Supplementary Material for: Application of the cytokinesis-block micronucleus assay for high dose exposures using imaging flow cytometry

The cytokinesis-block micronucleus (CBMN) assay is a well-established method to assess radiation-induced genetic damage in human cells. This assay has been adapted to imaging flow cytometry (IFC), allowing automated analysis of many cells, and eliminating the need to create microscope slides. Furthermore, to improve the efficiency of assay performance, a small-volume method previously developed was employed. Irradiated human blood samples were cultured, stained, and analysed by IFC to produce images of the cells. Samples were run using both manual and 96-well plate automated acquisition. Multiple parameter-based image features were collected for each sample and the results were compared to confirm that these acquisition methods are functionally identical. This paper details the multi-parametric analysis developed, and the resulting calibration curves up to 10 Gy. The calibration curves were created using a quadratic random coefficient model with Poisson errors, as well as a logistic discriminant function. The curves were then validated with blinded, irradiated samples, using relative bias and relative mean square error. Overall, the accuracy of the dose estimates was adequate for triage dosimetry (within 1 Gy of the true dose) over 90% of the time for lower doses and about half the time for higher doses, with the lowest success rate between 5 and 6 Gy where the calibration curve reached its peak and there was the smallest change in MN/BNC with dose. This work describes the application of novel multi-parametric analysis that fits the calibration curves and allows dose estimates up to 10 Gy, which were previously limited to 4 Gy. Furthermore, it demonstrates that the results from samples acquired manually and with the autosampler are functionally similar.

胞质分裂阻滞微核（cytokinesis-block micronucleus, CBMN）试验是评估人类细胞中辐射诱导遗传损伤的成熟可靠方法。该试验已适配成像流式细胞术（imaging flow cytometry, IFC），可实现大量细胞的自动化分析，且无需制备显微镜玻片。此外，为提升检测效率，本研究采用了此前开发的小体积检测方案。对受辐射的人类血液样本进行培养、染色，并通过成像流式细胞术分析以获取细胞图像。样本分别采用手动采集与96孔板自动化采集两种方式上机检测。为每份样本提取多参数图像特征，并对两种采集方式的结果进行对比，以确认二者功能等效。本研究详述了所开发的多参数分析方法，以及最高剂量达10 Gy的校准曲线。校准曲线分别基于带泊松误差的二次随机系数模型与逻辑判别函数构建。随后采用相对偏倚与相对均方误差，通过盲法受辐射样本对校准曲线进行验证。总体而言，该剂量估算精度可满足应急辐射剂量评估需求：低剂量范围内，90%以上的估算值与真实剂量偏差在1 Gy以内；高剂量范围内该比例约为50%；而在5~6 Gy区间，校准曲线达到峰值且MN/BNC随剂量的变化幅度最小，此时成功率最低。本研究阐述了新型多参数分析方法的应用，该方法可拟合校准曲线并实现最高10 Gy的剂量估算——此前此类方法的剂量上限仅为4 Gy。此外，本研究证实手动采集与自动进样器采集的样本检测结果功能相似。