/0 0.98 -0.30
P10 0 500 2 0 3 0.60 ± 0.35 498/1/1/0 1.66 12.85
P10 3 500 14 0 9 1.80 ± 0.60 491/9/0 0.98 -0.27
P11 0 500 1 0 1 0.20 ± 0.20 499/1/0 1.00 0.00
P11 4 1186 39 4 19 1.60 ± 0.37 1170/14/1/1/0 1.41 10.2
P12 0 500 0 0 0 0.00 ± 0.36 500/0 0.00 0.00
P12 3 500 13 2 9 1.80 ± 0.60 491/9/0 0.98 -0.27
P13 0 500 0 0 0 0.00 ± 0.36 500/0 0.00 0.00
P13 3 500 11 4 13 2.60 ± 0.72 488/11/1/0 1.13 2.14
P14 0 500 3 0 0 0.00 ± 0.36 500/0 0.00 0.00
P14 3 500 16 3 15 3.00 ± 0.77 485/15/0 0.97 -0.46
P15 0 500 1 0 4 0.80 ± 0.40 498/1/0/1/0 2.50 27.30
P15 4 500 17 1 12 2.40 ± 0.69 488/12/0 0.98 -0.36
P16 0 500 23 4 30 6.00 ± 1.10 471/28/1/0 1.01 0.14
P16 4 500 50 11 44 8.80 ± 1.33 458/40/2/0 1.00 0.08
P17 0 500 18 8 29 5.80 ± 1.08 472/27/1/0 1.01 0.21
P17 3 500 27 4 30 6.00 ± 1.10 472/26/2/0 1.08 1.21
P18 0 500 23 10 31 6.20 ± 1.11 469/31/0 0.94 -0.97
P18 3 500 38 12 38 7.60 ± 1.23 467/30/2/0/1/0 1.35 5.57
P19 0 500 24 3 18 3.60 ± 0.85 483/16/1/0 1.08 1.26
P19 4 500 23 8 45 9.00 ± 1.34 463/31/4/2/0 1.36 5.71
P20 0 500 22 5 28 5.60 ± 1.06 475/22/3/0 1.16 2.58
P20 3 500 35 9 47 9.40 ± 1.37 456/41/3/0 1.04 0.57
P21 0 500 42 10 41 8.20 ± 1.28 462/35/3/0 1.07 1.06
P21 3 500 55 13 45 9.00 ± 1.34 456/43/1/0 0.96 -0.70
P22 0 500 12 11 36 7.20 ± 1.20 469/27/3/1/0 1.26 4.23
P22 3 500 17 8 43 8.60 ± 1.31 458/41/1/0 0.96 -0.60
P23 0 500 8 5 15 3.00 ± 0.77 486/13/1/0 1.11 1.73

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Radiat Environ Biophys

Table 4 continued
Patient no. Date, days No of cells ace rc dic ? rc (dic ? rc)/100 Distribution (dic ? rc) r2/Y* U**
cells ± SEM in the cells

P23 3 514 28 4 24 4.67 ± 0.95 492/20/2/0 1.12 2.00

P24 0 500 49 3 36 7.20 ± 1.20 467/30/3/0 1.10 1.55
P24 3 500 38 7 45 9.00 ± 1.34 458/39/3/0 1.05 0.73
* Dispersion factor
** The U–statistic value (Edwards et al. 1979)

Table 5 Aberration yield and cytogenetic dose estimate for DTC patients treated one time using the frequency of dicentrics plus centric rings
Patient no. Pre-treatment T, h Post-treatment G(T) Dose, Gy (CI*) Dose, Gy at
G(T) = 1, (CI*)
Cells (dic ? rc)/100 ± SEM Cells (dic ? rc)/100 ± SEM

