A modification of the filtration method for studying the content of nonfilterable cells in erythrocyte suspension

The results of filtration assays provide estimates of the deformability of erythrocytes averaged over the entire suspension. These assays do not distinguish whether the entire population or only its small fraction exhibits abnormal rheological properties. We developed a simple method using a filtrometer to determine the percentage of non-filterable (under given conditions) cells in the erythrocyte suspension. Membrane filters made of a polyethylene terphthalate film had the mean pore diameter of 3.1 microns and the length of cylindrical micropores of 7 microns. The buffer flow rate tb depends on the number of free pores in a filter. The plot of the number of pores clogged by non-filterable cells versus the total number of erythrocytes passed through the filter had a linear portion whose slope represents the relative content Z of non-filterable cells in the suspension. We determined Z for various medium osmolarities u. These data were used to derive the distribution of erythrocytes in ucr, the value of u at which an erythrocyte cannot pass through a pore of a given filter because of geometric limitations. The distribution maximum corresponded to 190-200 mOsm/kg for erythrocytes from the normal blood. This means that normal erythrocytes have the median values of their surface area and area-to-volume ratio of 155-151 microns2 and 1.72-1.68 microns-1, respectively. The half-width of the distribution was approximately 30 mOsm/kg. This finding suggests that the normal blood contains a certain fraction of erythrocytes with a decreased area-to-volume ratio. Our results showed that the distribution is altered in various forms of anemia and in ATP-depleted erythrocyte suspensions.     

Distributions of rheological parameters in populations of human erythrocytes

We have previously proposed the osmofiltration method based on a modified Hanss hemorheometer to analyze distributions of erythrocytes in their ability to pass through membrane filters with 3 microns pores. Upon decrease in medium osmolality (u) the erythrocyte volume increases. When cell volume becomes V = Vcr at u = ucr, such cell loses its ability to pass through a 3 microns pore. The flow rate of erythrocyte suspension containing cells with different ucr through a filter gradually decreases with decreasing medium osmolality. This rate becomes zero at some u = omega, when the number of non-filterable cells in the applied sample approaches the number of pores in filter. Experimental determination of the dependencies of the filtration rate on medium osmolality for various hematocrit values allows to obtain omega for each hematocrit and, thereby, to assess the distribution of erythrocytes in ucr. Here, we propose a simplified version of this method, which allows screening of the erythrocytes in heterogeneous suspensions for the distribution in ucr by measuring omega for only two hematocrit values, 0.1% and 1%. Applications of the proposed method are exemplified by analysing the erythrocyte populations of healthy donors, of patients with microspherocytosis, hemochromatosis and normal erythrocyte populations in an acidic environment.     

Mathematical analysis of the effects of geometric parameters and mechanical properties of erythrocytes on the filterability of nonuniform suspensions

Approaches to determination of the pattern of erythrocytes distribution with regard to the rates of their passage through pores (3 microm in diameter) of a membrane filter by processing the data on changes in the flow rates of erythrocyte suspensions with time (filtration curves) are discussed. We considered the case when the suspension consisted of two subpopulations of erythrocytes differing in a single parameter. Using a model describing the erythrocyte passage through a pore and a model describing filtration of a nonuniform suspension, we analyzed the dependences of filtration kinetics of such suspensions on the relative contents of the subpopulations and their rheological characteristics. It has been shown that the filtration rates of the major subpopulation and the minor abnormal subpopulation, and their relative contents can be determined from the analysis of filtration curves. This can be done when the filtration rate of cells from the minor subpopulation is at least one order of magnitude lower than the filtration rate of cells from the major subpopulation. Thus we can register the presence of the minor subpopulation in the range of 0.5-1%. If filtration rates are recorded at different osmolalities, their analysis makes it possible to determine the surface area, intracellular viscosity, and membrane rigidity of cells of the major subpopulation and, in certain cases, the same parameters for the cells of the minor subpopulation.     

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.     

