Accepted Manuscript

Flow Cytometric Detection of Gamma-H2AX To Evaluate DNA Damage by

Low Dose Diagnostic Irradiation

Kainat Khan, Shikha Tewari, Namrata Punit Awasthi, Surendra Prasad Mishra,
Gaurav Raj Agarwal, Madhup Rastogi, Nuzhat Husain

PII: S0306-9877(18)30220-2
DOI: https://doi.org/10.1016/j.mehy.2018.03.016
Reference: YMEHY 8834

To appear in: Medical Hypotheses

Received Date: 22 February 2018

Accepted Date: 25 March 2018

Please cite this article as: K. Khan, S. Tewari, N.P. Awasthi, S.P. Mishra, G.R. Agarwal, M. Rastogi, N. Husain,
Flow Cytometric Detection of Gamma-H2AX To Evaluate DNA Damage by Low Dose Diagnostic Irradiation,
Medical Hypotheses (2018), doi: https://doi.org/10.1016/j.mehy.2018.03.016

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Title: Flow Cytometric Detection of Gamma-H2AX To Evaluate DNA Damage by Low Dose Diagnostic

Irradiation

Authors: Kainat Khan*β (M.Sc.), Shikha Tewari*β (PhD), Namrata Punit Awasthi* (MD), Surendra Prasad

Mishra† (PhD), Gaurav Raj Agarwalα (MD) , Madhup Rastogi†(MD) , Nuzhat Husain* # (MD)

Affiliation:
*
Department of Pathology, Dr Ram Manohar Lohia Institute of Medical sciences, Lucknow, INDIA-226010.
†
Department of Radiation Oncology, Dr Ram Manohar Lohia Institute of Medical sciences, Lucknow, INDIA-

226010.
α
Department of Radio diagnosis, Dr Ram Manohar Lohia Institute of Medical sciences, Lucknow,
β
The first and the second author contributed equally in the work

Corresponding Author:

Prof. Nuzhat Husain,

Head of the Department,

Department of Pathology,

Dr. Ram Manohar Lohia Institute of Medical Sciences,

Lucknow, U.P. India-226010

Phone :9415333729

Fax: 0522-6692055

Email: [email protected]

Declaration of Interest: None

Acknowledgement and Funding Statement:

The work was supported by the Science and Engineering Research Board (SERB), Department of Science and

Technology, New Delhi India Young scientist research grant provided to Dr Shikha Tewari (Grant No.

SB/YS/LS-208/2013,). The senior research fellowship of Mrs. Kainat Khan was supported by the Maulana Azad

National Fellowship, University Grant Commission, India. The authors would also like to thank Abdul Kalam

Technical University for providing Ph.D. registration to Mrs. Kainat Khan.

1
 HYPOTHESIS

In the present study we hypothesize that while undergoing diagnostic radiations multiple

times, double strand DNA breaks are induced which can be easily observed in peripheral

blood of exposed patients. Here we utilize the gamma h2ax assay to study the phenomenon.

The study also compares the cumulative damage which is hypothesized to occur and

accumulate depending on the radiation dose and exposure status.

This study discusses that blood can be utilized as surrogate marker to study DNA strand

break and repair phenomenon especially in field of radiation dosimetry and patient health

assessment.

2
 ABSTRACT

Introduction: γH2AX Assay has been used for DNA damage assessment at higher doses of

radiation exposure. Its expression has not been studied in cases with diagnostic low dose

radiation exposure. Concerns have been raised about the after-effects of radiation in

diagnostic procedures like Computed Tomography (CT) scan, Angiography etc especially

when such scans are repeated within short span of time. The purpose of the present study was

to assess immediate DNA damage after exposure to low level of ionizing radiation by the

flow cytometric method of Gamma-H2AX.

