Shape Of Red Blood Cells Research Articles

SCD-101: A New Anti-Sickling Drug Reduces Pain and Fatigue and Improves Red Blood Cell Shape in Peripheral Blood of Patients with Sickle Cell Disease

Background: Sickle cell disease (SCD) is the most common inherited blood disorder in the United States, caused by polymerization of a mutated form of hemoglobin (HbS). HbS can form long polymers inside red blood cells (RBCs) that affect RBC shape and adhesion, resulting in RBC destruction, acute and chronic pain, inflammation and cumulative organ damage. SCD-101 is a botanical drug with in vitro and in vivo anti-sickling activity. The mechanism by which SCD-101 inhibits sickling is unknown; however, it does not bind directly to Hb or change the affinity of Hb for oxygen. We recently completed a dose-escalation clinical study of SCD-101 in adults with sickle cell disease in steady state, and other clinical studies are on-going.Methods: The initial prospective, open-label, Phase 1B dose-escalation study enrolled homozygous (SS) sickle cell patients and S/beta0 thalassemia patients ages 18-55 with baseline Hb levels 6.0-9.0 g/dL and hemoglobin F ≤10%. Admission within 30 days, transfusion within 90 days, and hydroxyurea within 6 months were exclusions. SCD-101 was dosed orally for 28-days with a 14-day follow-up visit. Doses administered during the dose escalation study were 550 mg, 1100 mg, 2200 mg and 4400 mg BID. The primary endpoint was safety, and the secondary endpoints were mean change in Hb, Hct, percent reticulocytes, LDH, indirect bilirubin, CRP, PROMIS fatigue questionnaire score, and percent circulating partially oxygenated sickle cells (POSCs) in venous whole blood fixed in 2% glutaraldehyde without exposure to air. A repeat cohort study is ongoing with dose adjusted to 2750 mg TID, a 6-minute walk test added as a functional endpoint and measurement of inflammatory cytokines by ELISA to explore the effect of SCD-101 on inflammation.Results: As of August 4, 2016, 26 patients with sickle cell disease have been enrolled, 20 patients have completed the 28-day dose-escalation study and 3 were discontinued before receiving their first dose of SCD-101. Three patients are enrolled in the 28-day repeat cohort study. There have been no dose reductions or interruptions due to drug-related effects. SCD-101 was generally well-tolerated with mild-moderate bloating and flatulence at the highest dose on a BID schedule in the dose-escalation study, which were not observed with a TID schedule in the repeat cohort study. There were no significant changes in safety laboratory tests and no significant changes in hemoglobin, hematocrit, LDH, unconjugated bilirubin, or reticulocytes. No significant changes were observed in renal, hepatic or other safety chemistries or electrocardiograms. SCD-101 participants treated at higher doses reported a decrease in chronic pain and fatigue after 7-14 days, which returned post-treatment when measured on the 14-day follow-up visit. Participants at the two highest doses reported an increase in exercise capacity and an improvement in sleep. Two participants with ankle ulcers, enrolled at the two highest doses, showed improved healing. Analysis of peripheral blood smears revealed an improvement in the shape of circulating RBCs. There was no change in inflammatory cytokines in the first 7 days in the repeat cohort study, though the Day 28 data may show a different result.Conclusions: SCD-101 is a promising new drug for the treatment of sickle cell disease, based on the results from the studies reported here. SCD-101 was well tolerated over an 8-fold dose range, and dose-dependent anti-sickling effects on RBC were observed, though without significant changes in hemolysis. Clinical benefits included reduced chronic pain and fatigue, improved sleep and improved ulcer healing. While the identity of the active substance(s) in SCD-101 and the mechanism(s) of action are unknown, SCD-101 has a direct anti-sickling effect on RBCs, compatible with an intracellular or membrane effect on RBCs. We hypothesize that the observed effects could be explained by increased vascular flow or increased oxygen delivery or a reduction in inflammation. A placebo-controlled crossover study will be completed in 2016 including a 6-minute walk test as a primary clinical endpoint, and a multi-site Phase 2 study is planned for 2017. As a botanical drug, SCD-101 may be a useful treatment for sickle cell disease worldwide. [Display omitted] [Display omitted] [Display omitted] DisclosuresSwift:Global Blood Therapeutics: Equity Ownership; Mast Therapeutics: Equity Ownership; SCD Development: Employment, Equity Ownership.

