Measurement of biophysical properties of red blood cells by resistive pulse spectroscopy: volume, shape, surface area, and deformability

This paper presents a simple, new approach to the determination of size, shape, surface area, and deformability information for cells, notably red blood cells. The results are obtained by combining experimental measurements from resistive pulse spectroscopy (an extension of electronic cell-sizing methodology) with theoretical calculations for model cell systems. Assuming constancy of surface area and approximating red cell shapes by both prolate and oblate ellipsoids of revolution, values are determined for cell shape factor and volume under a variety of conditions. For red blood cells under low-stress conditions, shape factor, volume, and surface area results are found to be consistent with those available from the literature, when the oblate model is used. The applicability of this approach for determination of red cell properties under altered conditions is demonstrated by results for cell volume, at varying osmotic pressure and mechanical shear (tensile) stress. By quantitating the change in cell shape with stress, a new numerical scale for measuring cell deformability is also obtained, and data are presented on its variation for red cells at different osmolalities, over the range of 140 to 500 mOsm.     

Influence of counterions on the interaction of pyridinium salts with model membranes

The interaction of pyridinium salts (PS) with red blood cells and planar lipid membranes was studied. The aim of the work was to find whether certain cationic surfactant counterion influence its possible biological activity. The counterions studied were Cl-, Br-, I-, ClO4-, BF4- and NO3-. The model membranes used were erythrocyte and planar lipid membranes (BLM). At high concentration the salts caused 100% erythrocyte hemolysis (C100) or broke BLMs (CC). Both parameters describe mechanical properties of model membranes. It was found that the efficiency of the surfactant to destabilize model membranes depended to some degree on its counterion. In both, erythrocyte and BLM experiments, the highest efficiency was observed for Br-, the lowest for NO3-. The influence of all other anions on surfactant efficiency changed between these two extremities; that of chloride and perchlorate ions was similar. Some differences were found in the case of BF4- ion. Its influence on hemolytic possibilities of PS was significant while BLM destruction required relatively high concentration of this anion. Apparently, the influence of various anions on the destructive action of PS on the model membrane used may be attributed to different mobilities and radii of hydrated ions and hence, to different possibilities of particular anions to modify the surface potential of model membranes. This can lead to a differentiated interaction of PS with modified bilayers. Moreover, the effect of anions on the water structure must be taken into account. It is important whether the anions can be classified as water ordering kosmotropes that hold the first hydration shell tightly or water disordering chaotropes that hold water molecules in that shell loosely.     

pH regulatory Na/H exchange by Amphiuma red blood cells

In Amphiuma red blood cells, the Na/H exchanger has been shown to play a central role in the regulation of cell volume following cell shrinkage (Cala, P. M. 1980. Journal of General Physiology. 76:683- 708.) The present study was designed to evaluate the existence of pH regulatory Na/H exchange in the Amphiuma red blood cell. The data illustrate that when the intracellular pHi was decreased below the normal value of 7.00, Na/H exchange was activated in proportion to the degree of acidification. Once activated, net Na/H exchange flux persisted until normal intracellular pH (6.9-7.0) was restored, with a half time of approximately 5 min. These observations established a pHi set point of 7.00 for the pH-activated Na/H exchange of Amphiuma red blood cell. This is in contrast to the behavior of osmotically shrunken Amphiuma red blood cells in which no pHi set point could be demonstrated. That is, when activated by cell shrinkage the Na/H exchange mediated net Na flux persisted until normal volume was restored regardless of pHi. In contrast, when activated by cell acidification, the Na/H exchanger functioned until pHi was restored to normal and cell volume appeared to have no effect on pH-activated Na/H exchange. Studies evaluating the kinetic and inferentially, the molecular equivalence of the volume and pHi-induced Amphiuma erythrocyte Na/H exchanger(s), indicated that the apparent Na affinity of the pH activated cells is four times greater than that of shrunken cells. The apparent Vmax is also higher (two times) in the pH activated cells, suggesting the involvement of two distinct populations of the transporter in pH and volume regulation. However, when analyzed in terms of a bisubstrate model, the same data are consistent with the conclusion that both pH and volume regulatory functions are mediated by the same transport protein. Taken together, these data support the conclusion that volume and pH are regulated by the same effector (Na/H exchanger) under the control of as yet unidentified, distinct and cross inhibitory volume and pH sensing mechanisms.     

