Development of FAST-DOSE assay system for the rapid assessment of acute radiation exposure, individual radiosensitivity and injury in victims for a large-scale radiological incident

总结 在大规模放射性或核事件发生后，数十万人可能会受到 电离辐射，并需要随后的剂量依赖性医疗管理。关键是要收集 并在第一周内尽快分析人类生物流体（如血液，尿液，唾液）， 准确的剂量预测和早期分诊决策。需要FDA批准的体外诊断 高通量生物剂量测定装置，能够精确测定过去的辐射暴露 和准确性。在哥伦比亚大学中心的高辐射生物剂量学中心， 针对辐射医学对策（CMCR），我们开发了快速剂量（荧光 用于剂量测定的自动筛选工具）测定系统，以测量 人外周血样本用于辐射剂量的回顾性估计。蛋白质组还 包括反映血液学敏感性和损伤的血液白细胞亚型的生物标志物。快速- 剂量测定系统预期用作21 CFR 809.3定义的体外诊断器械（IVD）。的 该平台使用商业成像流式细胞术系统（ImageStream®X）和相关的图像数据库。 探索和分析软件（IDEAS®），以快速定量生物标志物表达水平的变化， 使用荧光成像和算法估计吸收剂量的特定细胞结构。的 这里计划的研究旨在开发和优化我们的快速剂量测定系统， 估算电离辐射后人体淋巴细胞的吸收剂量和评估造血损伤。的 第一个目标是建立在我们目前的生物标志物验证数据的基础上，通过 提交前程序。我们已经使用了人离体模型和人源化小鼠（Hu-NSG）和非人源化小鼠（Hu-NSG）。 人类灵长类动物（NHP）模型，以验证血液白细胞中的生物标志物表达和放射敏感性， 急性电离辐射照射。此处提出的具体目标旨在：优化测定 方案和鉴定生物标志物剂量/时间动力学，用于体外准确的剂量预测和测试1）供体间 变异性，2）供体内变异性和3）实验室间变异性（目标1）;测试特定 混杂因素：年龄和性别、生殖系BRCA 1/2致病性变异的遗传、炎症和创伤 在照射之前和之后（目标2），对生物标志物响应进行测量; 根据外周血白细胞计数和干细胞计数， Hu-NSG小鼠骨髓中的祖细胞水平（目的3），并开发数学模型（使用 机器学习和回归技术），以选择最佳的快速剂量生物标志物及其组合 用于基于这些生物标志物的离体和体内剂量响应生成剂量预测（目的4）。 我们对未来发展的愿景是开发一种更简单、更快速的快速定量分析系统 由此可以开发和转换生物标记物以用于护理点（POC）装置。

Summary Following a large scale radiological or nuclear event, hundreds of thousands of people may be exposed to ionizing radiation/s and require subsequent dose-dependent medical management. It will be crucial to collect and analyze human biofluids (such as blood, urine, saliva) as soon as possible within the first week for accurate dose prediction and early triage decision. There is a need for FDA-approved in vitro diagnostic high-throughput biodosimetry devices with the ability to determine past radiation exposure with precision and accuracy. At the Center for High Throughput Radiation Biodosimetry, the Columbia University Center for Medical Countermeasures against Radiation (CMCR), we have developed FAST-DOSE (Fluorescent Automated Screening Tool for Dosimetry) assay system, to measure radiation-responsive proteins in human peripheral blood samples for retrospective estimation of radiation dose. The protein panel also includes biomarkers for blood leukocyte subtypes to reflect hematological sensitivity and injury. The FAST- DOSE assay system is intended as an in vitro diagnostic device (IVD) as defined by 21 CFR 809.3. The platform uses a commercial imaging flow cytometry system (ImageStream®X) and associated Image Data Exploration and Analysis Software (IDEAS®) to rapidly quantify changes in biomarker expression levels within specific cellular structures using fluorescent imagery and algorithms for estimation of absorbed dose. The studies planned here are designed to develop and optimize our FAST-DOSE assay system to accurately estimate absorbed dose and assess hematopoietic injury in human lymphocytes after ionizing irradiation. The first objective is to build on our current biomarker validation data for early engagement with the FDA via the pre-submission process. We have used the human ex vivo model and humanized mouse (Hu-NSG) and non- human primate (NHP) models to validate biomarker expression and radiosensitivity in blood leukocytes after acute ionizing radiation exposure. The Specific Aims proposed here are designed to: optimize the assay protocol and identify biomarker dose/time kinetics for accurate dose predictions in vitro and test 1) inter-donor variation, 2) intra-donor variation and 3) inter-laboratory variability (Aim 1); test the effect of specific confounders: age and sex, inheritance with germline BRCA1/2 pathogenic variant, and inflammation and trauma on the biomarker response, before and after irradiation (Aim 2); measure biomarker levels and time kinetics in vivo and correlate with hematopoietic injury, based on peripheral blood leukocyte counts, and stem and progenitor cell levels in the bone marrow of Hu-NSG mice (Aim 3) and, develop mathematical models (using machine learning and regression techniques) to select the best FAST-DOSE biomarkers and their combinations for generating dose predictions based on the ex vivo and in vivo dose response of these biomarkers (Aim 4). Our vision for future development is to develop a more simplified, faster rapid FAST-DOSE assay system whereby the biomarkers could be developed and transitioned for use in a point-of-care (POC) device.