Red Blood Cells Filterability Research Articles

Metabolic rejuvenation upgrades circulatory functions of red blood cells stored under blood bank conditions.

Red blood cells (RBC) change upon hypothermic conservation, and storage for 6 weeks is associated with the short-term clearance of 15% to 20% of transfused RBCs. Metabolic rejuvenation applied to RBCs before transfusion replenishes energetic sources and reverses most storage-related alterations, but how it impacts RBC circulatory functions has not been fully elucidated. Six RBC units stored under blood bank conditions were analyzed weekly for 6 weeks and rejuvenated on Day 42 with an adenine-inosine-rich solution. Impact of storage and rejuvenation on adenosine triphosphate (ATP) levels, morphology, accumulation of storage-induced microerythrocytes (SMEs), elongation under an osmotic gradient (by LORRCA), hemolysis, and phosphatidylserine (PS) exposure was evaluated. The impact of rejuvenation on filterability and adhesive properties of stored RBCs was also assessed. Rejuvenation of RBCs restored intracellular ATP to almost normal levels and decreased the PS exposure from 2.78% to 0.41%. Upon rejuvenation, the proportion of SME dropped from 28.2% to 9.5%, while the proportion of normal-shaped RBCs (discocytes and echinocytes 1) increased from 47.7% to 67.1%. In LORCCA experiments, rejuvenation did not modify the capacity of RBCs to elongate and induced a reduction in cell volume. In functional tests, rejuvenation increased RBC filterability in a biomimetic splenic filter (+16%) and prevented their adhesion to endothelial cells (-87%). Rejuvenation reduces the proportion of morphologically altered and adhesive RBCs that accumulate during storage. Along with the improvement in their filterability, these data show that rejuvenation improves RBC properties related to their capacity to persist in circulation after transfusion.

Red Blood Cell Passage of Small Capillaries Is Associated with Transient Ca2+-mediated Adaptations.

When red blood cells (RBCs) pass constrictions or small capillaries they need to pass apertures falling well below their own cross section size. We used different means of mechanical stimulations (hypoosmotic swelling, local mechanical stimulation, passing through microfluidic constrictions) to observe cellular responses of human RBCs in terms of intracellular Ca2+-signaling by confocal microscopy of Fluo-4 loaded RBCs. We were able to confirm our in vitro results in a mouse dorsal skinfold chamber model showing a transiently increased intracellular Ca2+ when RBCs were passing through small capillaries in vivo. Furthermore, we performed the above-mentioned in vitro experiments as well as measurements of RBCs filterability under various pharmacological manipulations (GsMTx-4, TRAM-34) to explore the molecular mechanism of the Ca2+-signaling. Based on these experiments we conclude that mechanical stimulation of RBCs activates mechano-sensitive channels most likely Piezo1. This channel activity allows Ca2+ to enter the cell, leading to a transient activation of the Gardos-channel associated with K+, Cl−, and water loss, i.e., with a transient volume adaptation facilitating the passage of the RBCs through the constriction.

Stabilization of Human Erythrocytes for Lyophilization: Recovery of Biochemical Activities and Membrane Filterability.

