| With the progress of scientific technique, ray, microwave and electromagnetic radiation are more and more universal in human life, such as medical diagnose, medical treatment and commercial irradiance and so on. However, the progress of scientific technique also brings many negative effects on human society, among which the most important is human health. The actinic rays threaten human health severely and its injury on group genetic materials has been widely noticed. Many countries own their unified program to evaluate ray, microwave and electromagnetic radiation, among which chromosome aberration (CA) analysis and micronucleus test (MNT) are frequently used. The merits of CA analysis include precise and reliable results as well as providing abundant information. But CA analysis is time-consuming and needs great effort. Furthermore, cell culture is needed. Up to now, traditional MNT is dissatisfied for practical work because it needs counting the cell number by human under microscope. This technique is subjectivity and low sensitivity which needs a lot of work. Meanwhile, false positive and mock detection are common. Thus, developing automation technique to detect micronucleus is becoming an important method to monitor genetic injury in crowd quickly.With the progress of flow cytometric detection technology, the experts and scholars from home and abroad devoted all themselves to the research of automation. In 1992, the Sweden scientist Grawe developed the automatic detection technique which detect the micronucleated reticulocytes in mice peripheral blood using flow cytometry. In 1993 Caojia from our department improved this technique and made it come up to the practical standards. In 2003 Sun Liping also from our department established the automatic detection systems to analysis the micronucleated polychromatic erythrocytes(PCE) in rat marrow based on uni-laser flow cytometry. Meanwhile, he successfully applied the magnetic isolation technique assay into testing the reticulocyte micronuclear rates of patients who suffered form nasopharyngeal carcinoma and treated with radiotherapy. Furthermore, he also attempted to use this method as the biological counting meter to estimate the cumulative radiation dose.Based on our department's preliminary work and using mice as the animal model, we established flow cytometric automation testing method to detect low dose X ray-induced frequency of mcie marrow micronucleated reticulocytes. Then using this method and combing the new developed skills of magnetic isolation technique, we examined reticulocyte micronuclear rates in the peripheral blood of patients who suffered the head or the face cancerous and received low dose X ray treatment.The results of this study are as follow:1. Detection fMNPCE and fMNNCE of mice borrow after low dose X-ray treatment: Through the comparative analysis for the different parameter detections, it has been fixed that the FL1 for the ordinate and the FSC for the abscissa can be greatly distinguished every cell clones, including the TNC, PCE , MNP CE, NCE and the MNNCE. Conducting by the x-Rays after twenty hours, the FMNPCE was respectively 3.01±0.55‰,4.58±±0.83‰and 11.12±1.72‰;for one irradiation dose. For different accumulated dose 0.1Gy,0.15 Gy,0.2 Gy,0.3Gy,0.4Gy,0.8Gy,1.2Gy and 1.6Gy,the mouse marrow was 4.72±0.95‰,10.26±2.09‰,13.77±1.66‰,11.77±2.33‰,17.71±2.19‰,16. 43±1.33‰,21.85±3. 25‰and 22.17±3.48‰;The results displayed the good relationships including the Does-Reaction and the Time -Effect.2. Comparisons between FCM results and microscopic examination: Both results displayed good dose-reaction and time-response relationships. But FCM result was higher than microscopic results, there were no significant differences but had the good relevance between two methods. (R=0.984,P < 0.01).3. The enrich effect of magnetic isolation technique: seven craniofacial cancer patients and seven healthy volunteers were received the different accumulated radiotherapeutic dose and after that, CD71+ reticulocytes (Trf-Ret) were isolated. It was showed that the ratio of Trf-Ret varied between the distinct individuals with the ratio being 20% to 90%. It was not caused by the separation method but should attribute to the individual difference, such as the age, job and the received the X-rays dose. It can be proved that this method was simple, reliable and reproducible.4. Detection the MN-Trf-Ret in human peripheral blood dosed by FCM after X-ray radiation. Set FL4 ((indicating RNA fluorescence)) as ordinates and FSC (indicating cell size) as abscissa, we divided the enriched human peripheral blood samples into 5 subtypes, that is reticulocytes, reticulocytes with micronuclei, mature erythrocytes, mature erythrocytes with micronuclei and nucleated cells. We than detected the change of MN-Trf-Ret in peripheral blood of craniofacial cancer patients who had received low dose X-ray treatment. The results showed that the when fMN-Trf-Rets of craniofacial cancer patients increased quickly when they received radiation therapy. Along with the increasing accumulated dose,fMN-Trf-Ret was in high levels at all times. According to different accumulated radiotherapeutic dose 0Gy, 2Gy, 4Gy, 8Gy and 10Gy, the mean of fMN-Trf-Ret were 2.89±0.93‰, 16.2±2.31‰, 23.84±3.22‰, 32.17±6.95‰, 30.89±9.35‰and 35.74±9.95‰respectively.5. Curve estimation for biodosimetry: Based on the dose-response and time-response relationship between fMN-Trf-Ret and cumulative dose, the best curve equation was achieved through analysis by SPSS11.0, that is Y=2.595 + 8.874X - 1.005X2 +0.044X3(R=0.978, P<0.05). The equation can be tried and confirmed by estimating the biodosimetry of victims of radiation accidents within 0-10Gy.In conclusion, to speculate human fMN-Trf-Rets alteration after low-dose of X-ray radiation, we detected micronuclear rate of BM-RBC in mice after receiving low-dose of X-ray radiation using FCM. Our result indicates that FCM method exhibits a sensitive, rapid and convenient way of detecting the effect of low-dose of X-ray radiation. These are basics for micronuclear FCM promotion and application.
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