P1 500 0.0 ± 0.36 68 1131 0.97 ± 0.29 0.251 0.40 (0.0–0.65) 0.25 (0.0–0.40)
P2 500 0.20 ± 0.20 68 610 1.48 ± 0.49 0.251 0.54 (0.0–0.83) 0.32 (0.0–0.50)
P3 500 0.20 ± 0.20 92 500 2.20 ± 0.66 0.246 0.71 (0.21–1.02) 0.41 (0.13–0.59)
P4 500 0.0 ± 0.36 68 500 3.60 ± 0.85 0.251 0.96 (0.55–1.28) 0.54 (0.31–0.73)
P5 500 0.0 ± 0.36 68 300 1.33 ± 0.67 0.251 0.50 (0.0–0.87) 0.30 (0.0–0.52)
P6 500 0.0 ± 0.36 68 500 1.40 ± 0.53 0.251 0.52 (0.0–0.83) 0.31 (0.0–0.50)
P7 500 0.0 ± 0.36 68 500 1.40 ± 0.53 0.251 0.52 (0.0–0.83) 0.31 (0.0–0.50)
P8 500 0.0 ± 0.36 68 500 1.60 ± 0.57 0.251 0.57 (0.0–0.88) 0.34 (0.0–0.52)
P9 500 0.20 ± 0.20 68 500 2.00 ± 0.63 0.251 0.66 (0.13–0.97) 0.39 (0.09–0.57)
P10 500 0.60 ± 0.35 68 500 1.80 ± 0.60 0.251 0.62 (0.03–0.93) 0.36 (0.03–0.55)
P11 500 0.20 ± 0.20 92 1186 1.60 ± 0.37 0.246 0.57 (0.20–0.81) 0.34 (0.13–0.48)
P12 500 0.0 ± 0.36 68 500 1.80 ± 0.60 0.251 0.62 (0.03–0.92) 0.36 (0.03–0.55)
P14 500 0.0 ± 0.36 68 500 3.00 ± 0.77 0.251 0.86 (0.42–1.17) 0.49 (0.25–0.67)
P13 500 0.0 ± 0.36 68 500 2.60 ± 0.72 0.251 0.78 (0.32–1.09) 0.45 (0.19–0.63)
Mean ± SEM 500 0.11 ± 0.04 71 587 1.82 ± 0.15 0.250 0.62 (0.44–0.77) 0.37 (0.25–0.46)
* 95% confidence interval

exposure at G(T) = 1 is shown in Tables 5 and 6 for
comparison. Noteworthy, this dose was systemically lower
than the dose obtained in the model accounting for dose
protraction.
In addition to biological dosimetry, cytogenetic exami-
nation can provide useful individual details of radiation
sequela in DTC patients. The Papworth’s U-test detected 4
patients receiving RIT for the first time (P4, P10, P11 and
P13) and 6 re-treated patients (P15, P18, P19, P20, P22 and
P23) who displayed the departure of aberration frequencies
from Poisson distribution (|U| [ 1.96, Table 4). According
to the IAEA manual (IAEA 2011), the Papworth’s U-test
can determine whether irradiation of the body was homo-
geneous if cytogenetic analysis was performed just after
irradiation.
Thus, the inhomogeneous exposure due to RIT could be
expected in patients P4, P11, P13, P18, P19 and P23 in
whom unstable radiation markers were over-dispersed after
treatment. The over-dispersion of radiation markers
observed in P10 before RIT can result from computerized
tomography scan carried out for P10 three months before
RIT (Abe at al. 2015).
Inhomogeneous distribution of radiation markers has
been also associated with the presence of metastases (Fadel
et al. 2012). In this study, metastases were diagnosed in 7
of 10 patients with over-dispersion of radiation markers:
P4, P11, P15, P19, P20, P22 and P23 (Tables 1 and 4).
The relationship between the frequency of unstable ra-
diation markers and the cumulative administered activity of
131
I was analyzed in the subgroup of re-treated patients.
Figure 1 (the upper panel) shows the frequency of chro-
mosomal aberrations as a function of cumulative 131I
activity before the last RIT.
Apparently, two patients (P17 and P23) who received
the highest cumulative activity of 131I displayed relatively
low level of (dic ? rc). As for P17, the course of her RIT
was interrupted for 5 years in the middle of 14-year-long
treatment period, which could be the reason for the
reduction in aberration frequency. The cause of low fre-
quency of (dic ? rc) in P23 is unclear. Speculatively, this

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Table 6 Aberration yield and cytogenetic dose estimate for re-treated DTC patients using frequency of dicentrics plus centric rings
Patient no. Pre-treatment T, h Post-treatment Increment (dic ? rc)/ G(T) Dose, Gy (CI*) Dose, Gy at
100 ± SEM G(T) = 1, (CI*)
Cells (dic ? rc)/ Cells (dic ? rc)/
100 ± SEM 100 ± SEM

