Effects of dextran polydispersity on red blood cell aggregation

B 512 F dextran fractions of different polydispersities were prepared by fractionnal precipitation and preparative elution chromatography. By combination of low angle laser light scattering (LALLS) and gel permeation chromatography (GPC), absolute average molecular weights (MWs) and molecular weight distribution functions (MWDs) were determined. Static and thermodynamical properties in terms of polymer dimensions and second virial coefficient of dilute solutions of dextran in water have been investigated. The results indicate that dextran macromolecules in water are rather compact and impenetrable coils. Measuring the disaggregation shear stress of dextran-induced red blood cell aggregates by laser light reflectometry, the macromolecular bridging energy was shown to depend upon dextran sample polydispersity. This reflects the weak and reversible character of red blood cell aggregation by dextran chains in physiological saline solution.     

Preparation of red blood cell column for capillary electrochromatography.

A column packed with red blood cells (RBCs) was prepared for electrochromatography as a separation and reaction column. RBCs were kept inside a piece of fused silica capillary tubing with 2% agarose gel. In the column, RBCs were uniformly distributed in the agarose gel matrix and their electrophoretic movements due to an applied voltage were suppressed well. The durability of the biological function of the column under applied voltage was about 1 h, although it could remain for 2-3 days without applied voltage. The column could not be used when hemolysis of the RBCs was observed in the column. When the developed "RBC-gel column" was used, both pyridoxamine and serotonin were converted to other compounds through their direct contact with RBCs.     

Torocyte shapes of red blood cell daughter vesicles

The shape of the newly described torocyte red blood cell endovesicles induced by octaethyleneglycol dodecylether (C12E8) is characterized. A possible explanation for the origin and stability of the observed torocyte endovesicles is suggested. Three partly complementary mechanisms are outlined, all originating from the interaction of C12E8 molecules with the membrane. The first is a preferential intercalation of the C12E8 molecule into the inner membrane layer, resulting in a membrane invagination which may finally close, forming an inside-out endovesicle. The second is a preference of the C12E8-induced membrane inclusions (clusters) for small local curvature which would favour torocyte endovesicle shape with large regions of small or even negative membrane mean curvatures, the C12E8 membrane inclusion being defined as a complex composed of the embedded C12E8 molecule and some adjacent phospholipid molecules which are significantly distorted due to the presence of the embedded C12E8 molecule. The preference of the C12E8 inclusions for zero or negative local curvature may also lead to the nonhomogeneous lateral distribution of the C12E8 inclusions resulting in their accumulation in the membrane of torocyte endovesicles. The third possible mechanism is orientational ordering of the C12E8-induced inclusions in the regions of torocyte endovesicles with high local membrane curvature deviator.     

Penetration of laser light through red blood cell ghosts

Hemoglobin is the main absorber of visible light in blood and blood-perfused tissues. However, hemoglobin is released from a red blood cell (RBC) during hemolysis. Hemolysis may be caused by a large number of medical conditions, including photodynamic therapy (PDT) and this subsequently can affect passage of light through the treated biological structures. The purpose of the present study was to determine the penetration of a laser beam through a suspension of hemoglobin-free human red blood cells (RBCs) - ghosts. Although hemoglobin has been efficiently removed from the samples used in our experiments, our measurements show that the samples still effectively attenuate the radiant power of penetrating laser light. We established penetration depths of 12.6mm and 15.4mm for two different laser light wavelengths, 532nm and 630nm, respectively. The penetration depth of laser light was about one order of magnitude higher for hemoglobin-free RBC ghosts as compared to intact RBCs [8,10,12]. These results can be important in case of phototherapy or biostimulation, since all photons that penetrate in a biological object may interact with it and evoke biological response.     

