ORIGINAL PAPER

Ratio of c-H2AX level in lymphocytes to that in granulocytesdetected using flow cytometry as a potential biodosimeterfor radiation exposure

Zhidong Wang • Hailiang Hu • Ming Hu • Xueqing Zhang •

Qi Wang • Yulei Qiao • Haixiang Liu • Liping Shen •

Pingkun Zhou • Ying Chen

Received: 9 May 2013 / Accepted: 24 February 2014 / Published online: 1 April 2014

� Springer-Verlag Berlin Heidelberg 2014

Abstract This study aims to assess utilisation of the ratio

of c-H2AX in lymphocytes to that in granulocytes (RL/G

of c-H2AX) in blood as a rapid method for population

triage and dose estimation during large-scale radiation

emergencies. Blood samples from healthy volunteers

exposed to 0–10 Gy of 60Co irradiation were collected. The

samples were cultured for 0–24 h and then analysed using

flow cytometry to measure the levels of c-H2AX in lym-

phocytes and granulocytes. The basal RL/G levels of c-

H2AX in healthy human blood, the response of RL/G of c-

H2AX to ionising radiation and its relationship with doses,

time intervals after exposure and individual differences

were also analysed. The level of c-H2AX in lymphocytes

increased in a dose-dependent manner after irradiation,

whereas the level in granulocytes was not affected. A linear

dose–effect relationship with low inter-experimental and

inter-individual variations was observed. The RL/G of c-

H2AX may be used as a biomarker for population triage

and dose estimation during large-scale radiation emergen-

cies if blood samples can be collected within 24 h.

Keywords RL/G of c-H2AX � Radiation � Lymphocyte �Granulocyte � Flow cytometry

Introduction

Rapid identification of individuals who need immediate

treatment or further assessment for acute radiation syn-

drome is important during large-scale radiation emergen-

cies (Blakely et al. 2005; Flood et al. 2011). A simple, fast

and high-throughput biodosimetry assay is required for

population triage during the first few hours. The dicentric

assay is considered the gold standard for biodosimetry

because it is specific to ionising radiation and stable

enough for dose estimation several months after radiation.

However, the dicentric assay is not suitable for population

triage because it requires a 48-h culture period to obtain

metaphases before chromosome analysis (Roch-Lefevre

et al. 2010;IAEA 2001). c-H2AX, a biomarker of DNA

double-strand breaks (DSBs), may be useful for population

triage and dose estimation.

Double-strand breaks are induced by genotoxic agents,

including ionising radiation. Phosphorylation of H2AX at

serine 139 immediately after DSBs in DNA generates c-

H2AX. The use of an antibody to c-H2AX leads to the

formation of foci that cover large regions surrounding

DSBs. The scoring of c-H2AX foci is used to quantita-

tively evaluate DSBs and to estimate doses after radiation

exposure (Roch-Lefevre et al. 2010; Rothkamm and Lob-

rich 2003; Olive and Banath 2004; Lobrich et al. 2005;

Leatherbarrow et al. 2006; Rothkamm et al. 2007; Sak

et al. 2007). However, this method is labour intensive.

Developing a high-throughput method that can deal with a

large sample size is necessary. Flow cytometry (FCM)

allows rapid detection of c-H2AX in numerous cells.

Recent studies have established FCM methods to detect the

level of c-H2AX in human peripheral blood lymphocytes

exposed to radiation and found that the level of c-H2AX in

cells detected by FCM correlates well with radiation dose

Z. Wang � H. Hu � M. Hu � X. Zhang � Y. Qiao � H. Liu �L. Shen � P. Zhou (&) � Y. Chen (&)

Department of Radiation Toxicology and Oncology, Beijing

Institute of Radiation Medicine, 27 Taiping Road,

Beijing 100850, China

e-mail: zhoupk@nic.bmi.ac.cn

Y. Chen

e-mail: yingchen_29@163.com

Q. Wang

Beijing Consulting Centre of Biomedical Statistics, 27 Taiping

Road, Beijing 100850, China

123

Radiat Environ Biophys (2014) 53:283–290

DOI 10.1007/s00411-014-0530-0

(Hamasaki et al. 2007; Horn et al. 2011; Andrievski and

Wilkins 2009). FCM methods allow high-throughput ana-

lysis, but inter-experimental and inter-individual variations

are also high. These variations limit the application of this

method in radiation accidents because of the lack of non-

irradiated control samples for each individual. The reasons

for these high inter-experimental and inter-individual

variations remain unclear. Previous reports (Ismail et al.