Material and Methods: Study sample includes total 60, cases and controls with two groups

Group I-Normal controls (n=15); Group II- Low dose, further divided in three groups : Group

IIA- single CT scan (n=15); Group IIB- Multiple CT scans (n=15); and Group IIC-

angiography single exposure(n=15). For Low dose group blood was collected within 1 hour

after exposure in EDTA vaccutainers and immediately kept on ice. Lymphocytes were

isolated and were fixed in 80% chilled ethanol and stored at -20°C till further analysis. The

H2AX assay was done and 10,000 cells analyzed for gamma H2AX positivity in

flowcytometer.

Results: Significant gamma-H2AX positivity was found in cases versus control, the most

significant DNA damage amongst cases was observed in cases with multiple CT scans.

Conclusion: The exposure to multiple CT scans causes more double strand breaks as

compared to single scan.DNA damage can be studied by flow cytometric analysis of gamma-

H2AX in human peripheral lymphocytes.

3
 INTRODUCTION

Genomic DNA is well protected and packaged with histone octamer in the nucleus of the

cell. The histones H2A, H2B, H3 and H4 form the core of nucleosome and H1 is linker

protein (1). This nuclear DNA is subjected to various types of damage associated with

metabolism and cellular functions. Whenever any damage to DNA occurs the repair

machinery is activated to counteract and repair the DNA. The most severe type of breaks

produced are caused by ionizing radiation and DNA topoisomerase inhibitors as they produce

double strand break (DSB) in the DNA backbone, cutting through the phosphodiester bond

(2). Once the double strand break is produced a series of events are initiated followed by

phosphorylation of the second histone (H2A) protein at serine 139 residue converting it to

γH2AX (gamma-H2AX) , in nucleosome. With its phosphorylation DNA repair enzymes are

recruited at the site of the DSBs and repair machinery is activated. Induction of DSBs can

occur due to exposure to ionizing radiation or cytotoxic agents resulting in the formation of

gamma-H2AX foci, which can be used as a biomarker for DNA damage. It is important and

challenging to measure DNA damage as double strand breaks (DSBs) can be lethal at times

and the repair machinery might fail to repair the damage and then the cells may not undergo

the cell death pathway leading to replication of damaged DNA.

Human exposure to radiation by medical procedures has become frequent as numbers of

diagnostic scans have increased since last ten years (3). There are reports which highlight the

concern of using repeated diagnostic scans on patients at different age groups and relating it

to the probable outcome in future (4). Most diagnostic equipments use either X rays or

gamma rays for diagnostic and therapeutic purposes. The DNA disrupting nature of these

radiations has led to many studies for safety evaluation purpose. Several methods have been

4
reported for DNA damage detection in cells namely estimation of gamma-H2AX, comet

assay, pulse gel electrophoresis (5, 6, 7,8). Phosphorylation of histone protein at DNA DSBs

is a well established phenomenon (9). Formation of gamma-H2AX at ser 139 is an abundant

and fast event that correlates well with each DSB, hence quantifying gamma-H2AX remains

the most exact method as it evaluates formation of double strand breaks in 1:1 ratio (10).

When these radiation induce DNA damage, H2AX is phosphorylated and gamma-H2AX foci

equal to number of DSB are recruited (11). As per a study published in 1998, for an X ray

dosage of 1Gy, there is always around 1-2% of H2AX that becomes gamma-H2AX (10).

Gamma-H2AX assay has proved useful for detecting DNA damage at doses of radiation

down to 1 mGy and it is said to be 100 times more sensitive than other methods (12, 13).

Reportedly the gamma-H2AX can be quantified either by immunoflourescence or flow

cytometry (14). Studies have suggested gamma H2AX quantification by immunoflourescence

as a useful biomarker of human low level radiation exposure( 15). In immunofluorescence

method numbers of foci formed are individually counted by microscopic evaluation.

However, flow cytometric method can quickly quantify the gamma H2AX formation by

capturing fluorescence and giving the count of gamma-H2AX positive cells.

