Dielectric approach to investigation of erythrocyte aggregation. II. Kinetics of erythrocyte aggregation-disaggregation in quiescent and flowing blood

A method based on dielectric properties of dispersed systems was applied to investigate the kinetics of RBC aggregation and the break-up of the aggregates. Experimentally, this method consists of measuring the capacitance at a frequency in the beginning of the beta-dispersion. Two experimental protocols were used to investigate the aggregation process. In the first case, blood samples were fully dispersed and then the flow was decreased or stopped to promote RBC aggregation. It was found that the initial phases of RBC aggregation are not affected by the shear rate. This finding indicates that RBC aggregation is a slow coagulation process. In the second case, RBCs aggregated under flow conditions at different shear rates and after the capacitance reached plateau levels, the flow was ceased. The steady-state capacitance of the quiescent blood and the kinetics of RBC aggregation after stoppage of shearing depend on the prior shear rate. To clarify the reasons for this effect, the kinetics of the disaggregation process was studied. In these experiments, time courses of the capacitance were recorded under different flow conditions and then a higher shear stress was applied to break up RBC aggregates. It was found that the kinetics of the disaggregation process depend on both the prior and current shear stresses. Results obtained in this study and their analysis show that the kinetics of RBC aggregation in stasis consists of two consecutive phases: At the onset, red blood cells interact face-to-face to form linear aggregates and then, after an accumulation of an appropriate concentration of these aggregates, branched rouleaux are formed via reactions of ends of the linear rouleaux with sides of other rouleaux (face-to-side interactions). Branching points are broken by low shear stresses whereas dispersion of the linear rouleaux requires significantly higher energy.     

Effects of hemolysis, hematocrit, RBC swelling, and flow rate on light scattering by blood in a 0.26 cm ID transparent tube

The intensity of light scattering by blood in a tube of diameter 0.26 cm was measured with an apparatus devised by us at different angles on an incident cross-sectional plane. Changes in angular distribution of light intensity associated with hemolysis, and changes in hematocrit (Ht), red blood cell (RBC) swelling, and flow rate were plotted on polar coordinates. The dyssymmetry index, defined as the ratio of the intensity of light at 45 degrees to that at 135 degrees, was used to characterize the shape of the diagrams of light scattering. The index decreased with Ht, flow rate, but increased with RBC swelling. It is concluded that light scattering by blood requires intactness of the RBC membrane. Even when the cell membrane is intact, light scattering is subject to changes with the flow rate of blood, presumably due to RBC aggregation.     

Rheological properties of the blood influencing selectin-mediated adhesion of flowing leukocytes

We studied how the rheological properties of blood influenced capture and rolling adhesion of leukocytes as well as their margination in the bloodstream. When citrated, fluorescently labeled blood was perfused through glass capillaries coated with P-selectin, leukocytes formed numerous rolling attachments. The number of attached leukocytes increased as the hematocrit was increased between 10% and 30% and was essentially constant from 30% to 50%. In EDTA-treated blood, adhesion was absent, and the flux of marginated cells varied little with increasing hematocrit. However, the velocity of marginated leukocytes increased monotonically, whereas the volumetric flow rate was constant, implying that the flow velocity profile became blunted and wall shear rate increased. Thus increasing hematocrit promoted attachment for a given total flow rate, without increasing margination, even though wall shear rate and blood viscosity increased. Blood was diluted to 20% hematocrit with plasma, 40-kDa dextran (to reduce red blood cell aggregation), or 500-kDa dextran (to enhance aggregation). Increasing aggregation correlated with increasing leukocyte adhesion and with more slow-flowing leukocytes near the wall. Thus flowing erythrocytes promote leukocyte adhesion, either by causing margination of leukocytes or by initiating and stabilizing attachments that follow.     

