Microfluidic analysis of red blood cell deformability

A common indicator of rheological dysfunction is a measurable decrease in the deformability of red blood cells (RBCs). Decreased RBC deformability is associated with cellular stress or pathology and can impede the transit of these cells through the microvasculature, where RBCs play a central role in the oxygenation of tissues. Therefore, RBC deformability has been recognized as a sensitive biomarker for rheological disease. In the current study, we present a strategy to measure RBC cortical tension as an indicator of RBC deformability based on the critical pressure required for RBC transit through microscale funnel constrictions. By modeling RBCs as a Newtonian liquid drop, we were able to discriminate cells fixed with glutaraldehyde concentrations that vary as little as 0.001%. When RBCs were sampled from healthy donors on different days, the RBC cortical tension was found to be highly reproducible. Inter-individual variability was similarly reproducible, showing only slightly greater variability, which might reflect biological differences between normal individuals. Both the sensitivity and reproducibility of cortical tension, as an indicator of RBC deformability, make it well-suited for biological and clinical analysis of RBC microrheology.     

Hemoglobin-mediated selenium export from red blood cells

On the basis of the fact that selenium from selenite binds to hemoglobin (Hb), we investigated the missing process in the selenium export from red blood cells (RBCs), i.e., the transfer of selenium bound to Hb to RBC membrane proteins. To elucidate the molecular events of the Hb-associated selenium export from RBC, a Hb–Se complex was synthesized from thiol-exchange of Cys-β93 in Hb with penicillamine-substituted glutathione selenotrisulfide, as a model of major metabolic intermediates, and then interactions between the Hb–Se complex and RBC inside-out vesicles (IOVs) were examined. Selenium bound to Hb was transferred to the IOV membrane on the basis of the intrinsic interactions between Hb and the cytoplasmic domains of band 3 protein (CDB3). The observed selenium transfer was inhibited by the pretreatments of IOVs with iodoacetamide and the α-chymotrypsin digestion, indicating that the Hb mediates the selenium transfer to the thiol groups of CDB3. In addition, it was found that deoxygenated Hb, with a high binding affinity for CDB3, more favorably transferred selenium to the IOV membranes than oxygenated Hb, with a low affinity. When selenium export from RBC to the plasma was examined by continuously introducing nitrogen gas, the selenium export rate was promoted with an increase in the rate of deoxygenated Hb. Overall, these data suggested that Hb could possibly play a role in the selenium export from RBC treated with selenite in an oxygen-linked fashion. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Cryopreservation of red blood cells: Effect on rheologic properties and associated metabolic and nitric oxide related parameters

AimHigh glycerol cryopreservation of red blood cells (RBCs) reduces metabolic processes at ultralow temperatures but less is known regarding the effect of cryopreservation on RBC nitric oxide (NO) metabolism, haemorheological properties, structural behaviour and membrane fragility.MethodsBlood from ten healthy participants was sampled, glycerolized and stored at −80 °C (SB). Aliquots were thawed and further processed after 4, 8 and 12 weeks, respectively. At these time points, fresh blood (FB) was additionally sampled from each participant. FB/SB mixtures were prepared corresponding to transfusion of 1–3 blood bags. Additionally, mixtures were exposed to shear stress similar to that found in the circulation and deformability was measured to estimate possible behaviour of cryopreserved RBC in vivo.ResultsAgeing of RBC was reduced during cryopreservation. Markers for RBC metabolism (ATP, 2,3-DPG) were not altered but RBC sodium levels increased and potassium and calcium decreased, respectively. Mean cellular volume was higher and accordingly, mean cellular haemoglobin concentration was lower in SB. Deformability was altered during storage with less shear stress necessary to deform RBCs. Changes were also detectable in blood mixtures. Deformability remained unaltered in shear stress settings in FB and SB. RBC viscosity was reduced in SB. RBC-NOS content and phosphorylation sites as well as nitrite and RxNO levels seem not to be affected by the intervention.ConclusionCryopreservation maintains RBC metabolic function in vitro, but structure and function of cryopreserved RBC seems to be altered. Impact of these alterations in vivo seems to be less but needs further investigation.     