The flow cytometric method is fast and reliable, data is automatically calculated by the

instrument and the count number obtained in the assay is reasonably accurate because of the

automation. Antibody specific for gamma-H2AX is added and the positive cells are

quantified for damage analysis, through flow cytometry. Although flow cytometry has been

used to quantify DNA damage, it is still unexplored when it comes to low dose radiation and

its effects (16, 17). In the current study we have explored flow cytometry based gamma-

H2AX assay to detect immediate DNA damage in patients with partial body exposure to low

doses of radiation.

5
 MATERIAL AND METHODS

Study Sample:

The study included 60 samples including cases(n=45) and controls (n=15), which were

further divided as follows:

Group I-No exposure controls(NEC) : Patients who did not have a known previous history of

exposure to radiation since past 3 years and are also unexposed to other carcinogens, tobacco

and alcohol(n=15).

Group II (A, B, C): Low dose exposed population. Patients undergoing one or multiple partial

body radiation/scanning procedures, within a week (n=45) , which included cases undergoing

A: CT Scan-Low Dose Single exposure (LDS) n=15.

B: CT Scan-Low Dose Multiple exposure (LDM) n=15.

C: Angiography- Low Dose Angiograpgy (LDA) n=15.

The study was approved by the institution ethical committee (IEC No.7/14). Informed patient

consent was taken from all the recruited participants and patient history was recorded, any

past radiation exposure was also noted.

Exclusion criteria: Cases with history of taking alcohol, tobacco and other narcotics, cases

with family history of chromosomal genomic abnormalities, cases with previous radiological

diagnosis and treatment for malignancy, cases not giving consent to enter the study were

excluded from the study group.

Blood collection:. For patients undergoing diagnostic scans blood sample was collected

within 1 hour and was immediately kept on ice thus slowing down all enzymatic activity, as

37°C is the optimum temperature for all enzymatic reactions. For multiple exposures group,

6
cases undergoing two or more head scans within a week were recruited and radiation dose

was recorded for dose assessment, wherever available, previous CT scan dose was also noted

from the machine for total dose calculation. The normal blood samples were collected from

people who volunteered to be included in the study, met the inclusion criteria and had no

known radiation exposure.

Radiation Quantification of Exposure:

Group I: The patients with no previous radiation exposure history were recruited in this

group.

Group II: For Group II (A), the patients undergoing Multi Slice Computed Tomography

(MSCT) scan (64 Slice Philips make , Netherland) in the department of radiodiagnosis of the

Institute were recruited and the Dose Length Product (DLP) CT radiation dose in mGy*cm

received by the patient was recorded from the automated system generated dose projections

and blood sample was collected within 1 hour of MSCT scan. The effective radiation dose

was then calculated using the DLP obtained. For partial-body irradiation, effective dose is the

weighted summation of the absorbed dose, which was recorded as DLP to each specified

organ and multiplied by the ICRP-defined tissue-weighting factor(TWF) for that same organ

or tissue (ICRP-103). The Tissue Weighing Factor values used for calculation of effective

dose was as per the guidelines of the International Commission of Radiological Protection

(ICRP) publication 103(18).

The formula used for the calculation of the effective dose is:

E = ∑ { ∑ WT X HT }
Z T

7
where T is all ICRP-specified tissues and organs, WT is the ICRP-specified tissue-weighting

factor, HT is the dose to a particular organ or tissue, the inside summation T is over all

tissues, and the outside summation Z is over all irradiated slabs(19).

The effective dose may vary substantially depending on patient size, body part scanned,

number of sections taken and so forth and hence readings for every individual patient were

recorded. For Group II(B) the radiation dose of the previous scans was also recorded and

added to the radiation dose, 14 cases in this group underwent multiple head scans (twice or

thrice)and 1 underwent abdomen scan twice. Radiation dose for each scan was converted in

effective radiation dose and then added as total dose received within a week. For Group II(C)

individual patient dose was recorded in DLP and converted in effective radiation dose as

mentioned above. Out of the 15 cases in this group, 9 were cardiac angio, 4 were brain angio,

1 neck angio and 1 limb angio.