Evaluation of a method to assess red blood cell aggregation

Reversible aggregation of red blood cells (RBC) plays an important role in determining the flow properties of blood. To study different factors affecting RBC aggregation we used a new commercially available erythro-aggregameter (SEFAM, Nancy, France). The method allows the photometric quantitation of the kinetics of RBC aggregation and the estimation of the shear resistance of the aggregates. Modification of the hematocrit acts mostly on the determination of the disaggregation shear rate, while plasma composition strongly affects all measurements. Anticoagulants per se do no influence the aggregation process, but can alter the value of the parameters through a plasma dilution effect. Presence of white blood cells and platelets in the sample did not modify the data. Study on the effects of low concentration of heparin and low molecular weight heparin showed that at therapeutical doses these drugs did not alter the values of the aggregation parameters. Provided that precise guidelines are followed for the processing of blood samples, this method may serve to investigate RBC aggregation in various diseases and to search for adequate hemorheologic treatment.     

Rheology of Human Blood, near and at Zero Flow: Effects of Temperature and Hematocrit Level

Static normal human blood possesses a distinctive yield stress. When the yield stress is exceeded, the same blood has a stress-shear rate function under creeping flow conditions closely following Casson's model, which implies reversible aggregation of red cells in rouleaux and flow dominated by movement of rouleaux. The yield stress is essentially independent of temperature and its cube root varies linearly with hematocrit value. The dynamic rheological properties in the creeping flow range are such that the relative viscosity of blood to water is almost independent of temperature. Questions raised by these data are discussed, including red cell aggregation promoted by elements in the plasma.     

The superposition of steady on oscillatory shear and its effect on the viscoelasticity of human blood and a blood-like model fluid

To understand the pulsatility of human blood flow in vivo, it is necessary to separately investigate (1) steady shear and oscillatory flow, and (2) the superposition of steady shear flow on oscillatory flow performed under in vitro conditions. In this study a variable steady shear rate was superimposed in parallel on oscillatory shear at a constant frequency (0.5 Hz) for human blood (45% hematocrit), and an aqueous polyacrylamide polymer solution (AP 30E, concentration 300 ppm). The effect of superposition of the above two shear flows on the viscoelasticity of blood was more pronounced for the elastic (η′') than for the viscous (η′) component of viscoelasticity. With increasing superimposed shear rate, both η′ and η′' decreased, especially at the low shear region. This behavior can be explained by the viscoelastic properties of blood and the phenomena of blood aggregation and disaggregation. Quantitatively, the dependence of the viscous component of complex viscosity on superimposed shear for both blood and polymer solution is described by a modified Carreau equation. The elastic component of complex viscosity decreased exponentially with increasing superimposed shear, and it is described by an exponential model. © 1997 Elsevier Science Ltd     

Magnetic resonance microscopy determined velocity and hematocrit distributions in a Couette viscometer

Magnetic resonance microscopy is used to non-invasively measure the radial velocity distribution in Couette flow of erythrocyte suspensions of varying aggregation behavior at a nominal shear rate of 2.20 s(-1) in a 1 mm gap. Suspensions of red blood cells in albumin-saline, plasma and 1.48% Dextran added plasma at average hematocrits near 0.40 are studied, providing a range of aggregation ability. The spatial distribution of the red blood cell volume fraction, hematocrit, is calculated from the velocity distribution. The hematocrit profiles provide direct measure of the thickness of the aggregation and shear rate dependent red blood cell depletion at the Couette surfaces. At the nominal shear rate studied hematocrit distributions for the red blood cells in plasma show a depletion zone near the inner Couette wall but not the outer wall. The red blood cells in plasma with Dextran show cell depletion regions of approximately 100 mum at both the inner and outer Couette surfaces, with greater depletion at the inner wall, but approach the normal blood hematocrit distribution with a doubling of shear rate due to decreased aggregation. The material response of the blood is spatially dependent with the shear rate and the hematocrit distribution non-uniform across the gap.     

Assessment of red blood cell aggregation with dextran by ultrasonic interferometry.