It has much more advantages to preserve human red blood cells (RBC) in freezing and drying form than conventional blood storage, even though it is still under developing. In the present study, we successfully used 0.045% glutaraldehyde(GA) to fix RBCs under appropriate conditions prior to lyophilization and regained the bulk of RBCs membrane filterability. The phlebotomized whole blood was anti-coagulated with CPDA-1 and the plasma was removed by centrifugation. White blood cells were removed using Leucocytes filter (Pall Corporation). 1 x 10 9 cells/ml were separately incubated with 0.03%, 0.045% and 0.05% GA at 37 °C for 10 min; RBCs at concentrations 0.5 x 10 9, 1 x 10 9 and 2 x 10 9 cell/ml were incubated with 0.045% GA at 37 °C for 10 min; Bovine serum albumin (BSA), trehalose, hydroxyethyl starch and dextran were used as protective additive in freezing and drying RBCs, respectively; ethanolamine, glycine, lysine, BSA, glutamic acid and homocysteic acid were used to recover membrane deformability due to GA fixation. Recovery RBCs yield after reconstituion was determined by a cell counter. Fee hemoglobin , glutathione (GSH), 2,3-diphosphoglycerate (2,3-DPG), ATP, and Glucose-6-phosphate dehydrogenase (G-6-PDH) in RBCs were determined before and after lyophilization. Membrane flexibility was assayed by osmotic fragility test. RBCs filterability was determined using positive filter apparatus through 5 micrometer pore size nitrocellulose membrane. Our results suggested that RBCs at 1 x 10 9 cells/ml fixed by 0.045% GA for10 min at pH 7.8 were enough to resist freezing and drying damages and the bulk of RBCs's membrane filterability were remained. The effective fixation of GA is dependent on GA's concentration, RBCs concentration, incubation period and pH etc. BSA is the most potential additive in preserving RBCs. More interestingly, the following reagents can recover the reduced RBCs membrane filterability by GA fixation. The potency of recovery is in the order of : 10 mM ethanolamine > 5 mM lysine > 0.5%BSA > 10 mM glutamatic acid > 5 mM homcysteic acid > 2% glycine. After reconstitution, 85 ± 2.3% RBCs yield is achieved, 71 ± 4.1% of these RBCs can freely pass through 5 micrometer pore size filter membrane. Biochemical function indexes as GSH, 2,3-DPG, ATP, and G-6-PDH are saved by 20 ± 1.2%.Lyophilized human red blood cells yields from reconstitution by different treatment.1x 109 cells/ml fixed at0.03% glutaraldehyde44.7 ± 4.10.045%glutaraldehyde84.7 ± 5.80.05%glutaraldehyde88.7 ± 7.60.045% glutaraldehyde fix:0.5x 109 cells/ml91 ± 4.31x 109 cells/ml84.7 ± 5.82 x 109 cells/ml35.2 ± 2.3Effects of various treatment on lyophilized RBCs after reconstitution.GroupFilterability(%)1x9 cells/ml fixed by:0.03%glutaraldehyde88.3 ± 1.20.045%glutaraldehyde73.3 ± 1.80.05%glutaraldehyde44.7 ± 2.80.045%glutaraldehyde fixation at:0.5x109 cells/ml01x109 cells/ml73.3 ± 1.82x109 cells/ml83.9 ± 4.6N = 5. [Display omitted]

β‐Adrenergic agonists regulate cell membrane fluctuations of human erythrocytes

1. Mechanical fluctuations of the cell membrane (CMFs) in human erythrocytes reflect the bending deformability of the membrane-skeleton complex. These fluctuations were monitored by time-dependent light scattering from a small area ( approximately 0. 25 microm2) of the cell surface by a method based on point dark field microscopy. 2. Exposure of red blood cells (RBCs) to adrenaline (epinephrine) and isoproterenol (isoprenaline) resulted in up to a 45 % increase in the maximal fluctuation amplitude and up to a 35 % increase in the half-width of the amplitude distribution. The power spectra of membrane fluctuations of control and treated cells revealed that adrenaline stimulated only the low frequency component (0.3-3 Hz). Analysis of the dose-response curves of beta-adrenergic agonists yielded an EC50 of 5 x 10-9 and 1 x 10-11 M for adrenaline and isoproterenol, respectively. Propranolol had an inhibitory effect on the stimulatory effect of isoproterenol. These findings show a potency order of propranolol > isoproterenol > adrenaline. 3. The stimulatory effect of adrenaline was a temporal one, reaching its maximal level after 20-30 min but being abolished after 60 min. However, in the presence of 3-isobutyl-1-methylxanthine, a partial stimulatory effect was maintained even after 60 min. Pentoxifylline and 8-bromo-cAMP elevated CMFs. However, exposure of ATP-depleted erythrocytes to adrenaline or 8-bromo-cAMP did not yield any elevation in CMFs. These findings suggest that the beta-agonist effect on CMFs is transduced via a cAMP-dependent pathway. 4. Deoxygenation decreased CMFs and filterability of erythrocytes by approximately 30 %. The stimulatory effect of isoproterenol on CMFs was 2.2-fold higher in deoxygenated RBCs than in oxygenated cells. 5. Exposure of RBCs to adrenaline resulted in a concentration-dependent increase in RBC filterability, demonstrating a linear relationship between CMFs and filterability, under the same exposure conditions to adrenaline. These findings suggest that beta-adrenergic agonists may improve passage of erythrocytes through microvasculature, enhancing oxygen delivery to tissues, especially under situations of reduced oxygen tension for periods longer than 20 min.

