Deformability investigations of erythrocytes stored at different temperatures

The deformability changes of erythrocytes stored at different temperatures were characterized by a filtration procedure. The filterability index (FI) of erythrocytes cryopreserved at either -196 degrees C or -28 degrees C increased (deformability decreased) by about 9% and 40% respectively. For comparison with the cryopreserves the filterability of erythrocytes stored in liquid state at +4 degrees C were investigated. The filterability changes are discussed in comparison with the changes of MCHC during storage.     

Deformability of stored erythrocytes as a criteria for their in vivo survival rate

The loss of deformability observed in erythrocytes stored as whole blood for 36 days (ACD-AG) or as buffy-coat free erythrocyte concentrate (EK) was characterized by measuring their filterability. During the first 3 weeks the index of filterability for ACD-AG erythrocytes increased only slightly and rose to about 140% of its initial value on the 36th day. In contrast, a heavy loss of deformability (increase of the filterability index to more than 600%) was detected for erythrocytes from EK, which, from a rheological point of view, is apt to raise doubts of using this stored blood. An incubation of 1 hour at 37 degrees C in fresh plasma did not result in improving the deformability. A cell volume loss of more than 20% connected with an increase of the inner viscosity to more than 400% was found to be the cause of this decrease of deformability. These rheological differences are also reflected in the 24 hours in vivo survival rate (SR), if the "early loss" of damaged erythrocytes immediately after transfusion is taken into account. Whereas the SR values of 80% for whole blood erythrocytes do not change significantly, the SR values for EK values can be found to reach 54% approximately.     

Correlation between local cell membrane displacements and filterability of human red blood cells.

Local mechanical fluctuations of the cell membrane of human erythrocytes were shown to involve MgATP- and Mg(2+)-driven fast membrane displacements. We propose that these local bending deformations of the cell membrane are important for cell passage through capillaries. In order to verify this hypothesis, we examined cell membrane fluctuations and filterability of erythrocytes over a wide range of medium osmolalities (180-675 mosmol/kg H2O). The results indicate the existence of a positive correlation between the amplitude of local cell membrane displacements and cell filterability. We suggest that the occurrence of metabolically driven membrane displacements on the side surface of the red blood cell diminishes its bending stiffness and enables it to fold more efficiently upon entrance into blood capillaries. Thus, local cell membrane displacements seem to play an important role in microcirculation.     

The effects of ionophore A23187 on erythrocytes relationship of ATP and 2,3-diphosphoglycerate to calcium-binding capacity

The divalent cation ionophore, A23187, was employed as a means to load fresh human erythrocytes with calcium, and the capacity for accumulation was characterized. Erythrocytes exposed to A23187 in calcium-containing media rapidly accumulated calcium in millimolar quantities. The final cellular concentration was dependent upon medium calcium concentration and the size of the cellular organophosphate pool. When ATP and 2,3-diphosphoglycerate contents were depleted or repleted, the cellular calcium content changed proportionally. Calcium loading of fresh erythrocytes produced no discernible change in the cellular concentrations of ATP or 2,3-diphosphoglycerate. Calcium accumulation was also accompanied by loss of cellular potassium and H₂O, deterioration of cell filterability, and spheroechinocytic transformation.     

Changes in fluidity of erythrocyte membranes after storage of erythrocytes and regeneration of cellular ATP level

The membrane fluidity of freshly collected human erythrocytes, of erythrocytes stored for 3–4 weeks and of stored erythrocytes rejuvenated with glucose and inosine was investigated by measuring polarization of fluorescence emission of 1,6-diphenyl-1,3,5-hexatriene and $\text{N-}\text{phenyl}\text{-1-}\text{naphthylamine}$. The fluidity of membranes prepared from stored erythrocytes was higher than that of fresh erythrocytes. After rejuvenation of erythrocytes with glucose and with or without inosine the membrane fluidity decreased. These changes were probably due to variations of ATP levels in the erythrocytes.     

Decreased deformability in aging erythrocytes

Deformability of bovine erythrocytes separated according to density (and age) was estimated by a modified Teitel's filterability test, the centrifugational test of Sirs, and viscosity measurements of cell suspensions. Both youngest and oldest erythrocytes were found to be less deformable than middle-aged cells, a result speaking against any chief role for deformability in the recognition of senescent erythrocytes and their removal from the circulation.     