Aggregation of human red blood cells (RBCs) induced by dextrans of various molecular weight has been studied by using a new ultrasonic interferometry method. This method, based on A-mode echography, allowed for the measurement of the accumulation rate of particles on a solid plate which is related to their sedimentation rate (i.e., to their mean size). The initial aggregation process, the mean and the maximum sedimentation rate of aggregates and the packing of the sedimented RBCs have been investigated. Effects of hematocrit, molecular weight of dextrans and inhibition by dextran 40 on the RBC aggregation induced by dextran of higher molecular weight have been determined by analysing variations of the aggregate size. Results obtained confirm the aggregation effect of dextrans of molecular weights equal or higher than 70,000 dalton and disaggregation effect of dextran 40,000 dalton on aggregation by dextrans of higher molecular weight.     

Blood low shear rate rheometry: influence of fibrinogen level and hematocrit on slip and migrational effects.

Red blood cell (RBC) aggregation is of prime importance in vivo and in vitro for low flow rates. It may be estimated by rheometrical measurements at low shear rates, but these are perturbed by slip and migrational effects which have already been highlighted in the past. These effects lead to a torque decay with time so that the true value of the stress at low shear rates may be greatly underestimated. Elevated aggregation being associated with different diseases, pathological blood samples show more pronounced perturbing effects and a strong time dependency in low shear rate rheometry. To test the dependence of slip and migrational effects on RBC aggregation, and particularly to determine the way in which they depend upon fibrinogen concentration ([Fb]), a home-made measuring system with roughened internal and external walls (170 microns roughness) was used to study low shear rate rheometry for RBC suspensions in PBS buffer containing albumin (at 50 g/l) and fibrinogen at various concentrations. The influences of hematocrit, shear rate, and fibrinogen concentration were investigated. Particular attention was paid to data acquisition at low shear rates (10(-3) s-1 to 3 x 10(-2) s-1). The combined influence of hematocrit and fibrinogen was investigated by adjusting hematocrit to 44 or 57% and fibrinogen concentration ([Fb]) to 3.0-4.5-6.5 g/l. Microscopic observations of the blood samples at rest were performed. They showed that different structures were formed according to fibrinogen concentration. The rheometrical measurements indicated that torque decay with shearing duration was strongly dependent on fibrinogen concentration and on shear rate at fixed hematocrit. Migrational and slip effects were more pronounced as shear rate decreased, fibrinogen concentration was raised, and hematocrit was lowered. The results have been explained on the basis of the expected microstructure of flowing blood in relation to the microscopic observations at rest.     

Effects of flowing RBCs on adhesion of a circulating tumor cell in microvessels

Adhesion of circulating tumor cells (CTCs) to the microvessel wall largely depends on the blood hydrodynamic conditions, one of which is the blood viscosity. Since blood is a non-Newtonian fluid, whose viscosity increases with hematocrit, in the microvessels at low shear rate. In this study, the effects of hematocrit, vessel size, flow rate and red blood cell (RBC) aggregation on adhesion of a CTC in the microvessels were numerically investigated using dissipative particle dynamics. The membrane of cells was represented by a spring-based network connected by elastic springs to characterize its deformation. RBC aggregation was modeled by a Morse potential function based on depletion-mediated assumption, and the adhesion of the CTC to the vessel wall was achieved by the interactions between receptors and ligands at the CTC and those at the endothelial cells forming the vessel wall. The results demonstrated that in the microvessel of \(15\,\upmu \hbox {m}\) diameter, the CTC has an increasing probability of adhesion with the hematocrit due to a growing wall-directed force, resulting in a larger number of receptor–ligand bonds formed on the cell surface. However, with the increase in microvessel size, an enhanced lift force at higher hematocrit detaches the initial adherent CTC quickly. If the microvessel is comparable to the CTC in diameter, CTC adhesion is independent of Hct. In addition, the velocity of CTC is larger than the average blood flow velocity in smaller microvessels and the relative velocity of CTC decreases with the increase in microvessel size. An increased blood flow resistance in the presence of CTC was also found. Moreover, it was found that the large deformation induced by high flow rate and the presence of aggregation promote the adhesion of CTC.     

