Red Blood Cell Rigidity Research Articles

Nanocarriers' repartitioning of drugs between blood subcompartments as a mechanism of improving pharmacokinetics, safety, and efficacy

For medical emergencies, such as acute ischemic stroke, rapid drug delivery to the target site is essential. For many small molecule drugs, this goal is unachievable due to poor solubility that prevents intravenous administration, and less obviously, by extensive partitioning to plasma proteins and red blood cells (RBCs), which greatly slows delivery to the target. Here we study these effects and how they can be solved by loading into nanoscale drug carriers. We focus on fingolimod, a small molecule drug that is FDA-approved for treatment of multiple sclerosis, which has also shown promise in the treatment of stroke. Unfortunately, fingolimod has poor solubility and very extensive partitioning to plasma proteins and RBCs (in whole blood, 86% partitions to RBCs, 13.96% to plasma proteins, and 0.04% is free). We develop a liposomal formulation that slows the partitioning of fingolimod to RBCs and plasma proteins, enables intravenous delivery, and additionally prevents fingolimod toxicity to RBCs. The liposomal formulation nearly completely prevented fingolimod adsorption to plasma proteins (association with plasma proteins was 98.4 ± 0.4% for the free drug vs. 5.6 ± 0.4% for liposome-loaded drug). When incubated with whole blood in vitro, the liposomal formulation greatly slowed partitioning of fingolimod to RBCs and also eliminated deleterious effects of fingolimod on RBC rigidity, morphology, and hemolysis. In vivo, the liposomal formulation delayed fingolimod partitioning to RBCs for over 30 min, a critical time window for stroke. Fingolimod-loaded liposomes showed improved efficacy in a mouse model of post-stroke neuroinflammation, completely sealing the leaky blood-brain barrier (114 ± 11.5% reduction in albumin leak into the brain for targeted liposomes vs. 38 ± 16.5% reduction for free drug). This effect was only seen for liposomes modified with antibodies to enable targeted delivery to the site of action, and not in unmodified, long-circulating liposomes. Thus, loading fingolimod into liposomes prevented partitioning to RBCs and associated toxicities and enabled targeted delivery. This paradigm can be used for tuning the blood distribution of small molecule drugs for the treatment of acute illnesses requiring rapid pharmacologic intervention.

Influence of Hemoglobin Variants on Vaso-Occlusive Phenomena in Sickle Cell Anemia: A Review

Sickle cell anemia (SCA) is a genetic disorder characterized by the presence of abnormal hemoglobin S (HbS), leading to the sickling of red blood cells (RBCs) and subsequent vaso-occlusive crises (VOCs). These crises are responsible for acute pain episodes and potential organ damage, significantly affecting the quality of life for individuals with SCA. The clinical presentation of SCA can be modified by various hemoglobin variants, including hemoglobin C (HbC) and hemoglobin E (HbE), which influence the severity and frequency of VOCs through alterations in red blood cell morphology, oxygen affinity, and inflammatory responses. The presence of hemoglobin variants can affect red blood cell rigidity and aggregation, leading to enhanced vascular occlusion and increased susceptibility to VOCs. Hemoglobin C, for instance, results in more rigid RBCs that readily adhere to the endothelium, while hemoglobin E may reduce the degree of sickling due to its higher oxygen affinity. Furthermore, these variants can modulate the inflammatory response, influencing the recruitment of leukocytes and the activation of endothelial cells, thereby contributing to the overall pathophysiology of VOCs in SCA. Individualized treatment approaches, such as hydroxyurea therapy and emerging gene therapies, can be tailored based on the specific hemoglobin variant present in the patient. Continued research is crucial to elucidate the complex interactions between hemoglobin variants and VOCs, ultimately leading to improved patient outcomes and enhanced quality of life for those affected by sickle cell anemia. Keywords: Sickle cell anemia, hemoglobin variants, vaso-occlusive crises, hemoglobin S, hemoglobin C, hemoglobin E, vascular occlusion, inflammation, red blood cells, therapeutic strategies

Mechanical Characterization of the Erythrocyte Membrane Using a Capacitor-Based Technique.