2007; Hamasaki et al. 2007; Andrievski and Wilkins 2009)

indicated that the high variations do not correlate with

DNA damage level and radiosensitivity. In these reports,

the level of c-H2AX in lymphocytes was determined by

fluorescence intensities, which could be easily affected by

operating procedures. Therefore, we presumed that opera-

tor errors during sample labelling and measuring may

cause the high inter-experimental and inter-individual

variations, such as the different quantities of antibody and

parameters of flow cytometer. Blood cells with c-H2AX

levels that are not affected by radiation may help reduce

the variations. Radimet al. (2011) reported that granulo-

cytes in the blood of rats do not decrease at 24 h after

whole-body radiation. This finding indicates that the level

of c-H2AX in granulocytes might not be affected by

radiation. This observation was confirmed in the present

study.

Given that the level of c-H2AX in granulocytes is not

affected by radiation, we established a new FCM-based

method to measure c-H2AX in blood lymphocytes using

the ratio of c-H2AX in lymphocytes to that in granulocytes

(RL/G) of c-H2AX instead of fluorescence intensity. Our

assay showed that the RL/G of c-H2AX was linearly cor-

related with radiation dose. Inter-experimental and inter-

individual variations in the RL/G of c-H2AX at a given

radiation dose or background level were low. The RL/G of

c-H2AX may be used as a new dosimetry method for initial

triage and dose estimation.

Materials and methods

Blood collection and irradiation

After obtaining written informed consent from volunteers

and ethical approval from the Subcommittee on Human

Investigation of the Beijing Institute of Radiation Medicine

(2012–0128), we collected peripheral blood samples from

62 healthy volunteers (39 males, 23–56 years old; 23

females, 24–58 years old) using heparinised tubes. Healthy

human blood samples were not incubated before fixation to

obtain basal RL/G levels of c-H2AX. The blood samples

from the same three volunteers (two males, 35 and 36 years

old; one female, 36 years old) were used for all experi-

ments, except for the experiment in Fig. 2. The blood

sample was divided into 0.3 mL aliquots and then irradi-

ated at 37 �C with a cobalt-60 source (c-rays) at a dose rate

of 158 cGy/min (Beijing Institute of Radiation Medicine).

After irradiation, the aliquots were incubated at 37 �C for

1, 3, 6, 12 and 24 h to measure the kinetics and/or the

dose–effect relationship.

Fixation and immunofluorescence staining

The blood samples were processed using a previously

described protocol with some modifications (Andrievski

and Wilkins 2009). Briefly, 0.3 mL of the incubated blood

samples was added in 1.5 mL of 2 % paraformaldehyde/

phosphate-buffered saline (PFA/PBS) and then fixed for

30 min at room temperature. The samples were washed

with 1 mL PBS, centrifuged at 5009g for 5 min at room

temperature, and then mixed with 1.5 mL Triton

X-100(0.4 % v/v diluted in PBS). The samples were

incubated for 15 min at room temperature and then washed

twice with 1 mL PBS. Thereafter, the cells were incubated

with 100 lL monoclonal c-H2AX antibody(1:200 dilution,

Upstate-Millipore, USA) for 30 min at 37 �C, washed with

1 mL PBS, and then incubated with 100 lL fluorescein

isothiocyanate-conjugated goat anti-mouse secondary

antibody(1:200 dilution, Santa Cruz) for 30 min at 37 �C in

dark. After the reaction, the cells were washed twice with

1 mL PBS, resuspended in 0.5 mL 1 % PFA/PBS, and then

analysed using FCM. The samples incubated with sec-

ondary antibody only were used as control.

