Verma Jatin R, Harte Danielle S G, Shah Ume-Kulsoom, Summers Huw, Thornton Catherine A, Doak Shareen H, Jenkins Gareth J S, Rees Paul, Wills John W, Johnson George E
Institute of Life Science, Swansea University Medical School, Swansea University, Swansea.
College of Engineering, Swansea University, Swansea.
Mutagenesis. 2018 Oct 11;33(4):283-289. doi: 10.1093/mutage/gey021.
Use of imaging flow cytometry to assess induced DNA damage via the cytokinesis block micronucleus (CBMN) assay has thus far been limited to radiation dosimetry in human lymphocytes using high end, 'ImageStream X' series imaging cytometers. Its potential to enumerate chemically induced DNA damage using in vitro cell lines remains unexplored. In the present manuscript, we investigate the more affordable FlowSight® imaging cytometry platform to assess in vitro micronucleus (MN) induction in the human lymphoblastoid TK6 and metabolically competent MCL-5 cells treated with Methyl Methane Sulfonate (MMS) (0-5 µg/ml), Carbendazim (0-1.6 µg/ml), and Benzo[a]Pyrene (B[a]P) (0-6.3 µg/ml) for a period of 1.5-2 cell-cycles. Cells were fixed, and nuclei and MN were stained using the fluorescent nuclear dye DRAQ5™. Image acquisition was carried out using a 20X objective on a FlowSight® imaging cytometer (Amnis, part of Merck Millipore) equipped with a 488 nm laser. Populations of ∼20000 brightfield cell images, alongside DRAQ5™ stained nuclei/MN were rapidly collected (≤10 min). Single, in-focus cells suitable for scoring were then isolated using the IDEAS® software. An overlay of the brightfield cell outlines and the DRAQ5 nuclear fluorescence was used to facilitate scoring of mono-, bi-, tri-, and tetra-nucleated cells with or without MN events and in context of the cytoplasmic boundary of the parent cell.To establish the potential of the FlowSight® platform, and to establish 'ground truth' cell classification for the supervised machine learning based scoring algorithm that represents the next stage of our project, the captured images were scored manually. Alongside, MN frequencies were also derived using the 'gold standard' light microscopy and manual scoring. A minimum of 3000 bi-nucleated cells were assessed using both approaches. Using the benchmark dose approach, the comparability of genotoxic potency estimations for the different compounds and cell lines was assessed across the two scoring platforms as highly similar. This study therefore provides essential proof-of-concept that FlowSight® imaging cytometry is capable of reproducing the results of 'gold standard' manual scoring by light microscopy. We conclude that, with the right automated scoring algorithm, imaging flow cytometry could revolutionise the reportability and scoring throughput of the CBMN assay.
迄今为止，利用成像流式细胞术通过胞质分裂阻断微核（CBMN）试验评估诱导的DNA损伤，仅限于使用高端的“ImageStream X”系列成像细胞仪对人类淋巴细胞进行辐射剂量测定。其利用体外细胞系计数化学诱导的DNA损伤的潜力仍未得到探索。在本论文中，我们研究了更经济实惠的FlowSight®成像流式细胞术平台，以评估用甲磺酸甲酯（MMS）（0 - 5 µg/ml）、多菌灵（0 - 1.6 µg/ml）和苯并[a]芘（B[a]P）（0 - 6.3 µg/ml）处理1.5 - 2个细胞周期的人类淋巴母细胞TK6和具有代谢活性的MCL - 5细胞中的体外微核（MN）诱导情况。细胞被固定，细胞核和微核用荧光核染料DRAQ5™染色。使用配备488 nm激光的FlowSight®成像流式细胞仪（默克密理博旗下的Amnis公司）上的20倍物镜进行图像采集。快速收集约20000个明场细胞图像以及DRAQ5™染色的细胞核/微核（≤10分钟）。然后使用IDEAS®软件分离出适合评分的单个、聚焦清晰的细胞。明场细胞轮廓与DRAQ5核荧光的叠加用于辅助对有或无微核事件的单核、双核、三核和四核细胞进行评分，并结合母细胞的细胞质边界进行评分。为了确定FlowSight®平台的潜力，并为代表我们项目下一阶段的基于监督机器学习的评分算法建立“真实”细胞分类，对捕获的图像进行手动评分。同时，微核频率也使用“金标准”光学显微镜和手动评分得出。两种方法均评估了至少3000个双核细胞。使用基准剂量法，评估了在两个评分平台上不同化合物和细胞系的遗传毒性效力估计的可比性，结果高度相似。因此，本研究提供了重要的概念验证，即FlowSight®成像流式细胞术能够重现光学显微镜“金标准”手动评分的结果。我们得出结论，借助合适的自动评分算法，成像流式细胞术可能会彻底改变CBMN试验的报告性和评分通量。
