Center for Radiological Research, Columbia University Irving Medical Center, New York, New York, United States of America.
PLoS One. 2023 Aug 10;18(8):e0289634. doi: 10.1371/journal.pone.0289634. eCollection 2023.
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.
在广泛的放射性事件中，可能有成千上万的人暴露在广泛的电离辐射下。在这种不幸的情况下，需要快速筛选大量人群，以评估辐射暴露量并进行医疗分诊。在我们之前的工作中，我们使用人源化小鼠和非人类灵长类动物（NHP）模型，先前已经鉴定和验证了一组血液白细胞中的辐射敏感蛋白生物标志物。这项工作的目的是开发一种高通量的基于成像流式细胞术（IFC）的测定法，用于快速测量人外周血样本中蛋白质生物标志物的表达，这些样本是体外辐照的。在这个测定设计中，来自健康成年供体的外周人血样本在体外暴露于 0-5 Gy X 射线照射，并培养长达 2 天。用表面抗原（CD66b、CD20 和 CD3）混合物对样本进行染色，固定和透化，并对内源性 BAX（Bcl-2 相关 X）蛋白进行染色，这里将其用作代表性生物标志物。通过 IFC 对样本进行检测，并创建一个统一的分析模板，以在每个时间点测量异质和特定白细胞亚型群体中的生物标志物表达。在这种人血体外模型中，我们表明，在白细胞亚型的门控群体中，B 细胞对辐射高度敏感，存活分数最小，其次是 T 细胞和粒细胞。在淋巴细胞、B 细胞和 T 细胞群体中测量了剂量依赖性生物标志物反应，但在粒细胞中没有，淋巴细胞群体中的剂量反应曲线显示 BAX 蛋白表达的倍数增加，直到第 2 天。我们在这里展示了这种体外模型在开发候选蛋白生物标志物的剂量反应曲线方面的成功应用，用于未来的剂量重建和生物剂量学应用。