Pathological processes often change the mechanical properties of cells. Increased rigidity could be a marker of cellular malfunction. Erythrocytes are a type of cell that deforms to squeeze through tiny capillaries; changes in their rigidity can dramatically affect their functionality. Furthermore, differences in the homeostatic elasticity of the cell can be used as a tool for diagnosis and even for choosing the adequate treatment for some illnesses. More accurate types of equipment needed to study biomechanical phenomena at the single-cell level are very costly and thus out of reach for many laboratories around the world. This study presents a simple and low-cost technique to study the rigidity of red blood cells (RBCs) through the application of electric fields in a hand-made microfluidic chamber that uses a capacitor principle. As RBCs are deformed with the application of voltage, cells are observed under a light microscope. From mechanical force vs. deformation data, the elastic constant of the cells is determined. The results obtained with the capacitor-based method were compared with those obtained using optical tweezers, finding good agreement. In addition, P. falciparum-infected erythrocytes were tested with the electric field applicator. Our technique provides a simple means of testing the mechanical properties of individual cells.

Hypertonicity and/or Acidosis Induce Marked Rheological Changes Under Hypoxic Conditions in Sickle Trait Red Blood Cells

Hemoglobin S (HbS) is an abnormal hemoglobin resulting from a single nucleotide substitution in the beta-globin gene. Individuals who are heterozygous for the HbS genotype are regarded as carriers of sickle cell anemia (SCA; HbSS) and are said to have sickle cell trait (SCT; HbAS). Recent data have established SCT as a risk factor for venous thromboembolism (VTE), chronic kidney disease (CKD), and end-stage renal disease (ESRD) (Naik RP, 2018). These complications have a relative risk of approximately 1.5 - 2.0 compared to race- and age-matched controls, but the mechanisms that account for these complications are not well understood. To study potential mechanisms for renal disease, we evaluated red blood cell (RBC) deformability from SCT donors under conditions designed to mimic the hypertonic, acidemic, and hypoxemic environment of the inner renal medulla. RBC deformability was measured using the Laser Optical Rotational Red Cell Analyzer (LORRCA; RR Mechatronics, The Netherlands). In SCA, LORRCA has been used to monitor RBC rigidity at baseline and under progressive hypoxia by calculating the Elongation Index (EI; the longitudinal dimension of the RBC along the axis of applied shear stress divided by transverse dimension of the cell along the same axis). The OxygenScan function measures EI during a cycle of continuously decreasing pO2 from about 150 mmHg (ambient air) to near 20 mm Hg, before reversing in a re-oxygenation phase. In SS RBCs, EI decreases from a stable baseline as RBCs start to sickle; this ‘point of sickling (PoS)‘ is defined as the pO2 at which there is a >5% decrease in EI. Similarly, the Osmoscan function can record the RBC EI at ambient oxygenation while osmolality of the blood progressively increases from approximately 100 to 600 mOsm. First, OxygenScan profiles were performed utilizing modified LORRCA buffers to evaluate four separate conditions: isotonic + neutral pH, isotonic + pH 6.9, 460 mOsm + neutral pH, and 460 mOsm + pH 6.9. Buffers were modified by the addition of sodium chloride, hydrochloric acid, or both. HbS percentage (HbS%) and RBC indices were recorded for each subject. HbS% and RBC indices were compared to PoS for all samples and conditions. EI from AS and AA samples were compared at physiologic serum osmolality (290 mOsm) and at 460 mOsm. AA samples demonstrated no measurable PoS under any condition. Some (11/21) AS samples demonstrated sickling under hypoxia alone, with mean PoS of 32.26 mmHg. Mean PoS for AS samples in isotonic buffer + pH 6.9 was 40.73 mmHg, but with sickling detected in all samples. Mean PoS in hypertonic buffer (460 mOsm) significantly increased to 60.68 mmHg, with notable inter-individual heterogeneity (Fig. 1). Mean PoS in hypertonic buffer + pH 6.9 was 79.04 mmHg, with less inter-sample heterogeneity. In exploratory experiments, we have observed similar changes in the Townes AS murine model of SCT (not shown). PoS was compared to HbS% in the human samples under all conditions. A positive correlation was observed between HbS% and PoS in isotonic buffer at pH 6.9 (r=0.566, p=0.012). PoS was also compared to RBC indices, with a trend towards a positive correlation with mean corpuscular hemoglobin concentration (MCHC) under hypertonic + neutral pH conditions ( p=0.11). Next, utilizing the Osmoscan function, no difference in EI at physiologic osmolality (290 mOsm) was observed between AA (n= 7) vs. AS (n= 8). However, at 460 mOsm, EI trended lower in AS RBCs ( p=0.12) (Fig. 2). Given the presence of severe tissue hypoxia (as low as 10 mmHg), hypertonicity (as high as 1,200 mOsm), and acidemia (as low as pH 5.5) in the inner renal medulla, these data provide an explanation for RBC-mediated occlusion of the vasa recta that is known to occur in the kidneys of subjects with SCT. We demonstrate that extracellular hypertonicity contributes significantly to SCT RBC sickling in the presence of hypoxemia, and that there is significant inter-individual susceptibility to sickling under these harsh biophysical conditions that is only partially explained by HbS%.