Flow characteristics of red cell containing fluids through pores. The effect of filter plugging, a mathematical model

A mathematical model was developed that describes the effects of filter plugging on flow through 3 micron pore polycarbonate filters as a function of time, pressure, and cell concentration, both under stirring and nonstirring conditions. The mathematical constants for the model were derived from experimental data generated with a filtration apparatus, and were tested by using various concentrations of cells that are able to plug filter pores. A computer simulation program was written to test the model over a wide range of nonfilterable cell concentrations.     

Theoretical modeling of filtration of blood cell suspensions

A theoretical model of filtration of suspensions containing red blood cells (RBCs) and white blood cells (WBCs) has been developed. Equations are written for the pressure drop, the filtration flow and the fractions of filter pores containing RBCs (alpha) and WBCs (alpha*). Because the relative resistances (ratios of resistance of cell to resistance of suspending fluid) of RBCs (beta) and WBCs (beta*) through the filter pore are greater than one, the transit of these cells (especially WBCs) through the filter is slower than that of suspending fluid; this leads to values of alpha and alpha* higher than those simply expected from the hematocrit and leukocrit, respectively, in the entering and exiting suspensions. In the absence of pore plugging by the cells (steady flow), the pressure drop can be computed from alpha, alpha*, beta and beta*. In order to model unsteady flow, differential equations are written to include pore plugging and the subsequent unplugging by the rising filtration pressure at a constant flow. By specifying the fractions of entering RBCs (epsilon) and WBCs (epsilon*) which would plug the pores and the rate at which the plugged pores would unplug in response to pressure rise (epsilon u), as well as the fractions of entering RBCs (epsilon p) and WBCs (epsilon p*) that would plug the pores permanently, theoretical pressure-time curves can be generated by numerical integration, and the results fit the experimental data well. From such fitting of theoretical curve to experimental data, information can be deduced for epsilon, epsilon*, epsilon u, epsilon p and epsilon* p.     

Measurements of viscosity of synthetic erythrocyte suspensions

Measurements were made of the viscosity of suspensions of synthetic erythrocytes composed of hemoglobin solutions encapsulated in liposomes, as a function of shear rate, temperature, suspension concentration, lipid membrane composition, and the viscosity of the suspending medium. It was found that the viscous behavior of the synthetic erythrocyte suspensions was non-Newtonian and nearly the same as that of suspensions of natural erythrocytes prepared similarly, with the major difference being that synthetic erythrocyte suspensions are somewhat more viscous. Suspensions of Fluosol FC-43 prepared similarly were found to be essentially Newtonian fluids, and substantially different and more viscous than either erythrocyte suspension. The higher viscosity of synthetic erythrocyte suspensions probably accounts for the ability of these suspensions to maintain normal systemic vascular resistance in transfusion experiments, in spite of the fact that synthetic erythrocytes are smaller than natural erythrocytes.     

Theoretical and experimental study of the time dependent flow of red blood cell suspension through narrow pores

The Hemorheometer has been adapted to allow the recording of the flow rate during the filtration process. For newtonian fluids, the flow rate variation versus time through the pores is well approximated by Poiseuille's law. For dilute red blood cell suspensions, the same analysis can be applied by introducing the concept of "apparent filtration viscosity" which is higher than the usual viscosity measured by Couette viscometry. The apparent filtration viscosity parameter is related to the deformations undergone by red blood cells as they pass through the narrow pores. Apparent filtration viscosity can be used to obtain a precise determination of the erythrocyte deformability. Measurements performed, for a given blood sample, with pores of different diameters (5 microns, 8 microns and 12 microns) show that the error on the value of apparent filtration viscosity is less than 3%. As a result, the sensitivity of the filtration method allows to discriminate among normal blood samples. High concentrations of erythrocytes or leucocytes are found to modify the apparent filtration viscosity. These factors are apparent in the recorded filtration curves. Their effects on filtration measurements can be easily estimated.     

Physiological salines and the mechanical properties of trout red blood cells

Suspensions of rainbow trout erythrocytes in different physiological salines were compared with respect to their haematological and filtration properties.
A method is described for the suspension of erythrocytes in Cortland saline which has proved suitable for studies of their mechanical properties over periods of several hours.
Significant differences were found between whole blood samples taken during cannulation and after several days recovery, particularly mean cell volume, frequency distribution of red cell volumes and the pore passage time through nucleopore filters. These differences were also found using red cell suspensions of the same bloods. The pore passage time of whole blood sampled during cannulation or its suspensions is less than that of recovery blood although its mean cell volume is greater.     

