The micronucleus test (MNT) is the most widely applied short‐term assay to detect clastogens or spindle disruptors. The use of flow cytometry (FCM) has been reported for micronucleated erythrocytes scoring in peripheral blood. The aim of this study was to develop a novel and practical protocol for MNT in rat peripheral blood by FCM, with the method validation. CD71‐fluorescein isothiocyanate and DRAQ5 were adopted for the fluorescent staining of proteins and DNA, respectively, to detect micronuclei. To validate the method, groups of male Sprague–Dawley rats (five per group) received two oral gavage doses at 0 and 24 h of six chemicals (four positive mutagens: ethyl methanesulphonate [EMS], cyclophosphamide [CP], colchicine [COL], and ethyl nitrosourea [ENU]; two nongenotoxic chemicals: sodium saccharin and eugenol). Blood samples were collected from the tail vein before and on the five continuous days after treatments; all of which were analyzed for micronuclei presence by both the manual (Giemsa staining) and FCM methods. The FCM‐based method consistently demonstrated highly sensitive responses for micronucleus detection at all concentrations and all time points for EMS, CP, COL, and ENU. Sodium saccharin and eugenol could be identified as negative in this protocol. Results obtained with the FCM‐based method correlated well with the micronucleus frequencies (r = 0.659–0.952), and the proportion of immature erythrocytes (r = 0.915–0.981) tested by Giemsa staining. The method reported here, with easy operation, low background, and requirement for a regular FCM, could be an efficient system for micronucleus scoring.

中文翻译：

---

基于流式细胞术的大鼠外周血网织红细胞微核检测新方法的开发

微核试验 (MNT) 是应用最广泛的短期检测方法，用于检测断裂剂或纺锤体破坏剂。已有报道使用流式细胞术 (FCM) 对外周血中的微核红细胞进行评分。本研究的目的是开发一种新颖实用的 FCM 大鼠外周血 MNT 方案，并进行方法验证。CD71-异硫氰酸荧光素和DRAQ5分别用于蛋白质和DNA的荧光染色，以检测微核。为了验证该方法，雄性 Sprague-Dawley 大鼠组（每组 5 只）在 0 和 24 小时接受两次口服强饲剂量的六种化学物质（四种阳性诱变剂：甲磺酸乙酯 [EMS]、环磷酰胺 [CP]、秋水仙碱 [COL]和乙基亚硝基脲 [ENU]；两种非遗传毒性化学品：糖精钠和丁子香酚）。在治疗前和治疗后连续五天从尾静脉采集血样；所有这些都通过手动（吉姆萨染色）和 FCM 方法分析了微核的存在。基于 FCM 的方法在所有浓度和所有时间点对 EMS、CP、COL 和 ENU 的微核检测均表现出高度灵敏的响应。在该协议中，糖精钠和丁子香酚可以被确定为阴性。使用基于 FCM 的方法获得的结果与微核频率有很好的相关性（基于 FCM 的方法在所有浓度和所有时间点对 EMS、CP、COL 和 ENU 的微核检测均表现出高度灵敏的响应。在该协议中，糖精钠和丁子香酚可以被确定为阴性。使用基于 FCM 的方法获得的结果与微核频率有很好的相关性（基于 FCM 的方法在所有浓度和所有时间点对 EMS、CP、COL 和 ENU 的微核检测均表现出高度灵敏的响应。在该协议中，糖精钠和丁子香酚可以被确定为阴性。使用基于 FCM 的方法获得的结果与微核频率有很好的相关性（r = 0.659–0.952)，以及通过吉姆萨染色测试的未成熟红细胞的比例 ( r = 0.915–0.981)。此处报告的方法操作简单、背景低且需要常规 FCM，可能是一种有效的微核评分系统。