Inhibition of anion transport in the red blood cell membrane by anionic and non-anionic arginine-specific reagents

Arginine specific reagents are found to be powerful inhibitors of anion exchange in the red blood cell membrane. Some of these inhibitors such as cyclohexandione, phenylglyoxal and 2, 3-butandione are found to produce their inhibition by interacting covalently with band 3. In contrast to the action of these compounds, the inhibition caused by the phenylglyoxal derivative 4-hydroxy-3-nitrophenyl-glyoxal has been found to be completly reversible. In extending the studies on the mode of action of these compounds on sulfate exchange and to get some more information about their binding site, the degree of inhibition caused by different phenylglyoxal derivatives which have a similar core but differ in their substituent groups have been compared. The interaction between the binding sites of these compounds and other anion transport inhibitors have also been studied.     

Elastic properties of the red blood cell membrane that determine echinocyte deformability

The natural biconcave shape of red blood cells (RBC) may be altered by injury or environmental conditions into a spiculated form (echinocyte). An analysis is presented of the effect of such a transformation on the resistance of RBC to entry into capillary sized cylindrical tubes. The analysis accounts for the elasticity of the membrane skeleton in dilation and shear, and the local and nonlocal resistance of the bilayer to bending, the latter corresponding to different area strains in the two leaflets of the bilayer. The shape transformation is assumed to be driven by the equilibrium area difference (A₀, the difference between the equilibrium areas of the bilayer leaflets), which also affects the energy of deformation. The cell shape is approximated by a parametric model. Shape parameters, skeleton shear deformation, and the skeleton density of deformed membrane relative to the skeleton density of undeformed membrane are obtained by minimization of the corresponding thermodynamic potential. Experimentally, A₀ is modified and the corresponding discocyte–echinocyte shape transition obtained by high-pressure aspiration into a narrow pipette, and the deformability of the resulting echinocyte is examined by whole cell aspiration into a larger pipette. We conclude that the deformability of the echinocyte can be accounted for by the mechanical behavior of the normal RBC membrane, where the equilibrium area difference A₀ is modified.     

The kinetics of anion equilibrium exchange across the red blood cell membrane as measured by means of35S thiocyanate

Summary Up to a SCN^– concentration of about 110mm, the concentration dependence of SCN^– equilibrium exchange in human red cell ghosts can be represented by the superimposition of two flux components. One component shows saturation kinetics, the other does not. The saturable component has an activation enthalpy of 105 kJ/mole, exhibits arans acceleration by Cl^– and can be inhibited by H₂DIDS. The nonsaturable component has a much lower activation enthalpy of 33 kJ/mole, is slightly reduced intrans acceleration experiments with Cl^– and insensitive to H₂DIDS but susceptible to inhibition by phloretin. At SCN^– concentrations exceeding 110mm, the saturable component undergoes irreversible self inhibition while the nonsaturable component remains unaltered.The half saturation concentration of the saturable flux component increases with decreasing pH from 3.0mm at pH 7.4 to 13.3mm at pH 6.0. Over this pH range, the maximal flux is only slightly increased from 19×10^–12 to 22×10^–12 moles×cm^–2×sec^–1. The nonsaturable flux component also increases slightly.In accordance with previous observations of Wieth (J. Physiol. (London) 207:563–580, 1970), we find that SCN^– increases K⁺ and Na⁺ permeability. The induced cation-permeability is considerably smaller than the SCN^– exchange and the latter does not show the paradoxical temperature dependence that is known to pertain to the former.     