Inertial migration of rigid red blood cell particles in Poiseuille flow

Inertial migration of single and multiple red blood cell (RBC) particles in a tube Poiseuille flow is studied using the lattice Boltzmann method coupled with the Galilean invariant momentum exchange method. Different blockage ratios, Reynolds numbers, and solid volume fractions are considered. In the single particle case, the RBC particle migrating in tumbling and rolling modes is called the tumbling particle and rolling particle, respectively. Simulation results show that the tumbling particle has a higher equilibrium position and longer rotation period than the rolling particle. For both the tumbling and rolling particles, when the blockage ratio increases, the equilibrium position moves closer to the tube centerline, and the rotation period becomes longer. The higher Reynolds number strongly affects the angular velocity of the RBC particle, leading the RBC particle to rotate faster; however, the equilibrium position is insensitive to the Reynolds number based on the limited observation. In the multiparticle case, the second and third inner particle annuluses unexpectedly form inside the Segré–Silberberg annulus as the solid volume fraction increases. Furthermore, smaller particles form the perfect Segré–Silberberg annulus at a lower solid volume fraction than larger particles. The third inner annulus of smaller particles is located further from the tube centerline than that of larger particles. This work is beneficial for understanding the motion of RBC particles in Poiseuille flow. It can positively contribute to the design of microfluidic devices for focusing, separating, and transporting red blood cells.

Mathematical Analysis for the Flow of Sickle Red Blood Cells in Microvessels for Bio Medical Application.

Sickle cell disease (SCD) is an inherited monogenic disease which is characterized by distorted red blood cells (RBCs) that cause vaso-occlusion and vasculopathy. In the pathogenesis of SCD, polymerized hemoglobin turn RBCs into fragile, less deformable cells, and are subsequently more susceptible to endothelial adhesion after deoxygenation. Presently, electrophoresis and genotyping are used as routine tests for diagnosis of SCD. These techniques are expensive and require specialized laboratories. Lab-on-a-chip technology is a low-cost microfluidics-based diagnostic tool which holds significant promise for rapid screening of RBC deformability. To explore the sickle RBC mechanics for screening purposes, we present a mathematical model for the flow of single RBC with altered rheological properties and slip effect on capillary wall in microcirculation. We consider single-file flow of cells through the axis symmetrical cylindrical duct, applying lubrication theory as plasma trapped between successive red blood cells. The rheological parameters used from published literature for normal RBC and corresponding variation has been taken for the purpose of this simulation to present the condition of the disease. An analytical solution has been found for realistic boundary conditions and results are simulated using MATLAB. We found that the height of plasma film in the capillary increases with increase in cell deformability and compliance which affects the forward flow velocity in the capillary. Rigid RBCs with increased adhesion between cell and capillary wall shows reduction in velocity and occurrence of vaso-occlusion events in extreme conditions. These rheological properties of the cells coupled with microfluidics mechanics can mimic the physiological condition and provides unique insights with novel possibilities for the design of microfluidics base diagnostic kit towards effective therapeutic intervention of SCD.

