Microconfined flow behavior of red blood cells

Red blood cells (RBCs) perform essential functions in human body, such as gas exchange between blood and tissues, thanks to their ability to deform and flow in the microvascular network. The high RBC deformability is mainly due to the viscoelastic properties of the cell membrane. Since an impaired RBC deformability could be found in some diseases, such as malaria, sickle cell anemia, diabetes and hereditary disorders, there is the need to provide further insight into measurement of RBC deformability in a physiologically relevant flow field. Here, RBCs deformability has been studied in terms of the minimum apparent plasma-layer thickness by using high-speed video microscopy of RBCs flowing in cylindrical glass capillaries. An in vitro systematic microfluidic investigation of RBCs in micro-confined conditions has been performed, resulting in the determination of the RBCs time recovery constant, RBC volume and surface area and RBC membrane shear elastic modulus and surface viscosity. It has been noticed that the deformability of RBCs induces cells aggregation during flow in microcapillaries, allowing the formation of clusters of cells. Overall, our results provide a novel technique to estimate RBC deformability and also RBCs collective behavior, which can be used for the analysis of pathological RBCs, for which reliable quantitative methods are still lacking.     

胰蛋白酶对红细胞膜动态力学特性的影响

目的： 研究胰蛋白酶对人红细胞膜表面电荷密度及动态力学特性的影响。 方法： 用密度梯度离心法从健康成人血液中分离出“青年”和“老年”红细胞，37 ℃下与不同浓度的胰蛋白酶孵育60 min。利用Zeta电位分析仪测量红细胞膜表面电荷密度，利用快速显微动态图像分析技术测定单个活态红细胞的大小、形态和细胞膜弯曲弹性模量（Kc）和剪切弹性模量（μc）。 结果： （1）正常和用胰蛋白酶处理后“老年”红细胞的表面电荷密度分别显著小于正常和用胰蛋白酶处理后的“青年”红细胞（P<0.01）。（2）正常“青年”红细胞和正常“老年”红细胞在形态上没有显著区别，但正常“老年”红细胞的接触面积显著小于正常“青年”红细胞（P<0.01）；胰蛋白酶处理可减小红细胞的大小，但对形状规化因子没有显著影响。（3）正常“老年”红细胞膜Kc和μc均显著大于正常“青年”红细胞（P<0.05）；“青年”和“老年”红细胞膜Kc和μc均随胰蛋白酶浓度的增加而增大。结论： 胰蛋白酶处理可降低“青年”和“老年”红细胞膜表面电荷密度，增加红细胞膜的弯曲弹性模量，从而降低红细胞膜的力学变形性。     

Two-Dimensional Simulation of Red Blood Cell Deformation and Lateral Migration in Microvessels

A theoretical method is used to simulate the motion and deformation of mammalian red blood cells (RBCs) in microvessels, based on knowledge of the mechanical characteristics of RBCs. Each RBC is represented as a set of interconnected viscoelastic elements in two dimensions. The motion and deformation of the cell and the motion of the surrounding fluid are computed using a finite-element numerical method. Simulations of RBC motion in simple shear flow of a high-viscosity fluid show “tank-treading’’ motion of the membrane around the cell perimeter, as observed experimentally. With appropriate choice of the parameters representing RBC mechanical properties, the tank-treading frequency and cell elongation agree closely with observations over a range of shear rates. In simulations of RBC motion in capillary-sized channels, initially circular cell shapes rapidly approach shapes typical of those seen experimentally in capillaries, convex in front and concave at the rear. An isolated RBC entering an 8-μm capillary close to the wall is predicted to migrate in the lateral direction as it traverses the capillary, achieving a position near the center-line after traveling a distance of about 60 μm. Cell trajectories agree closely with those observed in microvessels of the rat mesentery.     

The adhesive sickle erythrocyte: cause and consequence of abnormal interactions with endothelium, monocytes/macrophages and model membranes

