The dynamics of heat production in erythrocytes of the scorpion fish (<Emphasis Type="Italic">Scorpaena porcus</Emphasis> Linnaeus, 1758) in vitro

Temperature measurements in a plastic tube isolated from external influences containing an erythrocyte suspension of the scorpion fish (Scorpaena porcus Linnaeus, 1758) showed that these red blood cells are able to generate heat. Heat release in the cell suspension was expressed by a linear temperature increase in the tube during the entire experiment. Addition of extracellular ATP (1 mg mL^–1) caused the effect of a thermal shift: a sharp temperature rise in the cell suspension for 30–60 s. We believe that the heat release was caused by hydrolysis of extracellular ATP by membrane ecto-ATPase. Inhibition of ecto-ATPase activity through the addition of EDTA (1 mM) to the erythrocyte suspension led to complete blockage of heat release; the effect of the thermal shift ceased. We assume that thermal properties of red blood cells play an important role in blood hemodynamics, especially in providing the “non-Newtonian” properties of blood. The thermal phenomena observed in suspensions of fish erythrocytes open new scientific directions in exploring the capabilities of multifunctional extracellular ATP.     

NMR water-proton spin-lattice relaxation time of human red blood cells and red blood cell suspensions

NMR water-proton spin-lattice relaxation times were studied as probes of water structure in human red blood cells and red blood cell suspensions. Normal saline had a relaxation time of about 3000 ms while packed red blood cells had a relaxation time of about 500 ms. The relaxation time of a red cell suspension at 50% hematocrit was about 750 ms showing that surface charges and polar groups of the red cell membrane effectively structure extracellular water. Incubation of red cells in hypotonic saline increases relaxation time whereas hypertonic saline decreases relaxation time. Relaxation times varied independently of mean corpuscular volume and mean corpuscular hemoglobin concentration in a sample population. Studies with lysates and resealed membrane ghosts show that hemoglobin is very effective in lowering water-proton relaxation time whereas resealed membrane ghosts in the absence of hemoglobin are less effective than intact red cells.     

Blood flow in the capillary bed

From arteries to veins, the blood has to go through the ‘capillary’ blood vessels. These blood vessels are so small that often their diameter is smaller than that of the red blood cells. Intimate interactions occur, therefore, between the blood cells and the blood vessels.

A general survey of recent works on capillary blood flow is given in this article. Some details are presented for two problems: the problem of deformation of the flexible red blood cells, their motion in the capillary blood vessels, and the pressure drop due to the red cell blood vessel interaction; and the problem of the flow of plasma ‘bolus’ between neighboring red cells.

The solution supplies many details about the microcirculation phenomenon. Taken together, a method is offered for the calculation of pressure drop in the capillary as a function of various physical parameters: the red cell volume per unit blood volume, (hematocrit), the ratio of the cell diameter to the blood vessel diameter, the ratio of the length of the blood vessel to its length, the volume of individual red cells, and a parameter relating the cell membrane elasticity, plasma viscosity and the cell velocity.     

Red blood cells initiate leukocyte rolling in postcapillary expansions: a lattice Boltzmann analysis

Leukocyte rolling on the vascular endothelium requires initial contact between leukocytes circulating in the blood and the vessel wall. Although specific adhesion mechanisms are involved in leukocyte-endothelium interactions, adhesion patterns in vivo suggest other rheological mechanisms also play a role. Previous studies have proposed that the abundance of leukocyte rolling in postcapillary venules is due to interactions between red blood cells (RBCs) and leukocytes as they enter postcapillary expansions, but the details of the fluid dynamics have not been elucidated. We have analyzed the interactions of red and white blood cells as they flow from a capillary into a postcapillary venule using a lattice Boltzmann approach. This technique provides the complete solution of the flow field and quantification of the particle-particle forces in a relevant geometry. Our results show that capillary-postcapillary venule diameter ratio, RBC configuration, and RBC shape are critical determinants of the initiation of cell rolling in postcapillary venules. The model predicts that an optimal configuration of the trailing red blood cells is required to drive the white blood cell to the wall.     

