

ORIGINAL ARTICLE 

Year : 2016  Volume
: 7
 Issue : 1  Page : 4 

Enhancing cytogenetic biological dosimetry capabilities of the philippines for nuclear incident preparedness
Celia O Asaad, Gloriamaris L Caraos, Gerardo Jose M Robles, Anie Day D. C. Asa, Maria Lucia C Cobar, AlAhmadgaid Asaad
Department of Science and Technology, Biomedical Research Section, Atomic Research Division, Philippine Nuclear Research Institute, Commonwealth Avenue, Diliman, Quezon City, Philippines
Date of Web Publication  30Dec2016 
Correspondence Address: Celia O Asaad Biomedical Research Section, Atomic Research Division, Philippine Nuclear Research Institute, Commonwealth Avenue, Diliman, Quezon City 1101 Philippines
Source of Support: None, Conflict of Interest: None  Check 
DOI: 10.4103/20419414.197163
The utility of a biological dosimeter based on the analysis of dicentrics is invaluable in the event of a radiological emergency wherein the estimated absorbed dose of an exposed individual is crucial in the proper medical management of patients. The technique is also used for routine monitoring of occupationally exposed workers to determine radiation exposure. An in vitro irradiation study of human peripheral blood lymphocytes was conducted to establish a doseresponse curve for radiationinduced dicentric aberrations. Blood samples were collected from volunteer donors and together with optically stimulated luminescence (OSL) dosimeters and were irradiated at 0, 0.1, 0.25, 0.5, 0.75, 1, 2, 4, and 6 Gy using a cobalt60 radiotherapy unit. Blood samples were cultured for 48 h, and the metaphase chromosomes were prepared following the procedure of the International Atomic Energy Agency's Emergency Preparedness and Response – Biodosimetry 2011 manual. At least 100 metaphases were scored for dicentric aberrations at each dose point. The data were analyzed using R language program. The results indicated that the distribution of dicentric cells followed a Poisson distribution and the doseresponse curve was established using the estimated model, Y_{dic} = 0.0003 (±0.0003) +0.0336 (±0.0115) × D + 0.0236 (±0.0054) × D^{2}. In this study, the reliability of the doseresponse curve in estimating the absorbed dose was also validated for 2 and 4 Gy using OSL dosimeters. The data were fitted into the constructed curve. The result of the validation study showed that the obtained estimate for the absorbed exposure doses was close to the true exposure doses. Keywords: Absorbed dose, biodosimetry, dicentric chromosomes, dose response, linear quadratic
How to cite this article: Asaad CO, Caraos GL, Robles GM, Asa AD, Cobar MC, Asaad AA. Enhancing cytogenetic biological dosimetry capabilities of the philippines for nuclear incident preparedness. Genome Integr 2016;7:4 
How to cite this URL: Asaad CO, Caraos GL, Robles GM, Asa AD, Cobar MC, Asaad AA. Enhancing cytogenetic biological dosimetry capabilities of the philippines for nuclear incident preparedness. Genome Integr [serial online] 2016 [cited 2019 Dec 9];7:4. Available from: http://www.genomeintegrity.org/text.asp?2016/7/1/4/197163 
Introduction   
The Philippine government has a national preparedness plan whose objective is to establish an organized response capability for timely and coordinated action in the event of an emergency. Nuclear incident is one of the four major categories of emergencies included in the plan. Realizing the critical role that the cytogenetics group will play during radiation emergency, the Philippine Nuclear Research Institute conducted a study to establish our own doseresponse calibration curve for dicentrics assay.
Biodosimetry is an essential tool for providing timely assessments of radiation exposure, particularly when physical dosimetry is unavailable or unreliable.^{[1]} Maznyk et al.^{[2]} argued that for masscasualty events involving public exposure to ionizing radiation, it is paramount to rapidly provide this dose information for medical management of casualties. For proper dose estimation, each biodosimetry laboratory should establish its own doseresponse curves to prevent any erroneous calculation.
Materials and Methods   
Subject recruitment
Eight healthy persons, three males and five females, aged 21–55 years old were recruited as blood donors. They were required to sign an informed consent form after being explained of the study. All donors were requested to accomplish a questionnaire to assess their general physical condition, lifestyle, previous Xray examinations, diets, use of medications, etc.
