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.     

Size determination of red blood cell aggregates induced by dextran using ultrasound backscattering phenomenon.

Different methods are commonly used to study the red blood cell aggregation phenomenon. The major interest of the ultrasonic method presently discussed is to assess the mean size of red blood cell (RBC) aggregates by measuring ultrasonic intensity backscattered by blood. Applying Rayleigh theory of sound to blood medium, one can show that the scattered ultrasonic intensity is proportional to the 6th power of the size of the RBC aggregates. The ultrasonic method is used to evaluate the mean size of RBC aggregates induced by dextrans. RBCs are suspended at various hematocrits H, in solution of dextrans of different molecular weights M and at different weight concentrations Cw. Results are presented by using the ultrasonic backscattering coefficient chi which is a relevant quantity in a scattering experiment. For suspensions of RBCs aggregated with dextran of molecular weight 70,000 dalton (dextran 70) at concentration Cw = 40 g/l, variations of chi as a function of H are similar to those obtained for normal blood. At a fixed hematocrit, variation of chi versus Cw for dextran 70 exhibits a maximum at 40 g/l. In the case of RBCs suspended at hematocrit 20% and aggregated with dextrans of molecular weight M, 70,000 less than or equal to M less than or equal to 2,000,000, the variations of chi versus molar concentration Cm are similar to those of the microscopic aggregation index defined by Chien (1). Finally, a statistical model of the blood structure previously described (2) is applied to evaluate the mean size of the aggregates. According to this model, the mean size of aggregates is independent of hematocrit for H less than or equal to 40% and independent of the molecular weight of dextran for M greater than or equal to 150,000 dalton.     

Analysis of erythrocyte aggregation mechanism in presence of dextran and magnetic field by ultrasound scattering in blood

Aggregation of erythrocytes is studied as function of time during their sedimentation process under the gravitational field. The method is based on ultrasonic scattering from the various blood samples in presence and absence of inhomogeneous magnetic field (IMF) and dextran 70 (10%). The experimental arrangement is consisting of ultrasonic transmitter and receiver probes placed in mutually perpendicular direction intersecting at the sampling volume of blood located at the centre of the blood column. The temporal kinetic process is represented in terms of histograms of amplitudes and number of scattering fluctuations related to the size and motion of aggregates. The results show that the application of IMF influences the aggregation and sedimentation of erythrocytes. The aggregates thus formed sediment faster than that of control sample. The aggregate formation and their movements in presence of dextran 70 are slower than that of normal blood which may be attributed to the enhanced suspending medium viscosity and their interaction with erythrocytes.     

The mechanism of erythrocyte sedimentation in Westergren's examination.

The authors deduced the equation that describes the sedimentation of erythrocytes as the function of time, hematocrit, hemoglobin and some plasma protein concentrations and the citrate viscosity and density. This values served to describe plasma and erythrocyte density, plasma viscosity, erythrocyte aggregation and the influence of suspension concentration on the erythrocyte sedimentation rate. The influence of citrate on blood dilution (the reduction of hematocrit and plasma protein concentrations) was also considered. A good agreement between the observed and predicted values was obtained.     

Aggregation of human RBC in binary dextran-PEG polymer mixtures

The present study was prompted by prior reports suggesting that small polymers can affect RBC aggregation induced by large macromolecules. Human RBC were washed and re-suspended in isotonic buffer solutions containing 72.5 kDa dextran (DEX 70, 2 g/dl) or 35.0 kDa poly(ethylene glycol) (PEG 35, 0.35 g/dl), then tested for aggregation in these solutions with and without various concentrations of smaller dextrans (10.5 and 18.1 kDa) or PEGs (3.35, 7.5 and 10.0 kDa). RBC aggregation was measured at stasis and at low shear using a photometric cone-plate system (Myrenne Aggregometer) and RBC electrophoretic mobility (EPM) in the various polymer solutions via an automated system (E4, HaSoTec GmbH). Our results indicate: (1) a heterogeneous effect with greater reduction of aggregation for small PEGs added to DEX 70 or for small dextrans added to PEG 35 than for small polymers of the same species; (2) for cells in DEX 70, aggregation decreased with increasing molecular mass and concentration of the small dextrans or PEGs; (3) for cells in PEG 35, small dextrans decreased aggregation with increasing molecular mass and concentration, whereas small PEGs had minimal effects with a minor influence of concentration and an inverse association between molecular mass and inhibition of aggregation. RBC EPM results indicated the expected polymer depletion for cells in DEX 70 or PEG 35, and that small PEGs yielded greater EPM values than small dextrans for cells in PEG 35 whereas the opposite was true for cells in DEX 70. Interpretation of our results in terms of the depletion model for RBC aggregations appears appropriate, and our findings are consistent with the assumption that inhibition of aggregation occurs because of an increase of small molecules in the depletion region. Our results thus suggest the merit of further studies of red blood cell aggregation in binary polymer systems.     