Co-expression of differentiation markers in hybrids between friend cells and lymphoid cells and the influence of the cell shape

We have studied the regulation of differentiation within the hemopoietic system by fusing mouse Friend cells (which can be induced to undergo red blood cell differentiation) to various mouse lymphomas and myelomas which express characteristic T and B lymphocyte surface antigens. Our results show that both erythroid and lymphoid differentiation markers can be co-expressed within the same cell. To determine whether this result applies to other differentiation states, we fused suspension Friend cells to three adherent fibroblast cell lines, and isolated both adherent and suspension hybrids. In fact, suspension hybrid clones were inducible for hemoglobin, whereas adherent clones were not. No obvious differences in overall chromosome balance were evident between the adherent and suspension hybrids. A similar correlation between suspension morphology and inducibility of hemoglobin was found in hybrids between suspension Friend cells and an adherent lymphoma line. These results show that different developmental programs can be coexpressed within the same hybrid cell; but the strongly adherent type of morphology is inconsistent with expression of the red blood cell phenotype, both in hybrid cells derived entirely from hemopoietic parental cells and in cells from widely different lineages.     

Unique ability of integrin alpha(v)beta 3 to support tumor cell arrest under dynamic flow conditions

Shear-resistant arrest of circulating tumor cells is required for metastasis from the blood stream. Arrest during blood flow can be supported by tumor cell interaction with attached, activated platelets. This is mediated by tumor cell integrin alpha(v)beta3 and cross-linking plasma protein ligands. To analyze the mechanism of tumor cell ligand interactions under dynamic flow conditions, we used real-time video microscopy and tested human melanoma cell binding to fibrinogen, von Willebrand Factor, or fibronectin matrices in a buffer perfusion system. When perfused at venous flow, melanoma cells arrested abruptly and began to spread immediately. This was uniquely mediated by integrin alpha(v)beta3 on all tested ligands, and required alpha(v)beta3 activation and actin polymerization. Under static conditions, alpha(v)beta3 cooperated with alpha(v)beta1 and alpha5beta1 in supporting melanoma cell adhesion to fibronectin. But even when activated, beta1 integrins did not contribute to melanoma cell arrest during flow. Soluble ligand served as a cross-linker between attached and circulating tumor cells and enhanced melanoma cell arrest. Cohesion of activated melanoma cells was restricted to the matrix surface and did not occur in suspension. We conclude that the presence of alpha(v)beta3 in a functionally activated state provides a unique advantage for circulating tumor cells by promoting tumor cell arrest in the presence of flow-dependent shear forces.     

Turbulent flow of red cells in dilute suspensions. Effect on kinetics of O2 uptake.

The turbulent flow properties of dilute (0.06% by volume) suspensions of human red blood cells in 4-mm-bore glass tubing were estimated by laser anemometry. The flow properties of the dilute red cell suspension were similar to those of a dilute suspension of polystyrene spheres (0.5 micron diameter) in isotonic NaCl solution. Flow was found to be laminar when the Reynolds number was below 2,000, transitional in the range of Reynolds numbers from 2,000 to 3,000, and fully turbulent above Reynolds number 3,000. These results differ from previous studies of more concentrated red cell suspensions. The length scales of the turbulence were also estimated: at a Reynolds number near 4,000 the macroscale is about 1.25 mm, the Taylor microscale is about 0.85 mm, and the Kolmogoroff scale is near 0.075 mm. The results are discussed in relation to previous measurements of the rate of oxygen uptake by dilute red cell suspensions in the flow-type rapid reaction apparatus. Our results suggest that under the conditions of most of these oxygen uptake measurements, the turbulent flow is characterized by eddies about 1 mm across, mixing with each other on a time scale of about 45 ms. Since most of the reported oxygen uptake measurements involve a similar time scale, it is possible that an effective "unstirred layer" influenced the reported rate of oxygen uptake.     

Lateral view flow system for studies of cell adhesion and deformation under flow conditions

Physical interactions between circulating cells and the vascular wall play a central role in inflammation, metastasis, atherosclerosis, and therapeutic cell delivery. Unfortunately, traditional in vitro flow assays cannot be used to visualize the details of cell-surface interactions in blood flow because of inappropriate geometry and the poor penetration of light in erythrocyte solutions. To overcome these obstacles, we have developed an agarose-cast cylindrical vessel system to examine the profiles of cells interacting with surfaces under flow conditions. This design allows observation and quantification of cell deformation as cells adhere to surfaces under dynamic flow conditions without modifying the microscope or optical path. Furthermore, our flow system is uniquely suited for monitoring the profiles of adherent leukocytes deforming in response to erythrocyte suspension flow. We have used this flow system to study the role of erythrocytes in leukocyte-substrate interactions. Our results show that the cell deformation index (the ratio of the cell length to cell height) is higher in erythrocyte solutions compared to erythrocyte-free saline. This novel lateral view flow system provides a powerful technique for visualizing and quantifying the morphological changes of cells in contact with substrates exposed to shear stress.     