Gamma-H2AX assay by Flow cytometry: Lymphocyte isolation was done by density

centrifugation method using 4 ml of peripheral blood (EDTA) and histopaque (Sigma,

10771). The lymphocytes were washed twice with sheath solution and then fixed in fixative

solution (BD Biosciences, 554655) for half an hour at 4°C. Then they were centrifuged and

resuspended in 80% chilled ethanol, the cells were then stored at -20°C till further analysis

(20). Cells were washed twice with stain buffer (BD Biosciences, 554656) and incubated in

CD45FITC (BD Biosciences, 347463) for 10 mins at 4°C, washed again in stain buffer and

permeabilized in perm buffer III (BD Biosciences, 558050) for 10mins. The cells were then

washed twice in stain buffer, and incubated in gamma-H2AX PE (BD Biosciences, 552377)

for 30 mins in dark. Finally cells were washed and read in stain buffer in flow cytometer

(Becton Dickinson). Isotype control for gamma-H2AX PE was also run to validate the assay.

Flow Cytometry Analysis: Cells stained with CD45 FITC and H2AX PE were analysed in a

2 laser 4 colour flow cytometer (Becton Dickinson, San Jose, CA) using 488nm excitation

8
(530/30 for FITC and 575/26 BP filter for PE). Bright CD45 expressing lymphocytes were

gated in R1 and analysed for gamma-H2AX expression (Figure 1). Integrated fluorescence

measurements for FITC and PE were recorded for 10,000 single non debris gated events.

FITC fluorescence was plotted against PE fluorescence using CELL QUEST PRO

ANALYSIS SOFTWARE. Gamma-H2AX positive cells were taken for individual samples

for result analysis. In Figure 2, representative histogram of the Gamma-H2AX expression

across all groups is shown demonstrating the shift in the Gamma-H2AX expression.

Statistical Analysis: The results are presented in frequencies, percentages and mean±SD. The

mean of positive gamma-H2AX cells was calculated and the findings were based on these

calculated values across all groups. Statistical difference (p< 0.05) between no exposure

controls and radiation exposed samples were determined by Bonferroni Post hoc test. Mann

Whitney U test of significance was done for age based analysis.

RESULTS

The mean age and the male to female ratio of the test and control group are depicted in Table

1. The age difference as well as the male to female ratio did not vary significantly across the

groups. The mean DLP radiation of test groups was calculated: Group II(A) received

1190±399 milliGray (mGy) of radiation, Group II(B) received 2658±1557, Group II(C)

received the highest among low dose, 3578±2129mGy. The mean effective radiation dose

was found to be 23.58 mSv in Group II-A, 55.36 mSv in Group II-B and 272.71 mSv in

Group II-C.

The mean percentage of gamma-H2AX positive cells was compared for all groups. Group I

had a mean value of 0.22% positive gamma-H2AX cell, which is the minimum observed

9
across all groups. The low dose or the diagnostic radiation group which was divided into

three sub groups showed different values: group II(A) had 2.25% gamma-H2AX positive

cells, group II(B) where the radiation dose was almost double from that of group II (A),

showed a five times increase in the gamma-H2AX positive cells where the mean was found

to be 10.13%(Table 3). However in group II(C) where the radiation was the maximum in low

dose group, the value observed was 6.69.% .The significance of difference between

subgroups of cases and the control by bonferroni post hoc analysis showed that no exposure

controls (group I) were significantly lower than the group involving multiple CT Scan

patients (LDM). Comparison of group I (NEC) with group II(C) (LDA) was not found to be

significant. Group II(A) (LDS) when compared with group II(B) (LDM) was lower but no

statistically significant difference was observed. .

