Effect of temperature on the deformability of a lamprey, Entosphenus japonicus, and Pacific salmon, Oncorhynchus keta, red blood cells, studied using a modified filtration method

The rates of filtration through Nuclepore filters (5 or 8 μm) of blood from lampreys and Pacific salmon have been studied using a method which visualizes the flow pattern. From these measurements, passage times for single red blood cells have been calculated and serve as an index of their deformability. The deformability increases as temperature is raised in vitro , but even at 5°C the passage time of lamprey blood is relatively rapid. The increase in deformability with a rise in temperature is small relative to that found in other fish such as yellowtail and carp.
The distribution of red cell volumes has shown the presence of a secondary peak for salmon blood taken during surgery which is reduced following recovery, the main peak being at a lower volume.     

Effect of carbonyl compounds on red blood cells deformability

The effect of Maillard reaction on red blood cells (RBC) deformability was investigated. Exposure of RBC to carbonyl compounds (dl-glyceraldehyde, glyoxal, glycolaldehyde, 3-deoxyglucosone, and d-glucose) leading to Maillard reaction caused a marked decrease in RBC deformability even at 1 mM level. The decrease rate depended on the kind of carbonyl compounds, in which both dl-glyceraldehyde and glyoxal significantly decreased the RBC deformability (p < 0.05). In addition, the decrease rate also differed among volunteers tested, indicating that the sensitivity against carbonyl compounds varies among them. In order to elucidate the mechanism of the decrease in RBC deformability, RBC was exposed to carbonyl compounds in the presence of aminoguanidine which is the inhibitor of AGE formation in Maillard reactions. Aminoguanidine inhibited the decrease in RBC deformability by dl-glyceraldehyde and glyoxal. When Hb which has a high reactivity with carbonyl compounds was incubated with those carbonyl compounds, dl-glyceraldehyde and glyoxal showed the high reactivity with Hb compared with other carbonyl compounds. These results indicate that Maillard reaction between RBC proteins and carbonyl compounds leads to the decrease in RBC deformability. On the other hand, generated by carbonyl compounds involved in lowering the deformability only to a negligible level.     

Separation and characterization of red blood cells with different membrane deformability using steric field-flow fractionation

Human red blood cells were treated in different ways to alter their membrane deformability, and the hydrodynamic behavior of these altered cells was studied using the steric field-flow fractionation (FFF) technique. The relationships between cell retention in the FFF channel, flow-rate of the carrier fluid and the applied field strength were studied for normal and glutaraldehyde-fixed human red cells, and separation conditions were optimized. The effect of flow-induced hydrodynamic lift forces on red cell retention in the steric FFF channel was studied, and the results suggest that the membrane deformability of the red cell is an important factor contributing to the lift force, besides other previously described effects due to density and flow velocity. Using steric FFF, a mixture of normal and glutaraldehyde-fixed human red cells was completely separated with a resolution twice that found in published d ata from gel permeation, another hydrodynamic separation technique. Partial loss of membrane deformability, induced by different degrees of glutaraldehyde-fixation, by diamide, or by a thermal treatment, has also been studied. Steric FFF is thus shown to have potential for rapid separation and differentiation of red cells with different density and membrane deformability, conditions known to be associated with, e.g., cell senescence and certain hematological diseases.     

Measurement of the distribution of red blood cell deformability using an automated rheoscope

BACKGROUND: Red blood cells (RBCs) have to deform markedly to pass through the smallest capillaries of the microcirculation. Techniques for measuring RBC deformability often result in an indication of the mean value. A deformability distribution would be more useful for studying diseases that are marked by subpopulations of less deformable cells because even small fractions of rigid cells can cause circulatory problems. METHODS: We present an automated rheoscope that uses advanced image analysis techniques to determine a RBC deformability distribution (RBC-DD) by analyzing a large number of individual cells in shear flow. The sensitivity was measured from density-separated fractions of one blood sample and from cells rendered less deformable by heat treatment. A preliminary experiment included the RBC-DDs of a patient with sickle cell anemia, one on dialysis and being treated with erythropoietin, and one with elliptocytosis. RESULTS: Measurement of the RBC-DD was highly reproducible. The sensitivity test showed markedly different deformability distributions of density-separated cells and yielded distinct RBC-DDs after each additional minute of heat treatment. CONCLUSION: The automated rheoscope enabled the determination of RBC-DDs from which less deformable subpopulations can be established. The shape of an RBC-DD may be valuable in assessing cell fractions with normal and anomalous deformability within pathologic blood samples.     

