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

心脏辅助装置（例如血泵或人工心脏瓣膜）经常用于治疗心血管疾病，作为移植的替代方案。尽管上述系统不断改进，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，因此给患者带来高风险，同时也导致高治疗和随访成本。溶血描述了红细胞（RBC）膜的破坏，其导致血红蛋白的损失。血红蛋白负责氧气的运输，因此对整个身体的氧气供应至关重要。目前，无论是计算机模拟还是体外技术都不允许评价心脏辅助器械中的溶血起源。因此，在器械开发后期或在患者治疗期间的最差情况下检测到主要溶血热点。荧光溶血检测（Fluorescent Hemolysis Detection，FHD）是一种基于血影细胞（Ghostcells，GC）的溶血检测方法，该血影细胞是一种由于细胞的可控溶解而缺乏血红蛋白的红细胞。在裂解过程中，GC装载有标记物，并加入到含有对标记物敏感的指示剂的人工血浆中。在溶血的情况下，GC内部的标记物被释放到人工血浆中，在那里它与荧光指示剂反应，突出溶血的起源。在DFG资助的先前项目中，FHD方法的主要方面得到了解决：负载的GC的大体积生产，GC对RBC流变学的流变学适应，与RBC相比负载的GC的变形能力，和GC相对于标记物的膜不透性。结果表明，加载GCs的流变性和抗渗透性与RBC相似，在后续的应用中，FHD方法将被验证为一种空间和时间分辨的方法，用于评估血液传导装置中的溶血。迄今为止，FHD方法仅检测到化学溶血。在本项目结束时，结合PIV测量和空间分辨溶血检测，将在离心血泵中检测到机械溶血。因此，将评估PIV颗粒的影响以及时间和地点的分辨率限制。此外，将GC溶血阈值与RBC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，因为结果不仅会改进体外测试方法，还会影响CFD模拟。仿真模型可以将新的见解纳入医疗器械溶血，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装置，从而改善患者护理和安全性。

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.