Red blood cell lingering modulates hematocrit distribution in the microcirculation

The distribution of red blood cells (RBCs) in the microcirculation determines the oxygen delivery and solute transport to tissues. This process relies on the partitioning of RBCs at successive bifurcations throughout the microvascular network, and it has been known since the last century that RBCs partition disproportionately to the fractional blood flow rate, therefore leading to heterogeneity of the hematocrit (i.e., volume fraction of RBCs in blood) in microvessels. Usually, downstream of a microvascular bifurcation, the vessel branch with a higher fraction of blood flow receives an even higher fraction of RBC flux. However, both temporal and time-average deviations from this phase-separation law have been observed in recent studies. Here, we quantify how the microscopic behavior of RBC lingering (i.e., RBCs temporarily residing near the bifurcation apex with diminished velocity) influences their partitioning, through combined in vivo experiments and in silico simulations. We developed an approach to quantify the cell lingering at highly confined capillary-level bifurcations and demonstrate that it correlates with deviations of the phase-separation process from established empirical predictions by Pries et al. Furthermore, we shed light on how the bifurcation geometry and cell membrane rigidity can affect the lingering behavior of RBCs; e.g., rigid cells tend to linger less than softer ones. Taken together, RBC lingering is an important mechanism that should be considered when studying how abnormal RBC rigidity in diseases such as malaria and sickle-cell disease could hinder the microcirculatory blood flow or how the vascular networks are altered under pathological conditions (e.g., thrombosis, tumors, aneurysm).

Sublingual Microcirculation Specificity of Sickle Cell Patients: Morphology of the Microvascular Bed, Blood Rheology, and Local Hemodynamics

Patients with sickle cell disease (SCD) have poorly deformable red blood cells (RBC) that may impede blood flow into microcirculation. Very few studies have been able to directly visualize microcirculation in humans with SCD. Sublingual video microscopy was performed in eight healthy (HbAA genotype) and four sickle cell individuals (HbSS genotype). Their hematocrit, blood viscosity, red blood cell deformability, and aggregation were individually determined through blood sample collections. Their microcirculation morphology (vessel density and diameter) and microcirculation hemodynamics (local velocity, local viscosity, and local red blood cell deformability) were investigated. The De Backer score was higher (15.9 mm-1) in HbSS individuals compared to HbAA individuals (11.1 mm-1). RBC deformability, derived from their local hemodynamic condition, was lower in HbSS individuals compared to HbAA individuals for vessels < 20 μm. Despite the presence of more rigid RBCs in HbSS individuals, their lower hematocrit caused their viscosity to be lower in microcirculation compared to that of HbAA individuals. The shear stress for all the vessel diameters was not different between HbSS and HbAA individuals. The local velocity and shear rates tended to be higher in HbSS individuals than in HbAA individuals, notably so in the smallest vessels, which could limit RBC entrapment into microcirculation. Our study offered a novel approach to studying the pathophysiological mechanisms of SCD with new biological/physiological markers that could be useful for characterizing the disease activity.

Cell-free layer development and spatial organization of healthy and rigid red blood cells in a microfluidic bifurcation.

Bifurcations and branches in the microcirculation dramatically affect blood flow as they determine the spatiotemporal organization of red blood cells (RBCs). Such changes in vessel geometries can further influence the formation of a cell-free layer (CFL) close to the vessel walls. Biophysical cell properties, such as their deformability, which is impaired in various diseases, are often thought to impact blood flow and affect the distribution of flowing RBCs. This study investigates the flow behavior of healthy and artificially hardened RBCs in a bifurcating microfluidic T-junction. We determine the RBC distribution across the channel width at multiple positions before and after the bifurcation. Thus, we reveal distinct focusing profiles in the feeding mother channel for rigid and healthy RBCs that dramatically impact the cell organization in the successive daughter channels. Moreover, we experimentally show how the characteristic asymmetric CFLs in the daughter vessels develop along their flow direction. Complimentary numerical simulations indicate that the buildup of the CFL is faster for healthy than for rigid RBCs. Our results provide fundamental knowledge to understand the partitioning of rigid RBC as a model of cells with pathologically impaired deformability in complex in vitro networks.