When the patients were sub categorized according to age groups of <50 and >=50, it was

observed that the percentage of gamma-H2AX positive cells was higher in the older group in

multiple exposure group(Table 4). In group II(A) (LDS) the values were 2.31 for <50 and

2.04 for >=50, the difference was not much in this group as the radiation received was

minimal. In group II (B) (LDM) the >=50 subgroup had 12.27% positivity for gamma-H2AX

whereas the younger group (<50) had a value of 8.24%. In group II(C) (LDA) where

radiation dose was single but high the older group (>=50) had 4.9% of gamma-H2AX

positivity whereas the younger group had a positivity of 9.3% (Figure 4).

DISCUSSION

In the present study we have attempted to quantify immediate DNA damage occurring in

individuals who have undergone diagnostic CT scan, single or multiple times and other

radiological investigations using gamma-H2AX as a marker for double strand breaks. The

DNA damage quantified was further analyzed with reference to quantity of radiation given

10
and repeated exposure within one week of the first diagnostic procedure. Clinical use of

radiation for therapeutic and diagnostic purposes has become an increasingly used modality

in the past 50 years. In the present study gamma-H2AX has been quantified using flow

cytometry to get an objective quantitative assessment. It was interesting to note that single

exposure to medical imaging did not cause as much change in gamma H2AX assay as

compared to cases with multiple diagnostic exposures to low dose irradiation within a short

period of time.

The gamma-H2AX flow cytometric assay has been reported to effectively detect DNA

damage caused by exposure to ionizing radiation. Several studies have used this method to

quantify damage due to large dose radiation exposure (21, 22, 23). This method is established

as a sensitive DNA damage biomarker (24) and a biomarker for DNA damage by ionizing

radiation (25). Estimation of gamma-H2AX can be done either by immunofluorescence or

by flow cytometry, the immunofluorescence technique has been used extensively and

effectively to study the DNA damage caused by ionizing radiation wherein gamma-H2AX

has emerged as a sensitive biomarker for DNA damage detection (26, 27, 28). The

immunoflorescence technique has been used to detect DSBs induced by low doses of

diagnostic radiation (15, 29). Flow cytometric evaluation of gamma-H2AX however has not

been explored in cases with low dose diagnostic radiation exposure in previous studies

although attempts have been made to quantify damage in patients undergoing radiotherapy

(30).

In the present study environmental exposure was taken to be same for all groups since the

study population was urban and from the same region. The natural background radiation of

2.4mSv was also common in all groups including cases and no exposure control. DNA

damage quantified in study subjects was therefore proportional to the amount of radiation

given and number of times the diagnostic procedure was repeated. The gamma-H2AX values

11
were correlated with the age and sex of the individual to have better understanding of damage

in relation to the age of the patient, in view of the fact that the repair mechanism becomes

weaker with ageing. A general trend of increased H2AX percentage value was observed in

cases more than 50 years of age in multiple exposure group. Our results show higher

susceptibility to DNA damage in individuals >50 years of age. This age related pattern of

DNA damage as assessed by H2AX was present, especially in study group with multiple

exposure (Figure 4). Study subjects in the higher age group are metabolically active, have

higher stress and compromised antioxidant status which may be a reason for higher number

of strand breaks occurring post exposure at same levels as compared to younger individuals

(31). There are studies suggesting that the cumulative effective radiation dose increases with

advancing age and also that the effects are higher in female patients than in male patients

(32). We have not observed any difference in gamma-H2AX values in males versus females.

Epidemiological evidence for age dependent radio sensitivity variations have been reported in

various studies (33, 34). A cohort study of Japanese atomic bomb survivors examined excess

relative risk (ERR) for radiation induced cancers as a function of age at exposure. The ERR

for cancer induction was higher during childhood and decreased at exposure age of 30 to 40.

The ERR rose up again at age higher than 40 years (35). Our study included individuals in

the age range of 17-80; the pediatric population has not been included.