Blood collection and irradiation
Ten milliliters of blood was drawn from each donor and 1 ml each was transferred to nine sterile glass vials. Two sets of optically stimulated luminescence nanoDot dosimeters per dose were also placed in separate glass vials. The vial with the blood sample and the vial containing the dosimeters were placed side by side in an improvised container filled with water just enough to immerse the vials with the temperature maintained at 37°C ± 1. The blood samples and the dosimeters were irradiated using the cobalt60 radiotherapy system of the Veterans Memorial Medical Center. The doses were 0.1, 0.25, 0.5, 0.75, 1, 2, 4, and 6 Gy at the dose rate of 91.98 cGy/min. Irradiation time was computed based on the dose rate of the source at the time of irradiation.
Blood culturing
About 0.5 ml of irradiated blood samples was then added to 5 ml PBMax Karyotyping medium within 2 h from irradiation time, in duplicates. Samples were cultured in an incubator with 5% CO_{2} for 48 h at 37°C.
Harvesting, slide spreading, and staining
Metaphase chromosomes were harvested, fixed, spread on slides, and stained as per guidelines of the International Atomic Energy Agency's (IAEA) Emergency Preparedness and Response: Biodosimetry 2011 manual.^{[1]}
Dicentric scoring
The slides were first scanned at low magnification, and when metaphase spreads were found, the microscope was switched to higher magnification. The images were captured using Nikon Microscope Eclipse E200 with a Motic DSFi1 camera attachment. Images were saved on a flash drive for counting of chromosomes and dicentric scoring in the computer.
Validation study
To validate the accuracy of the established doseresponse curve in estimating absorbed dose, blood samples from four donors, two males and two females, aged 23–52 years old were exposed to 2 Gy and 4 Gy. Processing, culturing, harvesting, slide preparation, and analysis were done following the procedure for the in vitro irradiation study of blood samples for the establishment of doseresponse curve for dicentrics in the IAEA guidelines.^{[1]}
Statistical analysis
The data were analyzed using R programming language, an opensource programming language designed by Ihaka and Gentleman,^{[3]} and the R programming language by R Core Team.^{[4]} The method used for estimating the coefficients is the iterative reweighted least squares, which is the method mentioned in the IAEA EPR 2011 manual ^{[1]} and stated that “the best fit value for each coefficient is achieved by assuming a Poisson distribution and maximizing the likelihood of the observations by the method of iteratively reweighted least squares.”
Results and Discussion   
Doseresponse curve
The data obtained [Table 1] showed an increase in the frequency of dicentric chromosomes as the irradiation dose is increased. This observation is more pronounced at higher doses (1.0, 2.0, and 4.0 Gy). For 6.0 Gy, the number of dicentrics decreased. It was also observed that the number of metaphase cells decreases as the irradiation dose is increased. This observed decrease in metaphase cells at 6.0 Gy can be inferred from the study of Pujol et al.^{[5]} who reported that most cells show difficulties in reaching mitosis at doses over 5 Gy and that this cannot be used to score dicentric chromosomes. It was also observed that the number of analyzable metaphase spreads decreased at the higher doses.
Statistical analysis showed that the cell distributions of dicentrics followed a Poisson distribution except for 0.1 Gy and 0.25 Gy, where both have not available (does not exist) Utest statistic. This is due to the dicentric count which is 1. As stated in IAEA EPR 2011 (equation 5, section 8.3),^{[1]} the number of dicentrics detected should have at least 2 counts to compute the Utest statistic. However, at lower doses, it is difficult to achieve higher dicentric scores and instead several thousand cells per point should be scored.^{[1]} Dicentric yield at 6 Gy showed overdispersed distribution considering that the estimated Utest statistic is more than 1.96. The doseresponse curve [Figure 1] follows the linearquadratic model. These results can be explained by the study of Vaurijoux et al.^{[6]} which stated that low linear energy transfer radiation (X or γ rays) produces many tracks containing few primary events with a more randomized distribution of tracks and a more uniform distribution of damage between cells. The same authors also reported that the doseeffect relationship is linear in the lowdose range and becomes quadratic at high doses.  Figure 1: Doseresponse curve: observed values dotted in blue and expected values in purple line; the 95% confidence interval in green line. The predicted values were extracted from the linearquadratic model with complete coefficient
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For [Table 2], all estimates except the constant, C, are significant base on its P value. The Chisquare P value indicated that the fitted data points were not statistically different from the observed ones, confirming a good fit.  Table 2: Curve fitting: The followings are the summary from linearquadratic model
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The doseresponse curve was established using the estimated linearquadratic model, Y = 0.0003298 + 0.0336027D + 0.0236312D^{2}.
