Interlaboratory validation of a CD71-based flow cytometric method (Microflow) for the scoring of micronucleated reticulocytes in mouse peripheral blood.
Interlaboratory validation of a CD71-based flow cytometric method (Microflow) for the scoring of micronucleated reticulocytes in mouse peripheral blood.
复制标题
基于 CD71 的流式细胞术（Microflow）对小鼠外周血中微核网织红细胞评分的实验室间验证。
DOI：
10.1002/em.20081
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发表时间：
2005
期刊：
影响因子：
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通讯作者：
Lynch,AnthonyM
中科院分区：
文献类型：
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作者：
Torous,DorotheaK;Hall,NikkiE;Illi-Love,AnneH;Diehl,MarilynS;Cederbrant,Karin;Sandelin,Kerstin;Ponten,Ingrid;Bolcsfoldi,George;Ferguson,LynnetteR;Pearson,Amira;Majeska,JennessB;Tarca,JamesP;Hynes,GeoffreyM;Lynch,AnthonyM
进行了一项实验室间研究，以验证基于抗CD 71/流式细胞术的小鼠外周血微核网织红细胞（MN-RET）计数技术。这些实验旨在通过评价流式细胞术（FCM）生成的MN-RET测量值与使用传统显微镜方法获得的测量值之间的对应程度，来解决遗传毒性试验程序国际研讨会验证标准。除了这些交叉方法数据外，还在两个不同的研究中心对每份血液样本进行了流式细胞术MN-RET测量，以评价实验室之间数据的重现性。在这些研究中，雄性CD-1小鼠组接受溶剂（生理盐水或植物油）、阴性对照（生理盐水或植物油）或四种剂量水平的五种已知遗传毒性物质（致染色体断裂剂：环磷酰胺、苯并[a]芘、5-氟尿嘧啶、甲氨蝶呤;非整倍体：硫酸长春新碱）处理。通过腹膜内注射连续3天暴露，并在最终处理后约24小时采集血样。基于对2，000（显微镜检查）和20，000（FCM）网织红细胞的分析，确定每份样本的MN-RET频率。无论使用何种方法，观察到每种遗传毒性物质均导致MN-RET频率出现统计学显著性增加，并且每种反应均以剂量依赖性方式发生。FCM与基于显微镜的MN‐RET测量（9次实验，252次配对测量）的斯皮尔曼相关系数（rs）为0.740，表明方法之间的高度一致性。在两个独立研究中心进行的所有流式细胞术MN‐RET测量的rs值为0.857（n = 248），表明自动化方法在实验室之间具有高度可转移性。此外，流式细胞仪系统相对于基于显微镜的评分具有优势，包括分析的细胞数量更多，分析时间更快，客观性更高。总之，本报告中提供的数据表明，使用流式细胞术评分程序可提高小鼠外周血微核试验的整体性能。Environ.摩尔诱变剂，2005.© 2004 Wiley利斯公司
An interlaboratory study was performed to validate an anti‐CD71/flow cytometry‐based technique for enumerating micronucleated reticulocytes (MN‐RETs) in mouse peripheral blood. These experiments were designed to address International Workshop on Genotoxicity Test Procedures validation criteria by evaluating the degree of correspondence between MN‐RET measurements generated by flow cytometry (FCM) with those obtained using traditional microscopy‐based methods. In addition to these cross‐methods data, flow cytometric MN‐RET measurements for each blood sample were performed at two separate sites in order to evaluate the reproducibility of data between laboratories. In these studies, groups of male CD‐1 mice were treated with vehicle (saline or vegetable oil), a negative control (saline or vegetable oil), or four dose levels of five known genotoxicants (clastogens: cyclophosphamide, benzo[a]pyrene, 5‐fluorouracil, methotrexate; aneugen: vincristine sulfate). Exposure occurred on 3 consecutive days via intraperitoneal injection, and blood samples were obtained approximately 24 hr after the final treatment. MN‐RET frequencies were determined for each sample based on the analysis of 2,000 (microscopy) and 20,000 (FCM) reticulocytes. Regardless of the method utilized, each genotoxic agent was observed to cause statistically significant increases in the frequency of MN‐RETs, and each response occurred in a dose‐dependent manner. Spearman's correlation coefficient (rs) for FCM versus microscopy‐based MN‐RET measurements (nine experiments, 252 paired measurements) was 0.740, indicating a high degree of correspondence between methods. The rsvalue for all flow cytometric MN‐RET measurements performed at the two independent sites was 0.857 (n = 248), suggesting that the automated method is highly transferable between laboratories. Additionally, the flow cytometric system offered advantages relative to microscopy‐based scoring, including a greater number of cells analyzed, much faster analysis times, and a greater degree of objectivity. Collectively, data presented in this report suggest that the overall performance of mouse peripheral blood micronucleus tests is enhanced by the use of the flow cytometric scoring procedure. Environ. Mol. Mutagen., 2005. © 2004 Wiley‐Liss, Inc.