MD/DPD Multiscale Framework for Predicting Morphology and Stresses of Red Blood Cells in Health and Disease.

Healthy red blood cells (RBCs) have remarkable deformability, squeezing through narrow capillaries as small as 3 microns in diameter without any damage. However, in many hematological disorders the spectrin network and lipid bilayer of diseased RBCs may be significantly altered, leading to impaired functionality including loss of deformability. We employ a two-component whole-cell multiscale model to quantify the biomechanical characteristics of the healthy and diseased RBCs, including Plasmodium falciparum-infected RBCs (Pf-RBCs) and defective RBCs in hereditary disorders, such as spherocytosis and elliptocytosis. In particular, we develop a two-step multiscale framework based on coarse-grained molecular dynamics (CGMD) and dissipative particle dynamics (DPD) to predict the static and dynamic responses of RBCs subject to tensile forcing, using experimental information only on the structural defects in the lipid bilayer, cytoskeleton, and their interaction. We first employ CGMD on a small RBC patch to compute the shear modulus, bending stiffness, and network parameters, which are subsequently used as input to a whole-cell DPD model to predict the RBC shape and corresponding stress field. For Pf-RBCs at trophozoite and schizont stages, the presence of cytoadherent knobs elevates the shear response in the lipid bilayer and stiffens the RBC membrane. For RBCs in spherocytosis and elliptocytosis, the bilayer-cytoskeleton interaction is weakened, resulting in substantial increase of the tensile stress in the lipid bilayer. Furthermore, we investigate the transient behavior of stretching deformation and shape relaxation of the normal and defective RBCs. Different from the normal RBCs possessing high elasticity, our simulations reveal that the defective RBCs respond irreversibly, i.e., they lose their ability to recover the normal biconcave shape in successive loading cycles of stretching and relaxation. Our findings provide fundamental insights into the microstructure and biomechanics of RBCs, and demonstrate that the two-step multiscale framework presented here can be used effectively for in silico studies of hematological disorders based on first principles and patient-specific experimental input at the protein level.

Red blood cell thickness is evolutionarily constrained by slow, hemoglobin-restricted diffusion in cytoplasm.

During capillary transit, red blood cells (RBCs) must exchange large quantities of CO2 and O2 in typically less than one second, but the degree to which this is rate-limited by diffusion through cytoplasm is not known. Gas diffusivity is intuitively assumed to be fast and this would imply that the intracellular path-length, defined by RBC shape, is not a factor that could meaningfully compromise physiology. Here, we evaluated CO2 diffusivity (DCO2) in RBCs and related our results to cell shape. DCO2 inside RBCs was determined by fluorescence imaging of [H+] dynamics in cells under superfusion. This method is based on the principle that H+ diffusion is facilitated by CO2/HCO3− buffer and thus provides a read-out of DCO2. By imaging the spread of H+ ions from a photochemically-activated source (6-nitroveratraldehyde), DCO2 in human RBCs was calculated to be only 5% of the rate in water. Measurements on RBCs containing different hemoglobin concentrations demonstrated a halving of DCO2 with every 75 g/L increase in mean corpuscular hemoglobin concentration (MCHC). Thus, to compensate for highly-restricted cytoplasmic diffusion, RBC thickness must be reduced as appropriate for its MCHC. This can explain the inverse relationship between MCHC and RBC thickness determined from >250 animal species.

Lennard-Jones type pair-potential method for coarse-grained lipid bilayer membrane simulations in LAMMPS

