Study of Solving the Bottleneck Problem of the Estimation of Biological Dose in the Window of Time of Medical Emergency in Large-scale Nuclear Radiation Accidents

Purpose In order to achieve the goal of rapid response, effective disposal and protection of life of large-scale radiation events, how to establish the uniform standard curve of biological dose estimation for chromosome aberration analysis becomes an urgent need. Chromosomal aberrations with different irradiation dose rates were used to analyze the biological dose curve and the share of the “dicentric + ring” caused by the dose rate at each dose point. The dose-rate effect of 60 Co-rays on peripheral blood lymphocytes was analyzed by statistical method . Irradiation dose is dominant ; At each dose point, “(dicentric chromosome + centric rings) /cell” is proportional to "dose rate", that is, Y = k X + b;Between 1-5Gy dose, “(dicentric chromosome + centric rings) /Cell ” holds a quadratic linear relationship with dose rate, that is,y = ax 2 + bx + c. The fraction of “dicentric + ring” caused by dose rate was calculated, if “Dose rate” is Z Gy•min − 1 , it corresponds to an increase in linear relationships. Biological


Introduction
In response to the problem of rapid and high-throughput accurate estimation of personal high biological dose exposure during nuclear emergencies and nuclear terrorism, we will focus on research to solve the bottleneck of large quantities of biological dose estimation problem in large-scale radiation medical emergency events within a window period quickly and dose estimation of the illuminated staff clearly to determine whether the staff are irradiated the radiation damage degree and the important basis of classi cation of early treatment measures to prevent the public panic, and the reasonable medical treatment. the chromosome "dicentric + ring"with the irradiation dose rate on the rise of share are calculated, Research and development of a uni ed medical emergency curve for nuclear and radiation accidents are carried forward based on human peripheral blood lymphocyte chromosome "dicentric + ring" analysis. Rapid response to large-scale radiation events is developed to achieve the goal of rapid response, effective disposal and protection of life.
The classi cation of potential victims in medical rescue of nuclear and radiation accidents will help to make better use of current medical resources and improve the e ciency of rescue.Biodosimetry (BIOdosimetry) is an effective method to evaluate the extent of external radiation damage.The application of biological dose estimation method in nuclear accident classi cation is of great signi cance for effective medical rescue in nuclear accidents.
For a long time, dose assessment after ionizing radiation (IR) exposure has been done by analyzing chromosomal aberrations in mitotic cells. Analysis of metaphase dicentric chromosomes in peripheral blood lymphocytes has allowed the development of biodosimetry and has become the preferred method for suspected IR overexposure (International Atomic Energy Agency (IAEA) 2011).Cytogenetic dosimetry: Applications in radiation emergency preparedness and response. [1] Each institution was required to establish a biological dose estimation curve for chromosome aberration analysis. Under these circumstances, we cannot form a joint response to a nuclear and radiological accident.
Collection, irradiation, and transportation of blood All donors give informed consent. No recent ionizing radiation exposure, no smoking. For each exercise, blood samples were drawn from each of 5-6 donors (ages 26-68 years), Blood was collected through vein and added into 20 ml of lithium heparinization tube by puncture. All blood samples were irradiated in vitro in a test tube at room temperature.
Irradiation at 8 different dose points between 0.0 and 5.0Gy. Peripheral blood of volunteers was collected about 20 mL and divided into 1-ml portions. At 37±1℃, each vial was exposed to different doses of 60 Co radiation (2.8 and 3.7 Gy). One vial was not irradiated and kept as a control.

Cell culture and harvest
Based on general guidelines provided by the International Atomic Energy Agency (IAEA 2001, 2011) and ISO 19238 and 21243 (ISO 2004, 2008.

Dicentric chromosome assay
Cells were incubated at 37℃ and 5% CO 2 for 50 h, and only the rst metaphase diffusion was used to count the dichotomies. The standard way to ensure that only the rst metaphase diffusion is scored is to add colchicine in advance. The whole blood culture method was adopted, and the ratio of blood to medium was 1:10.0.5mL heparin lithium was added to 5mL lymphocyte culture medium as anticoagulant. Prepare cell suspension. Cells were treated with 5 mL KCl hypotonic twice every 30 minutes, and then xed with Carnoy solution for 4 times for 5 minutes each time. Prepare and stain for Giemsa, air dry and code. Used for chromosome aberration analysis of lymphocytes. [1] Su cient mitotic phase is analyzed according to the following formula, where p is the ratio of "dicentric+ ring" aberrations cells, n is the number of cells to be analyzed, and p can be calculated after a certain number of aberrations cells are obtained by counting analysis. At least 100 "dicentric+ ring" or 1000 mitotic phases were analyzed for each sample and biological dose estimates were made.
n=(1-p)×96.04/p For example, for an accident exposed person, 100 metaphase mitotic cells were observed and 18 aberrant cells with double centromere and ring were found. When an error of 20% is allowed, calculate the number of cells to analyze. Formula:

