使用成像流式细胞术和人工智能实现微核检测自动化
Summary
微核 （MN） 测定是量化 DNA 损伤的成熟测试。然而，使用常规技术（如手动显微镜或基于特征的图像分析）对测定进行评分既费力又具有挑战性。本文描述了开发人工智能模型以使用成像流式细胞术数据对 MN 测定进行评分的方法。
Abstract
微核（MN）测定在全球范围内被监管机构用于评估化学品的遗传毒性。该测定可以通过两种方式进行：在一次分裂、细胞分裂阻断的双核细胞或完全分裂的单核细胞中对 MN 进行评分。从历史上看，光学显微镜一直是对测定进行评分的金标准方法，但它既费力又主观。近年来，流式细胞术已被用于对测定进行评分，但由于无法直观地确认细胞图像的关键方面而受到限制。成像流式细胞术（IFC）结合了高通量图像捕获和自动图像分析，已成功应用于快速获取MN检测中所有关键事件的图像并对其进行评分。最近，已经证明，基于卷积神经网络的人工智能（AI）方法可用于对IFC获取的MN测定数据进行评分。本文介绍了使用 AI 软件创建深度学习模型以对所有关键事件进行评分并应用此模型自动对其他数据进行评分的所有步骤。AI 深度学习模型的结果与手动显微镜相比效果很好，因此可以通过结合 IFC 和 AI 实现 MN 检测的全自动评分。
Introduction
微核 （MN） 测定是遗传毒理学的基础，用于评估人类使用的化妆品、药品和化学品开发中的 DNA 损伤1，2，3，4。微核由整个染色体或染色体片段形成，这些染色体或染色体片段在分裂后不会合并到细胞核中，并凝结成与细胞核分离的小圆形体。因此，MN可用作量化遗传毒性检测中DNA损伤的终点1。
定量 MN 的首选方法是通过使用细胞松弛素-B （Cyt-B） 阻断分裂在一次分裂的双核细胞 （BNC） 内。在此版本的测定中，还通过评分单核（MONO）和多核（POLY）细胞来评估细胞毒性。该测定也可以通过在未阻塞的单核细胞中对MN进行评分来进行，该评分更快，更容易评分，并使用暴露前和暴露后的细胞计数来评估细胞毒性以评估增殖5，6。
历来通过手动显微镜进行检测的物理评分，因为这允许目视确认所有关键事件。然而，手动显微镜具有挑战性且主观1。因此，已经开发了自动化技术，包括显微镜载玻片扫描和流式细胞术，每种技术都有自己的优点和局限性。虽然玻片扫描方法允许可视化关键事件，但必须以最佳细胞密度创建载玻片，这可能难以实现。此外，这种技术通常缺乏细胞质可视化，这可能会影响单核细胞和POLY细胞7，8的评分。虽然流式细胞术提供高通量数据采集，但必须裂解细胞，因此不允许使用Cyt-B形式的测定。此外，作为一种非成像技术，传统的流式细胞术不能提供关键事件的视觉验证9，10。
因此，已经研究了成像流式细胞术（IFC）来执行MN测定。ImageStreamX Mk II 将传统流式细胞术的速度和统计稳健性与显微镜的高分辨率成像功能结合在一个系统中11.研究表明，通过使用IFC，可以使用基于特征的12，13或人工智能（AI）技术14，15捕获并自动评分所有关键事件的高分辨率图像。通过使用IFC进行MN测定，与显微镜相比，可以在更短的时间内自动对更多的细胞进行评分。
这项工作偏离了前面描述的图像分析工作流程16 ，并讨论了使用Amnis AI软件（以下简称“AI软件”）开发和训练随机森林（RF）和/或卷积神经网络（CNN）模型所需的所有步骤。描述了所有必要的步骤，包括使用人工智能辅助标记工具填充真实数据，解释模型训练结果，以及应用模型对其他数据进行分类，允许计算遗传毒性和细胞毒性15。
Protocol
1. 使用成像流式细胞术采集数据 注意：请参考Rodrigues等人16 进行以下修改，注意使用IFC的采集区域可能需要修改以获得最佳图像捕获： 对于非Cyt-B方法，按照制造商的说明（参见 材料表）在培养前和恢复期后立即对每种培养物进行细胞计数。 如果在单相机成像流式细胞仪上运行样品，请将明场（BF）置?…
Representative Results
图 1 显示了使用 AI 软件创建 MN 检测模型的工作流程。用户将所需的 .daf 文件加载到 AI 软件中，然后使用 AI 辅助集群（图 2）和预测（图 3）标记算法将对象分配给真实模型类。一旦所有真实模型类都填充了足够的对象，就可以使用 RF 或 CNN 算法训练模型。训练后，可以使用包括类分布直方图、准确性统计和交互式混淆矩阵在内?…
Discussion
这里介绍的工作描述了使用深度学习算法来自动对 MN 测定进行评分。最近的几篇出版物表明，直观的交互式工具允许创建深度学习模型来分析图像数据，而无需深入的计算知识18，19。这项工作中描述的协议使用用户界面驱动的软件包，旨在很好地处理非常大的数据文件，并允许轻松创建深度学习模型。讨论了在AI软件包中创建和训练RF和CNN模型的所有?…
Divulgazioni
The authors have nothing to disclose.