Effect of erythrocyte aggregation on velocity profiles in venules

A recent whole organ study in cat skeletal muscle showed that the increase in venous resistance seen at reduced arterial pressures is nearly abolished when the muscle is perfused with a nonaggregating red blood cell suspension. To explore a possible underlying mechanism, we tested the hypothesis that red blood cell aggregation alters flow patterns in vivo and leads to blunted red blood cell velocity profiles at reduced shear rates. With the use of fluorescently labeled red blood cells in tracer quantities and a video system equipped with a gated image intensifier, we obtained velocity profiles in venous microvessels (45-75 microm) of rat spinotrapezius muscle at centerline velocities between 0.3 and 14 mm/s (pseudoshear rates 3-120 s(-1)) under normal (nonaggregating) conditions and after induction of red blood cell aggregation with Dextran 500. Profiles are nearly parabolic (Poiseuille flow) over this flow rate range in the absence of aggregation. When aggregation is present, profiles are parabolic at high shear rates and become significantly blunted at pseudoshear rates of 40 s(-1) and below. These results indicate a possible mechanism for increased venous resistance at reduced flows.     

Particle-fluid suspension model of blood flow through stenotic vessels with applications

The present study deals with the problem of blood flow through stenotic vessels when blood is represented by a particle-fluid suspension model, i.e. a suspension of red blood cells in plasma. The expressions for the dimensionless resistance to flow, the wall shear stress, and the shearing stress on the wall at the maximum height of the stenosis are derived. The results obtained in the analysis are discussed in brief, both qualitatively and quantitatively by comparison with other theories. It is observed that the magnitudes of the blood flow characteristics significantly increase with an increase in the red cell concentration. The importance of the decreasing vessel diameter is also pointed out. Finally, to observe the biological relevance of the analysis, the results obtained are used to compute the blood flow characteristics for normal and diseased blood using the experimental data from published literature and results are compared with those computed using the present theoretical approach.     

Particulate nature of blood determines macroscopic rheology: a 2-D lattice Boltzmann analysis

Historically, predicting macroscopic blood flow characteristics such as viscosity has been an empirical process due to the difficulty in rigorously including the particulate nature of blood in a mathematical representation of blood rheology. Using a two-dimensional lattice Boltzmann approach, we have simulated the flow of red blood cells in a blood vessel to estimate flow resistance at various hematocrits and vessel diameters. By including white blood cells (WBCs) in the flow, we also calculate the increase in resistance due to white cell rolling and adhesion. The model considers the blood as a suspension of particles in plasma, accounting for cell-cell and cell-wall interactions to predict macroscopic blood rheology. The model is able to reproduce the Fahraeus-Lindqvist effect, i.e., the increase in relative apparent viscosity as tube size increases, and the Fahraeus effect, i.e., tube hematocrit is lower than discharge hematocrit. In addition, the model allows direct assessment of the effect of WBCs on blood flow in the microvasculature, reproducing the dramatic increases in flow resistance as WBCs enter short capillary segments. This powerful and flexible model can be used to predict blood flow properties in any vessel geometry and with any blood composition.     

Numerical Simulation of Blood Flow Through Microvascular Capillary Networks

A numerical method is implemented for computing blood flow through a branching microvascular capillary network. The simulations follow the motion of individual red blood cells as they enter the network from an arterial entrance point with a specified tube hematocrit, while simultaneously updating the nodal capillary pressures. Poiseuille’s law is used to describe flow in the capillary segments with an effective viscosity that depends on the number of cells residing inside each segment. The relative apparent viscosity is available from previous computational studies of individual red blood cell motion. Simulations are performed for a tree-like capillary network consisting of bifurcating segments. The results reveal that the probability of directional cell motion at a bifurcation (phase separation) may have an important effect on the statistical measures of the cell residence time and scattering of the tube hematocrit across the network. Blood cells act as regulators of the flow rate through the network branches by increasing the effective viscosity when the flow rate is high and decreasing the effective viscosity when the flow rate is low. Comparison with simulations based on conventional models of blood flow regarded as a continuum indicates that the latter underestimates the variance of the hematocrit across the vascular tree.     