P15 500 0.80 ± 0.40 92 500 2.40 ± 0.69 1.60 ± 1.09 0.246 0.56 (0–1.04) 0.34 (0–0.61)
P16 500 6.00 ± 1.10 92 500 8.80 ± 1.33 2.80 ± 2.42 0.246 0.81 (0–1.56) 0.47 (0–0.87)
P17 500 5.80 ± 1.08 68 500 6.00 ± 1.10 0.20 ± 2.17 0.251 0.11 (0–1.15) 0.09 (0–0.67)
P18 500 6.20 ± 1.11 68 500 7.60 ± 1.23 1.40 ± 2.34 0.251 0.51 (0–1.36) 0.31 (0–0.78)
P19 500 3.60 ± 0.85 92 500 9.00 ± 1.34 5.40 ± 2.19 0.246 1.22 (0.26–1.8) 0.68 (0.16–0.99)
P20 500 5.60 ± 1.06 68 500 9.40 ± 1.37 3.80 ± 2.43 0.251 0.98 (0–1.66) 0.56 (0–0.93)
P21 500 8.20 ± 1.28 68 500 9.00 ± 1.34 0.80 ± 2.62 0.251 0.34 (0–1.35) 0.22 (0–0.77)
P22 500 7.20 ± 1.20 68 500 8.60 ± 1.31 1.40 ± 2.51 0.251 0.51 (0–1.40) 0.31 (0–0.80)
P23 500 3.00 ± 0.77 68 514 4.67 ± 0.95 1.67 ± 1.73 0.251 0.57 (0–1.23) 0.35 (0–0.71)
P24 500 7.20 ± 1.20 68 500 9.00 ± 1.34 1.80 ± 2.54 0.251 0.60 (0–1.45) 0.36 (0–0.83)
Mean ± SEM 500 5.36 ± 0.33 75 501 7.44 ± 0.39 2.08 ± 0.72 0.246 0.67 (0.03–1.0) 0.39 (0.03–0.59)
* 95% confidence interval

patient might have decreased individual radiation

sensitivity.
Overall, a rather weak correlation (R = 0.68,
p = 0.065) between the frequency of (dic ? rc) and
cumulative activity of 131I administered was found (Fig. 2).
This suggests that the frequency of unstable aberrations in
re-treated patients with DTC only partly depends on the
cumulative activity of administered radioactivity, and that
other factors may play a role.

Stable aberrations detected by FISH method

In contrast to one time-treated patients, retrospective esti-

mation of the dose accumulated from previous RIT is the

Fig. 1 Unstable (upper panel) and stable (lower panel) radiation

markers in blood lymphocytes of re-treated DTC patients before
(open bars) and after (hatched bars) RIT. Solid bars in lower panel
show age-dependent background. Patients are arranged in ascending
order according to the cumulative activity of 131I received before the
last administration. n.a. not available
Fig. 2 Frequency of unstable radiation markers in re-treated DTC
patients detected before the last RIT in relation to the cumulative
administered activity of 131I. Solid line is a linear regression, dashed
lines are the 95% confidence limits

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Table 7 Stable chromosomal

Patients no. Date, days No cells Translocations Insertions *FG(tc ? ti)/100 GE ± SEM
aberrations detected in blood
lymphocytes of re-treated DTC tc ti
patients by FISH method before
and after RIT P16 0 999 26 14 0 12.8 ± 2.0
P16 4 688 28 10 0 17.6 ± 2.9
P18 0 1000 17 8 0 8.0 ± 1.6
P18 3 1000 18 14 0 10.2 ± 1.8
P19 0 998 18 12 0 9.6 ± 1.8
P19 4 312 8 6 0 14.3 ± 3.8
P20 0 1000 12 7 0 6.1 ± 14
P20 3 1000 28 18 0 14.7 ± 2.2
P21 0 1200 25 13 1 10.1 ± 1.6
P21 3 1200 27 20 0 12.5 ± 1.8
P24 0 1000 33 13 0 14.7 ± 2.2
P24 3 1000 35 19 0 17.3 ± 2.3
* FG frequency of (tc ? ti) per 100 genome equivalent cells

