Erythrocyte swelling and membrane hole formation in hypotonic media as studied by conductometry.

Hypoosmotic swelling of erythrocytes and the formation of membrane holes were studied by measuring the dc conductance (G). In accordance with the theoretical predictions, these processes are manifested by a decrease in G followed by its increase. Thus, unlike the conventional osmotic fragility test, the proposed methodological approach allows investigations of both the kinetics of swelling and the erythrocyte fragility. It is shown that the initial rate of swelling and the equilibrium size of the cells are affected by the tonicity of a hypotonic solution and the membrane rheological properties. Because the rupture of biological membranes is a stochastic process, a time-dependent increase in the conductance follows an integral distribution function of the membrane lifetime. The main conclusion which stems from reported results is that information about rheological properties of red blood cell (RBC) membranes and the resistivity of RBCs to a certain osmotic shock may be extracted from conductance signals.

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. METHODS: Information about erythrocyte sedimentation is extracted from time records of the blood conductivity measured after a dispersion of RBC network into individual fragments. RESULTS: It was found that the sedimentation velocity of RBC network fragments correlates positively with the intensity of attractive intercellular interactions, whereas no effect of hematocrit (Hct) was observed. CONCLUSION: Thus, unlike Westergren erythrocyte sedimentation rate, sedimentation data obtained by the proposed method do not require correction for Hct.

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.

A novel technique for quantification of erythrocyte aggregation abnormalities in pathophysiological situations.

Red blood cell (RBC) aggregation in blood samples taken from healthy volunteers and from multiple myeloma (MM), iron deficiency (IDA) and beta-minor thalassemia (T) patients was studied by a novel method based on electrical properties of colloidal systems. It was found that RBC aggregation changes in the following order: MM > IDA > control > or = T. Comparison of aggregation data obtained by this and other techniques shows that the sensitivity of the proposed technique to detect abnormal changes in RBC aggregation is substantially higher. For example, the mean values of relative aggregation indices measured for MM by this method and that based on the phenomenon of light scattering are 13.0 and 4.2, respectively. The high sensitivity of this technique allows investigations of the effect of moderate aggregating agents (i.e., IgG) on RBC aggregation. It is assumed that the higher sensitivity of the proposed technique to abnormal changes in RBC aggregation may be helpful both in basic studies to improve the understanding of the reason(s) for these abnormal changes, and in clinical investigations for earlier diagnostics.

The mechanism of the dextran-induced red blood cell aggregation.

In order to clarify the mechanism of dextran-induced aggregation, the effect of the ionic strength (I) on the minimal shear stress (tau(c)) required to rupture RBC doublets was studied for suspensions with the external media containing 76 and 298 kDa dextrans. At low and high ionic strengths, tau(c) increases with increasing I, whereas at intermediate I values, tau(c) versus I dependencies reveal a plateau step. The non-monotonous shape of these curves disagrees with the depletion model of RBC aggregation and is consistent with the predictions of the bridging mechanism. Literature reports point out that elastic behavior of dextran molecules in low and high I regions is fairly typical of Hookean springs and hence predict an increase in tau(c) with increasing I. A plateau step is accounted for by the enthalpic component of the dextran elasticity due to the shear-induced chair-boat transition of the dextran's glucopyranose rings. A longer plateau step for suspensions with a higher molecular weight dextran is explained by a larger contribution of the enthalpic component to the dextran elasticity. Thus, the results reported in this study provide evidence that RBC aggregation is caused by the formation of dextran bridges between the cells.

The role of erythrocyte aggregation in the abnormal hemorheology of multiple myeloma patients.