Lipid bilayer membranes have been extensively studied by coarse-grained molecular dynamics simulations. Numerical efficiencies have been reported in the cases of aggressive coarse-graining, where several lipids are coarse-grained into a particle of size 4∼6nm so that there is only one particle in the thickness direction. Yuan et al. proposed a pair-potential between these one-particle-thick coarse-grained lipid particles to capture the mechanical properties of a lipid bilayer membrane, such as gel–fluid–gas phase transitions of lipids, diffusion, and bending rigidity Yuan et al. (2010). In this work we implement such an interaction potential in LAMMPS to simulate large-scale lipid systems such as a giant unilamellar vesicle (GUV) and red blood cells (RBCs). We also consider the effect of cytoskeleton on the lipid membrane dynamics as a model for RBC dynamics, and incorporate coarse-grained water molecules to account for hydrodynamic interactions. The interaction between the coarse-grained water molecules (explicit solvent molecules) is modeled as a Lennard-Jones (L-J) potential. To demonstrate that the proposed methods do capture the observed dynamics of vesicles and RBCs, we focus on two sets of LAMMPS simulations: 1. Vesicle shape transitions with enclosed volume; 2. RBC shape transitions with different enclosed volume. Finally utilizing the parallel computing capability in LAMMPS, we provide some timing results for parallel coarse-grained simulations to illustrate that it is possible to use LAMMPS to simulate large-scale realistic complex biological membranes for more than 1 ms. Program summaryProgram Title: fluidmembraneProgram Files doi:http://dx.doi.org/10.17632/4v53nkv5hc.1Licensing provisions: GNU GPLv3Programming language: C++.Nature of problem:A pair-potential function for simulating the shape transition of fluid vesicles and the resting shapes of red blood cells.Program Solution method:Nosé-Hoover thermostat and Berendsen pressure coupling algorithms.External routines/libraries: LAMMPSSubprograms used: create_rbc_with_water (MATLAB), bond_harmonic1 (C++), in_example (LAMMPS input script).

Effect of in vivo and in vitro exposure to electrostatic field on some hematological parameters in rats.

The aim of this study was to investigate the effect of the external electrostatic field (ESF) on some hematological parameters in rats. Both in vivo and in vitro experiments were carried out. In in vivo investigations, rats were exposed to ESF (200 kV/m) during short (1 h) and long periods (6 days, 6 h daily). For in vitro study, the blood of intact rats was exposed to ESF for 1 h. Blood hematology was measured using validated ABX Micros ESV 60 Veterinary Hematology Analyzer. DNA damage in blood leucocytes was detected by means of comet assay. ESF effect on blood cell count was mainly manifested in white blood cells (WBC) and platelets. Damage of WBC was shown both in vitro and in vivo despite alterations in the count. This means the observed increase in WBC count in some cases might be a result of WBC compensatory mobilization from the bone marrow. Red blood cell (RBC) count and related parameters were slightly affected by ESF. Nevertheless, alterations in the shape and size of RBC were manifested. All ESF effects were extinguished in 14 days after the end of exposure. Bioelectromagnetics. 37:513-526, 2016. © 2016 Wiley Periodicals, Inc.

Biomechanics of red blood cells in human spleen and consequences for physiology and disease

Red blood cells (RBCs) can be cleared from circulation when alterations in their size, shape, and deformability are detected. This function is modulated by the spleen-specific structure of the interendothelial slit (IES). Here, we present a unique physiological framework for development of prognostic markers in RBC diseases by quantifying biophysical limits for RBCs to pass through the IES, using computational simulations based on dissipative particle dynamics. The results show that the spleen selects RBCs for continued circulation based on their geometry, consistent with prior in vivo observations. A companion analysis provides critical bounds relating surface area and volume for healthy RBCs beyond which the RBCs fail the "physical fitness test" to pass through the IES, supporting independent experiments. Our results suggest that the spleen plays an important role in determining distributions of size and shape of healthy RBCs. Because mechanical retention of infected RBC impacts malaria pathogenesis, we studied key biophysical parameters for RBCs infected with Plasmodium falciparum as they cross the IES. In agreement with experimental results, surface area loss of an infected RBC is found to be a more important determinant of splenic retention than its membrane stiffness. The simulations provide insights into the effects of pressure gradient across the IES on RBC retention. By providing quantitative biophysical limits for RBCs to pass through the IES, the narrowest circulatory bottleneck in the spleen, our results offer a broad approach for developing quantitative markers for diseases such as hereditary spherocytosis, thalassemia, and malaria.