We found elevated gamma-H2AX values in all radiation exposed groups even in the group

where patients with a single CT scan were recruited as compared to the control. The mean

gamma-H2AX positive cells were 0.22 in controls, the positive cells increased to a mean

value of 2.25 in single exposure. This is a ten times increase suggesting that the gamma

H2AX is expressed at a higher level with recruitment of repair enzymes, and their active

participation in the DNA damage repair pathway. Within the low dose exposure group viz

group II, the radiation dose was highest in the group II-C (LDA) however the highest values

12
of gamma-H2AX positive cells were obtained in group II-B (LDM). This suggests that

cumulative damage caused by multiple exposures within a short span is more damaging than

a single exposure of higher dose. The toxic lymphocytes persist and the repeated exposure

enhances damage. Albert 2002 suggested that some lymphocytes can stay in the body for

years so any alteration in DNA can be toxic if replicated again and again(1). The radiation

risk from angiography have been estimated by some researchers who concluded that patients

exposed to radiation from cardiac CT angiography had evidence of DNA damage, which was

associated with programmed cell death and activation of genes involved in apoptosis and

DNA repair (36). In our study the radiation dose was the highest in the angiography group

amongst the low dose group and elevated levels of gamma-H2AX positive cells were found.

Several studies have suggested that there is an increased cancer risk by exposure to

diagnostic radiation (37, 38, 39). Huda et al estimated 1 in 650 is at a lifetime risk of cancer.

They came up with the findings that the average cancer induction risk in sensitive organs

from cardiac CT angiography for their patient cohort was 0.13%, with a female to male

cancer induction risk ratio of 2.6 (40).

Our research highlights the need to monitor and control multiple CT scans repeated over a

short span of time as it produces more double strand breaks which can lead to significantly

higher amount of DNA damage. In case of children higher precaution needs to be exercised.

Retrospective analyses have been done questioning pediatric CT as a matter of serious

concern and whether the benefit outweighs the risk. Brenner published a study in 2007

reporting that 62 million scans were done in United States and out of these 4 million were on

children (41). The scans being done on children who have mitotic cells and a larger span of

life to develop mutations and hence chances of developing malignancy, raise a serious matter

of concern (42, 43). Research focusing radiation effects support an increased cancer risk due

to diagnostic irradiation (44). However postulates on cancer and low dose radiation exposure

13
due to diagnostic procedures are controversial and another study from France has reported

that no significant increase was found in cancer incidence (45). Research on low dose of

radiation is being carried out extensively all over the world and India being a developing

country needs to be vigilant about the effects and after effects of scans involving radiation.

The present study is one such initiative to aware the clinicians, radiologists and the common

man, that unnecessary scans should not be repeated where any other test like ultrasound can

suffice.

In conclusion our study demonstrates that the multiple exposures to diagnostic radiation

within a span of few days’ causes more double strand DNA breaks as compared to unexposed

and single procedure exposures in peripheral blood lymphocytes of patients. The exposure at

higher age causes more damage as compared to younger individuals. The DNA damage and

repair phenomenon can be very well studied by flow cytometry using gamma-H2AX as a

marker in peripheral blood.

CONFLICT OF INTEREST

The authors declare that there is no conflict of interest.

ACKNOWLEDGEMENT AND FUNDING STATEMENT:

The work was supported by the Science and Engineering Research Board (SERB),

Department of Science and Technology, New Delhi India Young scientist research grant

provided to Dr Shikha Tewari (Grant No. SB/YS/LS-208/2013,). The senior research

fellowship of Mrs. Kainat Khan was supported by the Maulana Azad National Fellowship,

University Grant Commission, India. The authors would also like to thank Abdul Kalam

Technical University for providing Ph.D. registration to Mrs. Kainat Khan.