Stiffness of normal and pathological erythrocytes studied by means of atomic force microscopy

During recent years, atomic force microscopy has become a powerful technique for studying the mechanical properties (such as stiffness, viscoelasticity, hardness and adhesion) of various biological materials. The unique combination of high-resolution imaging and operation in physiological environment made it useful in investigations of cell properties. In this work, the microscope was applied to measure the stiffness of human red blood cells (erythrocytes). Erythrocytes were attached to the poly-L-lysine-coated glass surface by fixation using 0.5% glutaraldehyde for 1 min. Different erythrocyte samples were studied: erythrocytes from patients with hemolytic anemias such as hereditary spherocytosis and glucose-6-phosphate-dehydrogenase deficiency patients with thalassemia, and patients with anisocytosis of various causes. The determined Young's modulus was compared with that obtained from measurements of erythrocytes from healthy subjects. The results showed that the Young's modulus of pathological erythrocytes was higher than in normal cells. Observed differences indicate possible changes in the organization of cell cytoskeleton associated with various diseases.     

Theory of the Sphering of Red Blood Cells

A rigorous mathematical solution of the sphering of a red blood cell is obtained under the assumptions that the red cells is a fluid-filled shell and that it can swell into a perfect sphere in an appropriate hypotonic medium. The solution is valid for finite strain of the cell membrane provided that the membrane is isotropic, elastic and incompressible. The most general nonlinear elastic stress-strain law for the membrane in a state of generalized plane stress is used. A necessary condition for a red cell to be able to sphere is that its extensional stiffness follow a specific distribution over the membrane. This distribution is strongly influenced by the surface tension in the cell membrane. A unique relation exists between the extensional stiffness, pressure differential, surface tension, and the ratio of the radius of the sphere to that of the undeformed red cell. The functional dependence of this stiffness distribution on various physical parameters is presented. A critique of some current literature on red cell mechanics is presented.     

Volume regulation by Amphiuma red blood cells: strategies for identifying alkali metal/H+ transport

The regulation of cell volume in response to anisotonic media, and in a broader perspective electroneutral alkali/metal H+ exchange transport, are currently areas of general interest to transport physiologists. In this paper I outline the basic features of volume-sensitive ion fluxes as studied with Amphiuma red blood cells. As has been shown in previous studies the alkali metal ion fluxes that are responsible for volume regulation by these cells are electroneutral by virtue of obligatory counter coupling with H+. The criteria for establishing the existence of electroneutral alkali metal/H+ exchange in these cells will be reviewed and expanded on. In the process, behavior and phenomena consistent with, as well as those unique to, electroneutral alkali metal/H+ exchange will be introduced, illustrated with experimental data, and discussed. Finally, based on thermodynamic considerations, kinetic behavior will be evaluated in terms of electroneutral alkali metal/H+ transport.     

The effect of plasma viscosity on the resistance of erythrocyte movement along capillaries

By means of three-dimensional mathematical model describing movement of an asymmetric erythrocyte along the capillary of a small section the effect of the viscosity of the blood plasma on the resistance value to the erythrocyte movement in microvessels was studied. The model takes into account the elastic properties of the erythrocyte, the volume and area of its surface and the mobility of the membrane performing the rotation. It is shown that for characteristic conditions of hemodynamics experimentally observed in the microcirculation system the pressure gradient on the erythrocyte ends is 3-5-fold above the difference of pressures at the ends of plasma column of similar length. The dependence of the pressure gradient at the erythrocyte ends, which accounts for its movement along the capillary, on plasma viscosity is determined at different values of erythrocyte velocity.     