Osmolality dependence of erythrocyte sedimentation and aggregation in a strong magnetic field

In order to specify the major determinant of the magnetic enhancement of erythrocyte sedimentation observed previously, the dependence of erythrocyte sedimentation rate (ESR) on osmolality was measured under a strong magnetic field. Even at hypotonic osmolality, an increase in ESR due to aggregation was observed in plasma solution as compared with that without aggregation in saline solution. However, the magnetic field did not enhance ESR at hypotonic osmolality, when the cell shape was an isotropic sphere (spherocyte). Thus, we narrowed our search to a mechanism that would explain the enhanced ESR found specifically in anisotropic erythrocytes. It was concluded that the major determinant can only work for anisotropic erythrocytes and is a magnetic field-induced increase in an intermembrane adhesive area due to magnetic orientation of anisotropic erythrocytes.     

The mechanism of blood cell sedimentation. XVIII. Sedimentation effect of fibrinogen plasminolysis products]

Various high molecular weight plasmin degradation products of fibrinogen were isolated by chromatographic procedures and investigated to what extent they influence the sedimentation rate of erythrocytes in heparinized serum. Among the early plasminolysis products Fragment X accelerated the sedimentation rate considerably although it was less effective than fibrinogen. Fragment Y showed a weaker but clearly demonstrable agglomerine activity. The late plasminolysis products D and E were ineffective, if applied alone and in concentrations equivalent to the fibrinogen content of plasma. However, they promoted the sedimentation effect of fibrinogen. If present in considerably higher concentrations are Fragments D and E, in absence of fibrinogen, also accelerated the sedimentation rate of erythrocytes significantly. A mixture of D and E was not more effective than the single compounds.     

Effect of water-soluble polymers on the state of aggregation, vesicle size, and phase transformations in mixtures of phosphatidylcholine and sodium cholate.

The state of aggregation and the steady-state size of mixed aggregates made of phospholipids and surfactants are both determined by the surfactant/lipid ratio in the mixed aggregates (Re). Water-soluble polymers, such as dextrans and polyethylene glycols (PEGs) of different molecular weights, induce reversible aggregation of phospholipid vesicles, mostly due to dehydration of the vesicle surface and depletion forces, and only at much higher concentrations, PEGs (but not dextran) also induce irreversible size growth of the vesicles. Here we show that the water-soluble polymers dextrans and PEGs do not affect the vesicle-micelle phase boundaries in mixtures of phosphatidylcholine and the anionic surfactant sodium cholate. By contrast, these polymers affect markedly the steady-state size of cholate-containing vesicles. As compared with pure phosphatidylcholine vesicles, the cholate-containing vesicles have a lower tendency to undergo polymer-induced aggregation, probably due to the electrostatic repulsion between the negatively charged vesicles, but a higher tendency to undergo irreversible size growth at relatively low polymer concentrations. Such irreversible size growth was observed not only for PEG but also for dextran, which in the absence of cholate is incapable of inducing vesicle size growth. These findings are consistent with the prevailing concept that the polymer-induced size growth is due to the effect of large structural fluctuations in the bilayers of deformed aggregated vesicles, the surface of which is dehydrated by the polymer. The presence of cholate in the bilayers at sufficiently high concentrations induces such fluctuations, yielding irreversible size growth within the clusters of dehydrated vesicles formed upon mixing with polymers.     