Extracellular Vesicles from Sickle Red Blood Cells Activate Endothelial Cells and Trigger Von Willebrand Factor-Mediated Sickle Red Cell Adhesion

Introduction: The sickle mutation of hemoglobin (HbS) afflicts millions of people worldwide and is characterized by hemolytic anemia, inflammation, painful vaso-occlusive crises, significant morbidity, and early mortality. In sickle cell disease (SCD), HbS polymerization under deoxygenation increases red blood cell (RBC) rigidity, adhesivity and susceptibility to lysis. Products of intravascular hemolysis including heme and RBC-released extracellular vesicles (REVs) promote acute and chronic inflammation triggering endothelial activation and abnormal RBC adhesion. Extracellular vesicles (EVs) are nano- or micro-particles composed of a lipid bilayer, comprised of transmembrane proteins, and enclosing intracellular remnants, including cytosolic proteins and miRNAs. EVs are known to exchange biomaterial between cells, and may serve as surrogate markers for the activated state of the parent cell thus are of high clinical significance. Quantitatively, REVs are more abundant from sickle RBCs (SS RBCs) compared with normal HbA-containing RBCs (AA RBCs). SS REVs are known to promote endothelial cell (EC) activation through cell signaling and transcriptional regulation at longer terms. However, the SS REV-mediated short term non transcriptional response of EC is unclear. Methods: Here, we have developed the SCD-EV-BioChip using microfluidic technology. SCD-EV-BioChip assesses RBC adhesion as a biomarker for REV-mediated human pulmonary microvascular endothelial cells (HPMECs) dysfunction using functional biomarker of RBC adhesion. Utilizing SCD-EV-BioChip, Here, we examined the impact of SS REVs on acute microvascular EC activation and RBC adhesion at 2 hours. For in vitro studies, SS REVs and AA REVs were collected from pooled human SS RBCs and AA RBCs activated using calcium ionophore. The generated REVs were characterized using scanning electron microscopy (SEM) and nanoparticle tracking analyzer (NTA). HPMECs were cultured in microfluidic channels under controlled shear stress for more than 72 hours then treated with REVs at 37 ºC for 2 hours. Von Willebrand factor (vWF) expression on REVs-treated HPMECs were quantified. RBC adhesion tests were conducted with and without vWF protease ADAMTS13 and heme-binding protein hemopexin under shear stress at 1 dyne/cm2 followed by a rinse step and quantification of adhered RBCs. For in vivo study, HbSS-Townes mice and a dorsal skin-fold chamber model was used to determine if ADAMTS13 would reduce microvascular stasis in response to hemin. Results: Within 2 hours, SS REVs, but not AA REVs, promoted increased levels of vWF expression on HPMECs, indicating HPMEC activation (Fig. 1A-C). VWF is known to trigger SRBC adhesion and vaso-occlusion. Using SCD-EV-BioChip, we found significantly increased SS RBC adhesion to HPMECs exposed to SREVs, compared to SRBC adhesion to HPMECs exposed to AA REVs, and AA RBC adhesion to HPMECs exposed to SS REVs (Fig. 1D-G). SS RBC adhesion was significantly reduced on HPMECs treated with SS REVs pre-incubated with hemopexin. ADAMTS13 is a regulator which is responsible for cleaving ultra large vWF. In this study, SS RBC adhesion was significantly reduced by vWF cleaving protease ADAMTS13 to a level similar to HPMECs treated with AA REVs (Fig. 1G). Consistent with these observations, studies in SS mice with implanted dorsal skin-fold chambers found hemin-induced stasis was inhibited by ADAMTS13 (Fig. 1F). SRBC adhesion was variable within a total of 15 test subjects with SCD, and was higher with SRBCs from patients with increased markers of hemolysis (LDH and reticulocyte count) or a concomitant clinical diagnosis of deep vein thrombosis. Conclusions: This work demonstrated the acute, non-transcriptional contribution made by REVs to the microvascular EC activation and vWF-mediated SRBC adhesion. Results from SCD-EV-BioChip indicated the effect of hemopexin in mitigating SREV-mediated HPMEC activation. Additionally, results from in vitro SCD-EV BioChip tests agreed with in vivo stasis tests using SS mice, both of which indicated the effect of ADAMTS13 in mitigating SS RBC adhesion and vaso-occlusion. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