Anion transport systems in the plasma membrane of vertebrate cells

In the case of the red blood cell, anion transport is a highly specific one-for-one exchange catalyzed by a major membrane protein known as band 3 or as capnophorin. This red cell anion-exchange system mediates the Cl-(-)HCO3- exchange responsible for most of the bicarbonate transport capacity of the blood. The rapidly expanding knowledge of the molecular biology and the transport kinetics of this specialized transport system is very briefly reviewed in Section III. Exchange diffusion mechanisms for anions are found in many cells other than erythrocytes. The exchange diffusion system in Ehrlich cells has several similarities to that in red cells. In several cell types (subsection IV-B), there is evidence that intracellular pH regulation depends on Cl-(-)HCO3- exchange processes. Anion exchange in other single cells is described in Section IV, and its role in pH regulation is described in Section VII. Anion exchange mechanism operating in parallel with, and only functionally linked to Na+-H+ or K+-H+ exchange mechanisms can also play a role in cell volume regulation as described in Section VII. In the Ehrlich ascites cell and other vertebrate cells, electroneutral anion transfer has been found to occur also by a cotransport system for cations and chloride operating in parallel with the exchange diffusion system. The cotransport system is capable of mediating secondary active chloride influx. In avian red cells, the cotransport system has been shown to be activated by adrenergic agonists and by cyclic AMP, suggesting that the cotransport is involved in regulatory processes (see subsection V-A.). In several cell types, cotransport systems are activated and play a role during volume regulation, as described in Section V and in Section VII. It is also likely that this secondary active cotransport of chloride plays a significant role for the apparently active extrusion of acid equivalents from certain cells. If a continuous influx of chloride against an electrochemical gradient is maintained by a cotransport system, the chloride disequilibrium can drive an influx of bicarbonate through the anion exchange mechanism, as described in Section VII. Finally, even the electrodiffusion of anions is shown to be regulated, and in Ehrlich cells and human lymphocytes an activation of the anion diffusion pathway plays a major role in cell volume regulation as described in Section VI and subsection VII-B.(ABSTRACT TRUNCATED AT 250 WORDS)     

Basic haematological values in antelopes--IV. A comparison and concluding remarks

Comparison of mean erythrocyte counts, mean haematocrit values, mean haemoglobin content and mean corpuscular volume of the red cell is presented for 21 species of antelopes (four species being excluded because of the small number of animals investigated). A direct relationship of haematocrit values and haemoglobin content and an inverse relationship of red cell counts and mean corpuscular volume of the erythrocytes were noted. The significance of an increased total surface area for oxygen exchange is discussed and values obtained in the red blood picture are compared with those of domesticated animals (cattle, goat and sheep) taken from the literature. Mean leukocyte counts were found to be in the human range with the exception of two species, but significantly lower than in domesticated artiodactylids. Problems in assessing the general health, the age, the effect of diet and of environment in captivity are discussed. Problems of methodology, especially of blood sampling, are given comparing results in manually restrained and in sedated animals.     

Effects of stiffness and volume on the transit time of an erythrocyte through a slit

By using a fully coupled fluid–cell interaction model, we numerically simulate the dynamic process of a red blood cell passing through a slit driven by an incoming flow. The model is achieved by combining a multiscale model of the composite cell membrane with a boundary element fluid dynamics model based on the Stokes flow assumption. Our concentration is on the correlation between the transit time (the time it takes to finish the whole translocation process) and different conditions (flow speed, cell orientation, cell stiffness, cell volume, etc.) that are involved. According to the numerical prediction (with some exceptions), the transit time rises as the cell is stiffened. It is also highly sensitive to volume increase inside the cell. In general, even slightly swollen cells (i.e., the internal volume is increased while the surface area of the cell kept unchanged) travel dramatically slower through the slit. For these cells, there is also an increased chance of blockage.     

Osmotic properties of bovine erythrocytes aged in vivo

The osmotic properties of bovine erythrocytes aged in vivo were studied by the modified microhematocrit method. The osmotic fragility of older red cells decreases due to their larger relative osmotically non-active volume. Relative critical cell volume of bovine erythrocytes does not alter significantly with cell age. The age dependent change in the osmotic fragility of human red blood cells, the reverse of that found for bovine erythrocytes, is due to a different alteration of the critical cell volume during intravascular erythrocyte aging.     

Cell volume regulation by Amphiuma red blood cells. The role of Ca+2 as a modulator of alkali metal/H+ exchange

In response to osmotic perturbation, the Amphiuma red blood cell regulates volume back to "normal" levels. After osmotic swelling, the cells lose K, Cl, and osmotically obliged H2O (regulatory volume decrease [RVD] ). After osmotic shrinkage, cell volume is regulated as a result of Na, Cl, and H2O uptake (regulatory volume increase [RVI] ). As previously shown (Cala, 1980 alpha), ion fluxes responsible for volume regulation are electroneutral, with alkali metal ions obligatorily counter-coupled to H, whereas net Cl flux is in exchange for HCO3. When they were exposed to the Ca ionophore A23187, Amphiuma red blood cells lost K, Cl, and H2O with kinetics (time course) similar to those observed during RVD. In contrast, when cells were osmotically swollen in Ca-free media, net K loss during RVD was inhibited by approximately 60%. A role for Ca in the activation of K/H exchange during RVD was suggested from these experiments, but interpretation was complicated by the fact that an increase in cellular Ca resulted in an increase in the membrane conductance to K (GK). To determine the relative contributions of conductive K flux and K/H exchange to total K flux, electrical studies were performed and the correspondence of net K flux to thermodynamic models for conductive vs. K/H exchange was evaluated. These studies led to the conclusion that although Ca activates both conductive and electroneutral K flux pathways, only the latter pathways contribute significantly to net K flux. On the basis of observations that A23187 did not activate K loss from cells during RVI (when the Na/H exchange was functioning) and that amiloride inhibited K/H exchange by swollen cells only when cells had previously been shrunk in the presence of amiloride, I concluded that Na/H and K/H exchange are mediated by the same membrane transport moiety.     

