Simple linear regression analysis for Fig 3B.

In the event of a widespread radiological incident, thousands of people may be exposed to a wide range of ionizing radiation. In this unfortunate scenario, there will be a need to quickly screen a large number of people to assess the amount of radiation exposure and triage for medical treatment. In our earlier work, we previously identified and validated a panel of radiosensitive protein biomarkers in blood leukocytes, using the humanized-mouse and non-human primate (NHP) models. The objective of this work was to develop a high-throughput imaging flow-cytometry (IFC) based assay for the rapid measurement of protein biomarker expression in human peripheral blood samples irradiated ex vivo. In this assay design, peripheral human blood samples from healthy adult donors were exposed to 0–5 Gy X-irradiation ex vivo and cultured for up to 2 days. Samples were stained with a cocktail of surface antigens (CD66b, CD20, and CD3), fixed and permeabilized, and intracellularly stained for BAX (Bcl-2-associated X) protein, used here as a representative biomarker. Samples were interrogated by IFC, and a uniform analysis template was created to measure biomarker expression in heterogeneous and specific leukocyte subtype populations at each time point. In this human blood ex vivo model, we show that within gated populations of leukocyte subtypes, B-cells are highly radiosensitive with the smallest surviving fraction, followed by T-cells and granulocytes. Dose-dependent biomarker responses were measured in the lymphocytes, B-, and T-cell populations, but not in the granulocytes, with dose-response curves showing increasing fold changes in BAX protein expression up to Day 2 in lymphocyte populations. We present here the successful use of this ex vivo model for the development of radiation dose-response curves of a candidate protein biomarker towards future applications of dose reconstruction and biodosimetry.当发生大范围放射性事件时，数千人可能会暴露于多种电离辐射之中。在此类不幸场景下，亟需快速筛查大量人群，以评估其辐射暴露剂量并开展医疗救治分流。在本团队此前的研究中，我们已借助人源化小鼠与非人灵长类（non-human primate, NHP）模型，鉴定并验证了血液白细胞中的一组辐射敏感性蛋白生物标志物。本研究旨在开发一种基于高通量成像流式细胞术（imaging flow-cytometry, IFC）的检测方法，以快速定量体外（ex vivo）辐照的人外周血样本中的蛋白生物标志物表达水平。在该检测方法的设计中，我们将健康成年志愿者的外周血样本进行体外0~5戈瑞（Gy）X射线辐照，并培养长达2天。样本经表面抗原组合（CD66b、CD20与CD3）染色后，进行固定与透化处理，随后针对作为代表性生物标志物的BAX（Bcl-2相关X蛋白，Bcl-2-associated X）进行胞内染色。通过成像流式细胞术对样本进行检测，并构建统一的分析模板，以在每个时间点定量分析异质性及特定白细胞亚群中的生物标志物表达水平。在该人血体外模型中，我们发现，在门控的白细胞亚群中，B细胞的辐射敏感性最强，存活分数最低，其次为T细胞与粒细胞。在淋巴细胞、B细胞与T细胞群中可检测到生物标志物表达的剂量依赖性响应，而粒细胞中未出现该现象；剂量响应曲线显示，淋巴细胞中BAX蛋白的表达倍数变化在第2天仍呈上升趋势。本研究证实，该体外模型可成功用于构建候选蛋白生物标志物的辐射剂量响应曲线，为未来辐射剂量重建与生物剂量学应用奠定基础。