Endothelin-1 improves the impaired filterability of red blood cells through the activation of protein kinase C.

We previously showed that the deformability of human red blood cells (RBCs) is affected by intracellular signaling pathways by examining the effects of Ca2+ influx and the intracellular cAMP level on mechanically-impaired RBC filterability. In the present study, we investigated whether protein kinase C (PKC) participates in the regulation of RBC deformability by affecting membrane properties. The filterability of mechanically-stressed RBCs showed a V-shaped curve depending on the extracellular Ca2+ concentration; the maximum decrease was achieved at 20-40 microM. The PKC activity, as measured in the membrane-rich fraction by an ELISA method using an antibody for the phosphorylated PKC substrate, maximally increased at the extracellular Ca2+ concentration where the filterability showed a marked improvement following the bottom of the V-shaped curve of the impaired filterability. At this Ca2+ concentration, the PKC activator endothelin-1 increased the PKC activity, and a PKC inhibitor (calphostin C) decreased it. Endothelin-1 improved and calphostin C worsened the impaired filterability. A specific type-B endothelin receptor agonist (IRL 1620) also improved the impaired filterability. A Western blot analysis revealed the presence of endothelin receptors in the RBC membrane. These results indicate that PKC improves the impaired filterability and that RBCs are the target of endothelin-1.

Treatment of Myeloproliferative Disorders With Hydroxyurea: Effects on Red Blood Cell Geometry and Deformability

AbstractHydroxyurea (HU) is used in suppressing the bone marrow and producing fetal-like red blood cells (RBCs). These RBCs are large in size and may theoretically disturb the microcirculation. In five patients with myeloproliferative disorders (MPD), the RBC geometry and deformability were analyzed before and after 6 to 8 months of HU treatment. In untreated MPD, the RBC geometry and filterability was normal. After HU, the RBC membrane area increased 24% and the cell volume increased 39% (P < .005). This change resulted in a 12% increase in the minimum cylindrical diameter (MCD). From a static bending model of initial deformation, the RBC diametrical cross-section had a significantly increased section modulus. However, this increase in profile stiffness was compensated for by its larger projected cell area and, thus, pressure load on the RBC corpuscle. The resulting resistance to initial deformation therefore remained unchanged after HU. These findings were tested experimentally; with 3-μm filter membranes, HU treatment caused a significant increase in flow resistance (P < .02), in accordance with MCD. However, with 5-μm pores, no difference was seen, again in consonance with the theoretical findings of initial deformation. Because most capillaries are larger than 3 μm, we suggest that HU is acceptable from a perspective of cellular microrheology.

Treatment of Myeloproliferative Disorders With Hydroxyurea: Effects on Red Blood Cell Geometry and Deformability