Relations among variations in human red cell volume,density, membrane area,hemoglobin content and cation content

It is shown how variations in different properties of red cells can be inter-related provided relations exist among these properties at the single cell level. On the basis of the cell density dependence on cell volume and hemoglobin content, and the assumed volume dependence on red cell cation and hemoglobin content, nine relations among the variations in red cell volume, density, membrane area, hemoglobin content and cation content, and their correlations are derived. Values of seven correlation coefficients are theoretically predicted and are shown to be consistent with the experiments performed by density fractionated red blood cells. The cell volume dependence on cation and hemoglobin content obtained from relations among variations is compared with the predictions obtained by the existing model about the osmotic behavior of the red blood cell. Furthermore, it is shown that data on the variations of the red cell properties indicate the existence of the relation among cation content, hemoglobin content, and membrane area at the level of a single cell.     

Surface modification of silica core-shell nanocapsules: biomedical implications

In this article we present the synthesis of oil core silica shell nanocapsules with different shell thicknesses. The surface of the nanocapsules was modified with polyethyleoxide (PEO) and succinic anhydride. Two biomedical tests were then used to study the biocompatibility properties of these nanocapsules with different surface treatments, hemolysis and thromboelastography (TEG). PEO surface modification greatly reduced the damaging interactions of nanocapsules with red blood cells (RBCs) and platelets and attenuated particle size effects. It was found that the blood toxicity of charged particles increased with the acid strength on the surface. Experiments toward the assessment of detoxification of these nanocapsules in model drug overdose concentrations are currently underway.     

Effective bilayer expansion and erythrocyte shape change induced by monopalmitoyl phosphatidylcholine. Quantitative light microscopy and nuclear magnetic resonance spectroscopy measurements.

When human erythrocytes are treated with exogenous monopalmitoyl phosphatidylcholine (MPPC), the normal biconcave disk shape red blood cells (RBC) become spiculate echinocytes. The present study examines the quantitative aspect of the relationship between effective bilayer expansion and erythrocyte shape change by a newly developed method. This method is based on the combination of direct surface area measurement of micropipette and relative bilayer expansion measurement of 13C crosspolarization/magic angle spinning nuclear magnetic resonance (NMR). Assuming that 13C NMR chemical shift of fatty acyl chain can be used as an indicator of lateral packing of membrane bilayers, it is possible for us to estimate the surface area expansion of red cell membrane induced by MPPC from that induced by ethanol. Partitions of lipid molecules into cell membrane were determined by studies of shape change potency as a function of MPPC and red cell concentration. It is found that 8(+/- 0.5) x 10(6) molecules of MPPC per cell will effectively induce stage three echinocytes and yield 3.2(+/- 0.2)% expansion of outer monolayer surface area. Surface area of normal cells determined by direct measurements from fixed geometry of red cells aspirated by micropipette was 118.7 +/- 8.5 microns2. The effective cross-sectional area of MPPC molecules in the cell membrane therefore was determined to be 48(+/- 4) A2, which is in agreement with those determined by x-ray from model membranes and crystals of lysophospholipids. We concluded that surface area expansion of RBC can be explained by a simple consideration of cross-sectional area of added molecules and that erythrocyte shape changes correspond quantitatively to the incorporated lipid molecules.     

