Optical measurement of biomechanical properties of individual erythrocytes from a sickle cell patient

Sickle cell disease (SCD) is characterized by the abnormal deformation of red blood cells (RBCs) in the deoxygenated condition, as their elongated shape leads to compromised circulation. The pathophysiology of SCD is influenced by both the biomechanical properties of RBCs and their hemodynamic properties in the microvasculature. A major challenge in the study of SCD involves accurate characterization of the biomechanical properties of individual RBCs with minimum sample perturbation. Here we report the biomechanical properties of individual RBCs from a SCD patient using a non-invasive laser interferometric technique. We optically measure the dynamic membrane fluctuations of RBCs. The measurements are analyzed with a previously validated membrane model to retrieve key mechanical properties of the cells: bending modulus; shear modulus; area expansion modulus; and cytoplasmic viscosity. We find that high cytoplasmic viscosity at ambient oxygen concentration is principally responsible for the significantly decreased dynamic membrane fluctuations in RBCs with SCD, and that the mechanical properties of the membrane cortex of irreversibly sickled cells (ISCs) are different from those of the other types of RBCs in SCD.     

Effects of shape and size on red blood cell deformability: a static bending analysis

When flowing down a tapered tube, such as a narrow capillary, red blood cells (RBCs) are subject to deformation, the first event of which is folding in a pancake manner. The RBC deformability is reduced during cell ageing, a phenomenon that may reflect alterations in intracellular viscosity, membrane rigidity or RBC shape. Age related shape changes and their importance for increased RBC rigidity were theoretically analysed. The average empirically observed RBC profile is shown to offer little resistance to bending as compared to other, theoretically possible profiles of the same membrane area and RBC volume. Because of a decrease in projected area (diameter size), and therefore in pressure load, the pressure needed to initiate folding of an old RBC is between 20 and 55% higher than that required to fold a young one if, during RBC ageing, membrane area to cell volume ratio is constant as empirically observed. This difference exists whether the RBC is mathematically treated as a solid body or as a membrane shell.     

Use of Cell Transit Analyser pulse height to study the deformation of erythrocytes in microchannels

Information on microvascular rheology can be used to develop mathematical models of network hemodynamics in various contexts: tumor-induced angiogenesis, reaction to ischemia and collateralization, hypertension, ... The rheological method which probably most closely simulates red blood cell (RBC) flow behavior in the microcirculation is the determination of individual erythrocyte transit time and shape changes during flow through cylindrical micropores. However, these filtration experiments remain difficult to interpret in terms of cell flow properties, because the shape of the cell inside the pore is unknown. This paper uses the cell transit analyser (CTA) electrical pulse (especially the pulse height) to determine the relationship between the cell velocity and deformed shape in the pore, depending on the flow strength, suspending medium viscosity and cell membrane elasticity. As predicted by published numerical simulations for the flow of deformable particles through narrow channels, increasing pressure leads to increased deformation, but for a given pressure, increased viscosity leads to a slight increase in deformation. The sensitivity of filtration experiments to cell mechanical properties is improved when using low driving pressures and narrow and sufficiently long pores.     

Computational Biorheology of Human Blood Flow in Health and Disease

Hematologic disorders arising from infectious diseases, hereditary factors and environmental influences can lead to, and can be influenced by, significant changes in the shape, mechanical and physical properties of red blood cells (RBCs), and the biorheology of blood flow. Hence, modeling of hematologic disorders should take into account the multiphase nature of blood flow, especially in arterioles and capillaries. We present here an overview of a general computational framework based on dissipative particle dynamics (DPD) which has broad applicability in cell biophysics with implications for diagnostics, therapeutics and drug efficacy assessments for a wide variety of human diseases. This computational approach, validated by independent experimental results, is capable of modeling the biorheology of whole blood and its individual components during blood flow so as to investigate cell mechanistic processes in health and disease. DPD is a Lagrangian method that can be derived from systematic coarse-graining of molecular dynamics but can scale efficiently up to arterioles and can also be used to model RBCs down to the spectrin level. We start from experimental measurements of a single RBC to extract the relevant biophysical parameters, using single-cell measurements involving such methods as optical tweezers, atomic force microscopy and micropipette aspiration, and cell-population experiments involving microfluidic devices. We then use these validated RBC models to predict the biorheological behavior of whole blood in healthy or pathological states, and compare the simulations with experimental results involving apparent viscosity and other relevant parameters. While the approach discussed here is sufficiently general to address a broad spectrum of hematologic disorders including certain types of cancer, this paper specifically deals with results obtained using this computational framework for blood flow in malaria and sickle cell anemia.     

