Interaction Of Red Blood Cells Research Articles

Computational analysis on the mechanical interaction between a thrombus and red blood cells: Possible causes of membrane damage of red blood cells at microvessels

Previous studies investigating thrombus formation have not focused on the physical interaction between red blood cells (RBCs) and thrombus, although they have been speculated that some pathological conditions such as microangiopathic hemolytic anemia (MAHA) stem from interactions between RBCs and thrombi. In this study, we investigated the mechanical influence of RBCs on primary thrombi during hemostasis. We also explored the mechanics and aggravating factors of intravascular hemolysis. Computer simulations of primary thrombogenesis in the presence and the absence of RBCs demonstrated that RBCs are unlikely to affect the thrombus height and coverage, although their presence may change microvessel hemodynamics and platelet transportation to the injured wall. Our results suggest that intravascular hemolysis owing to RBC membrane damage would be promoted by three hemodynamic factors: (1) dispersibility of platelet thrombi, because more frequent spatial thrombus formation decreases the time available for an RBC to recover its shape and enforces more severe deformation; (2) platelet thrombus stiffness, because a stiffer thrombus increases the degree of RBC deformation upon collision; and (3) vessel size and hemocyte density, because a smaller vessel diameter and higher hemocyte density decrease the room for RBCs to escape as they come closer to a thrombus, thereby enhancing thrombus–RBC interactions.

Simulation of red blood cell motion in microvessels using modified moving particle semi-implicit method

Red Blood Cells (RBCs) are the main cells in human blood, with a main role in the mechanical properties of blood as a fluid. Several methods have been improved to simulate the mechanical behavior of RBC in micro-capillaries. Since, in microscopic scales, using discrete models is more preferred than continuum methods, the Moving Particle Semi-Implicit method (MPS), which is a recent innovative particle based method, can simulate micro-fluidic flows based on Navier–Stokes equations. Although, by recent developments, the MPS method has turned into a considerable tool for modeling blood flow in micro meter dimensions, some problems, such as a commitment to use small time step sizes, still restrict the method for large models and also for long time simulations. A new modified semi-implicit algorithm is developed and implemented on RBC motion through microvessels, in order to reduce calculation time by more than a factor of twenty, while the error of position and velocity remains constant. A two-dimensional, parallel plate, fluid flow is simulated based on the proposed method, and the effect of the calculation time decrement is evaluated. Findings indicate a reduction of 90 percent in simulation time compared to previous studies with the same results. This significant developed method could be applied to RBC interaction within micro-capillaries and constricted zones in blood flow.

Conserved regions of the Plasmodium falciparum rhoptry-associated protein 3 mediate specific host-pathogen interactions during invasion of red blood cells

Invasion of red blood cells (RBCs) by the Plasmodium falciparum malaria merozoite is mediated by parasite surface molecules and proteins contained within apical organelles that are capable of recognizing receptors on the membrane of RBCs. The identification and characterization of these P. falciparum invasion-associated proteins is the first step for unveiling potential new drug and vaccine target molecules to eradicate this deadly disease. Among the exclusive set of malarial vaccine candidates, the members of the rhoptry-associated protein (RAP) family have been associated with the parasite's binding to and invasion of RBCs. Remarkably, the third member of this family (named RAP-3) has been recently detected on the surface of non-infected RBCs exposed to free merozoites, therefore suggesting the participation of this protein during RBC infection. In this study, the sequence of RAP-3 was finely mapped using synthetic peptides in order to identify which are the specific binding regions involved in RAP3–RBC interactions. Two high-activity binding peptides (HABPs) established high affinity interactions with RBC surface molecules of about 27–90 kDa, which were differentially affected by different enzymatic treatments. RAP-1 and RAP-2 HABPs inhibited binding of RAP-3 HABPs to different extents, thus suggesting the recognition of similar binding sites on RBC membrane, as well as ability of RAP-3 HABPs to inhibit P. falciparum infection in vitro. Altogether, these functional analyses of RAP-3 HABPs strongly suggest a potential role for this protein in RBC invasion, and highlight its HABPs as potential targets to develop a fully protective minimal subunit-based malarial vaccine.