HNE produced by the malaria parasite Plasmodium falciparum generates HNE-protein adducts and decreases erythrocyte deformability

In Plasmodium falciparum-parasitized erythrocytes, hemozoin (HZ) formation was accompanied by enhanced formation of 4-hydroxynonenal (HNE)-protein adducts on the cell surface, reaching in the HZ-rich schizont forms the 16.8-fold amount of control non-parasitized cells. The addition of 1-100 microM exogenous HNE to control non-parasitized cells generated HNE-adducts on surface proteins in amounts similar to those found in schizonts. Parasitized as well as HNE-treated non-parasitized erythrocytes showed decreased cell deformability (measured as decreased filterability through cylindrical-pore filters) related to the amount of HNE adducts. In vivo, the HZ-containing trophozoites and schizonts are phagocytic targets for monocytes/macrophages. The reduced deformability of circulating erythrocytes carrying HNE-adducts may increase their phagocytic elimination. Uncontrolled HNE production by parasitized erythrocytes may additionally modify non-parasitized bystander erythrocytes, induce their phagocytosis, and contribute to malarial anemia, which is predominantly due to the removal of large numbers of indirectly damaged non-parasitized erythrocytes.     

The relationship of phosphorylation of membrane proteins with the osmotic fragility and filterability of Plasmodium berghei-infected mouse erythrocytes

Membrane from Plasmodium berghei-infected mouse erythrocytes showed a pattern of protein phosphorylation which was substantially altered from the normal pattern, with an increase in the phosphorylation of the protein with an apparent molecular weight of 43,000 (M 43), which increased from undetectable in uninfected cells to a maximum in the mature trophozoite stage. Phosphorylation levels of this and other minor bands were strongly correlated with osmotic fragility and filterability. The level of M 43 phosphorylation in membranes from cells which remained intact in a hypotonic medium was 3.82 +/- 0.59-times that of lysed cells, compared with the value of 0.76 +/- 0.07 calculated from distribution alone. Results found when intact erythrocytes were phosphorylated by incubation with [32P]Pi prior to partial lysis were similar to those found when membranes from the lysed and unlysed fractions were subsequently phosphorylated with [gamma-32P]ATP. Infected erythrocytes which could pass repeatedly through 3-micron polycarbonate filters had a much higher phosphorylation level for the M 43 region than whole infected cells with similar parasitemia and stage distribution. The phosphorylation change could play a role in the control of osmotic and mechanical properties of the infected erythrocytes during maturation.     

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.     

Increased susceptibility of stored erythrocytes to anti-band 3 IgG autoantibody binding

When human blood was stored in a citrate-phosphate-dextrose (CPD) solution at 4°C, the susceptibility of the erythrocytes to binding of autologous IgG increased. The autologous IgG binding was partially inhibited by purified Band 3 glycoprotein and its oligosaccharides. The susceptibility of the erythrocytes to binding of ¹²⁵I-labeled anti-band 3 IgG autoantibody similarly increased. The results indicate that the anti-band 3 binding sites composed of Band 3 oligosaccharides were generated on the cell surface. The rate of the increase in the susceptibility of the stored cells to the antibody binding was lowered when blood was stored in a CPD solution containing L-ascorbic acid or erythorbic acid, suggesting involvement of an oxidative mechanism in the generation of the binding sites. The cytoplasmic glutathione level of erythrocytes gradually decreased during the blood storage. Storing blood in a CPD solution containing glutathione monoethylester or glutathione monoisopropylester resulted in partial prevention of the decrease in cytoplasmic glutathione level and of the increase in the IgG-binding ability of the cells. Similar preventive effect of glutathione monoethylester was observed in the binding of ¹²⁵I-labeled anti-band 3 autoantibody to the stored erythrocytes. Thus, the increase in the susceptibility of the stored erythrocytes to anti-band 3 binding may be caused, at least partially, by an oxidative stress resulting in a decreased cytoplasmic glutathione level.     

Differences between cation-osmotic hemolysis and filterability in exaprolol- and glutaraldehyde-treated human red blood cells

The changes in human red blood cell microrheology in different glutaraldehyde (3.0 and 5.0 x 10(-6) mol x l(-1)) and exaprolol (2.5 and 5.0 x 10(-4) mol x l(-1)) concentrations were studied. The method of millipore filtration was compared with the method of cation-osmotic hemolysis. Both drugs prolonged the filtration time. Cation-osmotic hemolysis in glutaraldehyde-treated cells was significantly lower in comparison with the control group. On the other hand, there was a significant increase in cation-osmotic hemolysis in exaprolol-treated cells. Besides cation-osmotic hemolysis and filterability of erythrocytes, we evaluated the medium cell volume (MCV) and the medium cell hemoglobin concentration (MCHC). No changes in MCV and MCHC in glutaraldehyde-treated cells were observed. However, the MCV was significantly lower and the MCHC was significantly higher in exaprolol-treated cells. In conclusion, we suggest that the method of cation-osmotic hemolysis is more sensitive than the filtration method for determination of red blood cell microrheology.     