PSUN168 The relationship between vitamin C concentrations and reduced red blood cell deformability in diabetes

Abstract Background Microvascular angiopathy is a common complication for patients with diabetes. Reduced red blood cell (RBC) deformability in diabetes has been associated with micro- and macrovascular complications such as diabetic nephropathy and coronary artery disease. Preliminary studies in mice have shown an inverse relationship between RBC vitamin C concentrations and RBC deformability measures. This study investigated the relationship between RBC vitamin C concentration and cell deformability measures. Methods We analyzed RBC deformability and vitamin C level in a cross-sectional cohort study of individuals with diabetes (n=82) and non-diabetic controls (n=80). Subjects were studied outpatient, with samples obtained following an overnight fast. RBC rigidity parameters defined as SS1/2 (shear stress at which half of the maximum deformability occurs) was assessed using the laser diffraction viscometry ektacytometer (LORRCA, Mechatronics, Netherland). We also quantified osmotic sensitivity by measuring deformability under an osmotic gradient (osmoScan, o-hyper; osmolality corresponding to 50% of the Elmax). Vitamin C in plasma was measured using coulometric electrochemical detection with high performance liquid chromatography. We evaluated the association between these deformability parameters, vitamin C concentration. Results Compared to the non-diabetic cohort, the diabetes cohort had significantly lower RBC vitamin C concentration, respectively 28.1µM and 42.9µM (p=0.0013). Low RBC vitamin C concentration was significantly associated with reduce RBC deformability measured by increased SS1/2 (p=0.009). Additionally, low RBC vitamin C was associated with decreased o-hyper, suggesting a decrease dehydration of RBC (p=0.0250). Conclusion Our data demonstrate reduced vitamin c levels in the diabetic cohort. Reduced RBC deformability is associated with reduced RBC vitamin C concentration. Thus, reduced vitamin C levels in diabetic patients might increase red blood cell rigidity and reduce deformability. Presentation: Sunday, June 12, 2022 12:30 p.m. - 2:30 p.m.

Diverse Approaches to Gene Therapy of Sickle Cell Disease.

Sickle cell disease (SCD) results from a single base pair change in the sixth codon of the β-globin chain of hemoglobin, which promotes aggregation of deoxyhemoglobin, increasing rigidity of red blood cells and causing vaso-occlusive and hemolytic complications. Allogeneic transplant of hematopoietic stem cells (HSCs) can eliminate SCD manifestations but is limited by absence of well-matched donors and immune complications. Gene therapy with transplantation of autologous HSCs that are gene-modified may provide similar benefits without the immune complications. Much progress has been made, and patients are realizing significant clinical improvements in multiple trials using different approaches with lentiviral vector-mediated gene addition to inhibit hemoglobin aggregation. Gene editing approaches are under development to provide additional therapeutic opportunities. Gene therapy for SCD has advanced from an attractive concept to clinical reality.

Incidence of eye complications among sickle cell disease patients in Jeddah, Saudi Arabia: A cross-sectional study

IntroductionSickle cell disease (SCD) is a heritable blood disorder resulting in deformed, rigid red blood cells, rendering them more prone to vaso-occlusion. Ocular complications are known to affect multiple organs through the body's vasculature. Here, we evaluated the incidence of eye complications in patients with SCD at King Abdulaziz Medical City in Jeddah, Saudi Arabia. Materials and methodsWe used a cross-sectional approach and collected relevant medical data from nine patients with SCD. Ophthalmic assessment included visual acuity and an in-clinic dilated fundus examination. All patients were asked to attend the ophthalmology clinic to undergo optical coherence tomography (OCT) angiography, macular OCT scan, and fundus photography. The results of the imaging tests were interpreted by a certified ophthalmology consultant specializing in retinal diseases. Descriptive analyses of the results were also performed. ResultsThe mean age of the nine patients was 24.78 ± 10.9 years. All patients were non-hypertensive, one had type 1 diabetes, and another had type 2 diabetes. Five patients had normal visual acuity, two had slight impairments in at least one eye, and two had moderate impairment in at least one eye. One of the nine patients exhibited retinal ischemia in the right eye despite normal macular thickness (visual acuity, OD, 6/30; OS, 6/21). The remaining eight patients showed no ocular abnormalities. ConclusionOf the nine patients with SCD, five showed no symptoms of ocular complications. One patient showed retinal ischemia in the right eye, despite a normal macular thickness. This study's results suggest routine ophthalmologic examination may not be able to detect or monitor macular or retinal abnormalities unless augmented with detailed imaging techniques.