Table 8 Aberration yield and cytogenetic dose estimate for re-treated DTC patients using the frequency of complete plus incomplete
translocations
Patient no. Pre-treatment T, h Post-treatment Increment (tc ? ti)/ G(T) Dose, Gy (CI*) Dose, Gy at
100 GE cells ± SEM G(T) = 1, (CI*)
Cells (tc ? ti)/100 GE Cells (tc ? ti)/100 GE
cells ± SEM cells ± SEM

P16 999 12.8 ± 2.0 92 688 17.6 ± 2.9 4.85 ± 4.85 0.246 1.37 (0–2.59) 0.76 (0–1.51)
P18 1000 8.0 ± 1.6 68 1000 10.2 ± 1.8 2.24 ± 3.37 0.251 0.83 (0–1.94) 0.49 (0–1.17)
P19 998 9.6 ± 1.8 92 312 14.3 ± 3.8 4.73 ± 5.48 0.246 1.35 (0–2.71) 0.75 (0–1.57)
P20 1000 6.1 ± 1.4 68 1000 14.7 ± 2.2 8.63 ± 3.51 0.251 1.92 (0–2.70) 1.05 (0–1.57)
P21 1200 10.1 ± 1.6 68 1200 12.5 ± 1.8 2.40 ± 3.25 0.251 0.87 (0–1.93) 0.51 (0–1.17)
P24 1000 14.7 ± 2.2 68 1000 17.3 ± 2.3 2.60 ± 4.50 0.251 0.91 (0–2.23) 0.53 (0–1.33)
Mean ± SEM 1033 10.2 ± 0.7 76 867 14.2 ± 0.9 3.98 ± 1.62 0.249 1.21 (0–1.72) 0.68 (0–1.06)
* 95% confidence interval

vital task for re-treated patients. According to the IAEA
manual (2011), stable chromosomal aberrations should be
analyzed to this end.
Table 7 shows the numbers and GE frequencies of
complete and incomplete translocations (the radiation
markers) detected in re-treated DTC patients by FISH
method. Figure 1 (the lower panel) presents the frequency
of stable radiation markers before and after RIT along with
unstable radiation markers in the same patient. The fre-
quency of both unstable and stable aberrations before RIT
was found to be elevated as compared to spontaneous level.
The data obtained show different increments in unsta-
ble and stable radiation markers due to RIT, attesting to the
usefulness of cytogenetic analysis for biological dosimetry
of DTC patients.
The dose-protraction factor, G(T), for re-treated patients
was calculated in the same way as for patients treated one
time. As a result, the mean whole-body dose of re-treated
patients ranged 0.83–1.92 Gy with an average of 1.21 Gy
(Table 8). The dose calculated assuming instantaneous
exposure at G(T) = 1 is shown in Table 8 for comparison.
Again, it was far lower than the prediction of the model
accounting for dose protraction.
Retrospective biological dosimetry
Elevated frequency of stable and/or unstable radiation
markers in blood lymphocytes of re-treated patients, as
compared to spontaneous level, is a sign of radiation his-
tory. This frequency can be used for retrospective dose
assessment, and the cumulative 131I activity administered
to a patient in all previous courses of RIT should be taken
into account.
The cumulative administered activity of 131I before the
last treatment, the frequencies of unstable markers
(dic ? rc) and the induced translocations (tc ? ti) are
shown in Table 9. Using these frequencies, the retro-
spective dose was calculated based on in vitro calibration

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Table 9 Aberration yield, cytogenetic cumulative dose estimate and retrospective dose reconstruction for re-treated DTC patients based on the
frequency of unstable and stable chromosomal aberrations and administered cumulative activity of 131I
131
Patient Cumulative I, (dic ? rc)/100 D(dic ? rc), Gy bkg* FG(tc ? ti) D(tc ? ti), Gy DRETRO,
no. Gbq cells ± SEM ±SEM** Gy***
G(T) = 0 G(T) = 1 G(T) = 0 G(T) = 1

