Alexander Pribush

Kinetics of erythrocyte swelling and membrane hole formation in hypotonic media.

Red blood cell (RBC) swelling and membrane hole formation in hypotonic external media were studied by measuring the time-dependent capacitance, C, and the conductance, G, in the beginning of the beta-dispersion range. At high and moderate osmolarities of the external solution the capacitance reaches a steady-state whereas at low osmolarities it reveals a biphasic kinetics. Examination of RBC suspensions exposed to different concentrations of HgCl(2) demonstrates that water transport through mercury-sensitive water channel controls RBC swelling. Unlike the capacitance, an increase in the conductance to a stationary level is observed after a certain delay. A comparison of G(t) curves recorded for the suspensions of the intact cells and those treated with cytochalasin B or glutaraldehyde demonstrates the significant effect of the membrane viscoelasticity on the pore formation. It is shown that the stretched membrane of completely swollen RBC retains its integrity for a certain time, termed as the membrane lifetime, t(memb). Therefore, the resistivity of RBCs to a certain osmotic shock may be quantified by the distribution function of RBC(t(memb)).

Radiation Damage to the Erythrocyte Membrane: The Effects of Medium and Cell Concentrations

Human erythrocytes suspended in plasma, or in phosphate buffered saline (PBS), were exposed to ionizing radiation. Potassium leakage from irradiated erythrocytes is significantly higher in PBS than in plasma. The potassium leakage decreases when PBS is gradually replaced by plasma. These findings suggest that some of the plasma constituents have radioprotective properties. The potassium leakage per cell is independent of the hematocrit, Hct. The potassium leakage is attributed to the formation of radiation defects in the membrane. Analysis of the effect of radiation dose, plasma and cell concentrations on the product of the number and surface area of the radiation defects indicates that the radiation damage is mainly due to the direct formation of free radicals in the cell membrane. The radioprotective effect of plasma is attributed to surface reactions of these free radicals with plasma constituents adsorbed on the membrane.

The mechanism of erythrocyte sedimentation. Part 2: The global collapse of settling erythrocyte network

Results reported in the companion paper showed that erythrocytes in quiescent blood are combined into a network followed by the formation of plasma channels within it. This study is focused on structural changes in the settling dispersed phase subsequent to the channeling and the effect of the structural organization on the sedimentation rate. It is suggested that the initial, slow stage of erythrocyte sedimentation is mainly controlled by the gravitational compactness of the collapsed network. The lifetime of RBC network and hence the duration of the slow regime of erythrocyte sedimentation decrease with an increase in the intercellular pair potential and with a decrease in Hct. The gravitational compactness of the collapsed network causes its rupture into individual fragments. The catastrophic collapse of the network transforms erythrocyte sedimentation from slow to fast regime. The size of RBC network fragment is insignificantly affected by Hct and is mainly determined by the intensity of intercellular attractive interactions. When cells were suspended in the weak aggregating medium, the Stokes radius of fragments does not differ measurably from that of individual RBCs. The proposed mechanism provides a reasonable explanation of the effects of RBC aggregation, Hct and the initial height of the blood column on the delayed erythrocyte sedimentation.

The mechanism of erythrocyte sedimentation. Part 1: Channeling in sedimenting blood.

Despite extensive efforts to elucidate the mechanism of erythrocyte sedimentation, the understanding of this mechanism still remains obscure. In attempt to clarify this issue, we studied the effect of hematocrit (Hct) on the complex admittance of quiescent blood measured at different axial positions of the 2 mm x 2 mm cross-section chambers. It was found that after the aggregation process is completed, the admittance reveals delayed changes caused by the formation of cell-free zones within the settling dispersed phase. The delay time (tau(d)) correlates positively with Hct and the distance between the axial position where measurements were performed and the bottom and is unaffected by the gravitational load. These findings and literature reports for colloidal gels suggest that erythrocytes in aggregating media form a network followed by the formation of plasma channels within it. The cell-free zones form initially near the bottom and then propagate toward the top until they reach the plasma/blood interface. These channels increase the permeability of a network and, as a result, accelerate the sedimentation velocity. The energy of the flow field in channels is sufficiently strong to erode their walls. The upward movement of network fragments in channels is manifested by erratic fluctuations of the conductivity. The main conclusion, which may be drawn from the results of this study, is that the phase separation of blood is associated with the formation of plasma channels within the sedimenting dispersed phase.

Osmotic swelling and hole formation in membranes of thalassemic and spherocytic erythrocytes.

Osmotic swelling and kinetics of the pore formation in the membranes of spherocytic, thalassemic, and normal erythrocytes were studied by measuring the time-dependent capacitance and conductance at a frequency of 0.2 MHz. No significant difference between the swelling rate of control and spherocytic cells was observed, whereas slower kinetics of swelling were found for thalassemic cells. Time records of the conductance indicate that the probability of the pore formation in the stretched membrances varies in the following order: thalassemia < control < spherocytosis. Based on these findings it was concluded that the erythrocyte swelling is controlled by the initial cell shape, volume, intracellular hemoglobin concentration, and elastic membrane properties, whereas the kinetics of the pore formation depend solely on the resistivity of the stretched membrane of the swollen RBC to the osmotic shock. Therefore, it was assumed that investigations of the pore formation may be used not only for examinations of spherocytic and thalassemic cells, but also for normocytic, normochromic, biconcave-shaped RBCs with altered membrane elasticity.