Development of standard tests to examine viscoelastic properties of blood of experimental animals for pediatric mechanical support device evaluation

We investigated the applicability of measuring the viscoelasticity of bovine, ovine, and porcine whole blood for the evaluation of sublethal damage to red blood cells (RBCs). An increase in blood viscosity and elasticity without changes in hematocrit and plasma viscosity would signify a decrease in RBC deformability. Blood viscoelasticity was assessed using a Vilastic Scientific viscoelastometer. Due to the natural absence of RBC aggregation and small RBC size in normal bovine and ovine blood, viscoelastic properties are less readily detected. However, we found that adjustment of blood hematocrit to a standard level of 40-50% allows for sensitive assessment of viscoelasticity in these blood types demonstrating a marked non-Newtonian behavior mostly related to RBC deformability. Porcine blood showed a pronounced non-Newtonian behavior at all tested hematocrit values, which makes it rheologically comparable to human blood. Both viscosity and elasticity were elevated after blood exposure to a uniform mechanical stress. RBCs rigidified by heat exposure demonstrated a loss of viscoelasticity dependence on shear rate. Measurements of blood viscoelasticity can be meaningful in bovine, ovine, and, especially, porcine blood, and can be used for evaluation of sublethal blood damage during in vitro and animal trials of heart-assist devices.     

新型显微成像与分析系统在糖尿病患者红细胞定量研究中的应用

目的：基于新理论、研发新技术,用于定量检测高血糖引起的红细胞形态功能的变化,实现糖尿病的早期诊断和对糖尿病病程的监控。方法：利用新型显微成像技术与动静态图象分析软件,研究糖尿病患者红细胞形态和细胞膜的弹性变化。分别检测健康人、糖调节受损组和糖尿病不同病程组糖化血红蛋白、红细胞形态参数（细胞的接触面积、周长、长轴、短轴、圆度因子）和细胞膜的弹性参数（弯曲弹性模量、剪切弹性模量）。结果：糖尿病患者红细胞形态和功能在糖尿病病变过程中发生了变化,其形态参数和细胞膜弹性参数随糖化血红蛋白和病程的增加有不同程度的增加。红细胞形态变大必将影响其功能的改变,细胞膜弹性参数的增加使细胞刚性增加和细胞膜弹性功能减弱。结论：新型显微成像与分析系统可定量检测、分析糖尿病患者活态红细胞的形态和功能,是探索糖尿病发病机制的一种新技术。     

血液透析对尿毒症患者血液流变性的影响

研究单次及长期维持血液透析时尿毒症患者血液流变性改变的影响。结果表明，单次血透后，红细胞比积明显升高，红细胞刚性明显降低；血浆粘度、红细胞聚集指数、Ｃａｓｓｏｎ粘度、血液屈服应力及低切至高切的全血粘度无明显改变。维持血透患者的红细胞比积、血浆粘度、红细胞聚集指数、Ｃａｓｓｏｎ粘度、血液屈服应力均无明显改变。进一步研究发现，单次血透后血浆渗透比明显降低．这可能是引起红细胞比积升高、红细胞刚性降低的原因之一。     

Effect of therapeutic plasmapheresis on rheological properties of blood in patients with ischemic heart disease]

Patients with angina pectoris, functional classes III-IV, were examined for changes in the rheological blood properties (red blood cell aggregation ratio, fluidity limit, apparent viscosity, electrophoretic mobility of red blood cells, fibrinogen, total plasma protein and ADP, induced platelet aggregation) under the effect of continuous plasmapheresis (PA). PA was established to produce a selective effect on the rheological blood properties depending on the initial level of its disorders. The most remarkable clinical effect was produced by PA in patients with initially high characteristics of hemorheology. The first PA session ameliorated hemorheology at the expense of the plasma component, reduced high platelet aggregation whereas the repeated sessions largely affected red blood cell function.     