FCM analysis

For FCM analysis, data acquisition was set to analyse

10,000 lymphocytes on forward scatter versus side scatter

plot in the region R1 and granulocytes in the region R2

(Fig. 1a). The level of c-H2AX was measured by relative

c-H2AX fluorescence intensities, i.e. the ratio of the geo-

metric mean fluorescence of the cells incubated with

monoclonal c-H2AX antibody and secondary antibody to

that of the cells incubated only with secondary antibody.

All samples were analysed on a BD FACS Calibur flow

cytometer (BD Biosciences).

Statistical analysis

All data were expressed as mean ± standard deviation

(SD) unless stated otherwise. Differences between groups

were analysed using ANOVA. Statistical significance was

considered at P \ 0.05. The SD and coefficient of variation

(CV) of at least three independent measurements or three

individuals were calculated to test for inter-experimental

and inter-individual variations.

284 Radiat Environ Biophys (2014) 53:283–290

123

Results

c-Irradiation increased the c-H2AX level

in lymphocytes but did not affect the c-H2AX level

in granulocytes

Blood samples from three healthy volunteers were irradi-

ated with 0.5, 1, 2, 4, 6, 8 or 10 Gy of c-rays and then

incubated at 37 �C for 1, 3, 6, 12 or 24 h to determine the

effect of radiation on the c-H2AX level in blood lympho-

cytes and granulocytes. Figure 1a, b, c show an example of

FCM used for determining the c-H2AX levels in lym-

phocytes and granulocytes in 10 Gy irradiated blood or

control. As shown in Fig. 1d, h, the level of c-H2AX in

lymphocytes increased in a dose-dependent manner at 1, 3,

6, 12 or 24 h after irradiation, whereas that in granulocytes

was not affected. Thus, the level of c-H2AX in granulo-

cytes could be used as an internal control for the level of c-

H2AX in lymphocytes. In addition, the RL/G of c-H2AX

can be used for c-H2AX assay.

Basal levels of RL/G of c-H2AX in healthy human

blood

We analysed the basal RL/G of c-H2AX in non-irradiated

human blood samples of 62 healthy volunteers (23 females

and 39 males from Chinese population, Fig. 2) to study its

variation among individuals. The mean ± SD of the RL/G

of c-H2AX was 1.21 ± 0.12, with a range of 1.01–1.54, and

the CV was 9.94 % (Fig. 2a). No significant differences in

the RL/G of c-H2AX were found between age groups

(Fig. 2b, 1.18 ± 0.09, 1.22 ± 0.13, 1.20 ± 0.14 and

1.21 ± 0.09 for 21 years old (y) to 30y, 31y to 40y, 41y to

50y and 51y to 60y groups, respectively; P = 0.7372). The

mean value of RL/G of c-H2AX from 23 female volunteers

was similar to that of the 39 male volunteers (Fig. 2c,

1.20 ± 0.10 vs. 1.22 ± 0.15, P = 0.4531). Among the 62

volunteers, 12 have a smoking habit. The RL/G of c-H2AX

from individuals with smoking history (1.21 ± 0.12) was

not significantly higher than that of individuals without

smoking history (1.17 ± 0.08) (Fig. 2d; P = 0.2194).

Fig. 1 Flow cytometry analysis

of c-H2AX in lymphocytes and

granulocytes at 1, 3, 6, 12 and

24 h after c-irradiation.

a Gating on the lymphocytes

(R1) and granulocytes (R2)

based on forward and side light

scattering, which were used for

all subsequent analyses. b c-

H2AX histograms in

lymphocytes at 1 h after 0 Gy

(black line) or 10 Gy (grey

line). c c-H2AX histograms in

granulocytes at 1 h after 0 Gy

(black line) or 10 Gy (grey

line). d–h c-H2AX levels in

lymphocytes and granulocytes

at 1 h (d), 3 h (e), 6 h (f),12 h (g) and 24 h (h) after c-

irradiation at different doses.