Geometrical focusing of cells in a microfluidic device: an approach to separate blood plasma

It is well known that when a suspension of cells flows in small vessels (arterioles or venules), there exists a cell-free layer of a few microns adjacent to the vascular walls. Using an in vitro model, we show experimentally that for a fixed flow rate a geometrical constriction in the flow can artificially enhance the cell-free layer. Also, we show that rapid variation of the geometry coupled to the deformability of the cells can dramatically modify their spatial distribution in the channel. The effects of the constriction geometry, flow rate, suspending fluid viscosity, cell concentration, and cell deformability are studied and the results are interpreted in terms of a model of the hydrodynamic drift of an ellipsoidal cell in a shear flow. We propose a microfluidic application of this focusing effect for separation of the red blood cells from the suspending plasma.     

Mesoscale simulation of blood flow in small vessels

Computational modeling of blood flow in microvessels with internal diameter 20-500 microm is a major challenge. It is because blood in such vessels behaves as a multiphase suspension of deformable particles. A continuum model of blood is not adequate if the motion of individual red blood cells in the suspension is of interest. At the same time, multiple cells, often a few thousands in number, must also be considered to account for cell-cell hydrodynamic interaction. Moreover, the red blood cells (RBCs) are highly deformable. Deformation of the cells must also be considered in the model, as it is a major determinant of many physiologically significant phenomena, such as formation of a cell-free layer, and the Fahraeus-Lindqvist effect. In this article, we present two-dimensional computational simulation of blood flow in vessels of size 20-300 microm at discharge hematocrit of 10-60%, taking into consideration the particulate nature of blood and cell deformation. The numerical model is based on the immersed boundary method, and the red blood cells are modeled as liquid capsules. A large RBC population comprising of as many as 2500 cells are simulated. Migration of the cells normal to the wall of the vessel and the formation of the cell-free layer are studied. Results on the trajectory and velocity traces of the RBCs, and their fluctuations are presented. Also presented are the results on the plug-flow velocity profile of blood, the apparent viscosity, and the Fahraeus-Lindqvist effect. The numerical results also allow us to investigate the variation of apparent blood viscosity along the cross-section of a vessel. The computational results are compared with the experimental results. To the best of our knowledge, this article presents the first simulation to simultaneously consider a large ensemble of red blood cells and the cell deformation.     

Melittin lysis of red cells

Summary This paper describes experiments designed to explore interactions between human red blood cell membranes and melittin, the main component of bee venom. We found that melittin binds to human red cell membranes suspended in isotonic NaCl at room temperature, with an apparent dissociation constant of 3×10^–8 m and maximum binding capacity of 1.8×10⁷ molecules/cell. When about 1% of the melittin binding sites are occupied, cell lysis can be observed, and progressive, further increases in the fraction of the total sites occupied lead to progressively greater lysis in a graded manner. 50% lysis occurs when there are about 2×10⁶ molecules bound to the cell membrane. For any particular extent of melittin binding, lysis proceeds rapidly during the first few minutes but then slows and stops so that no further lysis occurs after one hour of exposure of cells to melittin. The graded lysis of erythrocytes by melittin is due to complete lysis of some of the cells, since both the density and the hemoglobin content of surviving, intact cells in a suspension that has undergone graded melittin lysis are similar to the values observed in the same cells prior to the addition of melittin. The cells surviving graded melittin lysis have an increased Na and reduced K, proportional to the extent of occupation of the melittin binding sites. Like lysis, Na accumulation and K loss proceed rapidly during the first few minutes of exposure to melittin but then stops so that Na, K and hemoglobin content of the cells remain constant after the first hour. These kinetic characteristics of both lysis and cation movements suggest that melittin modifies the permeability of the red cell membrane only for the first few minutes after the start of the interaction. Direct observation of cells by Nomarsky optics revealed that they crenate, become swollen and lyse within 10 to 30 sec after these changes in morphology are first seen. Taken together, these results are consistent with the idea that melittin produces lysis of human red cells at room temperature by a colloid osmotic mechanism.     

Measurement of the erythrocyte orientation in a flow by spin labeling

When a suspension of erythrocytes labeled in their membrane with a fatty acid paramagnetic molecule is allowed to flow in a flat quartz sample cell, the recorded electron paramagnetic spectra change as a function of the orientation of the cell in the magnetic field. This indicates that the red cells are themselves oriented in the flow. Such spectral variations have been reproduced by a numerical simulation procedure, which allowed us to quantify the proportion of oriented red blood cells by measuring the amplitude of some characteristic lines on the experimental spectra. Orientation rates were then measured as a function of various rheological parameters, such as shear rate, hematocrit and viscosity of the suspending medium. The kinetics of the disorientation process was determined by stopping the flow.     