The sickle RBC has a distinct propensity for abnormal adherence to vascular endothelial cells, monocytes, macrophages and aminophospholipid liposomes. Sickle RBC adherence to endothelium may be due to aberrant electrostatic forces, with a major contribution coming from factors in the RBC's plasma environment. The abnormal translocation of aminophospholipids in sickle RBC membranes has been implicated in their adherence to monocytes and liposomes. Abnormal interactions with macrophages (adherence and erythrophagocytosis) may be due to abnormal amounts of IgG on the sickle RBC surface and/or modification of the RBC membrane by dialdehyde byproducts of lipid peroxidation. However, the RBC membrane is an exceedingly complex structure, and these various abnormalities may well be highly interrelated. On the other hand, it is also possible that different abnormalities may be responsible for different cell-cell interactions. Precise identification of the adhesive factor(s) of the sickle RBC membrane will require additional investigation. Regardless of the mechanism(s) involved, data suggest that propensity for adherence of RBC to endothelial cells may correlate with clinical vaso-occlusive severity, perhaps helping to explain not only heterogeneity among patients but also temporal variability in disease severity for individual patients. Likewise, the abnormal adherence of sickle RBCs to monocytes/macrophages may help explain the attenuated survival of sickle RBCs.     

原子力显微镜对自由基损伤的红细胞膜表面精细结构的研究

目的　直接观察自由基损伤、加女贞子保护、H     

The effects of marathon running on expression of the complement regulatory proteins CD55 (DAF) and CD59 (MACIF) on red blood cells

Exercise is known to result in the haemolysis of red blood cells (RBCs). Although mechanical stressors such as footstrike and an increased velocity of blood flow may be involved, the biological mechanisms that underpin RBC haemolysis remain elusive. RBCs are potentially susceptible to lysis by autologous complement activation. RBCs are protected from the lytic effects of complement by regulatory proteins (CRPs) bound to the cell membrane via glycosylphosphatidylinositol (GPI) anchors. This study aimed to determine if marathon running would result in RBC haemolysis through a loss of membrane expression of the CRPs CD55 (decay accelerating factor) and CD59 (membrane attack complex inhibitory factor). Blood samples were obtained from 14 male runners before, within 30 min after, and 24 h after completion of the 2004 London Marathon. RBCs were assessed for cell surface CD55 and CD59 expression using indirect immunofluorescence assays and flow cytometry. No significant changes in the total RBC count, haematocrit or haemoglobin concentrations were found in response to running the marathon (P > 0.05). Blood bilirubin concentrations after the marathon were significantly greater than the pre-race values (P < 0.01). The relative fluorescent intensity (arbitrary units) of CD55 and CD59 expression on RBC membranes did not change in response to the marathon race (P > 0.05). In conclusion, marathon running did not alter the expression of CD55 or CD59 on RBCs, despite concomitant elevations in blood bilirubin concentrations. Consequently, any haemolysis of RBCs that occurred in response to the marathon was not likely due to a loss of membrane bound CRPs and subsequent cell lysis by autologous complement.     

Quantitative microscopy and nanoscopy of sickle red blood cells performed by wide field digital interferometry

We have applied wide-field digital interferometry (WFDI) to examine the morphology and dynamics of live red blood cells (RBCs) from individuals who suffer from sickle cell anemia (SCA), a genetic disorder that affects the structure and mechanical properties of RBCs. WFDI is a noncontact, label-free optical microscopy approach that can yield quantitative thickness profiles of RBCs and measurements of their membrane fluctuations at the nanometer scale reflecting their stiffness. We find that RBCs from individuals with SCA are significantly stiffer than those from a healthy control. Moreover, we show that the technique is sensitive enough to distinguish classes of RBCs in SCA, including sickle RBCs with apparently normal morphology, compared to the stiffer crescent-shaped sickle RBCs. We expect that this approach will be useful for diagnosis of SCA and for determining efficacy of therapeutic agents.     

Morphological changes in red blood cells of calves caused by Leptospira interrogans serovar pomona

Haemoglobinaemia is seen in certain hosts infected by certain serovars of Leptospira interrogans, but is absent from other serovar-host associations. Comparisons were made between calves infected with serovar pomona and those receiving a crude "toxin" prepared from the same organism. Red blood cells from "toxin"-injected calves showed discocyte-echinocyte transformation and contained portions of cytoplasm segregated within vacuoles. These animals showed increased sequestration of RBCs within the spleen but no overt haemoglobinaemia. Red blood cells from infected and haemoglobinaemic animals were spherical and pitted. They also showed vacuoles and tracts under the cell membrane in fully haemoglobinized RBCs and dark granular inclusions within the cytoplasm of those which were only partially haemoglobinized. Intracellular leptospires were not seen within the RBCs. Red blood cell sequestration and erythrophagocytosis were very pronounced within the spleen, liver and bone marrow. The changes in the RBCs are not easily explained by the previously proposed theory that RBC destruction is due to a phospholipase-like toxin acting directly upon the RBC membrane. A more appropriate hypothesis is that the RBC lesions are due to the adverse effects of leptospiral "toxin(s)" on the metabolism of the RBC causing the formation of defective portions of cytoplasm. These are then either degraded and expelled, leaving empty vacuoles, or are degraded and left within the cytoplasm as dark granular inclusions.     