温度对单个人红细胞膜力学特性的即时作用

目的：探讨温度对单个红细胞膜力学性质的即时影响。 方法： 利用静态显微图像分析技术和动态显微图像分析技术，在无损、实时、在位的情况下，观察和测量不同温度下单个人红细胞的形态、大小、膜弯曲弹性模量和剪切弹性模量的变化。 结果： 红细胞的接触面积和直径随温度的升高而减小，胞膜的弯曲弹性模量和剪切弹性模量都在生理温度37 ℃时最小，而温度低于或高于37 ℃时红细胞膜的弯曲弹性模量和剪切弹性模量都增大。 结论： 红细胞在生理温度37 ℃时有最好的形态和力学变形性，便于发挥其生理功能。     

Viscoelasticity of pediatric blood and its implications for the testing of a pulsatile pediatric blood pump

Red blood cell hematocrit, aggregation and deformability, and plasma protein concentration influence the viscosity and elasticity of whole blood. These parameters affect the flow properties, especially at low shear rates (< 50 s(-1)). In particular, we have previously shown that the viscoelasticity of fluid affects the inlet filling characteristics and regions of flow separation in small pulsatile blood pumps. Although the viscosity of pediatric blood has been thoroughly studied, its elasticity has not been previously measured. Here we present the viscosity and elasticity of pediatric blood against shear rate for hematocrits from 19-56, measured using an oscillatory rheometer. There is little effect of patient age on blood viscoelasticity. A statistical analysis showed that when compared at constant hematocrit, blood from adult and pediatric patients had similar viscoelastic properties. We present blood analog solutions, as a function of hematocrit, constructed on the basis of the pediatric measurements. Flow field results for viscoelastic analogs of 20, 40 and 60% hematocrit and a Newtonian analog will be compared in the initial, in vitro testing of the Penn State pediatric blood pump, to determine the importance of incorporating a viscoelastic analog into the desigh interaction.     

The association between erythrocyte internal viscosity, protein non-enzymatic glycosylation and erythrocyte membrane dynamic properties in juvenile diabetes mellitus.

The association of intracellular viscosity of red blood cells and the dynamic properties of erythrocyte membranes in children suffering from diabetes has been investigated by means of ESR spectroscopy. It has been revealed that the slight decrease in the ratio hw/hs of maleimide bound to membrane protein-SH groups of erythrocytes in diabetes may ensue from the enhanced membrane protein immobilization in the plane of lipid bilayer. These alterations were accompanied by a corresponding increase in the relative rotational correlation time (tau c) of iodoacetamide spin label, thus suggesting that the conformational changes in membrane proteins may occur at both the intrinsic and more exposed thiol groups. The membranes of diabetic red blood cells were more glycosylated than those of relevant controls, and the extent of glycosylation was found to correlate significantly with h + 1/h0 and tau c (r = -0.652, P < 0.01 and r = 0.609, P < 0.01). Further, the conformational alterations in erythrocyte membranes from diabetic subjects were accompanied by a significant increase in the mobility parameter (h + 1/h0) of haemoglobin molecules in diabetic erythrocytes. The latter changes correlated well with the enhanced intracellular viscosity of diabetic red blood cells and the level of glycosylated haemoglobin. We conclude that the alterations in membrane lipid-protein interactions together with the increased glycosylation-derived internal viscosity may consequently imply altered viscoelastic properties of erythrocyte membranes and, underlying the impaired deformability of red blood cells in the diabetic state, contribute to the development of late diabetic sequelae.     

Echinocytes in the blood of hyperproteinaemic mice with proteinuria. Is the effect of such cells on blood viscosity the cause of the proteinuria?

Early studies in this series failed to obtain evidence for the cause of hyperproteinaemic proteinuria although it was speculated that blood viscosity, increased because of high plasma protein levels, might play a significant role. As we had no means of measuring blood viscosity it was decided to investigate the effects of reducing blood viscosity in a small number of mice made anaemic before subjecting them to albumin overload. All but one of the mice given injections i.p. of 250 mg HSA on 2 successive days developed proteinuria within 24 h of the first injection. This result seemed to show that altered blood viscosity was not a factor in the mechanism of the hyperproteinaemic proteinuria. However, it has been shown that changes in the red cell environment can lead to red cell deformation resulting in an increase in blood viscosity. To check on this possibility another small group of mice were injected with HSA as previously. On the morning after their second injection the mice were bled by percutaneous heart puncture and the blood was examined by scanning electron microscopy. This showed that a vast preponderance of red cells, probably more than 95%, were echinocytes. Although no measurements of blood viscosity were made, it can be speculated that hyperproteinaemic proteinuria is caused by the intraglomerular effects of blood with increased viscosity (because of the red cell transformation) being made more viscous by glomerular filtration. Enhanced protein filtration would occur because of the increase in glomerular pressure needed to restore flow of viscous blood at the efferent arteriole.(ABSTRACT TRUNCATED AT 250 WORDS)     