Error bars represent SD

(n = 3). Significance testing

was performed against the

control for each corresponding

time point. *significant

difference compared with the

control, P \ 0.05

Radiat Environ Biophys (2014) 53:283–290 285

123

Kinetics of RL/G of c-H2AX after exposure

to radiation

Changes in the RL/G of c-H2AX in blood from three

healthy volunteers were analysed at 1, 3, 6, 12 and 24 h

after 2 Gy or 10 Gy irradiation. Results are shown in

Fig. 3, which is directly derived from the data shown in

Fig. 1. The RL/G of c-H2AX increased rapidly and

reached 2.78 ± 0.32 (2 Gy) or 5.79 ± 0.84 (10 Gy) at 1 h

after irradiation. This level was maintained up to 3 h after

radiation. The RL/G of c-H2AX then gradually decreased

with time. At 12 h after 2 Gy irradiation, the RL/G of c-

H2AX (1.81 ± 0.14) was higher than that of the non-

irradiated control (P = 0.0133). At 24 h after 2 Gy irra-

diation, the RL/G of c-H2AX (1.40 ± 0.01) was still

higher than that of the non-irradiated control (P = 0.0395).

However, this value (RL/G of c-H2AX at 24 h after 2 Gy

irradiation) was still lower than that of the maximum

background level. At 24 h after 10 Gy irradiation, the RL/

G of c-H2AX (2.67 ± 0.09) was still higher than that of

the non-irradiated control (P = 0.0006).

Inter-experimental and inter-individual variations

in the RL/G of c-H2AX

Three independent experiments were performed using

blood samples obtained at different times from the same

volunteers to study the inter-experimental variations. The

blood samples were analysed at 1 h after exposure to

0–10 Gy of c-rays. Table 1 shows the data from the same

volunteers in three independent experiments. The CVs were

\10 % at 1 h after 0–10 Gy irradiation. The RL/G of c-

H2AX in blood from three healthy volunteers was mea-

sured at 1, 3, 6, 12 and 24 h after 0–10 Gy irradiation to

quantify inter-individual variations. As shown in Table 2,

all CVs were\15 % except for the CVs at 1 h after 6 and

8 Gy irradiation (18.25 and 15.89, respectively).

Dose–effect response, dose–effect curves

and thresholds of detection

Figure 4a shows the relationship between the RL/G of c-

H2AX in blood and radiation doses (0–10 Gy) at 1, 3, 6, 12

and 24 h after irradiation. At each time point, the RL/G of

c-H2AX increased in a dose-dependent manner. At 1 and

3 h after irradiation, the RL/Gs of c-H2AX in blood

exposed to all doses indicated were higher than those of the

non-irradiated control (P \ 0.05). At 6 h after irradiation,

the RL/Gs of c-H2AX in blood exposed to 1, 2, 4, 6, 8 and

10 Gy were higher than those of the non-irradiated control

(P \ 0.05). At 12 h after irradiation, the RL/Gs of c-H2AX

in blood exposed to 2, 4, 6, 8 and 10 Gy were higher than

those of the non-irradiated control (P \ 0.05). At 24 h after

irradiation, the RL/Gs of c-H2AX in blood exposed to 4, 6,

8 and 10 Gy were higher than those of the non-irradiated

control (P \ 0.05). Five dose–effect curves were con-

structed as shown in Fig. 4b, c, d, f. For all curves, y is the

RL/G of c-H2AX and x is the dose.