A Critical Review of Published Methods for Analysis of Red Cell Antigen-Antibody Reactions by Flow Cytometry, and Approaches for Resolving Problems with Red Cell Agglutination

Flow cytometry operators often apply familiar white blood cell (WBC) methods when studying red blood cell (RBC) antigens and antibodies. Some WBC methods are not appropriate for RBCs, as the analysis of RBCs requires special considerations, for example, avoidance of agglutination. One hundred seventy-six published articles from 88 groups studying RBC interactions were reviewed. Three fourths of groups used at least one unnecessary WBC procedure for RBCs, and about one fourth did not use any method to prevent/disperse RBC agglutination. Flow cytometric studies were performed to determine the effect of RBC agglutination on results and compare different methods of preventing and/or dispersing agglutination. The presence of RBC agglutinates have been shown to be affected by the type of pipette tip used for mixing RBC suspensions, the number of antigen sites/RBC, the type and concentration of primary antibody, and the type of secondary antibody. For quantitation methods, for example, fetal maternal hemorrhage, the presence of agglutinates have been shown to adversely affect results (fewer fetal D+ RBCs detected).

Impact of cellular properties on red cell-red cell affinity in plasma-like suspensions

The reversible aggregation of human red blood cells (RBC) by proteins or polymers continues to be of biological and biophysical interest, yet the mechanistic details governing this process are still being explored. In this report an approach is described to compute the interaction energy between RBC by considering cellular properties as well as polymer properties. Cell-cell affinities were calculated as functions of glycocalyx thickness and glycocalyx volume concentration as well as bulk polymer concentration. Our theoretical predictions show that cell-cell affinities do not monotonically increase with polymer size and concentration, but rather demonstrate an optimum dextran molecular mass and concentration which depends on cellular properties of RBC. These results show qualitative agreement with recent experimental observations. In conclusion, our model not only confirms the concept of a depletion mechanism for RBC aggregation but also provides new insights which should help understanding how cellular properties control in vivo RBC interactions.

Computational fluid dynamics of aggregating red blood cells in postcapillary venules

Aggregate formation of red blood cells (RBCs) in a postcapillary venular bifurcation is investigated with three-dimensional computer simulations using the Chimera grid method. Interaction energy between the RBCs is modelled by a depletion interaction theory; RBCs are modelled as rigid oblate ellipsoids. The cell–cell interactions of RBCs are strongly dependent on vessel geometry and shear rates. The experimental data on vessel geometry, pseudoshear rates, and Dextran concentration obtained in our previous in vivo RBC aggregation study in postcapillary venules of the rat spinotrapezius muscle were used to simulate RBC aggregation. The computational results were compared to the experimental results from the in vivo study. The results show that cells have a larger tendency to form an aggregate under reduced flows. Aggregate formation also depends on the angle and location of the cells before they enter the bifurcation region. Comparisons with experimental data are discussed.

Role of Macromolecular Depletion in Red Blood Cell Adhesion

The adhesion of red blood cells (RBCs) to cells or surfaces is of current basic science and clinical interest yet the details governing this process are still being explored. In this study, the effects of nonadsorbing polymers on the adhesion of RBC to albumin-coated glass have been investigated employing interference reflection microscopy. Our experimental results indicate that adhesion can be induced in the presence of dextran with a molecular mass ≥70 kDa and that the induced forces are strong enough to significantly suppress membrane undulations. The overall dependence of the adhesion energies on the polymer concentration is consistent with the assumption that macromolecular depletion induces this attractive interaction. In conclusion, our results indicate that depletion interaction might play a significant role in RBC adhesion via initiating close contacts, and thus suggest the importance of depletion forces for RBC interactions and its relevance to a wide variety of in vitro and in vivo cell-cell and cell-surface interactions.