OBJECTIVES: The aim of this study is to clarify whether increased aggregation of red blood cells (RBCs) of multiple myeloma (MM) patients is caused by changes in plasma chemical composition or is associated with alterations in RBC properties and in addition, to suggest an approach to revert the enhanced aggregation in MM toward normal. MATERIALS AND METHODS: 40 blood samples of MM patients and suspensions of control RBCs in MM plasma were examined. In addition, RBC aggregation in MM blood was studied in the presence of dextrans with mean molecular weights of 9.6 and 40 kDa (D9.6 and D40). A method based on electrical and dielectric properties of cellular suspensions was used to study RBC aggregation. In this method, a lower aggregation index demonstrates a higher aggregability. RESULTS: The mean values of aggregation index for whole blood of healthy individuals, control cells in MM plasma and MM blood sample are 19.0, 7.2 and 3.2%, respectively. The kinetics of RBC aggregation slow down with the decrease in the fraction of MM plasma. No correlation was found between RBC aggregation and the immunoglobulin plasma level. Addition of D9.6 to MM blood reverts the enhanced aggregation toward normal. DISCUSSION: The findings that RBC aggregation changes in the following order: MM blood > normal RBCs in MM plasma > control blood sample, suggest that surface-active plasma molecules play a role in enhanced aggregation in MM. The surface concentration of these molecules and hence RBC aggregability is reduced in the presence of dextrans due to their competitive adsorption onto RBC membrane. Because the end-to-end distance of D40 is quite comparable with the Debye length, the effect of this particular dextran on RBC aggregation is negligible.

Conductometric study of shear-dependent processes in red cell suspensions. I. Effect of red blood cell aggregate morphology on blood conductance.

The conductance and capacitance of flowing and quiescent red blood cell (RBC) suspensions were measured at a frequency of 0.2 MHz. The results demonstrate that the time-dependent changes in the conductance recorded during the aggregation process differ in nature for suspensions of short linear rouleaux, branched aggregates and RBC networks. It is shown that the conductance of RBC suspensions measured during the aggregation and disaggregation processes follows the morphological transformations of the RBC aggregates. Thus, this method enables characterization of the morphology of RBC aggregates formed in whole blood and in suspensions with physiological hematocrits both under flow conditions and in stasis. These results in combination with previous ones suggest that this technique can be used for studies of dynamic RBC aggregation and probably for diagnostic use.

Conductometric study of shear-dependent processes in red cell suspensions. II. Transient cross-stream hematocrit distribution.

A novel experimental approach based on electrical properties of red blood cell (RBC) suspensions was applied to study the effects of the size and morphology of RBC aggregates on the transient cross-stream hematocrit distribution in suspensions flowing through a square cross-section flow channel. The information about the effective size of RBC aggregates and their morphology is extracted from the capacitance (C) and conductance (G) recorded during RBC aggregation, whereas a slower process of particle migration is manifested by delayed long-term changes in the conductance. Migration-induced changes in the conductance measured at low shear rates (< or =3.1 s(-1)) for suspensions of RBCs in a strongly aggregating medium reveal an increase to a maximum followed by a decrease to the stationary level. The ascending branch of G(t) curves reflects the aggregate migration in the direction of decreasing shear rate. A further RBC aggregation in the region of lower shear stresses leads to the formation of RBC networks and results in the transformation of the rheological behavior of suspensions from the thinning to the thickening. It is suggested that the descending branches of the G(t) curves recorded at low shear rates reflect an adjustment of the Hct distribution to a new state caused by a partial dispersion of RBC networks. For suspensions of non-aggregating RBCs it is found that depending on whether the shear rate is higher or lower compared with the prior value, individual RBCs migrate either toward the centerline of the flow or in the opposite direction.

The effect of low-molecular weight dextran on erythrocyte aggregation in normal and preeclamptic pregnancy.

Erythrocyte aggregation was determined by a novel method enabling the quantification of the aggregation process in whole blood. Blood samples of 47 healthy pregnant women and 39 preeclamptic patients were examined. Subjects within each group were matched for the gestational age. It was found that RBC aggregation increases with the gestational age in healthy pregnancy and further increases in preeclampsia. Addition of low-molecular weight dextran (MW = 9300) to blood samples of both healthy pregnant women and preeclamptic patients reduces RBC aggregation in a concentration-dependent manner. The obtained results indicate alterations in plasma composition as the primary factor for the increased RBC aggregation in both normal and pathological pregnancy. It is suggested that adsorption of low-molecular weight dextran on the RBC membrane reduces the surface concentration of plasma bridging molecules thereby reducing RBC aggregation toward normal.

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.

Dielectric approach to the investigation of erythrocyte aggregation: I. Experimental basis of the method.

