Establishment And Application Of The Automatic Assay Based On Acridine Orange Staining And Single Laser Flow Cytometer For Detecting Micronucleated Erythrocytes In Mice Bone Marrow

Micronucleus test(MNT) is an important assay used to screen chromosome damage, which is as sensitive, accurate and specific as chromosome analysis(CA), however, the former is more simple and rapid. Thus, MNT has been applied to assess genetic damage in many fields, such as medicine ,biology and environment , et al. As a kind of biomarker, MNT can monitor genetic damage of people explored to harmful environment. With daily serious environmental pollution, the more and more scientists being engaged in toxicological study have been showing solicitude for finding out an automatic assay for many years. The flow cytometry is popular because flow cytometers(FCM) have been widely used in many laboratories. Two kinds of staining based on FCM have been used ,the one is propidium iodide(PI) compounding CD71-FITC, the other is acridine orange(AO).The former uses Malaria-infected erythrocytes to act as biological standards to ensure reliable and consistent scoring of micronucleated erythrocytes. But the process is complex and time-consumed, while the latter is simple and rapid. But this technology was difficult to repeat. Our laboratory explored and improved this assay recently, detecting micronucleated (MN) erythrocytes either in rat bone marrow, or in human peripheral blood, and assessed chronic radiation damage as biodosimeter. This study developed the assay on mice model and realized discerning genetic damage resulted from CP, HAN, VCR three toxogen and X-ray. At the same time, our study improved manual microscopy inspection based on AO staining. The main results from our study are as follows:1. The results from both manual assay and automatic assay showed good dose-response relationship between the frequency of micronucleated polychromatic erythrocytes (fMNPCE) and CP doses. Good correlation coefficient was obtained between AO and Giemsa staining(r =0.978, P < 0.01). So was that of AO-FCM and Giemsa(r = 0.973,P < 0.01). %Ret decreased with dose increase. The correlation coefficient was 0.808(P < 0.01) between AO microscopic assay and AO-FCM automatic assay.2. The results from HAN treatment groups were not significantly different from the control level. Good correlation coefficient(r = 0.993, P < 0.01, r = 0.608, P < 0.01) were observed between the two methods of AO and AO-FCM.