Depletion of high molecular weight dextran from the red cell surface measured by particle electrophoresis

The reversible aggregation of human red blood cells (RBC) by proteins or polymers continues to be of biological and biophysical interest, yet the mechanistic details governing the process are still being explored. A depletion model has been proposed for aggregation by the neutral polyglucose dextran and its applicability at high molecular weights has been recently documented. In the present study the depletion of high molecular weight dextrans on the red cell surface was measured as a function of polymer molecular mass (40 kDa-28 MDa), ionic strength (5 and 15 mM NaCl) and polymer concentration (< or =0.9 g/dL). The experimental data clearly indicate an increasing depletion effect with increasing molecular weight: the effects of medium viscosity on RBC mobility were markedly overestimated by the Helmholtz-Smoluchowski relation, with the difference increasing with dextran molecular mass. These results agree with the concept of polymer depletion near the RBC surface and lend strong support to a "depletion model" mechanism for dextran-mediated RBC aggregation. Our findings provide important new insight into polymer-RBC interactions and suggest the usefulness of this model for fundamental studies of cell-cell affinity and for the development of new agents to stabilize or destabilize specific bio-fluids.

Red blood cell adhesion in diabetes mellitus is mediated by advanced glycation end product receptor and is modulated by nitric oxide

Red blood cell (RBC) adhesion to endothelium is increased in diabetes mellitus and is correlated with the severity of vascular complications. Microangiopathy is the most frequent complications in patients suffering from diabetes mellitus. Elevated glucose concentration increases the oxidation phenomenon and advanced glycation end product (AGE) formation. Plasma proteins, structural proteins and also RBC proteins can be glycated such as glycated hemoglobin and RBC membrane proteins. Interaction of plasmatic AGE or RBC bearing AGE with the receptor for AGE (RAGE) alters vascular function leading to a vascular hyperpermeability inflammatory reaction including oxidant stress and cytokine production. Reactive oxygen species (ROS) react with nitric oxide (NO) limiting its vasodilatory effect and NO synthase function is altered. All these factors may be at the origin of high blood pressure which is deleterious for the eye and kidney vasculature. AGE can act directly on vascular function but also through RAGE. AGE binding to RAGE alters endothelial cell function stimulating NADPH oxidase and reactive oxygen species production. Limiting oxidation, reducing AGE formation or interaction with RAGE is achievable by drugs already used for hypertension or diabetes, but new treatment by NO modulators may limit the deleterious effect of RBC adhesion to endothelium.

Sensitive and Simple Methodologies for Measuring of Red Blood Cell (RBC) Electrical Properties and Cell Aggregation.