Hydroxyurea (HU) is used in suppressing the bone marrow and producing fetal-like red blood cells (RBCs). These RBCs are large in size and may theoretically disturb the microcirculation. In five patients with myeloproliferative disorders (MPD), the RBC geometry and deformability were analyzed before and after 6 to 8 months of HU treatment. In untreated MPD, the RBC geometry and filterability was normal. After HU, the RBC membrane area increased 24% and the cell volume increased 39% (P &lt; .005). This change resulted in a 12% increase in the minimum cylindrical diameter (MCD). From a static bending model of initial deformation, the RBC diametrical cross-section had a significantly increased section modulus. However, this increase in profile stiffness was compensated for by its larger projected cell area and, thus, pressure load on the RBC corpuscle. The resulting resistance to initial deformation therefore remained unchanged after HU. These findings were tested experimentally; with 3-μm filter membranes, HU treatment caused a significant increase in flow resistance (P &lt; .02), in accordance with MCD. However, with 5-μm pores, no difference was seen, again in consonance with the theoretical findings of initial deformation. Because most capillaries are larger than 3 μm, we suggest that HU is acceptable from a perspective of cellular microrheology.

In vitro effects of in vivo activated leukocytes on RBC filterability and lipid peroxidation

Activated leukocytes are known to generate reactive oxygen species which may attack other cells in their surroundings. On the other hand, rheological properties of red blood cells (RBC) are atIected by oxidant attack. This study was designed to detetmine the intluence of activated leukocytes on RBC rheology in vitro. Activated leukocytes were isolated from the Guinea pigs with an induced E. Coli sepsis and incubated with RBC obtained from normal animals. After two hours of incubation with activated leukocytes, RBC deform ability indices were increased 1.23 ± 0.05 (Mean±Standart error) times (compared to the control RBC suspensions) while incubation with leukocytes obtained from healthy animals increased defOlmability indices 1.12 ± 0.03 times. Thiobarbituric acid reactive substances as a measure of lipid peroxidation were increased 2.95 ± 0.36 times in the RBC suspensions incubated with activated leucocytes, but 1.70 ± 0.10 times in suspensions incubated with nOlmal leukocytes. These results suggest that RBC mechanical properties might be affected by activated leukocytes and possibly an oxidative mechanism plays role in this interaction.

Atrial natriuretic peptide: Direct effects on human red blood cell dynamics

The ability to deform is an important feature of red blood cells (RBCs) for performing their function of oxygen delivery. Little is known about the hormonal regulation of RBC deformability. Here we report that human atrial natriuretic peptide (ANP) acts directly on human RBCs leading to the elevation of local bending fluctuations of the cell membrane. These changes are accompanied by an increase in the filterability of RBCs. These ANP effects were mimicked by cyclic GMP analogues, suggesting modulation of local membrane bending fluctuations and RBC filterability via a cyclic GMP-dependent pathway. The effect of ANP on the mechanical properties of RBCs suggests that ANP may increase the passage red blood cells through capillaries resulting in an improved oxygen delivery to the tissues.

Platelet-activating factor acetylhydrolase activity in red blood cell-stroma from patients with cerebral thrombosis.

Platelet-activating factor (1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine, PAF) is a bioactive phospholipid and inactivated by a specific enzyme, PAF acetylhydrolase (PAF-AH). We have measured PAF-AH activity in red blood cells (RBCs) from patients with a history of cerebral thrombosis and age-matched healthy controls. The activities in 34 patients and 34 controls were 1.29 +/- 0.28 and 1.64 +/- 0.26 nmol/10(9) RBCs/min (or, 30.0 +/- 5.8 and 35.1 +/- 4.7 nmol/g protein/min) (mean +/- SD), respectively, and the difference was significant (p less than 0.001). In patients, RBC PAF-AH activity correlated positively with RBC filterability, an index of RBC deformability (r = +0.501, p less than 0.05). The RBC activity may play a role in scavenging oxidation products of membrane phospholipids. Lower RBC PAF-AH activity may predispose to ischemic diseases by disturbing microcirculatory behavior of the RBCs.

Deformability of Mammalian and Fish Erythrocytes:Comparison of Mean Pore Transit Times of Cells and Estimation of Cellular Viscosity and Elasticity.