14
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TABLES

Table 1 : Age and Sex distribution of Cases and Controls

Group I
Characteristic (NEC) Group II (LD)
Group II (A) Group II (B) Group II (C)

Age (yrs)
Mean(±SD) 32.6(±4.64) 41.93(±3.21) 47.8(±13.96) 46.54(±16.02)
Range 26-48 17-75 24-70 17-70
Sex
Male/Female 08:07 11:04 10:05 11:04
*NEC-No exposure Control; LD-Low Dose

Table 2: Distribution of Radiation Dose and Gamma-H2AX Positive cells in

Cases and Control
H2AX Assay(Number of Positive Cells)

Radiation Dose Calculated Minimum Maximum Mean(±SD) Median

DLP (mGy) Effective dose mSv

(ICRP 103)

Group I (NEC) 0 0 0.08 0.52 0.22(±0.14) 0.15

Group II-A (LDS) 1190 23.58 0.12 10.29 2.25(±3.21) 0.62

Group II-B (LDM) 2658 55.36 0.45 45.21 10.12(±13.20) 4.61

Group II-C (LDA) 3578 272.71 0.18 50.63 6.69(±13.46) 0.80

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Table 3 : Comparison of Gamma-H2AX values in Cases and Controls

95% Confidence Interval

Mean Difference
(I) Groups (J) Groups (I-J) Std. Error Sig. Lower Bound Upper Bound

NEC IIA (Single) -2.03400 3.08419 1.000 -10.9743 6.9063

*
IIB (Multiple) -9.90067 3.08419 0.020* -18.8409 -0.9604

IIC(Angiography) -6.47133 3.08419 0.395 -15.4116 2.4689

IIA (Single) NEC 2.03400 3.08419 1.000 -6.9063 10.9743

IIB (Multiple) -7.86667 3.08419 0.129 -16.8069 1.0736

IIC(Angiography) -4.43733 3.08419 1.000 -13.3776 4.5029

IIB (Multiple) NEC 9.90067* 3.08419 0.020 0.9604 18.8409

IIA (Single) 7.86667 3.08419 0.129 -1.0736 16.8069

IIC(Angiography) 3.42933 3.08419 1.000 -5.5109 12.3696

IIC(Angiography) NEC 6.47133 3.08419 0.395 -2.4689 15.4116

IIA (Single) 4.43733 3.08419 1.000 -4.5029 13.3776

IIB (Multiple) -3.42933 3.08419 1.000 -12.3696 5.5109

Bonferroni Post hoc test : *(p-value <0.05 is significant). NEC-No exposure Control; LDS- Low Dose Single exposure; LDM- Low
Dose Multiple exposure; LDA- Low Dose Angiography.

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Table 4: Age based distribution and mean gamma H2AX of cases and controls

Groups Age groups Mean Std. Deviation N P value

Group I (NEC) < 50 years 0.2247 0.14745 15 NA

Total 0.2247 0.14745 15

Group IIA (LDS) < 50 years 2.3133 3.40860 12 0.901

>=50 years 2.0400 2.90331 3

Total 2.2587 3.21650 15

Group IIB (LDM) < 50 years 8.2438 10.87667 8 0.574

>=50 years 12.2757 16.07812 7

Total 10.1253 13.20130 15

Group IIC (LDA) < 50 years 9.3883 20.24632 6 0.535

>=50 years 4.9011 5.88153 9

Total 6.6960 13.08978 15

Mann Whitney U test for significance*(p-value <0.05 is significant)

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 FIGURE LEGENDS

Figure 1:A: Side scatter versus CD45 FITC intensity plot showing gating of

lymphocytes. The gated (R1) lymphocytes were used for Gamma H2AX analysis.

B-E: Intensity scatter plot of gated lymphocytes showing gamma H2AX positive

cells versus CD45 FITC cells.

Figure 2: Histogram showing counts of gated Gamma H2AX PE positive cells in

isotope control and across different experimental groups. Group I (NEC- No

Exposure Controls), Group IIA ( LDS- Low Dose Single Exposure) , Group IB

(LDM- Low Dose Multiple Exposure), Group IIC (LDA- Low Dose

Angiography).

Figure 3: A Bar chart representing mean gamma-H2AX positive cells at

different doses.

Figure 4: Age related values of mean gamma-H2AX positive cells in low dose

exposure.

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