Fig. 2 Basal level of RL/G of

c-H2AX in blood samples from

62 healthy volunteers (a). Effect

of age (b), gender (c) and

smoking habit (d) on the basal

RL/G level of c-H2AX. Error

bars represent SD

Fig. 3 Time course of radiation-induced RL/G of c-H2AX in blood

samples from three healthy individuals. The RL/G of c-H2AX was

measured at 1, 3, 6, 12 and 24 h after 2 or 10 Gy irradiation. Error

bars represent SD (n = 3). *Significant difference compared with the

control, P \ 0.05. (This figure is directly derived from the data shown

in Fig. 1)

286 Radiat Environ Biophys (2014) 53:283–290

123

The threshold of detection is the dose at which the RL/G

of c-H2AX is significantly different from the background

level. Since the level of RL/G of c-H2AX varies with time

after irradiation, the sensitivity of its detection is dependent

on time. We calculated the threshold of detection according

to the dose–effect curve constructed above. The thresholds

of detection at different time point are 0.19 Gy (1 h),

0.25 Gy (3 h), 0.96 Gy (6 h), 0.99 Gy (12 h) and 1.56 Gy

(24 h), respectively.

Discussion

In this study, we established a new FCM-based method for

measuring the level of c-H2AX in irradiated human lym-

phocytes. This method could be used in initial triage and

dose estimation for large-scale nuclear accidents. c-H2AX

focus formation has been used as a biomarker for DSBs

induced by exposure to genotoxic agents, such as ionisation

(Roch-Lefevre et al. 2010). Scoring of c-H2AX foci in

lymphocytes has also been used to estimate doses after

exposure to radiation in vivo and in vitro (Roch-Lefevre

et al. 2010; Redon et al. 2010). FCM methods for mea-

suring c-H2AX in lymphocytes were previously developed

in several laboratories. However, these methods could not

be used to estimate radiation doses because of high inter-

experimental and inter-individual variations in c-H2AX

between irradiated and non-irradiated lymphocytes. In this

study, we used the RL/G of c-H2AX based on the phe-

nomenon that the level of c-H2AX in granulocytes was not

affected by radiation.

The sensitivities of lymphocytes and granulocytes to

radiation are different. Lymphocytes rapidly decrease at

several hours after radiation, whereas granulocytes start to

decrease at several days after irradiation. At 24 h after

whole-body irradiation, the amount of lymphocytes in

blood of rats is significantly decreased, whereas that of

granulocytes is not significantly affected (Radim et al.

2011). In the present study, the level of c-H2AX in lym-

phocytes increased in a dose-dependent manner at 1, 3, 6,

12 or 24 h after 0–10 Gy c-ray irradiation, whereas that in

granulocytes was not significantly affected. A significant

difference in the induction of c-H2AX was observed

between lymphocytes and granulocytes. Human polymor-

phonuclear leucocytes have been reported to lack DNA-

dependent protein kinase (DNA-PK), which is composed of

Ku protein and the catalytic subunit DNA-PKcs, needed for

the generation of c-H2AX (Ajmani et al. 1995; Annahita

et al. 2004).Such report may explain the significant dif-

ference in the induction of c-H2AX between lymphocytes

and granulocytes observed in the present study. Given that

the level of c-H2AX in granulocytes was not significantly

affected by irradiation, we established a new FCM-based

method to measure the level of c-H2AX in lymphocytes in

blood using the RL/G of c-H2AX instead of fluorescence

intensity used in many reports.

Table 1 RL/G of c-H2AX from three volunteers at 1 h after exposure to 0–10 Gy irradiation

Donor Experiment Dose(Gy)