Alterations in rheologic properties of stored blood as criterion of the functional state of erythrocytes in haemocarboperfusion

Rheologic studies performed on rotational viscometers "Rheotest-2" and "VIR-78" within shear rates gamma interval of 0.1 s-1 less than gamma less than 1,312 s-1, revealed a substantial reduction in dynamic viscosity of the blood subjected to sorption treatment on activated charcoal. The importance of volume concentration of erythrocytes proper for dynamic viscosity is analysed. Special emphasis is placed on the necessity to take into account the dispersion of electrophoretic mobility of erythrocytes in analyzing surface properties of erythrocytes from stored blood before and after hemocarboperfusion. In addition, the normalization of electrosurface characteristics of erythrocytes and associated with them the aggregational property of blood after its purification by sorption is demonstrated. The restoration of the explored physical and chemical characteristics of blood, perfused through the sorbent, permits its more valuable transfusional medium to be considered in comparison to conventional stored blood with identical times of preservation.     

Changes in membrane properties of rat red blood cells induced by cadmium accumulating in the membrane fraction

When rat red blood cells were incubated in a cadmium (Cd)-free medium following 1-h pretreatment with 0.5 mM CdCl2, incorporated Cd was retained in the cell during 14-h incubation and progressively accumulated in the membrane fraction, especially in the cytoskeleton fraction. In parallel to this accumulation, red cell filterability decreased and echinocytic cells increased, although intracellular ATP was maintained at the control level. The echinocytic shape was maintained in ghosts and cytoskeletons prepared from the Cd-loaded cells. In addition, the association of bands 2.1, 3, 4.2, and 4.5 with cytoskeletons increased and dissociation of cytoskeletal networks at low ionic strength decreased as the incubation time increased. Pretreatment of red blood cells with Cd also induced a release of small vesicles. These vesicles contained hemoglobin but were depleted of spectrin and actin, showing a phospholipid composition similar to that of red cell ghosts. These results suggest that the organization of cell membranes, especially cytoskeletal networks, is altered by Cd accumulation in the cytoskeleton fraction, which results in acceleration of age-related changes of red blood cells such as shape change and decreased filterability.     

Lysis of erythrocytes from stored human blood by phospholipase C (Bacillus cereus).

The ability of phospholipase C (Bacillus cereus) to lyse erythrocytes from human blood that had been stored under Transfusion Service conditions for up to 16 weeks has been examined. When incubated at 20 degrees C with enzyme (0.03 mg/ml, 55 units/ml) for up to 1 h fresh erythrocytes were not lysed. After about 4 weeks of storage a population of very readily lysed erythrocytes appeared. The morphological changes in erythrocytes from blood stored up to 16 weeks were examined by scanning electron microscopy. The proportion of very readily lysed erythrocytes correlated well with the proportion of spheroechinocytes I. This morphological form was shown to be preferentially removed by phospholipase C and before lysis a transient appearance of smooth spheres occurred. The decrease in blood ATP concentrations on storage was measured and found to correlate with the disappearance of discoid erythrocyte forms, but not directly with the increased susceptibility of the erythrocytes to lysis by the enzyme. However, erythrocytes of up to at least 15 weeks of age could be made less susceptible to lysis by pre-incubation in a medium designed to cause intracellular regeneration of ATP. During the lysis of spheroechinocytes I by electrophoretically pure recrystallized phospholipase C a rapid degradation of phosphatidylcholine, phosphatidylethanolamine and phosphatidylserine + phosphatidylinositol) occurred together with a slower degradation of sphingomyelin.     

Maintenance of in vitro cultures of Babesia divergens and Babesia major at low temperatures

Cultures of erythrocytes parasitized by Babesia divergens and Babesia major were stored in medium cooled to 4 degrees C for up to 8 weeks. There was a marked decrease in parasitaemia and an increase in the number of free extra-erythrocytic, unagglutinated merozoites, during the cooled period. Cultures stored in this way and returned to 38 degrees C resumed growth, with or without sub-culture. At the low temperature, only one sub-culture is required per week.     

Use of polyester leukocyte elimination filters in blood filterability research

We tested a new routine to eliminate leukocytes for blood rheology measurements using commercial leukocyte absorbing filters (here PALL RC400). These filters were punched out and fitted in smaller chambers through which blood was filtered under controlled suction pressure (< 30 mm Hg). This technique resulted in a very effective leukocyte elimination to 0.0022% but also a platelet reduction to 0.2%. The process causes a small but significant hemolysis with free hemoglobin, of the order of 0.06% of the filtered erythrocytes. A small fraction of the erythrocytes were retained in the filter, versus plasma, to reduce the hematocrit on the order of 1.4%. The leukocyte filtration did not cause any detectable functional trauma to the erythrocytes, measured as micro-pore filterability of normal and glutaraldehyde (GA) hardened erythrocytes. However, when 10% of the erythrocytes were hardened with GA, which caused an increase in pore clogging slope (p < 0.05), the additional passage through the leukocyte elimination filter removed this measured change in clogging. This observation suggests that the leukocyte elimination filter may selectively remove, not only leukocytes and platelets, but also hardened erythrocytes. Reticulocyte counting did not reveal any selective removal of young erythrocytes. In general, we find the presented method reproducible, efficient and easy for eliminating leukocytes for blood rheology research although the risk of removing undeformable erythrocytes must be considered.     