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.     

Effect of lanthanum on red blood cell deformability

Prior reports describing the effects of lanthanum (La(3+)) on red blood cells (RBC) have focused on the effects of this lanthanide on cell fusion or on membrane characteristics (e.g., ion movement across membrane, membrane protein aggregation); the present study explores its rheological and biophysical effects. Normal human RBC were exposed to La(3+) levels up to 200 microM then tested for: (1) cellular deformability using a laser-based ektacytometer and an optical-based rheoscope; (2) membrane viscoelastic behavior via micropipettes; (3) surface charge via micro electrophoresis. La(3+) concentrations of 12.5 to 200 microM caused dose-dependent decreases of deformability that were greatest at low stresses: these rheological changes were completely reversible upon removing La(3+) from the media either by washing with La(3+)-free buffer or by suspending La(3+)-exposed cells in La(3+)-free media (i.e., viscous dextran solution). Both membrane shear elastic modulus and membrane surface viscosity were increased by 25-30% at 100 or 200 microM. As expected, La(3+) decreased RBC electrophoretic mobility (EPM), with EPM inversely but not linearly associated with deformability; changes of EPM were also completely reversible. These results thus indicate novel aspects of RBC cellular and membrane rheological behavior yet raise questions regarding specific mechanisms responsible for La(3+)-induced alterations.     

A simple method for the liquid scintillation counting of precipitated protein from red blood cells

A method for the liquid scintillation counting of precipitated protein from red cells in 0.1–1.0 ml of blood is described. Precipitate is incubated for 0.5 hr at 100°C with equal volumes of acetic acid, ethyl acetate, and hydrogen peroxide; an equal volume of hydrochlorie acid is then added, followed by a toluene/Triton X-100 scintillation mixture containing primary and secondary scintillators. Maximum counting efficiencies with precipitate from 0.2 ml of blood were 90% for ¹⁴C and 35% for ³H. Recovery of labeled amino acid was not less than 90%. Chemiluminescence decayed to not more than 15 cpm above background in 45 min.     

Morphology of glutaraldehyde-fixed preserved red blood cells and 24-hr post-transfusion survival

Liquid-stored red blood cells and washed, previously frozen red blood cells were studied to determine whether a correlation existed between morphology and post-transfusion survival. Red cell concentrates were stored at 4 °C in citrate-phosphate-dextrose (CPD) for 21 days or in CPD-adenine (CPDA-1, CPDA-2, or CPDA-3) for as long as 35 days as liquid-preserved red cells. Both nonrejuvenated and rejuvenated red blood cells were frozen with 40%$\text{w}\text{v}$ glycerol at ?80 °C and were washed prior to testing.Samples of fresh, liquid-stored, and washed, previously frozen red blood cells were fixed with a 2% veronal glutaraldehyde solution. Phase, light, and electron microscopy were used to measure the numbers of discocytes, discoechinocytes, echinocytes, echinospherocytes, and spherocytes in each sample. A morphology score was assigned, with 100 representing all discocytes and 500 all spherocytes. In all samples phase and light microscopy gave nearly identical scores (r = 0.94), and phase and electron microscopy gave highly similar scores (r = 0.83).The morphology score proved to be a good indicator of 24-hr post-transfusion survival in liquid-stored red blood cells but not in washed, previously frozen red blood cells. Red blood cells stored in the liquid state at 4 °C in CPD, CPDA-1, CPDA-2, or CPDA-3 showed a significant inverse correlation between morphology and 24-hr post-transfusion survival (r = ?0.611) and a significant correlation between red cell ATP and 24-hr post-transfusion survival (r = 0.742). We saw no significant correlation between morphology scores and 24-hr post-transfusion values or between ATP levels and post-transfusion survival values in nonrejuvenated or rejuvenated washed, previously frozen red blood cells.     

Effects of nitric oxide on red blood cell deformability

In addition to its known action on vascular smooth muscle, nitric oxide (NO) has been suggested to have cardiovascular effects via regulation of red blood cell (RBC) deformability. The present study was designed to further explore this possibility. Human RBCs in autologous plasma were incubated for 1 h with NO synthase (NOS) inhibitors [N(omega)-nitro-l-arginine methyl ester (l-NAME) and S-methylisothiourea], NO donors [sodium nitroprusside (SNP) and diethylenetriamine (DETA)-NONOate], an NO precursor (l-arginine), soluble guanylate cyclase inhibitors (1H-[1,2,4]oxadiazolo-[4,3-a]quinoxalin-1-one and methylene blue), and a potassium channel blocker [triethylammonium (TEA)]. After incubation, RBC deformability at various shear stresses was determined by ektacytometry. Both NOS inhibitors significantly reduced RBC deformability above a threshold concentration, whereas the NO donors increased deformability at optimal concentrations. NO donors, as well as the NO precursor l-arginine and the potassium blocker TEA, were able to reverse the effects of NOS inhibitors. Guanylate cyclase inhibition reduced RBC deformation, with both SNP and DETA-NONOate able to reverse this effect. These results thus indicate the importance of NO as a determinant of RBC mechanical behavior and suggest its regulatory role for normal RBC deformability.     