Optimal hematocrit theory during activity in the bullfrog (Rana catesbeiana).

1. There is an exponential relationship between blood viscosity (cP) and hematocrit (%) for the bullfrog; eta = 1.81 e0.033Hct. The in vitro optimal hematocrit calculated for blood flow through tubes, from this relationship for bullfrog blood, is 30%. 2. Amphibian blood is a non-Newtonian fluid with viscosity dependent on shear rate. It has a finite yield shear stress of about 1.5 dynes cm-2. 3. Hematocrit of bullfrogs was increased from 27% (control) to 57% by isovolemic erythrocythemia (constant volume blood-doping). There was a slight increase in systolic, diastolic and venous blood pressure with elevated hematocrit. 4. Systemic arch blood flow rate was inversely related to blood viscosity for erythrocythemic bullfrogs. The decrease in systemic arch blood flow at high hematocrits was due primarily to reduced pulse volume rather than reduced heart rate. 5. Systemic arch blood flow, when standardised between individuals, was inversely related to blood viscosity; Qbl = 0.185 + 3.73 eta -1. This relationship was significantly different from that predicted by the Poiseuille-Hagen flow formula. The in vivo optimal hematocrit calculated from this relationship was 41%. 6. Optimal hematocrit theory appears to be generally applicable for Rana catesbeiana in vitro and in vivo. Most individuals had an in vivo optimal hematocrit, but the absence of a clear optimal hematocrit for some individuals could reflect methodological variability, or in vivo physiological compensation for the increased blood viscosity at high hematocrit.     

Correlated measurement of platelet release and aggregation in whole blood

We have used a technique for the simultaneous measurement of platelet activation and aggregation in whole blood using two-color immunofluorescence and flow cytometry to study the relationship between the release reaction and aggregation. A monoclonal antibody specific for the alpha granule membrane protein GMP-140 was used to measure the release reaction, and a monoclonal antibody specific for platelet membrane glycoprotein Ib (GPIb) was used to identify platelets and platelet aggregates. Aggregates were identified as particles expressing both levels of GPIb and size larger than that of resting single platelets. Anticoagulated whole blood was incubated with platelet agonists. At various times samples of the blood were removed and immediately fixed with paraformaldehyde. Blood that had been anticoagulated with ethylenediamine tetraacetic acid showed progressive release of platelets but little or no aggregation. However, blood anticoagulated with citrate or heparin showed correlated release and aggregation. The degree of aggregation was greater in heparin than in citrate. The expression of GPIb and GMP-140 increased in direct proportion to the size of the aggregates. Aggregates were observed varying in apparent diameter up to approximately 20 microns. During prolonged incubation there was progressive disaggregation of adenosine diphosphate (ADP)-induced aggregates. After disaggregation the proportion of GMP-140 negative single platelets increased, indicating that both released and nonreleased platelets participated in the aggregation. There was little or no disaggregation of phorbol myristate acetate (PMA)-induced aggregates. The relatively small size and reversibility of platelet aggregates that we have observed in whole blood may be relevant to phenomena occurring in vivo and in extracorporeal circulation.(ABSTRACT TRUNCATED AT 250 WORDS)     

Influence of erythrocyte aggregation on radial migration of platelet-sized spherical particles in shear flow