Validation of doseresponse curve
Dose point estimate validation was performed using the estimated linearquadratic model and is given by:
Y = 0.0003298 + 0.0336027D + 0.0236312D^{2}
The calibration is done by solving for D above, which is given by:
Thus, for known dose 2 Gy [Figure 2], the estimate of the model for X = 33 (dicentrics) and N = 256 (cells), such that is  Figure 2: Interval estimates of dose for 2.0 Gy under 95% confidence interval with doseresponse regression curve under 95% confidence interval
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For known dose 4 Gy [Figure 3], such that is  Figure 3: Interval estimate of dose for 4.0 Gy under 95% confidence interval with doseresponse regression curve under 95% confidence interval
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Dose interval estimate
The uncertainty can be calculated using the 95% confidence limit of the Poisson variable. The confidence limit is the gray dashed line intersecting the confidence interval of the doseresponse curve. The value in the xaxis corresponds to the confidence limit [Figure 4] and [Figure 5]. Both 2.0 Gy and 4.0 Gy are inside the confidence limit, but 4.0 Gy has overdispersed distribution as indicated by its Utest statistics in [Table 3]. To account for this overdispersion, the confidence limit of 4 Gy was computed using some adjustments on its Y_{l} and Yu. These Y_{l} and Y u are the corresponding y values projected by the dashed lines onto the xaxis. The adjustment was done by multiplying a correction factor described in Equation 9 of EPRBiodosimetry 2011.^{[1]} To avoid overestimation of dose, 83% confidence limit of the regression curve was used instead of the 95% as suggested also in EPRBiodosimetry 2011.^{[1]} The statistical table from Crow and Gardner ^{[7]} was the reference used to obtain the Poisson 95% confidence limit of the dicentrics in 4 Gy of [Table 4].  Figure 4: Dose interval estimates for 2.0 Gy under 95% confidence interval with doseresponse regression curve under 83% confidence interval
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 Figure 5: Dose interval estimates for 4.0 Gy under 95% confidence interval with doseresponse regression curve under 83% confidence interval
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 Table 3: Distribution of dicentrics in blood samples exposed to 2.0 and 4.0 Gy
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Conclusion   
The established doseresponse curve follows the linearquadratic model. The goodness of fit test adjusted by the linearquadratic mo (Chisquare P = 0.24 with df = 6) indicated that the model is appropriate and has passed the validity test. The uncertainty of the dose estimate, especially in cases of overdispersion in the dicentric distribution, can be corrected by adjusting the confidence interval from 95% to a lower confidence interval like 83% as in this case.
Acknowledgments
This study was made possible through the financial and technical support of the International Atomic Energy Agency. The authors are greatly indebted to the eight volunteer blood donors for the doseresponse curve study and the four volunteer donors for the validation study. The management of the Veterans Memorial Medical Center is also acknowledged for allowing the research team to use their cobalt60 teletherapy machine in the irradiation of blood samples.
Financial support and sponsorship
This study was supported by International Atomic Energy Agency, Philippine Nuclear Research Institute.
Conflicts of interest
There are no conflicts of interest.
References   
1.  IAEA EPRBiodosimetry 2011. Cytogenetic Dosimetry: Applications in Preparedness for and Response to Radiation Emergencies. Vienna, Austria: IAEA; 2011. 
2.  Maznyk NA, Wilkins RC, Carr Z, Lloyd DC. The capacity, capabilities and needs of the WHO BioDoseNet member laboratories. Radiat Prot Dosimetry 2012;151:61120. 
3.  
4.  
5.  Pujol M, Barquinero JF, Puig P, Puig R, Caballín MR, Barrios L. A new model of biodosimetry to integrate low and high doses. PLoS One 2014;9:e114137. 
6.  
7.  Crow EL, Gardner RS. Confidence intervals for the expectation of a poisson variable. Biometrika Trust 1959;46:44153. 
[Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5]
[Table 1], [Table 2], [Table 3], [Table 4]