Choline phosphate functionalized cellulose membrane: A potential hemostatic dressing based on a unique bioadhesion mechanism

Wound dressings are a key component in provision of optimal conditions for bleeding control and wound healing. For absorbent dressings, electrostatic interactions are frequently utilized as one of the mechanisms driving dressing adhesion. Herein, a choline phosphate functionalized biocompatible cellulose membrane that can efficiently arrest human red blood cells was developed to have potential application in wound dressing. The bioadhesion is based on the unique multivalent electrostatic interaction between the head groups of phosphatidyl choline based lipids on the cell membrane and its inverse orientation but virtually identical structure, choline phosphate, coupled to the cellulose membrane. For functionalization, the cellulose membrane was decorated with polymer brushes bearing multiple choline phosphate groups via surface-initiator atom transfer radical polymerization followed by click chemistry. The modified cellulose membranes were characterized by ATR-FTIR and the molecular weight and the grafting density of polymer brushes grafted from the cellulose membrane surface were thoroughly evaluated by calibrated force-distance measurements with atomic force microscopy (AFM). This new method provides an approach to estimating polymer brush parameters on rough surfaces of unknown surface area based on the dependence of brush thickness on brush density and polymer molecular weight for a calibration set of brushes. The dependence of binding of human red blood cells (RBCs) to the cellulose membrane surface on the number density of choline phosphate groups (e.g. molecular weight) and the grafting density were investigated using this AFM-based approach. Bound RBCs showed “pseudopodia”-like membrane projections under scanning electron microscopy where cells contacted the microfibers of the cellulose, distorting the RBC shape, reflecting the multivalent interactions between the RBCs and the choline phosphate-doped cellulose membrane. We believe this efficient strategy provides a promising approach to blood conservation and trauma management. Statement of SignificanceUncontrolled bleeding can dramatically affect morbidity and mortality. Absorptive wound dressings provide either adherent or non-adherent layers to control bleeding. Our new adherent material is based on a universal adhesion reaction between cell membrane phosphatidyl choline (PC) headgroups and cellulose membranes (CM) decorated with polymer brushes carrying a CP group per monomer. The CP-PC multivalent interactions provide adherence to cut tissue margins and blood cells, blocking bleeding. We here demonstrate the strong specific binding of red cells to CM-CP but not CM-PC membranes and determine the requisite brush molecular weight and surface concentration via a new approach using atomic force microscopy, applicable to rough surfaces. We believe this strategy provides a promising approach to blood conservation and trauma management.

Shape of red blood cells in contact with artificial surfaces.

The phenomenon of physical contact between red blood cells and artificial surfaces is considered. A fully three-dimensional mathematical model of a bilayer membrane in contact with an artificial surface is presented. Numerical results for the different geometries and adhesion intensities are found to be in agreement with experimentally observed geometries obtained by means of digital holographic microscopy.

Trypanosoma vivax Adhesion to Red Blood Cells in Experimentally Infected Sheep.

Trypanosomosis, a globally occurring parasitic disease, poses as a major obstacle to livestock production in tropical and subtropical regions resulting in tangible economic losses. In Latin America including Venezuela, trypanosomosis of ruminants is mainly caused by Trypanosoma vivax. Biologically active substances produced from trypanosomes, as well as host-trypanosome cellular interactions, contribute to the pathogenesis of anemia in an infection. The aim of this study was to examine with a scanning electron microscope the cellular interactions and alterations in ovine red blood cells (RBC) experimentally infected with T. vivax. Ovine infection resulted in changes of RBC shape as well as the formation of surface holes or vesicles. A frequent observation was the adhesion to the ovine RBC by the trypanosome's free flagellum, cell body, or attached flagellum in a process mediated by the filopodia emission from the trypanosome surface. The observed RBC alterations are caused by mechanical and biochemical damage from host-parasite interactions occurring in the bloodstream. The altered erythrocytes are prone to mononuclear phagocytic removal contributing to the hematocrit decrease during infection.

Mature red blood cells: from optical model to inverse light-scattering problem.

We propose a method for characterization of mature red blood cells (RBCs) morphology, based on measurement of light-scattering patterns (LSPs) of individual RBCs with the scanning flow cytometer and on solution of the inverse light-scattering (ILS) problem for each LSP. We considered a RBC shape model, corresponding to the minimal bending energy of the membrane with isotropic elasticity, and constructed an analytical approximation, which allows rapid simulation of the shape, given the diameter and minimal and maximal thicknesses. The ILS problem was solved by the nearest-neighbor interpolation using a preliminary calculated database of 250,000 theoretical LSPs. For each RBC in blood sample we determined three abovementioned shape characteristics and refractive index, which also allows us to calculate volume, surface area, sphericity index, spontaneous curvature, hemoglobin concentration and content.