Photosensitization of red blood cell hemolysis by lutetium texaphyrin

Lutetium (III) texaphyrin photosensitizes postirradiation or "delayed" photohemolysis (DPH) of human and bovine red blood cells at 730 nm by a Type-2 pathway mediated by singlet molecular oxygen. The DPH rate increases with increasing incubation temperature and with the second power of the incident fluence. The experimental DPH curves are in good agreement with a multi-hit kinetics model based on target theory.     

[31P]NMR measurements of hexokinase activity in intact red blood cells with 2-deoxyglucose as substrate

[³¹P] NMR spectroscopy is demonstrated to be a suitable tool to follow the time course of 2-deoxyglucose-6-phosphate in intact human erythrocytes incubated with 2-deoxyglucose. It allowed to determine hexokinase V_max and K_m in near physiological conditions.     

Washing frozen red blood cell concentrates using hollow fibres

Today the cryopreservation of human blood products is routine. However, before reinfusion the cryoprotectant, often glycerol, has to be removed. We have designed a combined microfiltration diafiltration process using microporous hollow fibres for removing glycerol from frozen red blood cell concentrates. As the system can be closed to the atmosphere there is no possibility of infection of the “washed” blood. Thus the post-thaw shelf life of the blood may be greatly increased. The process has been optimized by minimizing both the processing time and diluent volume required. Finally a hollow fibre module capable of completing the entire washing process in 30 min has been developed. We show that such a module requires hollow fibres with an inside diameter of 200 μm. The design equations we present are generally applicable to the design of hollow fibre microfiltration systems.     

The effect of protease inhibitors on the two-dimensional electrophoresis pattern of red blood cell membranes

The last few years have brought dramatic improvements for sample preparation and solubilization of protein for electrophoretic analyses. The use of reagents such as thiourea and novel sulfobetaine surfactants increases the total number of proteins able to be visualized from a complex mixture such as a cell lysate and also allows more hydrophobic membrane proteins to be resolved. As the red blood cell (RBC) contain no organelles, it is an ideal source of relatively pure plasma membrane for protein solubilization studies. In addition, there are a number of diseases related to abnormalities of RBCs proteins, thus it is of medical relevance as well as a test sample for technology development. However, the procedure for purifying RBC membranes is rather time-consuming and is normally carried out under almost physiological conditions, which can be conducive to proteolytic degradation of the membrane proteins. Significant differences in two-dimensional (2-D) patterns with and without protease inhibitors in sample preparation are demonstrated. In addition, is shown that preparation of RBC membranes with sodium carbonate, pH 11, leads to multimeric complexes of hemoglobin and causes hemoglobin to be irreversibly attached to the membrane. When using immobilized pH gradients (IPG) as the first dimension, it is demonstrated that the spectrins (large, filamentous proteins of 280 kDa) are lost from the 2-D map unless active, instead of passive, sample hydration into the IPG strip is adopted.     

Dielectric spectroscopy of red blood cells in sickle cell disease

Hypoxia-induced polymerization of sickle hemoglobin and the related ion diffusion across cell membrane can lead to changes in cell dielectric properties, which can potentially serve as label-free, diagnostic biomarkers for sickle cell disease. This article presents a microfluidic-based approach with on-chip gas control for the impedance spectroscopy of suspended cells within the frequency range of 40 Hz to 110 MHz. A comprehensive bioimpedance of sickle cells under both normoxia and hypoxia is achieved rapidly (within ∼7 min) and is appropriated by small sample volumes (∼2.5 μL). Analysis of the sensing modeling is performed to obtain optimum conditions for dielectric spectroscopy of sickle cell suspensions and for extraction of single cell properties from the measured impedance spectra. The results of sickle cells show that upon hypoxia treatment, cell interior permittivity and conductivity increase, while cell membrane capacitance decreases. Moreover, the relative changes in cell dielectric parameters are found to be dependent on the sickle and fetal hemoglobin levels. In contrast, the changes in normal red blood cells between the hypoxia and normoxia states are unnoticeable. The results of sickle cells may serve as a reference to design dielectrophoresis-based cell sorting and electrodeformation testing devices that require cell dielectric characteristics as input parameters. The demonstrated method for dielectric characterization of single cells from the impedance spectroscopy of cell suspensions can be potentially applied to other cell types and under varied gas conditions.     

