| Transfusion could save life of patients with massive hemorrhage and trauma. Storage RBC, as a major member of band blood, has been widely used in clinic. Usually, RBC could be preserved at 4℃ for 42 days with CPDA. However, a series of changes occur during storage, such as the depletion of ATP, 2,3-DPG and NO, and increased oxygen affinity, all of which are called storage lesions. The clinical data indicate that the old blood transfusion is related to the morbidity and mortality of patients and researchers focus on the mechanism. The previous study has studied the effect and mechanism of old blood. For example, the structure of hemoglobin changes with storage, and the oxygen delivery capacity decreases; The membrane also changes with storage, which makes RBCs hemolysis, and induces iron overloading and inflammatory reaction; The deteriorative deformability makes it easy to block the microcirculation and further aggravates the anoxia. However, the detailed mechanism is unclear and needs further study.The study about mechanical properties of RBCs becomes hot with the progressive idea and gradually-perfect technology in the field of biomechanics. The study in recent years promotes the hypothesis that the mechanical changes of RBCs during storage may be the critical factor of adverse transfusion reaction. It is because that the mechanical changes during storage occur in early and are irreversible. And they are capable of regulating the hemorheology and hemodynamics, and determine the effect of microcirculatory perfusion and oxygen delivery of RBCs. Therefore, study about the mechanical changes of stored RBCs benefits illustrating the mechanism of adverse transfusion reaction, and improving the RBC storage quality and transfusion effect. The following scientific problems must be solved one by one.First, the change rule of RBCs’ mechanical properties during storage must be determined, which need the testing method of general applicability.Researchers have focused on the mechanical changes of stored RBC. However, the results are inconsistent with the test methods. The devices using in mechanical property test have been studied for years. The laser diffraction method is not good at representing the single cell. And the high-viscous buffer is required, which makes the result complex. The micropipette aspiration has the defection of poor operation in practice, high time-consuming and complex analysis method, which makes it not suitable for online detection. Recently, the microfluidic devices are often used to evaluate the mechanical properties of RBC. However, these devices could not simulate the microcirculation realistically and export the different results. Therefore, the test method for stored RBCs’ mechanical properties needs further research.Second, the effects of liquid environment on RBCs’ mechanical properties need to be determined.In clinic, the plasma expanders, such as Dextron, hydroxyethyl starch(HES) and gelatin, are usually transfused earlier than RBCs to expand blood volume. However, they affect the mechanical properties of RBCs, especially the aggregation. The macromolecules, including saccharides and protein, could adsorb on the RBCs surface to induce aggregation. The size and structure of macromolecules determine the aggregation extent. The effects of these plasma expanders on aggregation of stored RBCs in circulation are unclear. It is needed to determine which plasma expander combining with stored RBCs is advisable.Third, the effect of stored RBCs on blood flow has important implication for predicting the adverse transfusion reaction and needs further study.As the major component of blood(~45%), RBCs’ mechanical properties could affect the blood flow. For example, the enhanced aggregation and decrease deformability of RBCs will increase the flow resistance, even block capillary and induce microcirculation disturbance. Then, the changes of stored RBCs’ mechanical properties will regulate microcirculation and affect the transfusion effect. However, it is different to quantitate the relation between mechanical changes and blood flow parameters via previous technology. The computational fluid mechanics could establish the two-dimensional capillary, simulate the motion of stored RBCs in vessel and quantize the relation between mechanical properties and flow. Therefore, research should be carried out to determine the effects of mechanical changes of stored RBC on blood flow via software.Four, the changes of RBCs’ mechanical properties during storage may be result from the deletion of mechanics micro-environment. It is worth exploring if the lesions could recover by reset the mechanics micro-environment.The average lifetime of RBCs in vivo is 120 days, while it shortens to 42 days in CPDA in vitro. The preserving fluid provides enough energy substrate, but the storage lesions are unavoidable. The