Cryopreservation of rare pediatric red blood cells for support following bone marrow transplant.

A 2-year-old, 10.8kg male pediatric patient with X-linked chronic granulomatous disease (CGD) with McLeod syndrome (MLS) was scheduled for a hematopoietic stem cell transplant (HSCT). Identification of allogenic red blood cells (RBC) for post-transplant support was unsuccessful prompting the development of a customized method to collect and freeze rare autologous pediatric cells. A protocol was developed for the collection of small volume pediatric whole blood (WB) via peripheral venipuncture with collection into 10ml syringes containing anticoagulants. Additionally, a closed system RBC glycerolization and deglycerolization instrument was adapted to process small volume, non-leukoreduced WB. Both collection and WB processes were validated. In total 4 approximately 100 ml autologous units were collected and frozen. Two units were thawed, deglycerolized, and used for clinical transfusion support. To appreciate processing impacts on RBC rigidity, ektacytometry was performed on pre-processed and post-deglycerolization samples. Free hemoglobin (HGB) of validation units after thawing/deglycerolization was <150 mg/dL with an average red cell recovery of 85%. These units also showed little difference between pre-and post-processing Lorrca deformability curves or membrane rigidity. Two pediatric units were thawed and deglycerolized for transfusion. Free HGB was 70 mg/dL and 50 mg/dL post-thaw, and these RBCs had a slight decrease in deformability and increased membrane rigidity. Customized WB collection, glycerolization, freezing, and deglycerolization processes were developed to successfully support a pediatric patient with CGD and MLS after autologous HSCT. Both pediatric units showed increased membrane rigidity post-deglycerolization which may be a consequence of the CGD and MLS genetic background.

The effect of stiffened diabetic red blood cells on wall shear stress in a reconstructed 3D microaneurysm

Blood flow within the vasculature of the retina has been found to influence the progression of diabetic retinopathy. In this research cell resolved blood flow simulations are used to study the pulsatile flow of whole blood through a segmented retinal microaneurysm. Images were collected using adaptive optics optical coherence tomography of the retina of a patient with diabetic retinopathy, and a sidewall (sacciform) microaneurysm was segmented from the volumetric data. The original microaneurysm neck width was varied to produce two additional aneurysm geometries in order to probe the influence of neck width on the transport of red blood cells and platelets into the aneurysm. Red blood cell membrane stiffness was also increased to resolve the impact of rigid red blood cells, as a result of diabetes, in blood flow. Wall shear stress and wall shear stress gradients were calculated throughout the aneurysm domains, and the quantification of the influence of the red blood cells is presented. Average wall shear stress and wall shear stress gradients increased due to the increase of red blood cell membrane stiffness. Stiffened red blood cells were also found to induce higher local wall shear stress and wall shear stress gradients as they passed through the leading and draining parental vessels. Stiffened red blood cells were found to penetrate the aneurysm sac more than healthy red blood cells, as well as decreasing the margination of platelets to the vessel walls of the parental vessel, which caused a decrease in platelet penetration into the aneurysm sac.

Reusable Device for the Electrical Sensing of Red Blood Cells Rigidity Abnormalities, Based on A Reversible Microfluidic Assembly.