Morphofunctional peculiarities of erythrocytes in wild and farmed Coregonid fishes from Lake Baikal

Structure and cytometric indices of red blood cells (RBC) and hemoglobin content (Hb) and oxygen capacity of the blood (OCB) of omul, whitefish, and hybrids thereof captured in Lake Baikal (wild) and incubated and grown in a freshwater aquarium complex (FAC) (farmed) have been analyzed. Cytometric parameters of red blood cells of wild omul, whitefish, and hybrids thereof exceed those of the cells of fish reared in aquariums under identical conditions. The effect of aquarium rearing on the shape of red blood cells is the least pronounced in Siberian whitefish and F1 progeny of Siberian whitefish females and omul males (f Sw x m Om). The erythrocyte size in hybrids of female Lacustrine whitefish and male omul (f Lw x m Om) is determined by the size of these cells in female parents, since female Lacustrine whitefish have the largest erythrocytes. Cytometric parameters of erythrocytes of all Coregonid fishes investigated are higher in fish reared in warm aquaria than in conspecifics reared in aquaria with cold water. Erythrocyte nuclei are smaller in artificially propagated hybrids than in parent fish captured in the wild or in whitefish and omul reared in aquaria under the same conditions. A distinct pool of erythrocytes from whitefish captured in the wild have a 20–30% higher content of functionally active mitochondria than erythrocytes of whitefish reared in aquaria; a disrupted mitochondrial structure is also observed in erythrocytes from the latter population of fish. The results show that distinctive features of metabolism related to oxygen transport in the Baikal coregonid fish that were investigated are determined by adaptation to the conditions of the ecological niches occupied by the fish.

2E35 浸透圧によるヒト赤血球の膨張・溶血挙動

The swelling and hemolysis behavior of human red blood cells (RBCs) due to the osmotic pressure imbalance has been investigated. Adding the 0.1wt% NaCl solution as hypotonic solution into the isotonic RBC suspension, we experimentally observed an RBC swelling from the biconcave disk to the spheroidal and spherical shape. The time evolution of the surface area and the volume of the RBC was evaluated from the lateral shape on the assumption of the axisymmetric geometry. The results show that the volume significantly increases with shape variation under the hypotonic condition, and then it decreases in a short time. Finally, the inclusion such as the hemoglobin leaks out of the RBC keeping the spherical membrane shape. Corresponding to the experimental observation, we theoretically and numerically calculated the variation in the hydrostatic pressure difference through the RBC membrane during the osmotic swelling. The calculated results enable an evaluation of the hydrostatic pressure differences at arbitrary moment in the swelling process by comparing the RBC shapes obtained experimentally and theoretically in terms of the ratio of major to minor radii.

Influence of erythrocyte shape on platelet scattering towards vessel wall

Red Blood Cell (RBC) geometry forms unique distinguished shape. This shape is thought to have a major influence on both the function and the dynamics of the moving erythrocyte. The shape of the RBC is hypothesised to cause platelet scattering and diffusion towards vessel wall. The current study is built to provide a detailed review of the previous theories that discuss the influence of RBC shape on its functionality and dynamics, a detailed focus on the influence of RBC dynamics on platelet migration towards the vessel wall is presented. Afterwards, computational results representing the influence of RBC shape on platelet migration to the vessel wall are listed. The results involve comparison between circular and elliptical RBCs using low Reynolds number channel flow regime. It is shown that this migration process is primarily affected by the diameter of the RBC and not by its shape.

Shape matters: the effect of red blood cell shape on perfusion of an artificial microvascular network.