Alterations of uninfected red blood cells in malaria

Red blood cell (RBC) negative charges and resistance to linoleic acid (LNA)-induced lysis were studied inPlasmodium yoelii-infected mice and in malaria (P. falciparum)-affected individuals. RBCs from mice infected withP. yoelii showed a progressive decrease in the net surface negative charges at 24 h after infection, reaching a minimal value on day 3, followed by a second phase that was characterised by a recovery to normal levels on day 6. Resistance to linoleic acid follows similar kinetics. These alterations preceded the appearance of parasites in the peripheral blood. A similar increase in LNA-induced lysis was observed in RBCs from malaria-affected individuals. These early membrane alterations of uninfected RBCs could be responsible for spreading of infection and RBC lysis during infection.     

Influence of polymer architecture on antigens camouflage, CD47 protection and complement mediated lysis of surface grafted red blood cells

Hyperbranched polyglycerol (HPG) and polyethylene glycol (PEG) polymers with similar hydrodynamic sizes in solution were grafted to red blood cells (RBCs) to investigate the impact of polymer architecture on the cell structure and function. The hydrodynamic sizes of polymers were calculated from the diffusion coefficients measured by pulsed field gradient NMR. The hydration of the HPG and PEG was determined by differential scanning calorimetry analyses. RBCs grafted with linear PEG had different properties compared to the compact HPG grafted RBCs. HPG grafted RBCs showed much higher electrophoretic mobility values than PEG grafted RBCs at similar grafting concentrations and hydrodynamic sizes indicating differences in the structure of the polymer exclusion layer on the cell surface. PEG grafting impacted the deformation properties of the membrane to a greater degree than HPG. The complement mediated lysis of the grafted RBCs was dependent on the type of polymer, grafting concentration and molecular size of grafted chains. At higher molecular weights and graft concentrations both HPG and PEG triggered complement activation. The magnitude of activation was higher with HPG possibly due to the presence of many hydroxyl groups per molecule. HPG grafted RBCs showed significantly higher levels of CD47 self-protein accessibility than PEG grafted RBCs at all grafting concentrations and molecular sizes. PEG grafted polymers provided, in general, a better shielding and protection to ABO and minor antigens from antibody recognition than HPG polymers, however, the compact HPGs provided greater protection of certain antigens on the RBC surface. Our data showed that HPG 20kDa and HPG 60kDa grafted RBCs exhibited properties that are more comparable to the native RBC than PEG 5kDa and PEG 10kDa grafted RBCs of comparable hydrodynamic sizes. The study shows that small compact polymers such as HPG 20kDa have a greater potential in the generation of functional RBC for therapeutic delivery applications. The intermediate sized polymers (PEG or HPG) which showed greater antigen camouflage at lower grafting concentrations have significant potential in transfusion as universal red blood donor cells.     

Red cell morphology in leukemia, hypoplastic anemia and myelodysplastic syndrome

Leukemic patients of different classifications are associated with anemia. Such clinical conditions are often referred to as refractory anemia, paraoxymal nocturnal hemoglobinuria, hemolytic uremia and autoimmune hemolytic anemia, all of which could be categorized as the cancer cachexia. In the present work, we have studied the overall morphology of intact red cells in different leukemic patients along with patients of hypoplastic anemia (HPA) by scanning electron microscopy. We have also studied the ultrastructure of the red cell surface membranes by transmission electron microscopy. For all experiments, erythrocytes from normal individuals served as controls. We have shown direct evidence of the altered red cell (RBC) membrane morphology irrespective of the hemoglobin status of the patients which includes (1) presence of large central holes in RBCs of acute myeloid leukemia (AML), (2) presence of thorn- and horn-like structure in RBCs of acute lymphoblastic leukemia (ALL) and chronic myeloid leukemia (CML) and (3) flaccid appearance of RBCs in chronic lymphocytic leukemia (CLL) patients. A mixture of the above mentioned structures were found in the red cells of patients suffering from myelodysplastic syndrome (MDS) and in case of patients of HPA the RBCs lost the normal biconcave structures. TEM studies revealed presence of pores with diameters ranging from 100 to 200 nm on the RBC membrane surface of myeloid leukemia with AML being the most prominent among others. Such pathophysiological alterations of the RBC morphology in leukemic patients could be identified as characteristic signature of the onset of anemia associated with the disease.     

