Determination of the content of nonfilterable cells in erythrocyte suspensions as a function of the medium osmolality

The results of most filtration assays for deformability of erythrocytes do not distinguish whether the entire population or only its small fraction exhibits abnormal rheological properties. We developed a simple filtration method for determination of the percentage of nonfilterable cells in erythrocyte suspension using membrane filters with mean pore diameter of 3.1 microns. This method makes it possible to detect even minor abnormal subpopulations in erythrocyte suspensions. The flow rate of buffer depends on the number of free pores of a filter. The plot of the number of pores clogged by nonfilterable cells vs the total number of erythrocytes that were allowed to pass through the filter had a linear portion, with a slope representing the relative content, Z%, of nonfilterable cells in the suspension. We determined Z% for various medium osmolalities u and used the data to derive the distribution of erythrocytes in ucr (ucr is the maximum value of u at which an erythrocyte cannot pass through a pore of a given filter because of geometric limitations). The distribution of ucr in suspension of normal erythrocytes has a maximum of about 200 mOsm/kg and a half-width of about 20 mOsm/kg. The distributions of ucr are altered in normal erythrocyte suspensions at decreased pH values, in cryopreserved and ATP-depleted erythrocyte suspensions and in erythrocytes from a xerocytosis patient.     

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.     

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.     

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.     

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.     

Viscoelasticity of packed erythrocyte suspensions subjected to low amplitude oscillatory deformation.

Concentrated adult erythrocyte suspensions were subjected to low amplitude oscillatory shear in a Weissenberg rheogoniometer equipped with a cone-and-plate assembly. The dynamic viscoelastic properties of the suspension were measured over a broad range of frequency by a numerical solution that accounted for fluid inertia. Variation of shear amplitude and cell volume percent, and comparison of buffered saline, plasma, and dextran as suspending media showed that the cellular elements had undergone small bending and shearing deformations. Studies of normal adult erythrocytes, hypotonically swollen cells, temperature-altered cells, and erythrocyte ghosts suggested that the method was evaluating membrane material properties. The normal membrane was found to exhibit a shear rate dependent elastic modulus that increased by more than a factor of 20 over a frequency range from 0.0076 Hz to 60 Hz. The membrane viscosity showed a substantial drop with frequency indicative of a frequency thinning phenomenon. At high frequency of deformation the viscous response of normal erythrocytes was no longer indicative of a membrane property due to the dominant influence of the internal hemoglobin solution. The studies generally supported the ability of the method to quantify relative membrane material properties and detect changes in membrane structure.     

Immunologic properties of mouse thymus cells: membrane antigen patterns associated with various cell subpopulations

The membrane antigen components of mouse thymus cells and fractions derived from BSA density gradient centrifugation were assayed by quantitative cytotoxicity tests. Two subpopulations were identified on the basis of average density and antigen patterns. The major subpopulation consisted of small lymphoid cells and comprised 80%–90% of all cells, was of high relative density and rich in θ, TL, G_IX, Ly-A, Ly-B, and Ly-C, but contained little or no H-2. The minor subpopulation was chiefly large lymphoid cells, comprised 10%–15% of cells, was of low relative density, was relatively rich in H-2 but low in θ and Ly antigens, and contained no detectable TL or G_IX. This minor subpopulation was identical in density and antigen patterns to those cells remaining in the thymus after short-term cortisone treatment or whole-body irradiation. It could also be reproduced by treating whole thymus with anti-TL or anti-θ sera. The antigenic attributes of this minor subpopulation differed from those of spleen lymphocytes only with respect to average density.     