P15 1.48 0.80 ± 0.40 0.56 0.22 0.10 – – – 0.35

P16 25.9 6.00 ± 1.10 4.2 0.72 0.20 12.6 ± 2.0 8.0 1.25 6.2
P17 42.8 5.80 ± 1.08 4.1 0.69 0.36 – – – 10.2
P18 11.1 6.20 ± 1.11 4.3 0.73 0.18 7.8 ± 1.6 5.0 0.99 2.6
P19 26.9 3.60 ± 0.85 2.5 0.54 0.20 9.4 ± 1.8 6.0 1.10 6.4
P20 21.7 5.60 ± 1.06 3.9 0.70 0.24 5.9 ± 14 3.7 0.84 5.2
P21 34.6 8.20 ± 1.28 5.8 0.85 1.37 8.7 ± 1.6 5.5 1.05 8.2
P22 23.0 7.20 ± 1.20 5.1 0.79 1.34 – – – 5.5
P23 39.0 3.00 ± 0.77 2.1 0.49 0.78 – – – 9.3
P24 23.0 7.20 ± 1.20 5.1 0.79 0.63 14.1 ± 2.2 8.9 1.35 5.5
* bkg—age-dependent background of (tc ? ti) per 100 genome equivalent cells (Sigurdson et al. 2008)
** FG—radiation-induced genome equivalent frequency of (tc ? ti) per 100 genome equivalent cells adjusted for age-dependent background
*** DRETRO—retrospective estimation of accumulated dose

curves (Table 3). In addition, according to the IAEA
manual (IAEA 2011), the following two scenarios of
irradiation were considered: prolonged exposure with low
dose rate when G(T) & 0 and instantaneous exposure
when G(T) & 1. Eventually, the retrospective whole-body
dose based on unstable markers ranged 0.56–5.8 Gy at
G(T) = 0 and 0.22–0.85 Gy at G(T) = 1. In turn, the
retrospective dose based on stable markers ranged
3.7–8.9 Gy at G(T) = 0 and 0.84–1.35 Gy at G(T) = 1
(Table 9).
The cytogenetic dose was compared with retrospective
physical dose estimation based on cumulative 131I activity
administered in all previous RITs. For this purpose, the
mean dose per unit activity was estimated using the
administered activities of 131I (Table 1) and absorbed dose
shown in Tables 5 and 8. This resulted in the mean whole-
body dose per activity unit (the kD) of 0.238±0.020 mGy/
MBq, and physical estimates of retrospective dose ranged
0.35–10.2 Gy (Table 9). This dose is expected to be
received by re-treated DTC patients as a result of all pre-
vious RIT courses.
Discussion
The primary focus of cytogenetic biodosimetry is the
excess of a radiation marker yield over background level.
The background frequency of unstable radiation markers
(dicentrics) is rather low, just about 0.1 per 100 cells
(IAEA 2011), and it does not depend on age or gender. The
background frequency of stable radiation markers
(translocations) is relatively higher. This frequency is age
dependent, increasing from 0.04 to 1.6 per 100 GE cells
during man’s lifetime up to 75 years old (Sigurdson et al.
2008).
Our work demonstrated the usefulness of cytogenetic
biodosimetry as a tool for dose assessment of DTC patients
receiving RIT. In our study, the group-average frequency
of dicentrics plus centric rings per 100 cells was
1.82 ± 0.15 in patients treated one time, and the increment
of 2.08 ± 0.72 was found in re-treated patients after
administration of 2–4 GBq 131I. Based on this data, we
estimated the mean whole-body dose per activity unit to be
0.238±0.020 mGy/MBq.
It should be noted that for improving the accuracy of
retrospective dose estimation, it would be useful to take
into account individual scheme of RIT re-treatment and
inherent individual radiosensitivity of a patient whenever
possible. However, this problem could not be addressed in
this work. In addition, the calibration curve used could be
defined more precisely by scoring more cells per dose
points, up to 500–2000 GE cells, as recommended by the
IAEA (IAEA 2011).
Our estimate of the mean whole-body dose per activity
unit is broadly in agreement with other estimates obtained
using different methods, that are 0.23 mGy/MBq according
to the Medical Internal Radiation Dose (MIRD) procedures
(de Keizer et al. 2004), 0.18 mGy/MBq assessed by chro-
mosome aberration analysis (Violot et al. 2005) and
0.2 mGy/MBq assessed by the MN assay, (Serna
et al. 2008; Kinashi et al. 2007). Thus, one-time adminis-
tration of 10 GBq of 131I (270 mCi) may result in the
whole-body dose up to 2 Gy (cutoff of myelotoxicity) if
kD = 0.2 mGy/MBq. In clinical practice, administration of
such a high activity of 131I is not unusual (Kulkarni et al.
2006).