Light scattering of human red blood cells during metabolic remodeling of the membrane

We present the light scattering properties of individual human red blood cells (RBCs). We show that both the RBC static and dynamic scattering signals are altered by adenosine 5'-triphosphate (ATP)-driven membrane metabolic remodeling. To measure the light scattering signal from individual RBCs, we use diffraction phase microscopy together with a Fourier transform light scattering technique. RBC cytosolic ATPs are both chemically and metabolically depleted, and the corresponding scattering signals are compared with the light scattering signal of normal RBCs having physiologic levels of ATP.     

Zastosowanie cytometrii przepływowej oraz analizy proteomicznej w ocenie erytrocytarnych białek błonowych podczas przechowywania koncentratów krwinek czerwonych

For over 100 years the erythrocyte cell membrane attracted interest of transfusiologists mainly due to the antigens localized on their surface and associated risks of patient alloimmunisation and therefore the need of serological selection of donor's and recipient's blood. Presently it is known that RBC antigens and other membrane proteins play important transport and protective functions, and are involved in adhesion, maintenance of cell shape and in the process of aging and phagocytosis. Since the available results of retrospective clinical observations suggest an adverse effect of transfusion on selected groups of patients, it is important to undertake studies on the changes taking place within the cell membrane of erythrocytes stored in blood banks. Flow cytometric analysis of stored leucodepleted or non-leucodepleted erythrocyte concentrates, revealed significant changes in the level of expression of many RBC surface molecules: CD44, CD47, CD55, CD58, CD59, CD235a (GPA). In parallel, a significant development of proteomic analysis of stored RBCs is observed. Stored RBCs offer less variability of biological material, caused by drugs, illnesses, etc. when compared with clinical proteomics studies; however, the complexity of the methodology and the lack of uniform and comparable procedures may cause misinterpretation and even create artifacts. Still, modern research methods offer hope for the elaboration of aging biomarkers of stored RBCs as well as for raising the level of transfusion medicine quality.     

Mechanisms behind adhesive erythrocytes in sickle-cell disease

Erythrocytes containing primarily hemoglobin S (SS RBCs) are abnormally adherent to a number of ligands, including normal constituents of the extracellular matrix as well as those present on the surfaces of other blood cells and endothelial cells. However, SS RBCs are not really very different from normal (AA) RBCs, except that they are younger. In general, they express the same range of adhesion receptors, and the levels of expression of these receptors, although increased in some cases, are not sufficient to explain the difference between the adhesivity of SS and AA RBCs. However, SS RBCs have activated adhesion receptors, and the signaling pathways responsible for this activation are also upregulated in SS RBCs. In addition, SS RBCs convey different extracellular signals, which are also likely to affect their adhesion to other blood cells and endothelial cells. Together, SS RBCs induce cell–cell interactions mediated by the B-CAM/LU, ICAM4 (LW), CD44 (In), CD47, α4β1 integrin, and CD36 adhesion molecules on SS RBCs with target ligands on leukocytes and endothelial cells. Other, as yet unidentified cell surface moieties on SS RBCs are probably also responsible for SS RBC interactions with P-selectin. Interestingly, at least some of these RBC adhesion receptors may also mediate adhesion and adverse physiologic events in the setting of transfusion of stored normal RBCs. Therefore, understanding the mechanisms behind SS RBC adhesion may offer opportunities for improving both therapy of sickle-cell disease as well as transfusion practices.     

Cell Registration and Flickering Detection for the Complexity Analysis of Red Blood Cell Dynamics with GSM Exposure

Red blood cells(RBC)' flickering present the dynamic properties of the cytomembrane. Its complexity could be used for aging analysis or the evaluation for the storage quality. The flickering activity is a kind of reversible perpendicular motion of the specified pixel. Therefore, the complexity analysis depends on the reliable detection of temporal variation for the gray-scale values from each pixel of the cells. In this paper, we improved our previous work on the screening of the horizontal drifted cells with a surface based on cell registration method and the effect of GSM exposure to the dynamic properties of the RBCs in terms of multi-scale sample entropy was presented in the paper.     

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.     