Combining microfluidic with sensors enables the development of smart analysis systems. Microelectrodes can be embedded within the microchannels network for electrical sensing, electrochemical analysis or impedance measurement. However, at the laboratory scale, the assembly between microfluidic network and electrical parts on the substrate remains an issue. This paper first discusses the principles of biosensing, and then proposes an original device integrating microfluidics with microelectrodes for the analysis of red blood cells transit in a structure mimicking micro-vascular flow. Some results concerning red blood cells discrimination of sickle cell disease are discussed with statistical analysis.Clinical relevance- This paper introduces a portable reusable device combining a microfluidic blood vessel mimicking network with microelectrodes for the biosensing of RBC.

To deform or not to deform: the evolutionary basis of mammalian red blood cell deformability

To deform or not to deform: the evolutionary basis of mammalian red blood cell deformability

Classification of Red Blood Cell Rigidity from Sequence Data of Blood Flow Simulations Using Neural Networks

Numerical models for the flow of blood and other fluids can be used to design and optimize microfluidic devices computationally and thus to save time and resources needed for production, testing, and redesigning of the physical microfluidic devices. Like biological experiments, computer simulations have their limitations. Data from both the biological and the computational experiments can be processed by machine learning methods to obtain new insights which then can be used for the optimization of the microfluidic devices and also for diagnostic purposes. In this work, we propose a method for identifying red blood cells in flow by their stiffness based on their movement data processed by neural networks. We describe the performed classification experiments and evaluate their accuracy in various modifications of the neural network model. We outline other uses of the model for processing data from video recordings of blood flow. The proposed model and neural network methodology classify healthy and more rigid (diseased) red blood cells with the accuracy of about 99.5% depending on the selected dataset that represents the flow of a suspension of blood cells of various levels of stiffness.

The Erythrocyte Sedimentation Rate and Its Relation to Cell Shape and Rigidity of Red Blood Cells from Chorea-Acanthocytosis Patients in an Off-Label Treatment with Dasatinib.

Background: Chorea-acanthocytosis (ChAc) is a rare hereditary neurodegenerative disease with deformed red blood cells (RBCs), so-called acanthocytes, as a typical marker of the disease. Erythrocyte sedimentation rate (ESR) was recently proposed as a diagnostic biomarker. To date, there is no treatment option for affected patients, but promising therapy candidates, such as dasatinib, a Lyn-kinase inhibitor, have been identified. Methods: RBCs of two ChAc patients during and after dasatinib treatment were characterized by the ESR, clinical hematology parameters and the 3D shape classification in stasis based on an artificial neural network. Furthermore, mathematical modeling was performed to understand the contribution of cell morphology and cell rigidity to the ESR. Microfluidic measurements were used to compare the RBC rigidity between ChAc patients and healthy controls. Results: The mechano-morphological characterization of RBCs from two ChAc patients in an off-label treatment with dasatinib revealed differences in the ESR and the acanthocyte count during and after the treatment period, which could not directly be related to each other. Clinical hematology parameters were in the normal range. Mathematical modeling indicated that RBC rigidity is more important for delayed ESR than cell shape. Microfluidic experiments confirmed a higher rigidity in the normocytes of ChAc patients compared to healthy controls. Conclusions: The results increase our understanding of the role of acanthocytes and their associated properties in the ESR, but the data are too sparse to answer the question of whether the ESR is a suitable biomarker for treatment success, whereas a correlation between hematological and neuronal phenotype is still subject to verification.

Characterizing bulk rigidity of rigid red blood cell populations in sickle-cell disease patients

In this work, we utilized a parameterization model of ektacytometry to quantify the bulk rigidity of the rigid red blood cell (RBC) population in sickle cell disease (SCD) patients. Current ektacytometry techniques implement laser diffraction viscometry to estimate the RBC deformability in a whole blood sample. However, the diffraction measurement is an average of all cells present in the measured sample. By coupling an existing parameterization model of ektacytometry to an artificially rigid RBC model, we formulated an innovative system for estimating the average rigidity of the rigid RBC population in SCD blood. We demonstrated that this method could more accurately determine the bulk stiffness of the rigid RBC populations. This information could potentially help develop the ektacytometry technique as a tool for assessing disease severity in SCD patients, offering novel insights into the disease pathology and treatment.