Crosslinking of membrane proteins in red blood cells from vitamin E-deficient lead-poisoned rats

Red blood cells from vitamin E-deficient rats lose their filterability after incubation in vitro and concurrent lead poisoning of the rats accelerates this decline. This decreased red cell filterability is associated with an increased crosslinking of red cell membrane proteins. Previous reports by others suggested an association between red cell glutathione levels and filterability, but we found no such association in red cells treated with N-ethylmaleimide. Increased aggregation of red cell membrane proteins may play a role in the discocyte/ spherocyte shape change that accounts for the impaired filterability of red blood cells from vitamin E-deficient lead-poisoned rats.     

Homocysteinylation score of high-molecular weight plasma proteins

Ultrafiltration rates (filterability) of protein-bound homocysteine (Hcy), unlike glutathione (Glt), are significantly decreased in patients with cardiovascular disorders, end-stage renal disease, and prothrombotic conditions. Reduced filterability of Hcy through 300,000 MWCO PES membranes in these groups of patients was observed, regardless of hyperhomocysteinemia (HHcy) degree. Filterability of Hcy, but not of glutathione, was impaired in plasma preparations. It is possible that the patients with impaired filterability of Hcy in mixed disulfide state are characterized by increased ability of partial Hcy retention by proteins and complexes with higher than albumin molecular weight. These findings led us to conclude that evaluation of protein-bound Hcy filterability may provide more complete diagnostic data, to interpret clinical significance of HHcy. The proposed Hcy filterability test may be performed as a simple laboratory procedure, in addition to conventional tests for total Hcy.     

Filtration, haze and foam characteristics of fermented wort mediated by yeast strain

AIMS: To investigate the influence of the choice of yeast strain on the haze, shelf life, filterability and foam quality characteristics of fermented products. METHODS AND RESULTS: Twelve strains were used to ferment a chemically defined wort and hopped ale or stout wort. Fermented products were assessed for foam using the Rudin apparatus, and filterability and haze characteristics using the European Brewing Convention methods, to reveal differences in these parameters as a consequence of the choice of yeast strain and growth medium. CONCLUSIONS: Under the conditions used, the choice of strain of Saccharomyces cerevisiae effecting the primary fermentation has an impact on all of the parameters investigated, most notably when the fermentation medium is devoid of macromolecular material. SIGNIFICANCE AND IMPACT OF THE STUDY: The filtration of fermented products has a large cost implication for many brewers and wine makers, and the haze of the resulting filtrate is a key quality criterion. Also of importance to the quality of beer and some wines is the foaming and head retention of these beverages. The foam characteristics, filterability and potential for haze formation in a fermented product have long been known to be dependant on the raw materials used, as well as other production parameters. The choice of Saccharomyces cerevisiae strain used to ferment has itself been shown here to influence these parameters.     

Effects of temperature on water diffusion in human erythrocytes and ghosts--nuclear magnetic resonance studies

The temperature-dependence of water diffusion across human erythrocyte membrane was studied on isolated erythrocytes and resealed ghosts by a doping nuclear magnetic resonance technique. The conclusions are the following: (1) The storage of suspended erythrocytes at 2 degrees C up to 24 h or at 37 degrees C for 30 min did not change the water exchange time significantly, even if Mn2+ was present in the medium. This indicates that no significant penetration of Mn2+ is taking place under such conditions. (2) In case of cells previously incubated at 37 degrees C for longer than 30 min with concentrations of p-chloromercuribenzene sulfonate (PCMBS) greater than 0.5 mM, the water-exchange time gradually decreased if the cells were stored in the presence of Mn2+ for more than 10 min at 37 degrees C. (3) When the Arrhenius plot of the water-exchange time was calculated on the basis of measurements performed in such a way as to avoid a prolonged exposure of erythrocytes to Mn2+ no discontinuity occurred, regardless of the treatment with PCMBS. (4) No significant differences between erythrocytes and resealed ghosts regarding their permeability and the activation energy of water diffusion (Ea,d) were noticed. The mean value of Ea,d obtained on erythrocytes from 35 donors was 24.5 kJ/mol. (5) The value of Ea,d increased after treatment with PCMBS, in parallel with the percentage inhibition of water diffusion. A mean value of 41.3 kJ/mol was obtained for Ea,d of erythrocytes incubated with 1 mM PCMBS for 60 min at 37 degrees C and 28.3 kJ/mol for ghosts incubated with 0.1 mM PCMBS for 15 min, the values of inhibition being 46% and 21% respectively.