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Radiat Environ Biophys
An estimate of the absorbed dose received by DTC
patients can be obtained using the MIRD procedures
(Stabin and Brill 2008). According to those, the kD of a
‘‘reference’’ man ranges 0.035–0.12 mGy/MBq when thy-
roid uptake is 0–55% (Brill et al. 2006). However, because
of total thyroidectomy, this ‘‘reference’’ model is not
applicable to DTC patients. Instead, the individual exam-
ination of DTC patients using a whole-body counter and/or
measuring circulating radioiodine are required to assess the
dose to the blood.
Based on the MIRD procedures, individual absorbed
doses have been determined; the kD for red bone marrow
was 0.16 and 0.23 mGy/MBq for the blood (de Keizer et al.
2004). Haenscheid et al. (2006) estimated kD to the blood
of 0.167 mGy/MBq in the group of DTC patients deprived
of thyroid hormone prior to treatment. In similar studies,
Gonzalez et al. (2013) estimated the whole-body kD of
0.201 mGy/MBq, while Frigo et al. (2009) reported kD to
red bone marrow of 0.10 mGy/MBq. So far, there is some
discordance between dose estimates from RIT in DTC
patients seen as a high variance of estimates of the dose per
activity unit obtained using different assays.
The whole-body dose assessment using MN assay and/
or chromosome aberration analysis in blood lymphocytes
have been performed in a number of groups of DTC
patients who underwent total thyroidectomy and were
treated with 131I (Lloyd et al. 1976; M’Kacher et al.
1996, 1997, 1998; Watanabe et al. 1998, 2004; Monteiro
Gil et al. 2000; Nascimento et al. 2010; Catena et al. 2000;
Monsieurs et al. 1999, 2001; Serna et al. 2008). In all these
studies, blood samples were collected twice: before 131I
administration, and from 3 days to a half of a year after
RIT. Usually DTC patients were administered 3.7 GBq
(100 mCi), but the maximum one-time activity was
8.51 GBq (230 mCi). The study groups included both one
time-treated and re-treated DTC patients who received the
cumulative activities up to 25.9 GBq (700 mCi). As a
result, the group-average frequency of dicentrics per 100
cells ranged from 0.42 (Monteiro Gil et al. 2000) to 4.8
(Violot et al. 2005). The use of MN assay resulted in a wide
variation in MN counts. In the study (Watanabe et al.
2004), the increment of MN in B-lymphocytes and
peripheral blood lymphocytes after 3.7 GBq of 131I was
31.2 and 20.0 per 1000 binuclear cells (BNC), respectively.
In contrast, Serna (2008) reported an increment of 9.2 MN
per 1000 BNC after treatment with 3.7 GBq of 131I. A very
large excess of MN was obtained by Kinashi (2007) in 14
DTC patients who received 3.3–5.6 GBq of 131I: the mean
increment of MN was up to 105 per 1000 BNC.
Finally, Watanabe et al. (1998, 2004) obtained the kD of
0.089 and 0.12 mGy/MBq in two studies using MN assay.
The same assay was used by Serna et al. (2008), who
reported the kD of 0.197 mGy/MBq. On the whole, the
estimates of kD using MN assay ranged from 0.060
(Monsieurs et al. 2001) to 0.197 mGy/MBq (Serna et al.
2008), and those using chromosome aberration analysis
ranged from 0.073 (Nascimento et al. 2010) to 0.34 mGy/
MBq (Lloyd et al. 1976).
Thus, the group-average estimate of kD ranges
0.060–0.34 mGy/MBq depending on the assay and study
group. Moreover, individual values of kD can differ from
each other 2–4-fold under the same administered activity of
131
I. The difference in cytogenetic estimations of kD may
result from the variation of individual parameters of DTC
patients, the assay used and the method of dose calculation.
Besides, there is no agreed biokinetic model for DTC
patients receiving high doses of 131I. In addition, different
stage of disease and occasional mixing of data from follow-
up procedures, thyroid remnant ablation and RIT could be
possible explanations for the discordance in kD estimate
(Kulkarni et al. 2006).
Within the framework of cytogenetic biodosimetry, the
absorbed dose depends on the frequency of chromosomal
aberrations. In the course of RIT, the rate at which the dose
is delivered affects the frequency of chromosomal aberra-
tion. Low dose rate allows DNA repair to take place during
irradiation thus modifying the frequency of aberrations
which is used for dose reconstruction. That is why the
dose-rate effect should be taken into account when cyto-
genetic biodosimetry is used for examination of DTC
patients (Lloyd et al. 1976; Serna et al. 2008; Monteiro Gil
et al. 2000; Kinashi et al. 2007). The main reason for this is
systematic under-estimation of absorbed dose when dose-
rate effect is neglected.
The MIRD procedure and cytogenetic biodosimetry are
the main methods of dose assessment in DTC patients. A
comparative analysis of cytogenetic dosimetry and physi-
cal MIRD procedure has been reported by Lloyd et al.
(1976), Monsieurs et al. (2001) and Nascimento et al.
(2010). For re-treated patients with neuroblastoma and
carcinoid tumors who received 7.4 or 8.5 GBq of 131I, the
whole-body dose has been calculated using both MN assay
and MIRD formalism based on bi-planar scans (Monsieurs
et al. 2001). A reasonable correlation (R = 0.87) between
MIRD dose and cytogenetic dose was obtained. The slope
of the linear regression fit was 0.75 that means 25%
underestimation of dose by cytogenetic assay. The authors
suggested that the reason of this underestimation was
neglecting the dose-rate effect in the course of in vitro
calibration (Monsieurs et al. 2001).
By contrast, a dose-rate effect has been taken into
account in several cytogenetic studies (Lloyd et al. 1976;
Monteiro Gil et al. 2000; Serna et al. 2008). In such a way,
the dose received by DTC patients due to RIT was calcu-
lated avoiding underestimation. Our analysis demonstrate
that the extent of possible underestimation may be 1.2–2.1-
123