Cytokinesis block micronucleus assay
Two hours after exposure, 9 ml medium (80% RPMI-1640 and 20% FBS) was added to 1 ml of blood. Ph -M (20 mg/mL) was stimulated and incubated at 37℃. [2,3] At the 44th hour, cyto-b was added 6 mg/mL and cultured for 28h.After incubation for 72 hours, cells were collected with a pre-cooled hypotonic solution (0.075M) and xed with Carnoy's solution (methanol/acetic acid 5:1).For each dose of irradiated blood sample, multiple sections were cast, air-dried, and coded.
In the following dose ranges, the obtained data were tted by the method of minimum sum of squares according to the four mathematical models provided by WHO, the signi cance test of the regression coe cient was carried out, and the correlation index (R 2 ) test of the tting degree of the equation was carried out. According to the regression coe cient signi cance test (P), the degree of t (R 2 ), the difference between a value and the spontaneous aberration rate, the optimal regression equation was selected for each dose range. 0-0.5Gy(Dic+r) Y=3.99D 1.1033 ;0-0.5Gy(The total distortion),Y=6.5328D 1.0196 0.5-5.0Gy(Dic+ r),Y=7.1466D 1.8933 ;0-5.0Gy(Dic+r),Y=8.9846D 1.632 . 0-6.0Gy (Dic+r),Y=9.23D 1.6606 .
Biological dose estimation and error analysis were performed on the national assessment samples in 2015 and 2016 by using the dose curves established by different dose rates in different laboratories.
The dose curves with different exposure dose rates established by different laboratories were used to estimate the biological dose of the national assessment samples in 2015 and 2016. The error analysis is shown in table 2 3. Table 3 shows that the absolute value of the error increases with the increase of the radiation dose rate, which is signi cant with the increase of the radiation dose. It indicates that the irradiation dose is dominant and the irradiation dose rate is secondary.
Analysis of double + ring (%) and irradiation dose results of chromosome aberrations in peripheral blood irradiated by different dose rates in different laboratories (see table 4) Table4 shows that the original data of the biological dose estimation curve of chromosome aberration established by different laboratories have certain differences when comparing the dose-effect curve of each laboratory. Although there was an increasing trend of different dose rates at the same dose, there were also cases in which the high dose rate irradiation of "dicentric+ring" (%) of chromosome aberration was lower than that of low dose rate irradiation. The unit of x is Gy·min -1 At each dose point, "Dic + r/Cell" is proportional to "dose rate", that is, Y=k X +b, It shown table 5, gure1, 2.
The dose rate caused by the "dicentric+ring" increase in the share of the analysis, it shown table 6, 7 and Figure 3 The fraction of "dicentric+ring" caused by dose rate was calculated, if "Dose rate" is Z Gy·min -1 , it corresponds to an increase in linear relationships.