Role of Surface Electric Charge in Red Blood Cell Interactions

The role of the surface charge of human red blood cells (RBC's) in affecting RBC aggregation by macromolecules was studied by comparing the behavior of normal RBC's with that of RBC's treated with neuraminidase, which removes the sialic acids from the cell membrane and reduces the zeta potential. RBC aggregation in dextrans with different molecular weights (Dx 20, Dx 40, and Dx 80) was quantified by microscopic observation, measurement of erythrocyte sedimentation rate, and determination of low-shear viscosity. Dx 20 did not cause aggregation of normal RBC's, but caused considerable aggregation of neuraminidase-treated RBC's. Neuraminidase-treated RBC's also showed stronger aggregation than normal RBC's in Dx 40 and 80. Together with the electron microscopic findings that the intercellular distance in the RBC rouleaux varies with the molecular size of dextrans used, the present study indicates that the surface charge of RBC's inhibits their aggregation by dextrans and that the electrostatic repulsive force between cell surfaces may operate over a distance of 20 nm.     

Mechanism of erythrocyte aggregate formation in presence of magnetic field and dextrans as analyzed by laser light scattering

The mechanism of erythrocyte aggregation has been studied in normal plasma, dextran 40 and dextran 70 suspensions in presence and absence of magnetic field at a concentration of 5 percent by laser light scattering. The inhomogeneous magnetic field enhances the aggregating tendency of normal erythrocytes. The growth of aggregates due to dextran 70 is enhanced in presence of magnetic field. On the other hand the disaggregating effect of dextran 40 is reduced due to this field. The induced changes due to magnetic field during the development of erythrocyte aggregates in these media are determined.     

Influence of malaria parasite (P. vivax) on erythrocyte aggregation: a study based on dynamic imaging and analysis

Malaria due to P. vivax (PV) is prevalent in many countries. The present work is aimed to determine the cell-cell interaction through formation of aggregates under dynamic conditions. Blood samples are obtained from patients (n=11) suffering from PV malaria, and the normal subjects (n=10) in test tubes containing citrate phosphate dextrose (10:1.4), as an anticoagulant. The signature analysis of infected erythrocytes shows significant alterations in their shape and membrane. For aggregation analysis, erythrocyte suspension in plasma at hematocrit 5%, was placed in a glass chamber and mounted vertically on the stage of the video-microscope system. The aggregate images thus acquired show erythrocytes adhering with each other to form mash-like structures. With increase in parasitaemia, the erythrocytes show hyper-aggregation compared to that of normal cells. By processing of the sequence of recorded images during sedimentation, the various aggregation parameters are obtained. These parameters show that the formed aggregates are compact which produce distinct changes in sedimentation pattern with significantly higher sedimentation velocity compared to that in healthy blood samples. These changes in malaria could partly be responsible for alteration in blood flow through microcirculatory system.     

Synthesis of soluble dextran-hemoglobin complexes of different molecular sizes.

Experimental conditions were defined that determined the synthesis of dextran-hemoglobin complexes through the alklation of hemoglobin by N-bromoacetylaminoethylaminodextran. Using appropriate concentrations of the two reactants, over 90% yield of dextran-hemoglobin was obtained for dextrans of average molecular weight of 200 000 110000, 70000, 400000, and 20000. Extensive viscosity increase due to crosslinking could be avoided, and a large molar excess of dextran over hemoglobin made unnecessary, under the optimal conditions.     

Effect of protein adsorption on the transport characteristics of asymmetric ultrafiltration membranes.

The effects of bovine serum albumin adsorption on the transport characteristics of asymmetric poly(ether sulfone) ultrafiltration membranes were determined using polydisperse dextrans with gel permeation chromatography. Actual dextran sieving coefficients were evaluated from observed sieving data for both the clean and preadsorbed membranes using a stagnant film model. The flux dependence of the actual dextran sieving coefficients was used to evaluate the intrinsic membrane hindrance factors for convective (i.e., sieving) and diffusive transport for the different molecular weight dextrans using classical membrane transport theory. Protein adsorption caused a reduction in both dextran sieving and diffusion, with the magnitude of the reduction a function of the dextran molecular weight and pore size. The effects of adsorption on the specific pore area and the membrane porosity were then determined using a recent model for solute transport through asymmetric ultrafiltration membranes. The data indicate that protein adsorption occurs preferentially in the larger membrane pores, causing a greater reduction in solute sieving compared to the membrane hydraulic permeability and porosity than would be predicted on the basis of either a simple pore blockage or pore constriction model.     