fold for one-time RIT assuming the dose-protraction factor
G(T) ranges from 0.1 to 0.58 (Supplementary materials).
Serna et al. (2008) applied MN assay to 25 DTC patients
receiving RIT (Serna et al. 2008). To overcome the diffi-
culty in MN count when it was close to or lower than
background level, the Bayesian statistical approach was
used. The group-average blood dose received by DTC
patients after 3 days of exposure was 0.73 ± 0.5 Gy con-
sidering a dose-protraction factor G(T) = 0.1.
Cytogenetic examination of 19 DTC patients treated
with 2.59 GBq of 131I has been carried out using MN assay
and chromosome aberration analysis at 1 and 6 months
post-treatment (Monteiro Gil et al. 2000). The whole-body
dose has been obtained using the linear in vitro dose–effect
with G(T) = 0. The group-average whole-body dose was
0.2–0.3 Gy based on a dicentric assay and *0.2 Gy based
on a MN analysis.
Lloyd et al. (1976) performed cytogenetic examination
of 5 one-time and 6 re-treated DTC patients to compare
physical and cytogenetic doses. Patients were sampled
before treatment and two weeks post-treatment with 2.96 or
7.4 GBq of 131I. The whole-body dose was obtained using
calibration curve established by in vitro irradiation human
lymphocytes with 60Co c-rays at a very low dose rate of
3 mGy/min (Lloyd et al. 1975). Comparing the quadratic
coefficients of low- and high dose-rate curves, the dose-
protraction factor G(T) was determined to be 0.58. The
group-average dose was 1.02 Gy for DTC patients treated
one time and 0.48 Gy for re-treated DTC patients (Lloyd
et al. 1976).
In the present study, the mean whole-body dose after
3 or 4 days of RIT was 0.63 Gy or 0.238 mGy/MBq. The
dose-protraction factor G(T) * 0.25 was used to account
for the dose-rate effect of exposure. This factor is in the
range of other estimates, such as G(T) = 0 in the MN assay
(Monteiro Gil et al. 2000); G(T) = 0.1 in another MN
assay (Serna et al. 2008), and G(T) = 0.58 in the chro-
mosomal aberration analysis (Lloyd et al. 1976). In addi-
tion, we performed dose calculation assuming
instantaneous irradiation at G(T) = 1 to evaluate the order
of dose underestimation. The results shown in Table 5
demonstrate 1.7-fold lower doses for one-time adminis-
tration of 2–4 GBq of 131I. The dose underestimation ran-
ges from 2.6- to 6.8-fold (Tables 6 and 8) for re-treatment
with the cumulative activity of 1.48–42.8 GBq of 131I.
Despite the dose rate is time dependent during RIT, the
necessity of accounting for its effect has been challenged
(Lassmann et al. 2005). The authors studied biokinetics of
radioiodine in 9 DTC patients to estimate the uptake, dis-
tribution and clearance of 131I in order to calculate the dose
to the blood using MIRD procedures (Luster et al. 2003).
As a result, the dose ranged from 0.32 to 0.47 Gy after
administration of 3.7 GBq of 131I. In parallel, Watanabe
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Radiat Environ Biophys
et al. (1998, 2004), using MN assay, reported the group-
average doses 0.33 and 0.45 Gy in 25 and 22 DTC patients,
respectively, due to administration 3.7 GBq of 131I. Nota-
bly, Watanabe et al. (2004) admitted that reported doses
were underestimated because the calibration curve was
based on the high dose-rate exposure. But Lassmann et al.
(2005) argued this point because the values reported by