The shape of human red blood cells (RBCs) deteriorates progressively throughout hypothermic storage, with echinocytosis being the most prevalent pathway of this morphologic lesion. As a result, each unit of stored blood contains a heterogeneous mixture of cells in various stages of echinocytosis and normal discocytes. Here we studied how the change in shape of RBCs following along the path of the echinocytic transformation affects perfusion of an artificial microvascular network (AMVN). Blood samples were obtained from healthy consenting volunteers. RBCs were leukoreduced, resuspended in saline, and treated with various concentrations of sodium salicylate to induce shape changes approximating the stages of echinocytosis experienced by RBCs during hypothermic storage (e.g., discocyte, echinocyte I, echinocyte II, echinocyte III, spheroechinocyte, and spherocyte). The AMVN perfusion rate was measured for 40% hematocrit suspensions of RBCs with different shapes. The AMVN perfusion rates for RBCs with discocyte and echinocyte I shapes were similar, but there was a significant decline in the AMVN perfusion rate between RBCs with shapes approximating each subsequent stage of echinocytosis. The difference in AMVN perfusion between discocytes and spherocytes (the last stage of the echinocytic transformation) was 34%. The change in shape of RBCs from normal discocytes progressively through various stages of echinocytosis to spherocytes produced a substantial decline in the ability of these cells to perfuse an AMVN. Echinocytosis induced by hypothermic storage could therefore be responsible for a similarly substantial impairment of deformability previously observed for stored RBCs.

Automated tracking of temporal displacements of a red blood cell obtained by time-lapse digital holographic microscopy.

Red blood cell (RBC) phase images that are numerically reconstructed by digital holographic microscopy (DHM) can describe the cell structure and dynamics information beneficial for a quantitative analysis of RBCs. However, RBCs investigated with time-lapse DHM undergo temporal displacements when their membranes are loosely attached to the substrate during sedimentation on a glass surface or due to the microscope drift. Therefore, we need to develop a tracking algorithm to localize the same RBC among RBC image sequences and dynamically monitor its biophysical cell parameters; this information is helpful for studies on RBC-related diseases and drug tests. Here, we propose a method, which is a combination of the mean-shift algorithm and Kalman filter, to track a single RBC and demonstrate that the optical path length of the single RBC can be continually extracted from the tracked RBC. The Kalman filter is utilized to predict the target RBC position in the next frame. Then, the mean-shift algorithm starts execution from the predicted location, and a robust kernel, which is adaptive to changes in the RBC scale, shape, and direction, is designed to improve the accuracy of the tracking. Finally, the tracked RBC is segmented and parameters such as the RBC location are extracted to update the Kalman filter and the kernel function for mean-shift tracking; the characteristics of the target RBC are dynamically observed. Experimental results show the feasibility of the proposed algorithm.

Commonly Used Clinical Intravenous Fluid Formulations Differentially Affect Sickle Red Blood Cell Stiffness and Transit Time