key reason may be the deletion of mechanics micro-environment. RBCs are under blood circulation and complex mechanical environment, which regulate the functions of RBCs. First, some cytokines, CD47 expression, is regulated by shear stress. Second, shear stress can regulate the expression of glucose transporter in membrane to determine the metabolism of RBCs. Therefore, we hypotheses that defect of mechanics micro-environment during storage may be the critical factor of storage lesions. Research should be designed to explore the hypothesis.To sum up, the mechanical changes of stored RBCs are significant for illustrating the effects and mechanism of storage lesions on adverse transfusion reaction. The present study will optimize the test method of RBCs’ mechanical properties, and determine the effects of storage and external fluid environment on RBCs mechanical properties. Via the computational fluid mechanics experiments, we will explore the effect of these changes on blood flow. These experiments will benefit determining the mechanism of adverse transfusion reaction. Moreover, the present study suggests that mechanical micro-environment may be an effective strategy to improve the stored RBCs quality and explore it. The research contents were studied as follows. Chapter I, establishing the RBC storage lesion model of mouseThe appropriate RBCs storage lesion model is needed for the study of mechanical properties. The mouse RBC storage lesion model has been widely used. The mouse modal has the advantage in lower cost, better availability and shorter storage cycle than human RBC. What is more important is that the stored mouse RBCs could be transfused and used to carry out further experiment in vivo, which makes it fit for the present study. Therefore, the study focuses on establishing the RBC storage lesion model of C57 mouse. RBCs were mixed with CPDA-1 and stored at 4℃. Then, the biochemical, morphological and functional changes were detected on the time point of 0d, 7d and 14 d. We also detected the changes of hemoglobin structure using Raman spectroscopy. The results suggest that the RBCs storage lesion model and Raman spectroscopy method are established successfully. The biochemical parameters, the blood oxygenation, the hematology parameters and the Hb function and structure in RBCs of C57 mice change significantly during storage. These lesions in RBCs of C57 mice after storage for 14 d are similar with those in RBCs of human after storage for 42 d. The RBCs storage lesion model in chapter I is suitable for the subsequent biomechanical study. Chapter II, determining the change rule of mechanical property during RBCs storage.To explore the change rule of stored RBCs’ deformability, the present study plans to determine the appropriate mechanical properties test method. Then the classic viscometric assay is used to evaluate RBC aggregation to explore the effect of storage and external liquid environment.First, Optimizing the AFM detection method of stored RBCs. We produced the microwell in the basement. The stored RBCs were fixed physically into the microwell on the basement and then tested via AFM. The RBCs Young’s modulus decreased with storage time. These results are not accordance with the theory. We think it is because RBCs have the specific shape. However, the present method could be used to test other suspension cells.Second, Establishing the microfluidic test method of stored RBCs’ mechanical properties. The RBCs were adhered to the base via PLL and PBS was used to shear RBCs. We analyzed the shape of stored RBCs after shearing and obtained the mechanical parameters. It does not require high-speed camera. The advantages of this method are the small sample needed, high-throughput and easy-use, which makes it promising for the quality monitoring of RBCs. Using the novel microfluidic method to test RBCs and the results showed decreased DI, changed morphology distribution, decreased surface charge and increased Young’s modulus with storage time. These results suggest the method is capable of representing the mechanical properties and can distinguish the mechanical changes of the RBCs during storage.Finally, The change rule of viscosity and AI in stored RBCs with or without plasma expanders. We tested the viscosity and calculated the AI of RBCs after storing for 0d, 7d, and 14 d. Then, the stored RBCs were mixed with plasma expanders, such as HES40、HES130、HES200、GEL. The viscosity and AI were detected, too. The blood viscosity and AI of RBCs increased at first and then decreased with storage time. The plasma expanders didn’t affect the fresh RBCs, while have significant effects on viscosity and AI of stored RBCs, which change with the storage time. Chapter III, the digital simulation for the effect of stored RBCs’ mechanical heterogeneity on blood flowTo determine the effects of mechanical changes