Blood platelets when activated are involved in the mechanisms of hemostasis and thrombosis, and their migration toward injured vascular endothelium necessitates interaction with red blood cells (RBCs). Rheology co-factors such as a high hematocrit and a high shear rate are known to promote platelet mass transport toward the vessel wall. Hemodynamic conditions promoting RBC aggregation may also favor platelet migration, particularly in the venous system at low shear rates. The aim of this study was to confirm experimentally the impact of RBC aggregation on platelet-sized micro particle migration in a Couette flow apparatus. Biotin coated micro particles were mixed with saline or blood with different aggregation tendencies, at two shear rates of 2 and 10 s⁻¹ and three hematocrits ranging from 20 to 60%. Streptavidin membranes were respectively positioned on the Couette static and rotating cylinders upon which the number of adhered fluorescent particles was quantified. The platelet-sized particle adhesion on both walls was progressively enhanced by increasing the hematocrit (p < 0.001), reducing the shear rate (p < 0.001), and rising the aggregation of RBCs (p < 0.001). Particle count was minimum on the stationary cylinder when suspended in saline at 2 s⁻¹ (57 ± 33), and maximum on the rotating cylinder at 60% hematocrit, 2 s⁻¹ and the maximum dextran-induced RBC aggregation (2840 ± 152). This fundamental study is confirming recent hypotheses on the role of RBC aggregation on venous thrombosis, and may guide molecular imaging protocols requiring injecting active labeled micro particles in the venous flow system to probe human diseases.     

The "black hole" phenomenon in ultrasonic backscattering measurement under pulsatile flow with porcine whole blood in a rigid tube

The "black hole" phenomenon was further investigated with porcine whole blood under pulsatile flow conditions in a straight rigid tube 120 cm long and of 0.95 cm diameter. A modified Aloka 280 commercial scanner with a 7.5 MHz linear array was used to collect the radio frequency (RF) signal of backscattering echoes from the blood inside the tube. The transducer was located downstream from the entrance and parallel to the longitudinal direction of the tube. The experimental results showed that higher hematocrits enhanced the black hole phenomenon, leading to a more apparent and larger diameter black hole. The black hole was not apparent at hematocrits below 23%. The highest hematocrit used in the experiment was 60%. Beat rates of 20, 40 and 60 beats per minute (bpm) were used, and the black hole became weaker in amplitude and smaller in diameter when the peak flow velocity was increased at each beat rate. These results are consistent with the suggestion in previous work that the black hole arises from insufficient aggregation of red blood cells (RBCs) at the center of the tube because of the low shear rate. At 20 and 40 bpm, the peak flow velocity ranges were 10 approximately 25 cm/s and 18 approximately 27 cm/s, respectively. The black hole was very clear at the minimal peak flow velocity but almost disappeared at the maximal velocities for each beat rate. At 60 bpm, experiments were only performed at one peak flow velocity of 31 cm/s and the black hole was clear. The results showed that the black hole was more pronounced at higher beat rates when the peak velocity was the same. This phenomenon cannot be explained by previous hypotheses. Acceleration seems to be the only flow parameter that varies at different beat rates when peak velocities are the same. Therefore, the influence of acceleration on the structural organization and orientation of RBC rouleaux might be another factor involved in the formation of the black hole in addition to the shear rate. As the entrance length was changed from 110 to 15 diameters (D) in seven steps at the hematocrit of 60%, it was found that a position farther downstream yielded a black hole with a greater contrast relative to the surrounding region, while the backscattering power at the central hypoechoic zone did not increase with increasing entrance length.     

Effect of enhanced red blood cell aggregation on blood flow resistance in an isolated-perfused guinea pig heart preparation

The role of red blood cell (RBC) aggregation as a determinant of in vivo blood flow is still unclear. This study was designed to investigate the influence of a well-controlled enhancement of RBC aggregation on blood flow resistance in an isolated-perfused heart preparation. Guinea pig hearts were perfused through a catheter inserted into the root of the aorta using a pressure servo-controlled pump system that maintained perfusion pressures of 30 to 100 mmHg. The hearts were beating at their intrinsic rates and pumping against the perfusion pressure. RBC aggregation was increased by Pluronic (F98) coating of RBC at a concentration 0.025 mg/ml, corresponding to about a 100% increment in RBC aggregation as measured by erythrocyte sedimentation rate. Isolated heart preparations were perfused with 0.40 l/l hematocrit unmodified guinea pig blood and with Pluronic-coated RBC suspensions in autologous plasma. At high perfusion pressures there were no significant differences between the flow resistance values for the two perfusates, with differences in flow resistance only becoming significant at lower perfusion pressures. These results can be interpreted to reflect the shear dependence of RBC aggregation: higher shear forces associated with higher perfusion pressures should have dispersed RBC aggregates resulting in blood flow resistances similar to control values. Experiments repeated in preparations in which the smooth muscle tone was inhibited by pre-treatment with papaverine indicated that significant effects of enhanced RBC aggregation could be detected at higher perfusion pressures, underlining the compensatory role of vasomotor control mechanisms.     

