A quantitative viability metric for liver transplantation using Resonance Raman Spectroscopy

项目摘要 器官移植是治疗终末期器官疾病的一种挽救生命的治疗方法;然而， 只有约36%的等待名单上的患者接受移植。与此同时，许多重要 可移植的器官被丢弃-一些估计表明，多达25%的回收器官被丢弃。 最终没有移植，还有一些未恢复的器官， 损坏然而，由于其不确定的生存能力和不成功移植的高成本， 这些潜在的器官被使用，而仅仅一小部分的移植就可能大大减少器官的数量。 短缺 一个特别大的未开发的供体器官的目标是轻微损伤的热缺血器官， 循环性死亡后捐献（DCD）。虽然这些器官与较低的生存率相关， 移植后并发症增加，机器灌注技术有望修复 其中一些器官，从而大大增加了可用性。然而，一个关键的瓶颈， 器官修复的实验和临床进展正在开发非客观的指标， 在移植前确定一个器官是否可行。临床上，由于器官分配已经 由于受到时间的严格限制，需要为每个器官迅速地并且最好是实时地确定这些标准。 从实验上看，使用灵活的平台将增加研究人员的规模和数量， 这些难题。为了满足这一需求，我们建议开发一种新型的成像平台， 共振拉曼光谱（RRS），用于量化组织表面/活检组织上的线粒体氧化还原状态， 线粒体的分解产物。我们的方法在器官移植评估中是独一无二的 因为：1）在不到3分钟内获得结果，并且是非破坏性的，2）提供所需的灵活性 为不同的器官系统，大大增加了拟议的工作的影响。 虽然我们将针对不同的可移植器官，但我们将通过执行两个 明确的目标。我们将开发一种台式RRS设备，该设备能够集成多种类型的 对肝脏的测量，包括活组织检查、表面测量和灌注液样本。在具体目标1中， 将应用并验证活检单元和可扩展臂，使肝脏活检和表面测量成为可能 计算还原的线粒体与总线粒体的比率（定义为共振拉曼还原的线粒体 比率，3RMR）。在特定目标2中，我们将应用并验证与相同的灌注液池兼容的灌注液池。 台式RRS装置，以实时定量来自灌注液的线粒体分解产物（定义为 灌注液活力指数PVI）。在SA 1 -2中，我们将定义3RMR/PVI阈值，如果超过该阈值， 将表明不可逆的肝损伤，在测量来自啮齿动物的样本之前， 在机器灌注期间丢弃的人DCD肝脏。

PROJECT SUMMARY Organ transplantation is a life-saving treatment for end-stage organ disease; however, there is a severe shortage of donor organs whereby only ~36% of wait-listed patients receive a transplant. At the same time, many vital organs that may be transplantable are discarded - some estimates suggest that up to 25% of recovered organs are not ultimately transplanted, and there are additional pools of unrecovered organs that are only marginally damaged. However, because of their uncertain viability and the high cost of an unsuccessful transplant, none of these potential organs are used while transplantation of just a fraction could dramatically reduce the organ shortage. A particularly large target of untapped donor organs are marginally injured warm ischemic organs from Donation after Circulatory Death (DCD). While these organs have been correlated to lower survival rates and increased post-transplant complications, machine perfusion technology holds the promise of reconditioning some of these organs, thereby dramatically increasing availability. However, a critical bottleneck that impacts experimental and clinical advances in reconditioning organs are developing non-objective metrics that can definitively ascertain if an organ is viable prior to transplantation. Clinically, since organ allocation is already severely restricted by time, these criteria need to be defined for each organ rapidly and preferably in real-time. Experimentally, access to flexible platforms would increase the scale and number of researchers that can tackle these difficult problems. In response to this need, we propose to develop a novel imaging platform that will use Resonance Raman Spectroscopy (RRS) to quantify mitochondrial redox state on tissue surfaces/biopsies and mitochondrial breakdown products in the perfusate. Our approach is unique in organ transplantation assessment since: 1) results are obtained in less than 3 minutes and are non-destructive, and 2) offers the flexibility required for diverse organ systems, significantly increasing the impact of the proposed work. While we will target diverse transplantable organs, we focus our initial efforts on liver through execution of two specific aims. We will develop a benchtop RRS device with the capacity to integrate several types of measurements on livers, including biopsies, surface measurements, and perfusate samples. In Specific Aim 1, we will apply and validate the biopsy cell and extendable arm that enables liver biopsies and surface measurements to calculate the ratio of reduced to total mitochondria (defined as Resonance Raman Reduced Mitochondrial Ratio, 3RMR). In Specific Aim 2, we will apply and validate the perfusate cell that is compatible with the same benchtop RRS device to quantify mitochondrial breakdown products in real-time from the perfusate (defined as the Perfusate Viability Index, PVI). In SA1-2, we will define the 3RMR/PVI threshold values that if exceeded will indicate irreversible liver injury, beginning with rigorous studies in rodents before measuring samples from discarded human DCD livers during machine perfusion.