Background:Disruption of red blood cell (RBC) volume regulation and water homeostasis is a major component of sickle cell pathophysiology. As such, hydration is a mainstay of prevention and treatment of vaso-occlusive crises (VOC) in patients with sickle cell disease (SCD). However, evidence for guiding clinicians' choice for intravenous (IV) fluid hydration in the acute setting is lacking (Okomo, Coch Data Syst Rev, 2012). Pediatric hematologists typically discourage rapid infusion of hypotonic fluid given the risk of hyponatremia, although such fluids as 5% dextrose with 34meq/L or 77meq/L sodium are often used after stabilizing various acute clinical situations relevant to SCD. Although altered sodium concentration and osmolarity have been shown to affect erythrocyte swelling and rheology, dextrose-containing fluids used clinically such as D5 ¼ and D5 ½ normal saline (NS), have not been studied (Reinhart et al., Mic Res, 2015; Hijiya et al., J Lab Clin Med, 1991). To those ends, we sought to investigate the effect of different clinically relevant IV fluid formulations on normal and sickle RBC stiffness and deformability.Methods:Fresh blood was obtained from healthy volunteers and patients with sickle cell anemia (SS) on hydroxyurea and not transfused for at least 100 days. Sterile, clinical grade fluids stored at room temperature were used for the experiments (Baxter, Figure 1A). Our laboratory has previously published a description of a microfluidic device comprised of multiple parallel microchannels 5μm wide recapitulating the in vivo geometry of capillaries (Rosenbluth et al., Lab Chip, 2008). This microvasculature-on-a-chip enables measurements of single-RBC transit times, which correlate with cell stiffness (Figure 1B,C). For the transit time experiments, a master silicon wafer was used to mold the microfluidic channels out of polydimethylsiloxane (PDMS) silicone. Centrifuged RBCs were washed with phosphate-buffered saline (PBS) and then diluted to 0.5% hematocrit (HCT) in the various fluids prior to flow. Cell suspensions were perfused into a microfluidic device pre-coated with 2% bovine serum albumin (BSA) at an average linear flow rate of 0.50 mm s-1 in the smallest channels with a syringe pump (Harvard Apparatus) and then imaged at 20x at 20 frames per second. Images were recorded for future analysis. For the experiments assessing RBC shape, washed RBCs were diluted with PBS to 0.5% HCT and imaged at 40x in plastic wells at time 0. PBS was removed and 100 μL of each clinical fluid formulation was added to the wells and images were then obtained over time. RBC circularity was calculated using custom-written scripts in Matlab (Figure 1D).Results:RBC transit times in both healthy and sickle blood were affected by osmolarity and the various solute concentrations (Figures 2A,B). Transit times of sickle RBCs in all IV fluid formulations were significantly higher, over 10 times, than that of RBCs from healthy donors. Transit times for both healthy and sickle donors were least in the D5 ¼ NS solution. Of note, sickle RBC transit time was greatest in the NS solution. Sickle RBC circularity also changed with solute concentration and osmolarity with statistical significance (Figure 2C). Cells with the highest change in circularity from baseline were also those exposed to the D5 ¼ NS solution.Conclusion:Our results suggest the stiffness of sickle RBCs is affected by different formulations of clinical IV fluids. Increased transit time of sickle RBCs in NS through our device may in part be explained by the decreased circularity, indicating that RBCs adopt more irregular shapes in this fluid. This, in turn, could lead to increased propensity of microchannel obstruction. Although the exact mechanisms are unclear, this begs the question of whether NS is an appropriate choice for initial fluid resuscitation for VOC and other SCD-related complications as it could exacerbate the already high stiffness and shape irregularity of sickle RBCs, further increasing microvascular occlusion. As these in vitro results have significant clinical implications, ongoing experiments involve investigating how these fluid-dependent effects may alter sickle RBC adhesion in 'endothelialized' microfluidic devices, how different oxygen tensions affect these fluid-mediated effects, potential differences on and off of hydroxyurea, and the underlying mechanisms of this IV fluid formulation-dependent effect. [Display omitted] [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Role of Red Cells in Thrombosis and Hemostasis

Until recently it has been assumed that red blood cells (RBCs) play mainly a passive role in hemostasis and thrombosis, but now some studies have uncovered several novel roles for them in both of these processes. The best-known effects of RBCs in clotting in vivo are rheological, involving laminar shearing plus aggregation and deformability of RBCs. In addition, RBCs have specific receptors for platelets and fibrin(ogen) that may be involved in thrombosis. Furthermore, in the contraction of blood clots, the trapping of RBCs in the platelet-fibrin network and subsequent physical changes may be important for hemostasis and thrombosis. Contracted blood clots develop a remarkable structure, with a meshwork of fibrin and platelet aggregates on the exterior of the clot and a close-packed, tessellated array of compressed polyhedral RBCs within. The structure and properties of contracted clots vary depending on the relative amounts of platelets, fibrinogen and RBCs and the conditions of clotting. Such close-packed arrays of polyhedral erythrocytes, or polyhedrocytes, have also been observed in human arterial and especially venous thrombi taken from patients. The mechanical nature of this RBC shape change was confirmed by polyhedrocyte formation from the forces of centrifugation of blood without clotting. Many experiments suggest that polyhedrocytes are more prevalent under conditions associated with thrombosis and could be a marker of prothrombotic conditions. Consistent with this hypothesis, RBC membranes have been found to be a major source of exposure of phosphatidylserine, which forms a procoagulant surface to generate thrombin. Platelets (with their cytoskeletal motility proteins) and fibrin(ogen) (as the substrate bridging platelets for contraction) are required to generate the forces necessary to segregate platelets/fibrin from RBCs and to compress these cells into a tightly packed array. Clots formed after puncture of the mouse saphenous vein are composed primarily of polyhedrocytes, suggesting that they may be important in venous hemostasis. Moreover, the permeability of contracted clots is nearly the same as that of the endothelial cell lining, consistent with their functional significance. These results demonstrate how contracted clots form an impermeable barrier important for hemostasis and wound healing and also to restore flow past obstructive thrombi. On the other hand, they also help explain how fibrinolysis is greatly retarded after clots contract, as a result of these striking structural changes. In summary, RBCs may perform a dual role, both helping to stem bleeding but at the same time contributing to thrombosis in a variety of ways, including when the tightly packed array of RBCs in a contracted clot blocks blood flow and resists dissolution. Disclosures No relevant conflicts of interest to declare.

