A quantitative viability metric for liver transplantation using Resonance Raman Spectroscopy

项目总结 器官移植是治疗终末期器官疾病的一种挽救生命的疗法；然而，目前还存在严重的短缺。 仅有约36%的候补患者接受器官移植。与此同时，许多至关重要的 可能可以移植的器官被丢弃--一些估计表明，高达25%的回收器官是 最终不会被移植，而且还有更多的未被发现的器官 损坏了。然而，由于它们的生存能力不确定，以及移植失败的高昂成本，没有一个 这些潜在的器官被使用，而仅移植一部分可能会极大地减少器官 短缺。 未开发的供体器官的一个特别大的目标是来自 循环性死亡后的捐献(DCD)。虽然这些器官与较低的存活率和 移植后并发症增加，机器灌流技术有望修复 其中的一些器官，从而极大地增加了可获得性。然而，一个关键的瓶颈是影响 修复器官的实验和临床进展正在开发非客观指标，可以 在移植前确定一个器官是否可以存活。在临床上，由于器官分配已经 由于时间的严格限制，需要迅速、最好是实时地为每个器官确定这些标准。 在实验上，使用灵活的平台将增加研究人员的规模和数量 这些难题。针对这一需求，我们建议开发一种新的成像平台，该平台将使用 共振拉曼光谱(RRS)定量检测组织表面/活检组织中线粒体的氧化还原状态 灌流液中的线粒体分解产物。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分钟的时间内获得结果，并且是非破坏性的；2)提供所需的灵活性 对于不同的器官系统，大大增加了拟议工作的影响。 虽然我们将针对不同的可移植器官，但我们最初的努力是通过执行两个 明确的目标。我们将开发一款桌面RRS设备，能够集成几种类型的RRS 肝脏测量，包括活组织检查、表面测量和灌流液样本。在具体目标1中，我们 将应用和验证活组织细胞和可伸展手臂，以实现肝脏活组织检查和表面测量 计算还原线粒体与总线粒体的比率(定义为共振拉曼还原线粒体 比率，3RMR)。在特定目标2中，我们将应用和验证与其兼容的灌流液单元 台式RRS设备，用于实时量化来自灌流液的线粒体分解产物(定义为 灌流液活性指数(PVI)。在SA1-2中，我们将定义超过的3RMR/PVI阈值 将表明不可逆转的肝脏损伤，从对啮齿动物的严格研究开始，然后测量来自 在机器灌流过程中丢弃人的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.