Red blood cells (RBCs) of various species of mammals and fish were subjected to a modified Nuclepore filtration test, which determines average time required for cells to pass through filter pores under constant suction. The values so far obtained, when plotted against the ratio of mean cell diameter to pore diameter, fall in a region given by a formula, which has been derived from a simple viscoelastic model for the deformation of a cell, for a small range of cellular viscosity (1.0-2.0 x 10(3) dyn.s/cm for mammals at 37 degrees C, 3.0-5.0 x 10(-3) dyn.s/cm for fish at 15 degrees C). Furthermore, estimates of cellular elasticity given from pore transit times at the cell-pore diameter ratio beyond 2.0 also fall in a similar range (0.5-1.0 dyn/cm) for both the mammalian and fish cells. It is thus suggested that RBC filterability may not differ greatly, when compared at a comparative temperature, between different species studied so far, including mammals and fish, for a relatively large range of the extent to which cells are caused to deform. A question then arises about the shape and cytoplasm factors which have been accepted as the primary determinants of RBC deformability.

Quantitative relationship between Heinz body formation and red blood cell deformability [see comments

The ultimate cause of destruction of red blood cells (RBCs) after oxidative damage with Heinz body formation is not well understood. We correlated the changes in RBC morphology and membrane protein composition after oxidant treatment with the alterations in deformability of whole cells and cell membranes. The incubation of RBCs with phenylhydrazine concentrations of 0.3 to 100 mg/dL at 37 degrees C for one hour led to a dose-dependent formation of Heinz bodies, ranging from isolated Heinz bodies at 1 mg/dL to a confluent coating of the inner membrane surface at 100 mg/dL phenylhydrazine. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed the presence of a large quantity of hemoglobin bound to the ghost membrane of treated RBCs. Electrophoresis with and without dithiothreitol indicated that disulfide bridges are abundant between hemoglobin molecules and are also present among membrane proteins but are not the major bond between hemoglobin and membrane. Changes of spectrin, ankyrin, band 3, and band 6 and the appearance of a 260,000-dalton complex were also observed. With phenylhydrazine concentrations below 30 mg/dL, even in the presence of multiple Heinz bodies, the RBC deformability measured by filtration through 2.6-, 4.5-, and 6.8-microns pores and the membrane deformability determined by a filter aspiration technique were not altered. With 100 mg/dL phenylhydrazine, when the entire membrane was coated with Heinz bodies, RBC filterability and membrane deformability were drastically reduced. These results indicate that oxidative damage of RBCs with discrete Heinz body formation causes focal membrane rigidification but does not affect the global cellular deformability until the Heinz bodies nearly cover the entire cell endoface.

Quantitative Relationship Between Heinz Body Formation and Red Blood Cell Deformability

Abstract The ultimate cause of destruction of red blood cells (RBCs) after oxidative damage with Heinz body formation is not well understood. We correlated the changes in RBC morphology and membrane protein composition after oxidant treatment with the alterations in deformability of whole cells and cell membranes. The incubation of RBCs with phenylhydrazine concentrations of 0.3 to 100 mg/dL at 37 degrees C for one hour led to a dose-dependent formation of Heinz bodies, ranging from isolated Heinz bodies at 1 mg/dL to a confluent coating of the inner membrane surface at 100 mg/dL phenylhydrazine. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed the presence of a large quantity of hemoglobin bound to the ghost membrane of treated RBCs. Electrophoresis with and without dithiothreitol indicated that disulfide bridges are abundant between hemoglobin molecules and are also present among membrane proteins but are not the major bond between hemoglobin and membrane. Changes of spectrin, ankyrin, band 3, and band 6 and the appearance of a 260,000-dalton complex were also observed. With phenylhydrazine concentrations below 30 mg/dL, even in the presence of multiple Heinz bodies, the RBC deformability measured by filtration through 2.6-, 4.5-, and 6.8-microns pores and the membrane deformability determined by a filter aspiration technique were not altered. With 100 mg/dL phenylhydrazine, when the entire membrane was coated with Heinz bodies, RBC filterability and membrane deformability were drastically reduced. These results indicate that oxidative damage of RBCs with discrete Heinz body formation causes focal membrane rigidification but does not affect the global cellular deformability until the Heinz bodies nearly cover the entire cell endoface.