Watanabe et al. (1998, 2004) were well within the dose
range determined earlier by Luster et al. (2003) for a
similar group of patients. Based on this, Lassmann et al.
(2005) concluded that the dose-rate effect should not play a
role for biodosimetry of DTC patients. Apparently, this
reasoning leaves ample room for further discussions
because of considerable variance of dose estimates repor-
ted by different researchers. A good agreement between
group-average doses obtained by Watanabe et al.
(1998, 2004) and Luster et al. (2003) might have occurred
by chance due to uncertainties of dosimetric approaches.
Notwithstanding methodological difficulties, the under-
estimation of whole-body dose to DTC patients is of great
practical relevance in the field of RIT. It may be especially
alarming when a large cumulative activity of 131I is
administered in the course of multiple re-treatments.
Conclusions
The cytogenetic test provides a substantial improvement of
dose assessment in DTC patients after RIT as a personal-
ized approach allowing for inherent individual radiosensi-
tivity. The cytogenetic examination of DTC patients
evaluates individual response to radiation in terms of a
balance between radiation-induced DNA damage and
repair capacity. Therefore, it can be used for estimation of
individual radiation risk due to RIT.
Chromosomal aberrations detected in blood lympho-
cytes of DTC patients is a reliable sign of exposure, and
the cytogenetic examination can be used to determine if
the cutoff of whole-body dose of 2 Gy has been excee-
ded. Our work demonstrates that the dose-rate effect
should be taken into account to avoid underestimation of
the absorbed dose. If it is ignored, the ‘‘safe’’ aberration
frequency threshold goes up leading to possible underes-
timation of the risk from RIT. Further studies are neces-
sary to clarify the importance of the dose-rate effect in
RIT setting.
With regard to retrospective dose assessment from pre-
vious exposure(s), which are of a significant concern, ele-
vated frequency of stable radiation markers can be
effectively used. From a clinical point of view, cytogenetic
examination of re-treated DTC patients may provide useful
information regarding the possibility of hematological side
effects before further RIT.
Radiat Environ Biophys
Acknowledgements The authors would like to thank Prof. Vitali
Moiseenko and Prof. David Lloyd for helpful discussions, critical
reading of the manuscript and valuable comments. We are grateful to
Dr. Natalya Shepel and Olga Korovchuk for performing conventional
and FISH analysis. This study was funded in part by the Russian
Humanitarian Scientific Fund and the Government of Kaluga Region,
Project No 15-16-40011 a(p).
Compliance with ethical standards
Conflict of interest The authors declared no potential conflict of
interest with respect to the research, authorship, and/or publication of
this study.
Ethical approval All procedures performed in studies involving
human participants were in accordance with the ethical standards of
the institutional and/or national research committee and with the 1964
Helsinki declaration and its later amendments or comparable ethical
standards.
Informed consent Informed consent was obtained from all partici-
pants of the study.
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