The red blood cell membrane contains proteins and glycoproteins embedded in a fluid lipid bilayer that confers viscoelastic behaviour. Sialylated glycoproteins of the RBC membrane are responsible for a negatively charged surface, which creates a repulsive electric zeta potential (ζ) between the cells.The compact layer of charge or Stern consists of ions rigidly bonded to the cell and the double layer includes ions diffusely distributed around the cell. Measurement of RBC layer of charge and zeta potential can be use as a marker of RBC membrane injury. Study of membrane electrical properties and forces and bindings between cells, can provide a better comprehension of membrane injury presented in RBC unit storage, after RBC unit irradiation, cell senescence process and in addition, also to improve the sensitivity and specificity of the hemoagglutination reaction. The aim of this study was to measure the RBC double layer thickness of the charge (DLC) around the cell, zeta potential (ζ) and cell aggregation force, using optical tweezers.To measure DLC (d) and zeta potential (ζ) a special chamber was built consisting of 2 platinum electrodes separated by a channel. An external electrical field (E) was applied with a voltage power supply connected to the electrodes. The optical tweezers consisted of Nd:YAG laser beam focused through the 100x objective of a microscope equipped with a minicam, which registered the trapped cell image in real time. These images were recorded in VHS and captured in a computer where they could be analyzed using Image Pro Plus software. Optical tweezers utilizes the light momentum transfer to trap the silica beads with pico Newtons forces (10−12). Ten RBC units from healthy blood donors were collected in CPDA-1. Samples were diluted (1:1000μl) in AB plasma. Silica beads were added to a RBC solution (10μl). DLC thickness (d) was obtained measuring, in the special chamber, the force on the silica bead attached to a single RBC in response to an applied voltage (50–100V). Zeta potential (ζ) was obtained by measuring the RBC terminal velocity (Tv) after being released from the optical trap at a constant voltage applied supply (100V) Tv=(εE/η) ζ; ε= medium dielectric constant; η= viscosity). The DLC was calculated by the equation Fop= (Aεζ/d)E (Fop= optical force; A= RBC area). DLC median size (d) was 0,85 μm (min 0.4/max 1.3) and zeta potential was ζ=−12,5 mV (min- 9.3/max −15). These results were in accordance with the data in literature, where other methods to measure the same property were used.For measuring RBC aggregation, a silica bead attached to RBCs was trapped and the force needed to slide one RBC over the other, as a function of the velocities (2,5,7,12,18,32X10−7m/s) was determined. RBC O Rh(D) positive were diluted (1:1000μl) in plasma AB (control group) and in agglutination potentiator solutions, used in immunohematological tests (dextran 0.15%, low ionic strength solution-LISS, bromelain and papain enzymes). Aggregation was also analyzed using anti-D antibodies serum (5μl). The median aggregation force obtained of 20 RBC control samples was 1x10−3 (0.1–2.5) poise.cm. Samples analyzed with dextran 0.15%, LISS and bromelain lacking antibodies showed no agglutination. RBCs treated with papain enzyme presented high adherence preventing the capability of separating cells. The 20 samples analyzed with anti-D serum showed median aggregation force 2x10−3 (1.0–4.0) poise.cm. These results obtained from RBC with antibodies were statistically higher (p<0.001) when compared to control. The method revealed to be sensitive and results were in accord with serological tests used in transfusionroutine, where false positive results could be frequently found in RBC treated with papain, however rarely obtained with dextran and bromelain. In conclusion, the methodologies proposed are simple and may provide specific information on RBC interaction in addition to cell aggregation. Individual cell analyze methods are always more sensitive to small differences than those based on average values. This is the first time that the size of the double layer was measured using optical tweezers and the ability of the optical trap also allowed us to obtain the zeta potential and DLC thickness for the same RBC.

Modification of Single Walled Carbon Nanotube Surface Chemistry to Improve Aqueous Solubility and Enhance Cellular Interactions

Single walled carbon nanotubes (SWNTs) continue to demonstrate the potential of nanoscaled materials in a wide range of applications. The ability to modulate the mechanical or electrical properties of a material by varying the SWNT component may result in diverse "application tunable" materials. Similarly, biomaterials used in tissue engineering applications may benefit from these characteristics by varying electrical and mechanical properties to enhance or direct tissue specific regeneration. The interactions between SWNTs and cellular systems need to be optimized to integrate these highly hydrophobic nanoparticles within an aqueous environment while maintaining their unique properties. We assessed solubility, conductance, and cellular interactions between four different SWNT preparations (unrefined, refined, and SWNT with either albumin or human plasma adsorbed). Initial interactions between cells and SWNTs were assessed within a 3D environment using a red blood cell lysis model, with longer-term interactions assessing the effects on PC12 and 3T3 fibroblast function when cultured on SWNT-collagen composite hydrogels. After SWNT purification, the lytic effect on red blood cells (RBCs) is significantly reduced from 11% to 0.7%, indicating manufacturing contaminants play a significant role in undesirable cell interactions. Nanotubes with either human plasma or albumin physisorbed onto the nanotube surface were significantly more hydrophilic than either unrefined or refined preparations and displayed improved RBC interactions. Despite improved dispersion, purification, and adsorption of either plasma or albumin, SWNTs caused a significant reduction in conductance. Although the molecular interactions occurring at the cell membrane remain unclear, these investigations have identified two main factors contributing to membrane failure: manufacturing impurities and to a lesser extend the material's innate hydrophobicity. Although purification is a critical step to remove toxic manufacturing contaminants, care must be taken to ensure improved aqueous dispersion does not compromise desirable mechanical and electrical attributes.