0 0.5 1 2 4 6 8 10

1 Time1 1.21 1.67 1.95 2.69 3.88 4.57 5.53 6.28

Time2 1.25 1.71 2.18 3.12 3.95 5.04 5.56 6.47

Time3 1.24 1.69 2.26 3.13 4.16 5.15 5.91 6.07

Mean 1.23 1.69 2.13 2.98 3.99 4.92 5.67 6.27

SD 0.02 0.02 0.16 0.25 0.15 0.31 0.21 0.20

CV (%) 1.87 1.25 7.65 8.42 3.65 6.27 3.71 3.23

2 Time1 1.18 1.50 1.96 2.45 3.14 3.34 4.14 4.85

Time2 1.21 1.53 1.91 2.44 3.04 3.66 4.23 4.81

Time3 1.15 1.58 1.86 2.35 3.11 3.63 3.95 4.80

Mean 1.18 1.53 1.91 2.41 3.09 3.54 4.10 4.82

SD 0.03 0.04 0.05 0.05 0.05 0.18 0.14 0.02

CV (%) 2.42 2.44 2.63 2.10 1.73 5.02 3.46 0.48

3 Time1 1.16 1.65 2.07 3.09 4.03 4.76 5.53 6.22

Time2 1.19 1.60 2.20 3.01 4.07 4.84 5.64 6.57

Time3 1.15 1.64 2.17 2.83 3.87 4.81 5.51 6.12

Mean 1.17 1.63 2.15 2.98 3.99 4.80 5.56 6.30

SD 0.02 0.03 0.06 0.13 0.10 0.04 0.07 0.24

CV (%) 1.87 1.74 3.02 4.47 2.61 0.86 1.21 3.78

Radiat Environ Biophys (2014) 53:283–290 287

123

In several laboratories, high variations in the level of c-

H2AX were observed in irradiated lymphocytes between

individuals or experiments, which limited the use of c-

H2AX as a biological dosimeter. Assessment of the inter-

experimental and inter-individual variations in the RL/G of

c-H2AX in irradiated or non-irradiated blood samples is

important for biodosimetry. After measuring the basal

levels of RL/G of c-H2AX in 62 healthy control volun-

teers, we observed that the mean RL/G of c-H2AX ± SD

was 1.21 ± 0.12, with a range of 1.01–1.54, and the CV

was 9.94 %. The basal level of the RL/G of c-H2AX was

not affected by age, gender and smoking habit. Low inter-

experimental and inter-individual variations in the RL/G of

c-H2AX of non-irradiated or irradiated blood were

observed in our study (Fig. 1; Tables 1, 2).

With regard to the changes in the RL/G of c-H2AX over

time, the RL/G of c-H2AX increased rapidly up to 1 h after

2 or 10 Gy irradiation. A maximum response was observed

at 3 h after irradiation. At 12 h after 2 Gy irradiation, the

RL/G of c-H2AX was still higher than that of the non-

irradiated control. At 24 h after 10 Gy irradiation, the RL/

G of c-H2AX was also higher than that of the non-irradi-

ated control. Similar results were reported in other studies,

with the maximum response achieved at 1–2 h after

exposure to radiation and the basal level achieved at 24 h

after exposure (Hamasaki et al. 2007; Andrievski and

Wilkins 2009). The most critical issue for the use of the

RL/G of c-H2AX as biological dosimeters is the c-H2AX

signal loss. Inhibition of c-H2AX signal loss is possible for

up to 24 h by incubating whole blood on ice (Roch-Lefevre

Table 2 RL/G of c-H2AX from three volunteers at 1, 3, 6, 12 and 24 h after exposure to 0–10 Gy

Time (h) Donors Dose(Gy)