Sugar moiety of cardiac glycosides is essential for the inhibitory action on the palytoxin-induced K+ release from red blood cells

Palytoxin (PTX), a highly toxic and sugar-containing substance isolated from Palythoa tuberculosa, caused K+ release from rabbit red blood cells. Cardiac glycosides, such as ouabain, convallatoxin, cymarin, digoxin and digitoxin, inhibited the PTX-induced K+ release. Their corresponding aglycones did not inhibit the K+ release, but antagonized the inhibitory effect of the glycosides. All these cardiotonic steroids equally inhibited the activity of (Na+ + K+)-ATPase prepared from hog cerebral cortex. These results suggest that the sugar moiety of the cardiac glycosides is important for the inhibitory effect on the K+ release induced by PTX and that the inhibition is not related to their inhibitory potency on the (Na+ + K+)-ATPase activity.     

Signaling mechanisms in red blood cells: A view through the protein phosphorylation and deformability

Intracellular signaling mechanisms in red blood cells (RBCs) involve various protein kinases and phosphatases and enable rapid adaptive responses to hypoxia, metabolic requirements, oxidative stress, or shear stress by regulating the physiological properties of the cell. Protein phosphorylation is a ubiquitous mechanism for intracellular signal transduction, volume regulation, and cytoskeletal organization in RBCs. Spectrin-based cytoskeleton connects integral membrane proteins, band 3 and glycophorin C to junctional proteins, ankyrin and Protein 4.1. Phosphorylation leads to a conformational change in the protein structure, weakening the interactions between proteins in the cytoskeletal network that confers a more flexible nature for the RBC membrane. The structural organization of the membrane and the cytoskeleton determines RBC deformability that allows cells to change their ability to deform under shear stress to pass through narrow capillaries. The shear stress sensing mechanisms and oxygenation-deoxygenation transitions regulate cell volume and mechanical properties of the membrane through the activation of ion transporters and specific phosphorylation events mediated by signal transduction. In this review, we summarize the roles of Protein kinase C, cAMP-Protein kinase A, cGMP-nitric oxide, RhoGTPase, and MAP/ERK pathways in the modulation of RBC deformability in both healthy and disease states. We emphasize that targeting signaling elements may be a therapeutic strategy for the treatment of hemoglobinopathies or channelopathies. We expect the present review will provide additional insights into RBC responses to shear stress and hypoxia via signaling mechanisms and shed light on the current and novel treatment options for pathophysiological conditions.     

Diffusional water permeability of mammalian red blood cells

An extensive programme of comparative nuclear magnetic resonance measurements of the membrane diffusional permeability for water (P_d) and of the activation energy (E_a,d) of this process in red blood cells (RBCs) from 21 mammalian species was carried out. On the basis of P_d, these species could be divided into three groups. First, the RBC's from humans, cow, sheep and “large” kangaroos (Macropus giganteus and Macropus rufus) had P_d values 5 × 10⁻³ cm/s at 25°C and 7 × 10⁻³ cm/s at 37°C. The RBCs from other marsupial species, mouse, rat, guinea pig and rabbit, had P_d values roughly twice higher, whereas echidna RBCs were twice lower than human RBCs. The value of E_a,d was in most cases correlated with the values of P_d. A value of E_a,d -26 kJ/mol was found for the RBCs from humans and the species having similar P_d values. Low values of E_a,d (ranging from 15 to 22 kJ/mol) appeared to be associated with relatively high values of P_d. The highest value of E_a,d (33 kJ/mol) was found in echidna RBCs. This points to specialized channels for water diffusion incorporated in membrane proteins; a relatively high water permeability of the RBC membrane could be due to a greater number of channel proteins. There are, however, situations where a very high water permeability of RBCs is associated with a high value of E_a,d (above 25 kJ/mol) as in the case of RBCs from mouse, rat and tree kangaroo. Moreover, it was found that P_d in different species was positively correlated to the RBC membrane phosphatidylcholine and negatively correlated to the sphingomyelin content. This suggests that in addition to the number of channel proteins, other factors are involved in the water permeability of the RBC membrane.     