Overview of the diversity of erythrocyte blood group antigens

During the differentiation and maturation of erythrocytes, the surface molecules of erythrocytes are gradually expressed and stabilized. These molecules are to be antigenic in addition to their functions of maintaining cell membrane structural stability, material transport and exchange of cells and signal transmission between cells. The antigenic molecules on the erythrocyte surface are called erythrocyte blood group antigens. The blood group antigens and their corresponding blood group antibodies in vivo are important indicators for clinical blood transfusion and organ transplantation, and also form the basis for research on blood group related diseases. Three hundred and sixty-eight erythrocyte blood group antigens have been confirmed so far, which are classified into 39 blood group systems, 5 blood group collections and 2 blood group series. Based on the diversity of blood group antigens and their composition of glycolipids, glycoproteins and other molecules, this study mainly reviews the classification, molecular structure, antibody response and gene regulation of blood group antigens, and explains the main reasons for the diversity of blood group antigens.     

Kinetics of decrease in erythrocyte filterability under the effect of ephazol]

The effect of palladium-containing complex Ephazol on the filtration rate of erythrocyte suspensions through nuclear filters was studied by the constant-pressure filtration method. It was shown that the filterability of red blood cells incubated with ephazol decreased. If the time necessary for a fixed volume of red blood cell suspension to pass through a filter was plotted against the time of incubation with Ephazol or against its initial concentration, the curves typical of autoaccelerated processes were obtained. From analysis of kinetic models, it was concluded that the effects observed are due to the nonlinear dependence of the filtration rate w on the rate at which an erythrocyte passes through a pore and the influence of Ephazol on the distribution of erythrocytes with respect to w. Several models describing changes in the distribution of erythrocytes with respect to w in the presence of Ephazol and possible mechanisms relating the filtration kinetics to the incubation parameters are discussed.     

Aluminium toxicity induced poikilocytosis in an air-breathing teleost, Clarias batrachus (Linn.)

The present study was undertaken to assess the toxicity of acid alone and two different sublethal concentrations of aluminium, (25% and 75% dose of 96 hr LC50 value in acidified soft water of pH 5) on red blood cells of a stenohaline catfish, C. batrachus for an acute exposure of 5 days. The scanning electron microscopic studies on all the three treated groups revealed several kinds of erythrocyte alterations and modifications with abnormal morphology. These included abnormal surface-wrinkling accompanied with excessive roughness on the membrane, erythrocytes with surface granulation in higher dose and finally the appearance of morphologically abnormal forms, the codocyte (target cell) and the stomatocyte. The results suggest that abnormality in the shape of erythrocytes could be linked to altered surface membrane area to volume ratio, decrease in cytoplasmic volume owing to reduced Hb content or increase in the amount of water content within the cell resulting from osmotic disequilibrium. In this context, the abnormal surface membrane morphology could be attributed to cytoskeleton fragility and defects in structural proteins. Further, the acid group exhibited a striking behavior of cellular adhesion and bonding to adjoining cell surfaces, culminating in several bunches which thereby reduces the surface area for gaseous exchange and could produce blocking effect while flowing through microcirculation.     

Effect of polyamines on the electrokinetic properties of red blood cells.

At the physiological pH 7.4, the zeta potential of the normal red blood cell in 1.5% glycine buffer was found to be ?52 mv, whereas that of sickling erythrocytes is ?45 mv. Addition of spermidine to normal red blood cells reduced the zeta potential by approximately 20 mv. In sickling red blood cells, where the polyamine content is determined to be 5 to 6 times greater than in the normal erythrocyte, addition of spermidine reduced the zeta potential by only 5 mv, indicating that little more polyamine binding occurs. The polyamine content of whole blood taken from 24 patients having sickle cell anemia was found to be more than ten times that of whole blood from normal donors. Binding of polyamines to the normal red blood cell was analyzed from the surface charge potential variation as a function of polyamine concentration and the apparent binding constant determined to be 130 d1/g. The difference in the electrokinetic properties of normal and sickling red blood cells in this system may be attributed, in part, to a variation in the polyamine content of the two types of erythrocytes.     

A comparative study of the effect of refining on charge of various pulps

This work studied the influence of refining on various pulp properties such as freeness, specific surface area, specific volume, water retention value, surface charge, total charge and tensile index of five different pulps. At a freeness level the wheat straw pulp exhibited higher surface charge and total charge compared to other pulps. The specific surface area, specific volume and water retention value of the pulps increased with increased refining. The total fibre charge as determined by conductometric titrations was not affected by refining. However, the surface charge as determined by titrations with poly-DADMAC increased with refining. The increases in specific surface area of pulps by refining resulted in a higher fibre surface charge and also better fibre-fibre bonding. The linear regression models developed using experimental data were found to be accurate. The comparison of model predicted data and the experimental data showed an excellent agreement between them.