Interactions of chrysotile and crocidolite asbestos with red blood cell membranes. Chrysotile binds to sialic acid

Chrysotile and crocidolite are commonly used forms of asbestos. Hemolysis has been widely used as a test of membrane injury, and it has been shown previously that chrysotile causes rapid breakdown of red blood cells (RBCs), whereas crocidolite is only weakly hemolytic. A reasonable hypothesis set forth to explain the cytotoxic effects of chrysotile maintains that positively charged chrysotile fibers bind to negatively charged sialic acid residues on RBC membranes causing clustering of membrane proteins and increased cell permeability to Na and K ions. Our studies presented here provide two lines of evidence in direct support of this hypothesis. (a) Morphologic--Ultrastructural techniques showed that both chrysotile and crocidolite asbestos bind to and distort more than 85% of RBCs treated for 15 minutes. The distorting effects of chrysotile, but not crocidolite, were almost totally ablated by pretreating the cells with neuraminidase. In addition, gold-conjugated wheat germ agglutinin was used to label the distribution of sialic acid groups on RBC membranes. Pretreatment of the RBCs with chrysotile, but not crocidolite, reduced the number of gold-conjugated wheat germ agglutinin-labeled sites to less than 30% of the control level. (b) Biochemical--The thiobarbituric acid assay was used to determine the percentage of sialic acid that remained with the cell pellet after neuraminidase and/or asbestos treatment. Asbestos treatment alone caused no release of sialic acid from the cells. Neuraminidase treatment for 3.5 hours removed more than 80% of the sialic acid from cell surfaces. Chrysotile, but not crocidolite, asbestos prevented neuraminidase-mediated removal of sialic acid from RBCs. In addition, x-ray energy spectrometry of freeze-dried cells showed that RBCs distorted by chrysotile, but not by crocidolite, exhibited significant alterations in intracellular Na:K ratios. The morphologic and biochemical data strongly support the hypothesis that chrysotile asbestos binds to sialic acid groups on RBC membranes. Consequently, the sialic acid residues are redistributed on the surfaces of distorted cells which then are unable to maintain a normal Na:K balance with the surrounding medium.     

Computer Modeling of Red Blood Cell Rheology in the Microcirculation: A Brief Overview

One of the major functions of the cardiovascular system is to deliver blood to the microcirculation where exchange of mass and energy can take place. In the present article, we will provide an overview of the state-of-the-art computational methods for modeling of red blood cell (RBC) rheology and dynamics in the microcirculation. While significant progress has been made in simulation of single-file motion of deformable RBCs in capillaries and of diluted sheared suspensions of RBCs in infinite domains, detailed understanding of the mechanics of blood flow in intermediate diameter microvessels (8–1000μm) has presented formidable challenges. The difficulties are largely due to modeling the motion of multiple, interacting, highly deformable particles. The current computational tools consist mainly of three-dimensional (3D) boundary-integral methods for single RBC dynamics and deformation; and for rheology of large systems of droplets at large volume fractions using periodic boundary conditions and novel adaptive computational meshes. Further advances will result from combination of these tools to produce new algorithms capable of describing the motion and deformation of large systems of RBCs in microvessels at physiologically relevant volume fractions.     

红细胞非惯性升力对血液流动的影响

目的 为准确模拟血流,研究红细胞变形性对血液流动的影响。方法 基于血液流变特性和红细胞力学特性分析,对现有血液两相流流动模型进行改进,改进模型中考虑了易变形红细胞受剪切流场或血管壁面作用而产生的非惯性升力的影响。利用改进模型对多个不同直径血管内的血液流动进行模拟。结果 由红细胞所受非惯性升力导致的径向运动对血管内红细胞体积分数、运动速度分布有明显影响;当血管直径为0.1~3.0 mm时,用改进模型得到的血液相对黏度的模拟值与测量值接近。结论 非惯性升力是血流呈现Fahraeus-Lindqvist效应的主要原因之一。考虑非惯性升力的改进模型可以准确模拟血液流动,为循环系统诊疗机制和细胞分选等过程的模拟提供更为准确的方法。     

新生婴儿与老年人红细胞物理特性对比实验

目的 通过比较新生婴儿与80岁以上老年人红细胞物理特性(细胞表面积、体积和剪切弹性模量)及与总胆固醇、糖化血红蛋白等生理生化参数的相关关系,分析不同年龄和生化参数对红细胞物理特性的影响.方法 采用微量吸管技术测量新生婴儿和80岁以上老年人的红细胞表面积、体积和剪切弹性模量,对数据进行统计分布分析、相关性分析和回归分析....