Stability of membrane pores in hypotonically dialyzed erythrocytes: coencapsulation of different stokes radius probes can occur for at least 21 days in human erythrocytes

Hypotonic dialysis of human erythrocytes results in porous cell stability for several days. Hypotonic cells stored for 1 week are essentially normal with respect to the preparation of carrier erythrocytes. Afterward, cells begin to irreversibly hemolyze resulting in decreased cell recoveries and decreased encapsulation percentages of two probes, sucrose and inulin. The holes generated by controlled hypotonic dialysis (100 mOsm/kg) are unlike the single rupture hole generated by dialysis to 10-20 mOsm/kg. The minimum pore size of resealed, annealed carrier cells is confirmed to be less than 5.2 A.     

Na, k, and nonspecific solute requirements for induction and function of galactose active transport in an antarctic psychrophilic marine bacterium

Strong adherence of bacteria, yeast, erythrocytes, leukocytes, platelets, spores, and polystyrene spheres to membrane filter materials was noted during filtration through membranes with pore size diameters much larger than the particles themselves. Quantitative recovery on the membrane filters of these particles from low-concentration suspensions was achieved during gravity- or vacuum-assisted filtration through membranes with pore diameters as much as 30 times that of the filtered particles. Mechanical sieving was not responsible. The phenomenon was judged to be electrostatic. It could be partially blocked by pretreating the filter with a nonionic surfactant (Tween 20), and elution of adherent particles was achieved with 0.05% Tween 20. Gram-positive cocci were removed from suspension more efficiently than gram-negative rods. The commonly used cellulose membranes adsorbed more bacteria, blood cells, and other particles than did polycarbonate filters. Of lesser adsorptive capacity were vinyl acetate, nylon, acrylic, and Teflon membranes. Backwashing with saline, serum, 6% NaCl, dextran solutions, or phosphate buffers of varying molality and pH removed only a fraction of adherent particles. Tween 20 (0.05%) eluted up to 45% of adherent particles in a single back-filtration. Selected filters quantitatively removed the particles tested, which then could be washed and subjected to reagents for a variety of purposes. It is important to anticipate the removal of particles during membrane filtration, since it is not a simple mechanical event.     

Adherence of bacteria, yeast, blood cells, and latex spheres to large-porosity membrane filters.

Strong adherence of bacteria, yeast, erythrocytes, leukocytes, platelets, spores, and polystyrene spheres to membrane filter materials was noted during filtration through membranes with pore size diameters much larger than the particles themselves. Quantitative recovery on the membrane filters of these particles from low-concentration suspensions was achieved during gravity- or vacuum-assisted filtration through membranes with pore diameters as much as 30 times that of the filtered particles. Mechanical sieving was not responsible. The phenomenon was judged to be electrostatic. It could be partially blocked by pretreating the filter with a nonionic surfactant (Tween 20), and elution of adherent particles was achieved with 0.05% Tween 20. Gram-positive cocci were removed from suspension more efficiently than gram-negative rods. The commonly used cellulose membranes adsorbed more bacteria, blood cells, and other particles than did polycarbonate filters. Of lesser adsorptive capacity were vinyl acetate, nylon, acrylic, and Teflon membranes. Backwashing with saline, serum, 6% NaCl, dextran solutions, or phosphate buffers of varying molality and pH removed only a fraction of adherent particles. Tween 20 (0.05%) eluted up to 45% of adherent particles in a single back-filtration. Selected filters quantitatively removed the particles tested, which then could be washed and subjected to reagents for a variety of purposes. It is important to anticipate the removal of particles during membrane filtration, since it is not a simple mechanical event.     

Theory of sound attenuation in the blood and erythrocyte suspensions]

Mechanisms of the ultrasound attenuation in blood and erythrocyte suspension in the long wave range are examined. It is shown that the theory proposed for dilute suspension of structured microobjects has a good coincidence with the known experimental data, both on erythrocyte suspensions and on blood. The contribution of viscous losses to attenuation are decreased with frequency and reach 44% in water suspensions of erythrocytes and 24% in the whole blood at 1 MHz.     