0 0.5 1 2 4 6 8 10

1 1 1.23 1.67 1.95 2.69 3.88 4.57 5.53 6.28

2 1.18 1.50 1.96 2.45 3.14 3.34 4.14 4.85

3 1.16 1.65 2.07 3.09 4.03 4.76 5.53 6.22

Mean 1.19 1.61 1.99 2.74 3.68 4.22 5.07 5.78

SD 0.04 0.09 0.07 0.33 0.47 0.77 0.81 0.81

CV (%) 3.06 5.59 3.44 11.88 12.87 18.25 15.89 14.04

3 1 1.16 1.89 2.53 3.99 5.17 5.97 6.72 6.30

2 1.26 1.85 2.58 3.32 5.21 5.42 5.41 6.34

3 1.33 1.82 2.46 3.02 4.36 4.91 5.41 6.21

Mean 1.25 1.85 2.52 3.44 4.91 5.43 5.85 6.28

SD 0.08 0.04 0.06 0.50 0.48 0.53 0.76 0.06

CV (%) 6.67 2.07 2.36 14.44 9.74 9.74 12.98 1.03

6 1 1.51 1.64 1.87 2.17 2.63 2.65 3.28 3.64

2 1.56 1.54 1.76 2.08 2.53 2.69 3.41 3.32

3 1.40 1.51 1.72 1.87 2.38 2.82 3.52 2.95

Mean 1.49 1.56 1.79 2.04 2.51 2.72 3.40 3.30

SD 0.08 0.07 0.08 0.15 0.12 0.09 0.12 0.35

CV (%) 5.44 4.38 4.45 7.49 4.94 3.31 3.62 10.48

12 1 1.36 1.33 1.68 1.95 2.32 2.62 3.06 3.14

2 1.45 1.43 1.52 1.80 2.20 2.68 3.10 2.87

3 1.30 1.26 1.45 1.67 1.88 2.35 2.48 2.69

Mean 1.37 1.34 1.55 1.81 2.13 2.55 2.88 2.90

SD 0.07 0.09 0.12 0.14 0.23 0.18 0.35 0.23

CV (%) 5.42 6.69 7.68 7.73 10.67 6.95 12.00 7.83

24 1 1.17 1.19 1.27 1.41 1.60 1.89 1.80 2.21

2 1.28 1.23 1.11 1.41 1.66 1.91 1.93 2.38

3 1.22 1.20 1.28 1.39 1.49 1.89 2.01 2.21

Mean 1.23 1.21 1.22 1.40 1.58 1.89 1.91 2.27

SD 0.06 0.02 0.10 0.01 0.09 0.01 0.11 0.09

CV (%) 4.69 1.73 7.92 0.84 5.54 0.69 5.74 4.15

288 Radiat Environ Biophys (2014) 53:283–290

123

et al. 2010). In the present study, the signals of the RL/G of

c-H2AX in samples labelled with antibody were stable up

to 90 h(data not shown). Dose–effect curves for the RL/G

of c-H2AX were constructed using the data obtained at 1,

3, 6, 12 and 24 h after irradiation. At these time points, the

RL/G of c-H2AX showed a linear dose–effect relationship

in the dose range of 0–10 Gy. This result is consistent with

those in previous studies (Hamasaki et al. 2007; Andrievski

and Wilkins 2009).

In case of a large-scale radiation accident, rapid identi-

fication of radiation-exposed individuals who need emer-

gency medical treatment is critical. The dicentric assay is

the gold standard method for biological dosimeters in recent

cases of overexposure to ionising radiation. However, the

dicentric assay is not suitable for population triage during

the first few hours because a 48-h culture period is needed to

obtain metaphases before chromosome scoring. Lympho-

cyte depletion rate is a simple, fast and high-throughput

assay, but it cannot be observed within 12 h after irradiation

(Flood et al. 2011). In the present study, we developed a

method that requires only 2–3 h to perform (from blood

sampling to dose estimation). Measurement of the RL/G of

c-H2AX can be used as a potential, rapid method for pop-

ulation triage and dose estimation. Rapid decline in the c-

H2AX signal may limit the application of the method to the

first 12 h (or 24 h at[4 Gy), and knowledge about the time

of exposure is required.

In summary, we present a potential biodosimetry

method based on the RL/G of c-H2AX in blood. The linear

dose–effect relationship, high throughput and low inter-

experimental and inter-individual variations of this method

are advantageous for the use of the RL/G of c-H2AX as a

biological dosimeter. Therefore, the developed method

may be used as a potential dosimeter for population triage

and dose estimation during large-scale radiation emergen-

cies if blood samples can be collected within 24 h. Addi-

tional studies, such as in vivo experiments, are needed to

validate this method further.

Fig. 4 Dose–effect response of

the RL/G of c-H2AX in blood at

different times after 0–10 Gy

radiation (a). Dose–effect

calibration curves for different

time intervals (b, c, d, e and f).Error bar represent SD (n = 3)

Radiat Environ Biophys (2014) 53:283–290 289

123

Conflict of interest The authors declare that they have no conflict

of interest.

Ethical standard The written informed consent from volunteers

and ethical approval from the Subcommittee on human investigation

of the Beijing Institute of Radiation Medicine (2012-0128) were

obtained before experiment.

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