Free radical formation and erythrocyte membrane alterations during MetHb formation induced by the BHA metabolite, tert-butylhydroquinone

Erythrocyte membranes are altered as a consequence of oxidative stress following the incubation of intact erythrocytes with one of the major metabolites of the antioxidant butylated hydroxyanisole (BHA), tert-butylhydroquinone(tBHQ). A ratherpersistentsemiquinone radical was observed by electron spin resonance (ESR) spectroscopy when tBHQ was incubated with either homogeneous oxyhemoglobin solutions or suspensions of intact erythrocytes. Erythrocyte ghosts prepared from fresh control erythrocytes and ghosts from erythrocytes preincubated with BHA and its metabolite, tBHQ, were subjected to polyacrylamide gel electrophoresis (SDS-PAGE). Only minor changes of the electrophoresis pattern relative to the control was observed in the BHA incubations whereas tBHQ significantly increased the amount of high molecular weight degradation products of erythrocyte membrane constituents. These changes were only observed when incubations were performed in the presence of oxygen. In control experiments where heme oxygen was replaced by carbon monoxide, no membrane degradation products appeared. These observations can be interpreted in terms of metabolic activation of the antioxidant BHA via tBHQ to the tert-butylsemiquinone free radical and finally to the corresponding quinone, thereby leading to harmful effects on erythrocyte membrane structures. Moreover, deleterious effects on other biological membranes are also likely to occur.     

Identification of Human Erythrocyte Cytosolic Proteins Associated with Plasma Membrane During Thermal Stress

The influence of thermal stress on the association between human erythrocyte membranes and cytosolic proteins was studied by exposing erythrocyte suspensions and whole blood to different elevated temperatures. Membranes and cytosolic proteins from unheated and heat-stressed erythrocytes were analyzed by electrophoresis, followed by mass spectrometric identification. Four major (carbonic anhydrase I, carbonic anhydrase II, peroxiredoxin VI, flavin reductase) and some minor (heat shock protein 90α, heat shock protein 70, α-enolase, peptidylprolyl cis–trans isomerase A) cytosolic proteins were found to be associated with the erythrocyte membrane in response to in vitro thermal stress. Unlike the above proteins, catalase and peroxiredoxin II were associated with membranes from unheated erythrocytes, and their content increased in the membrane following heat stress. The heat-induced association of cytosolic proteins was restricted to the Triton shells (membrane skeleton/cytoskeleton). Similar results were observed when Triton shells derived from unheated erythrocyte membranes were incubated with an unheated erythrocyte cytosolic fraction at elevated temperatures. This is a first report on the association of cytosolic catalase, α-enolase, peroxiredoxin VI, peroxiredoxin II and peptidylprolyl cis–trans isomerase A to the membrane or membrane skeleton of erythrocytes under heat stress. From these results, it is concluded that specific cytosolic proteins are translocated to the membrane in human erythrocytes exposed to heat stress and they may play a novel role as erythrocyte membrane protectors under stress by stabilizing the membrane skeleton through their interactions with skeletal proteins.     

Altered expression of Plasmodium knowlesi variant antigen on the erythrocyte membrane in splenectomized rhesus monkeys

Variant antigens are present on the surface of Plasmodium knowlesi malaria-infected erythrocytes as detected by the schizont-infected cell agglutination (SICA) assay. We found that parasitized erythrocytes passaged in splenectomized monkeys did not agglutinate with immune sera. On the first passage from intact to splenectomized monkeys, the SICA titers decreased 4- to 16-fold; after the second and subsequent passages in splenectomized monkeys, the infected cells became nonagglutinable to all sera tested, including sera from animals infected with the nonagglutinating parasites. This loss of agglutinability could have resulted from selection of a genetically distinct subpopulation of the original parasites or the ability of the original parasites to alter their phenotypic expression. We have designated the new nonagglutinable phenotype, SICA [-], and the agglutinable phenotype, SICA [+]. The loss of agglutinability indicates that the variant antigen normally expressed on the erythrocyte membrane of infected cells is altered or absent. Because SICA [-] parasites developed in the absence of the spleen, the major organ of host defense against malaria, then this organ may in some manner influence or modulate antigenic expression in P. knowlesi and possibly other malaria parasites.     