Radial dispersion of red blood cells in blood flowing through glass capillaries: The role of hematocrit and geometry

The flow properties of blood in the microcirculation depend strongly on the hematocrit (Hct), microvessel geometry, and cell properties. Previous in vitro studies have measured the radial displacement of red blood cells (RBCs) at concentrated suspensions using conventional microscopes. However, to measure the RBCs motion they used transparent suspensions of ghost red cells, which may have different physical properties than normal RBCs. The present study introduces a new approach (confocal micro-PTV) to measure the motion of labeled RBCs flowing in concentrated suspensions of normal RBCs. The ability of confocal systems to obtain thin in-focus planes allowed us to measure the radial position of individual RBCs accurately and to consequently measure the interaction between multiple labeled RBCs. All the measurements were performed in the center plane of both 50 and 100 μm glass capillaries at Reynolds numbers ( Re) from 0.003 to 0.005 using Hcts from 2% to 35%. To quantify the motion and interaction of multiple RBCs, we used the RBC radial dispersion ( D yy ). Our results clearly demonstrate that D yy strongly depends on the Hct. The RBCs exhibited higher D yy at radial positions between 0.4 and 0.8 R and lower D yy at locations adjacent to the wall (0.8–1 R) and around the middle of the capillary (0–0.2 R). The present work also demonstrates that D yy tends to decrease with a decrease in the diameter. The information provided by this study not only complements previous investigations on microhemorheology of both dilute and concentrated suspensions of RBCs, but also shows the influence of both Hct and geometry on the radial dispersion of RBCs. This information is important for a better understanding of blood mass transport mechanisms under both physiological and pathological conditions.

An Integrated Mathematical and Experimental Model for Nitric Oxide uptake by Red Blood Cells

Endothelium‐derived nitric oxide (NO) is a potent vasodilator despite the presence of hemoglobin (Hb) encapsulated in red blood cells (RBC) which has high affinity for scavenging NO (k~ 107 M−1 s−1). NO uptake by RBC‐Hb is reported to be several orders lesser than that of free Hb. Reduced uptake by RBC encapsulated Hb is attributed to several diffusional resistances for NO to react with Hb including extracellular resistance, cell free layer resistance near the vessel wall, resistance in unstirred layer surrounding RBCs and resistance by RBC membrane. The extent of contribution from each diffusional layer towards reduced NO uptake by RBCs is under intense investigation. We developed a novel stirred bioreactor with a headspace to maintain controlled NO concentrations. Using this bioreactor along with an electrochemical NO sensor, we measured the bulk NO concentration in RBC suspensions DI water for various NO concentrations in the bioreactor headspace. We developed a steady state one dimensional mathematical model representing NO biotransport using the data obtained from above experiments to predict NO concentration profile and to determine the role of extracellular diffusional resistance, resistance offered by unstirred layer surrounding RBCs and resistance offered by RBC membrane. This integrated analysis will provide quantitative information about NO_RBC interaction parameters.