Effect of blood storage on erythrocyte/wall interactions: implications for surface charge and rigidity

In this report, we study, under flow conditions, the interactions of stored erythrocytes with an artificial surface: a microelectrode whose charge density ranges from –15 to +27 μC/cm². Interactions consist of red cells slowly circulating on the microelectrode and exerting a real contact with the electrode. Interaction is detected and measured by transient fluctuations of the electrolyte resistance obtained by impedance measurement of the microelectrode. Effects of aging induced by storage of whole blood at 4 °C show that the surface charge of erythrocytes rapidly decreases when blood is stored for more than 6 days under our experimental conditions. In comparison with trypsin-treated erythrocytes, an eight day storage induces a 60% decrease in the surface charge of red cells. After two weeks of storage, red cells are no longer negatively charged, presumably be cause of removal of sialic acid. Cells rigidity is significant after 6 days of storage and influences the electrical contact. Membrane rigidity increase could arise from the surface charge decrease. Finally, the surface charge decrease could be of importance in the use of stored blood. Received: 30 October 1996 / Accepted: 12 November 1996     

Effects of membrane reference state on shape memory of a red blood cell

By using a three-dimensional continuum model, we simulate the shape memory of a red blood cell after the remove of external forces. The purpose of this study is to illustrate the effect of membrane reference state on cell behavior during the recovery process. The reference state of an elastic element is the geometry with zero stress. Since the cell membrane is composed of cytoskeleton and lipid bilayer, both the reference states of cytoskeleton (RSC) and lipid bilayer (RSL) are considered. Results show that a non-spherical RSC can result in shape memory. The energy barrier due to non-spherical RSC is determined by the ratio of the equator length to the meridian length of the RSC. Thus different RSCs can have similar energy barrier and leading to identical recovery response. A series of simulations of more intermediate RSCs show that the recovery time scale is inversely proportional to the energy barrier. Comparing to spherical RSL, a spheroid RSL contributes to the energy barrier and recovery time. Furthermore, we observe a folding recovery due to the biconcave RSL which is different from the tank treading recovery. These results may motivate novel numerical and experimental studies to determine the exact RSC and RSL.     

Resistance to the Brownian movement of red blood cells on flat horizontal surfaces

The movements of red blood cells (RBC), suspended in plasma, on plastic, glass, rhodium metal plate, siliconized glass, and siliconized rhodium were recorded on cinéfilm and analyzed. Values for the drag coefficient were calculated, using Einstein's theory of Brownian movement, and compared with the theoretical Stokes' hydrodynamic drag. The difference between the computed and Stokes' values gave the frictional coefficient or resistance resulting from the interaction of the cells, with the test surface. Of the three uncoated test surfaces, plastic was found to have the least interaction with the RBC. The frictional coefficient for plastic was found to be 1.75×10⁻⁷ N s m⁻¹ compared with a value of 2.82×10⁻⁷ N s m⁻¹ for rhodium metal, which had the largest interaction. Upon siliconization of the test surfaces, the interaction decreased by 40%. Reduction in the pH of the suspending plasma increased the interaction between the cells and the uncoated test surfaces, but the pH effect of diminished when the surfaces were siliconized.     

Three-dimensional focusing of red blood cells in microchannel flows for bio-sensing applications

Three-dimensional (3D) focusing of particles in microchannels has been a long-standing issue in the design of biochemical/biomedical microdevices. Current microdevices for 3D cell or bioparticle focusing involve complex channel geometries in view of their fabrication because they require multiple layers and/or sheath flows. This paper proposes a simple method for 3D focusing of red blood cells (RBCs) in a single circular microcapillary, without any sheath flows, which is inspired from the fluid dynamics phenomenon in that a spherical particle lagging behind a Poiseuille flow migrates toward and along the channel axis. More explicitly, electrophoresis of RBCs superimposed on the pressure-driven flow is utilized to generate an RBC migration mode analogous to this phenomenon. A particle-tracking scheme with a sub-pixel resolution is implemented to spatially position red blood cells flowing through the channel, so that a probability density function (PDF) is constructed to evaluate the tightness of the cell focusing. Above a specific strength of the electric field, approximately 90% of the sheep RBCs laden in the flow are tightly focused within a beam diameter that is three times the cell dimension. Particle shape effect on the focusing is discussed by making comparisons between the RBCs and the spherical particles. The lateral migration velocity, predicted by an existing theoretical model, is in good agreement with the present experimental data. It is noteworthy that 3D focusing of non-spherical particles, such as RBCs, has been achieved in a circular microchannel, which is a significant improvement over previous focusing methodologies.     