A method based on dielectric properties of dispersed systems was developed to investigate red blood cell (RBC) aggregation in blood and RBC suspensions. Measurements of capacitance and resistance were made in a rectangular channel at low (0.2 MHz) and high (14 MHz) frequencies relative to the mid-point of the beta-dispersion range. Compared to capacitance, minimal post-shearing changes of resistance were observed; capacitance changes at 0.2 MHz were two orders of magnitude larger than those at 14 MHz and hence subsequent measurements were carried out at the lower frequency. It is shown that post-shearing changes in the capacitance are affected by the recovery of RBC shape and relaxation processes at the electrode-suspension interface. However, the dominant factor contributing to time-dependent changes in the capacitance is the dynamic process of RBC aggregation. It is experimentally shown that the time record of the capacitance at 0.2 MHz quantitatively reflects the aggregation process in RBC-plasma suspensions with hematocrit up to 0.56 (v/v) and in suspensions of RBCs in artificial aggregating media. It is concluded that a dielectric approach to the study of RBC aggregation in whole blood offers great potential for basic studies and for diagnostic use.

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.

Conductometric study of erythrocytes during centrifugation. I. Size distribution of erythrocytes.

Sedimentation of hardened erythrocytes in a centrifugal field was studied by time recording of the current chamber in the longitudinal and the transversal directions relative to the cells' movement. The results clearly indicate the existence of an erythrocyte concentration profile during centrifugation. The rates of both longitudinal and transversal current alteration increase with centripetal acceleration and with falling cell concentration. The pellet formed from hardened cells represents virtually incompressible body. It is shown that erythrocyte shape affects the pellet conductivity. Analysis of the data using the modified Stokes' law enables calculation of the cell size distribution. The modal size of macrocytes, normal erythrocytes and two samples of microcytes thus measured was 3.40, 3.01, 2.63 and 2.83 microns, respectively. These data demonstrate that conductometric analysis is useful for investigating abnormalities in erythrocyte size.

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.

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.

[Changes in cellular radiosensitivity by modifying the cytoplasmic membranes]. / Izmenenie radiochuvstvitel'nosti kletok putem modifikatsii tsitoplazmaticheskikh membran.

The radiosensitivity of mouse myeloma and E. coli cells in the presence of Mg2+ and UO2(2+) ions has been investigated. It has been shown that Mg2+ ions (10(-4) M) do not influence the viability of E. coli and mouse myeloma cells. The presence of Mg2+ ions during irradiation reduces the survival rate of E. coli cells, but the addition of Mg2+ ions after irradiation does not influence the radiosensitivity of E. coli cells. Comparison of the results on the influence of Mg2+ ions upon cells and bilayer lipid membranes (BLM) permits us to suppose that Mg2+ ions increase the positive charge of the membranes thus promoting the increase in the number of short-lived radiolysis products which impair membranes and increase cell radiosensitivity. UO2(2+) ions (10(-4) M) increase the radioresistance of E. coli cells which can be associated with the increase in the lateral membrane viscosity, as it was shown in the studies on BLM.

[Pulsed radiolysis of aqueous solutions of serum albumin containing naphthoquinones]. / Impul'snyi radioliz vodnykh rastvorov syvorotochnogo al'bumina, soderzhashchikh naftokhinony.

As was shown by the pulse radiolysis method the simultaneous presence of naphthoquinone and human serum albumin molecules in an aqueous solution leads to the adsorption of the former on the surface of the latter. It is suggested that in these conditions the protein tertiary structure changes. New conformation reduces the reactivity of albumin toward the hydrated electron.

[The effect of the frequency of periodic irradiation on the rate of inactivation of E. coli cells]. / Vliianie chastoty periodicheskogo oblucheniia na skorost' inaktivatsii kletok E. coli.

It was shown that the periodic radiation source frequency influences the rate of E. coli cell inactivation. The impulse duration from 2 microseconds to 40 ns does not influence the survival of E. coli. The analysis of the survival curves obtained permitted to assess the contribution of the direct and indirect effects of radiation to microorganism inactivation.

[The use of the pulse radiolysis method in the study of rapid processes in suspensions of Escherichia coli]. / Issledovanie metodom impul'snogo radioliza bystroprotekaiushchikh protsessov v suspenzii Escherichia coli.

It was proved that the method of pulse radiolysis can be used to study the processes rapidly occurring in a microorganism suspension. It was shown that optically active particles formed after irradiation in an E. coli cell were involved into the first-(pseudo-first)-order reaction the rate constant being approximately 30 s-1. The presence of oxygen did not influence the kinetics of disappearance of these particles.