Conductometric study of erythrocytes during centrifugation. II. Erythrocyte deformability

Erythrocyte deformability was studied by continuous reading of sediment conductance during centrifugation. The decrease in sediment conductivity during centrifugation reflects erythrocyte deformation in the pellet. The degree of erythrocyte deformation depends on the duration of centrifugation and the magnitude of centripetal acceleration. When constant centrifugal force is applied over an extended period of time, a gradual decrease in pellet conductivity occurs. Stepwise enhancement of centripetal acceleration during centrifugation induces a rapid increase in erythrocyte deformation. After centrifugation, the relaxation of erythrocyte deformation is observed. However, the relaxation and the recovery of cell shape are incomplete. The difference in compressibility of previously centrifuged and noncentrifuged cells demonstrates that centrifugation causes irreversible alteration in erythrocyte deformability. The results show that the time-dependent resistance of erythrocyte sediment during centrifugation may serve as a useful index for the kinetics of erythrocyte deformation.

A novel approach for assessments of erythrocyte sedimentation rate.

Introduction:  Previous studies have shown that the dispersed phase of sedimenting blood undergoes dramatic structural changes: Discrete red blood cell (RBC) aggregates formed shortly after a settling tube is filled with blood are combined into a continuous network followed by its collapse via the formation of plasma channels, and finally, the collapsed network is dispersed into individual fragments. Based on this scheme of structural transformation, a novel approach for assessments of erythrocyte sedimentation is suggested.

Methodological aspects of erythrocyte aggregation.

The aim of this paper is to analyze merits and demerits of methodological approaches designed for investigations of erythrocyte aggregation--a process, which plays a crucial role in rheological and transport properties of blood. Ideally, erythrocyte aggregation should be characterized in terms of the time-dependent gyration radius of the aggregates and their fractal dimension. Among various experimental techniques suggested so far, only imaging analysis meets this requirement. However, because this technique is designed for investigations of the aggregation process in thin layers of dilute erythrocyte suspensions, aggregation data are affected by cell-wall interactions and, in addition, problems arise when attempts are made to extend these data to whole blood. Interpretation of results obtained by light scattering techniques faces problems associated with effects of multiple scattering, a design of experimental setups and the wavelength on the kinetics of recorded signals. A method based on electric and dielectric properties of blood is advantageous over other methodological approaches, because it provides reliable information about time-dependent and steady-state size and morphology of the aggregates at physiological hematocrits. A common drawback of most methodological approaches is that interpretation of experimental results is based on simplified theoretical models of blood. To avoid complicated physical problems posed by optical, ultrasound, electrical and dielectrical properties of blood, it is suggested to use the adhesion energy as a measure of RBC aggregability.

The effect of the prior flow velocity on the structural organization of aggregated erythrocytes in the quiescent blood.

Usually, investigations of erythrocyte aggregation at rest are focused on effects of the strength of erythrocyte-erythrocyte attractive interactions and the volume fraction of the cells, whereas the role of prior flow velocity has not been thoroughly investigated. The aim of this study is to fill this gap. The main conclusions extracted from time records of the complex admittance of blood are as follows: (1) Dispersion of blood in a prior flow into discrete aggregates increases the mesh size of network, which, as has been recently shown, is formed in the quiescent blood. (2) If the energy of the flow field is sufficient to prevent the formation of face-to-side intercellular links, so that the dispersed phase consists of linear rouleaux, changes in the mesh size correlate positively with the length of rouleaux. (3) At slower prior flow velocities, the cells are combined into branched aggregates. As the degree of branching increases, the effect becomes less important. (4) The effects of the length of linear rouleaux and the degree of branching of ramified aggregates on the mesh size are qualitatively similar for suspensions with different aggregating media. (5) Erythrocytes suspended in strongly aggregating media form at low flow conditions a network-like structure. In this case, unlike high and moderate prior flow regimes, the mesh size of RBC network at rest is less than that formed after the stoppage of completely dispersed blood.

Study of red blood cell aggregation by admittance measurements.

A method based on dielectric properties of cellular suspensions was developed to study red blood cell (RBC) aggregability. The time-dependent current in a Couette-type viscometer was recorded after abrupt stoppage of shearing. Since the current reaches steady state 2 min after the end of shearing, the observed effects were quantified by the relative current difference, delta Irel = (I(2min)-I5s)/I2min, where subscripts designate the time of measurements. delta Irel increases with hematocrit, plasma and fibrinogen concentration. The dependence of delta Irel and of RBC aggregability on the concentration of dextran were similar. The experimental data and their analysis indicate that in suspensions with aggregating media, the delta Irel value measured in the field of the beta-dispersion reflects the difference between the size of aggregates under steady-state conditions and that of dispersed particles 5 s after the end of shearing. Therefore, this value may serve as a measure of RBC aggregability.