Effects of antioxidants and haemoglobin status on the t-butyl hydroperoxide-induced oxygen uptake by red blood cells

Oxygen uptake by erythrocytes exposed to t-butyl hydroperoxide (t-BHP) exhibited an induction period. The rate of oxygen consumption can be reduced by antioxidants and blood plasma. The induction time was not appreciably modified by the antioxidants tested, however, plasma increased it by a factor of two. The in vivo pretreatment with diethyl maleate (0.6 g kg-1) produced increased rates of oxygen uptake without changes in the induction period, while vitamin E (12.5 mg kg-1) elicited lower oxygen consumption rates and longer induction times, compared to those observed in cells from control rats upon addition of the hydroperoxide. These results suggest that the antioxidants tested on the t-BHP lipid peroxidation in erythrocyte suspensions act as inhibitors and/or retarders of the process. Furthermore, lipid peroxidation induced in these conditions seems to depend upon the haemoglobin status of the cells as oxygen uptake, malondialdehyde production and chemiluminescence were significantly higher in methaemoglobin-containing cells than in those containing oxyhaemoglobin.     

Studies on the methodology of the carboxyfluorescein assay and on the mechanism of liposome stabilization by red blood cells in vitro

The stability of small unilamellar liposomes was investigated in human blood, in vitro. Using the carboxyfluorescein technique, interaction between the dye, the detergent Triton X-100, and an as yet unidentified component of human serum grossly interferes with the experiment and necessitates the use of other detergents, preferably sodium deoxycholate. Separation of liposomes and blood cells by centrifugation induces a small leakage from the liposomes and can lead to an underestimation of the real liposome stability. Upon incubation with whole blood, intact liposomes are absorbed nonspecifically to erythrocytes and internalized by leukocytes, the extent and kinetics of the former process being insenstive to the presence of metabolic inhibitors. The stability of liposomes is significantly enhanced in whole blood or in serum containing washed erythrocytes. Similarly, liposome stability in serum could be augmented be presaturating the serum lipoproteins with excess phospholipid. Our work adds support to previous notions that stable liposomes with high affinities for certain blood-cell components might be developed as suitable carrier systems for drug targetting in pathological disorders within the blood stream.     

Properties of the basal calcium influx in human red blood cells

The basal ⁴⁵Ca²⁺ influx in human red blood cells (RBC) into intact RBC was measured. ⁴⁵Ca²⁺ was equilibrated with cells with t_1/2=15-20 s and the influx reached the steady state value in about 90-100 s and the steady state level was 1.5±0.2 μmol/l_{packed cells} (n=6) at 37 °C. The average value of the Ca²⁺ influx rate was 43.2±8.9 μmol/l_{packed cells} hour. The rate of the basal influx was pH-dependent with a pH optimum at pH 7.0 and on the temperature with the temperature optimum at 25 °C. The basal Ca²⁺ influx was saturable with Ca²⁺ up to 5 mmol/l but at higher extracellular Ca²⁺ concentrations caused further increase of basal Ca²⁺ influx. The ⁴⁵Ca²⁺ influx was stimulated by addition of submicromolar concentrations of phorbol esters (phorbol 12-myristate-13-acetate (PMA) and phorbol-12,13-dibutyrate (PDBu)) and forskolin. Uncoupler (3,3′,4′,5-tetrachloro-salicylanilide (TCS) 10⁻⁶-10⁻⁵ mol/l) inhibited in part the Ca²⁺ influx. The results show that the basal Ca²⁺ influx is mediated by a carrier and is under control of intracellular regulatory circuits. The effect of uncoupler shows that the Ca²⁺ influx is in part driven by the proton-motive force and indicates that the influx and efflux of Ca²⁺ are coupled via the RBC H⁺ homeostasis.     