Non-classical processes in surface hemostasis: mechanisms for the poly-N-acetyl glucosamine-induced alteration of red blood cell morphology and surface prothrombogenicity

It is well established that platelets and the intrinsic plasma coagulation pathway can be activated when blood contacts artificial surfaces. Experiments were performed to assess the effect of hemostatic poly-N-acetyl glucosamine (pGlcNAc) nanofibers on red blood cells. The pGlcNAc nanofibers, isolated from a marine diatom, interact with red blood cells (RBCs) to produce stomatocytes. The stomatocytes could be converted to echinocytes by treatment with echinocytic reagents, as measured by electron microscopy. Electrophoretic and Western blot analysis of RBC surface proteins demonstrated that pGlcNAc fibers were bound to band 3 of the RBC. An important and unique result of the interaction of RBCs with pGlcNAc fibers was the activation of the intrinsic coagulation cascade. This prothrombotic effect was associated with the presentation of phosphatidylserine on the outer layer of the surface membrane of nanofiber bound RBCs. The results demonstrate that RBCs can play a direct and important role in achieving surface hemostasis by accelerating the generation of thrombin, and add to the growing body of evidence that RBCs can strongly interact with hemostatic systems.

Sickle Cell Vaso-Occlusion Is Triggered by E-Selectin Ligand Signaling and Propagated by the Leukocyte Integrin Mac-1.

Vasoocclusion (VOC) is the leading cause of morbidity and mortality in patients with sickle cell disease (SCD). Intravital microscopy studies in a murine model of SCD have revealed that capture of sickle red blood cells (RBC) by intravascular adherent leukocytes (WBC) plays an important role in VOC, and that deficiency in both P-and E-selectins protect from VOC. Here, we have investigated the cellular and molecular mechanisms leading to sickle RBCs interactions with adherent WBCs. Intravital microscopy analyses of the individual role of P- or E-selectin revealed, unexpectedly, a profound reduction in RBC-WBC interactions in Berkeley sickle mice lacking E-selectin (Sele−/−; >97% reduction), whereas the protection was only partial in the absence of endothelial P-selectin. Since E-selectin is expressed exclusively on the endothelium, and its deficiency does not alter WBC recruitment, we hypothesized that E-selectin might provide activation signals to neutrophils that allow them to capture RBCs. During our studies, we observed that RBC-WBC interactions are not exclusive of sickle animals but are also present in wild-type B6 mice treated with TNF-α, suggesting that this phenomenon accompanies a physiological inflammatory response. We found that RBC-WBC interactions in B6 mice occur at a lower frequency than in sickle mice and that these interactions are also reduced in Sele−/− mice (60% reduction; p<0.05). We thus reasoned that B6 mice might provide a convenient model to gain molecular insight into RBC-WBC interactions in vivo. Since PSGL-1, CD44 and ESL-1 harbor the entire E-selectin ligand activity on neutrophils in vivo (Hidalgo et al., Immunity 2007), we investigated which of these glycoproteins mediates the signals allowing RBC capture. High speed digital multichannel fluorescence intravital microscopy analyses revealed that RBC-WBC interactions were only markedly reduced in the absence of ESL-1 (63% reduction, p<0.001), but not in the absence of PSGL-1 or CD44. Further detailed image analyses mapped RBC captures at the leading edge of adherent neutrophils, an area where chemokine receptors and integrins may accumulate. Since selectin-mediated signaling is known to activate β2 integrins, we tested the role of Mac-1, whose expression and affinity are elevated in neutrophils from SCD patients. We found that RBC-WBC interactions were virtually absent in mice deficient in Mac-1 (97% reduction; p<0.0001). To determine whether E-selectin/ESL-1-mediated signaling promoted Mac-1 activation, we developed an assay to assess Mac-1 activity in real time on adherent WBCs in vivo. Albumin-coated fluorobeads bound to subsets of adherent leukocytes in TNF-α-stimulated venules of B6 mice. These interactions were Mac-1-dependent since they were ablated in Itgam−/− mice. Absence of E-selectin or ESL-1, but not P-selectin, PSGL-1 or CD44, significantly reduced Mac-1 activity (by 36% and 52%, respectively; p<0.05). In contrast, Mac-1 activation on adherent leukocytes was dramatically increased in inflamed venules of sickle mice (2.5-fold; p<0.001) and was restricted to a subset of adherent neutrophils. Preliminary experiments in which Mac-1 function is blocked in sickle animals with a monoclonal antibody revealed a reduction of RBC-WBC interactions (by 57%) compared to an isotype control antibody. Our results indicate that the binding of E-selectin to neutrophil ESL-1 promotes Mac-1 activation, which in turn mediates the capture of sickle RBCs. These findings provide attractive therapeutic targets to alleviate this devastating disease.