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.     

Glutaraldehyde crosslinking of red blood cells by using a hemodialyzer

Summary Human red blood cells (RBC) were crosslinked with glutaraldehyde (GA) by using a hemodialyzer which is used as an artificial kidney. Human RBC, which was in a flow of 2 ml/min, was extensively crosslinked with 50 mM GA solution of 10 ml/min flow rate. The crosslinked RBC showed high stability against osmotic pressure. The oxygen transport activity of the crosslinked RBC was similar to unmodified RBC. This crosslinking method could be used for the development of an efficient reactor which produces a stable and active RBC.     

Purification of hemoglobin from red blood cells using tangential flow filtration and immobilized metal ion affinity chromatography

Two methods for purifying hemoglobin (Hb) from red blood cells (RBCs) are compared. In the first method, red blood cell lysate is clarified with a 50 nm tangential flow filter and hemoglobin is purified using immobilized metal ion affinity chromatography (IMAC). In the second method, RBC lysate is processed with 50 nm, 500 kDa, and 50-100 kDa tangential flow filters, then hemoglobin is purified with IMAC. Our results show that the hemoglobins from both processes produce identical Hb products that are ultrapure and retain their biophysical properties (except for chicken hemoglobin, which shows erratic oxygen binding behavior after purification). Therefore, the most efficient method for Hb purification appears to be clarification with a 50 nm tangential flow filter, followed by purification with IMAC, and sample concentration/polishing on a 10-50 kDa tangential flow filter.     

Assessment of oxidant susceptibility of red blood cells in various species based on cell deformability

The present study was designed to investigate the oxidant susceptibility of red blood cells (RBC) from four species (echidna, human, koala, Tasmanian devil) based on changes in cellular deformability. These species were specifically chosen based on differences in lifestyle and/or biology associated with varied levels of oxidative stress. The major focus was the influence of superoxide radicals generated within the cell (phenazine methosulfate, PMS, 50 μM) or in the extracellular medium (xanthine oxidase-hypoxanthine, XO-HX, 0.1 U/ml XO) on RBC deformability at various shear stresses (SS). RBC deformability was assessed by laser-diffraction analysis using a "slit-flow ektacytometer". Both superoxide-generating treatments resulted in significant increases of methemoglobin for all species (p < 0.01), with Tasmanian devil RBC demonstrating the most sensitivity to either treatment. PMS caused impaired RBC deformability for all species, but vast interspecies variations were observed: human and koala cells exhibited a similar sigmoid-like response to SS, short-beaked echidna values were markedly lower and only increased slightly with SS, while Tasmanian devil RBC were extremely rigid. The effect of XO-HX on RBC deformability was less when compared with PMS (i.e., smaller increase in rigidity) with the exception of Tasmanian devil RBC which exhibited essentially no deformation even at the highest SS; Tasmanian devil RBC response to XO-HX was thus comparable to that observed with PMS. Our findings indicate that ektacytometry can be used to determine the oxidant susceptibility of RBC from different species which varies significantly among mammals representing diverse lifestyles and evolutionary histories. These differences in susceptibility are consistent with species-specific discrepancies between observed and allometrically-predicted life spans and are compatible with the oxidant theory of aging.     

Effects of calcium and A23187 on deformability and volume of human red blood cells

Human red blood cells treated in vitro with Ca2+ plus A23187 in low K+ medium exhibited significantly decreased cell volume and deformability, the latter determined by ektacytometry. These effects of Ca2+ plus A23187 were prevented in the presence of high K+ medium. Increased K+ permeability mediated by increased intracellular Ca2+ (Gardos effect) was apparently responsible for decreased cell volume and deformability in low K+ medium. Although it is commonly accepted that Ca2+ accumulation and/or ATP depletion per se cause decreased red blood cell deformability, the present results demonstrate that acutely induced changes in red blood cell volume as promoted by Ca2+ are a more important determinant of red blood cell deformability.