In Vitro Impact of Treatment With Aqueous Extract of Cassia Fistula on Red Blood Cell Sickling in Individuals With Sickle Cell Trait

Background: Sickle cell anemia is an autosomal recessive genetic blood disorder caused by a replacement of an amino acid in the hemoglobin structure, followed by disturbance in its electrical charge. In this disorder, the solubility of hemoglobin is reduced, in hypoxic condition, and formation of crystals occurs, leading to sickling the shape of red blood cells (RBC). The Cassia Fistula (C. fistula) (Flous Plant), as a flowering plant in the fabaceae family, is used as a traditional medicine in the treatment of sickle cell anemia. Objectives: The aim of this study was to evaluate the impact of aqueous extract of C. fistula (the ripped fruit) on red cells in sickle cell anemia. Materials and Methods: The aqueous extract of C. fistula fruit was obtained by vacuum distillation method, using different dilutions of 1:2, 1:4, 1:8, 1:16, 1:50, 1:100, 1:200. The RBC from individuals presenting the hemoglobin S (HbS) trait were washed and treated with different dilutions. After 24 hours incubation, they were evaluated by sickling test (sodium metabisulfite test). The Wilcoxon signed ranks test was used for data analysis. Results: Sickle cells were observed among 20% of subjects with the dilution of 1:50 and 70% of subjects with the dilution of 1:100. Mean percentage of sickle red cells with dilutions of 1:50, 1:100 and 1:200 were 3.75%, 25.75% and 43.0%, respectively. Conclusions: It is suggested that C. fistula may protect RBC against sickling, in hypoxic conditions, in individuals with HbS gene defect.

Synthesis, characterization, and cytotoxicity in human erythrocytes of multifunctional, magnetic, and luminescent nanocrystalline rare earth fluorides.

Multifunctional nanoparticles exhibiting red or green luminescence properties and magnetism were synthesized and thoroughly analyzed. The hydrothermal method was used for the synthesis of Eu3+- or Tb3+-doped GdF3-, NaGdF4-, and BaGdF5-based nanocrystalline materials. The X-ray diffraction patterns of the samples confirmed the desired compositions of the materials. Transmission electron microscope images revealed the different morphologies of the products, including the nanocrystal sizes, which varied from 12 nm in the case of BaGdF5-based nanoparticles to larger structures with dimensions exceeding 300 nm. All of the samples presented luminescence under ultraviolet irradiation, as well as when the samples were in the form of water colloids. The highest luminescence was observed for BaGdF5-based materials. The obtained nanoparticles exhibited paramagnetism along with probable evidence of superparamagnetic behavior at low temperatures. The particles’ magnetic characteristics were also preserved for samples in the form of a suspension in distilled water. The cytotoxicity studies against the human erythrocytes indicated that the synthesized nanoparticles are non-toxic because they did not cause the red blood cells shape changes nor did they alter their membrane structure and permeabilization.Electronic supplementary materialThe online version of this article (doi:10.1007/s11051-015-3191-2) contains supplementary material, which is available to authorized users.

An approximation to red blood cells with a model of three-center-combined shells

Red blood cells present a biconcave shape and bear an inner pressure (osmotic pressure) when they are in the static state. In this paper, a model of “three-center-combined shells”, which consists of two spherical shells and a toroidal shell, is employed to describe the geometric shape of red blood cells. Surface area and volume of the combined shells model are very close to those measured from experiment. The stress distribution in the cell membrane is formulized as a closed form according to the Novozhilov's theory of the three-center-combined shells. Calculating results in terms of Novozhilov's formula give a good agreement with the numerical results given by ABAQUS when using actual measurements. It is concluded that the combined shells model can well approximate to the biconcave structure of red blood cells. In addition, stress calculation shows that the membrane of biconcave red blood cells can carry bending moments, and the moments reach a maximum value in the vicinity of joint line of the spherical shell and the toroidal shell in the combined shells model.