during RBCs storage on blood flow, the present study aims to establish the models of RBC and capillary(size, 50 μm × 10 μm,inlet velocity, 0.001 m/s) via computational fluid dynamics software. The Young’s moduli of stored RBCs obtained in chapter II were substituted intocomputational fluid dynamics software to determine the effects of stored RBCs’ mechanical properties on RBCs’ motion and blood flow. Via the simulated calculationof single RBC circulating in capillary, we identify that the changes of RBCsmechanical properties during storage could induce the higher pressure drop, narrowerCFL width and higher wall shear stress, which may be one of the mechanisms forinvalid transfusion and adverse reactions from old blood. Chapter IV, the effects of shear stress on RBCs storage lesionThe present study aims to explore the effects of mechanical micro-environmenton stored RBCs quality. We sheared stored RBCs briefly(1.384 Pa for 30 min) anddetected the morphology, the expression and location of membrane proteins, and thefunction of hemoglobin stored RBCs. RBCs become acanthocyte with the storagetime gradually. However, they become biconcave-disk again after subjecting to shearstress; Band 4.9+5 increases significantly with the storage time, while band 3decreases and aggregates in membrane of RBCs in 14 d group. After shearing, band4.9+5 decrease and band 3 distribution recovers. These data suggest that shear stressmay act as a candidate measure to improve the quality of stored RBCs and be able todetermine transfusion effect and reduce adverse reactions. SummaryThe present studies focus on the mechanical changes of stored RBCs and theirroles in blood flow and RBCs function. We study the specific scientific problems, andthe results are as follows.First, we established the test method of Raman spectrum for stored RBCs. TheRaman spectrum could represent the structure and function of stored RBCs, the spinstate of iron and center bore of porphyrin ring. The Raman spectrum has advantagesin high sensitivity and easy to use, which is hopeful to replace the classic tests and usein clinic.Second, the stored RBCs’ mechanical properties were determined via two testmethods, the AFM test and microfluidic test. The result suggested that AFM test withthe microwell on the basement might not be suitable for RBCs. However, it might beappropriate for the other suspension cell; via the novel microfluidic test method, theDI distribution and shape distribution were obtained. And the downstream curvatureof stored RBCs under physiological shear could represent the surface charge. We alsodetermined the Young’s modulus of stored RBCs via model calculation. Themicrofluidic method has the advantages in easy to use and could distinguish the mechanical properties of stored RBCs via multiply parameters without any high-speed camera. The present microfluidic method is hopeful to use to evaluate the quality of stored RBCs in clinic.Third, the effects of storage and plasma expanders on AI of stored RBCs were determined. The results showed that blood viscosity and AI of stored RBCs changed with the storage time and plasma expander type. The results suggest that the doctors should choose the plasma expanders according to the hemorheology of patients when transfusion in clinic. The study may give some guidance for the emergency treatment.Four, the RBC and capillary model were established via the computational fluid mechanics software to illustrate the effects of the mechanical changes during RBC storage on blood flow. The present study obtains the exact data via the simulation calculation and illustrates the correction between abstract mechanical parameters and blood flow parameters. These results could be used to determine the mechanism for the adverse transfusion reaction resulting from old RBCs transfusion and predict the transfusion effect and adverse reaction.Finally, we further explore the problem if mechanics micro-environment could improve the function of stored RBCs. The results showed transient shear stress could regulate the expression and distribution of proteins in stored RBCs, which suggests that shear stress is a potential strategy to improve the functions of stored RBCs. The results are in contrast to the classic theory, and provide a new strategy for recovering the lesions of stored RBCs.In conclusion, the present study established the appropriate test method for stored RBCs’ mechanical properties, determined the change rule of mechanical properties during storage, illustrated the direct correction between mechanical properties and blood flow, and explored the new strategy for improving the quality of stored RBCs. Our results have innovativeness and practicability, and benefit the improvement of stored RBCs quality and accurate prediction of adverse transfusion reaction. |
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