Bryce Steven M, Bernacki Derek T, Bemis Jeffrey C, Spellman Richard A, Engel Maria E, Schuler Maik, Lorge Elisabeth, Heikkinen Pekka T, Hemmann Ulrike, Thybaud Véronique, Wilde Sabrina, Queisser Nina, Sutter Andreas, Zeller Andreas, Guérard Melanie, Kirkland David, Dertinger Stephen D
Litron Laboratories, Rochester, New York.
Pfizer Worldwide Research and Development, Groton, Connecticut.
Environ Mol Mutagen. 2017 Apr;58(3):146-161. doi: 10.1002/em.22083.
We previously described a multiplexed in vitro genotoxicity assay based on flow cytometric analysis of detergent-liberated nuclei that are simultaneously stained with propidium iodide and labeled with fluorescent antibodies against p53, γH2AX, and phospho-histone H3. Inclusion of a known number of microspheres provides absolute nuclei counts. The work described herein was undertaken to evaluate the interlaboratory transferability of this assay, commercially known as MultiFlow DNA Damage Kit-p53, γH2AX, Phospho-Histone H3. For these experiments, seven laboratories studied reference chemicals from a group of 84 representing clastogens, aneugens, and nongenotoxicants. TK6 cells were exposed to chemicals in 96-well plates over a range of concentrations for 24 hr. At 4 and 24 hr, cell aliquots were added to the MultiFlow reagent mix and following a brief incubation period flow cytometric analysis occurred, in most cases directly from a 96-well plate via a robotic walk-away data acquisition system. Multiplexed response data were evaluated using two analysis approaches, one based on global evaluation factors (i.e., cutoff values derived from all interlaboratory data), and a second based on multinomial logistic regression that considers multiple biomarkers simultaneously. Both data analysis strategies were devised to categorize chemicals as predominately exhibiting a clastogenic, aneugenic, or nongenotoxic mode of action (MoA). Based on the aggregate 231 experiments that were performed, assay sensitivity, specificity, and concordance in relation to a priori MoA grouping were ≥ 92%. These results are encouraging as they suggest that two distinct data analysis strategies can rapidly and reliably predict new chemicals' predominant genotoxic MoA based on data from an efficient and transferable multiplexed in vitro assay. Environ. Mol. Mutagen. 58:146-161, 2017. © 2017 Wiley Periodicals, Inc.
我们之前描述了一种基于流式细胞术分析的多重体外遗传毒性检测方法，该方法用于分析经去污剂处理后释放的细胞核，这些细胞核同时用碘化丙啶染色，并用抗p53、γH2AX和磷酸化组蛋白H3的荧光抗体进行标记。加入已知数量的微球可提供绝对的细胞核计数。本文所述的工作旨在评估这种检测方法（商业上称为MultiFlow DNA损伤试剂盒-p53、γH2AX、磷酸化组蛋白H3）在不同实验室之间的可转移性。在这些实验中，七个实验室研究了来自84种参考化学物质中的一组，这些物质代表了致断裂剂、非整倍体诱导剂和非遗传毒性剂。将TK6细胞在96孔板中暴露于一系列浓度的化学物质中24小时。在4小时和24小时时，将细胞 aliquots 加入到MultiFlow试剂混合物中，经过短暂孵育期后进行流式细胞术分析，在大多数情况下直接通过机器人自动数据采集系统从96孔板中进行分析。使用两种分析方法评估多重反应数据，一种基于全局评估因子（即从所有实验室间数据得出的临界值），另一种基于同时考虑多种生物标志物的多项逻辑回归。两种数据分析策略都旨在将化学物质分类为主要表现出致断裂、非整倍体诱导或非遗传毒性作用模式（MoA）。基于总共进行的231次实验，与先验MoA分组相关的检测灵敏度、特异性和一致性≥92%。这些结果令人鼓舞，因为它们表明两种不同的数据分析策略可以基于高效且可转移的多重体外检测数据快速可靠地预测新化学物质主要的遗传毒性MoA。《环境与分子突变》58:146 - 161, 2017年。©2017威利期刊公司。