Effects of sedimentation of small red blood cell aggregates on blood flow in narrow horizontal tubes

The flow properties of aggregating red cell suspensions flowing at low flow rates through horizontal tubes are analyzed using a theoretical model. The effects of sedimentation of small aggregates, which will be formed at comparatively high flow rates, on the relative apparent viscosity are considered. In the case in which a large number of small aggregates are formed in a suspension flowing through a horizontal tube, it seems that red cells are transported as a concentrated suspension through the bottom part of the tube because of sedimentation of aggregates. A two-layer flow model is used for the distribution of red cells. It consists of plasma in the upper part and a concentrated red cell suspension in the bottom part of the tube divided by a smooth and horizontal interface. It is assumed that the suspension is a Newtonian fluid whose viscosity increases exponentially with hematocrit. The velocity distribution, the relative apparent viscosity and the flux of red cells are calculated as functions of width of plasma layer for a different discharge hematocrit. The theoretical results are compared with the results obtained from experimental data. The relative apparent viscosity increases rapidly with an increasing degree of sedimentation over a wide range of plasma layer widths.     

Studies of fluids simulating blood-like rheological properties and applications in models of arterial branches

We studied several non-Newtonian fluids to determine how closely they simulate the flow behavior of human blood. The viscous and viscoelastic properties of these fluids were compared with human blood samples in steady flow and transient flow Couette viscometers and in an oscillatory tube flow viscoelasticity analyzer. We examined: 1) A polyacrylamide suspension (Separan AP30 and AP45) to which we added 4% isopropanol and 0.01% magnesium chloride. 2) A suspension of 2% Dextran with 16% by weight biconcave disc-shaped particles simulating red blood cells. 3) 40% ghost cells prepared according to Dodge in Tri (hydroxymethyl) aminomethane. These ghost cells were used to simulate the two-phase flow behavior of blood. 4) A suspension of 5% Dextran (70,000) with 12% polystyrene particles (diameter of 1 micron) and 10 mMol calcium chloride. All these fluids closely approximate the flow behavior of blood and can be used in a variety of different experimental situations. To measure velocity distribution using a laser-Doppler-anemometer, we used fluids #1 and #3 in a rigid T-junction simulating the first septal branch of the left descending coronary artery. The measurements were done in steady and pulsatile flow experiments at different flow rate ratios. The fluids showed large differences in velocity profiles compared to Newtonian fluids.     

Microfluidic Study of Enhanced Deposition of Sickle Cells at Acute Corners

Sickle cell anemia is a blood disorder, known to affect the microcirculation and is characterized by painful vaso-occlusive crises in deep tissues. During the last three decades, many scenarios based on the enhanced adhesive properties of the membrane of sickle red blood cells have been proposed, all related to a final decrease in vessels lumen by cells accumulation on the vascular walls. Up to now, none of these scenarios considered the possible role played by the geometry of the flow on deposition. The question of the exact locations of occlusive events at the microcirculatory scale remains open. Here, using microfluidic devices where both geometry and oxygen levels can be controlled, we show that the flow of a suspension of sickle red blood cells around an acute corner of a triangular pillar or of a bifurcation, leads to the enhanced deposition and aggregation of cells. Thanks to our devices, we follow the growth of these aggregates in time and show that their length does not depend on oxygenation levels; instead, we find that their morphology changes dramatically to filamentous structures when using autologous plasma as a suspending fluid. We finally discuss the possible role played by such aggregates in vaso-occlusive events.     

Interaction of cationic carbosilane dendrimers and their complexes with siRNA with erythrocytes and red blood cell ghosts

We have investigated the interactions between cationic NN16 and BDBR0011 carbosilane dendrimers with red blood cells or their cell membranes. The carbosilane dendrimers used possess 16 cationic functional groups. Both the dendrimers are made of water-stable carbon–silicon bonds, but NN16 possesses some oxygen–silicon bonds that are unstable in water. The nucleic acid used in the experiments was targeted against GAG-1 gene from the human immunodeficiency virus, HIV-1.By binding to the outer leaflet of the membrane, carbosilane dendrimers decreased the fluidity of the hydrophilic part of the membrane but increased the fluidity of the hydrophobic interior. They induced hemolysis, but did not change the morphology of the cells. Increasing concentrations of dendrimers induced erythrocyte aggregation. Binding of short interfering ribonucleic acid (siRNA) to a dendrimer molecule decreased the availability of cationic groups and diminished their cytotoxicity. siRNA–dendrimer complexes changed neither the fluidity of biological membranes nor caused cell hemolysis. Addition of dendriplexes to red blood cell suspension induced echinocyte formation.