Neutral and DEAE dextrans as tracers for assessing lung microvascular barrier permeability and integrity.

Steady-state lymph-to-plasma concentration ratios (L/Ps) of neutral dextrans, cationic DEAE dextrans, and endogenous proteins were determined under normal and increased permeability conditions in six unanesthetized yearling sheep prepared with chronic lung lymph fistulas. Fluorescent dextrans with radii ranging from 1 to 30 nm were intravenously infused, and after 24 h, perilla ketone (PK) was given to alter permeability while the dextran infusion was maintained. Plasma and lymph samples were collected before and after PK administration and analyzed for dextran and protein concentrations after high-performance liquid chromatography size separation. Under both baseline and increased permeability conditions, DEAE dextrans had higher L/Ps than neutral dextrans of similar size but lower L/Ps than proteins of similar size. Comparison of L/Ps before and after PK revealed that the percentage change in permeability for neutral and DEAE dextrans was significantly larger than that for proteins. These results suggest that 1) the pulmonary microvascular barrier behaves as a net negative barrier, 2) some transport mechanisms for proteins and dextrans are different, and 3) neutral and cationic dextrans are more sensitive markers than proteins of the same size for assessing changes in pulmonary capillary permeability.     

Effects of red blood cell aggregation on myocardial hematocrit gradient using two approaches to increase aggregation

The normal transmyocardial tissue hematocrit distribution (i.e., subepicardial greater than subendocardial) is known to be affected by red blood cell (RBC) aggregation. Prior studies employing the use of infused large macromolecules to increase erythrocyte aggregation are complicated by both increased plasma viscosity and dilution of plasma. Using a new technique to specifically alter the aggregation behavior by covalent attachment of Pluronic F-98 to the surface of the RBC, we have determined the effects of only enhanced aggregation (i.e., Pluronic F-98-coated RBCs) versus enhanced aggregation with increased plasma viscosity (i.e., an addition of 500 kDa dextran) on myocardial tissue hematocrit in rapidly frozen guinea pig hearts. Although both approaches equally increased aggregation, tissue hematocrit profiles differed markedly: 1) when Pluronic F-98-coated cells were used, the normal transmyocardial gradient was abolished, and 2) when dextran was added, the hematocrit remained at subepicardial levels for about one-half the thickness of the myocardium and then rapidly decreased to the control level in the subendocardial layer. Our results indicate that myocardial hematocrit profiles are sensitive to both RBC aggregation and to changes of plasma viscosity associated with increased RBC aggregation. Furthermore, they suggest the need for additional studies to explore the mechanisms affecting RBC distribution in three-dimensional vascular beds.     

Depletion flocculation and depletion stabilization of erythrocytes.

At dextran (Mw approximately 500,000) concentrations from 2 to approximately 10%, suspensions of normal human erythrocytes flocculate in small convex agglutinates. At dextran concentrations greater than 10%, the erythrocytes resegregate in a stable monodisperse suspension. At all these dextran concentrations, the erythrocytes are coated with considerable amounts of dextran. It can be argued that at dextran concentrations from 2 to 10%, as well as at dextran concentrations greater than 10%, there is a thin layer, which is depleted of dextran, between the dextran layer adsorbed onto the erythrocytes and the bulk dextran solution. It can also be shown that there is a repulsive interaction between the two layers of dextran: one adsorbed and one free. When the adsorbed dextran layer is the most concentrated, stability must ensue, and when the dextran in free solution is the most concentrated, flocculation should occur. Below 7% dextran, the concentration of free dextran is higher than the adsorbed concentration; above 10% dextran that situation is reversed. These data correlate well with the depletion flocculation predicted for the lower concentration and the depletion stabilization predicted for the higher dextran concentration.     