Rheology of red blood cell aggregation by computer simulation

The aggregation of red blood cells (RBC) induced by the interactions between RBCs is a dominant factor of the in vitro rheological properties of blood, and existing models of blood do not contain full cellular information. In this work, we introduce a new three-dimensional model that couples Navier–Stokes equations with cell interactions to investigate RBC aggregation and its effect on blood rheology. It consists of a depletion mediated aggregation model to describe the interactions of RBCs and an immersed continuum model to track the deformation/motion of RBCs in blood plasma. To overcome the large deformation of RBCs, the meshfree method is used to model the RBCs. Three important phenomena in blood rheology are successfully captured and studied via this approach: the shear rate dependence of blood viscosity, the influence of cell rigidity on blood viscosity, and the Fahraeus–Lindqvist effect. As a microscopic illustration of the shear-rate dependence of the blood’s viscoelasticity, the disaggregation of an RBC rouleau at shear rates varying between 0.125 and 24 s −1 is modeled. Lower RBC deformability and higher shear rates above 0.5 s −1 are found to facilitate disaggregation. The effective viscosities at different shear rates and for cells with different deformabilities are simulated. The numerical results are shown to agree with the reported experimental measurements. The Fahraeus–Lindqvist effect is, for the first time, studied through three-dimensional numerical simulations of blood flow through tubes with different diameters and is shown to be directly linked to axial-migration of deformable cells. This study shows that cell–cell interaction and cell deformability have significant effects on blood rheology in capillaries.

Rheologic behavior of sickle and normal red blood cell mixtures in sickle plasma: implications for transfusion therapy

Guidelines for transfusion in sickle cell disease usually define an upper hematocrit (Hct) limit of 0.30 to 0.35 to avoid blood hyperviscosity. In vitro viscosity studies of normal (AA) and sickle (SS) red blood cell (RBC) mixtures in buffer appear to confirm that this Hct limit is optimal for oxygen delivery to vascular beds as judged by the ratio of Hct to viscosity, with this ratio often termed "oxygen or RBC transport effectiveness." In the absence of plasma, however, effects due to RBC-RBC interactions mediated by plasma proteins cannot be assessed. To investigate the optimal Hct-to-viscosity ratio of RBCs in plasma, the rheologic effects of Hct (0.20-0.40), the proportion of SS RBCs (0-100%), and shear rate (1-1000/sec) for mixtures of oxygenated and deoxygenated SS and AA RBCs were evaluated in sickle plasma at 37 degrees C. RBC suspension viscosity was shear-dependent (i.e., viscosity decreased with increasing shear rate) and increased with Hct and proportion of SS RBCs. An "optimal" Hct level (defined as a maximal of the Hct-to-viscosity ratio) was seen only at shear rates above 50/sec. At lower shear rates (e.g., 5/sec), where plasma-mediated RBC-RBC interactions predominate, any increment in Hct was offset by a proportionally greater increase in viscosity, thus leading to a lower Hct-to-viscosity ratio. These results indicate the importance of plasma-mediated RBC interactions and suggest that the benefits of transfusion may vary depending on local flow rates (i.e., shear rates) and organ-specific hemodynamics.

Non-Hemolytic Antibody Induced Loss of RBC Antigen during Transfusion of Crossmatch Incompatible Blood.