Appearance of an erythrocyte population with decreased deformability and hemoglobin content following sepsis

With the use of the cecal ligation and puncture model in mice, this study tested whether sepsis-induced decreased erythrocyte deformability is restricted to a subpopulation of cells. Erythrocyte subpopulations were isolated by centrifugal elutriation. Lineweaver-Burk conversion of deformability-response curves to shear stress was used to determine the shear stress at half-maximal cell elongation (K(EI)) and maximal cell elongation (EI(max)). Sepsis decreased erythrocyte deformability in whole blood. K(EI) values were elevated (2.7 vs. 2.1 Pa) and EI(max) values decreased (0.56 vs. 0.50) in sepsis compared with sham mice. K(EI) values for cells eluted at 7 ml/min (smallest and oldest cells) were similar; however, K(EI) values for cells eluted at 8 ml/min were greater in septic than sham animals (2.50 vs. 2.10). Younger and larger subpopulations of erythrocytes (eluted at 9, 10, and 11 ml/min) also showed a tendency of decreased deformability in sepsis. Mean corpuscular hemoglobin content was decreased in cells eluted at 7 and 8 ml/min in sepsis (4.5 and 10.2 pg) compared to sham (7.4 and 11.4 pg) mice. This study indicates that an erythrocyte subpopulation that represents 20% of circulating cells shows the most pronounced decrease in cell deformability during sepsis. Increased rigidity together with decreased corpuscular hemoglobin content in these cells may contribute to microcirculatory dysfunction and immune modulation during sepsis.     

A single partitioning step in aqueous polymer two-phase systems reduces hypotonized rat erythrocyte heterogeneity

Rat carrier erythrocytes prepared by hypotonic dialysis (80 mOsm/kg) are a heterogeneous cell population that can be fractionated into two-well-defined cell subpopulations by a single partition step, in charge-sensitive dextran-poly(ethylene glycol) aqueous two-phase systems. One subpopulation (65% of total cells) has a decreased cell surface charge and is partitioned at the interface in a single step and then fractionated by counter-current distribution as a low-G subpopulation. The other subpopulation (35% of total cells) has charge surface properties more like those of the untreated control rat erythrocytes. These last cells are partitioned in the top phase in a single step and then fractionated by counter-current distribution as a high-G subpopulation. Partitioning is more effective in reducing cell heterogeneity in hypotonized rat erythrocyte populations than is density separation in Ficoll-paque which only separates a small less dense cell subpopulation (5% of total cells), with the most fragile cells, from a larger and more dense cell subpopulation (95% of total cells), with a mixture of fragile and normal cells. This simple cell separation procedure quickly reduces carrier erythrocyte heterogeneity in a single partitioning step so it can be used to prepare cells for in vivo studies.     

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.     

Free radical formation and erythrocyte membrane alterations during MetHb formation induced by the BHA metabolite,tert-butylhydroquinone

Erythrocyte membranes are altered as a consequence of oxidative stress following the incubation of intact erythrocytes with one of the major metabolites of the antioxidant butylated hydroxyanisole (BHA), tert-butylhydroquinone(tBHQ). A ratherpersistentsemiquinone radical was observed by electron spin resonance (ESR) spectroscopy when tBHQ was incubated with either homogeneous oxyhemoglobin solutions or suspensions of intact erythrocytes. Erythrocyte ghosts prepared from fresh control erythrocytes and ghosts from erythrocytes preincubated with BHA and its metabolite, tBHQ, were subjected to polyacrylamide gel electrophoresis (SDS-PAGE). Only minor changes of the electrophoresis pattern relative to the control was observed in the BHA incubations whereas tBHQ significantly increased the amount of high molecular weight degradation products of erythrocyte membrane constituents. These changes were only observed when incubations were performed in the presence of oxygen. In control experiments where heme oxygen was replaced by carbon monoxide, no membrane degradation products appeared. These observations can be interpreted in terms of metabolic activation of the antioxidant BHA via tBHQ to the tert-butylsemiquinone free radical and finally to the corresponding quinone, thereby leading to harmful effects on erythrocyte membrane structures. Moreover, deleterious effects on other biological membranes are also likely to occur.     