Study of erythrocyte sedimentation behavior by piezoelectric crystal impedance sensor

Based on the impedance characteristic of erythrocytes at high frequency, the response of piezoelectric crystal impedance (PCI) sensor in the erythrocyte suspension was derived and verified experimentally. A method of using PCI sensor to investigate erythrocyte aggregation-sedimentation phenomenon was proposed. From the frequency response of the PCI sensor, the erythrocyte aggregation time and sedimentation rate could be obtained during erythrocyte aggregation and sedimentation. With the present method, the effects of the erythrocyte deformability, the osmotic pressure and the coexisting macromolecules on the erythrocyte sedimentation rate were studied. The results show that the PCI sensor possesses some advantages, such as good sensitivity, simplicity of use and no thermal effect for the impedance study of erythrocyte aggregation and sedimentation.     

Interaction of glucosyltransferase from Streptococcus mutans with various glucans

Cell-free glucosyltransferase of Streptococcus mutans strain B13 (serotype d) exclusively synthesized water-insoluble glucan from sucrose. The insoluble glucan possessed strong glucan-associated glucosyltransferase activity even after extensive washing and lyophilization. Furthermore, cell-free glucosyltransferase became bound to heat-treated water-insoluble glucan or to heat-treated S. mutans B13 cells grown in Todd Hewitt broth, and the resulting glucan and cells adhered to a glass surface in the presence of exogenous sucrose. No other water-insoluble glucans bound significant quantities of glucosyltransferase. Glucan synthesis by free or glucan-bound glucosyltransferase was stimulated by low concentrations (1 to 5 mg ml-1) of isomaltose or water-soluble dextrans of various molecular weights, but higher concentrations (10 mg ml-1) inhibited glucan synthesis. The glucan synthesized in the presence of primer dextrans exhibited a reduced ability to adhere to a glass surface. Certain sugars such as maltose and fructose significantly lowered the yield of insoluble glucans. Preincubation of glucosyltransferase with the low molecular weight dextran T10 increased subsequent binding to S. mutans B13 insoluble glucan, whereas preincubation with higher molecular weight dextrans significantly inhibited the glucosyltransferase binding.     

Glutathione reductase from human erythrocytes. Molecular weight, subunit composition and aggregation properties.

Glutathione reductase from human erythrocytes exists predominatly as an entity of 100 000 molecular weight under various conditions of pH and ionic strength. The S20,W of 5.5 S and D20W of 50 mum2/s correlate with the molecular weight determined by sedimentation equilibrium. The homogeneity of this species is primarily dependent on the presence of thiols and secondarily on high concentrations of salt. The amino-acid composition of the enzyme shows similarities both with glutathione reductases from other sources and with lipoamide dehydrogenase. From the flavin content and dodecylsulphate-polyacrylamide electrophoresis it is inferred that the native enzyme is a dimer composed of similar subunits of 50 000 molecular weight. In the absence of thiols, glutathione reductase shows a tendency to form tetramers and larger aggregates. Although these larger species are also catalytically active, under cellular conditions the presence of its product, reduced glutathione, should maintain the enzyme as the dimeric entity.     

Effect of electric field on erythrocyte sedimentation rate. II. Dependence on electric current

We measured the electric current dependence of sedimentation curves of swine erythrocytes in a saline solution at the volume fraction of erythrocytes H = 0.091 and 0.220. The sedimentation curve fitted well to the exponential type equation l = a[1-exp(-bt)] at the upward initial electric current I0 = 0.50 mA, 1.01 mA and 1.50 mA, where l is the length of the medium layer at time t, and a and b are phenomenological parameters. The initial slope v0 of sedimentation curve was enhanced from 0.68 mm/hr at I0 = 0 mA to 2.85 mm/hr, 3.87 mm/hr and 5.50 mm/hr at I0 = 0.50 mA, 1.01 mA and 1.50 mA, respectively, for H = 0.220. We also made sedimentation measurements of erythrocytes in their own plasma at H = 0.220 and 0.316. Sedimentation curves coincided with the sigmoidal type equation l = l infinity/[1 + (t50/t)beta] at I0 = 0 mA and 0.50 mA, where l infinity is l at t----infinity, t50 is the time when the plasma level falls to l infinity/2 and beta is a constant. The maximum slope vmax of sedimentation curve increased from 13.29 mm/hr at I0 = 0 mA to 18.65 mm/hr at I0 = 0.50 mA for H = 0.220.