Acknowledgements
没有。
Materials
| 15 mL centrifuge tube | Falcon | 352096 | |
| Cleanser – Coulter Clenz | Beckman Coulter | 8546931 | Fill container with 200 mL of Cleanser. https://www.beckmancoulter.com/wsrportal/page/itemDetails?itemNumber=8546931#2/10//0/25/ 1/0/asc/2/8546931///0/1//0/ |
| Colchicine | MilliporeSigma | 64-86-8 | |
| Corning bottle-top vacuum filter | MilliporeSigma | CLS430769 | 0.22 µm filter, 500 mL bottle |
| Cytochalasin B | MilliporeSigma | 14930-96-2 | 5 mg bottle |
| Debubbler – 70% Isopropanol | MilliporeSigma | 1.3704 | Fill container with 200 mL of Debubbler. http://www.emdmillipore.com/US/en/product/2-Propanol-70%25-%28V%2FV%29-0.1-%C2%B5m-filtred,MDA_CHEM-137040?ReferrerURL=https%3A%2F%2Fwww.google.com%2F |
| Dimethyl Sulfoxide (DMSO) | MilliporeSigma | 67-68-5 | |
| Dulbecco's Phosphate Buffered Saline 1X | EMD Millipore | BSS-1006-B | PBS Ca++MG++ Free |
| Fetal Bovine Serum | HyClone | SH30071.03 | |
| Formaldehyde, 10%, methanol free, Ultra Pure | Polysciences, Inc. | 04018 | This is what is used for the 4% and 1% Formalin. CAUTION: Formalin/Formaldehyde toxic by inhalation and if swallowed. Irritating to the eyes, respiratory systems and skin. May cause sensitization by inhalation or skin contact. Risk of serious damage to eyes. Potential cancer hazard. http://www.polysciences.com/default/catalog-products/life-sciences/histology-microscopy/fixatives/formaldehydes/formaldehyde-10-methanol-free-pure/ |
| Guava Muse Cell Analyzer | Luminex | 0500-3115 | A standard configuration Guava Muse Cell Analyzer was used. |
| Hoechst 33342 | Thermo Fisher | H3570 | 10 mg/mL solution |
| Mannitol | MilliporeSigma | 69-65-8 | |
| MEM Non-Essential Amino Acids 100X | HyClone | SH30238.01 | |
| MIFC – ImageStreamX Mark II | Luminex, a DiaSorin company | 100220 | A 2 camera ImageStreamX Mark II eqiped with the 405 nm, 488 nm, and 642 nm lasers was used. |
| MIFC analysis software – IDEAS | Luminex, a DiaSorin company | 100220 | "Image analysis sofware" The companion software to the MIFC (ImageStreamX MKII) |
| MIFC software – INSPIRE | Luminex, a DiaSorin company | 100220 | "Image acquisition software" This is the software that runs the MIFC (ImageStreamX MKII) |
| Amnis AI software | Luminex, a DiaSorin company | 100221 | "AI software" This is the software that permits the creation of artificial intelligence models to analyze data |
| Mitomycin C | MilliporeSigma | 50-07-7 | |
| NEAA Mixture 100x | Lonza BioWhittaker | 13-114E | |
| Penicllin/Streptomycin/Glutamine solution 100X | Gibco | 15070063 | |
| Potassium Chloride (KCl) | MilliporeSigma | P9541 | |
| Rinse – Ultrapure water or deionized water | NA | NA | Use any ultrapure water or deionized water. Fill container with 900 mL of Rinse. |
| RNase | MilliporeSigma | 9001-99-4 | |
| RPMI-1640 Medium 1x | HyClone | SH30027.01 | |
| Sheath – PBS | MilliporeSigma | BSS-1006-B | This is the same as Dulbecco's Phosphate Buffered Saline 1x Ca++MG++ free. Fill container with 900 mL of Sheath. |
| Sterile water | HyClone | SH30529.01 | |
| Sterilizer – 0.4%–0.7% Hypochlorite | VWR | JT9416-1 | This is assentually 10% Clorox bleach that can be made by deluting Clorox bleach with water. Fill container with 200 mL of Sterilzer. |
| T25 flask | Falcon | 353109 | |
| T75 flask | Falcon | 353136 | |
| TK6 cells | MilliporeSigma | 95111735 |
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Citazione di questo articolo
Rodrigues, M. A., Gracia García Mendoza, M., Kong, R., Sutton, A., Pugsley, H. R., Li, Y., Hall, B. E., Fogg, D., Ohl, L., Venkatachalam, V. Automation of the Micronucleus Assay Using Imaging Flow Cytometry and Artificial Intelligence. J. Vis. Exp. (191), e64549, doi:10.3791/64549 (2023).