Table_1_A Novel Fragmentation Sensitivity Index Determines the Susceptibility of Red Blood Cells to Mechanical Trauma.DOCX

Supraphysiological shear stresses (SSs) induce irreversible impairments of red blood cell (RBC) deformability, overstretching of RBC membrane, or fragmentation of RBCs that causes free hemoglobin to be released into plasma, which may lead to anemia. The magnitude and exposure tisme of the SSs are two critical parameters that determine the hemolytic threshold of a healthy RBC. However, impairments in the membrane stability of damaged cells reduce the hemolytic threshold and increase the susceptibility of the cell membrane to supraphysiological SSs, leading to cell fragmentation. The severity of the RBC fragmentation as a response to the mechanical damage and the critical SS levels causing fragmentation are not previously defined. In this study, we investigated the RBC mechanical damage in oxidative stress (OS) and metabolic depletion (MD) models by applying supraphysiological SSs up to 100 Pa by an ektacytometer (LORRCA MaxSis) and then assessed RBC deformability. Next, we examined hemolysis and measured RBC volume and count by Multisizer 3 Coulter Counter to evaluate RBC fragmentation. RBC deformability was significantly impaired in the range of 20–50 Pa in OS compared with healthy controls (p < 0.05). Hemolysis was detected at 90–100 Pa SS levels in MD and all applied SS levels in OS. Supraphysiological SSs increased RBC volume in both the damage models and the control group. The number of fragmented cells increased at 100 Pa SS in the control and MD and at all SS levels in OS, which was accompanied by hemolysis. Fragmentation sensitivity index increased at 50–100 Pa SS in the control, 100 Pa SS in MD, and at all SS levels in OS. Therefore, we propose RBC fragmentation as a novel sensitivity index for damaged RBCs experiencing a mechanical trauma before they undergo fragmentation. Our approach for the assessment of mechanical risk sensitivity by RBC fragmentation could facilitate the close monitoring of shear-mediated RBC response and provide an effective and accurate method for detecting RBC damage in mechanical circulatory assist devices used in routine clinical procedures.

超生理剪切应力（Supraphysiological shear stresses, SSs）会引发红细胞（red blood cell, RBC）变形能力不可逆受损、红细胞膜过度拉伸或红细胞碎裂，进而导致游离血红蛋白释放至血浆中，最终可能引发贫血。超生理剪切应力的强度与暴露时长，是决定健康红细胞溶血阈值的两个关键参数。然而，受损红细胞的膜稳定性下降会降低溶血阈值，使细胞膜更易受超生理剪切应力影响，进而引发细胞碎裂。此前尚未明确红细胞针对机械损伤发生碎裂的严重程度，以及引发碎裂的临界剪切应力水平。本研究通过激光衍射红细胞变形仪（ektacytometer, LORRCA MaxSis）施加最高达100 Pa的超生理剪切应力，在氧化应激（oxidative stress, OS）与代谢耗竭（metabolic depletion, MD）模型中探究红细胞的机械损伤情况，并评估红细胞变形能力。随后，研究人员借助库尔特多通道3型细胞计数仪（Multisizer 3 Coulter Counter）检测溶血情况、测量红细胞体积与计数，以评估红细胞碎裂程度。与健康对照组相比，氧化应激组红细胞在20~50 Pa范围内的变形能力显著受损（p < 0.05）。代谢耗竭组在90~100 Pa剪切应力水平下可检测到溶血，氧化应激组在所有施加的剪切应力水平下均出现溶血。超生理剪切应力会使两种损伤模型与对照组的红细胞体积均出现升高。对照组与代谢耗竭组在100 Pa剪切应力下的碎裂红细胞数量增加，氧化应激组在所有施加的剪切应力水平下碎裂红细胞数量均上升，且伴随溶血现象。对照组在50~100 Pa、代谢耗竭组在100 Pa、氧化应激组在所有施加的剪切应力水平下，碎裂敏感性指数均出现升高。据此，本研究提出将红细胞碎裂作为受损红细胞发生机械损伤并最终碎裂前的新型敏感性指数。本研究通过红细胞碎裂评估机械风险敏感性的方法，可助力精准监测剪切应力介导的红细胞应答反应，同时为常规临床操作中使用的机械循环辅助装置内的红细胞损伤检测，提供一种高效且准确的手段。