Red blood cell ghosts and intact red blood cells as complementary models in photodynamic cell research

Recent research on erythrocytes as model cells for photodynamic therapy showed differing behaviour of certain photosensitisers in erythrocytes compared to other cells. Differences of dye accumulation in the cell membrane were proposed to be the reason for the distinct photodynamic effects. Using pheophorbide a as an example, the combination of erythrocyte ghosts as models to follow the dye accumulation in the cell membrane and intact erythrocytes as model cells to show the photodynamic damage is provided. Evidence for the correctness of the combination of erythrocyte ghosts and intact erythrocytes as a functioning model system in photodynamic cell research is provided using the confocal laser scanning microscopy on intact, pheophorbide a loaded erythrocytes.     

Osmolarity effects on red blood cell elution in sedimentation field-flow fractionation.

Field-flow fractionation (FFF) is an analytical technique particularly suitable for the separation, isolation, and characterization of macromolecules and micrometer- or submicrometer-sized particles. This chromatographic-like methodology can modulate the retention of micron-sized species according to an elution mode described to date as "steric hyperlayer". In such a model, differences in sample species size, density, or other physical parameters make particle selective elution possible depending on the configuration and the operating conditions of the FFF system. Elution characteristics of micron-sized particles of biological origin, such as cells, can be modified using media and carrier phases of different osmolarities. In these media, a cells average size, density, and shape are modified. Therefore, systematic studies of a single reference cell population, red blood cells (RBCs), are performed with 2 sedimentation FFF systems using either gravity (GrFFF) or a centrifugational field (SdFFF). However, in all cases, normal erythrocyte in isotonic suspension elutes as a single peak when fractionated in these systems. With carrier phases of different osmolarities, FFF elution characteristics of RBCs are modified. Retention modifications are qualitatively consistent with the "steric-hyperlayer" model. Such systematic studies confirm the key role of size, density, and shape in the elution mode of RBCs in sedimentation FFF for living, micronsized biological species. Using polymers as an analogy, the RBC population is described as highly "polydisperse". However, this definition must be reconsidered depending on the parameters under concern, leading to a matricial concept: multipolydispersity. It is observed that multipolydispersity modifications of a given RBC population are qualitatively correlated to the eluted sample band width.     

Effects of saikosaponin metabolites on the hemolysis of red blood cells and their adsorbability on the cell membrane

The hemolytic properties and the adsorbability on red blood cells of saikosaponin a, saikosaponin d and 13 metabolites formed in the alimentary tract were investigated. Among these compounds, saikosaponin d and its intestinal product, prosaikogenin G, which possess an alpha-hydroxyl function at C16, showed the strongest hemolytic activity at the dose range of 1.0 to 5.0 micrograms/ml. Saikosaponin a and its intestinal product, prosaikogenin F, which possess a beta-hydroxyl function at C16, showed activity above 10 micrograms/ml. In this case, the monoglycoside, prosaikogenin F, showed the stronger activity than the diglycoside, saikosaponin a. Among the gastric products whose ether ring was cleaved to produce a carbinol, the monoglycosides, prosaikogenin A and prosaikogenin H, showed a slight activity above 25 micrograms/ml, and the saikogenins except saikogenin A were inactive. Saikogenin A, however, had hemolytic activity at a dose of 15 micrograms/ml. The adsorbabilities of these compounds on red blood cell membranes closely paralleled their degrees of hemolytic activity.     