Separation of mouse thymocytes into two subpopulations by the use of peanut agglutinin.

A new method for the separation of cell subpopulations using a lectin as a reversible probe, is described. We have found that the major immature thymocyte subpopulation can be readily separated from the immunocompetent minor subpopulation by agglutination with peanut agglutinin (PNA) and can be recovered as viable single cells by dissociation of the agglutinated cells with d-galactose.The two subpopulations were characterized by their content of H-2 and θ antigens, their graft versus host activity and their mitogenic response to phytohemagglutinin and concanavalin A. Binding studies with [¹²⁵I]PNA indicate that attachment of sialic acid residues to the PNA receptor may be an important step in the maturation of the murine thymocytes.     

Synchronized human diploid fibroblasts: progression capabilities of a subpopulation that fails to keep pace with the predominant, rapidly dividing cohort of cells

Highly synchronized cultures of HSF-55 human diploid fibroblasts contain subpopulations of cells with intact plasma membranes that do not participate in the parasynchronous division wave. To determine the fate of these laggard cells, cultures were incubated with BrdU for variable periods to label newly replicated DNA in both the readily synchronizable and nonsynchronizable subpopulations. The kinetics of labeling with BrdU were determined with a two-laser flow cytometric technique that did not employ antibody to BrdU, but instead monitored emission of fluorescence from DNA-specific stains that differed in the degree of BrdU-induced quenching of their fluorescence signals. Approximately 90% of the cells rapidly incorporated BrdU and later divided within a 3 hr period. The remaining 10% of the cells, however, were found to reside within a minority subpopulation that maintained the capacity to traverse the cell cycle, but at a greatly reduced rate relative to the progression capacity of the majority of cells. Cells were viably sorted from these cohorts within the synchronized culture, and their kinetic behavior was determined through direct measurement of their growth rates and plating efficiencies. As predicted by the BrdU labeling studies, the sorted cells from the minority, slowly traversing subpopulation divided at a rate that was 30 to 50% lower than that obtained with cells sorted from the readily synchronizable subpopulation. From consideration of the kinetics of entry into S-phase of the majority and minority subpopulations, protocols are described that should allow preparation of relatively pure populations of both early- and late-replicating species of human DNA.     

The dynamics of heat production in erythrocytes of the scorpion fish (<Emphasis Type="Italic">Scorpaena porcus</Emphasis> Linnaeus, 1758) in vitro

Temperature measurements in a plastic tube isolated from external influences containing an erythrocyte suspension of the scorpion fish (Scorpaena porcus Linnaeus, 1758) showed that these red blood cells are able to generate heat. Heat release in the cell suspension was expressed by a linear temperature increase in the tube during the entire experiment. Addition of extracellular ATP (1 mg mL^–1) caused the effect of a thermal shift: a sharp temperature rise in the cell suspension for 30–60 s. We believe that the heat release was caused by hydrolysis of extracellular ATP by membrane ecto-ATPase. Inhibition of ecto-ATPase activity through the addition of EDTA (1 mM) to the erythrocyte suspension led to complete blockage of heat release; the effect of the thermal shift ceased. We assume that thermal properties of red blood cells play an important role in blood hemodynamics, especially in providing the “non-Newtonian” properties of blood. The thermal phenomena observed in suspensions of fish erythrocytes open new scientific directions in exploring the capabilities of multifunctional extracellular ATP.     

Changes of membrane permeability due to extensive cholesterol depletion in mammalian erythrocytes

55% of the total membrane cholesterol could be removed from porcine, bovine and human erythrocytes by incubating the cells in suspensions of lecithin liposomes. Up to 30% depletion, membrane permeability remained unaltered; more extensive depletion induced a marked increase of the transfer rates of nonelectrolytes and of organic acids penetrating by nonionic diffusion. This biphasic response of permeability to cholesterol depletion, which has not been observed in artificial lipid membranes, may be related to the heterogeneity of the erythrocyte membrane lipids or to a pool of cholesterol not interacting with the phospholipids.