Neutrophil aggregation is beta 2-integrin- and L-selectin-dependent in blood and isolated cells.

Neutrophil aggregation in response to formyl peptide was analyzed in blood and isolated cells by fluorescence flow cytometry. The isolated leukocyte aggregates and the leukocytes in blood were identified with the vital nucleic acid stain LDS-751. This method enabled us to discriminate nucleated cells from other blood cells and to detect granulocyte aggregates without isolation or E lysis. Cells isolated in the absence of endotoxin retained the characteristics of cells in blood and exhibited similar aggregation kinetics and dose-response to formyl peptide. We show that it is possible to analyze epitope expression in blood with homogeneous flow cytometric assays and that carefully isolated neutrophils retain the expression characteristics of those in blood. The expression of CD18 was at its lowest levels in unstimulated cells, while the rate of formyl peptide stimulated aggregation was most rapid in these cells. Aggregation in isolated cells as well as blood preceded an increase in receptor expression. After stimulation, L-selectin expression decreased in both blood and isolated cells over a time frame similar to disaggregation. The aggregation response in blood was blocked by pretreatment with antibody to CD18 over a concentration range consistent with the amount of antibody bound. Aggregation was also blocked in isolated cells and blood by antibodies DREG-200 and DREG-56 to L-selectin, but not by isotype controls or anti-LFA-1. The results are discussed in terms of the roles of adhesive receptor expression and recognition in neutrophil aggregation. The methods validated here permit linkage between isolated cells and in vivo studies.     

Aggregation of human red blood cells after moderate heat treatment

The aggregation behaviour of normal and heat treated (48.4 degrees C, 48.8 degrees C, 49.5 degrees C) red blood cells (RBCs) suspended in dextran-saline solutions (Dx 70, Dx 173) was investigated by a laser light reflectometric method over a wide range of bridging energies. The characteristic times of rouleau formation were found to be increased after RBC heat treatment. The disaggregation shear stress is not significantly different between normal RBCs and heat treated RBCs. The loss of cell deformability is nevertheless shown to improve slightly the dissociation efficiency of the flowing liquid in a shear flow resulting in a small reduction of the disaggregation shear rate after heat treatment. Heat treatment is also shown to alter the structure of RBC network at equilibrium. These results indicate that heat induced alterations of erythrocytes only affects the mechanical properties of the cell membrane without significant changes in the macromolecular bridging energy.     

Time-dependent rheological behaviour of blood flow at low shear in narrow horizontal tubes

Magnitude and time-dependence of the effects of red cell aggregation and sedimentation on the rheology of human blood were studied during low shear (tau W 2.5 to 92 mPa) flow through horizontal tubes (ID 25 to 105 microns). Immediately following reduction of perfusion pressure to a low value the red cell concentration near the tube walls decreases as a result of red cell aggregation. This is associated with a transient increase of centerline velocity. Simultaneously, sedimentation begins to occur and eventually leads to the formation of a cell-free supernatant plasma layer. Time-course and extent of this sedimentation process are strongly affected by wall shear stress variation, particularly in the larger tubes. At the lower shear stresses, centerline velocity decreases (flow resistance increases) with time following the initial acceleration period, due to sedimentation of red cells. This is followed by a further increase of resistance caused by the elevation of hematocrit occurring because of the reduction of cell/plasma velocity ratio. The time dependence of blood rheological behaviour under these flow conditions is interpreted to reflect the net effect of the partially counteracting phenomena of sedimentation and red cell aggregation.