Discussion
Biodosimetry has been used for many years to estimate the amount of ionizing radiation an individual receives. This information is of vital importance to the medical community as it helps to develop effective and timely treatment plans for potential patients.
Several biomarkers have been developed to measure radiation damage. Traditionally, dicentric chromosome testing (DCA) is a dose estimation method based on the frequency of dicentric chromosomes in peripheral blood lymphocytes. As the background dual center frequency is low and stable (0.5& NDASH; DCA is very sensitive (IAEA 2011) and is particularly sensitive to ionizing radiation damage. In this experiment, when the dose level was as low as 0.1&Ndash. 0.2Gy, 500& NDASH; 1000 intermediate extensions were analyzed, but this required many hours of analysis. Peripheral blood lymphocytes are usually G0 at rest and must be stimulated to enter metaphase. During this process, cells must overcome the different cell cycle checkpoints that control proper progression. When a cell suffers DNA damage, DNA repair mechanisms are activated by other signaling pathways, inducing delays in cellular processes, and triggering programmed cell death when necessary. At checkpoints, G 2 /M ensures that cells do not enter mitosis before they have a chance to repair damaged DNA (Jeggo and Lobrich 2006Jeggo PA, Lobrich M. 2006).The role of DNA repair and cell cycle checkpoint blocking in maintaining genomic stability. DNA repair.Now,92 -1198.
However, in mass casualty incidents, only patients treated with 2.0 Gy or more did not need such sensitivity.
In these cases, the sensitivity of the analysis can be reduced by reducing the number of metaphase cells, greatly reducing the time required for analysis. Standard shunt DCA analysis only analyzes 50 mid-term diffusions now, providing a detection threshold of 1/2 Gy. Still su cient to guide the treatment of acute radiation syndrome (ARS) (Lloyd 1997, Lloyd et al. 2000, Voisin et al. 2001 2008;GB/T 28236-2011) By introducing a scoring technique called &Lsquo, the time e ciency of sorting based scoring can be greatly improved without losing the accuracy of dose estimation. DCA quick scan; (Flegal et al., 2010(Flegal et al., ,2012.This approach is not based on counting individual centromeres, but checking for obvious damage of intermediate diffusion simply and quickly, thereby eliminating the count of individual chromosomes as traditional DCA (CDCA) methods do to ensure the integrity of the cell which is analyzed, This approach has been shown to be as accurate as traditional triage scoring, while reducing grading time by approximately 6 times (Flegal et al., 2012). [5] Another strategy to improve biodosimetric throughput is the development of a network of biodosimetric laboratories. A number of networks have been established to improve dose estimation throughput, such as the National Biological Dose Response Program (NBDRP) in Canada (Miller et al. 2007), the Biological dosimetry Network in Latin America (Garcia et al. 1995) and the Chromosome network in Japan (Yoshida et al. 2007).In addition, the European Network, the European Biodosimetry Network (RENEB), is being gradually established (Kulka et al. 2012).When a network is established, regular comparisons must be made between the various laboratories of the network to maintain and evaluate accuracy and throughput. Many one-off crosscomparisons have been carried out over the past few years, with each country having a different design within two laboratory networks (Garcia et al. 1995) and between laboratories in different networks or between countries (Roy et al. 2004, Wilkins et al. 2008,Di et al. 2011,Beinke et al. 2013.The purpose of this paper is to describe the comparative results of Canada's National Biological dose Response Program (NBDRP) over the past 6 years. Experiments of similar design are conducted annually, including DCA, routine and rapid scanning, and CBMN analysis. These efforts involve four Canadian reference laboratories and, occasionally, two biodosimetry laboratories in the United States. The lessons learned from these comparisons will be discussed and the importance of repeating the comparison exercise will be emphasized. [6][7][8][9][10][11] In order to accurately estimate the human biological dose at different dose rates, low, medium and high dose rates were irradiated to human peripheral blood to prepare chromosome samples, and the dose response curves were established according to the double-center frequency and loop frequency. The results show that at the same dose level, the distortion frequency increases with the increase of dose rate, and there is an obvious dose rate effect. The estimated absorbed dose of low dose -reaction curve is signi cantly higher than that of high dose -reaction curve. Therefore, the in uence of dose rate should be considered in dose estimation, and the approximate dose rate and dose response curve should be selected to make the estimation results credible.

Conclusion
Establish a unity standard curve of biological dose estimation for the analysis of chromosome aberration.
The fraction of "dicentric + ring" caused by dose rate was calculated, if "Dose rate" is Z Gy·min − 1 , it corresponds to an increase in linear relationships.
These ndings provide con dence to the medical community. Application of estimated biodosimetry to medical rescue during nuclear and radiation accidents is widespread, the public and government believes that in the event of a nuclear accident, biodosimetry can be applied to manage and medically treat casualties to ensure the minimization of health risks.
On this basis, the chromosome karyotype analysis pretreatment system and chromosome automatic scan analysis system were used to realize the automation of biological dose estimation. Joint laboratories in different parts of the world can respond to nuclear and radiation accidents in a timely manner.

Declarations
Ethics approval and consent to participate The research was approved by the Bioethics Committee at the Gansu provincial center for disease control and prevention. This article does not contain any studies with animals performed by any of the authors.

Consent for publication
Agreed to publish Availability of data and materials Not applicable

Competing interests
The authors declare that they have no con ict of interest.

Funding
This study was funded by the Lanzhou science and technology bureau (2018-1-127) and Gansu provincial health commission (GSWSKY2018-48).   Abbreviation: Dic, dicentric chromosome; r, centric rings Table 7 The dose rate caused by the "dicentric+ring" increase in the share of the analysis (2) Dose rate  Figure 1 The relationship between radiation dose and k Value Figure 2 The relationship between radiation dose and b Value Biological dose estimation curve