Study On High-speed Biological-dose-diagnosis Of Acute Radiation Sickness Using Micronucleated Reticulocytes

Objective Ionizing radiation could cause genetic damage. When human body was exposed to ionizing radiation, the degree of genetic damage was related to irradiation dose. Acute radiation sickness is a systemic disease caused by a dose of radiation,ususaly more than 1Gy, and often received in a short period of time. Generally speaking, after uniformity exposure of 1Gy or more, human could suffer from acute radiation sickness. People who received less than 6Gy dose of ionizing radiation can be cured with prompt therapeutic measure. The sooner one was treated, the less pathological change of all systemic occurred. Patients exposed to more than dose of 8Gy had no record of long term survival. Thus, treatment of acute radiation sickness need a rapid accurate diagnosis of exposure dosage.Micronucleus test has become essential test in fields of radiation biological dose estimation and genetoxic evaluation with physical or chemical materials in recent decade. Cytokinesis-block micronucleus assay as contiditional micronucleus test need to culture cell and count the cell number by human under microscope. So it was time-costing and personnel-costing. Automatic micronucleated reticlucytes test with flow cytometry is established in current years, and it markedly accelerate rapidity of detection. Our test aimed to establish automatic method for micronucleus test, and supply evidence for rapid accurate diagnosis to the exposure dose of acute radiation sickness paitients.Methods1. Establishment of a mouse model of acute total body irradiation: one hundred and twenty male BALB / c mice were randomized divided into ten groups which were exposed to multiple doses of 60Co-γray( 0,0.5,1,2,3,4,5,6,8,10Gy). They were scared after 24h or 48h of exposure toγ-ray, than their bone marrow specimens were detected.2. To establish our automatic scoring method based on the flow cytometer for detection of frequencies of micronucleated polychromatic erythrocytes from mice bone marrow. Results of FCM were compared with those of microscope to test accuracy of the automatic method.3. If the results of automatic testing have doses-effect relationship within a certain range,we will fitting doses-effect curve according to the results of automatic testing of the frequencies of micronucleated polychromatic erythrocytes from mice bone marrow after radiation.4. To establish the method of testing the frequencies of micronucleated polychromatic erythrocytes from human bone marrow and peripheral reticulocytes with FCM. Results of FCM were compared with those of microscope to test accuracy of the automatic method.5. The automatic method was applied to detect peripheral blood specimen from those patients who were exposed to chronic low dosage radiation for years, results of automatic method were compared with those of microscope to test accuracy of the automatic method.6. The automatic method also was apply to detect peripheral blood specimen of persons who may had been exposed in the leakage of Japanese nuclear power plant, and the results of this method were compared with those of Cytokinesis-block micronucleus method .Results:1. we have established an automatic micronucleus test method for detection of specimens, including mice bone marrow, human bone marrow,and human peripheral blood.2. The results of automatic FCM testing are obviously correlated with those of artificial microscopy findings in examination of bone marrow specimens in mice. The correlation coefficient is 0.987. Although results of FCM are seemingly little higher, but the difference between two methods is not significant statistically (p > 0.05). FCM is the time-saving and labor-saving. Even more FCM can avoid the subjectivity of artificial microscopic detection.3. After exposure to 60Co–γrays within a range of 0-6Gy, the frequencies of micronucleated polychromatic erythrocytes from mice bone marrow are well represented the radiation biologic dosages. The results and the irradiation dose have good doses-effect relationship. Results of the specimen which were collected 48h after exposed toγrays are significantly higher than those of 24h, which indicated that the frequencies of micronucleated polychromatic erythrocytes from mice bone marrow presents good time-effect relationship with span of time from exposure to obtainment of specimen..4. We used the automatic FCM method and control method of CBMN to detect human peripheral blood specimens from suspicious stuff, and defined the dosages are 0.15 Gy or so. It suggest that the frequencies of micronucleated reticulcytes is a sensitive radiation biology dose marker.Conclusion:1. We have established an automatic micronucleus test method for detection of specimens including mice bone marrow, human bone marrow, and human peripheral blood. Also we proof that the frequencies of micronucleated reticulcytes is a sensitive radiation biology dose marker, and it can be used for automatic diagnosis biological of dosages of acute radiation sickness, with the advantage of saving a lot of time and manpower.2. We proved the automatic method used FCM and dying cell with acridine orange to detect the specimens is less time-consuming, labor- consuming and more objective than the traditional microscopic method. The automatic method is more suitable for rapid diagnosis of dosages of individuals who have exposed to ionizing radiation in radiation accident, especially for large mass of people in large-scale radiation accident.. We recommend to use this method to replace the traditional artificially microscopy method in further research.3. We defined minimum value of the frequencies of micronucleated reticulcytes donation members and occupational radiation workers in Health Surveillance which are suspicious of being exposed by detecting their peripheral blood specimens with this automatic method. It provides experimental basis for further research on rapid dosage diagnosis of human acute radiation sickness.4. We have established an automatic micronucleus test method for detection human bone marrow specimens for the first time. It was unnecessary to enrich reticulocytes to detect human bone marrow specimens, so that we can save more time for testing, and increase relevance ratio of micronucleated reticulcytes. This part of test provides experimental basis for further research on rapid dosage diagnosis of human acute radiation sickness patients.