A new integrated system combining atomic force microscopy and micro-electrode array for measuring the mechanical properties of living cardiac myocytes

In this paper we present a new experimental set-up which combines the surface characterization capabilities of atomic force microscopy at the sub-micrometer scale with non-invasive electrophysiological measurements obtained by using planar micro-electrode arrays. In order to show the potential of the combined measurements we studied the changes in cell topography and elastic properties of cardiac muscle cells as during the contraction-relaxation cycle. The onset of each beating cycle was precisely identified by the use of the extracellular potential signal, allowing us to combine nanomechanical measurements from multiple cardiomyocyte contractions in order to analyze the time-dependent variation of cell morphology and elasticity. Moreover, by estimating the elastic modulus at different indentation depths in a single location on the cell membrane, we observed a dynamic mechanical behavior that could be related to the underlying myofibrillar structure dynamics.     

The Effects of Substrate Elastic Modulus on Neural Precursor Cell Behavior

The spinal cord has a limited capacity to self-repair. After injury, endogenous stem cells are activated and migrate, proliferate, and differentiate into glial cells. The absence of neuronal differentiation has been partly attributed to the interaction between the injured microenvironment and neural stem cells. In order to improve post-injury neuronal differentiation and/or maturation potential, cell–cell and cell–biochemical interactions have been investigated. However, little is known about the role of stem cell–matrix interactions on stem cell-mediated repair. Here, we specifically examined the effects of matrix elasticity on stem cell-mediated repair in the spinal cord, since spinal cord injury results in drastic changes in parenchyma elasticity and viscosity. Spinal cord-derived neural precursor cells (NPCs) were grown on bis-acrylamide substrates with various rigidities. NPC growth, proliferation, and differentiation were examined and optimal in the range of normal spinal cord elasticity. In conclusion, limitations in NPC growth, proliferation, and neuronal differentiation were encountered when substrate elasticity was not within normal spinal cord tissue elasticity ranges. These studies elucidate the effect injury mediated mechanical changes may have on tissue repair by stem cells. Furthermore, this information can be applied to the development of future neuroregenerative biomaterials for spinal cord repair.     

Influence of osmolarity on the optical properties of human erythrocytes

Plasma osmolarity influences the volume and shape of red blood cells (RBCs). The volume change is inversely related to the hemoglobin concentration and as a consequence to the complex refractive index within the cell. These morphological changes can be linked to changes in the optical behavior of the cells. The optical parameters, absorption coefficient μa, scattering coefficient μs, and effective scattering phase function of red blood cells are investigated in dependence on osmolarity in the spectral range from 250 to 1100 nm. Integrating sphere measurements of light transmittance and reflectance in combination with inverse Monte-Carlo simulations are carried out for osmolarities from 225 to 400 mosmol/L. Osmolarity changes have a significant influence on the optical parameters, which can in part be explained by changes in the complex refractive index, cell shape, and cell volume. Spherical forms of RBCs induced by low osmolarity show reduced scattering effects compared to the normal RBC biconcave disk shape. Spinocytes, which are crenated erythrocytes induced by high osmolarity, show the highest scattering effects. Even only a 10% change in osmolarity has a drastic influence on the optical parameters, which appears to be of the same order as for 10% hematocrit and oxygen saturation changes.     

Comparison of three rheological models of shear flow behavior studied on blood samples from post-infarction patients

Quantitative analysis of blood viscosity was performed on the basis of mathematical models of non-Newtonian fluid shear flow behavior (Casson, Ree-Eyring and Quemada). A total of 100 blood samples were drawn from clinically stable survivors of myocardial infarction, treated with aspirin or acenocoumarol and controls to these drugs. Whole blood and plasma viscosity were measured at a broad range of shear rates using a rotary-oscillating viscometer Contraves LS40. Numerical analysis of the experimental data was carried out by means of linear (for Casson) and non-linear regression for the remaining models. In the evaluation of the results, both the fit quality and physical interpretation of the models’ parameters were considered. The Quemada model fitted most precisely with the experimental findings and, despite the controversies concerning the relationship between in vivo tissue perfusion and in vitro rheological measurements, seemed to be a valuable method enhancing investigation possibilities of cardiovascular patients. Our results suggest that aspirin does not affect blood rheological properties, while acenocoumarol may slightly alter red cell deformability and rouleaux formation.     

