Fluorescent Hemolysis Detection (FHD): Vaidation of the in-vitro test method

心脏辅助装置(如血泵或人工心脏瓣膜)经常用于心血管疾病的治疗，作为移植的替代方案。尽管上述系统得到了改善，但溶血(红细胞破坏)、血栓形成或出血事件等严重并发症仍然频繁发生，因此对患者来说风险很高，同时导致高昂的治疗和后续费用。溶血是指红细胞膜的破坏，导致血红蛋白的损失。血红蛋白负责氧气的运输，因此对全身的氧气供应至关重要。目前，无论是硅胶技术还是体外技术都不能评估心脏辅助装置的溶血来源。因此，主要的溶血热点是在设备开发的后期或在最糟糕的情况下在患者治疗期间发现的。针对空间分辨溶血检测方法的不足，提出了一种基于幽灵细胞(GC)的荧光溶血检测方法，GCs是由于细胞裂解受到控制而缺乏血红蛋白的红细胞。在裂解过程中，GC被加载一个标记，并加入到含有对该标记敏感的指示剂的人造血浆中。在之前的DFG资助项目中，FHD方法的主要方面包括：大量生产负载GC，GC的流变学适应红细胞的流变性，负载GC相对于RBCs的变形能力，以及GCs相对于标志物的膜不通透性。在后续的应用中，FHD方法将被验证为一种用于评价血液传导装置中溶血的时空分辨方法。到目前为止，FHD法只检测到化学溶血。在该项目结束时，将结合PIV测量和空间分辨溶血检测，在离心式血泵中检测到机械性溶血。因此，将评估PIV粒子的影响以及关于时间和地点的分辨率限制。此外，GC溶血阈值将与红细胞进行比较，以建立定量溶血分析。这项工作的结果将允许在溶血方面对医疗器械进行先验评估，因为结果不仅将改进体外测试方法，还将影响CFD模拟。模拟模型可以将新的见解融入到医疗器械的溶血中，并将显着提高其溶血预测。从长远来看，这种方法的结果将改善血液传导设备，从而提高患者的护理和安全性。

Cardiac assist devices (e.g. blood pumps or artificial heart valves) are frequently used for the treatment of cardiovascular diseases as an alternative for transplantations. Despite the improvement of the aforementioned systems, severe complications like hemolysis (red blood cell destruction), thrombosis or bleeding events still occur frequently and therefore bear high risks for patients and cause high therapy and follow-up costs at the same time.Hemolysis describes red blood cells’ (RBCs) membrane destruction, which leads to the loss of hemoglobin. Hemoglobin is responsible for the oxygen transport and is thus crucial for the oxygen supply of the whole body. Currently, neither in-silico nor in-vitro techniques allow for evaluating the hemolysis origin in cardiac assist devices. Hence, major hemolysis hotspots are detected late during device development or in the worst case during patient treatment. A new approach for overcoming the deficit of spatially resolved hemolysis detection was offered by the Fluorescent Hemolysis Detection (FHD) method.The FHD method is based on ghostcells (GCs), which are RBCs lacking hemoglobin due to controlled lyses of the cells. During lyses, GCs are loaded with a marker and added into an artificial plasma, containing an indicator sensitive to the marker. In case of hemolysis, the marker from the GCs’ interior is released into the artificial plasma where it reacts with the fluorescence indicator, highlighting the origin of hemolysis.During the previous DFG-funded project, main aspects of the FHD method were addressed: large volume production of loaded GCs, rheology adaption of GCs to RBCs’ rheology, deformation ability of loaded GCs compared with RBCs, and membrane impermeability of GCs with respect to the marker. It was shown that loaded GCs properties are similar to RBCs in terms of rheology and impermeability.Within the follow-up application, the FHD method will be validated as a spatially and temporally resolved method for the evaluation of hemolysis in blood conducting devices. Up to now, only chemical hemolysis was detected by the FHD method. At the end of this project, mechanical hemolysis will be detectable in a centrifugal blood pump, combined with PIV measurements and spatially resolved hemolysis detection. Therefore, the impact of PIV particles as well as the resolution limits with regard to time and place will be evaluated. Additionally, the GC hemolysis threshold will be compared to RBCs to establish a quantitative hemolysis analysis. The results of this work will allow for a-priori evaluation of medical devices in terms of hemolysis, as results will not only improve in-vitro test methods but also influence CFD simulations. Simulation models can incorporate new insights into medical devices hemolysis and will improve their hemolysis prediction significantly. In the long run, results of this method will improve blood conducting devices and therewith patient care and safety.