Membrane pores in hypotonically dialyzed sheep erythrocytes remain open after three weeks of storage: entrapment of different stokes radius probes, [14C]sucrose and [3H]inulin

Sheep carrier erythrocytes were prepared from dialyzed cells stored for 3 weeks. The initial pore size in freshly dialyzed cells exceeds the Stokes radius of that for hemoglobin. Hypotonically dialyzed erythrocytes are then very stable in a porous state. Two probes of different Stokes radius were used to determine the relative size of the pores. Sheep erythrocytes entrap inulin to a greater extent than sucrose, a much smaller molecule. With storage, a greater fraction of dialyzed cells become impermeable to inulin than to sucrose indicative of pore size greater than 5.2 less than 20 A. Since hemoglobin content did not change relative to storage, the pore size was less than the Stokes radius of hemoglobin. Pores generated by controlled hypotonic dialysis are unlike the single rupture pore found in erythrocyte ghosts.     

A new simple method for determination of erythrocyte filtrability.

A new simple filtration technique designed for measuring red cell filtrability in the routine laboratory use was developed. The suspension of the whole blood in saline (1:20,000 dilution) was processed on the Sartorius filter membranes, pore size 8 micron. The percentage of passed erythrocytes indicating red cell filtrability was determined. The suitability and perspective applicability of this method for studying various hematological disorders is proposed.     

Indices of filterability of red blood cell suspensions

A number of different experimental techniques have been devised in recent years to use microsieving as a test of the filterability of suspensions of red blood cells. Various indices have been proposed to express the results of these tests. In the present paper a correlation is made of the intrinsic increase in resistance at the level of a single pore in the filter to the macroscopically observed pressure and flow through the entire filter. Further it is shown how a number of different tests may be used to derive the same index. The results apply only to situations in which there is no plugging of pores.     

Application of some droplet sedimentation theories to layered erythrocyte suspensions

The applicability of some existing droplet sedimentation theories to erythrocyte suspensions was investigated using glutaraldehyde-fixed erythrocytes from horse, canine, pig, chicken, and human. The Svensson criteria were shown to underestimate the load supportable by a density gradient. The available theories on droplet formation times could not predict each other, and the data on erythrocyte suspensions, especially at high suspension concentrations. It was finally argued that, since the behavior of erythrocytes was not controlled by the diffusion process, the erythrocyte suspensions were not expected to exhibit lasting or absolute stability even when the particle load was small.     

Dewatering of algal suspension using microfiltration with cross flow in the presence of magnetite as a filter aid

Dewatering algal suspensions is an important step in the extraction of oil and other useful substances from algae. In this study, spherical Nannochloropsis sp. and ellipsoidal Monoraphidium sp. suspensions were dewatered in the presence of different amounts of 350-nm magnetite particles using a microfiltration membrane with 360-nm pores in cross-flow mode. Magnetite functions as a filter aid by reducing the deformation of the cake of filtered algae on the membrane and providing paths for water to flow through the filtration cake of algae. In the case of Nannochloropsis sp., the highest dewatering rate was obtained when the number ratio, defined based on the size and ideal density, between Nannochloropsis sp. and magnetite was 1:12.5, but the addition of magnetite had no observable effect on the filtration of ellipsoidal Monoraphidium sp. suspensions through the membrane. After dewatering, magnetite was effectively separated from the concentrated algal suspension by the application of a magnetic field in an open flow system. Magnetite has the potential to enhance dewatering performance using a cross-flow membrane system.     

Application of some droplet sedimentation theories to layered erythrocyte suspensions

The applicability of some existing droplet sedimentation theories to erythrocyte suspensions was investigated using glutaraldehydefixed erthrocytes from horse, canine, pig, chicken, and human. The Svensson criteria were shown to underestimate the load supportable by a density gradient. The available theories on droplet formation times could not predict each other, and the data on erythrocyte suspensions, especially at high suspension concentrations. It was finally argued that, since the behavior of erythrocytes was not controlled by the diffusion process, the erythrocyte suspensions were not expected to exhibit lasting or absolute stability even when the particle load was small.