Effect of solution environment on the permeability of red blood cells

The cell membrane permeability governs the rate of solute transport into and out of the cell, significantly affecting the cell's metabolic processes, viability, and potential usefulness in both biotechnological applications and physiological systems. Most previous studies of the cell membrane permeability have neglected the possible effects of suspending medium on membrane transport, even though there is extensive experimental evidence that suspending phase composition can significantly affect other properties related to the cell membrane (e.g., cell deformability, fragility, and aggregation rate). This study examined the effects of suspending phase composition (both proteins and electrolytes) on the permeability of human red blood cells to the metabolites creatinine and uric acid. Data were obtained using a stirred ultrafiltration device with direct cell- and proteinfree sampling through a semipermeable membrane. Both the uric acid and creatinine permeabilities were strongly affected by the suspending phase composition, with the permeabilities in different buffer solutions varying by as much as a factor of three. The predominant factors affecting the permeability were the presence (or absence) of chloride, phosphate/adenine, and proteins, although the magnitude and even the direction of these effects were significantly different for creatinine and uric acid transport. The dramatic differences in behavior for uric acid and creatinine reflect the different transport mechanisms for these solutes, with uric acid transported by a carrier-mediated mechanism and creatinine transported by passive diffusion through the lipid bilayer. These results provide important insights into the effects of solution environment on cell membrane transport and other cell membrane-mediated properties. (c) 1994 John Wiley & Sons, Inc.     

Deglycerolization of red blood cells: A new dilution-filtration system

In this work, we present a new version of the dilution-filtration system for rapidly deglycerolizing a large volume of cryopreserved blood. In our earlier system, one of the major problems was the damage induced to the red blood cells (RBCs) due to high osmolality change at the dilution point. Therefore, we devised a new system to solve this problem. First, we theoretically simulated the osmolality variation in the new system and the variation of the maximum and minimum volumes of the RBCs at the dilution point to examine the effects of operating parameters/conditions. Next, we experimentally validated the effects of these operating parameters by deglycerolizing porcine blood. The results show that when the initial NaCl concentration in the hypertonic solution is 18%, the volume of the hypertonic solution is 200 mL, and the flow rate of the filtrate is 50 mL/min, the system can effectively remove glycerin from 200 mL of porcine blood in 30 min, with ∼87% RBC survival rate and ∼73% RBC recovery rate. Our results indicated that in the new system the concentration and the volume of the hypertonic solution used to dilute the blood are the important parameters that need to be adjusted to reduce osmotic damage to the RBCs. In addition, a fast filtrate flow rate is highly recommended. This work can significantly contribute to the development of a more efficient and effective system for deglycerolizing large volumes of cryopreserved blood in clinic.     

Visualization study of motion and deformation of red blood cells in a microchannel with straight,divergent and convergent sections

The size of red blood cells (RBC) is on the same order as the diameter of microvascular vessels. Therefore, blood should be regarded as a two-phase flow system of RBCs suspended in plasma rather than a continuous medium of microcirculation. It is of great physiological and pathological significance to investigate the effects of deformation and aggregation of RBCs on microcirculation. In this study, a visualization experiment was conducted to study the microcirculatory behavior of RBCs in suspension. Motion and deformation of RBCs in a microfluidic chip with straight, divergent, and convergent microchannel sections have been captured by microscope and high-speed camera. Meanwhile, deformation and movement of RBCs were investigated under different viscosity, hematocrit, and flow rate in this system. For low velocity and viscosity, RBCs behaved in their normal biconcave disc shape and their motion was found as a flipping motion: they not only deformed their shapes along the flow direction, but also rolled and rotated themselves. RBCs were also found to aggregate, forming rouleaux at very low flow rate and viscosity. However, for high velocity and viscosity, RBCs deformed obviously under the shear stress. They elongated along the flow direction and performed a tank-treading motion.