Background: Transfusion of red blood cells (RBC) into patients with anti-donor RBC antibodies (crossmatch incompatible transfusion) can result in antibody mediated hemolysis. Less well appreciated is the ability of anti-RBC antibodies to specifically remove their target antigen from donor RBCs without compromising cell survival. This phenomenon has now been reported for the major clinically significant blood group antigens, including Rh, Kell, Kidd and Duffy. Although this has been described multiple times in humans, no mechanistic elucidation has been accomplished. In an effort to investigate the mechanism of this process, we describe the first animal model of non-hemolytic antibody induced RBC antigen loss.Methods: mHEL mice express the model antigen Hen Egg Lysozyme (HEL) as a cell surface protein on RBC. Since mHEL mice are on a C57BL/6 background, the mHEL antigen represents a single antigenic difference between donor RBC and recipient mice. Immunizing C57BL/6 mice with HEL/CFA results in the generation of high titer IgG anti-HEL responses rendering the mice crossmatch incompatible with mHEL RBC. This system was utilized to study the effects of transfusing mHEL RBC into crossmatch incompatible recipients.Results: Similar to the antibody induced antigen loss observed in humans, transfusion of donor mHEL RBC into crossmatch incompatible mice results in selective loss of HEL antigen from donor RBC without affecting other blood group antigens or reducing the circulatory lifespan of the donor RBC. In addition, recovered RBC that have lost their antigen have normal morphology. This process is antigen specific and occurs in mice that have received passive injections of anti-HEL antisera. A spleen is not required for antigen loss to occur. However, antigen loss does not occur in animals with a targeted deletion of the FcγIII receptor. Although polyclonal anti-HEL antisera consistently causes antigen loss, and IgG1 and IgG2b are the predominant subclasses of anti-HEL IgG in the antisera, no antigen loss is observed in response to purified monoclonal anti-HEL antibodies of the IgG1 and IgG2b subclass.Conclusion: These studies demonstrate that antibody induced antigen loss is a process that involves interaction of RBC, anti-RBC IgG and FcγIII receptors, thus providing mechanistic insight into the phenomenon of antigen loss during incompatible transfusion. The lack of antigen loss in response to monoclonal anti-HEL IgG1 or IgG2b suggests that antigen loss occurs in response to a minor IgG subtype in antisera, depends upon biological properties of the antibody (such as affinity), or that additional serum cofactors are involved.

Dynamics of Interaction of RBC with optical tweezers

It has recently been shown that a red blood cell (RBC) can be used as optically driven motor. The mechanism for rotation is however not fully understood. While the dependence on osmolarity of the buffer led us to conclude that the osmolarity dependent changes in shape of the cell are responsible for the observed rotation, role of ion gradients and folding of RBC to a rod shape has been invoked by Dharmadhikari et al to explain their observations. In this paper we report results of studies undertaken to understand the dynamics of a RBC when it is optically tweezed. The results obtained support our earlier conjecture that osmolarity dependent changes in shape of the cell are responsible for the observed rotation.

Influence of beta-amyloid fibrils on the interactions between red blood cells and endothelial cells

Alzheimer's disease is associated with vascular amyloidosis. As blood flows through the microcirculation, red blood cells (RBCs) come in contact with the vasculature. RBCs as well as endothelial cells (ECs) are known to bind beta amyloid fibrils. This suggests that a potential effect of amyloidosis may involve the interactions of RBCs with ECs lining the wall of the blood vessels mediated by amyloid fibrils. We have studied the effect of β-amyloid peptide[1–40] (Aβ1–40) fibrils on the interactions of murine RBCs with ECs derived from bovine lung microvascular endothelium (BLMVEC) as well as bovine pulmonary arterial endothelium (BPAEC) in culture. We show that the initial incorporation of Aβ fibrils onto either RBCs or ECs cause RBCs to adhere to the ECs with greater affinity for the microvascular cells than the arterial cells. In addition, there is a transfer of Aβ fibrils between the RBCs and the ECs. Both the transfer and adhesion occurs when the amyloid fibrils are on the ECs or on the RBCs. However, with the amyloid fibrils on the RBCs, the adhesion and the transfer are greater than with the fibrils on the ECs. These results suggest that amyloidosis may affect the flow of RBCs through the microcirculation and that RBCs may play a role in propagating amyloidosis through the vasculature.