Further characterization of cation channels present in the chicken red blood cell membrane

In this paper, we provide an update on cation channels in nucleated chicken erythrocytes. Patch-clamp techniques were used to further characterize the two different types of cation channels present in the membrane of chicken red blood. In the whole-cell mode, with Ringer in the bath and internal K+ saline in the pipette solution, the membrane conductance was generated by cationic currents, since the reversal potential was shifted toward cations equilibrium when the impermeant cation NMDG was substituted to small cations. The membrane conductance could be increased by application of mechanical deformation or by the addition of agonists of the cAMP-dependent pathway. At the unitary level, two different types of cationic channels were revealed and could account for the cationic conductance observed in whole-cell configuration. One of them belongs to the family of stretch-activated cationic channel showing changes in activity under conditions of membrane deformation, whereas the second one belongs to the family of the cAMP activated cationic channels. These two channels could be distinguished according to their unitary conductances and drug sensitivities. The stretch-activated channel was sensitive to Gd(3+) and the cAMP-dependent channel was sensitive to flufenamic acid. Possible role of these channels in cell volume regulation process is discussed.     

Adiabatic compressibility of red blood cell membrane: influence of skeleton

Measurements of ultrasound velocity and density were used for determination of the adiabatic compressibility of red blood cells (RBC) during detachment of the membrane skeleton. Skeleton detachment was induced by addition of nystatin into a low ionic strength RBC suspension resulting in an increase (10%) of the ultrasound velocity concentration increment, [u], while the specific volume of cells, phi(v) did not change significantly. Changes of the concentration increment had rather long kinetics and were not completed even after 60 min. Both [u] and phiV values were used for calculation of the specific apparent adiabatic compressibility of RBC, phiK/beta0. The value of the specific apparent compressibility decreases following addition of nystatin. This corresponds to an increase in the volume elastic modulus of RBC membranes during detachment of the membrane skeleton. Control experiments with large unilamellar liposomes at conditions similar to that performed with the RBC did not reveal significant changes of [u] after the addition of nystatin. Our results show that the role of the membrane skeleton probably consists in maintaining higher compressibility of the RBC membranes. This may partly provide conditions for conformational changes of RBC membrane proteins.     

Cytoskeleton mediated effective elastic properties of model red blood cell membranes

The plasma membrane of human red blood cells consists of a lipid bilayer attached to a regular network of underlying cytoskeletal polymers. We model this system at a dynamic coarse-grained level, treating the bilayer as an elastic sheet and the cytoskeletal network as a series of phantom entropic springs. In contrast to prior simulation efforts, we explicitly account for dynamics of the cytoskeletal network, both via motion of the protein anchors that attach the cytoskeleton to the bilayer and through breaking and reconnection of individual cytoskeletal filaments. Simulation results are explained in the context of a simple mean field percolation model and comparison is made to experimental measurements of red blood cell fluctuation amplitudes.     

Investigation of red blood cell fractionation by gravitational field-flow fractionation.

Gravitational field-flow fractionation is used for the separation of particles according to their sizes in the range 1-100 microns: larger particles elute before smaller ones. This phenomenon can be explained as a result of the steric exclusion of the particles from the vicinity of the channel walls, and/or hydrodynamic effects supposedly associated with the inertia of the liquid. The method was used for the investigation of red blood cells. The dependence of the retention ratio on the flow-rate, sample volume, concentration of blood and relaxation time was studied. Analysis of fifteen individual fractions by Coulter counter and reinjection of three other fractions were studied in order to verify fractionation of red blood cells.     

Mechanical property analysis of stored red blood cell using optical tweezers

The deformation of human red blood cells subjected to direct stretching by optical tweezers was analyzed. The maximum force exerted by optical tweezers on the cell via a polystyrene microbead 5 μm in diameter was 315 pN. Digital image correlation (DIC) method was introduced to calculate the force and the deformation of the cell for the first time. Force–extension relation curves of the biconcave cell were quantitatively assessed when erythrocytes were stored in Alsever's Solution for 2 days, 5 days, 7 days and 14 days respectively. Experiment results demonstrated that the deformability of red blood cells was impaired with the stored time.