三种莨菪药对急性肾衰大鼠血流变性改变的影响

本文研究了油酸致大鼠急性肾衰时血液流变性的改变。观察了樟柳碱，东莨菪碱及山莨菪碱对这些改变的影响。结果表明，急性肾衰时，低切至高切变率时的全血粘度均明显增高，红细胞比积，血浆粘度及红细胞聚集指数也明显高于对照组。樟柳碱及东莨菪碱能降低血浆粘度、红细胞聚集指数及全血粘度；山莨菪碱能降低血浆粘度及红细胞聚集指数。结果说明，急性肾衰时血液的流变性有明显障碍，三种莨菪药均能改善急性肾衰时血液的流变性。     

Stability of spiculated red blood cells induced by intercalation of amphiphiles in cell membrane

The stability of speculated red blood cells, induced by intercalation of amphiphilic molecules into the cell membrane, is studied. It is assumed that the stable red blood cell shape corresponds to the minimum of its membrane elastic energy, which consists of the local and non-local bilayer bending energies and of the skeleton shear elastic energy. The cell volume and the membrane area are kept constant. It is calculated that the number of spicules of the stable echinocytic shape is larger when the amphiphile concentration is higher, which is in agreement with experimental observations. Also, it is established that, in explaining the stability of the echinocytic shape of the red blood cell, it is necessary to include the membrane skeleton shear elasticity.     

In vitro growth and chloroquine sensitivity of Plasmodium falciparum (FCR-3 strain) in red blood cells containing HbC

Freshly drawn AA and CC red cells were more suitable for in vitro development of Plasmodium falciparum than red blood cells (RBC) stored for 13 days before use. Growth rate inhibition in CC red cell cultures reached 31% in freshly drawn red cells and 57% in aged red cells of the same donor. Ultrastructural studies of CC cells revealed very important irregular cavities sometimes occupied by a granular content. Parasites in CC cells were generally normal but occasionally showed signs of functional impairment. P. falciparum growing in CC red cells was less sensitive in vitro to chloroquine than in AA red cells. This phenomenon may be explained either by the type of the hemoglobin of the host cell or to abnormal haematological parameters of the HbC homozygote donor, particularly the high proportion of neocytes. As metabolism of reduced glutathione is higher in young RBC and as chloroquine lyses parasitized RBC by reducing the regeneration capacities of this compound, the increased rate of young RBC in the CC red cell population was probably related to the decreased chloroquine sensitivity of P. falciparum growing in these cells.Technical collaboration: A. Wattez     

Connections between single-cell biomechanics and human disease states: gastrointestinal cancer and malaria

We investigate connections between single-cell mechanical properties and subcellular structural reorganization from biochemical factors in the context of two distinctly different human diseases: gastrointestinal tumor and malaria. Although the cell lineages and the biochemical links to pathogenesis are vastly different in these two cases, we compare and contrast chemomechanical pathways whereby intracellular structural rearrangements lead to global changes in mechanical deformability of the cell. This single-cell biomechanical response, in turn, seems to mediate cell mobility and thereby facilitates disease progression in situations where the elastic modulus increases or decreases due to membrane or cytoskeleton reorganization. We first present new experiments on elastic response and energy dissipation under repeated tensile loading of epithelial pancreatic cancer cells in force- or displacement-control. Energy dissipation from repeated stretching significantly increases and the cell's elastic modulus decreases after treatment of Panc-1 pancreatic cancer cells with sphingosylphosphorylcholine (SPC), a bioactive lipid that influences cancer metastasis. When the cell is treated instead with lysophosphatidic acid, which facilitates actin stress fiber formation, neither energy dissipation nor modulus is noticeably affected. Integrating recent studies with our new observations, we ascribe these trends to possible SPC-induced reorganization primarily of keratin network to perinuclear region of cell; the intermediate filament fraction of the cytoskeleton thus appears to dominate deformability of the epithelial cell. Possible consequences of these results to cell mobility and cancer metastasis are postulated. We then turn attention to progressive changes in mechanical properties of the human red blood cell (RBC) infected with the malaria parasite Plasmodium falciparum. We present, for the first time, continuous force-displacement curves obtained from in-vitro deformation of RBC with optical tweezers for different intracellular developmental stages of parasite. The shear modulus of RBC is found to increase up to 10-fold during parasite development, which is a noticeably greater effect than that from prior estimates. By integrating our new experimental results with published literature on deformability of Plasmodium-harbouring RBC, we examine the biochemical conditions mediating increases or decreases in modulus, and their implications for disease progression. Some general perspectives on connections among structure, single-cell mechanical properties and biological responses associated with pathogenic processes are also provided in the context of the two diseases considered in this work.